#

Lower Limbs BMD Upper Limbs-BMD Total BMD

**Figure 3.** Analysis of bone mineral density (BMD) in high and low level paraplegics and controls. Diagram modified

**Figure 2.** Whole body and regional distribution of fat mass, lean mass, bone mineral content (BMC) and bone mineral density (BMD) from controls male subject using whole body DEXA Norland X-36 and values of measured parameters. Modified and translated with permission from Dionyssiotis, Doctoral Dissertation, Laboratory for Research of the Mus‐ culoskeletal System, University of Athens, 2008 [39].

#### **3. Physiopathological context**

Spinal cord injury (SCI) always results in substantial and rapid bone loss predominately in areas below the neurological level of injury. The predominant finding of SCI on bone is a large loss of bone during the first year of injury [5] and an ongoing demineralisation 3 years after trauma in tibia [52] with a progressive bone loss over 12 to 16 months prior to stabilizing [53] was demonstrated.

Cancellous bone is more affected than cortical bone after SCI. In a prospective study, six acute tetraplegics were followed up for 12 months, and the trabecular and cortical BMD's of the tibia were found to be decreased by 15 and 7% [54], while in paraplegics trabecular metaphysicalepiphyseal areas of the distal femur and the proximal tibia are the most affected sites [55]. A cross-sectional study [56] in SCI subjects demonstrated a significant demineralization at the distal femur (-52%) and the proximal tibia (-70%), respectively.

There is no demineralization of the upper limbs in paraplegics. On the contrary, a minor increase of BMD (6%) in the humerus was reported in a cross-sectional study of 31 male chronic paraplegics 1 year post injury. With reliance on the upper limbs to provide movement for activities of daily living in the SCI population, this area could be subjected to greater sitespecific loading, and thus increasing osteogenesis, than in the corresponding able-bodied population. At the lumbar spine, the trabecular bone demineralization remains relatively low compared to the cortical bone demineralization of long bones [56]. Normal [52, 57] or even higher than normal [58] values of BMD in the lumbar spine have been reported a phenomenon is named "dissociated hip and spine demineralization" [54]. One reason for preservation of bone mass in the vertebral column is because of its continued weight-bearing function in paraplegics. In a cross-sectional study of 135 SCI men, BMD in the lumbar spine was found to be stable with an insignificant decline in the tetraplegic population at 1±5 years post injury in the 20–39-year age group, whereas in the 40-59-year age group and the 60+-year age group, bone mass in the lumbar spine remained unchanged or even increased with age [49]. However, several factors may affect the results of BMD measurement: lumbar spine arthrosis, bone callus, vertebral fracture, aortic calcification, osteosynthesis material, etc. Degenerative changes in the spine may be the most possible reason to give falsely higher values of BMD [56]. An interesting question is why we don't see osteoporotic vertebral fractures in SCI patients to the extent it occurs in post-menopausal osteoporotic women or senile osteoporotic men?

muscle mass would be overestimated by prediction models that assume that muscle represents all or a certain proportion of the fat-free soft tissue mass, i.e. in spinal cord injured subjects [7]. DXA technique has been used in assessment of SCI and appears to be

**Figure 2.** Whole body and regional distribution of fat mass, lean mass, bone mineral content (BMC) and bone mineral density (BMD) from controls male subject using whole body DEXA Norland X-36 and values of measured parameters. Modified and translated with permission from Dionyssiotis, Doctoral Dissertation, Laboratory for Research of the Mus‐

Spinal cord injury (SCI) always results in substantial and rapid bone loss predominately in areas below the neurological level of injury. The predominant finding of SCI on bone is a large loss of bone during the first year of injury [5] and an ongoing demineralisation 3 years after trauma in tibia [52] with a progressive bone loss over 12 to 16 months prior to stabilizing [53]

Cancellous bone is more affected than cortical bone after SCI. In a prospective study, six acute tetraplegics were followed up for 12 months, and the trabecular and cortical BMD's of the tibia were found to be decreased by 15 and 7% [54], while in paraplegics trabecular metaphysicalepiphyseal areas of the distal femur and the proximal tibia are the most affected sites [55]. A cross-sectional study [56] in SCI subjects demonstrated a significant demineralization at the

There is no demineralization of the upper limbs in paraplegics. On the contrary, a minor increase of BMD (6%) in the humerus was reported in a cross-sectional study of 31 male chronic

distal femur (-52%) and the proximal tibia (-70%), respectively.

Figure 1. Whole body and regional distribution of fat mass, lean mass, bone mineral content (BMC) and bone mineral density (BMD) from paraplegic subject thoracic 6 (left picture) using whole body DXA (Norland X-36, Fort Atkinson, Wisconsin, USA) and values of measured parameters. Modified and translated with permission from: Dionyssiotis Y. (2008a). Doctoral Dissertation, Laboratory for Research of the Musculoskeletal System,

tolerated well by this population [49, 50, 51].

culoskeletal System, University of Athens, 2008 [39].

**3. Physiopathological context**

was demonstrated.

160 Topics in Paraplegia

University of Athens, Greece,

Figure 3 depicts the analysis of bone mineral density (BMD) in high and low level paraple‐ gics and controls. A statistically significant reduction in total BMD (p<0.001) and lower limbs BMD in body composition compared to able-bodied males was observed. On the con‐ trary, upper limbs BMD was higher in low paraplegics and controls, an unexpected finding explained in the paper of Dionyssiotis et al. [19].

**Figure 3.** Analysis of bone mineral density (BMD) in high and low level paraplegics and controls. Diagram modified and translated from Dionyssiotis Y [39].

The neurological level of the lesion i.e. the extent of impairment of motor and sensory function is important, because tetraplegics are more likely to lose more bone mass throughout the skeleton than paraplegics [60]. In paraplegics legs' BMC was reduced vs. controls, independently of the neurological level of injury and negatively correlated with the duration of paralysis in total paraplegic group, but after investigation according to the neurological level of injury this correlation was due to the strong correlation of high paraplegics' legs BMC with the duration of paralysis, meaning that the neurological level of injury determines the extent of bone loss [61]. The similar severity of demineralization in the sublesional area was shown between paraplegics and tetraplegics, and the extent of the bone loss may be variable [56, 60, 62].

extensor muscles [65]. Other investigators have not established a correlation between BMD

Body Composition in Paraplegia http://dx.doi.org/10.5772/58539 163

Studies also emphasize the contribution of aging to bone loss in complete SCI patients. Moderate correlation between age and femoral BMD was observed in a cross-sectional study of 30 patients with SCI of 1-year duration or less [69]. On the other side bone loss in eight pairs of identical male twins with SCI of duration ranging from 3 to 26 years appeared to be

Muscular loading of the bones has been thought to play a role in the maintenance of bone density. The ability to stand or ambulate itself does not improve BMD and does not prevent osteoporosis after SCI, although exercise increases site-specific osteogenesis in able-bodied individuals [71]. There was only one study demonstrating that standing might reduce the loss of trabecular bone after SCI. In this prospective study of 19 acute SCΙ patients, the patients involved in early loading intervention exercise lost almost no bone mineral, whereas the

Muscles rather than body weight are causing the greatest loads on bone [72]. It is diffi‐ cult to translate in vivo bone strains from animal work to a gross loading environment for humans. However, the pioneering work in animal models [73] suggests that if the active– resistive standing exercise can indeed transmit loads at an appropriate frequency and strainrate, compressive loads approaching 240 % body weight may have the potential to be

FES cycling [75] and quadriceps muscle training [76] have been able to increase forcegenerating capability and to improve muscular endurance with training after SCI [75]. Conversely, cycling with FES has been reported to induce only small improvements in BMD [77, 78] as well as have no effect [78] on lower extremity BMD measurements in individu‐ als with SCI. Additionally, neither passive standing, ambulation with long-leg braces, nor ambulation with FES have yet to exhibit any improvement in lower extremity BMD in chronically injured subjects [79]. The subject populations of previous BMD studies were comprised almost exclusively of individuals with chronic rather than acute SCI. these interventional BMD studies may have utilized sub-threshold mechanical stimuli. The use of relatively low bone loading regimens is not unexpected due to the extensive atrophy of chronically paralyzed muscle [80, 81] and concerns of fracture, which have been reported

The role of leptin: The hormone leptin is secreted by fat cells and help regulate body weight and energy consumption [84]. The amount of leptin in the circulation is positively correlated with the percentage of fat in people [85]. In paraplegics, when compared with healthy subjects, higher levels of leptin have been found, possibly due to greater fat tissue storage [86, 87]. Leptin activates the sympathetic nervous system (SNS) through a central administration. The disruption of the sympathetic nervous system may modify the secretion and activity of the leptin, because the sympathetic preganglionic neurons become atrophic in high paraplegics [88, 89]. The irritation thus, below the neurological level of injury, from the leptin is disturbed. In addition, extensive obesity is known to reduce lipolytic sensitivity [89, 90, 91]. Given that

immobilization patients lost 6.9 to 9.4% of trabecular bone [66].

to occur with physical interventions [82, 83].

and muscle spasticity [68].

independent of age [70].

osteogenic [73, 74].

In addition, in those SCI individuals with complete lesions (an absence of sensory or motor function below the neurological level, including the lowest sacral segment) bone loss is more severe than subjects with incomplete lesions (partial preservation of motor and/or sensory function below the neurological level, including the lowest sacral segment) [62, 63]. In a crosssectional study of 11 patients with complete SCI and 30 patients with incomplete SCI noticed a significant osteopenia in patients with complete SCI than in patients with incomplete SCI [62].

The duration of paralysis has an inverse relationship with leg percentage-matched BMD and trunk percentage-matched BMD [64]. In addition in complete paraplegics, with high (thoracic 4-7) and low (thoracic 8-12) neurological level of injury, upper limbs FM and lower limbs BMD were correlated with the duration of paralysis in total paraplegic group but after investigation according the neurological level of injury this correlation was due to the strong correlation of high paraplegics' lower limbs BMD with the duration of paralysis. The explanation of this strong correlation could possibly lie on higher incidence of standing in the group of low paraplegics and direct effect of loading lower limbs while standing and walking with orthotic equipment. Moreover, the association of the duration of paralysis with parameters below and above the neurological level of injury (upper limbs FM) raises the question of the existence of a hormonal mechanism as an influential regulator in paraplegics' body composition [19, 61, 65].

Is there a time after injury where bone loss ceases? Some authors reported that approximately 2 years after SCI, a new steady state level between bone resorption and formation would be re-established [52, 57], whereas others [66] found that there was no sign of a new steady state in bone formation in the lower extremities 2 years after the SCI. If a new steady state of bone remodelling is re-established after SCI still remains controversial.

Inconsistent results have been reported regarding the effect of muscle spasms on BMD in SCI patients. Those with spasticity were found with higher BMD when compared with flaccid individuals [62], and a significant correlation between the degree of spasticity measured with modified Ashworth scale and BMD was reported. Thus, it was concluded that spasticity may be protective against bone loss in SCI patients [67]; however, without any preserving effect in the tibia [65, 67]. A possible explanation for that could lie in the fact paraplegics to be above thoracic (T)12 level with various degrees of spasticity according to the Ashworth scale. In addition, muscle spasms affecting the lower leg would mainly be extension spasms resulting in plantar flexion thus creating little resistance to the contracting muscles. Furthermore, the measuring sites of the tibia did not include any muscle insertions of either the knee or the ankle extensor muscles [65]. Other investigators have not established a correlation between BMD and muscle spasticity [68].

The neurological level of the lesion i.e. the extent of impairment of motor and sensory function is important, because tetraplegics are more likely to lose more bone mass throughout the skeleton than paraplegics [60]. In paraplegics legs' BMC was reduced vs. controls, independently of the neurological level of injury and negatively correlated with the duration of paralysis in total paraplegic group, but after investigation according to the neurological level of injury this correlation was due to the strong correlation of high paraplegics' legs BMC with the duration of paralysis, meaning that the neurological level of injury determines the extent of bone loss [61]. The similar severity of demineralization in the sublesional area was shown between paraplegics and tetraplegics, and the extent of

In addition, in those SCI individuals with complete lesions (an absence of sensory or motor function below the neurological level, including the lowest sacral segment) bone loss is more severe than subjects with incomplete lesions (partial preservation of motor and/or sensory function below the neurological level, including the lowest sacral segment) [62, 63]. In a crosssectional study of 11 patients with complete SCI and 30 patients with incomplete SCI noticed a significant osteopenia in patients with complete SCI than in patients with incomplete SCI [62]. The duration of paralysis has an inverse relationship with leg percentage-matched BMD and trunk percentage-matched BMD [64]. In addition in complete paraplegics, with high (thoracic 4-7) and low (thoracic 8-12) neurological level of injury, upper limbs FM and lower limbs BMD were correlated with the duration of paralysis in total paraplegic group but after investigation according the neurological level of injury this correlation was due to the strong correlation of high paraplegics' lower limbs BMD with the duration of paralysis. The explanation of this strong correlation could possibly lie on higher incidence of standing in the group of low paraplegics and direct effect of loading lower limbs while standing and walking with orthotic equipment. Moreover, the association of the duration of paralysis with parameters below and above the neurological level of injury (upper limbs FM) raises the question of the existence of a hormonal mechanism as an influential regulator in paraplegics' body composition [19, 61, 65]. Is there a time after injury where bone loss ceases? Some authors reported that approximately 2 years after SCI, a new steady state level between bone resorption and formation would be re-established [52, 57], whereas others [66] found that there was no sign of a new steady state in bone formation in the lower extremities 2 years after the SCI. If a new steady state of bone

Inconsistent results have been reported regarding the effect of muscle spasms on BMD in SCI patients. Those with spasticity were found with higher BMD when compared with flaccid individuals [62], and a significant correlation between the degree of spasticity measured with modified Ashworth scale and BMD was reported. Thus, it was concluded that spasticity may be protective against bone loss in SCI patients [67]; however, without any preserving effect in the tibia [65, 67]. A possible explanation for that could lie in the fact paraplegics to be above thoracic (T)12 level with various degrees of spasticity according to the Ashworth scale. In addition, muscle spasms affecting the lower leg would mainly be extension spasms resulting in plantar flexion thus creating little resistance to the contracting muscles. Furthermore, the measuring sites of the tibia did not include any muscle insertions of either the knee or the ankle

the bone loss may be variable [56, 60, 62].

162 Topics in Paraplegia

remodelling is re-established after SCI still remains controversial.

Studies also emphasize the contribution of aging to bone loss in complete SCI patients. Moderate correlation between age and femoral BMD was observed in a cross-sectional study of 30 patients with SCI of 1-year duration or less [69]. On the other side bone loss in eight pairs of identical male twins with SCI of duration ranging from 3 to 26 years appeared to be independent of age [70].

Muscular loading of the bones has been thought to play a role in the maintenance of bone density. The ability to stand or ambulate itself does not improve BMD and does not prevent osteoporosis after SCI, although exercise increases site-specific osteogenesis in able-bodied individuals [71]. There was only one study demonstrating that standing might reduce the loss of trabecular bone after SCI. In this prospective study of 19 acute SCΙ patients, the patients involved in early loading intervention exercise lost almost no bone mineral, whereas the immobilization patients lost 6.9 to 9.4% of trabecular bone [66].

Muscles rather than body weight are causing the greatest loads on bone [72]. It is diffi‐ cult to translate in vivo bone strains from animal work to a gross loading environment for humans. However, the pioneering work in animal models [73] suggests that if the active– resistive standing exercise can indeed transmit loads at an appropriate frequency and strainrate, compressive loads approaching 240 % body weight may have the potential to be osteogenic [73, 74].

FES cycling [75] and quadriceps muscle training [76] have been able to increase forcegenerating capability and to improve muscular endurance with training after SCI [75]. Conversely, cycling with FES has been reported to induce only small improvements in BMD [77, 78] as well as have no effect [78] on lower extremity BMD measurements in individu‐ als with SCI. Additionally, neither passive standing, ambulation with long-leg braces, nor ambulation with FES have yet to exhibit any improvement in lower extremity BMD in chronically injured subjects [79]. The subject populations of previous BMD studies were comprised almost exclusively of individuals with chronic rather than acute SCI. these interventional BMD studies may have utilized sub-threshold mechanical stimuli. The use of relatively low bone loading regimens is not unexpected due to the extensive atrophy of chronically paralyzed muscle [80, 81] and concerns of fracture, which have been reported to occur with physical interventions [82, 83].

The role of leptin: The hormone leptin is secreted by fat cells and help regulate body weight and energy consumption [84]. The amount of leptin in the circulation is positively correlated with the percentage of fat in people [85]. In paraplegics, when compared with healthy subjects, higher levels of leptin have been found, possibly due to greater fat tissue storage [86, 87]. Leptin activates the sympathetic nervous system (SNS) through a central administration. The disruption of the sympathetic nervous system may modify the secretion and activity of the leptin, because the sympathetic preganglionic neurons become atrophic in high paraplegics [88, 89]. The irritation thus, below the neurological level of injury, from the leptin is disturbed. In addition, extensive obesity is known to reduce lipolytic sensitivity [89, 90, 91]. Given that in the high level of neurological paraplegia there is a problem of disorder of the autonomic nervous system and in combination with the existence of scientific evidence that the hormone leptin activates the sympathetic nervous system through central control, was formulated, that the closure <of paths> of the central nervous system disrupts the effect of leptin and possibly increases the risk of obesity in paraplegic patients with high-level injury [92, 93].

injury [80] but it is greater a year or more after SCI (Hillegass and Dudley, unpublished observations). The greater fatigue, when evident, was partially attributed to lower metabolic

Body Composition in Paraplegia http://dx.doi.org/10.5772/58539 165

Muscular loading of the bones has been thought to play a role in the maintenance of bone density [65, 66]. However, the ability to stand or ambulate itself does not improve BMD or

Other important issues according alterations of body composition are the completeness of lesions (an absence of sensory or motor function below the neurological level, including the lowest sacral segment), because body composition seems to be worse than subjects with incomplete lesions (partial preservation of motor and/or sensory function below the neuro‐ logical level, including the lowest sacral segment) and aging which contributes to major

In disabled subjects the most important issue according to body composition is how to promote optimal body weight to reduce risk of diseases such as coronary heart disease, non-insulin dependent diabetes mellitus, lipid abnormalities and fractures because of bone loss. Dietary changes, individualized physical activity programs and medication should be taken in mind in therapy when we deal with this subgroup of subjects. However, self-management of dietary changes to improve weight control and disease should be the case, which means they need to follow diets with lower energy intake and at the same time to eat regularly foods rich in

We need to take in mind that healthy BMI values often underestimate body fat and may mask the adiposity and spasticity did not defend skeletal muscle mass and bone, supporting the concept that in neurologic disabilities the myopathic muscle could not recognize correctly the stimulation because of the neurogenic injury. Moreover, disabled subjects mostly transfer much of the weight-bearing demands of daily activities to their upper extremities reducing the weight-bearing of the affected paralyzed muscles triggering a cycle of added muscle atrophy which interacts with the continuous catabolic action caused by the neurogenic factor. Finally, an irreversible (once established) decline in bone mineral density, bone mineral content as well as geometric characteristics of bone is expected and the duration of lesion-injury is

Further research about body composition is needed in all physical disabilities and more longitudinal studies to quantitate and monitor body composition changes and to modify our therapeutic interventions. However, prevention rather than treatment may have the greatest potential to alleviate these major complications. Therapies should focus on how to perform weight bearing, standing or therapeutically walking activities early in the rehabilitation

enzyme levels [95].

**4. Conclusions**

nutrients [26].

prevent osteoporosis after SCI.

alterations of body composition [62, 63].

positively correlated with the degree of bone loss.

program to gain benefits according to muscles and bones.

However, after separation of SCI subjects into those with an injury above or below Thoracic (T) 6, leptin levels were significantly higher in the former group. T6 appears to be the lowest level of injury in most patients with SCI to develop autonomic dysreflexia. With SCIs above the level of T6, there is reduced SNS outflow and supraspinal control to the splanchnic outflow and the lower-extremity blood vessels. Multiple regression analysis showed that serum leptin levels in men with SCI correlated not only with BMI but also with the neurologic deficit. This finding supports the notion that decentralization of sympathetic nervous activity relieves its inhibitory tone on leptin secretion, because subjects with tetraplegia have a more severe deficit of sympathetic nervous activity [94].

Actually, little is known regarding the nature and time frame of the influence of complete SCI on human skeletal muscle because published data are coming from cross-sectional studies, where different groups with few subjects have been examined at different times, usually in the chronic phase of paralysis. Disuse was thought to be the mechanism responsible for the skeletal muscle atrophy in paraplegics, but muscle fibres following SCI begin to change their functional properties early post injury. Muscle fiber cross-sectional area (CSA) has been suggested to decline from 1 to 17 months after injury and thereafter to reach its nadir. Con‐ version to type II fibers has been suggested to occur between 4 months and 2 years after injury, resulting in even slow-twitch muscle becoming predominantly fast twitch thereafter (Castro et al 1999). Metabolic enzymes levels in skeletal muscle might be expected to be reduced after SCI because of inactivation. In support of this contention, succinic dehydrogenase (SDH) activity, a marker of aerobic-oxidative capacity, has been reported to be 47–68% below control values in fibers of tibialis anterior muscle years after injury in support of this contention [95].

The muscle atrophy in SCI is of central type and depends on the disuse and loss of upper connections of the lower motor neuron, sometimes associated to the loss of anterior horn cells and transinaptic degeneration. The last alteration may be responsible for the denervation changes seen in early stages post SCI. In the later stages (10-17 months post SCI) diffuse muscle atrophy with reduction of the muscle fascicle dimension is associated to fat infiltration and endomysial fibrosis. In all stages post SCI, almost all patients showed myopathic changes, as internal nuclei, fibre degeneration and cytoplasmic vacuolation due to lipid accumulation [95].

It is evident that other co-factors as spasticity and microvascular damage, contribute to the induction of the marked morphological and enzyme histochemical changes seen in the paralyzed skeletal muscle [95]. Small fibers, predominantly fast-twitch muscle, and low mitochondrial content have been reported years after injury in cross-sectional studies. These data have been interpreted to suggest that human skeletal muscle shows plasticity [48].

On the contrary, force loss during repetitive contractions evoked by surface electrical stimu‐ lation (ES) of skeletal muscle in humans does not appear to be altered within a few months of injury [80] but it is greater a year or more after SCI (Hillegass and Dudley, unpublished observations). The greater fatigue, when evident, was partially attributed to lower metabolic enzyme levels [95].

Muscular loading of the bones has been thought to play a role in the maintenance of bone density [65, 66]. However, the ability to stand or ambulate itself does not improve BMD or prevent osteoporosis after SCI.

#### **4. Conclusions**

in the high level of neurological paraplegia there is a problem of disorder of the autonomic nervous system and in combination with the existence of scientific evidence that the hormone leptin activates the sympathetic nervous system through central control, was formulated, that the closure <of paths> of the central nervous system disrupts the effect of leptin and possibly

However, after separation of SCI subjects into those with an injury above or below Thoracic (T) 6, leptin levels were significantly higher in the former group. T6 appears to be the lowest level of injury in most patients with SCI to develop autonomic dysreflexia. With SCIs above the level of T6, there is reduced SNS outflow and supraspinal control to the splanchnic outflow and the lower-extremity blood vessels. Multiple regression analysis showed that serum leptin levels in men with SCI correlated not only with BMI but also with the neurologic deficit. This finding supports the notion that decentralization of sympathetic nervous activity relieves its inhibitory tone on leptin secretion, because subjects with tetraplegia have a more severe deficit

Actually, little is known regarding the nature and time frame of the influence of complete SCI on human skeletal muscle because published data are coming from cross-sectional studies, where different groups with few subjects have been examined at different times, usually in the chronic phase of paralysis. Disuse was thought to be the mechanism responsible for the skeletal muscle atrophy in paraplegics, but muscle fibres following SCI begin to change their functional properties early post injury. Muscle fiber cross-sectional area (CSA) has been suggested to decline from 1 to 17 months after injury and thereafter to reach its nadir. Con‐ version to type II fibers has been suggested to occur between 4 months and 2 years after injury, resulting in even slow-twitch muscle becoming predominantly fast twitch thereafter (Castro et al 1999). Metabolic enzymes levels in skeletal muscle might be expected to be reduced after SCI because of inactivation. In support of this contention, succinic dehydrogenase (SDH) activity, a marker of aerobic-oxidative capacity, has been reported to be 47–68% below control values in fibers of tibialis anterior muscle years after injury in support of this contention [95].

The muscle atrophy in SCI is of central type and depends on the disuse and loss of upper connections of the lower motor neuron, sometimes associated to the loss of anterior horn cells and transinaptic degeneration. The last alteration may be responsible for the denervation changes seen in early stages post SCI. In the later stages (10-17 months post SCI) diffuse muscle atrophy with reduction of the muscle fascicle dimension is associated to fat infiltration and endomysial fibrosis. In all stages post SCI, almost all patients showed myopathic changes, as internal nuclei, fibre degeneration and cytoplasmic vacuolation due to lipid accumulation [95].

It is evident that other co-factors as spasticity and microvascular damage, contribute to the induction of the marked morphological and enzyme histochemical changes seen in the paralyzed skeletal muscle [95]. Small fibers, predominantly fast-twitch muscle, and low mitochondrial content have been reported years after injury in cross-sectional studies. These data have been interpreted to suggest that human skeletal muscle shows plasticity [48].

On the contrary, force loss during repetitive contractions evoked by surface electrical stimu‐ lation (ES) of skeletal muscle in humans does not appear to be altered within a few months of

increases the risk of obesity in paraplegic patients with high-level injury [92, 93].

of sympathetic nervous activity [94].

164 Topics in Paraplegia

Other important issues according alterations of body composition are the completeness of lesions (an absence of sensory or motor function below the neurological level, including the lowest sacral segment), because body composition seems to be worse than subjects with incomplete lesions (partial preservation of motor and/or sensory function below the neuro‐ logical level, including the lowest sacral segment) and aging which contributes to major alterations of body composition [62, 63].

In disabled subjects the most important issue according to body composition is how to promote optimal body weight to reduce risk of diseases such as coronary heart disease, non-insulin dependent diabetes mellitus, lipid abnormalities and fractures because of bone loss. Dietary changes, individualized physical activity programs and medication should be taken in mind in therapy when we deal with this subgroup of subjects. However, self-management of dietary changes to improve weight control and disease should be the case, which means they need to follow diets with lower energy intake and at the same time to eat regularly foods rich in nutrients [26].

We need to take in mind that healthy BMI values often underestimate body fat and may mask the adiposity and spasticity did not defend skeletal muscle mass and bone, supporting the concept that in neurologic disabilities the myopathic muscle could not recognize correctly the stimulation because of the neurogenic injury. Moreover, disabled subjects mostly transfer much of the weight-bearing demands of daily activities to their upper extremities reducing the weight-bearing of the affected paralyzed muscles triggering a cycle of added muscle atrophy which interacts with the continuous catabolic action caused by the neurogenic factor. Finally, an irreversible (once established) decline in bone mineral density, bone mineral content as well as geometric characteristics of bone is expected and the duration of lesion-injury is positively correlated with the degree of bone loss.

Further research about body composition is needed in all physical disabilities and more longitudinal studies to quantitate and monitor body composition changes and to modify our therapeutic interventions. However, prevention rather than treatment may have the greatest potential to alleviate these major complications. Therapies should focus on how to perform weight bearing, standing or therapeutically walking activities early in the rehabilitation program to gain benefits according to muscles and bones.

#### **Author details**

Yannis Dionyssiotis1,2\*

1 Rehabilitation Center "Aghios Loukas o Iatros", Trikala Thessaly, Greece

2 University of Athens, 1st Department of Orthopaedics, General University Hospital Attikon, Athens, Greece

[11] Dionyssiotis Y. Spinal cord injury-related bone impairment and fractures: an upda‐ teon epidemiology and physiopathological mechanisms. J Musculoskelet Neuronal

Body Composition in Paraplegia http://dx.doi.org/10.5772/58539 167

[12] Smeltzer SC, Zimmerman V, Capriotti T. Osteoporosis risk and low bone mineral density in women with physical disabilities. Arch Phys Med Rehabil. 2005;86:582-6.

[13] Coupaud S, McLean AN, Allan DB. Role of peripheral quantitative computed tomog‐ raphy in identifying disuse osteoporosis in paraplegia. Skeletal Radiol. 2009;

[14] Gorgey AS, Dudley GA. Skeletal muscle atrophy and increased intramuscular fat af‐

[15] Jones LM, Legge M, Goulding A. Healthy body mass index values often underesti‐ mate body fat in men with spinal cord injury. Arch Phys Med Rehabil.

[16] Bulbulian R, Johnson RE, Gruber JJ, Darabos B. Body composition in paraplegic male

[17] Maggioni M, Bertoli S, Margonato V, Merati G, Veicsteinas A, Testolin G. Body com‐ position assessment in spinal cord injury subjects. Acta Diabetol. 2003;40:S183-6.

[18] Mamoun L, Puech AM, Manetta J, Badiou S, Paris F, Ohanna F, Rossi M, Sultan C. Circulating leptin concentrations can be used as a surrogate marker of fat mass in

[19] Dionyssiotis Υ, Petropoulou Κ, Rapidi CA, Papagelopoulos PJ, Papaioannou N, Gal‐ anos A, Papadaki P, and Lyritis GP. Body Composition in Paraplegic Men. Journal of

[20] National Institutes of Health Consensus Development Conference Statement. Health implications of obesity. Natl Inst Health Consens Dev Conf Consens Statement.

[21] Schulte PA, Wagner GR, Ostry A, Blanciforti LA, Cutlip RG, Krajnak KM, Luster M, Munson AE, O'Callaghan JP, Parks CG, Simeonova PP, Miller DB. Work, obesity,and

[22] Kocina P. Body composition of spinal cord injured adults. Sports Medicine.

[23] Gupta N, White KT, Sandford PR. Body mass index in spinal cord injury -- a retro‐

[24] McDonald CM, Abresch-Meyer AL, Nelson MD, Widman LM. Body mass index and body composition measures by dual x-ray absorptiometry in patients aged 10 to 21

occupational safety and health. Am J Public Health. 2007;97:428-36.

years with spinal cord injury. J Spinal Cord Med. 2007;30:S97-104.

ter incomplete spinal cord injury. Spinal Cord. 2007;45:304–9.

acute spinal cord injury patients. Metabolism. 2004;53:989-94.

athletes. Med Sci Sports Exerc. 1987;19:195-201.

Clinical Densitometry. 2008;11: 437-43.

spective study. Spinal Cord. 2006;44:92-4.

Interact. 2011;11:257-65.

38:989-95.

2003;84:1068-71.

1985;5:1-7.

1997;23:48-60.

#### **References**


[11] Dionyssiotis Y. Spinal cord injury-related bone impairment and fractures: an upda‐ teon epidemiology and physiopathological mechanisms. J Musculoskelet Neuronal Interact. 2011;11:257-65.

**Author details**

166 Topics in Paraplegia

Athens, Greece

**References**

Spinal Cord. 1998;36:637-40

Am J Epidemiol. 1998;147:42-8.

York: Springer-Verlag; 1987.

1996;156:958-63.

Yannis Dionyssiotis1,2\*

1 Rehabilitation Center "Aghios Loukas o Iatros", Trikala Thessaly, Greece

2 University of Athens, 1st Department of Orthopaedics, General University Hospital Attikon,

[1] Jones LM, Goulding A, Gerrard DF. DEXA: a practical and accurate tool to demon‐ strate total and regional boneloss, lean tissue loss and fat mass gain in paraplegia.

[2] Seidell JC, Verschuren WM, van Leer EM, Kromhout D. Overweight, underweight, and mortality. A prospective study of 48.287 men and women. Arch Intern Med.

[3] Bender R, Trautner C, Spraul M, Berger M. Assessment of excess mortality in obesity.

[4] Van Der Ploeg GE, Withers RT, Laforgia J. Percent body fat via DEXA: comparison

[5] Spungen AM, Adkins RH, Stewart CA, Wang J, Pierson RN Jr, Waters RL, Bauman WA. Factors influencing body composition in persons with spinal cord injury: a

[6] Clarys JP, Martin AD, Drinkwater DT. Gross tissue weights in the human body by

[7] Modlesky CM, Bickel CS, Slade JM, Meyer RA, Cureton KJ, Dudley GA. Assessment of skeletal muscle mass in men with spinal cord injury using dual-energy X-ray ab‐

[8] Forbes GB. Human body composition: growth, aging, nutrition, and activity. New

[9] Mojtahedi MC, Valentine RJ, Arngrímsson SA, Wilund KR, Evans EM. The associa‐ tion between regional body composition and metabolic outcomes in athletes with

[10] Dionyssiotis Y. (2011). Bone Loss in Spinal Cord Injury and Multiple Sclerosis. In: JH Stone, M Blouin, editors. International Encyclopedia of Rehabilitation, av. online:

sorptiometry and magnetic resonance imaging. J Appl Physiol. 2004;96:561-5.

with a four-compartment model. J Appl Physiol. 2003;94:499-506.

cross-sectional study. J Appl Physiol. 2003;95: 2398–2407.

cadaver dissection. Hum Biol. 1984;56:459-73.

spinal cord injury. Spinal Cord. 2008;46:192-7.

http://cirrie.buffalo.edu/encyclopedia/en/article/340/


[25] Spungen AM, Wang J, Pierson RN, Jr., Bauman WA. Soft tissue body composition differences in monozygotic twins discordant for spinal cord injury. J Appl Physiol. 2000;88:1310-5.

[39] Dionyssiotis Y. Changes in bone density and strength of the tibia and alterations of lean and fat mass in chronic paraplegic men. Doctoral Dissertation Laboratory for

Body Composition in Paraplegia http://dx.doi.org/10.5772/58539 169

[40] Peppler WW, Mazess RB. 1981. Total body bone mineral and lean body mass by du‐

[41] Pietrobelli A, Formica C, Wang AM, Heymsfield SB. 1996. Dual-energy X-ray absorp‐ tiometry body composition model: review of physical concepts. Am J Physiol 271

[42] Ferretti J.L., Cointry G.R., Capozza R.F., Zanchetta J.R. Dual energy X-ray absorpti‐ ometry. Skeletal Muscle: Pathology, Diagnosis and Management of Disease. V.R.Pre‐

edy, T.J.Peters (eds),Greenwich Medical Media, Ltd., London, 2001; p.451-458.

[43] Bauman WA, Spungen AM, Raza M, Rothstein J, Zhang RL, Zhong YG, Tsuruta M, Shahidi R, Pierson RN Jr, Wang J, et al. Coronary artery disease: metabolic risk fac‐ tors and latent disease in individuals with paraplegia. Mt Sinai J Med. 1992;59:163-8.

[44] Bauman WA, Spungen AM. Disorders of carbohydrate and lipid metabolism inveter‐ ans with paraplegia or quadriplegia: A model of premature aging. Metabolism.

[45] Garland DE, Stewart CA, Adkins RH, Hu SS, Rosen C, Liotta FJ, Weinstein DA Os‐

[46] Heymsfield SB, Wang J, Heshka S, Kehayias JJ, Pierson RN. Dual-photon absorpti‐ ometry: comparison of bone mineral and soft tissue mass measurements in vivo with

[47] Laskey MA. Dual-energy X-ray absorptiometry and body composition. Nutrition.

[48] Castro MJ, Apple DF Jr, Staron RS, Campos GE, Dudley GA. Influence of complete spinal cord injury on skeletal muscle within 6 mo of injury. J Appl Physiol.1999;

[49] Szollar SM, Martin EM, Parthemore JG, Sartoris DJ, Deftos LJ. Densitometric pattern‐

[50] Uebelhart D, Demiaux-Domenech B, Roth M, Chantraine A. Bone metabolism in spi‐ nal cord injured individuals and in others who have prolonged immobilisation. A re‐

[51] Chow YW, Inman C, Pollintine P, Sharp CA, Haddaway MJ, el Masry W, Davie MW.Ultrasound bone densitometry and dual energy X-ray absorptiometry in pa‐ tients with spinal cord injury: a cross-sectional study. Spinal Cord. 1996;34:736-41.

[52] Biering-Sorensen F, Bohr H, Schaadt O. Bone mineral content of the lumbar spine and lower extremities years after spinal cord lesion. Paraplegia. 1988 ;26:293-301.

sof spinal cord injury associated bone loss. Spinal Cord. 1997;35:374-82.

teoporosis after spinal cord injury. J Orthop Res. 1992;10:371-8.

established methods. Am J Clin Nutr.1989;49:1283-9.

Research of the Musculoskeletal System, University of Athens, Athens 2008.

al-photon absorptiometry. Calcif Tissue Int 33:353-359

(Endocrinol Metab 34): E941-E951

1994;43:749-56.

1996;12:45-51.

view.Paraplegia. 1995;33:669-73.

86:350-8.


[39] Dionyssiotis Y. Changes in bone density and strength of the tibia and alterations of lean and fat mass in chronic paraplegic men. Doctoral Dissertation Laboratory for Research of the Musculoskeletal System, University of Athens, Athens 2008.

[25] Spungen AM, Wang J, Pierson RN, Jr., Bauman WA. Soft tissue body composition differences in monozygotic twins discordant for spinal cord injury. J Appl Physiol.

[26] Groah SL, Nash MS, Ljungberg IH, Libin A, Hamm LF, Ward E, Burns PA, Enfield G. Nutrient intake and body habitus after spinal cord injury: an analysis by sex and lev‐

[27] Maimoun L, Fattal C, Micallef JP, Peruchon E, Rabischong P. Bone loss in spinal cord-injured patients: from physiopathology to therapy. Spinal Cord. 2006;44:203-10.

[28] Lussier L, Knight J, Bell G, Lohman T, Morris AF. Body composition comparison in

[29] Spungen AM, Bauman WA, Wang J, Pierson RN. Reduced quality of fat free mass in

[30] Womersley J, Durnin JV, Boddy K, Mahaffy M. Influence of muscular development, obesity, and age on the fat-free mass of adults. J Appl Physiol. 1976;41:223-9.

[31] Fuller NJ, Sawyer MB, Laskey MA, Paxton P, Elia M: Prediction of body composition

[32] Hildreth HG, Johnson RK, Goran MI, Contompasis SH. Body composition in adults with cerebral palsy by dual-energy X-ray absorptiometry, bioelectrical impedance analysis, and skinfold anthropometry compared with the 18O isotope-dilution tech‐

[33] Liu LF, Roberts R, Moyer-Mileur L, Samson-Fang L. Determination of body composi‐ tion in children with cerebral palsy: bioelectrical impedance analysis and anthropom‐

[34] Kuperminc MN, Stevenson RD. Growth and nutrition disorders in children with cer‐

[35] Mitsiopoulos N, Baumgartner RN, Heymsfield SB, Lyons W, Gallagher D, and Ross R. Cadaver validation of skeletal muscle measurement by magnetic resonance imag‐

[36] Mahon AK, Flynn MG, Iglay HB, Stewart LK, Johnson CA, McFarlin BK, Campbell WW. Measurement of body composition changes with weight loss in postmenopaus‐

[37] LaForgia J, Dollman J, Dale MJ, Withers RT, Hill AM. Validation of DXA body com‐ position estimates in obese men and women. Obesity (Silver Spring). 2009;17:821-6.

[38] Rittweger J, Beller G, Ehrig J, Jung C, Koch U, Ramolla J, Schmidt F, Newitt D, Ma‐ jumdar S, Schiessl H, Felsenberg D. Bone-muscle strength indices for the human low‐

etry vs dual-energy x-ray absorptiometry. J Am Diet Assoc. 2005;105:794-7.

ing and computerized tomography. J Appl Physiol. 1998;85:115–22.

al women: comparison of methods. J Nutr Health Aging. 2007;11:203-13.

two elite female wheelchair athletes. Paraplegia. 1983;21:16-22.

in elderly men over 75 years of age. Ann Hum Biol. 1996;23:127-47.

2000;88:1310-5.

168 Topics in Paraplegia

el of injury. J Spinal Cord Med. 2009;32:25-33.

paraplegia. Clin Research. 1992;40:280A.

nique. Am J Clin Nutr. 1997;66:1436-42.

er leg. Bone. 2000;27:319-26.

ebral palsy. Dev Disabil Res Rev. 2008;14:137-46.


[53] Lazo MG, Shirazi P, Sam M, Giobbie-Hurder A, Blacconiere MJ, Muppidi M. Osteo‐ porosis and risk of fracture in men with spinal cord injury. Spinal Cord. 2001;39:208-14.

[67] Eser P, Frotzler A, Zehnder Y, Schiessl H, Denoth J. Assessment of anthropometric, systemic, and lifestyle factors influencing bone status in the legs of spinal cord in‐

Body Composition in Paraplegia http://dx.doi.org/10.5772/58539 171

[68] Löfvenmark I, Werhagen L, Norrbrink C. Spasticity and bone density after a spinal

[69] Kiratli BJ, Smith AE, Nauenberg T, Kallfelz CF, Perkash I. Bone mineral and geomet‐ ric changes through the femur with immobilization due to spinal cord injury. J Reha‐

[70] Wood DE, Dunkerley AL, Tromans AM. Results from bone mineral density scans in

[71] Kunkel CF, Scremin AM, Eisenberg B, Garcia JF, Roberts S, Martinez S. Effect of ''standing'' on spasticity, contracture, and osteoporosis in paralyzed males. Arch

[72] Frost HM. Bone "mass" and the "mechanostat": a proposal. Anat Rec. 1987;219:1-9.

[73] Frost HM. Skeletal structural adaptations to mechanical usage (SATMU): 2. Redefin‐

[74] Frost HM. Perspectives: on a "paradigm shift" developing in skeletal science. Calcif

[75] Ragnarsson KT, Pollack S, O'Daniel W Jr, Edgar R, Petrofsky J, Nash MS. Clinical evaluation of computerized functional electrical stimulation after spinal cord injury:

[76] Belanger M, Stein RB, Wheeler GD, Gordon T, Leduc B. Electrical stimulation: can it increase muscle strength and reverse osteopenia in spinal cord injured individuals?

[77] Bloomfield SA, Mysiw WJ, Jackson RD. Bone mass and endocrine adaptations to

[78] BeDell KK, Scremin AM, Perell KL, Kunkel CF. Effects of functional electrical stimu‐ lation-induced lower extremity cycling on bone density of spinal cord-injured pa‐

[79] Needham-Shropshire BM, Broton JG, Klose KJ, Lebwohl N, Guest RS, Jacobs PL. Evaluation of a training program for persons with SCI paraplegia using the Parastep 1 ambulation system: part 3. Lack of effect on bone mineral density. Arch Phys Med

[80] Shields RK. Muscular, skeletal, and neural adaptations following spinal cord injury. J

twenty-two complete lesion paraplegics. Spinal Cord. 2001;39:145–8.

ing Wolff's law: the remodeling problem. Anat Rec. 1990;226:414-22.

a multicenter pilot study. Arch Phys Med Rehabil. 1988;69:672-7.

training in spinal cord injured individuals. Bone. 1996;19:61-8.

jured individuals. Osteoporos Int. 2005;16:26-34.

cord injury. J Rehabil Med. 2009;41:1080-4.

bil Res Dev. 2000;37:225-33.

Phys Med Rehabil. 1993;74:73–8.

Arch Phys Med Rehabil. 2000;81:1090-8.

tients. Am J Phys Med Rehabil. 1996;75:29-34.

Orthop Sports Phys Ther. 2002;32:65-74.

Tissue Int. 1995;56:1-4.

Rehabil. 1997;78:799-803.


[67] Eser P, Frotzler A, Zehnder Y, Schiessl H, Denoth J. Assessment of anthropometric, systemic, and lifestyle factors influencing bone status in the legs of spinal cord in‐ jured individuals. Osteoporos Int. 2005;16:26-34.

[53] Lazo MG, Shirazi P, Sam M, Giobbie-Hurder A, Blacconiere MJ, Muppidi M. Osteo‐ porosis and risk of fracture in men with spinal cord injury. Spinal Cord.

[54] Frey-Rindova P, de Bruin ED, Stussi E, Dambacher MA, Dietz V. Bone mineral densi‐ ty in upper and lower extremities during 12 months after spinal cord injury meas‐ ured by peripheral quantitative computed tomography. Spinal Cord. 2000;38:26–32.

[55] Jiang SD, Dai LY, Jiang LS. Osteoporosis after spinal cord injury. Osteoporos Int.

[56] Dauty M, Perrouin Verbe B, Maugars Y, Dubois C, Mathe JF. Supralesional and sub‐ lesional bone mineral density in spinal cord-injured patients. Bone. 2000;27:305-9. [57] Chantraine A, Nusgens B, Lapiere CM. Bone remodelling during the development of

[58] Ogilvie C, Bowker P, Rowley DI. The physiological benefits of paraplegic orthotically

[59] Leslie WD, Nance PW. Dissociated hip and spine demineralization: a specific finding

[60] Tsuzuku S, Ikegami Y, Yabe K. Bone mineral density differences between paraplegic and quadriplegic patients: a cross-sectional study. Spinal Cord. 1999; 37:358-61. [61] Dionyssiotis Y, Lyritis GP, Papaioannou N, Papagelopoulos P, Thomaides T. Influ‐ ence of neurological level of injury in bones, muscles, and fat in paraplegia. J Rehabil

[62] Demirel G, Yilmaz H, Paker N, Onel S. Osteoporosis after spinal cord injury. Spinal

[63] Sabo D, Blaich S, Wenz W, Hohmann M, Loew M, Gerner HJ. Osteoporosis in pa‐ tients with paralysis after spinal cord injury: a cross sectional study in 46 male pa‐ tients with dual-energy X-ray absorptiometry. Arch Orthop Trauma Surg.

[64] Clasey JL, Janowiak AL, Gater DR Relationship between regional bone density meas‐ urements and the time since injury in adults with spinal cord injuries. Arch Phys

[65] Dionyssiotis Y, Lyritis GP, Mavrogenis AF, Papagelopoulos PJ. Factors influencing‐

[66] de Bruin ED, Vanwanseele B, Dambacher MA, Dietz V, Stussi E. Long-term changes in the tibia and radius bone mineral density following spinal cord injury. Spinal

osteoporosis in paraplegia. Calcif Tissue Int. 1986;38:323-7.

in spinal cord injury. Arch Phys Med Rehabil. 1993; 74:960-4.

aided walking. Paraplegia. 1993;31:111-5.

Res Dev. 2009;46:1037-44.

Med Rehabil. 2004;85:59–64

Cord. 2005;43:96-101.

bone loss in paraplegia. Hippokratia. 2011;15:54-9.

Cord. 1998;36:8

2001;121:75–8.

2001;39:208-14.

170 Topics in Paraplegia

2006;17:180-92.


[81] Shields RK, Dudley-Javoroski S. Musculoskeletal adaptations in chronic spinal cord injury: effects of long-term soleus electrical stimulation training. Neurorehabil Neu‐ ral Repair. 2007;21:169-79.

[94] Wang YH, Huang TS, Liang HW, Su TC, Chen SY, Wang TD. Fasting serum levels of adiponectin, ghrelin, and leptin in men with spinal cord injury. Arch Phys Med Re‐

Body Composition in Paraplegia http://dx.doi.org/10.5772/58539 173

[95] Scelsi R. Skeletal Muscle Pathology after Spinal Cord Injury: Our 20 Year Experience and Results on Skeletal Muscle Changes in Paraplegics, Related to Functional Reha‐

habil. 2005;86:1964-8.

bilitation Basic Appl Myol. 2001;11:75-85.


[94] Wang YH, Huang TS, Liang HW, Su TC, Chen SY, Wang TD. Fasting serum levels of adiponectin, ghrelin, and leptin in men with spinal cord injury. Arch Phys Med Re‐ habil. 2005;86:1964-8.

[81] Shields RK, Dudley-Javoroski S. Musculoskeletal adaptations in chronic spinal cord injury: effects of long-term soleus electrical stimulation training. Neurorehabil Neu‐

[82] Valayer-Chaleat E, Calmels P, Giraux P, Fayolle-Minon I. Femoral fracture and iatro‐

[83] Frey Law LA, Shields RK. Femoral loads during passive, active, and active-resistive stance after spinal cord injury: a mathematical model. Clin Biomech. (Bristol, Avon).

[84] Fruhbeck G, Jebb SA, Prentice AM. Leptin: physiology and pathophysiology. Clin

[85] Maffei M, Halaas J, Ravussin E, Pratley RE, Lee GH, Zhang Y, Fei H, Kim S, Lallone R, Ranganathan S, et al. Leptin levels in human and rodent: measurement of plasma leptin and ob RNA in obese and weight-reduced subjects. Nat Med. 1995;1:1155-61.

[86] Bauman WA, Spungen AM, Zhong YG, Mobbs CV. Plasma leptin is directly related to body adiposity in subjects with spinal cord injury. Horm Metab Res. 1996;28:732-6.

[87] Correia ML, Morgan DA, Mitchell JL, Sivitz WI, Mark AL, Haynes WG. Role of corti‐ cotrophin-releasing factor in effects of leptin on sympathetic nerve activity and arte‐

[88] Elias CF, Lee C, Kelly J, Aschkenasi C, Ahima RS, Couceyro PR, Kuhar MJ, Saper CB, Elmquist JK. Leptin activates hypothalamic CART neurons projecting to the spinal

[89] Haque MS, Minokoshi Y, Hamai M, Iwai M, Horiuchi M, Shimazu T. Role of the sympathetic nervous system and insulin in enhancing glucose uptake in peripheral tissues after intrahypothalamic injection of leptin in rats. Diabetes. 1999;48:1706-12.

[90] Horowitz JF, Coppack SW, Paramore D, Cryer PE, Zhao G, Klein S. Effect of shortterm fasting on lipid kinetics in lean and obese women. Am J Physiol.

[91] Horowitz JF, Klein S. Whole body and abdominal lipolytic sensitivity to epinephrine is suppressed in upper body obese women. Am J Physiol Endocrinol Metab.

[92] Krassioukov AV, Bunge RP, Pucket WR, Bygrave MA. The changes in human spinal sympathetic preganglionic neurons after spinal cord injury. Spinal Cord.

[93] Jeon JY, Steadward RD, Wheeler GD, Bell G, McCargar L, Harber V. Intact sympa‐ thetic nervous system is required for leptin effects on resting metabolic rate in people

with spinal cord injury. J Clin Endocrinol Metab. 2003;88:402-7.

genic hyperthyroidism in spinal cord injury. Spinal Cord. 1998;36:593-5.

ral Repair. 2007;21:169-79.

Physiol. 1998;18:399-419.

rial pressure. Hypertension. 2001;38:384-8.

cord. Neuron. 1998;21:1375-85.

1999;276:E278-84.

2000;278:E1144-52.

1999;37:6-13.

2004;19:313-21.

172 Topics in Paraplegia

[95] Scelsi R. Skeletal Muscle Pathology after Spinal Cord Injury: Our 20 Year Experience and Results on Skeletal Muscle Changes in Paraplegics, Related to Functional Reha‐ bilitation Basic Appl Myol. 2001;11:75-85.

**Chapter 8**

**Paraplegia Related Osteoporosis**

Additional information is available at the end of the chapter

Osteoporosis is characterized by low bone mass and destruction of the micro architecture of

The World Health Organisation (WHO) created an operational definition of postmenopausal osteoporosis based on a bone mineral density (BMD)-based T-score measurement. The most widely validated technique to measure BMD is dual energy X-ray absorptiometry (DXA), and diagnostic criteria based on the T-score for BMD are a recommended entry criterion for the development of pharmaceutical interventions in osteoporosis. (2) The ranking system of the WHO is commonly used in the literature and in all discussions with respect to bone diseases. According to WHO criteria, the general categories for making a diagnosis are the following: 1) normal: BMD of not less than one standard deviation (SD) than the average young adult (Tscore>-1), 2) osteopenia: BMD between one and 2.5 SD below the average for young adults (-1<T-score<-2.5), 3) osteoporosis: BMD 2.5 SD or more below the average for young adults (Tscore>-2.5) and 4) severe or established osteoporosis: BMD 2.5 SD or more below the average

Because of the unique and individually-based approach needed in the management of each disabled subject with a spinal cord lesion and their complications according to bone loss the new term "paraplegia-related bone impairment, (Para-related BI)" is used throughout this chapter. The term bone impairment is more appropriate than bone disorder because includes terminology from Rehabilitation Science a specialty which interferes with all complications of spinal cord injury (SCI) and follows these patients during aging with paralysis. It is not used here for the 1st time. Very experienced scientists and researchers chose this term to describe

> © 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

bone tissue, resulting in increased bone fragility and susceptibility to fractures. [1]

for young adults and the presence of one or more fractures. [2, 3]

Yannis Dionyssiotis

**1. Introduction**

"osteoporosis" in SCI. [4]

http://dx.doi.org/10.5772/57969

#### **Chapter 8**

### **Paraplegia Related Osteoporosis**

#### Yannis Dionyssiotis

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/57969

#### **1. Introduction**

Osteoporosis is characterized by low bone mass and destruction of the micro architecture of bone tissue, resulting in increased bone fragility and susceptibility to fractures. [1]

The World Health Organisation (WHO) created an operational definition of postmenopausal osteoporosis based on a bone mineral density (BMD)-based T-score measurement. The most widely validated technique to measure BMD is dual energy X-ray absorptiometry (DXA), and diagnostic criteria based on the T-score for BMD are a recommended entry criterion for the development of pharmaceutical interventions in osteoporosis. (2) The ranking system of the WHO is commonly used in the literature and in all discussions with respect to bone diseases. According to WHO criteria, the general categories for making a diagnosis are the following: 1) normal: BMD of not less than one standard deviation (SD) than the average young adult (Tscore>-1), 2) osteopenia: BMD between one and 2.5 SD below the average for young adults (-1<T-score<-2.5), 3) osteoporosis: BMD 2.5 SD or more below the average for young adults (Tscore>-2.5) and 4) severe or established osteoporosis: BMD 2.5 SD or more below the average for young adults and the presence of one or more fractures. [2, 3]

Because of the unique and individually-based approach needed in the management of each disabled subject with a spinal cord lesion and their complications according to bone loss the new term "paraplegia-related bone impairment, (Para-related BI)" is used throughout this chapter. The term bone impairment is more appropriate than bone disorder because includes terminology from Rehabilitation Science a specialty which interferes with all complications of spinal cord injury (SCI) and follows these patients during aging with paralysis. It is not used here for the 1st time. Very experienced scientists and researchers chose this term to describe "osteoporosis" in SCI. [4]

© 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

#### **2. Paraplegia related bone impairment**

#### **2.1. Epidemiology**

According to the literature, spinal cord injury-related bone impairment (SCI-related BI) occurs in 75% of patients with complete SCI. [5] Twenty five out of 41 patients with SCI (61%) met WHO criteria for osteoporosis; eight (19.5%) were osteopenic and only eight (19.5%) showed normal values.[6] In SCI children (boys and girls), values for BMD at the hip were approxi‐ mately 60% of normal, or had a Z-score that indicated a 1.6-1.8 SD reduction in BMD compared with age-and sex-matched peers. [7] The decrease in BMD was probably the dominant cause for the high prevalence of SCI-related BI in the long femur or proximal tibia and explains why these areas are often fracture site. [6, 8, 9] For example, a reduction in bone mineral density in the femoral neck of about 0.1 g/cm2 increases fracture risk by 2.2 times. This decrease in bone mass is associated with alterations in bone material, reduced bone elasticity and is connected to the origin of pathological fractures with minimal injury, in which these patients are vulnerable and exposed. [8, 9]

remodeling keeping the total BMD similar. Concerning cortical geometric properties the results had shown an increased endosteal circumference between both paraplegic groups vs. controls leading to reduction of cortical thickness, 19.78% vs. 16.98% in paraplegic groups

Paraplegia Related Osteoporosis http://dx.doi.org/10.5772/57969 177

**Figure 1.** Peripheral quantitative computed tomography (p QCT) tibia slices in control (a) and paraplegic subject (b), (scanner XCT 3000 Stratec, Medizintechnik, Pforzheim, Germany). Areas in red represent trabecular bone, while areas in grey represent fat; pQCT allows the measurements of true volumetric densities at a minimum exposure to X-rays, assess cortical and trabecular bone density separately as well as to evaluate the geometrical properties of long bones

Regarding tetraplegic patients statistically significant differences were found in BMD of the spine, trochanteric region and upper limbs between paraplegic and tetraplegic patients but not in the femoral neck, pelvis, and lower extremities. [16] Indeed, the effects on spinal BMD differed from previously published work in which the investigation was mainly focused in

The importance of mechanical loading and site specificity to maintain or increase BMD is already shown. [20] According to bone loss there are some interesting features in spinal cord injured subjects; demineralization is area dependent, occurs exclusively in the areas below the level of injury, affecting mainly paralyzed extremities and increasing from proximal to distal regions i.e. in paraplegics weight bearing skeleton regions, as the distal end of femur and proximal tibia, which are rich in cancellous bone, while region of the diaphysis of the femur

non-invasively, adapted from: Dionyssiotis Y. (15) (with permission).

paraplegics. [17-19]

respectively, whereas periosteal circumference was comparable to controls (Fig. 1).

#### **2.2. Bone mineral density**

In individuals with SCI bone loss begins immediately after injury. [10, 11] SCI-related BI below the level of injury is much greater compared with other conditions (i.e. age, immobilization, bed rest, lack of gravity environment). A reduction of bone mineral content during the first years after the injury of 4% per month in regions rich in cancellous bone, and 2% per month on sites containing mainly cortical bone is reported. [12] According to another study 25 out of 41 patients with SCI (61%) met WHO's criteria for osteoporosis, eight (19.5%) were osteopenic and only eight (19.5%) showed normal values. [6] In SCI children (boys and girls) values for BMD at the hip were approximately 60% of normal, or had a Z-score that indicated a 1.6-1.8 SD reduction in BMD compared with age-and sex-matched peers. [7]

Bone loss measured with peripheral quantitative computed tomography (p QCT) in SCI subjects in the femur's and tibia's epiphyses was 50% and 60% vs. 35% and 25% in the diaphyses, respectively, meaning that bone loss in the epiphyses almost doubled the loss in the diaphyses. [13] This study also showed that bone loss between trabecular and cortical bone compartment differs in mechanism, i.e. in the epiphyses bone is lost due to the decrease in trabecular, while in diaphysis, the cortical bone density is maintained and bone is lost due to endocortical resorption. In line with the previous study, another p QCT study, performed in complete paraplegics with high (thoracic 4-7) and low (thoracic 8-12) neurological level of injury at the tibia, found a loss of trabecular (57.5% vs. 51%, in high vs. low paraplegics, respectively) and cortical bone (3.6% and 6.5%, respectively), suggesting that trabecular bone is more affected during the years of paralysis in comparison with cortical bone. [14] In the same study both paraplegic groups had a similar loss of total BMD (46.90% vs. 45.15%, in high vs. low paraplegics, respectively) suggesting that a homogenously deficit pattern occurs in the epiphyseal area, especially in the group of low paraplegics because the central and the peripheral of the cross sectional area of bone were similarly affected. On the contrary, in high paraplegics' group trabecular bone loss was higher suggesting an increasing endocortical remodeling keeping the total BMD similar. Concerning cortical geometric properties the results had shown an increased endosteal circumference between both paraplegic groups vs. controls leading to reduction of cortical thickness, 19.78% vs. 16.98% in paraplegic groups respectively, whereas periosteal circumference was comparable to controls (Fig. 1).

**2. Paraplegia related bone impairment**

the femoral neck of about 0.1 g/cm2

vulnerable and exposed. [8, 9]

**2.2. Bone mineral density**

According to the literature, spinal cord injury-related bone impairment (SCI-related BI) occurs in 75% of patients with complete SCI. [5] Twenty five out of 41 patients with SCI (61%) met WHO criteria for osteoporosis; eight (19.5%) were osteopenic and only eight (19.5%) showed normal values.[6] In SCI children (boys and girls), values for BMD at the hip were approxi‐ mately 60% of normal, or had a Z-score that indicated a 1.6-1.8 SD reduction in BMD compared with age-and sex-matched peers. [7] The decrease in BMD was probably the dominant cause for the high prevalence of SCI-related BI in the long femur or proximal tibia and explains why these areas are often fracture site. [6, 8, 9] For example, a reduction in bone mineral density in

mass is associated with alterations in bone material, reduced bone elasticity and is connected to the origin of pathological fractures with minimal injury, in which these patients are

In individuals with SCI bone loss begins immediately after injury. [10, 11] SCI-related BI below the level of injury is much greater compared with other conditions (i.e. age, immobilization, bed rest, lack of gravity environment). A reduction of bone mineral content during the first years after the injury of 4% per month in regions rich in cancellous bone, and 2% per month on sites containing mainly cortical bone is reported. [12] According to another study 25 out of 41 patients with SCI (61%) met WHO's criteria for osteoporosis, eight (19.5%) were osteopenic and only eight (19.5%) showed normal values. [6] In SCI children (boys and girls) values for BMD at the hip were approximately 60% of normal, or had a Z-score that indicated a 1.6-1.8

Bone loss measured with peripheral quantitative computed tomography (p QCT) in SCI subjects in the femur's and tibia's epiphyses was 50% and 60% vs. 35% and 25% in the diaphyses, respectively, meaning that bone loss in the epiphyses almost doubled the loss in the diaphyses. [13] This study also showed that bone loss between trabecular and cortical bone compartment differs in mechanism, i.e. in the epiphyses bone is lost due to the decrease in trabecular, while in diaphysis, the cortical bone density is maintained and bone is lost due to endocortical resorption. In line with the previous study, another p QCT study, performed in complete paraplegics with high (thoracic 4-7) and low (thoracic 8-12) neurological level of injury at the tibia, found a loss of trabecular (57.5% vs. 51%, in high vs. low paraplegics, respectively) and cortical bone (3.6% and 6.5%, respectively), suggesting that trabecular bone is more affected during the years of paralysis in comparison with cortical bone. [14] In the same study both paraplegic groups had a similar loss of total BMD (46.90% vs. 45.15%, in high vs. low paraplegics, respectively) suggesting that a homogenously deficit pattern occurs in the epiphyseal area, especially in the group of low paraplegics because the central and the peripheral of the cross sectional area of bone were similarly affected. On the contrary, in high paraplegics' group trabecular bone loss was higher suggesting an increasing endocortical

SD reduction in BMD compared with age-and sex-matched peers. [7]

increases fracture risk by 2.2 times. This decrease in bone

**2.1. Epidemiology**

176 Topics in Paraplegia

**Figure 1.** Peripheral quantitative computed tomography (p QCT) tibia slices in control (a) and paraplegic subject (b), (scanner XCT 3000 Stratec, Medizintechnik, Pforzheim, Germany). Areas in red represent trabecular bone, while areas in grey represent fat; pQCT allows the measurements of true volumetric densities at a minimum exposure to X-rays, assess cortical and trabecular bone density separately as well as to evaluate the geometrical properties of long bones non-invasively, adapted from: Dionyssiotis Y. (15) (with permission).

Regarding tetraplegic patients statistically significant differences were found in BMD of the spine, trochanteric region and upper limbs between paraplegic and tetraplegic patients but not in the femoral neck, pelvis, and lower extremities. [16] Indeed, the effects on spinal BMD differed from previously published work in which the investigation was mainly focused in paraplegics. [17-19]

The importance of mechanical loading and site specificity to maintain or increase BMD is already shown. [20] According to bone loss there are some interesting features in spinal cord injured subjects; demineralization is area dependent, occurs exclusively in the areas below the level of injury, affecting mainly paralyzed extremities and increasing from proximal to distal regions i.e. in paraplegics weight bearing skeleton regions, as the distal end of femur and proximal tibia, which are rich in cancellous bone, while region of the diaphysis of the femur and tibia, rich in cortical bone is reserved. [13, 14, 21] Moreover, bone loss between trabecular and cortical bone compartment differs in mechanism, i.e. in the epiphyses is due to decrease in trabecular but in diaphysis cortical bone is maintained and bone is lost through endocortical resorption by reducing cortical wall thickness. [13, 14]

bone matrix. Osteocalcin is a non-collagen protein which is a primary constituent of osteo‐ blasts, and may also be released during apoptosis of osteoclasts and indicates either formation when resorption and formation are coupled or turnover in decoupling. [26, 27] Urinary excretion of cross-linked pyridoline type I collagen is recognised as a sensitive marker of bone resorption, and pyridoline quality tests including measurement of the aminoterminal (NTx) and carboxyterminal (CTx) intermolecular cross-linking domain of bone type-1 collagen provide a good indicator of bone resorption. [28, 29] Others studied markers of bone metab‐ olism for six months after acute spinal cord injury and observed an increase in ionised serum calcium above the upper limit of normal and suppression of serum PTH. [30] The indices of bone resorption (total pyridoline, deoxypiridoline [total and free] and NTx) recorded a significant increase (even 10 times above the upper limit of normal) after acute immobilisation, with the highest values found 10 to 16 weeks after injury. The markers of bone formation (total alkaline phosphatase and osteocalcin) showed an insignificant increase, which remained within the normal limits. [10] Moreover, Nance et al. observed that values of NTx in the urine were lower during the first months in patients receiving pamidronate compared with the control group, but this finding did not reach significance. [31] Regarding the lack or insuffi‐ ciency of vitamin D, it has been reported that 64% of paraplegics are deficient (<15ng/ml). [32]

Paraplegia Related Osteoporosis http://dx.doi.org/10.5772/57969 179

Mechanical unloading (paralysis) in acute SCI subjects causes greater sclerostin levels than those observed in the able bodied. This increase is associated with reduced bone formation during the acute phase of SCI. The ability to walk (mechanical loading) modulates the response of bone to paralysis by causing a smaller increase in sclerostin levels, thereby partially protecting against bone loss. In the chronic phase, bone wasting results in lower sclerostin levels than those observed in the able bodied. This effect is due to the reduction of sclerostinproducing osteocytes in the osteoporotic bone. In the chronic phase, similar to the acute phase, the ability to walk partially protects against bone loss. Sclerostin causes up-regulation of RANKL (key factor that promotes the differentiation of osteoclasts) and down regulation of osteoprotegerin (a key inhibitor of osteoclast differentiation) expression in osteocytes, which

The duration of paralysis affects the degree of bone loss in regions below the level of injury. A study of 21 men with SCI with an average duration of 10.6 years, using DEXA, expressed at various levels of injury an inverse relationship between BMD in the legs and the duration of the lesion, while others found a weaker relationship regarding the microarchitecture of the

In a study which included paraplegics with duration of paralysis of 14 ± 11.5 years a positive correlation between the duration of paralysis and the degree of bone loss was found. [13] The length of immobilization in the acute posttraumatic period increased bone loss in the legs, particularly in the proximal tibia; over 50% of bone mass was lost (in the affected areas) in the period of ten years after the injury. [21] When subjects categorized depending on the length of the lesion (0-1, 1-5, 6-9, 10-19, 20-29, 30-39, 40-49, and 50-59 years after the injury), in all age

leads to increased osteoclast activity and bone resorption. [33]

*2.2.3. Duration of paralysis and bone steady state*

distal end of tibia. [34, 35]

#### *2.2.1. The additional risk factor of feminine gender*

Women with disabilities have a higher risk of losing bone mass compared to men because of the inevitable reduction in estrogen levels that occurs at menopause. Findings that women with serious disabilities have low bone density are not surprising and are probably related to the lack of activity (reduced mobility, reduced loading on bone) and worsening of the disability. [22] Regarding women with complete SCI, the initial bone loss in the lumbar spine is negligible. Post injury over a period of years BMD in SCI women is maintained or increases compared with non-injured age-matched women, in whom BMD decreases during aging.

#### *2.2.2. Biochemical changes in bone after spinal cord injury*

After SCI, osteoblast activity slightly increases, while a significant increase in osteoclast activity within a maximum of 10 weeks after injury and at level up to 10 times greater than normal is present. The imbalance between bone resorption and bone formation below the level of the lesion or injured area may be due to decreased blood flow and venous stasis, arteriovenous anastomoses and tissue oxidation. [23] SCI-related BI can be enhanced by a lack of muscular tension on bone or other neuronal factors associated with the lesion. The parathyroid glands are inactive with low levels of parathyroid hormone (PTH) observed up to one year after injury. The hypercalcemia that occurs immediately after injury is responsible for low levels of PTH. Gradually, in a range of one to nine years after injury, the function of the parathyroid is restored. The result is an increase in bone resorption associated with dysfunction of the parathyroid glands in the chronic phase of injury. This mechanism of SCI-related BI during the chronic phase tends to be balanced by an increase in bone mineral density (BMD) in areas of the body with increased loading (upper limbs, spinal column) and adds bone density (transferring bone mineral) compared to a loss in the chronic non-loadable areas of the skeleton (pelvis, lower limbs and upper limbs in tetraplegics). Hormonal changes (parathyroid hormone, glucocorticoids and calcitonin) and metabolic disorders (increased alkaline phos‐ phatase, hypercalcemia/hypercalciuria and hydroxyproline excretion) may be secondary to the loss of bone density. [10, 24] Hypercalciuria is seen in the first 10 days after neurological injury and reaches its maximum value after one to six months and is two to four times greater that the hypercalciuria observed after prolonged bed rest. The significant increase of calcium in the urine is the result of an imbalance between bone formation and bone resorption. [25] The rate of formation or resorption of bone matrix can be determined by quantifying the enzyme activity of bone cells or by measuring the components of the matrix that are released into the circulation during the process of absorption. It should be noted that these indices of bone activity are somewhat non-specific. The intact procollagen I N-terminal propeptide (PINP) molecule is the amino end of type I procollagen before excision and the formation of fibrils and is a measure of the total synthesis of collagen in the body, all of which is related to bone matrix. Osteocalcin is a non-collagen protein which is a primary constituent of osteo‐ blasts, and may also be released during apoptosis of osteoclasts and indicates either formation when resorption and formation are coupled or turnover in decoupling. [26, 27] Urinary excretion of cross-linked pyridoline type I collagen is recognised as a sensitive marker of bone resorption, and pyridoline quality tests including measurement of the aminoterminal (NTx) and carboxyterminal (CTx) intermolecular cross-linking domain of bone type-1 collagen provide a good indicator of bone resorption. [28, 29] Others studied markers of bone metab‐ olism for six months after acute spinal cord injury and observed an increase in ionised serum calcium above the upper limit of normal and suppression of serum PTH. [30] The indices of bone resorption (total pyridoline, deoxypiridoline [total and free] and NTx) recorded a significant increase (even 10 times above the upper limit of normal) after acute immobilisation, with the highest values found 10 to 16 weeks after injury. The markers of bone formation (total alkaline phosphatase and osteocalcin) showed an insignificant increase, which remained within the normal limits. [10] Moreover, Nance et al. observed that values of NTx in the urine were lower during the first months in patients receiving pamidronate compared with the control group, but this finding did not reach significance. [31] Regarding the lack or insuffi‐ ciency of vitamin D, it has been reported that 64% of paraplegics are deficient (<15ng/ml). [32]

Mechanical unloading (paralysis) in acute SCI subjects causes greater sclerostin levels than those observed in the able bodied. This increase is associated with reduced bone formation during the acute phase of SCI. The ability to walk (mechanical loading) modulates the response of bone to paralysis by causing a smaller increase in sclerostin levels, thereby partially protecting against bone loss. In the chronic phase, bone wasting results in lower sclerostin levels than those observed in the able bodied. This effect is due to the reduction of sclerostinproducing osteocytes in the osteoporotic bone. In the chronic phase, similar to the acute phase, the ability to walk partially protects against bone loss. Sclerostin causes up-regulation of RANKL (key factor that promotes the differentiation of osteoclasts) and down regulation of osteoprotegerin (a key inhibitor of osteoclast differentiation) expression in osteocytes, which leads to increased osteoclast activity and bone resorption. [33]

#### *2.2.3. Duration of paralysis and bone steady state*

and tibia, rich in cortical bone is reserved. [13, 14, 21] Moreover, bone loss between trabecular and cortical bone compartment differs in mechanism, i.e. in the epiphyses is due to decrease in trabecular but in diaphysis cortical bone is maintained and bone is lost through endocortical

Women with disabilities have a higher risk of losing bone mass compared to men because of the inevitable reduction in estrogen levels that occurs at menopause. Findings that women with serious disabilities have low bone density are not surprising and are probably related to the lack of activity (reduced mobility, reduced loading on bone) and worsening of the disability. [22] Regarding women with complete SCI, the initial bone loss in the lumbar spine is negligible. Post injury over a period of years BMD in SCI women is maintained or increases compared with non-injured age-matched women, in whom BMD decreases during aging.

After SCI, osteoblast activity slightly increases, while a significant increase in osteoclast activity within a maximum of 10 weeks after injury and at level up to 10 times greater than normal is present. The imbalance between bone resorption and bone formation below the level of the lesion or injured area may be due to decreased blood flow and venous stasis, arteriovenous anastomoses and tissue oxidation. [23] SCI-related BI can be enhanced by a lack of muscular tension on bone or other neuronal factors associated with the lesion. The parathyroid glands are inactive with low levels of parathyroid hormone (PTH) observed up to one year after injury. The hypercalcemia that occurs immediately after injury is responsible for low levels of PTH. Gradually, in a range of one to nine years after injury, the function of the parathyroid is restored. The result is an increase in bone resorption associated with dysfunction of the parathyroid glands in the chronic phase of injury. This mechanism of SCI-related BI during the chronic phase tends to be balanced by an increase in bone mineral density (BMD) in areas of the body with increased loading (upper limbs, spinal column) and adds bone density (transferring bone mineral) compared to a loss in the chronic non-loadable areas of the skeleton (pelvis, lower limbs and upper limbs in tetraplegics). Hormonal changes (parathyroid hormone, glucocorticoids and calcitonin) and metabolic disorders (increased alkaline phos‐ phatase, hypercalcemia/hypercalciuria and hydroxyproline excretion) may be secondary to the loss of bone density. [10, 24] Hypercalciuria is seen in the first 10 days after neurological injury and reaches its maximum value after one to six months and is two to four times greater that the hypercalciuria observed after prolonged bed rest. The significant increase of calcium in the urine is the result of an imbalance between bone formation and bone resorption. [25] The rate of formation or resorption of bone matrix can be determined by quantifying the enzyme activity of bone cells or by measuring the components of the matrix that are released into the circulation during the process of absorption. It should be noted that these indices of bone activity are somewhat non-specific. The intact procollagen I N-terminal propeptide (PINP) molecule is the amino end of type I procollagen before excision and the formation of fibrils and is a measure of the total synthesis of collagen in the body, all of which is related to

resorption by reducing cortical wall thickness. [13, 14]

*2.2.2. Biochemical changes in bone after spinal cord injury*

*2.2.1. The additional risk factor of feminine gender*

178 Topics in Paraplegia

The duration of paralysis affects the degree of bone loss in regions below the level of injury. A study of 21 men with SCI with an average duration of 10.6 years, using DEXA, expressed at various levels of injury an inverse relationship between BMD in the legs and the duration of the lesion, while others found a weaker relationship regarding the microarchitecture of the distal end of tibia. [34, 35]

In a study which included paraplegics with duration of paralysis of 14 ± 11.5 years a positive correlation between the duration of paralysis and the degree of bone loss was found. [13] The length of immobilization in the acute posttraumatic period increased bone loss in the legs, particularly in the proximal tibia; over 50% of bone mass was lost (in the affected areas) in the period of ten years after the injury. [21] When subjects categorized depending on the length of the lesion (0-1, 1-5, 6-9, 10-19, 20-29, 30-39, 40-49, and 50-59 years after the injury), in all age groups bone mineral density of the proximal femur declined and was detected a year after the injury. [24]

injury), the type of injury (complete SCI subjects have more fractures than incomplete), low

Paraplegia Related Osteoporosis http://dx.doi.org/10.5772/57969 181

Spinal cord injury is a dynamic process that is related to alterations in both the central and peripheral sympathetic nervous system (SNS). Sympathetic denervation in SCI may cause arteriovenous shunts and a slowdown of intraosseous blood flow, thus increasing bone resorption. [44] With high-level spinal cord lesions the SNS is disproportionately involved when compared with the parasympathetic nervous system. In a complete high-level SCI, functioning in the isolated spinal cord below the lesion becomes independent of supraspinal control and has been termed ˝decentralization˝ of the sympathetic nervous system. [45]

Loss of supraspinal control leads to dysregulation of those homeostatic mechanisms normally influenced by the SNS through loss of facilitation or lack of inhibition. [46] Today there is clinical evidence that the sympathetic regulation of bone does exist in humans and plays a clinically important role in diseases characterized by excessive sympathetic activity. [47] The scientific finding about sympathetic innervations of bone tissue and its role in the regulation of bone remodelling is of major interest in situations where uncoupling between osteoclasts

So far, spasticity has been considered by many researchers as a prophylactic factor for bone. It is well known that voluntary muscle contraction is effective in the prevention of osteoporosis. [51, 52] Although muscle loading plays a vital role in maintaining bone density, conflicting results regarding the effect of muscle spasms in the form of spasticity have been reported in SCI patients. [53-56] Controversial results have also been reported regarding the effect of spasticity on BMD in paraplegics. A cross-sectional study of 41 paraplegics reported less reduction of BMD in the spastic compared to the flaccid paraplegic SCI patients. [53-55] Other investigators suggested that muscle spasms can slow bone loss based on the theory of a single basic muscle/bone unit. [56] Muscle spasms and muscle tension in the presence of spasticity put force on bone. This is likely to play a regulatory role in maintaining bone density. These studies concluded that spasticity may be a protective factor against bone loss in SCI. Other researchers, however, could not find a correlation between bone density and spasticity. [55] Moreover, in 18 motor complete SCI men matched for time since injury, gender and age (nine had severe spasticity and nine had spasticity that was either mild or not present) no difference was found in BMD depending on the level of spasticity. [57] A pQCT study investigating the tibia in complete paraplegics above the thoracic 12 (T12) level with various degrees of spasticity according to the Ashworth scale found no effect on volumetric BMD measurements.[41] Others have reported that spasticity may be protective against bone loss in SCI patients, however, without any preserving effect on the tibia. [55] A possible explanation for this could lie in the fact that studies include various SCI subjects with various degrees of spasticity. In addition,

body mass index (BMI) and low bone density in the tibia. [6, 24, 43]

*2.2.4. The role of central nervous system*

*2.2.4.1. Sympathetic denervation in SCI*

and osteoblasts occurs. [48-50]

*2.2.4.2. Spasticity*

Using DXA and QUS (quantitative ultrasound) measurements in 100 men with SCI, aged 18 to 60 years, it was found that bone density decreases over time in all measured points, while bone loss followed a linear pattern in the femoral neck and distal epiphysis, stabilized within three years after the injury. On the contrary, Z-scores of the distal region of the diaphysis of the tibia continued to decrease even beyond ten years after the injury. [36] Duration of paralysis related bone loss in the legs of monozygotic twins with chronic paraplegia in comparison with their able-bodied co-twins has been also reported. [37]

The results of a comparison of chronic complete paraplegic men vs. controls in another study found a reduction of BMD in paraplegics' legs independent of the neurological level of lesion. BMD of the legs was negatively correlated with the duration of paralysis in the total paraplegic group, but after investigation according to the neurological level this correlation was due to the strong correlation of high paraplegics' legs BMD with the duration of paralysis, suggesting a possible influence of the neurological level of injury on the extent of bone loss. [38] A significant inverse relationship between percentage-matched in BMD leg, arm and trunk values and time since injury was found when varying levels of SCI were analyzed. [34]

Studies are supporting the concept of a new bone steady state at 16-24 months after injury, especially for bone metabolic process, but BMD decreases over the years at different areas and is inversely related to the time of the injury, which means continuous bone loss beyond the first two years after the injury (Fig. 2). [11, 13, 14, 24, 38-41]

**Figure 2.** The duration of paralysis was inversely related with trabecular bone loss in spinal cord injured subjects. Ex‐ ponential correlation between volumetric trabecular bone mineral density BMD trab and duration of paralysis in high paraplegics was found to fit best. On the contrary no significant decrease in BMD cort of the diaphyses was found in total paraplegic group. BMD parameters were measured by pQCT in 31 paraplegic men in chronic stage (>1.5 years of injury). Spinal cord injury paraplegic men were allocated into 2 subgroups based on the neurological level of injury; subgroup A (n=16, Thoracic (T) 4 –T 7 neurological level of injury) and subgroup B (n=15, T8-T12 neurological level of injury). BMDtrab: BMD trabecular; BMDcort: BMD cortical; (adapted from Dionyssiotis et al. [41] with permission).

The role played by factors such as race or gender of patients is not yet clear documented, but studies indicated more loss in women than men. [42] Loss of bone is closing fracture threshold from 1 to 5 years after injury and risk factors for fractures after spinal cord injury are gender (women are more at risk than men), age and duration of injury (increasing age and duration of injury increases the risk of fracture with a statistically significant increase in 10 years after injury), the type of injury (complete SCI subjects have more fractures than incomplete), low body mass index (BMI) and low bone density in the tibia. [6, 24, 43]

#### *2.2.4. The role of central nervous system*

groups bone mineral density of the proximal femur declined and was detected a year after the

Using DXA and QUS (quantitative ultrasound) measurements in 100 men with SCI, aged 18 to 60 years, it was found that bone density decreases over time in all measured points, while bone loss followed a linear pattern in the femoral neck and distal epiphysis, stabilized within three years after the injury. On the contrary, Z-scores of the distal region of the diaphysis of the tibia continued to decrease even beyond ten years after the injury. [36] Duration of paralysis related bone loss in the legs of monozygotic twins with chronic paraplegia in comparison with

The results of a comparison of chronic complete paraplegic men vs. controls in another study found a reduction of BMD in paraplegics' legs independent of the neurological level of lesion. BMD of the legs was negatively correlated with the duration of paralysis in the total paraplegic group, but after investigation according to the neurological level this correlation was due to the strong correlation of high paraplegics' legs BMD with the duration of paralysis, suggesting a possible influence of the neurological level of injury on the extent of bone loss. [38] A significant inverse relationship between percentage-matched in BMD leg, arm and trunk values and time since injury was found when varying levels of SCI were analyzed. [34]

Studies are supporting the concept of a new bone steady state at 16-24 months after injury, especially for bone metabolic process, but BMD decreases over the years at different areas and is inversely related to the time of the injury, which means continuous bone loss beyond the

**Figure 2.** The duration of paralysis was inversely related with trabecular bone loss in spinal cord injured subjects. Ex‐ ponential correlation between volumetric trabecular bone mineral density BMD trab and duration of paralysis in high paraplegics was found to fit best. On the contrary no significant decrease in BMD cort of the diaphyses was found in total paraplegic group. BMD parameters were measured by pQCT in 31 paraplegic men in chronic stage (>1.5 years of injury). Spinal cord injury paraplegic men were allocated into 2 subgroups based on the neurological level of injury; subgroup A (n=16, Thoracic (T) 4 –T 7 neurological level of injury) and subgroup B (n=15, T8-T12 neurological level of injury). BMDtrab: BMD trabecular; BMDcort: BMD cortical; (adapted from Dionyssiotis et al. [41] with permission).

The role played by factors such as race or gender of patients is not yet clear documented, but studies indicated more loss in women than men. [42] Loss of bone is closing fracture threshold from 1 to 5 years after injury and risk factors for fractures after spinal cord injury are gender (women are more at risk than men), age and duration of injury (increasing age and duration of injury increases the risk of fracture with a statistically significant increase in 10 years after

their able-bodied co-twins has been also reported. [37]

first two years after the injury (Fig. 2). [11, 13, 14, 24, 38-41]

injury. [24]

180 Topics in Paraplegia

#### *2.2.4.1. Sympathetic denervation in SCI*

Spinal cord injury is a dynamic process that is related to alterations in both the central and peripheral sympathetic nervous system (SNS). Sympathetic denervation in SCI may cause arteriovenous shunts and a slowdown of intraosseous blood flow, thus increasing bone resorption. [44] With high-level spinal cord lesions the SNS is disproportionately involved when compared with the parasympathetic nervous system. In a complete high-level SCI, functioning in the isolated spinal cord below the lesion becomes independent of supraspinal control and has been termed ˝decentralization˝ of the sympathetic nervous system. [45]

Loss of supraspinal control leads to dysregulation of those homeostatic mechanisms normally influenced by the SNS through loss of facilitation or lack of inhibition. [46] Today there is clinical evidence that the sympathetic regulation of bone does exist in humans and plays a clinically important role in diseases characterized by excessive sympathetic activity. [47] The scientific finding about sympathetic innervations of bone tissue and its role in the regulation of bone remodelling is of major interest in situations where uncoupling between osteoclasts and osteoblasts occurs. [48-50]

#### *2.2.4.2. Spasticity*

So far, spasticity has been considered by many researchers as a prophylactic factor for bone. It is well known that voluntary muscle contraction is effective in the prevention of osteoporosis. [51, 52] Although muscle loading plays a vital role in maintaining bone density, conflicting results regarding the effect of muscle spasms in the form of spasticity have been reported in SCI patients. [53-56] Controversial results have also been reported regarding the effect of spasticity on BMD in paraplegics. A cross-sectional study of 41 paraplegics reported less reduction of BMD in the spastic compared to the flaccid paraplegic SCI patients. [53-55] Other investigators suggested that muscle spasms can slow bone loss based on the theory of a single basic muscle/bone unit. [56] Muscle spasms and muscle tension in the presence of spasticity put force on bone. This is likely to play a regulatory role in maintaining bone density. These studies concluded that spasticity may be a protective factor against bone loss in SCI. Other researchers, however, could not find a correlation between bone density and spasticity. [55] Moreover, in 18 motor complete SCI men matched for time since injury, gender and age (nine had severe spasticity and nine had spasticity that was either mild or not present) no difference was found in BMD depending on the level of spasticity. [57] A pQCT study investigating the tibia in complete paraplegics above the thoracic 12 (T12) level with various degrees of spasticity according to the Ashworth scale found no effect on volumetric BMD measurements.[41] Others have reported that spasticity may be protective against bone loss in SCI patients, however, without any preserving effect on the tibia. [55] A possible explanation for this could lie in the fact that studies include various SCI subjects with various degrees of spasticity. In addition, in studies examining the lower leg, muscle spasms affecting the lower leg would mainly be extension spasms resulting in plantar flexion, thus creating little resistance to the contracting muscles. Furthermore, the measuring sites of the tibia did not include any muscle insertions of either the knee or the ankle extensor muscles. Patients without spasticity usually have more fractures. At the same time, excessive spasticity may cause fractures through uncontrolled limb movements, i.e. in a wheelchair. Therefore, the effect of spasticity on bone is probably twosided: a low grade of spasticity is beneficial while a high grade is harmful. [41]

#### **3. Interventions for prevention of bone impairment**

#### **3.1. Weight bearing activities – Body weight supported treadmill – Cycling**

The effect of standing in bone after SCI has been investigated by many researchers. A beneficial effect on bone mass using passive mechanical loading has been shown on preservation of bone mass in the region of the femoral shaft, but not at the proximal hip of standing and nonstanding patients and relatively better-preserved densities in patients standing with braces than in those using a standing frame or standing wheelchair. [58] A slower rate of bone loss in paraplegic subjects who did standing was expressed in a prospective study of 19 patients in acute SCI phase participated in early standing training program which showed benefits concerning the reduction of cancellous bone loss compared to immobilized subjects, while no correlation for passive standing-training to bone status was found in another p QCT study. [59, 60] Protection afforded by standing in the femoral diaphysis stands in contrast with the loss of bone in the proximal femur. This suggests that the transmission of forces through trabecular and cortical bone varies; so the less effective strain for the initiation of bone remodeling reaches faster cortical bone. [61] Others also supported the concept of different strain thresholds during bone remodeling control. [62-64] There is level 2 evidence (from 1 non-randomized prospective controlled trial) that Functional Electrical Stimulation (FES)-cycling did not improve or maintain bone at the tibial midshaft in the acute phase. [65] Moreover, there is level 4 evidence (from 1 pre-post study) that 6 months of FES cycle ergometry increased regional lower extremity BMD over areas stimulated. [66] Body weight supported treadmill training (BWSTT) did not alter the expected pattern of change in bone biochemical markers over time and bone density at fracture-prone sites. [67]

therapy program unchanged or to receive 9 minutes of side-alternating WBV (Vibraflex Home Edition II®, Orthometrix Inc) no effect on areal BMD at the lumbar spine was observed, while areal BMD seemed to decrease somewhat in the cortical region of the femoral diaphysis. Authors explained that mechanical stimulation increases intracortical bone remodeling and thereby cortical porosity; moreover changes occurred in ways that are not reflected by areal BMD, but might be detectable by more sophisticated techniques such as such as peripheral quantitative computed tomography. [70] Low-intensity vibration (LIV) has shown to be associated with improvement in bone mineral density in post-menopausal women and children with cerebral palsy. Seven non-ambulatory subjects with SCI and ten able-bodied controls underwent transmission of a plantar-based LIV signal (0.27+/-0.11 g; 34 Hz) from the feet through the axial skeleton as a function of tilt-table angle (15, 30, and 45 degrees). SCI subjects and controls demonstrated equivalent transmission of LIV, with greater signal transmission observed at steeper angles of tilt which supports the possibility of the utility of LIV as a means to deliver mechanical signals in a form of therapeutic intervention to prevent/

**Figure 3.** Weight bearing in disabled subjects; using standing frames, functional walking with orthoses between bars and crutches, even push-ups in the wheelchair (in case of multiple sclerosis with a clinical equivalent like tetraplegia)

Paraplegia Related Osteoporosis http://dx.doi.org/10.5772/57969 183

bone can be loaded and bone loss rate would be slower (unpublished photos of Dionyssiotis Y).

reverse skeletal fragility in the SCI population. [71]

#### **3.2. Whole body vibration**

At a meeting of the American Society for Bone and Mineral Research the results of a small randomised, placebo-controlled study among 20 children with cerebral palsy who used a similar, commercially available vibrating platform for 10 min per day, 5 days per week for 6 months, reported a significant increase in tibial, but not lumbar-spine bone density in the treated group despite the simplicity, short duration of the "vibration, the young age of the children and the poor compliance. [68, 69]

After 6 months of whole body vibration (WBV) therapy in twenty children (14 boys-6 girls) with cerebral palsy (age 6.2 to 12.3 years) randomized to either continue their school physio‐

in studies examining the lower leg, muscle spasms affecting the lower leg would mainly be extension spasms resulting in plantar flexion, thus creating little resistance to the contracting muscles. Furthermore, the measuring sites of the tibia did not include any muscle insertions of either the knee or the ankle extensor muscles. Patients without spasticity usually have more fractures. At the same time, excessive spasticity may cause fractures through uncontrolled limb movements, i.e. in a wheelchair. Therefore, the effect of spasticity on bone is probably two-

The effect of standing in bone after SCI has been investigated by many researchers. A beneficial effect on bone mass using passive mechanical loading has been shown on preservation of bone mass in the region of the femoral shaft, but not at the proximal hip of standing and nonstanding patients and relatively better-preserved densities in patients standing with braces than in those using a standing frame or standing wheelchair. [58] A slower rate of bone loss in paraplegic subjects who did standing was expressed in a prospective study of 19 patients in acute SCI phase participated in early standing training program which showed benefits concerning the reduction of cancellous bone loss compared to immobilized subjects, while no correlation for passive standing-training to bone status was found in another p QCT study. [59, 60] Protection afforded by standing in the femoral diaphysis stands in contrast with the loss of bone in the proximal femur. This suggests that the transmission of forces through trabecular and cortical bone varies; so the less effective strain for the initiation of bone remodeling reaches faster cortical bone. [61] Others also supported the concept of different strain thresholds during bone remodeling control. [62-64] There is level 2 evidence (from 1 non-randomized prospective controlled trial) that Functional Electrical Stimulation (FES)-cycling did not improve or maintain bone at the tibial midshaft in the acute phase. [65] Moreover, there is level 4 evidence (from 1 pre-post study) that 6 months of FES cycle ergometry increased regional lower extremity BMD over areas stimulated. [66] Body weight supported treadmill training (BWSTT) did not alter the expected pattern of change in bone biochemical markers over time and bone

At a meeting of the American Society for Bone and Mineral Research the results of a small randomised, placebo-controlled study among 20 children with cerebral palsy who used a similar, commercially available vibrating platform for 10 min per day, 5 days per week for 6 months, reported a significant increase in tibial, but not lumbar-spine bone density in the treated group despite the simplicity, short duration of the "vibration, the young age of the

After 6 months of whole body vibration (WBV) therapy in twenty children (14 boys-6 girls) with cerebral palsy (age 6.2 to 12.3 years) randomized to either continue their school physio‐

sided: a low grade of spasticity is beneficial while a high grade is harmful. [41]

**3.1. Weight bearing activities – Body weight supported treadmill – Cycling**

**3. Interventions for prevention of bone impairment**

density at fracture-prone sites. [67]

children and the poor compliance. [68, 69]

**3.2. Whole body vibration**

182 Topics in Paraplegia

**Figure 3.** Weight bearing in disabled subjects; using standing frames, functional walking with orthoses between bars and crutches, even push-ups in the wheelchair (in case of multiple sclerosis with a clinical equivalent like tetraplegia) bone can be loaded and bone loss rate would be slower (unpublished photos of Dionyssiotis Y).

therapy program unchanged or to receive 9 minutes of side-alternating WBV (Vibraflex Home Edition II®, Orthometrix Inc) no effect on areal BMD at the lumbar spine was observed, while areal BMD seemed to decrease somewhat in the cortical region of the femoral diaphysis. Authors explained that mechanical stimulation increases intracortical bone remodeling and thereby cortical porosity; moreover changes occurred in ways that are not reflected by areal BMD, but might be detectable by more sophisticated techniques such as such as peripheral quantitative computed tomography. [70] Low-intensity vibration (LIV) has shown to be associated with improvement in bone mineral density in post-menopausal women and children with cerebral palsy. Seven non-ambulatory subjects with SCI and ten able-bodied controls underwent transmission of a plantar-based LIV signal (0.27+/-0.11 g; 34 Hz) from the feet through the axial skeleton as a function of tilt-table angle (15, 30, and 45 degrees). SCI subjects and controls demonstrated equivalent transmission of LIV, with greater signal transmission observed at steeper angles of tilt which supports the possibility of the utility of LIV as a means to deliver mechanical signals in a form of therapeutic intervention to prevent/ reverse skeletal fragility in the SCI population. [71]

in BMD effects of PEMFs may relate to the features of the subjects. People with spinal cord injury are younger than osteoporosis patients, the osteoblasts and osteoclasts of patients with spinal cord injury may be more sensitive to the PEMFs stimulation than that of the old people.

**Clinical examination and management of bone loss in paraplegia**

**Table 1.** An algorithm for the screening and management of osteoporosis in subjects with spinal cord injury (should be read top to bottom starting with the left column); adapted from: Dionyssiotis Y. (84) (with permission).

Calcitonin in varying doses and methods of administration has given variable results in paraplegia (preferred dosage regimen, treatment duration, and administration route for adequate efficacy in SCI patients' remains unclear). [75, 76] Likewise, the outcome using bisphosphonates has been variable. Etidronate produced long-term benefit in lower limb bone mineral density (BMD) in selected walking SCI patients; whereas tiludronate appeared effective in reducing bone resorption and preserving bone mass in a histomorphometric study in 20 paraplegic patients. [77, 78] Intravenous pamidronate has been shown to attenuate bone loss in SCI and normalize serum calcium in immobilization hypercalcemia. [79] Alendronate

• pharmacological treatment with bisphosphonates p.os and i.v. that have been studied in patients with spinal cord injuries and had positive effects on bone parameters. • Use of calcium supplements (monitoring renal function) and vitamin D.

Paraplegia Related Osteoporosis http://dx.doi.org/10.5772/57969 185

• Education on falls prevention • Counseling regarding osteoporosis and related factors and identification of fractures in regions of impaired sensation.

• physical therapy including: a) range of motion exercises, b) loading of the skeleton to reduce bone loss, d) therapeutic standing-walking with orthoses, e) passiveactive cycling

• dietary interventions to improve dietary intake of calcium and nutrition indices.

• history of the patient (co morbidities, neurologic complications, use of drugs which impair bone metabolism, alcohol, smoking and information about the level of injury, duration of paralysis, immobilization period, onset of rehabilitation, use of assistive devices and orthoses).

• anthropometric parameters (age, weight, body mass index, BMI)• clinical examination (level of injury according to American Spinal Injury Association Impairment Scale, AIS) and assessment of spasticity)

• imaging (bone densitometry by DXA at the hip and spine, and if possible, p QCT at the the tibia or femur)

• measurement of bone turnover indices in the serum (parathyroid hormone, alkaline phosphatase, calcium, vitamin D, PINP molecule, osteocalcin) and urinary excretion of 24 hour (calcium, hydroxyproline, aminoterminal (NTx) and carboxylterminal (CTx) intermolecular cross-linking domain of bone type-1 collagen), which provide a good indicator of bone resorption.

**3.4. Drugs**

**Figure 4.** The Galileo Delta A TiltTable offers a wide variety of applications from relaxation to muscle training for a diverse range of patients who are unable to stand without support. The motor driven adjustable tilt angle of the Gali‐ leo Delta TiltTable (90°) allows vibration training with reduced body weight from 0 to 100%. This is ideal for decondi‐ tioned and disabled patients for gradually increasing training weights up to full body weight. System for application in adults (max. body height: 1.90 m) and children (max. body height: 1.50 m).The Galileo Delta A TiltTable is exclusively available from the manufacturer Novotec Medical GmbH. (published with permission).

#### **3.3. Pulsed Electromagnetic Fields (PEMF)**

Huang et al recently reviewed the effects of low-frequency pulsed electromagnetic fields (PEMFs) on chronic bony pain, bone mineral density (BMD), bone strength and biochemical markers of bone metabolism in the patients of osteoporosis. [72] Two studies are analyzed in SCI subjects: In a study that consisted of 6 male patients with complete spinal cord injury of a minimum of 2 years duration the time of therapy of PEMFs continued for 6 months and at 3 months BMD increased in the stimulated knees by 5.1% and declined in the control knees by 6.6% (*P* <0.05 and *P* <0.02, respectively). By 6 months the BMD returned to near baseline values and at 12 months both knees had lost bone at a similar rate. It was demonstrated that PEMFs can delay bone loss and there may exist both a local and a systemic response. [73] Another study consisted of 24 patients with SCI who were then divided into two groups, BMD of the total proximal femur and trochanter of patients in the treatment group were increased significantly compared with the control group. [74] Both of the trials indicated that the increase in BMD effects of PEMFs may relate to the features of the subjects. People with spinal cord injury are younger than osteoporosis patients, the osteoblasts and osteoclasts of patients with spinal cord injury may be more sensitive to the PEMFs stimulation than that of the old people.


**Table 1.** An algorithm for the screening and management of osteoporosis in subjects with spinal cord injury (should be read top to bottom starting with the left column); adapted from: Dionyssiotis Y. (84) (with permission).

#### **3.4. Drugs**

**Figure 4.** The Galileo Delta A TiltTable offers a wide variety of applications from relaxation to muscle training for a diverse range of patients who are unable to stand without support. The motor driven adjustable tilt angle of the Gali‐ leo Delta TiltTable (90°) allows vibration training with reduced body weight from 0 to 100%. This is ideal for decondi‐ tioned and disabled patients for gradually increasing training weights up to full body weight. System for application in adults (max. body height: 1.90 m) and children (max. body height: 1.50 m).The Galileo Delta A TiltTable is exclusively

Huang et al recently reviewed the effects of low-frequency pulsed electromagnetic fields (PEMFs) on chronic bony pain, bone mineral density (BMD), bone strength and biochemical markers of bone metabolism in the patients of osteoporosis. [72] Two studies are analyzed in SCI subjects: In a study that consisted of 6 male patients with complete spinal cord injury of a minimum of 2 years duration the time of therapy of PEMFs continued for 6 months and at 3 months BMD increased in the stimulated knees by 5.1% and declined in the control knees by 6.6% (*P* <0.05 and *P* <0.02, respectively). By 6 months the BMD returned to near baseline values and at 12 months both knees had lost bone at a similar rate. It was demonstrated that PEMFs can delay bone loss and there may exist both a local and a systemic response. [73] Another study consisted of 24 patients with SCI who were then divided into two groups, BMD of the total proximal femur and trochanter of patients in the treatment group were increased significantly compared with the control group. [74] Both of the trials indicated that the increase

available from the manufacturer Novotec Medical GmbH. (published with permission).

**3.3. Pulsed Electromagnetic Fields (PEMF)**

184 Topics in Paraplegia

Calcitonin in varying doses and methods of administration has given variable results in paraplegia (preferred dosage regimen, treatment duration, and administration route for adequate efficacy in SCI patients' remains unclear). [75, 76] Likewise, the outcome using bisphosphonates has been variable. Etidronate produced long-term benefit in lower limb bone mineral density (BMD) in selected walking SCI patients; whereas tiludronate appeared effective in reducing bone resorption and preserving bone mass in a histomorphometric study in 20 paraplegic patients. [77, 78] Intravenous pamidronate has been shown to attenuate bone loss in SCI and normalize serum calcium in immobilization hypercalcemia. [79] Alendronate (1000 times more potent than etidronate), in an open observational study, reversed BMD loss in men with established SCI increased both axial and trabecular bone density and has proven efficacy and safety in men treated for osteoporosis, prevents hypercalciuria and bone loss after bed rest and lower leg fracture. [80, 81] Six months after using zolendronic acid in the treatment group BMD showed differences in the response to treatment between the mixed trabecular/ cortical regions (narrow neck and intertrochanteric) and the purely cortical shaft. With respect to cross-sectional geometry, bone cross-sectional area and sectional modulus (indices of resistance to axial and bending loads, where higher values would indicate a positive effect of treatment) increased at the hip and buckling ratio (an index of the instability of thin-walled cross sections, where lower values would suggest that the treatment is improving stability) decreased consistent with improved bone outcomes; at 12 months, narrow-neck femur values declined and intertrochanteric and femoral shaft BMD was maintained vs. placebo group which showed a decrease in bone outcomes and an increase in buckling ratio at the hip at 6 and 12 months, while with respect to bone prevention 4 mg i.v. were effective and welltolerated to prevent BMD loss at the total hip and trochanter for up to 12 months following SCI. [82, 83]

[6] Lazo MG, Shirazi P, Sam M, Giobbie-Hurder A, Blacconiere MJ, Muppidi M. Osteo‐ porosis and risk of fracture in men with spinal cord injury. Spinal Cord

Paraplegia Related Osteoporosis http://dx.doi.org/10.5772/57969 187

[7] Lauer R, Johnston TE, Smith BT, Mulcahey MJ, Betz RR, Maurer AH. Bone mineral density of the hip and knee in children with spinal cord injury. J Spinal Cord Med.

[8] Ragnarsson KT, Sell GH. Lower extremity fractures after spinal cord injury: a retro‐

[9] Nottage WM. A review of long-bone fractures in patients with spinal cord injuries.

[10] Uebelhart D, Demiaux-Domenech B, Roth M, Chantraine A. Bone metabolism in spi‐ nal cord injured individuals and in others who have prolonged immobilisation. A re‐

[11] Bauman WA, Garland DE, Schwartz E. Calcium metabolism and osteoporosis in in‐ dividuals with spinal cord injury. Top Spinal Cord Inj Rehabil 1997;2:84-96.

[12] Wilmet E, Ismail AA, Heilporn A, Welraeds D, Bergmann P. Longitudinal study of bone mineral content and of soft tissue composition after spinal cord injury. Paraple‐

[13] Eser P, Frotzler A, Zehnder Y, Wick L, Knecht H, Denoth J, Schiessl H. Relationship between the duration of paralysis and bone structure: a pQCT study of spinal cord

[14] Dionyssiotis Y, Trovas G, Galanos A, Raptou P, Papaioannou N, Papagelopoulos P, Petropoulou K, Lyritis GP. Bone loss and mechanical properties of tibia in spinal

[15] Dionyssiotis Y. Spinal cord injury-related bone impairment and fractures: an update on epidemiology and physiopathological mechanisms. J Musculoskelet Neuronal In‐

[16] Tsuzuku S, Ikegami Y, Yabe K. Bone mineral density differences between paraplegic and quadriplegic patients: a cross-sectional study. Spinal Cord 1999;37:358-61.

[17] Biering-Sorensen F, Bohr HH, Schaadt OP. Longitudinal study of bone mineral con‐ tent in the lumbar spine, the forearm and the lower extremities after spinal cord in‐

[18] Biering-Sorensen F, Bohr HH, Schaadt OP. Bone mineral content of the lumbar spine and lower extremities years after spinal cord lesions. Paraplegia 1988;26:293-301.

[19] Leslie WD, Nance PW. Dissociated hip and spine demineralization: a specific finding

in spinal cord injury. Arch Phys Med Rehabil 1993;74:960-4.

cord injured men. J Musculoskelet Neuronal Interact 2007;7:62-8.

spective study. Arch Phys Med Rehab 1981;62: 418–23.

2001;39:208-14.

2007;30 Suppl 1:S10-4.

Clin Orthop 1981;155:65–70.

gia 1995;33: 674–7.

view. Paraplegia 1995;33:669–73.

injured individuals. Bone 2004;34:869-80.

teract. 2011 Sep;11(3):257-65.

jury. Europ J Clin Invest 1991;20:330-5.

#### **Author details**

Yannis Dionyssiotis1,2

1 Rehabilitation Center "Aghios Loukas o Iatros", Trikala Thessaly, Greece

2 University of Athens, 1st Department of Orthopaedics, General University Hospital Attikon, Athens, Greece

#### **References**


[6] Lazo MG, Shirazi P, Sam M, Giobbie-Hurder A, Blacconiere MJ, Muppidi M. Osteo‐ porosis and risk of fracture in men with spinal cord injury. Spinal Cord 2001;39:208-14.

(1000 times more potent than etidronate), in an open observational study, reversed BMD loss in men with established SCI increased both axial and trabecular bone density and has proven efficacy and safety in men treated for osteoporosis, prevents hypercalciuria and bone loss after bed rest and lower leg fracture. [80, 81] Six months after using zolendronic acid in the treatment group BMD showed differences in the response to treatment between the mixed trabecular/ cortical regions (narrow neck and intertrochanteric) and the purely cortical shaft. With respect to cross-sectional geometry, bone cross-sectional area and sectional modulus (indices of resistance to axial and bending loads, where higher values would indicate a positive effect of treatment) increased at the hip and buckling ratio (an index of the instability of thin-walled cross sections, where lower values would suggest that the treatment is improving stability) decreased consistent with improved bone outcomes; at 12 months, narrow-neck femur values declined and intertrochanteric and femoral shaft BMD was maintained vs. placebo group which showed a decrease in bone outcomes and an increase in buckling ratio at the hip at 6 and 12 months, while with respect to bone prevention 4 mg i.v. were effective and welltolerated to prevent BMD loss at the total hip and trochanter for up to 12 months following

1 Rehabilitation Center "Aghios Loukas o Iatros", Trikala Thessaly, Greece

2 University of Athens, 1st Department of Orthopaedics, General University Hospital Attikon,

[1] NIH Consensus Development Panel on Osteoporosis JAMA 285 (2001): 785-95

[2] World Health Organisation. Assessment of fracture risk and its implication to screen‐ ing for postmenopausal osteoporosis: Technical report series 843. Geneva: WHO,

[3] Sambrook, P., Schrieber, L., Taylor, T., & Ellis, A. (2001). The musculoskeletal system.

[4] Garland DE, Adkins RH, Stewart CA. 2013. Bone Impairment and Spinal Cord In‐ jury. In: JH Stone, M Blouin, editors. International Encyclopedia of Rehabilitation.

[5] Claus-Walker J, Halstead LS. Metabolic and endocrine changes in spinal cord injury: IV. Compounded neurologic dysfunctions. Arch Phys Med Rehabil 1982;63:632-8.

Available online: http://cirrie.buffalo.edu/encyclopedia/en/article/108/

SCI. [82, 83]

186 Topics in Paraplegia

**Author details**

Athens, Greece

**References**

1994.

Edinburgh: Churchill Livingstone.

Yannis Dionyssiotis1,2


[20] Lanyon LE, Rubin CT, Baust G. Modulation of bone loss during calcium insufficiency by controlled dynamic loading. Calcif Tissue Int 1986;38:209-16.

[34] Clasey JL, Janowiak AL, Gater DR. Relationship between regional bone density measurements and the time since injury in adults with spinal cord injuries. Arch

Paraplegia Related Osteoporosis http://dx.doi.org/10.5772/57969 189

[35] Modlesky CM, Bickel CS, Slade JM, Meyer RA, Cureton KJ, Dudley GA. Assessment of skeletal muscle mass in men with spinal cord injury using dual-energy X-ray ab‐

[36] Zehnder Y, Lüthi M, Michel D, Knecht H, Perrelet R, Neto I, Kraenzlin M, Zäch G, Lippuner K. Long-term changes in bone metabolism, bone mineral density, quantita‐ tive ultrasound parameters, and fracture incidence after spinal cord injury: a crosssectional observational study in 100 paraplegic men. Osteoporos Int. 2004;15:180-9.

[37] Bauman WA, Spungen AM, Wang J, Pierson RN Jr, Schwartz E. Continuous loss of bone during chronic immobilization: a monozygotic twin study. Osteoporos Int

[38] Dionyssiotis Y, Petropoulou K, Rapidi CA, Papagelopoulos P, Papaioannou N, Gala‐ nos A, Papadaki P, Lyritis GP. Body composition in paraplegic men. J Clin Densitom

[39] Coupaud S, McLean AN, Allan DB. Role of peripheral quantitative computed tomog‐ raphy in identifying disuse osteoporosis in paraplegia. Skeletal Radiol 2009; 38(10):

[40] Clasey JL, Janowiak AL, Gater DR. Relationship between regional bone density measurements and the time since injury in adults with spinal cord injuries. Arch

[41] Dionyssiotis Y, Lyritis GP, Mavrogenis AF, Papagelopoulos PJ. Factors influencing

[42] Garland DE, Adkins RH, Stewart CA, Ashford R, Vigil D. Regional osteoporosis in women who have a complete spinal cord injury. J Bone Joint Surg Am 2001;83 A:

[43] Garland DE, Foulkes G, Adkins RH, Hu SS, Rosen C, Liotta FJ, Weinstein DA. Re‐ gional osteoporosis following incomplete spinal cord injury. J Orthop Res

[44] Chantraine A, van Ouwenaller C, Hachen HJ, Schinas P. Intra-medullary pressure

[45] Karlsson AK, Friberg P, Lonnroth P, Sullivan L, Elam M Regional sympathetic func‐ tion in high spinal cord injury during mental stress and autonomic dysreflexia. Brain

and intra-osseous phlebography in paraplegia. Paraplegia 1979;17:391-9.

sorptiometry and magnetic resonance imaging. J Appl Physiol 2004;96:561-5.

Phys Med Rehabil 2004;85:59-64.

1999;10:123–7.

2008;11:437-43.

Phys Med Rehabil 2004;85:59-64.

bone loss in paraplegia Hippokratia 2011;15:54-9.

989-95.

1195-200.

1992;10:371-8.

1998;121:1711–9.


[34] Clasey JL, Janowiak AL, Gater DR. Relationship between regional bone density measurements and the time since injury in adults with spinal cord injuries. Arch Phys Med Rehabil 2004;85:59-64.

[20] Lanyon LE, Rubin CT, Baust G. Modulation of bone loss during calcium insufficiency

[21] Dauty M, Perrouin Verbe B, Maugars Y, Dubois C, Mathe JF. Supralesional and sub‐ lesional bone mineral density in spinal cord-injured patients. Bone 2000;27:305-9. [22] Smeltzer SC, Zimmerman V, Capriotti T. Osteoporosis risk and low bone mineral density in women with physical disabilities. Arch Phys Med Rehabil. 2005;86:582-6.

[23] Rauch F, Rittweger J. What is new in neuro-musculoskeletal interactions? J Muscu‐

[24] Szollar SM, Martin EM, Sartoris DJ, Parthemore JG, Deftos LJ. Bone mineral density and indexes of bone metabolism in spinal cord injury. Am J Phys Med Rehabil

[25] Bauman WA, Spungen AM, Morrison N, Zhang RL, Schwartz E. Effect of a vitamin D analog on leg bone mineral density in patients with chronic spinal cord injury. J

[26] Delmas PD. Markers of bone formation and resorption. In: Favus MJ, editor. Primer on the metabolic bone diseases and disorders of mineral metabolism. 2d ed. New

[27] Raisz LG, Kream BE, Lorenzo JA. Metabolic bone disease. In: Wilson JD, Foster DW, Kronenberg HM, Larsen PR, editors. Williams textbook of endocrinology. 9th ed.

[28] Gertz BJ, Shao P, Hanson DA, Quan H, Harris ST, Genant HK, Chesnut CH 3rd, Eyre DR. Monitoring bone resorption in early postmenopausal women by an immunoas‐ say for cross-linked collagen peptides in urine. J Bone Miner Res 1994;9:135-42. [29] Rosen HN, Dresner-Pollak R, Moses AC, Rosenblatt M, Zeind AJ, Clemens JD, Greenspan SL. Specificity of urinary excretion of cross-linked N-telopeptides of type

[30] Roberts D, Lee W, Cuneo RC, Wittmann J, Ward G, Flatman R, McWhinney B, Hick‐ man PE. Longitudinal study of bone turnover after acute spinal cord injury. J Clin

[31] Nance PW, Schryvers O, Leslie W, Ludwig S, Krahn J, Uebelhart D. Intravenous pa‐ midronate attenuates bone density loss after acute spinal cord injury. Arch Phys Med

[32] Bauman WA, Zhong YG, Schwartz E Vitamin D deficiency in veterans with chronic

[33] Morse LR, Sudhakar S, Danilack V, Tun C, Lazzari A, Gagnon DR, Garshick E, Batta‐ glino RA. Association between sclerostin and bone density in chronic spinal cord in‐

Philadelphia (PA): W.B. Saunders Company; 1998. p. 1220-21.

I collagen as a marker of bone turnover. Calcif Tissue Int 1994;54:26-9.

by controlled dynamic loading. Calcif Tissue Int 1986;38:209-16.

loskelet Neuronal Interact 2005;5:91-4.

Rehabil Res Dev. 2005;42:625-34.

York: Raven Press; 1993. p. 108-12.

Endocrinol Metab 1998;83:415-22.

spinal cord injury. Metabolism 1995;44:1612–6.

jury. J Bone Miner Res. 2012;27:352-9.

Rehabil. 1999;80:243-51.

1998;77:28-35.

188 Topics in Paraplegia


[46] Teasell RW, Arnold JM, Krassioukov A, Delaney GA. Cardiovascular consequences of loss of supraspinal control of the sympathetic nervous system after spinal cord in‐ jury. Arch Phys Med Rehabil. 2000;81:506-16.

[59] de Bruin ED, Frey-Rindova P, Herzog RE, Dietz V, Dambacher MA, Stüssi E.Changes of tibia bone properties after spinal cord injury: effects of early intervention. Arch

Paraplegia Related Osteoporosis http://dx.doi.org/10.5772/57969 191

[60] Frey-Rindova P, de Bruin ED, Stüssi E, Dambacher MA, Dietz V. Bone mineral densi‐ ty in upper and lower extremities during 12 months after spinal cord injury meas‐ ured by peripheral quantitative computed tomography. Spinal Cord 2000;38:26-32.

[61] Frost HM. Bone's mechanostat: a 2003 update. Anat Rec A Discov Mol Cell Evol Biol

[62] LeBlanc AD, Spector ER, Evans HJ, Sibonga JD. Skeletal responses to space flight and the bed rest analog: a review. J Musculoskelet Neuronal Interact 2007;7:33-47.

[63] LeBlanc AD, Spector ER, Evans HJ, Sibonga JD. Skeletal responses to space flight and the bed rest analog: a review. J Musculoskelet Neuronal Interact 2007;7:33-47.

[64] Smith SM, Zwart SR, Heer MA, Baecker N, Evans HJ, Feiveson AH, Shackelford LC, Leblanc AD. Effects of artificial gravity during bed rest on bone metabolism in hu‐

[65] Eser P, de Bruin ED, Telley I, Lechner HE, Knecht H, Stüssi E. Effect of electrical stimulation-induced cycling on bone mineral density in spinal cord-injured patients.

[66] Chen SC, Lai CH, Chan WP, Huang MH, Tsai HW, Chen JJ. Increases in bone miner‐ al density after functional electrical stimulation cycling exercises in spinal cord in‐

[67] Giangregorio LM, Thabane L, Debeer J, Farrauto L, McCartney N, Adachi JD, Pa‐ paioannou A. Body weight-supported treadmill training for patients with hip frac‐

[68] Ward K, Alsop C, Caulton J, Rubin C, Adams J, Mughal Z. Low magnitude mechani‐ cal loading is osteogenic in children with disabling conditions. J Bone Miner Res

[69] Eisman JA. Good, good, good... good vibrations: the best option for better bones?

[70] Ruck J, Chabot G, Rauch F. Vibration treatment in cerebral palsy: A randomized con‐

[71] Asselin P, Spungen AM, Muir JW, Rubin CT, Bauman WA. Transmission of low-in‐ tensity vibration through the axial skeleton of persons with spinal cord injury as a potential intervention for preservation of bone quantity and quality. J Spinal Cord

[72] Huang LQ, He HC, He CQ, Chen J, Yang L. Clinical update of pulsed electromagnet‐

trolled pilot study. J Musculoskelet Neuronal Interact 2010;10:77-83.

ic fields on osteoporosis. Chin Med J (Engl). 2008;121:2095-9.

ture: a feasibility study. Arch Phys Med Rehabil 2009;90:2125-30.

Phys Med Rehabil 1999 Feb;80:214-20.

mans. J Appl Physiol (1985) 2009;107:47-53.

jured patients. Disabil Rehabil 2005 30;27:1337-41.

Eur J Clin Invest 2003;33:412-9.

2004;19:360-9.

Lancet 2001 8;358:1924-5.

Med 2011;34:52-9.

2003 ;275:1081-101.


[59] de Bruin ED, Frey-Rindova P, Herzog RE, Dietz V, Dambacher MA, Stüssi E.Changes of tibia bone properties after spinal cord injury: effects of early intervention. Arch Phys Med Rehabil 1999 Feb;80:214-20.

[46] Teasell RW, Arnold JM, Krassioukov A, Delaney GA. Cardiovascular consequences of loss of supraspinal control of the sympathetic nervous system after spinal cord in‐

[47] Schwarzman RJ. New treatments for reflex sympathetic dystrophy. N Engl J Med

[48] Takeda S, Elefteriou F, Levasseur R, Liu X, Zhao L, Parker KL, Armstrong D, Ducy P, Karsenty G. Leptin regulates bone formation via the sympathetic nervous system.

[49] Kondo H, Nifuji A, Takeda S, Ezura Y, Rittling SR, Denhardt DT, Nakashima K, Kar‐ senty G, Noda M. Unloading induces osteoblastic cell suppression and osteoclastic cell activation to lead to bone loss via sympathetic nervous system. J Biol Chem.

[50] Levasseur R, Sabatier JP, Potrel-Burgot C, Lecoq B, Creveuil C, Marcelli C. Sympa‐ thetic nervous system as transmitter of mechanical loading in bone. Joint Bone Spine

[51] Engelke K, Kemmler W, Lauber D, Beeskow C, Pintag R, Kalender WA. Exercise maintains bone density at spine and hip EFOPS: a 3-year longitudinal study in early

[52] Kraemer WJ. Endocrine responses and adaptations to strength training. In: Strength & Power in Sport, P.V. Komi (Editor). Oxford: Blackwell Scientific Publications, 1992;

[53] Demirel G, Yilmaz H, Paker N, Onel S. Osteoporosis after spinal cord injury. Spinal

[54] Frey-Rindova P, de Bruin ED, Stussi E, Dambacher MA, Dietz V. Bone mineral densi‐ ty in upper and lower extremities during 12 months after spinal cord injury meas‐ ured by peripheral quantitative computed tomography. Spinal Cord 2000;38:26-32.

[55] Eser P, Frotzler A, Zehnder Y, Schiessl H, Denoth J. Assessment of anthropometric, systemic, and lifestyle factors influencing bone status in the legs of spinal cord in‐

[56] Rittweger J, Gerrits K, Altenburg T, Reeves N, Maganaris CN, de Haan A. Bone adaptation to altered loading after spinal cord injury: a study of bone and muscle

[57] Löfvenmark I, Werhagen L, Norrbrink C. Spasticity and bone density after a spinal

[58] Goemaere S, Van Laere M, De Neve P, Kaufman JM. Bone mineral status in paraple‐ gic patients who do or do not perform standing. Osteoporos Int 1994 May;4:138-43.

postmenopausal women. Osteoporos Int 2006;17:133-42.

jured individuals. Osteoporos Int 2005;16:26-34.

cord injury. J Rehabil Med. 2009; 41: 1080-4.

strength. J Musculoskelet Neuronal Interact 2006;6:269-76.

jury. Arch Phys Med Rehabil. 2000;81:506-16.

2000;343:654–6.

190 Topics in Paraplegia

Cell 2002;111:305-17

2005;280:30192-200.

2003;70:515-9

pp. 291-304.

Cord 1998;36:822-5.


[73] Garland DE, Adkins RH, Matsuno NN, Stewart CA. The effect of pulsed electromag‐ netic fields on osteoporosis at the knee in individuals with spinal cord injury. J Spi‐ nalCord Med 1999;22:239-45.

**Chapter 9**

**Estimating Renal Function in Paraplegia**

The National Kidney Disease Education Program recommends using either the Cockcroft– Gault creatinine clearance (CLCG) or Modification of Diet in Renal Disease (MDRD) equation when determining dosages of drugs that are primarily eliminated by the kidneys [1]. Both methods attempt to better predict creatinine clearance (CLCR) or glomerular filtration rate (GFR) by taking into account different variables such as age, weight, gender, race, and se‐ rum creatinine (SCr), however neither equation captures the key factor of paraplegia. Over time, individuals with paraplegia develop low SCr concentrations relative to their actual CLCR due to significantly reduced muscle mass as a result of chronic immobility and muscle atrophy. Both Cockcroft–Gault (CG) and MDRD formulas have SCr in their denominator in‐ versely proportional to CLCR or GFR, therefore low SCr in paraplegia would result in gross overestimation of their renal function. Based on falsely high CLCR or GFR, clinicians could potentially prescribe renally eliminated medications at dosages higher than recommended, resulting in undesirably high drug concentrations leading to drug toxicity and/or adverse drug reactions (ADRs). For example, supratherapeutic vancomycin and aminoglycosides (AG) serum concentrations, especially if combined with other nephrotoxic and/or ototoxic medications, could drastically increase the risk of nephrotoxicity and/or ototoxicity. This could be devastating to many individuals with paraplegia who have existing renal insuffi‐

In addition to high prevalence of traditional risk factors for CKD such as advanced age, dia‐ betes, hypertension, and cardiovascular disease, individuals with paraplegia have elevated incidence of recurrent and chronic urinary tract infections, neurogenic bladder dysfunction, and nephrolithiasis that put them at risk for developing CKD [2-6]. Fischer et al. conducted cross-sectional analyses of data on 9333 Veterans with spinal cord injury and disorder (SCI/D) and found that the prevalence of CKD in SCI/D was approximately 35%, considera‐ bly higher based on the modified MDRD for SCI/D than 10% based on the original MDRD

> © 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Additional information is available at the end of the chapter

Jennifer Pai Lee

**1. Introduction**

ciency.

http://dx.doi.org/10.5772/57231


### **Estimating Renal Function in Paraplegia**

#### Jennifer Pai Lee

[73] Garland DE, Adkins RH, Matsuno NN, Stewart CA. The effect of pulsed electromag‐ netic fields on osteoporosis at the knee in individuals with spinal cord injury. J Spi‐

[74] Cong F, Ji SR, Xu JB, Su GD, Du Y, Chang H, et al. Effect ofpulsed electromagnetic fields on bone mineral density of spinal cord injuried patients. Chin J Rehabil Theory

[75] Chantraine A, Heynen G, Franchimont P. Bone metabolism, parathyroid hormone,

[76] Minaire P, Depassio J, Berard E, Meunier PJ, Edouard C, Pilonchery G, Goedert G.Ef‐ fects of clodronate on immobilization bone loss. Bone 1987;8 Suppl 1:S63-8.

[77] Roux C, Oriente P, Laan R, Hughes RA, Ittner J, Goemaere S, Di Munno O, Pouillès JM, Horlait S, Cortet B. Randomized trial of effect of cyclical etidronate in the preven‐ tion of corticosteroid-induced bone loss. Ciblos Study Group. J Clin Endocrinol Met‐

[78] Chappard D, Minaire P, Privat C, Berard E, Mendoza-Sarmiento J, Tournebise H, Basle MF, Audran M, Rebel A, Picot C, et al. Effects of tiludronate on bone loss inpar‐

[79] Bauman WA, Wecht JM, Kirshblum S, Spungen AM, Morrison N, Cirnigliaro C, Schwartz E. Effect of pamidronate administration on bone in patients with acute spi‐

[80] Moran de Brito CM, Battistella LR, Saito ET, Sakamoto H. Effect of alendronate on bone mineral density in spinal cord injury patients: a pilot study. Spinal Cord

[81] Zehnder Y, Risi S, Michel D, Knecht H, Perrelet R, Kraenzlin M, Zäch GA, Lippuner K. Prevention of bone loss in paraplegics over 2 years with alendronate. J Bone Miner

[82] Bubbear JS, Gall A, Middleton FR, Ferguson-Pell M, Swaminathan R, Keen RW. Early treatment with zoledronic acid prevents bone loss at the hip following acute spinal

[83] Shapiro J, Smith B, Beck T, Ballard P, Dapthary M, BrintzenhofeSzoc K, Caminis J. Treatment with zoledronic acid ameliorates negative geometric changes in the proxi‐

[84] Dionyssiotis Y. Bone loss in paraplegia: A diagnostic and therapeutic protocol. Os‐

mal femur following acute spinal cord injury. Calcif Tissue Int 2007;80:316-22.

and calcitonin in paraplegia. Calcif Tissue Int 1979;27:199-204.

aplegic patients. J Bone Miner Res 1995;10:112-8.

nal cord injury. J Rehabil Res Dev 2005;42:305-13.

cord injury. Osteoporos Int 2011;22:271-9.

teoporos Int 2009;20:S23-S176.

nalCord Med 1999;22:239-45.

192 Topics in Paraplegia

Practice (Chin) 2005; 11: 250-1.

ab 1998;83:1128-33.

2005;43:341-8.

Res 2004;19:1067-74.

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/57231

#### **1. Introduction**

The National Kidney Disease Education Program recommends using either the Cockcroft– Gault creatinine clearance (CLCG) or Modification of Diet in Renal Disease (MDRD) equation when determining dosages of drugs that are primarily eliminated by the kidneys [1]. Both methods attempt to better predict creatinine clearance (CLCR) or glomerular filtration rate (GFR) by taking into account different variables such as age, weight, gender, race, and se‐ rum creatinine (SCr), however neither equation captures the key factor of paraplegia. Over time, individuals with paraplegia develop low SCr concentrations relative to their actual CLCR due to significantly reduced muscle mass as a result of chronic immobility and muscle atrophy. Both Cockcroft–Gault (CG) and MDRD formulas have SCr in their denominator in‐ versely proportional to CLCR or GFR, therefore low SCr in paraplegia would result in gross overestimation of their renal function. Based on falsely high CLCR or GFR, clinicians could potentially prescribe renally eliminated medications at dosages higher than recommended, resulting in undesirably high drug concentrations leading to drug toxicity and/or adverse drug reactions (ADRs). For example, supratherapeutic vancomycin and aminoglycosides (AG) serum concentrations, especially if combined with other nephrotoxic and/or ototoxic medications, could drastically increase the risk of nephrotoxicity and/or ototoxicity. This could be devastating to many individuals with paraplegia who have existing renal insuffi‐ ciency.

In addition to high prevalence of traditional risk factors for CKD such as advanced age, dia‐ betes, hypertension, and cardiovascular disease, individuals with paraplegia have elevated incidence of recurrent and chronic urinary tract infections, neurogenic bladder dysfunction, and nephrolithiasis that put them at risk for developing CKD [2-6]. Fischer et al. conducted cross-sectional analyses of data on 9333 Veterans with spinal cord injury and disorder (SCI/D) and found that the prevalence of CKD in SCI/D was approximately 35%, considera‐ bly higher based on the modified MDRD for SCI/D than 10% based on the original MDRD

© 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

formula [7]. Underrecognition of CKD in paraplegia makes it more crucial to use accurate tools to estimate renal function in this population.

**Characteristics Macdi-armid**

**SCI/D: Paraplegics (P) Tetraplegics (T) Non-SCI/D (control)**

**Age (yr.) (mean ± SD)**

**Race (n [%]) White and other**

**BMI (kg/m2) (mean ±**

**CLCG (mL/min) (mean**

**MDRD GFR (mL/min/ 1.73 m2) (mean ± SD)**

**SCr (mg/dL) (mean ±**

**Methodology** CLCG vs. CL24H

vs. measured CLCR by 99mTc-DTPA

CL24H more accurate than CLCG

**Black**

**SD)**

**[SD])**

**SD)**

**Findings/**

**Recommen-dations**

**et al. (2000)**

**Mirah-madi et al. (1983)** [10]

P: 48 ± 17 T: 47 ± 14

58 [100] Control: 11 [50]

P: 82 ± 46 T: 70 ± 23

T: 0.8 ± 0.3

Correction factor: 0.8 for paraplegic 0.6 for tetraple-

gic

**Table 2.** Review of the Current Literature on Assessing Renal Function in Paraplegia


**Chikkalingaiah et al. (2010)** [15]



CLCG vs. CL24H CLCG vs. CL24H vs. MDRD

> Correction factor: 0.7 for MDRD 0.8 for CLCG


**Lee and Dang (2011)** [16]

**Lavezo et al. (1995)** [18]

Estimating Renal Function in Paraplegia http://dx.doi.org/10.5772/57231

> 14 -- -- 14

140 [99] 87 [100]

41 [35]

91 ± 37 63 ± 26 93 ± 47

0.8 ± 0.4 Control: 1.1 ± 0.3

SCI/D vs. Non-SCI/D CLVANCO

↑half-life in SCI/D

0.74 ± 0.29 SCI/D:

CLCG vs. CLM vs. CL24H vs. MDRD vs. CKD-EPI

All methods over-estimate CLDRUG (P< 0.001). Devel-opment of CLSCI

63 ± 1 66 ± 11 53 ± 12 65 ± 16

**Lee and Yang (2013)** [19] 195

71 [82] 16 [18]

0.88 ± 0.40

CLSCI vs. CLCG vs. CLM vs. CL24H vs. MDRD vs. CKD-EPI

Verifi-cation of CLSCI: CLSCI un-biased and more precise.

[9]

38 (24-68)

**Male (n [%])** -- SCI/D:

Currently, there is no accepted standard method for determining renal dosing regimens for patients with paraplegia, and data on estimating renal function in such population is scarce. However clearance of drugs primarily eliminated by the kidneys such as vancomycin and AG nearly mirror that of the creatinine, hence could be used to assess renal function in paraplegia.

The aims of this chapter are: (1) to review the current literature on assessing renal function in paraplegia, (2) to evaluate different methods of estimating CLCR or GFR compared with patientspecific vancomycin and AG clearance (CLDRUG) in individuals with paraplegia, (3) to assess whether there is a difference in the estimation of renal function between the two anatomical degrees of SCI/D when compared with CLDRUG, and (4) to present the "Spinal Cord Injury Equation" that more accurately estimates renal function in paraplegia.

#### **2. Review of the current literature on assessing renal function in paraplegia**

Tables 1 and 2 show, respectively, comparison of equations to predict CLCG or GFR from SCr and review of the current literature on assessing renal function in paraplegia. Each equation and study will be discussed in detail below.

#### **Equation 1: Cockcroft-Gault equation (CLCG) [8]**

GFR = CLCR (mL/min) = [(140 – age) x IBW in kg] / (72 x SCr); (multiply 0.85 for females)

#### **Equation 2: Modified Cockcroft-Gault equation (CLM)** [16]

GFR = CLCR (mL/min) = [(140 – age) x IBW in kg] / (72 x SCr); (multiply 0.85 for females)

SCr rounded to 1 mg/dL for patients with SCr < 1 mg/dL while using the actual SCr for

patients with SCr ≥ 1 mg/dL

#### **Equation 3: MDRD equation** [11-13\*]

GFR (mL/min/1.73 m2) = 175 x standardized SCr-1.154 x age-0.203 x 1.212 (if black) x 0.742 (if female)

#### **Equation 4: CKD-EPI equation** [14\*]

GFR (mL/min/1.73 m2) = 141 x min (SCr/**ĸ**, 1)<sup>α</sup>x max (SCr/**ĸ**, 1)-1.209 x 0.993Age x 1.018 [if female] x 1.159 [if black]

where **ĸ** is 0.7 for females and 0.9 for males, α is -0.329 for females and -0.411 for males, min indicates the minimum of SCr/**ĸ** or 1, & max indicates the maximum of SCr/**ĸ** or 1.

#### **Equation 5: 24-Hour endogenous creatinine clearance (CL24H )** [8]

GFR = CL24H (mL/min) = [urine creatinine x urine volume (mL)] / [SCr x time (hours) x 60]

\*To enable the expression of comparisons among different methods in the same unit (mL/min), GFR values normal‐ ized to a BSA of 1.73 m2 need to be converted to uncorrected values.

**Table 1.** Comparison of Equations to Predict Creatinine Clearance (CLCR) or Glomerular Filtration Rate (GFR) from Serum Creatinine Concentration


**Table 2.** Review of the Current Literature on Assessing Renal Function in Paraplegia

formula [7]. Underrecognition of CKD in paraplegia makes it more crucial to use accurate

Currently, there is no accepted standard method for determining renal dosing regimens for patients with paraplegia, and data on estimating renal function in such population is scarce. However clearance of drugs primarily eliminated by the kidneys such as vancomycin and AG nearly mirror that of the creatinine, hence could be used to assess renal function in paraplegia.

The aims of this chapter are: (1) to review the current literature on assessing renal function in paraplegia, (2) to evaluate different methods of estimating CLCR or GFR compared with patientspecific vancomycin and AG clearance (CLDRUG) in individuals with paraplegia, (3) to assess whether there is a difference in the estimation of renal function between the two anatomical degrees of SCI/D when compared with CLDRUG, and (4) to present the "Spinal Cord Injury

**2. Review of the current literature on assessing renal function in paraplegia**

Tables 1 and 2 show, respectively, comparison of equations to predict CLCG or GFR from SCr and review of the current literature on assessing renal function in paraplegia. Each equation

Equation" that more accurately estimates renal function in paraplegia.

GFR = CLCR (mL/min) = [(140 – age) x IBW in kg] / (72 x SCr); (multiply 0.85 for females)

GFR = CLCR (mL/min) = [(140 – age) x IBW in kg] / (72 x SCr); (multiply 0.85 for females) SCr rounded to 1 mg/dL for patients with SCr < 1 mg/dL while using the actual SCr for

GFR = CL24H (mL/min) = [urine creatinine x urine volume (mL)] / [SCr x time (hours) x 60]

minimum of SCr/**ĸ** or 1, & max indicates the maximum of SCr/**ĸ** or 1.

**Equation 5: 24-Hour endogenous creatinine clearance (CL24H )** [8]

ized to a BSA of 1.73 m2 need to be converted to uncorrected values.

GFR (mL/min/1.73 m2) = 175 x standardized SCr-1.154 x age-0.203 x 1.212 (if black) x 0.742 (if female)

GFR (mL/min/1.73 m2) = 141 x min (SCr/**ĸ**, 1)<sup>α</sup>x max (SCr/**ĸ**, 1)-1.209 x 0.993Age x 1.018 [if female] x 1.159 [if black] where **ĸ** is 0.7 for females and 0.9 for males, α is -0.329 for females and -0.411 for males, min indicates the

\*To enable the expression of comparisons among different methods in the same unit (mL/min), GFR values normal‐

**Table 1.** Comparison of Equations to Predict Creatinine Clearance (CLCR) or Glomerular Filtration Rate (GFR) from

tools to estimate renal function in this population.

194 Topics in Paraplegia

and study will be discussed in detail below.

**Equation 2: Modified Cockcroft-Gault equation (CLM)** [16]

**Equation 1: Cockcroft-Gault equation (CLCG) [8]**

patients with SCr ≥ 1 mg/dL **Equation 3: MDRD equation** [11-13\*]

**Equation 4: CKD-EPI equation** [14\*]

Serum Creatinine Concentration

#### **a. The Cockcroft-Gault (CG) equation (CLCG)**

CLCG(mL/min) = (140 – age) x IBW in kg / (72 x SCr); (multiply 0.85 for females)

The CG equation was derived from a study of 236 males aged 18-92 years based on their 24 hour creatinine excretion. Since the publication in 1976, it has been exclusively used to estimate CLCR based on SCr to calculate dosing regimens for renally cleared medications including vancomycin and AG. However it may not extrapolate to individuals with paraplegia because the CG study excluded 31 patients with 24-h creatinine excretion < 10 mg/kg, and it didn't reveal whether the study population included paraplegia and to what extent [8].

A more recently developed MDRD has been widely used to estimate GFR in the nephrology arena. It is one of the two equations recommended by The National Kidney Disease Education

The MDRD equation was derived from a study of a relatively young non- paraplegic popula‐ tion (mean age 51±13 years) with chronic kidney disease, primarily to stage kidney disease [11-12]. The original 6-variable MDRD formula integrates patient parameters including age, gender, race, blood urea nitrogen (BUN), SCr, and serum albumin [11-12]. The performance of this equation can be limited by variability among clinical laboratories in calibrating SCr assays [13]. Thus, the formula was re-expressed as the 4-variable MDRD equation based on standardized SCr assays as shown above [13]. Despite SCr calibration, the accuracy of the equation remains compromised at levels of GFR >60 mL/min/1.73 m2 [12-14]. Nevertheless, MDRD stands useful for GFR <60 mL/min/1.73 m2 in non- paraplegia and is endorsed by the

National Kidney Disease Foundation for estimating GFR in CKD patients [1, 11-12].

+ (SD of the difference)2

Chikkalingaiah et al. compared the performance of the 4-variable MDRD and CG equations with CL24H in 64 patients with chronic paraplegia of greater than 6 months duration and stages II-V CKD [15]. Precision and bias of MDRD and CG formulas were measured by combined root mean square error (CRMSE) calculated as the square root of [(mean difference of estimated

ance of the prediction equations, a correction factor of 0.7 for MDRD and 0.8 for CG were applied which resulted in a decrease in their CRMSE values to 11.4 and 13 mL/min/1.73m2

respectively [15]. Accuracy of both prediction equations was evaluated by the percentage of patients who did not deviate >15%, 30%, or 50% from measured CL24H. Respective percentages for MDRD were 12.5, 25, and 48.4 before the correction, and 25, 42, 68 after the correction [15]. Respective percentages for CG were 22, 37.5, and 58 before the correction, and 25, 50, 75 after the correction [15]. On the whole, the CG equation had less bias and was more precise and more accurate than the MDRD equation, however still overestimated GFR in subjects with

factors markedly improved in the overall bias, precision, and accuracy of both MDRD and CG equations shown by both decreased CRMSE values and increased percentage of subjects in

where *ĸ* is 0.7 for females and 0.9 for males, α is -0.329 for females and -0.411 for males, min

In order to overcome the known bias of the MDRD equation for GFR values of ≥60 mL/min/

the CKD-EPI equation, to define dose modification across the GFR range in patients with and without CKD [14]. The data showed that the CKD-EPI equation was more precise and accurate

the researchers pooled the data from 26 studies to develop and validate a new equation,

indicates the minimum of SCr/*ĸ* or 1, & max indicates the maximum of SCr/*ĸ* or 1.

) = 141 x min (SCr/*ĸ*, 1)<sup>α</sup> x max (SCr/*ĸ*, 1)-1.209 x 0.993Age x 1.018 [if female]

]. Respective CRMSE values for original

. In order to improve the perform‐

Estimating Renal Function in Paraplegia http://dx.doi.org/10.5772/57231

. Application of the correction

, however it is not frequently used

,

197

Program for drug dosing [1].

GFR and measured CL24H)

2

MDRD and CG equations were 29 and 19.3 mL/min/1.73m2

chronic paraplegia with measured CL24H < 90 mL/min/1.73m2

compared to MDRD, especially at GFR > 60 mL/min/1.73 m2

**c. The CKD-EPI equation (CKD-EPI)**

GFR (mL/min/1.73 m2

x 1.159 [if black]

1.73 m2 ,

whom GFR did not deviate >15%, 30%, or 50% from measured CL24H [15].

The review of current literature reports significant overestimation of renal function by CLCG, thus does not recommend using the original equation in paraplegia [9-10].

Macdiarmid et al. studied 25 paraplegic and 11 tetraplegic patients and sought to com‐ pare their CLCG and 24-hour endogenous creatinine clearance (CL24H) to the measured CLCR by 99mTc-DTPA clearance technique [9]. The investigators found that the CG method did not correlate well with that of the CL24H (r=0.426) or 99mTc-DTPA clearance (r=0.366) [9]. The mean difference between CLCG and CL24H was 41.9%, and the difference between CLCG and 99mTc-DTPA clearance 50.7% where CG formula overestimated CLCR [9]. On the other hand, the difference between CL24H and 99mTc-DTPA clearance was 17.7% with good correlation (r=0.71) [9]. The authors concluded that the CG formula significantly overestimates CLCR thus not recommended, however CL24H is an accurate method of determining renal function in paraplegia [9].

A study by Mirahmadi et al. investigated 58 male hospitalized patients with SCI/D and 22 ambulatory subjects, and compared their measured CL24H by autoanalyzer method versus the predicted by CLCG [10]. The authors found that the predicted CLCG and measured CL24H values closely matched in the ambulatory group while the predicted values consistently exceeded the measured values in SCI/D [10]. Between the two anatomical degrees of SCI/D, the paraplegic group had a markedly higher SCr (1.0 ± 0.4 mg/dL) and 24-hour urinary creatinine excretion (16 ± 9 mg/kg) compared to the tetraplegic group where the respec‐ tive values were 0.8 ± 0.3 mg/dL and 11 ± 4.6 mg/kg [10]. The authors modified the original CG formula using a correction factor of 0.8 for paraplegics and 0.6 for tetraplegics to overcome significant overestimation by CLCG [10]. The correction factors improved the accuracy and precision of the predicted CLCG shown by the difference between the predicted and measured CLCR approaching zero and the slope of a linear correlation between the predicted and measured values approaching one with decreased Y-intercept values (p < 0.01) [10].

#### **b. The MDRD equation (MDRD)**

#### 4-Variable MDRD:

GFR (mL/min/1.73 m2 ) = 175 x standardized SCr-1.154 x age-0.203 x 1.212 (if black) x 0.742 (if female) A more recently developed MDRD has been widely used to estimate GFR in the nephrology arena. It is one of the two equations recommended by The National Kidney Disease Education Program for drug dosing [1].

The MDRD equation was derived from a study of a relatively young non- paraplegic popula‐ tion (mean age 51±13 years) with chronic kidney disease, primarily to stage kidney disease [11-12]. The original 6-variable MDRD formula integrates patient parameters including age, gender, race, blood urea nitrogen (BUN), SCr, and serum albumin [11-12]. The performance of this equation can be limited by variability among clinical laboratories in calibrating SCr assays [13]. Thus, the formula was re-expressed as the 4-variable MDRD equation based on standardized SCr assays as shown above [13]. Despite SCr calibration, the accuracy of the equation remains compromised at levels of GFR >60 mL/min/1.73 m2 [12-14]. Nevertheless, MDRD stands useful for GFR <60 mL/min/1.73 m2 in non- paraplegia and is endorsed by the National Kidney Disease Foundation for estimating GFR in CKD patients [1, 11-12].

Chikkalingaiah et al. compared the performance of the 4-variable MDRD and CG equations with CL24H in 64 patients with chronic paraplegia of greater than 6 months duration and stages II-V CKD [15]. Precision and bias of MDRD and CG formulas were measured by combined root mean square error (CRMSE) calculated as the square root of [(mean difference of estimated GFR and measured CL24H) 2 + (SD of the difference)2 ]. Respective CRMSE values for original MDRD and CG equations were 29 and 19.3 mL/min/1.73m2 . In order to improve the perform‐ ance of the prediction equations, a correction factor of 0.7 for MDRD and 0.8 for CG were applied which resulted in a decrease in their CRMSE values to 11.4 and 13 mL/min/1.73m2 , respectively [15]. Accuracy of both prediction equations was evaluated by the percentage of patients who did not deviate >15%, 30%, or 50% from measured CL24H. Respective percentages for MDRD were 12.5, 25, and 48.4 before the correction, and 25, 42, 68 after the correction [15]. Respective percentages for CG were 22, 37.5, and 58 before the correction, and 25, 50, 75 after the correction [15]. On the whole, the CG equation had less bias and was more precise and more accurate than the MDRD equation, however still overestimated GFR in subjects with chronic paraplegia with measured CL24H < 90 mL/min/1.73m2 . Application of the correction factors markedly improved in the overall bias, precision, and accuracy of both MDRD and CG equations shown by both decreased CRMSE values and increased percentage of subjects in whom GFR did not deviate >15%, 30%, or 50% from measured CL24H [15].

#### **c. The CKD-EPI equation (CKD-EPI)**

**a. The Cockcroft-Gault (CG) equation (CLCG)**

(multiply 0.85 for females)

196 Topics in Paraplegia

in paraplegia [9].

0.01) [10].

4-Variable MDRD:

GFR (mL/min/1.73 m2

**b. The MDRD equation (MDRD)**

CLCG(mL/min) = (140 – age) x IBW in kg / (72 x SCr);

The CG equation was derived from a study of 236 males aged 18-92 years based on their 24 hour creatinine excretion. Since the publication in 1976, it has been exclusively used to estimate CLCR based on SCr to calculate dosing regimens for renally cleared medications including vancomycin and AG. However it may not extrapolate to individuals with paraplegia because the CG study excluded 31 patients with 24-h creatinine excretion < 10 mg/kg, and it didn't

The review of current literature reports significant overestimation of renal function by CLCG,

Macdiarmid et al. studied 25 paraplegic and 11 tetraplegic patients and sought to com‐ pare their CLCG and 24-hour endogenous creatinine clearance (CL24H) to the measured CLCR by 99mTc-DTPA clearance technique [9]. The investigators found that the CG method did not correlate well with that of the CL24H (r=0.426) or 99mTc-DTPA clearance (r=0.366) [9]. The mean difference between CLCG and CL24H was 41.9%, and the difference between CLCG and 99mTc-DTPA clearance 50.7% where CG formula overestimated CLCR [9]. On the other hand, the difference between CL24H and 99mTc-DTPA clearance was 17.7% with good correlation (r=0.71) [9]. The authors concluded that the CG formula significantly overestimates CLCR thus not recommended, however CL24H is an accurate method of determining renal function

A study by Mirahmadi et al. investigated 58 male hospitalized patients with SCI/D and 22 ambulatory subjects, and compared their measured CL24H by autoanalyzer method versus the predicted by CLCG [10]. The authors found that the predicted CLCG and measured CL24H values closely matched in the ambulatory group while the predicted values consistently exceeded the measured values in SCI/D [10]. Between the two anatomical degrees of SCI/D, the paraplegic group had a markedly higher SCr (1.0 ± 0.4 mg/dL) and 24-hour urinary creatinine excretion (16 ± 9 mg/kg) compared to the tetraplegic group where the respec‐ tive values were 0.8 ± 0.3 mg/dL and 11 ± 4.6 mg/kg [10]. The authors modified the original CG formula using a correction factor of 0.8 for paraplegics and 0.6 for tetraplegics to overcome significant overestimation by CLCG [10]. The correction factors improved the accuracy and precision of the predicted CLCG shown by the difference between the predicted and measured CLCR approaching zero and the slope of a linear correlation between the predicted and measured values approaching one with decreased Y-intercept values (p <

) = 175 x standardized SCr-1.154 x age-0.203 x 1.212 (if black) x 0.742 (if female)

reveal whether the study population included paraplegia and to what extent [8].

thus does not recommend using the original equation in paraplegia [9-10].

GFR (mL/min/1.73 m2 ) = 141 x min (SCr/*ĸ*, 1)<sup>α</sup> x max (SCr/*ĸ*, 1)-1.209 x 0.993Age x 1.018 [if female] x 1.159 [if black]

where *ĸ* is 0.7 for females and 0.9 for males, α is -0.329 for females and -0.411 for males, min indicates the minimum of SCr/*ĸ* or 1, & max indicates the maximum of SCr/*ĸ* or 1.

In order to overcome the known bias of the MDRD equation for GFR values of ≥60 mL/min/ 1.73 m2 , the researchers pooled the data from 26 studies to develop and validate a new equation, the CKD-EPI equation, to define dose modification across the GFR range in patients with and without CKD [14]. The data showed that the CKD-EPI equation was more precise and accurate compared to MDRD, especially at GFR > 60 mL/min/1.73 m2 , however it is not frequently used in current clinical practice when determining dosages of drugs that are primarily eliminated by the kidneys due to need for further validation. Furthermore, the sample population used to develop the CKD-EPI formula did not include paraplegia, thus its use in paraplegia may be misleading.

equation (CLM) better estimated CLDRUG in SCI/D, compared with other frequently employed methods for predicting GFR. The mean difference between CLDRUG and CLM was smallest among the equations evaluated where overestimation by CLM was approximately 40%. Almost 65% of the patients had prediction of CLDRUG within 30 mL/min when using CLM to estimate empiric dosing for vancomycin and AG (p < 0.001) [16]. Despite pronounced improvement by

Abbreviations: GFR, glomerular filtration rate; CLDRUG, actual drug clearance; MDRD, the Modification of Diet in Renal Disease equation; CKD-EPI, Chronic Kidney Disease Epidemiology Collaboration equation; CL24H, 24-hour endogenous creatinine clearance; CLCG, the Cockcroft-Gault formula; CLM, modified Cockcroft-Gault formula; S.D., standard devia‐

**4. Estimation of renal function between the two anatomical degrees of SCI**

As previously mentioned, Mirahmadi et al. reported that both SCr and mean urinary creatinine excretion were markedly lower in paraplegics compared with ambulatory subjects [10]. The authors recommended an adjustment of the original CG equation by 20% for paraplegics to correct for reduction of muscle mass relative to the total body weight in such population [10]. Chikkalingaiah et al. found that both prediction equations (MDRD and CG) overestimated GFR in the paraplegic group with an overestimation by MDRD to a higher degree [15]. The fractional prediction error (FPE = (variable 1-variable 2) x 100/variable 1) for MDRD and CG were, respectively, 48.5% and 29.5% for paraplegic subjects, where an FPE greater than 20% was considered to be clinically unacceptable [15]. A correction factor of 0.7 for MDRD and 0.8 for CG proposed by the authors decreased the FPE to 3.9% and 3.6%, respectively, for the

Lee and Dang sought to evaluate various methods to predict CLDRUG for different anatomical degrees of SCI/D (Table 4) [16]. The mean difference between CLSCI and CLDRUG was not statistically significant when separated into paraplegia and tetraplegia [16]. Similar finding was noted for CLM and CL24H [16]. On the other hand, the mean differences between CLCG, CKD-EPI, and MDRD and CLDRUG were statistically significant between the two anatomical

**Difference from CLDRUG (mL/min) P-Value**

Estimating Renal Function in Paraplegia http://dx.doi.org/10.5772/57231 199

modification of CG, overestimation may not be clinically acceptable.

**CLDRUG** 49.77 ± 19.97 0 -- **MDRD** 119.76 ± 61.49 69.99 <0.001 **CKD-EPI** 90.71 ± 27.44 40.94 <0.001 **CL24H** 85.16 ± 33.88 35.39 <0.001 **CLCG** 91.24 ± 36.90 41.47 <0.001 **CLM** 69.38 ± 13.49 19.61 <0.001

**Mean ± S.D. (mL/min)**

tion. Published with permission of Lee [16].

**when compared with CLDRUG**

paraplegic group [15].

**Table 3.** Evaluation of Different Methods to Estimate GFR

**(N=141)**

#### **d. 24-Hour endogenous creatinine clearance (CL24H)**

CL24H (mL/min) = [urine creatinine x urine volume (mL)] / [SCr x time (hours) x 60]

Current literature reports that CL24H better predicts renal function compared to CLCG and MDRD in paraplegia, however this method is not routinely utilized for drug dosing due to the impracticability of collecting multiple urine samples as well as the propensity for error from serial collections [8, 16].

### **3. Evaluation of different methods of estimating CLCR or GFR compared with patient-specific vancomycin and aminoglycoside (AG) clearance (CLDRUG ) in individuals with SCI/D**

Data on the application of methods of estimating renal function compared with patient-specific CLDRUG in paraplegia is scarce.

Lavezo et al. compared the pharmacokinetics of vancomycin in 14 SCI/D and 14 non-SCI/D control patients with their age, weight, pharmacokinetic parameters of total body clearance, volume of distribution, and mean predicted dosages matched. Demographic data between the groups differed only in mean SCr where the values were 0.8 ± 0.4 in the SCI/D group and 1.1 ± 0.3 in the able-bodied control group (p=0.04). The investigators obtained the pharmacokinetic parameters via two steady-state vancomycin serum concentrations by the Sawchuk and Zaske method [17] and found that compared to the control group, mean elimination rate constant was significantly smaller, therefore mean elimination half-life significantly longer in patients with SCI/D [18]. The authors concluded that patients with SCI/D may require longer dosing intervals of vancomycin compared to non-SCI/D [18].

In 2011, Lee and Dang published the results of a retrospective pharmacokinetic analysis of data on 141 patients with long-term SCI/D in the Veterans Affairs (VA) hospital with the largest inpatient SCI center in the VA system. The investigators evaluated frequently employed methods to estimate GFR (CLCG, modified CG, CL24H, MDRD, and CKD-EPI) against patientspecific drug clearance of vancomycin and AG (CLDRUG) [16]. Table 3 shows that all methods overestimate CLDRUG (p <0.001). The mean difference between CLDRUG and MDRD is largest where overestimation by MDRD is more than two-fold. Almost 70% of the patients had overestimation of CLDRUG by greater than 30 mL/min when using MDRD to predict empiric dosing for vancomycin and AG (p < 0.001) [16]. The authors modified the original CG equation by rounding SCr to 1 mg/dL for patients with SCr < 1 mg/dL while using the actual SCr for patients with SCr ≥ 1 mg/dL in attempts to account for low SCr in SCI/D and to overcome gross overestimation of renal function by CLCG [16]. The investigators found that the modified CG equation (CLM) better estimated CLDRUG in SCI/D, compared with other frequently employed methods for predicting GFR. The mean difference between CLDRUG and CLM was smallest among the equations evaluated where overestimation by CLM was approximately 40%. Almost 65% of the patients had prediction of CLDRUG within 30 mL/min when using CLM to estimate empiric dosing for vancomycin and AG (p < 0.001) [16]. Despite pronounced improvement by modification of CG, overestimation may not be clinically acceptable.


Abbreviations: GFR, glomerular filtration rate; CLDRUG, actual drug clearance; MDRD, the Modification of Diet in Renal Disease equation; CKD-EPI, Chronic Kidney Disease Epidemiology Collaboration equation; CL24H, 24-hour endogenous creatinine clearance; CLCG, the Cockcroft-Gault formula; CLM, modified Cockcroft-Gault formula; S.D., standard devia‐ tion. Published with permission of Lee [16].

**Table 3.** Evaluation of Different Methods to Estimate GFR

in current clinical practice when determining dosages of drugs that are primarily eliminated by the kidneys due to need for further validation. Furthermore, the sample population used to develop the CKD-EPI formula did not include paraplegia, thus its use in paraplegia may be

Current literature reports that CL24H better predicts renal function compared to CLCG and MDRD in paraplegia, however this method is not routinely utilized for drug dosing due to the impracticability of collecting multiple urine samples as well as the propensity for error from

**3. Evaluation of different methods of estimating CLCR or GFR compared with patient-specific vancomycin and aminoglycoside (AG) clearance**

Data on the application of methods of estimating renal function compared with patient-specific

Lavezo et al. compared the pharmacokinetics of vancomycin in 14 SCI/D and 14 non-SCI/D control patients with their age, weight, pharmacokinetic parameters of total body clearance, volume of distribution, and mean predicted dosages matched. Demographic data between the groups differed only in mean SCr where the values were 0.8 ± 0.4 in the SCI/D group and 1.1 ± 0.3 in the able-bodied control group (p=0.04). The investigators obtained the pharmacokinetic parameters via two steady-state vancomycin serum concentrations by the Sawchuk and Zaske method [17] and found that compared to the control group, mean elimination rate constant was significantly smaller, therefore mean elimination half-life significantly longer in patients with SCI/D [18]. The authors concluded that patients with SCI/D may require longer dosing

In 2011, Lee and Dang published the results of a retrospective pharmacokinetic analysis of data on 141 patients with long-term SCI/D in the Veterans Affairs (VA) hospital with the largest inpatient SCI center in the VA system. The investigators evaluated frequently employed methods to estimate GFR (CLCG, modified CG, CL24H, MDRD, and CKD-EPI) against patientspecific drug clearance of vancomycin and AG (CLDRUG) [16]. Table 3 shows that all methods overestimate CLDRUG (p <0.001). The mean difference between CLDRUG and MDRD is largest where overestimation by MDRD is more than two-fold. Almost 70% of the patients had overestimation of CLDRUG by greater than 30 mL/min when using MDRD to predict empiric dosing for vancomycin and AG (p < 0.001) [16]. The authors modified the original CG equation by rounding SCr to 1 mg/dL for patients with SCr < 1 mg/dL while using the actual SCr for patients with SCr ≥ 1 mg/dL in attempts to account for low SCr in SCI/D and to overcome gross overestimation of renal function by CLCG [16]. The investigators found that the modified CG

CL24H (mL/min) = [urine creatinine x urine volume (mL)] / [SCr x time (hours) x 60]

**d. 24-Hour endogenous creatinine clearance (CL24H)**

**(CLDRUG ) in individuals with SCI/D**

intervals of vancomycin compared to non-SCI/D [18].

misleading.

198 Topics in Paraplegia

serial collections [8, 16].

CLDRUG in paraplegia is scarce.

### **4. Estimation of renal function between the two anatomical degrees of SCI when compared with CLDRUG**

As previously mentioned, Mirahmadi et al. reported that both SCr and mean urinary creatinine excretion were markedly lower in paraplegics compared with ambulatory subjects [10]. The authors recommended an adjustment of the original CG equation by 20% for paraplegics to correct for reduction of muscle mass relative to the total body weight in such population [10].

Chikkalingaiah et al. found that both prediction equations (MDRD and CG) overestimated GFR in the paraplegic group with an overestimation by MDRD to a higher degree [15]. The fractional prediction error (FPE = (variable 1-variable 2) x 100/variable 1) for MDRD and CG were, respectively, 48.5% and 29.5% for paraplegic subjects, where an FPE greater than 20% was considered to be clinically unacceptable [15]. A correction factor of 0.7 for MDRD and 0.8 for CG proposed by the authors decreased the FPE to 3.9% and 3.6%, respectively, for the paraplegic group [15].

Lee and Dang sought to evaluate various methods to predict CLDRUG for different anatomical degrees of SCI/D (Table 4) [16]. The mean difference between CLSCI and CLDRUG was not statistically significant when separated into paraplegia and tetraplegia [16]. Similar finding was noted for CLM and CL24H [16]. On the other hand, the mean differences between CLCG, CKD-EPI, and MDRD and CLDRUG were statistically significant between the two anatomical degrees of SCI where tetraplegics had a gross overestimation of CLDRUG compared with paraplegics [16]. The investigators stated that such difference may have risen from rounding SCr up to 1 mg/dL for patients with SCr < 1 mg/dL and using a ratio of urine creatinine to SCr done in CLM and CL24H, respectively, contrary to using the actual SCr in the other equations [16].

Figures 1 and 2 depict, respectively, plots of actual drug clearance versus modified CG predicted drug clearance and linear regression plots of actual drug clearance versus predicted drug clearance using the CLSCI equation [16]. The slope of a linear correlation between the predicted and measured CLV values approach one, and the Y-intercept of a linear correlation

Estimating Renal Function in Paraplegia http://dx.doi.org/10.5772/57231 201

The CLSCI equation was tested against other methods through a retrospective analysis of 87 hospitalized patients with long-term SCI/D [19]. The study population had similar baseline characteristics to the previous population by Lee and Dang, exclusively elderly, overweight, males with similar SCr. The authors used the Sheiner and Beal method [20] for determining predictive performance (precision and bias) to evaluate the predictive ability of the CLSCI equation in estimating vancomycin clearance, relative to five alternative methods (CLCG, modified CG, CL24H, MDRD, and CKD-EPI). Compared with other equations, the CLSCI equation was found to be less biased and more precise, with the smallest calculated mean prediction error (ME) and square root of the mean squared prediction error (RMSE) values (p < 0.005) (Table 5) [19]. Predictive performance of the CLSCI relative to each of the other five methods was measured by change in ME (relative bias between two methods) and change in MSE (relative precision) (Table 6). Negative values for changes in ME and MSE indicate an advantage favoring the comparator; a greater negative value signifies a greater magnitude of error. The five alternative equations significantly overestimated CLV, by 45-92% (p < 0.05) (Table 7) [19]. The CLSCI equation underestimated CLV by approximately 6%, however not to a significant degree (p = 0.06) [19]. The results of their finding were consistent with the previous

**Figure 1.** Plots of Actual Drug Clearance versus Modified Cockcroft–Gault Predicted Drug Clearance. Published with

between the predicted and measured CLV values is minimum [16].

study by Lee and Dang.

permission of Lee [16].

Individuals with paraplegia have variable functionality and range of mobility and movement depending on the injury levels. Degree of paralysis of lower body and legs and upper body strength could affect muscle mass therefore potentially alter SCr and CLCR or GFR. For example, one with high paraplegia (>T7) may have weaker upper body strength and balance compared to the one with low (T7-T12) paraplegia thus may have lower muscle mass and SCr resulting in a falsely low estimation of renal function compared to the low paraplegia. Unfortunately, there has yet been a study that assesses renal function between different anatomical levels or severity of injury in paraplegia.


Abbreviations: CLDRUG, actual drug clearance; SCI, spinal cord injury; CLSCI, spinal cord injury equation; CLM, modified Cockcroft-Gault formula; CL24H, 24-hour endogenous creatinine clearance; CLCG, the Cockcroft-Gault formula; CKD-EPI, Chronic Kidney Disease Epidemiology Collaboration equation; MDRD, the Modification of Diet in Renal Disease equa‐ tion; S.D., standard deviation. Published with permission of Lee [16].

**Table 4.** Evaluation of Methods to Predict CLDRUG for Different Anatomical Degrees of SCI/D

#### **5. The "Spinal Cord Injury Equation" (CLSCI)**

Gross overestimation of CLDRUG by the frequently employed methods for estimating GFR prompted the authors Lee and Dang to develop an alternative method of estimating CLDRUG in SCI/D, the "spinal cord injury equation" (henceforth referred to as the CLSCI equation):

CLSCI (mL/min) = 2.3 X CLM 0.7

where CLSCI and CLM denote, respectively, clearance values determined via use of the CLSCI equation and the CLM formula [16]. The CLSCI equation yields a value along the *line of best fit* (the straight trend line depicting the line of least variability in all points on a scatterplot of data derived by regression analysis of two variables) between CLM and patient-specific vancomycin clearance (CLV) values [16].

Figures 1 and 2 depict, respectively, plots of actual drug clearance versus modified CG predicted drug clearance and linear regression plots of actual drug clearance versus predicted drug clearance using the CLSCI equation [16]. The slope of a linear correlation between the predicted and measured CLV values approach one, and the Y-intercept of a linear correlation between the predicted and measured CLV values is minimum [16].

degrees of SCI where tetraplegics had a gross overestimation of CLDRUG compared with paraplegics [16]. The investigators stated that such difference may have risen from rounding SCr up to 1 mg/dL for patients with SCr < 1 mg/dL and using a ratio of urine creatinine to SCr done in CLM and CL24H, respectively, contrary to using the actual SCr in the other equations [16]. Individuals with paraplegia have variable functionality and range of mobility and movement depending on the injury levels. Degree of paralysis of lower body and legs and upper body strength could affect muscle mass therefore potentially alter SCr and CLCR or GFR. For example, one with high paraplegia (>T7) may have weaker upper body strength and balance compared to the one with low (T7-T12) paraplegia thus may have lower muscle mass and SCr resulting in a falsely low estimation of renal function compared to the low paraplegia. Unfortunately, there has yet been a study that assesses renal function between different

**Mean Difference from CLDRUG ± S.D. (mL/min) p-Value**

**Tetraplegics (n = 89)**

Abbreviations: CLDRUG, actual drug clearance; SCI, spinal cord injury; CLSCI, spinal cord injury equation; CLM, modified Cockcroft-Gault formula; CL24H, 24-hour endogenous creatinine clearance; CLCG, the Cockcroft-Gault formula; CKD-EPI, Chronic Kidney Disease Epidemiology Collaboration equation; MDRD, the Modification of Diet in Renal Disease equa‐

Gross overestimation of CLDRUG by the frequently employed methods for estimating GFR prompted the authors Lee and Dang to develop an alternative method of estimating CLDRUG in SCI/D, the "spinal cord injury equation" (henceforth referred to as the CLSCI equation):

where CLSCI and CLM denote, respectively, clearance values determined via use of the CLSCI equation and the CLM formula [16]. The CLSCI equation yields a value along the *line of best fit* (the straight trend line depicting the line of least variability in all points on a scatterplot of data derived by regression analysis of two variables) between CLM and patient-specific vancomycin

**CLSCI** -3.11 ± 13.14 -5.39 ± 21.16 0.48 **CLM** 21.04 ± 13.81 18.76 ± 22.26 0.5 **CL24H** 32.60 ± 30.78 37.02 ± 35.29 0.45 **CLCG** 27.26 ± 20.56 49.76 ± 38.55 <0.001 **CKD-EPI** 27.52 ± 25.50 48.77 ± 24.76 <0.001 **MDRD** 40.68 ± 40.71 50.64 ± 64.56 <0.001

**Table 4.** Evaluation of Methods to Predict CLDRUG for Different Anatomical Degrees of SCI/D

anatomical levels or severity of injury in paraplegia.

tion; S.D., standard deviation. Published with permission of Lee [16].

**5. The "Spinal Cord Injury Equation" (CLSCI)**

CLSCI (mL/min) = 2.3 X CLM 0.7

clearance (CLV) values [16].

**Paraplegics (n = 52)**

200 Topics in Paraplegia

The CLSCI equation was tested against other methods through a retrospective analysis of 87 hospitalized patients with long-term SCI/D [19]. The study population had similar baseline characteristics to the previous population by Lee and Dang, exclusively elderly, overweight, males with similar SCr. The authors used the Sheiner and Beal method [20] for determining predictive performance (precision and bias) to evaluate the predictive ability of the CLSCI equation in estimating vancomycin clearance, relative to five alternative methods (CLCG, modified CG, CL24H, MDRD, and CKD-EPI). Compared with other equations, the CLSCI equation was found to be less biased and more precise, with the smallest calculated mean prediction error (ME) and square root of the mean squared prediction error (RMSE) values (p < 0.005) (Table 5) [19]. Predictive performance of the CLSCI relative to each of the other five methods was measured by change in ME (relative bias between two methods) and change in MSE (relative precision) (Table 6). Negative values for changes in ME and MSE indicate an advantage favoring the comparator; a greater negative value signifies a greater magnitude of error. The five alternative equations significantly overestimated CLV, by 45-92% (p < 0.05) (Table 7) [19]. The CLSCI equation underestimated CLV by approximately 6%, however not to a significant degree (p = 0.06) [19]. The results of their finding were consistent with the previous study by Lee and Dang.

**Figure 1.** Plots of Actual Drug Clearance versus Modified Cockcroft–Gault Predicted Drug Clearance. Published with permission of Lee [16].

**ΔME (CI) (mL/min) ΔMSE (CI) (mL<sup>2</sup>/min<sup>2</sup>)**

Estimating Renal Function in Paraplegia http://dx.doi.org/10.5772/57231 203

**specific CLV (ml/min) p-value**

**CLSCI vs. CLM** -24.5 (-26.8 TO -22.3) -513.8 (-709.8 to -317.8) **CLSCI vs. CL24H** -35.5 (-42.6 TO -28.5) -1690.0 (-2436.5 to -943.5) **CLSCI vs. CKD-EPI** -36.1 (-41.3 TO -30.9) -1477.5 (-1922.8 to -1032.3) **CLSCI vs. CLCG** -47.5 (-55.9 TO -39.1) -3214.9 (-4331.1 to -2098.7) **CLSCI vs. MDRD** -50.6 (-59.1 TO -42.1) -3525.1 (-4645.0 to -2405.2)

Modification of Diet in Renal Disease equation. Published with permission of Lee [19].

**N = 87 Mean ± S.D. (ml/min) Difference from patient-**

**CLSCI** 45.2 ± 9.1 -3.1 0.06 **CLM** 69.7 ± 19.7 21.5 < 0.05 **CL24H** 82.8 ± 36.0 34.6 < 0.05 **CKD-EPI** 81.2 ± 30.4 33.0 < 0.05 **CLCG** 92.7 ± 47.0 44.4 < 0.05 **MDRD** 95.7 ± 45.2 47.5 < 0.05

**Table 6.** Relative Predictive Performance of Vancomycin Clearance

Renal Disease equation. Published with permission of Lee [19].

**Table 7.** Evaluation of Different Methods to Estimate CLV

**6. Conclusion**

Abbreviations: ΔME, the difference in mean errors; ΔMSE, the difference in mean squared errors; CI, confidence interval; CLSCI, spinal cord injury equation; CLM, modified Cockcroft-Gault formula; CL24H, 24-hour endogenous creatinine clearance; CKD-EPI, Long-term Kidney Disease Epidemiology Collaboration equation; CLCG, the Cockcroft-Gault formula; MDRD, the

Abbreviations: CLV, patient-specific vancomycin clearance ; S.D., standard deviation; CLSCI, spinal cord injury equation; CLM, modified Cockcroft-Gault formula; CL24H, 24-hour endogenous creatinine clearance; CKD-EPI, Long-term Kidney Disease Epidemiology Collaboration equation; CLCG, the Cockcroft-Gault formula; MDRD, the Modification of Diet in

SCr determinations are used to estimate the dose of potentially toxic drugs eliminated primarily by the kidneys. Due to immobility and muscle atrophy, individuals with longduration paraplegia have lower SCr levels relative to their CLCR; this could lead to substantial overestimation of GFR resulting in higher than desired concentrations of medications that increase the risk of toxicity and/or ADRs, especially in persons with existing renal insufficien‐ cy. To date, there is no accepted standard method that can reliably predict renal function in paraplegia. Review of the current literature shows that the most widely used CG and MDRD equations overestimate GFR thus not recommended in paraplegia. Although CL24H better predicts renal function compared to CLCG and MDRD in paraplegia, unpracticality of collecting

**Figure 2.** Linear Regression Plots of Actual Drug Clearance versus Predicted Drug Clearance Using the Spinal Cord In‐ jury Equation. The red line, y=x, represents a line with a slope of 1 that indicates a perfectly one-to-one association between the actual and predicted drug clearance. Published with permission of Lee [16].


Abbreviations: ME, mean error; CI, confidence interval; MSE, mean squared error; RMSE, root mean squared error; CLSCI, spinal cord injury equation; CLM, modified Cockcroft-Gault formula; CL24H, 24-hour endogenous creatinine clear‐ ance; CKD-EPI, Long-term Kidney Disease Epidemiology Collaboration equation; CLCG, the Cockcroft-Gault formula; MDRD, the Modification of Diet in Renal Disease equation. Published with permission of Lee [19].

**Table 5.** Absolute Predictive Performance of Vancomycin Clearance


Abbreviations: ΔME, the difference in mean errors; ΔMSE, the difference in mean squared errors; CI, confidence interval; CLSCI, spinal cord injury equation; CLM, modified Cockcroft-Gault formula; CL24H, 24-hour endogenous creatinine clearance; CKD-EPI, Long-term Kidney Disease Epidemiology Collaboration equation; CLCG, the Cockcroft-Gault formula; MDRD, the Modification of Diet in Renal Disease equation. Published with permission of Lee [19].

**Table 6.** Relative Predictive Performance of Vancomycin Clearance


Abbreviations: CLV, patient-specific vancomycin clearance ; S.D., standard deviation; CLSCI, spinal cord injury equation; CLM, modified Cockcroft-Gault formula; CL24H, 24-hour endogenous creatinine clearance; CKD-EPI, Long-term Kidney Disease Epidemiology Collaboration equation; CLCG, the Cockcroft-Gault formula; MDRD, the Modification of Diet in Renal Disease equation. Published with permission of Lee [19].

**Table 7.** Evaluation of Different Methods to Estimate CLV

#### **6. Conclusion**

**Figure 2.** Linear Regression Plots of Actual Drug Clearance versus Predicted Drug Clearance Using the Spinal Cord In‐ jury Equation. The red line, y=x, represents a line with a slope of 1 that indicates a perfectly one-to-one association


235.2 748.9 1925.2 1712.7 3450.1 3760.3

15.3 27.4 43.9 41.4 58.7 61.3

Abbreviations: ME, mean error; CI, confidence interval; MSE, mean squared error; RMSE, root mean squared error; CLSCI, spinal cord injury equation; CLM, modified Cockcroft-Gault formula; CL24H, 24-hour endogenous creatinine clear‐ ance; CKD-EPI, Long-term Kidney Disease Epidemiology Collaboration equation; CLCG, the Cockcroft-Gault formula;

MDRD, the Modification of Diet in Renal Disease equation. Published with permission of Lee [19].

**Table 5.** Absolute Predictive Performance of Vancomycin Clearance

13.0 to 17.4 23.7 to 30.6 34.5 to 51.6 35.7 to 46.4 48.3 to 67.6 51.4 to 69.9

168.9 to 301.4 562.7 to 935.2 1191.2 to 2659.2 1271.6 to 2153.8 2334.8 to 4565.3 2640.1 to 4880.5

**Parameter CLSCI CLM CL24H CKD-EPI CLCG MDRD**

**ME (mL/min)** -3.1 21.5 32.5 33.0 44.5 47.5

between the actual and predicted drug clearance. Published with permission of Lee [16].

**Bias**

202 Topics in Paraplegia

95% CI (mL/min)

**Precision** MSE (mL2/min2)

95% CI (mL2/min2)

RMSE (mL/min)

95% CI (mL/min) SCr determinations are used to estimate the dose of potentially toxic drugs eliminated primarily by the kidneys. Due to immobility and muscle atrophy, individuals with longduration paraplegia have lower SCr levels relative to their CLCR; this could lead to substantial overestimation of GFR resulting in higher than desired concentrations of medications that increase the risk of toxicity and/or ADRs, especially in persons with existing renal insufficien‐ cy. To date, there is no accepted standard method that can reliably predict renal function in paraplegia. Review of the current literature shows that the most widely used CG and MDRD equations overestimate GFR thus not recommended in paraplegia. Although CL24H better predicts renal function compared to CLCG and MDRD in paraplegia, unpracticality of collecting multiple urine samples as well as the propensity for error from serial collections make this method clinically unfeasible. Different authors have recommended different modification of existing methods. Until more studies become available, the following methods can serve as valuable tools in estimating CLDRUG and renal function in individuals with paraplegia: 0.8 CG, 0.7 MDRD, or CLSCI equations.

[3] Rogers WH, Kazis LE, Miller DR, et al. Comparing the health status of VA and non-VA ambulatory patients: the veterans' health and medical outcomes studies. The

Estimating Renal Function in Paraplegia http://dx.doi.org/10.5772/57231 205

[4] Kazis LE, Miller DR, Clark J, et al: Health-related quality of life in patients served by the Department of Veterans Affairs: results from the Veterans Health Study. Ar‐

[5] Myers J, Lee M, Kiratli J: Cardiovascular disease in spinal cord injury: an overview of prevalence, risk, evaluation, and management. American Journal of Physical Medi‐

[6] Vaziri ND, Cesarior T, Mootoo K, et al. Bacterial infections in patients with chronic renal failure: occurrence with spinal cord injury. Archives of Internal Medicine

[7] Fischer MJ, Krishnamoorthi VR, Smith BM, et al. Prevalence of chronic kidney dis‐ ease in patients with spinal cord injuries/disorders. American Journal of Nephrology

[8] Cockcroft DW, Gault MH. Prediction of Creatinine Clearance from Serum Creatinine.

[9] Macdiarmid SA, Mcintyre WJ, Anthony A, et al. Monitoring of renal function in pa‐ tients with spinal cord injury. The British Journal of Urology 2000; 85: 1014-1018.

[10] Mirahmadi MK, Byrne C, Barton C, et al. Prediction of creatinine clearance from se‐ rum creatinine in spinal cord injury patients. Paraplegia 1983; 21(1) 23-29.

[11] Levey AS, Bosch JP, Lewis JB, et al. A more accurate method to estimate glomerular filtration rate from serum creatinine: a new prediction equation. Annals of Internal

[12] Stevens LA, Coresh J, Feldman HI, et al. Evaluation of the modification of diet in re‐ nal disease study equation in a large diverse population. Journal of the American So‐

[13] Levey AS, Coresh J, Greene T, et al. Expressing the Modification of Diet in Renal Dis‐ ease Study Equation for Estimating Glomerular Filtration Rate with Standardized Se‐

[14] Levey AS, Stevens LA, Schmid CH, et al. A New Equation to Estimate Glomerular

[15] Chikkalingaiah KBM, Grant ND, Mangold TM et al. Performance of Simplified Mod‐ ification of Diet in Renal Disease and Cockcroft-Gault Equations in Patients With Chronic Spinal Cord Injury and Chronic Kidney Disease. The American Journal of

rum Creatinine Values. Clinical Chemistry 2007; 53(4) 766-772.

Filtration Rate. Annals of Internal Medicine 2009; 150(9) 604-12.

Journal of Ambulatory Care Management 2004;27: 249–262.

chives of Internal Medicine 1998;158: 626-632.

cine and Rehabilitation 2007;86: 142-152.

1982;142: 1273-1276.

2012;36: 542-548.

Nephron 1976; 16: 31-41.

Medicine 1999; 130 (6) 461-70.

ciety of Nephrology 2007; 18(10) 2749-57.

the Medical Sciences 2010; 339(2) 108-16.

#### **Acknowledgements**

Supported in full by Grant # 2867 from the PVA Research Foundation.

#### **Author details**

Jennifer Pai Lee\*

Address all correspondence to: jennifer.lee4332a@va.gov

Pharm.D., BCPS, Pharmacy, Veterans Affairs Long Beach Healthcare System, Long Beach, USA

#### **References**


[3] Rogers WH, Kazis LE, Miller DR, et al. Comparing the health status of VA and non-VA ambulatory patients: the veterans' health and medical outcomes studies. The Journal of Ambulatory Care Management 2004;27: 249–262.

multiple urine samples as well as the propensity for error from serial collections make this method clinically unfeasible. Different authors have recommended different modification of existing methods. Until more studies become available, the following methods can serve as valuable tools in estimating CLDRUG and renal function in individuals with paraplegia: 0.8 CG,

Pharm.D., BCPS, Pharmacy, Veterans Affairs Long Beach Healthcare System, Long Beach,

[1] National Institutes of Diabetes and Digestive and Kidney Disease and National Kid‐ ney Disease Education Program. Chronic kidney disease and drug dosing: informa‐ tion for providers 2009. www.nkdep.nih.gov/professionals/drug-dosing-

[2] McKinely WO, Jackson AB, Cardenas DD, et al. Long-term medical complications af‐ ter traumatic spinal cord injury: a regional model systems analysis. Archives of Phys‐

Supported in full by Grant # 2867 from the PVA Research Foundation.

Address all correspondence to: jennifer.lee4332a@va.gov

information.htm (accessed 10 May 2011).

ical Medicine and Rehabilitation 1999;80: 140-210.

0.7 MDRD, or CLSCI equations.

**Acknowledgements**

204 Topics in Paraplegia

**Author details**

Jennifer Pai Lee\*

USA

**References**


[16] Lee JP, Dang AT. Evaluation of Methods to Estimate Glomerular Filtration Rate ver‐ sus Actual Drug Clearance in Patients with Chronic Spinal Cord Injury. Spinal Cord. 2011; 1-6.

**Section 4**

**Research in Paraplegia**


### **Research in Paraplegia**

[16] Lee JP, Dang AT. Evaluation of Methods to Estimate Glomerular Filtration Rate ver‐ sus Actual Drug Clearance in Patients with Chronic Spinal Cord Injury. Spinal Cord.

[17] Sawchuk RJ, Zaske DE, Cipolle RJ et al. Kinetic model for gentamicin dosing with the use of individual patient parameters. Clinical Pharmacology and Therapeutics 1977;

[18] Lavezo LA, Davis RL. Vancomycin pharmacokinetics in spinal cord injured patients: a comparison with age-matched, able-bodied controls. The Journal of Spinal Cord

[19] Lee JP, Wang YJ. Testing the Predictive Ability of the "Spinal Cord Injury Equation" in Estimating Vancomycin Clearance. American Journal of Health-System Pharmacy

[20] Sheiner LB, Beal SL. Scientific Commentary—Some Suggestions for Measuring Pre‐ dictive Performance. Journal of Pharmacokinetics and Biopharmaceutics 1981;

2011; 1-6.

206 Topics in Paraplegia

21: 362-369.

Medicine 1995; 16: 233-235.

2013 70(8) 669-674.

9:503-12.

**Chapter 10**

**Animal Models in**

http://dx.doi.org/10.5772/57189

resolving this fundamental problem.

**1. Introduction**

SCIs.

**Traumatic Spinal Cord Injury**

Mahdi Sharif-Alhoseini and Vafa Rahimi-Movaghar

Traumatic spinal cord injury (SCI) causes high mortality, severe disability, expensive cure, extensive rehabilitation, and a high economic burden. There has been no definite treatment for SCI, but numerous studies including experimental modeling are being performed to assist

The first reported SCI model was presented by Allen in 1911 where a mass was dropped from a prescribed height onto the dorsal surface of the canine dura. After that, the animal models of SCI from simple lamprey to non-human primates were used to develop pathophysiological

Currently, to choose an animal model, some factors are considered depending upon the proposed aim of the study. Transections and contusions of the spinal cord are the most commonly used methods for animal modeling of SCI. While transection models provide an idealized setting for studying spinal cord regeneration across a complete lesion, but transected spinal cords are rarely encountered in human SCI. In other words, most injured spinal cords maintain some tissue continuity across the area of injury. But contusion and compression models are more clinically relevant. These models can create graded injuries and characterized by hemorrhagic necrosis, ischemia, inflammation, and central cavitation. Besides, compression models contribute to simulate the persistent spinal canal occlusion that is common in human

The ongoing development of SCI animal models reflects the need to review all types of them and gauge about their advantages or disadvantages. The purpose of this chapter is to review

> © 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Additional information is available at the end of the chapter

knowledge on cell injury and repair process of spinal cord.

animal models in SCI from studies indexed in Medline.

#### **Chapter 10**

## **Animal Models in Traumatic Spinal Cord Injury**

Mahdi Sharif-Alhoseini and Vafa Rahimi-Movaghar

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/57189

#### **1. Introduction**

Traumatic spinal cord injury (SCI) causes high mortality, severe disability, expensive cure, extensive rehabilitation, and a high economic burden. There has been no definite treatment for SCI, but numerous studies including experimental modeling are being performed to assist resolving this fundamental problem.

The first reported SCI model was presented by Allen in 1911 where a mass was dropped from a prescribed height onto the dorsal surface of the canine dura. After that, the animal models of SCI from simple lamprey to non-human primates were used to develop pathophysiological knowledge on cell injury and repair process of spinal cord.

Currently, to choose an animal model, some factors are considered depending upon the proposed aim of the study. Transections and contusions of the spinal cord are the most commonly used methods for animal modeling of SCI. While transection models provide an idealized setting for studying spinal cord regeneration across a complete lesion, but transected spinal cords are rarely encountered in human SCI. In other words, most injured spinal cords maintain some tissue continuity across the area of injury. But contusion and compression models are more clinically relevant. These models can create graded injuries and characterized by hemorrhagic necrosis, ischemia, inflammation, and central cavitation. Besides, compression models contribute to simulate the persistent spinal canal occlusion that is common in human SCIs.

The ongoing development of SCI animal models reflects the need to review all types of them and gauge about their advantages or disadvantages. The purpose of this chapter is to review animal models in SCI from studies indexed in Medline.

© 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

#### **2. Why animal models?**

Animal model refers to the use of a living, non-human animal to simulate the human disease or injury, for better understanding the disease where it is practically or ethically difficult to use humans. It is used to learn more about a disease, its pathophysiologic changes, diagnosis and treatment. Animal models are often preferable for experimental disease or injury research because of their unlimited supply, ease of manipulation, the possibility to standardize the condition, the capability to use more invasive procedures to observe the effects of treatment, and no concern for the patients' safety [1, 2]. In fact, many potential therapies require testing for safety and efficacy in animals before it is possible to move to a clinical trial.

canal [9]. In 1976, Eidelberg created an SCI model in rats caused by direct epidural compression [10]. New techniques were developed and improved, e.g. spinal cord stabilization and precise distribution of strengths involved on impact, the use of mechanisms able to measure the strength to which an animal's spinal cord is exposed, as well as the invention of pneumatic

Animal Models in Traumatic Spinal Cord Injury

http://dx.doi.org/10.5772/57189

211

Because the weight-drop techniques deliver a single, rapid blow to the spinal cord, neither model simulates ongoing cord compression secondary to residual spinal column displace‐ ment. Thus, in 1978, Rivlin and Tator introduced a clip compression model of SCI in rats, in which the spinal cord was compressed for variable durations between the arms of a modified aneurysm clip [12]. This model demonstrated the relation between the severity of neurologic

Afterwards, more technical devices such as Ohio State University's electromagnetic spinal cord injury device (OSU impactor) and New York University (NYU) impactor came into use. In 1987, the researchers at Ohio State University applied a computer feedback-controlled electromagnetic force to create contusion and concussion in the spinal cord of rats [13]. In this model, after laminectomy, the OSU impactor probe is slowly screwed down to the dural surface, which it contacts and displaces 30 micrometers with a force of approximately 3000 dynes. This is meant to provide a consistent starting point from which to initiate the injury. The system then is triggered, and the device rapidly impacts the cord for a predetermined amount of displacement before releasing [14]. Because the OSU impactor is actively with‐ drawn, there is no bouncing of the impactor back onto the cord, which is a probable basis of variation in a weight-drop technique. NYU impactor was at first described by Gruner in 1992 and then refined by a consortium of eight spinal cord laboratories in the United States called MASCIS (Multicenter Animal Spinal Cord Injury Study). The NYU-MASCIS weight-drop model standardizes grades of contusive spinal cord injury by dropping a 10g rod from specific heights of 6.25 (mild), 12.5 (moderate), 25 (severe) or 50 mm (very severe) upon the exposed dorsal surface of the spinal cord [15]. Usage of the recent impactors requires intense training, extensive maintenance and sophisticated software which give more room to exclude the post-

In addition to traumatic SCI, spinal cord ischemia remains an underappreciated clinical dilemma which mostly occurs after aortic problems. Therefore, experimental models of spinal cord ischemia have been developed in different animals with variable reproducibility [16-19].

In the last decades, transection has been favored to study approaches of nerve fiber regener‐ ation and cell transplantation that are likely to be most appropriate to the subacute stage.

The majority of reported human injuries occurs at the cervical level, often secondary to vertebral fracture, producing compression or contusion of the spinal cord [20]. Functional deficits after cervical injury are a result of damage to both white and gray matter. At this level,

impact mechanisms [11].

injury and the length of compression.

operative animals being used for the experiments.

**4. Level of SCI**

To serve as a useful model of a human condition, a modelled disease or injury not only must be similar in the etiology and function to the human equivalent but also has to offer advantages over direct clinical observation and experiment [2, 3].

On the other hand, spinal cord injury (SCI), as a fundamental problem in medicine, causes high mortality, severe disability, expensive cure, extensive rehabilitation, and a high economic burden. So far management of SCI is challenging and there has been no definite treatment for it. But numerous studies including experimental modeling are being performed to assist understanding the anatomical and biological consequences of injury and repair, and testing the efficacy and the risk-to-benefit ratio of a proposed therapy [3]. Animal models have been developed with the aim of recreating features of either complete or incomplete SCI to increase the knowledge about disease mechanisms and evolution of injury, and provides a clinically relevant platform for developing and evaluating therapies in SCI [4, 5]. Animal models have also some other benefits over their human equivalent; e.g. the specified tissue needed can be used and processed for histological purposes to investigate co-localization of proteins of interest, mRNA analysis (microarray) to give expression of proteins and protein analysis (western blotting) to give levels of protein [6].

#### **3. History**

Various methods for induction of experimental SCI have been used in the past. The first reported SCI model was presented by Allen in 1911 where a mass was dropped from a prescribed height onto the dorsal surface of the canine dura. He used a simple irrefutable logic that when a known weight dropped from a constant height shall produce same impact force on all occasions. Based on this concept, he prepared a metal tube with pores. A rod of 10 g was inserted into the tube and can be stopped at various heights using a pin inserted into the pores on the tube at regular intervals. By aiming the tube over a surgically exposed spinal cord and by withdrawing the pin holding the rod, a reproducible impact force would be created when the rod get dropped on the spinal cord. For unknown reasons, most data available concerning experimentally induced SCI are modifications of an injury model proposed by Allen [7].

In 1936, the load throw devices were used to make a spinal cord contusion [8]. In 1953, a model was created in which a dog had its spinal cord injured by an inflated balloon inside the spinal canal [9]. In 1976, Eidelberg created an SCI model in rats caused by direct epidural compression [10]. New techniques were developed and improved, e.g. spinal cord stabilization and precise distribution of strengths involved on impact, the use of mechanisms able to measure the strength to which an animal's spinal cord is exposed, as well as the invention of pneumatic impact mechanisms [11].

Because the weight-drop techniques deliver a single, rapid blow to the spinal cord, neither model simulates ongoing cord compression secondary to residual spinal column displace‐ ment. Thus, in 1978, Rivlin and Tator introduced a clip compression model of SCI in rats, in which the spinal cord was compressed for variable durations between the arms of a modified aneurysm clip [12]. This model demonstrated the relation between the severity of neurologic injury and the length of compression.

Afterwards, more technical devices such as Ohio State University's electromagnetic spinal cord injury device (OSU impactor) and New York University (NYU) impactor came into use. In 1987, the researchers at Ohio State University applied a computer feedback-controlled electromagnetic force to create contusion and concussion in the spinal cord of rats [13]. In this model, after laminectomy, the OSU impactor probe is slowly screwed down to the dural surface, which it contacts and displaces 30 micrometers with a force of approximately 3000 dynes. This is meant to provide a consistent starting point from which to initiate the injury. The system then is triggered, and the device rapidly impacts the cord for a predetermined amount of displacement before releasing [14]. Because the OSU impactor is actively with‐ drawn, there is no bouncing of the impactor back onto the cord, which is a probable basis of variation in a weight-drop technique. NYU impactor was at first described by Gruner in 1992 and then refined by a consortium of eight spinal cord laboratories in the United States called MASCIS (Multicenter Animal Spinal Cord Injury Study). The NYU-MASCIS weight-drop model standardizes grades of contusive spinal cord injury by dropping a 10g rod from specific heights of 6.25 (mild), 12.5 (moderate), 25 (severe) or 50 mm (very severe) upon the exposed dorsal surface of the spinal cord [15]. Usage of the recent impactors requires intense training, extensive maintenance and sophisticated software which give more room to exclude the postoperative animals being used for the experiments.

In addition to traumatic SCI, spinal cord ischemia remains an underappreciated clinical dilemma which mostly occurs after aortic problems. Therefore, experimental models of spinal cord ischemia have been developed in different animals with variable reproducibility [16-19].

In the last decades, transection has been favored to study approaches of nerve fiber regener‐ ation and cell transplantation that are likely to be most appropriate to the subacute stage.

#### **4. Level of SCI**

**2. Why animal models?**

210 Topics in Paraplegia

Animal model refers to the use of a living, non-human animal to simulate the human disease or injury, for better understanding the disease where it is practically or ethically difficult to use humans. It is used to learn more about a disease, its pathophysiologic changes, diagnosis and treatment. Animal models are often preferable for experimental disease or injury research because of their unlimited supply, ease of manipulation, the possibility to standardize the condition, the capability to use more invasive procedures to observe the effects of treatment, and no concern for the patients' safety [1, 2]. In fact, many potential therapies require testing

To serve as a useful model of a human condition, a modelled disease or injury not only must be similar in the etiology and function to the human equivalent but also has to offer advantages

On the other hand, spinal cord injury (SCI), as a fundamental problem in medicine, causes high mortality, severe disability, expensive cure, extensive rehabilitation, and a high economic burden. So far management of SCI is challenging and there has been no definite treatment for it. But numerous studies including experimental modeling are being performed to assist understanding the anatomical and biological consequences of injury and repair, and testing the efficacy and the risk-to-benefit ratio of a proposed therapy [3]. Animal models have been developed with the aim of recreating features of either complete or incomplete SCI to increase the knowledge about disease mechanisms and evolution of injury, and provides a clinically relevant platform for developing and evaluating therapies in SCI [4, 5]. Animal models have also some other benefits over their human equivalent; e.g. the specified tissue needed can be used and processed for histological purposes to investigate co-localization of proteins of interest, mRNA analysis (microarray) to give expression of proteins and protein analysis

Various methods for induction of experimental SCI have been used in the past. The first reported SCI model was presented by Allen in 1911 where a mass was dropped from a prescribed height onto the dorsal surface of the canine dura. He used a simple irrefutable logic that when a known weight dropped from a constant height shall produce same impact force on all occasions. Based on this concept, he prepared a metal tube with pores. A rod of 10 g was inserted into the tube and can be stopped at various heights using a pin inserted into the pores on the tube at regular intervals. By aiming the tube over a surgically exposed spinal cord and by withdrawing the pin holding the rod, a reproducible impact force would be created when the rod get dropped on the spinal cord. For unknown reasons, most data available concerning experimentally induced SCI are modifications of an injury model proposed by Allen [7].

In 1936, the load throw devices were used to make a spinal cord contusion [8]. In 1953, a model was created in which a dog had its spinal cord injured by an inflated balloon inside the spinal

for safety and efficacy in animals before it is possible to move to a clinical trial.

over direct clinical observation and experiment [2, 3].

(western blotting) to give levels of protein [6].

**3. History**

The majority of reported human injuries occurs at the cervical level, often secondary to vertebral fracture, producing compression or contusion of the spinal cord [20]. Functional deficits after cervical injury are a result of damage to both white and gray matter. At this level, white matter disruption leads to spastic paralysis below the injury, sensory loss/chronic pain, cardiovascular, gastrointestinal, and sexual dysfunction. Motor neurons controlling the upper limb musculature reside there, and their loss induces flaccid paralysis [21]. But so far, thoracic SCI is the most commonly used location in animal models. Since gray matter loss at this spinal level causes less identifiable functional loss, thoracic SCI could contribute to isolate and study white matter deficits. In addition, high cervical levels can result in diaphragm dysfunction due to interruption of bulbospinal respiratory drive to phrenic motoneuron pools (C3–C5) [22, 23]. Thus thoracic SCI models are obviously reliable and easy to reproduce [24, 25].

However, due to differences in spinal cord diameter, the distance of injury from both the neuronal cell body and the original targets of innervations, the relative dedication of the cord to specific ascending and descending systems and their different termination sites, the degree of vascularization, the size of the sensory and motor neuron populations, the level of their importance in locomotion, and white/gray matter distribution, histological, behavioral, and therapeutic findings in the thoracic spinal cord, may not be so readily applicable to the cervical level [26].

**Figure 1.** A: NYU Impactor. B: OSU Impactor. C: IH Impactor.

monitoring the dynamics of the impact [30].

precision to produce lesions more reliably [32].

cervical contusion is often utilized for motor functional analysis [21].

convinced force to induce mild, moderate or severe injury [33, 34].

compression model would be appropriate. (Figure 2)

impactor retracts [31].

The most widely used device is the NYU impactor which concurrent recording of kinematic

Animal Models in Traumatic Spinal Cord Injury

http://dx.doi.org/10.5772/57189

213

OSU impactor electromagnetically drives an impounder tip onto the cord until a desired displacement of the cord surface is reached. After a defined time, the tip is retracted and the pressure released [29]. This computer controlled contusion model consists of an animal trap that reproducibly delivers a defined weight to the exposed spinal cord, with a computer

In a similar way operates the only commercially available device, the Infinite Horizon (IH) impactor. A stepping motor applies a defined force to the cord. Once the force is reached, the

The NYU impactor is rather easier to use, but the OSU impactor and IH impactor have more

Hemicontusion: Hemicontusion or unilateral contusion is used in cervical spinal cord, because life-threatening adverse effects could occur in cervical contusion. Since motor dysfunction appears in the forelimbs, pain related behavior is difficult to estimate, and for this reason,

**•** *Compression:* Compression models contribute to simulate the persistent spinal canal occlusion that is common in human SCIs and investigate the effects of compression or the optimal timing of decompression. For this reason, a clip, balloon, spacer, or forceps

Clip compression injury is similar to spinal contusion injury at the point of the injury caused by pressure to the spinal cord. Following laminectomy, a vascular clip is dorsoventrally closed over the entire cord. With this method, the spinal cord becomes ischemic and mimics common clinical injuries and outcomes. Compressive injury is induced with clips calibrated to exert a

parameters of the impounded probe allows the validation of the injury process.

On the other hand, rats do not use their hindlimbs as skilfully as their forelimbs. Also the hindlimb paw and digit use cannot be evaluated as carefully as the forelimb paws and digits. Thus, forelimb evaluation could superiorly assess the efficacy of potential therapies, especial‐ ly in mild degrees of improvement. Therefore, some scientists tried to characterize cervical SCI in rats [26, 27]. In 2001, Soblosky et al. characterized a unilateral cervical contusion SCI model which allowed the contralateral side to serve as a within-subject control [24]. In this model, the injurydidnot causeovertbladderdysfunction,whichsignificantlyreducedtheneedfor chronic intensive care after SCI. In 2005, this model has been further standardizes by Gensel et al. [21].

#### **5. Injury paradigms**

In general, experimental models can be naturally occurring (e.g. injured dogs in road traffic crashes), congenital disease (e.g. a spontaneous mutant), or induced (surgical, genetically engineered) that is similar to a human condition. SCI models are mostly created based on surgical methods which are determined by the experimental aims of a particular research. Every injury techniques concentrate on a special question, and hence each carries their own pros and cons:

**•** *Contusion:* If the pathophysiology of secondary injury is the main part of research interest, a contusion and/or compression model could be selected; because most human SCIs involve contusive or compressive injury [28]. Contusion is the oldest and most widely used for SCI models. The contusive models can create graded injuries and characterized by hemorrhagic necrosis, ischemia, inflammation, and central cavitation. It elicits both motor and sensory dysfunction, such as tactile allodynia, neuropathic pain, and thermal hyperalgesia.

Somedevices existtocreate contusionina controlledwaytolimitthevariationbetweenanimals and allow the comparison between results obtained in different laboratories. (Figure 1)

**Figure 1.** A: NYU Impactor. B: OSU Impactor. C: IH Impactor.

white matter disruption leads to spastic paralysis below the injury, sensory loss/chronic pain, cardiovascular, gastrointestinal, and sexual dysfunction. Motor neurons controlling the upper limb musculature reside there, and their loss induces flaccid paralysis [21]. But so far, thoracic SCI is the most commonly used location in animal models. Since gray matter loss at this spinal level causes less identifiable functional loss, thoracic SCI could contribute to isolate and study white matter deficits. In addition, high cervical levels can result in diaphragm dysfunction due to interruption of bulbospinal respiratory drive to phrenic motoneuron pools (C3–C5) [22,

However, due to differences in spinal cord diameter, the distance of injury from both the neuronal cell body and the original targets of innervations, the relative dedication of the cord to specific ascending and descending systems and their different termination sites, the degree of vascularization, the size of the sensory and motor neuron populations, the level of their importance in locomotion, and white/gray matter distribution, histological, behavioral, and therapeutic findings in the thoracic spinal cord, may not be so readily applicable to the cervical

On the other hand, rats do not use their hindlimbs as skilfully as their forelimbs. Also the hindlimb paw and digit use cannot be evaluated as carefully as the forelimb paws and digits. Thus, forelimb evaluation could superiorly assess the efficacy of potential therapies, especial‐ ly in mild degrees of improvement. Therefore, some scientists tried to characterize cervical SCI in rats [26, 27]. In 2001, Soblosky et al. characterized a unilateral cervical contusion SCI model which allowed the contralateral side to serve as a within-subject control [24]. In this model, the injurydidnot causeovertbladderdysfunction,whichsignificantlyreducedtheneedfor chronic intensive care after SCI. In 2005, this model has been further standardizes by Gensel et al. [21].

In general, experimental models can be naturally occurring (e.g. injured dogs in road traffic crashes), congenital disease (e.g. a spontaneous mutant), or induced (surgical, genetically engineered) that is similar to a human condition. SCI models are mostly created based on surgical methods which are determined by the experimental aims of a particular research. Every injury techniques concentrate on a special question, and hence each carries their own

**•** *Contusion:* If the pathophysiology of secondary injury is the main part of research interest, a contusion and/or compression model could be selected; because most human SCIs involve contusive or compressive injury [28]. Contusion is the oldest and most widely used for SCI models. The contusive models can create graded injuries and characterized by hemorrhagic necrosis, ischemia, inflammation, and central cavitation. It elicits both motor and sensory

dysfunction, such as tactile allodynia, neuropathic pain, and thermal hyperalgesia.

Somedevices existtocreate contusionina controlledwaytolimitthevariationbetweenanimals and allow the comparison between results obtained in different laboratories. (Figure 1)

23]. Thus thoracic SCI models are obviously reliable and easy to reproduce [24, 25].

level [26].

212 Topics in Paraplegia

**5. Injury paradigms**

pros and cons:

The most widely used device is the NYU impactor which concurrent recording of kinematic parameters of the impounded probe allows the validation of the injury process.

OSU impactor electromagnetically drives an impounder tip onto the cord until a desired displacement of the cord surface is reached. After a defined time, the tip is retracted and the pressure released [29]. This computer controlled contusion model consists of an animal trap that reproducibly delivers a defined weight to the exposed spinal cord, with a computer monitoring the dynamics of the impact [30].

In a similar way operates the only commercially available device, the Infinite Horizon (IH) impactor. A stepping motor applies a defined force to the cord. Once the force is reached, the impactor retracts [31].

The NYU impactor is rather easier to use, but the OSU impactor and IH impactor have more precision to produce lesions more reliably [32].

Hemicontusion: Hemicontusion or unilateral contusion is used in cervical spinal cord, because life-threatening adverse effects could occur in cervical contusion. Since motor dysfunction appears in the forelimbs, pain related behavior is difficult to estimate, and for this reason, cervical contusion is often utilized for motor functional analysis [21].

**•** *Compression:* Compression models contribute to simulate the persistent spinal canal occlusion that is common in human SCIs and investigate the effects of compression or the optimal timing of decompression. For this reason, a clip, balloon, spacer, or forceps compression model would be appropriate. (Figure 2)

Clip compression injury is similar to spinal contusion injury at the point of the injury caused by pressure to the spinal cord. Following laminectomy, a vascular clip is dorsoventrally closed over the entire cord. With this method, the spinal cord becomes ischemic and mimics common clinical injuries and outcomes. Compressive injury is induced with clips calibrated to exert a convinced force to induce mild, moderate or severe injury [33, 34].

For certain applications, partial transection can be a viable alternative to complete transection. In other words, because the lesion that results from a complete transection creates such a hostile tissue environment, injury paradigms have been developed that decrease the physical damage to the cord and the consequential cavitation and physical separation. Thus researchers can selectively interrupt certain pathways with partial transections to hold a tissue bridge between the proximal and distal ends of the cord, and maintain tissue continuity [39]. A dorsal hemisection for selective transection of the corticospinal tract can be performed with some feedback from the change in color and texture between the white and gray matter, giving a sign of the entirety of the hemisection [2, 37]. But dorsal hemisection cannot be used rigorously to assess true axon regeneration [39]. Dorsolateral quadrant lesions are used to interrupt the rubrospinal tract, and lateral hemisections disrupt all tracts on one side but spare some or all

Animal Models in Traumatic Spinal Cord Injury

http://dx.doi.org/10.5772/57189

215

**•** *Photochemical model:* This model was developed by Watson et al. in 1986 [40] and was proven to be one of the most reliable and reproducible graded ischemic experimental models of SCI [41]. With the exposed spinal column intact, irradiation of the translu‐ cent dorsal surface induces excitation of the systemically injected dye (e.g. rose Bengal) in the spinal cord microvasculature. The resultant photochemical reaction leads to vascular stasis, hemorrhagic necrosis of the central grey matter, edematous pale-stain‐ ing white matter tracts and vascular congestion. The main benefit of this technique is that the resulting injury does not induce mechanical trauma to the cord, because there is no need for laminectomy. On the other hand, an intravascular photochemical reac‐ tion occurs through the use of a dye that is activated by an argon ion laser to produce single oxygen molecules at the endothelial surface of spinal cord vessels. This leads to a severe platelet reaction, subsequent vessel occlusion, and parenchymal tissue infarc‐

**•** *Ischemic model:* Initial studies used the methods described by Lang-Lazdunski et al. [16]. This method uses an anterior sternotomy with temporary aortic occlusion created by aneurysm

**•** *Excitotoxic model:* Following intraspinal or intrathecal injection of some excitotoxins (e.g. Ametabotropic receptor agonist quisqualic acid or other excitatory amino acids such as glutamate, N-methylasparate, and kainic acid), the cascade of events described following ischemic and traumatic SCI, including prominent inflammation, neuronal loss, astrocytic scaring, cavity formation, syringomyelia, long-lasting spontaneous pain, and mechanical allodynia occur. This model can correlate specific areas of tissue damage with behavioral changes. But almost all animals develop varying degrees of hypersensitivity to mechanical

**•** *Combination:* For some particular goals, a combination of models might be designed. For example, the early stages of an experimental study that explores axon regeneration may use transection paradigms to definitely reveal regenerated axons and recognize the most

promising therapies, which can then be examined in contusion models [37].

tion. Also, the degree of injury is hard to control [38].

clips sited at the aortic arch plus left subclavian artery [42].

tracts on the opposite side.

and thermal stimuli [38, 43].

**Figure 2.** A: Aneurysm clips. B: Fogarty catheter. C: Spacer. D: Forceps [2].

The balloon-induced method has been used because it is a simple method that does not cause any damage to the surrounding structures. The volume of balloon inflation must be measured several times and used in combination with the size of the experimental animals when determining a sufficient amount of injury to inflict [35]. A Fogarty catheter is inserted into the dorsal epidural space through a small hole made in vertebral arch, advanced cranially to one or two higher spinal levels. Spinal cord damage is graded by increasing the volume of saline used to inflate the balloon.

To use a spacer, at first the average anteroposterior spinal canal diameter should be determined from the spines of animals of similar weight and age. This allowed for the determination of the spacer size needed to produce a precise degree of narrowing of the spinal canal diameter [36].

The calibrated forceps can produce a lateral compression injury by inserting on either side of the spinal cord and closing together to induce a central hemorrhagic necrosis and displacement of the centrally located, damaged tissue in cranial and caudal directions [2].

**•** *Transection:* The transected spinal cords are rarely encountered in human SCI, but transec‐ tion models provide an idealized setting for studying hypotheses that concern regeneration, degeneration, tissue engineering strategies, or plasticity on an axonal level. These types of lesions are most usefully combined with neuroanatomical tract tracing and electrophysio‐ logical studies [32, 37]. Transection models are also increasingly used to model the effects of scaffolds, biomaterials, neurotrophic factors, and combinatorial therapies on axon regeneration after injury [6]. To allow for regeneration, sterile gel foams have been placed between the two ends of transected cords with variable degree of success [38]. Besides, if a device is to be implemented, a partial or complete transection model might be best suited for device placement. Many studies have reported bilateral muscle spasms, neuropathic pains, mechanical allodynia and thermal hyperalgesia at same, above, and/or below the level of the lesion following complete spinal transection model [38].

Spinal cord transection is performed after laminectomy with fine surgical scissors (iridectomy scissors) that allows the targeted interruption of a particular nerve fiber systems such as motor tracts (corticospinal tract, rubrospinal tract) or sensory tracts (dorsal columns), or even complete interruption of the spinal cord [32].

For certain applications, partial transection can be a viable alternative to complete transection. In other words, because the lesion that results from a complete transection creates such a hostile tissue environment, injury paradigms have been developed that decrease the physical damage to the cord and the consequential cavitation and physical separation. Thus researchers can selectively interrupt certain pathways with partial transections to hold a tissue bridge between the proximal and distal ends of the cord, and maintain tissue continuity [39]. A dorsal hemisection for selective transection of the corticospinal tract can be performed with some feedback from the change in color and texture between the white and gray matter, giving a sign of the entirety of the hemisection [2, 37]. But dorsal hemisection cannot be used rigorously to assess true axon regeneration [39]. Dorsolateral quadrant lesions are used to interrupt the rubrospinal tract, and lateral hemisections disrupt all tracts on one side but spare some or all tracts on the opposite side.

The balloon-induced method has been used because it is a simple method that does not cause any damage to the surrounding structures. The volume of balloon inflation must be measured several times and used in combination with the size of the experimental animals when determining a sufficient amount of injury to inflict [35]. A Fogarty catheter is inserted into the dorsal epidural space through a small hole made in vertebral arch, advanced cranially to one or two higher spinal levels. Spinal cord damage is graded by increasing the volume of saline

**Figure 2.** A: Aneurysm clips. B: Fogarty catheter. C: Spacer. D: Forceps [2].

To use a spacer, at first the average anteroposterior spinal canal diameter should be determined from the spines of animals of similar weight and age. This allowed for the determination of the spacer size needed to produce a precise degree of narrowing of the

The calibrated forceps can produce a lateral compression injury by inserting on either side of the spinal cord and closing together to induce a central hemorrhagic necrosis and displacement

**•** *Transection:* The transected spinal cords are rarely encountered in human SCI, but transec‐ tion models provide an idealized setting for studying hypotheses that concern regeneration, degeneration, tissue engineering strategies, or plasticity on an axonal level. These types of lesions are most usefully combined with neuroanatomical tract tracing and electrophysio‐ logical studies [32, 37]. Transection models are also increasingly used to model the effects of scaffolds, biomaterials, neurotrophic factors, and combinatorial therapies on axon regeneration after injury [6]. To allow for regeneration, sterile gel foams have been placed between the two ends of transected cords with variable degree of success [38]. Besides, if a device is to be implemented, a partial or complete transection model might be best suited for device placement. Many studies have reported bilateral muscle spasms, neuropathic pains, mechanical allodynia and thermal hyperalgesia at same, above, and/or below the

Spinal cord transection is performed after laminectomy with fine surgical scissors (iridectomy scissors) that allows the targeted interruption of a particular nerve fiber systems such as motor tracts (corticospinal tract, rubrospinal tract) or sensory tracts (dorsal columns), or even

of the centrally located, damaged tissue in cranial and caudal directions [2].

level of the lesion following complete spinal transection model [38].

complete interruption of the spinal cord [32].

used to inflate the balloon.

214 Topics in Paraplegia

spinal canal diameter [36].


#### **6. Species of animals used**

Rodents are the most common type of mammal employed in SCI experimental studies, and widespread research have been conducted using rats, mice, gerbils, guinea pigs, and hamsters [1]. Other animal experiments include cats, non-human primates, goats, pigs, and dogs [1, 4, 35, 44-49]. Of course, larger mammals such as cats, dogs, or pigs are also used but very rarely and are less experienced models based in SCI research, requiring expensive after care and housing as well as stringent ethical considerations [6, 37]. Other models include invertebrates, such as eels [50], whose unique regenerative capacities have been studied in efforts to apply novel strategies to human SCI.

**•** *Pig:* Because of large size and greater likeness to human physiology, pig models are becoming more important as a preclinical model that is intermediate in size between rodents

Animal Models in Traumatic Spinal Cord Injury

http://dx.doi.org/10.5772/57189

217

**•** *Dog:* Dogs can be surveyed after naturally occurring SCIs e.g. following road traffic accidents or disc degenerations. The mechanisms of injury in clinical SCI in dogs are similar to those in human patients: vertebral fracture–luxation and disc extrusions – both of which produce the mixed contusion-compression lesion to the ventral aspect of the cord that is problem‐ atical to model in the laboratory [45]. To date, dogs have been used to study spinal cord injuries because neurological examinations could be carried out easily, and more detailed pathophysiological studies could be conducted [35, 46]. Compared to analysis of trials in

human patients, dogs have the advantage that there is less of an ethical problem.

to basic discoveries that might not be identified in rodent models [4].

**•** *Non-human primate:* Non-human primate models are limited by extremely high costs related to the intensive animal care and ethically challenging, but may be imperative to prove safety and efficacy on a small scale prior to human experimentation, particularly for strategies involving device implantation [25]. Because of similar anatomy and pathology to human, a primate model could provide greater positive predictive value to human therapies, and lead

Behavioral outcome in experimental SCI models is the most important factor for evaluating the extent of injury and treatment efficacy. It is directly related to the extent of neuronal damage in the gray matter at the injury site, the loss of ascending and descending axons in the white matter, and the reorganization of the remaining nervous system [57, 58]. Sedy et al. categorized the behavioral tests as: locomotor tests (testing the locomotor apparatus of the animal), motor tests (analyzing the strength, coordination and other abilities of the skeletal muscles), sensory tests (evaluating proprioception, touch, pain or temperature sensing), sensory–motor tests (testing the proper connection between the sensory and motor systems), autonomic tests (evaluating the function of the sympathetic and parasympathetic systems), and reflex-

Rahimi-Movaghar et al. showed usefulness of the tail-flick reflex in the prognosis of functional recovery in paraplegic rats [59]. Although there has been an abundant interest in locomotion in animal studies, the connection between locomotion and spinal cord integrity at the site of injury in the animal is not at all easy. In particular, behavioral measurements in the context of lateral or dorsal hemisection are even more difficult [2]. Table 2 shows recommended testing

and humans [51].

**7. Outcome assessments**

response based tests [58]. (Table 1)

methods for SCI models.

**• Behavior**


Among rodents, the majority of genetic studies, especially those involving disease, have employed mice, not only because their genomes are so similar to that of humans, but also because of their availability, ease of handling, high reproductive rates, and relatively low cost of use [30, 54, 55]. Using mice with a knockout of a target molecule has become the goldstandard for functional testing, and Cre-Lox technology along with increasing numbers of transgenic mice have provided greater temporo-spatial control of the knockout strategy that has proven invaluable for providing mechanistic insights into the cellular and molecular processes of axon regeneration [51].

**•** *Cat:* Use of cats can clarify the histopathologic features of acute and chronic stages of SCI. Their larger size allows implementation of more intensive therapeutic regimens, such as implantation of electrical stimulators, than is possible when smaller animal models are studied [52]. Cats have been a popular model for spinal cord electrophysiologists [56].


#### **7. Outcome assessments**

#### **• Behavior**

**6. Species of animals used**

216 Topics in Paraplegia

novel strategies to human SCI.

quadrupeds not bipeds.

processes of axon regeneration [51].

Rodents are the most common type of mammal employed in SCI experimental studies, and widespread research have been conducted using rats, mice, gerbils, guinea pigs, and hamsters [1]. Other animal experiments include cats, non-human primates, goats, pigs, and dogs [1, 4, 35, 44-49]. Of course, larger mammals such as cats, dogs, or pigs are also used but very rarely and are less experienced models based in SCI research, requiring expensive after care and housing as well as stringent ethical considerations [6, 37]. Other models include invertebrates, such as eels [50], whose unique regenerative capacities have been studied in efforts to apply

**•** *Rat:* Rat models are most widely used to study SCI. They are inexpensive, friendly, easy to care for, and can be studied in large numbers. They have a well understood anato‐ my and few surgical infections. There are also well-established functional analysis techniques in rats. Early mortality of them is not costly [37, 38]. In addition, rats develop large fluid-filled cystic cavities at the injury site, similar to the human pathology. Therefore those are preferable for studies where mimicking the human pathology is important, including preclinical studies that focus on the efficacy of novel cellular and/or pharmacological therapies [51]. Rats can be used when the size is of less importance [52]. The corticospinal tract of rat is mostly dorsal. As two disadvantages of the rat models, the corticospinal tract lesions would not significantly create disability, and rats are

**•** *Mouse:* In SCI research, mouse models have also been implemented increasingly, but the small working size prohibits many surgical maneuvers and device implantations [37, 38]. The injury site in mice is densely packed with cells and actually decreases in size over time (that do not have a cyst). Thus to gain mechanistic insights into the basic cellular and

Among rodents, the majority of genetic studies, especially those involving disease, have employed mice, not only because their genomes are so similar to that of humans, but also because of their availability, ease of handling, high reproductive rates, and relatively low cost of use [30, 54, 55]. Using mice with a knockout of a target molecule has become the goldstandard for functional testing, and Cre-Lox technology along with increasing numbers of transgenic mice have provided greater temporo-spatial control of the knockout strategy that has proven invaluable for providing mechanistic insights into the cellular and molecular

**•** *Cat:* Use of cats can clarify the histopathologic features of acute and chronic stages of SCI. Their larger size allows implementation of more intensive therapeutic regimens, such as implantation of electrical stimulators, than is possible when smaller animal models are studied [52]. Cats have been a popular model for spinal cord electrophysiologists [56].

molecular biology of SCI, mouse models may have more to offer [51, 53].

Behavioral outcome in experimental SCI models is the most important factor for evaluating the extent of injury and treatment efficacy. It is directly related to the extent of neuronal damage in the gray matter at the injury site, the loss of ascending and descending axons in the white matter, and the reorganization of the remaining nervous system [57, 58]. Sedy et al. categorized the behavioral tests as: locomotor tests (testing the locomotor apparatus of the animal), motor tests (analyzing the strength, coordination and other abilities of the skeletal muscles), sensory tests (evaluating proprioception, touch, pain or temperature sensing), sensory–motor tests (testing the proper connection between the sensory and motor systems), autonomic tests (evaluating the function of the sympathetic and parasympathetic systems), and reflexresponse based tests [58]. (Table 1)

Rahimi-Movaghar et al. showed usefulness of the tail-flick reflex in the prognosis of functional recovery in paraplegic rats [59]. Although there has been an abundant interest in locomotion in animal studies, the connection between locomotion and spinal cord integrity at the site of injury in the animal is not at all easy. In particular, behavioral measurements in the context of lateral or dorsal hemisection are even more difficult [2]. Table 2 shows recommended testing methods for SCI models.


**Behavioral tests Tests Reflects**

Narrow beam Balance

Grooming Sensory–motor

Foot slip Sensory–motor

Grid walking Sensory–motor

coordination

coordination

**Sensory–motor**

**Reflex responsebased tests**

**Autonomic tests**

\* Modified by: Mahdi Sharif-Alhoseini

**Table 1.** Main behavioral methods for testing SCI models\*

**tests**

**Lesion severity**

**Moderate**

**Severe**

**Mild**

Cold sensitivity-based Temperature Simple False positivity

Von Frey filaments Mechanical allodynia Simple Low sensitivity

Paw compression Pain Simple, cheap High chance of

Withdrawal reflexes Reflex Simple Low sensitivity

Rope walk testing Balance Simple, cheap Low sensitivity

Toe spread reflex Reflex Simple, cheap Low sensitivity

Contact placing response Reflex Simple, cheap False positivity

Righting reflex Reflex Simple, cheap Low sensitivity

Non-contact erection Erection Unique data Low sensitivity

Ex copula erection Erection Unique data Subjectivity

Mating Erection Unique data Subjectivity

Telemetric monitoring Micturition erection Precise Equipment

Autonomic dysreflexia Autonomic dysreflexia Unique data Equipment

[58]

**Pros Cons**

Animal Models in Traumatic Spinal Cord Injury

http://dx.doi.org/10.5772/57189

219

Uncovers discrete changes

Uncovers discrete changes

Uncovers discrete changes

connection Simple, cheap Subjectivity

mistakes

Requires training

Requires training

False-positives or negatives


\* Modified by: Mahdi Sharif-Alhoseini

**Behavioral tests Tests Reflects**

**Locomotor tests**

218 Topics in Paraplegia

**Motor tests**

**Lesion severity**

**Moderate**

**Severe**

**Mild**

Primary open-field Locomotion Simple, cheap Low sensitivity

BBB Locomotion Simple, cheap Subjective

Open-field activity Locomotion Unique data Depends on

Automated walkway Locomotion Precise Equipment

Kinematic analysis Locomotion Detailed Equipment

Footprint analysis Motor coordination Precise

Swim Swimming ability Spontaneous

Inclined plane Muscle strength Simple, cheap

Limb hanging Grasping Unique data

**Sensory tests** Hot plate-based Temperature Simple Risk of injury

Limb grip strength Muscle strength Precise Equipment

Thoracolumbar height Weight support

Forelimb asymmetry Paw preference

Rearing Paw preference

Food pellet reaching Motor coordination

Eshkol–Wachmann

notation

**Pros Cons**

Examines only

characteristic

Sensitive to chronic deficits

Sensitive to selective limb

Fine motor

use

locomotion Subjective

one

Locomotion Detailed Requires training

motivation

Environmentdependent

Equipment

of scientist

Not standard among laboratories

Not for severe injuries

Not for severe injuries

Not for severe injuries

function test Food deprivation

**Table 1.** Main behavioral methods for testing SCI models\* [58]


[67]. It should be done under anesthesia and mechanically ventilation [58]. After the stimula‐ tion of the limb electrodes, a signal in the somatosensory cortex and/or subcortical sensory areas can be recorded. This method makes it possible to distinguish between the recovery of

Animal Models in Traumatic Spinal Cord Injury

http://dx.doi.org/10.5772/57189

221

*Magnetic Resonance Imaging (MRI):* MRI findings of parenchymal hemorrhage/contusion, edema, and spinal cord disruption in acute and subacute SCI may contribute to the under‐

*MRI-Diffusion Weighted Imaging (MRI-DWI):* It is an MRI-based imaging modality that deter‐ mines the free diffusion of water molecules, enabling the recognition of imaging information beyond the resolution of conventional MRI methods [69]. MRI-DWI can be utilized to measure

*Computerized Tomography (CT):* The assessment of the bone loss following SCI in an animal

Recently, several studies used neuroanatomical tracing procedures to study axonal remodel‐

Histological outcome measures, including sparing at the lesion epicenter, sparing throughout the extent of the lesion, quantification of myelin loss rostral and caudal to the lesion, and motor

Hematoxylin and Eosin (H&E) is useful as a general structural stain in most tissues. But the

Cresyl violet stains both neurons and glia. It bonds well with acidic parts of cells such as ribosomes, nuclei and nucleoli and demonstrates the nissl substance. It stains cell bodies a

Luxol Fast Blue gives particularly good delineation of nerve tracts in the CNS. It is probably

Osmium tetroxide is both a stain and a fixative. While it's primarily used these days as a fixative in electron microscopy, since it binds to lipids strongly, it's particularly well suited to reveal

Eriochrome cyanine (EC) staining protocol for differentiation of white matter and cell bodies

To choose an animal model, the proposed aim of the study must precisely be noted. The researchers involved in scientific work with animals should know the ethical standards in

standing of severity of injury and prognosis for neurological improvement [67].

response to various cellular therapy interventions after experimental SCI [67].

neuron counts, are demonstrated via staining sections of the spinal cord [21].

one of the most popular stains for the demonstration of normal myelin.

is used to calculate the amount of spared tissue in sections of injured cords.

high lipid content of nervous tissue makes it less suited to H&E than most others.

model could be done by high-resolution CT images [70].

ing after cell transplantation in experimental SCI models [71-76].

sensory and motor function [68].

**• Neuroanatomical tracing**

the details of myelin in nerves.

**8. Considerations**

**• Histology**

blue/violet.

**Table 2.** Recommended testing methods for SCI models [58].

#### **• Electrophysiology**

Electrophysiological assessments via the evoked potentials are useful to survey the neural substrates underlying deficits and functional recovery. They are also used to examine neural pathway integrity [58, 60].

*Somatosensory evoked potentials* (SSEP) are valuable for the assessment of sensory spinal axon conduction. They involve electrical stimulation of the paws with electrodes temporarily inserted into them, and the recording of evoked potentials from electrodes previously implanted in the cranium over the somatosensory cortex [61].

*Magnetically evoked inter-enlargement responses* (MIER) are helpful for the evaluation of pro‐ priospinal conduction. The MIER procedure involves noninvasive magnetic stimulation at the animal's hip or knee and the recording of evoked potentials with EMG electrodes temporarily inserted into forelimb and masseter muscles [62].

*Motor evoked potentials* (MEP) assess supraspinal axon conduction with EMG electrodes temporarily inserted into hindlimb muscles [63]. The MEP offers a precious insight into the physiological status of motor tracts within the spinal cord and is appropriate to animal studies. It is seen as complementary to SSEP monitoring rather than an alternative for it [64].

All evoked potential methods take a few minutes and cause only slight pain and distress and so could be done without anesthesia. But there are the restricted information content, and the need for rigorous electrophysiological interpretation of the resulting signals [64].

*Electromyography (EMG):* EMG can be elicited both by intramedullary manipulation and rapidly applied transaxial spinal cord compression. Presumably, rapid deformation of spinal motor tracts generates descending volleys which can bring to firing threshold lumbar motor neurons [65]. It can also be used to survey autonomic dysreflexia [66].

#### **• Neuroimaging**

*Functional magnetic resonance imaging(fMRI):* fMRI is an accurate but challenging technique which could measure the anatomic functional/metabolic correlates of sensory-motor activities [67]. It should be done under anesthesia and mechanically ventilation [58]. After the stimula‐ tion of the limb electrodes, a signal in the somatosensory cortex and/or subcortical sensory areas can be recorded. This method makes it possible to distinguish between the recovery of sensory and motor function [68].

*Magnetic Resonance Imaging (MRI):* MRI findings of parenchymal hemorrhage/contusion, edema, and spinal cord disruption in acute and subacute SCI may contribute to the under‐ standing of severity of injury and prognosis for neurological improvement [67].

*MRI-Diffusion Weighted Imaging (MRI-DWI):* It is an MRI-based imaging modality that deter‐ mines the free diffusion of water molecules, enabling the recognition of imaging information beyond the resolution of conventional MRI methods [69]. MRI-DWI can be utilized to measure response to various cellular therapy interventions after experimental SCI [67].

*Computerized Tomography (CT):* The assessment of the bone loss following SCI in an animal model could be done by high-resolution CT images [70].

#### **• Neuroanatomical tracing**

Recently, several studies used neuroanatomical tracing procedures to study axonal remodel‐ ing after cell transplantation in experimental SCI models [71-76].

#### **• Histology**

**Level of injury First choice Second choice Third choice Cervical** Forelimb asymmetry Footprint analysis BBB

Compression BBB Hot plate Inclined plane Contusion BBB Electrophysiology Von Frey, Hot plate Transection BBB Electrophysiology Kinematic analysis Hemisection BBB Electrophysiology Hot plate, Von Frey Excitotoxic Hot plate Cold testing Von Frey

Ischemic BBB Electrophysiology Inclined plane, Hot plate

**Other** BBB Electrophysiology Hot plate, Grid walk

Electrophysiological assessments via the evoked potentials are useful to survey the neural substrates underlying deficits and functional recovery. They are also used to examine neural

*Somatosensory evoked potentials* (SSEP) are valuable for the assessment of sensory spinal axon conduction. They involve electrical stimulation of the paws with electrodes temporarily inserted into them, and the recording of evoked potentials from electrodes previously

*Magnetically evoked inter-enlargement responses* (MIER) are helpful for the evaluation of pro‐ priospinal conduction. The MIER procedure involves noninvasive magnetic stimulation at the animal's hip or knee and the recording of evoked potentials with EMG electrodes temporarily

*Motor evoked potentials* (MEP) assess supraspinal axon conduction with EMG electrodes temporarily inserted into hindlimb muscles [63]. The MEP offers a precious insight into the physiological status of motor tracts within the spinal cord and is appropriate to animal studies.

All evoked potential methods take a few minutes and cause only slight pain and distress and so could be done without anesthesia. But there are the restricted information content, and the

*Electromyography (EMG):* EMG can be elicited both by intramedullary manipulation and rapidly applied transaxial spinal cord compression. Presumably, rapid deformation of spinal motor tracts generates descending volleys which can bring to firing threshold lumbar motor

*Functional magnetic resonance imaging(fMRI):* fMRI is an accurate but challenging technique which could measure the anatomic functional/metabolic correlates of sensory-motor activities

It is seen as complementary to SSEP monitoring rather than an alternative for it [64].

need for rigorous electrophysiological interpretation of the resulting signals [64].

neurons [65]. It can also be used to survey autonomic dysreflexia [66].

**Thoracic**

220 Topics in Paraplegia

**• Electrophysiology**

**• Neuroimaging**

pathway integrity [58, 60].

**Table 2.** Recommended testing methods for SCI models [58].

implanted in the cranium over the somatosensory cortex [61].

inserted into forelimb and masseter muscles [62].

Histological outcome measures, including sparing at the lesion epicenter, sparing throughout the extent of the lesion, quantification of myelin loss rostral and caudal to the lesion, and motor neuron counts, are demonstrated via staining sections of the spinal cord [21].

Hematoxylin and Eosin (H&E) is useful as a general structural stain in most tissues. But the high lipid content of nervous tissue makes it less suited to H&E than most others.

Cresyl violet stains both neurons and glia. It bonds well with acidic parts of cells such as ribosomes, nuclei and nucleoli and demonstrates the nissl substance. It stains cell bodies a blue/violet.

Luxol Fast Blue gives particularly good delineation of nerve tracts in the CNS. It is probably one of the most popular stains for the demonstration of normal myelin.

Osmium tetroxide is both a stain and a fixative. While it's primarily used these days as a fixative in electron microscopy, since it binds to lipids strongly, it's particularly well suited to reveal the details of myelin in nerves.

Eriochrome cyanine (EC) staining protocol for differentiation of white matter and cell bodies is used to calculate the amount of spared tissue in sections of injured cords.

#### **8. Considerations**

To choose an animal model, the proposed aim of the study must precisely be noted. The researchers involved in scientific work with animals should know the ethical standards in animal experiments and investigate what animals are appropriate for each area of study in their models. Reproducible experimental SCI requires suitable training, animal care, experi‐ ence with animal spine surgery, and proper surgical equipment. A standard housing envi‐ ronment with ad libitum access to food and water is a necessity for animal experiments. Pretraining and habituation of animals are important. When a behavioral testing is planned, animals must be trained inadequate sessions pre-operatively. Anesthetizing, surgery, and/or sacrificing have to been performed based on confirmed methods, attentively. All animals should be inspected regularly for wound healing, weight loss, dehydration, infection, auto‐ phagia and any discomfort [77]. Animal models, particularly complete SCI ones, need to serious care including preparation of supportive fluids, analgesia, and antibiotics, and also continuous bladder and bowel care. Appropriate veterinary care was provided as needed. All behavioral, histological, etc. analysis should be precisely selected before beginning a study and conducted by personnel blind to groups of study.

**References**

[1] Simmons D. The use of animal models in studying genetic disease: transgenesis and

Animal Models in Traumatic Spinal Cord Injury

http://dx.doi.org/10.5772/57189

223

[2] Blight AR. Animal models of spinal cord injury. Topics in Spinal Cord Injury Reha‐

[3] Blight AR, Tuszynski MH. Clinical trials in spinal cord injury. J Neurotrauma

[4] Nout Y, Rosenzweig E, Brock J, Strand S, Moseanko R, Hawbecker S, et al. Animal Models of Neurologic Disorders: A Nonhuman Primate Model of Spinal Cord Injury.

[5] Rahimi-Movaghar V, Rasouli MR, Smith H, Vaccaro AR. An evidence-based spinal cord injury decompression in experimental animals and human studies. In: Berkov‐ sky TC. (ed.) Handbook of Spinal Cord Injuries: Types, Treatments, and Prognosis.

[6] Kundi S, Bicknell R, Ahmed Z. Spinal cord injury: Current mammalian models.

[7] Vijayaprakash K, Sridharan N. An experimental spinal cord injury rat model using customized impact device: A cost-effective approach. J Pharmacol Pharmacother

[8] Amako T. Surgery of spinal injuries due to impact: II experimental study. J Jap Surg

[9] Tarlov I, Klinger H, Vitale S. Spinal cord compression studies: I. experimental techni‐ ques to produce acute and gradual compression. Archives of Neurology and Psy‐

[10] Eidelberg E, Staten E, Watkins J, McGraw D, McFadden C. A model of spinal cord

[11] Braga-Silva J, Gehlen D, Roman JA, Machado DC, Costa JCd, Faúndez M, et al. Ex‐ perimental model of spinal cord injury in rats with a device for local therapeutic

[12] Rivlin AS, Tator CH. Effect of duration of acute spinal cord compression in a new

[13] Bresnahan JC, Beattie MS, Todd FD, 3rd, Noyes DH. A behavioral and anatomical analysis of spinal cord injury produced by a feedback-controlled impaction device.

induced mutation. Nature Education 2008;1(1).

bilitation 2000;6(2) 1-13.

Neurotherapeutics 2012;9(2) 380-92.

NewYork: NOVA; 2009. p635-63

American Journal of Neuroscience 2013;4(1) 1.

2006;23(3-4) 586-93.

2013;4(3) 211-3.

Soc 1936;37 1843-74.

chiatry 1953;70(6) 813.

injury. Surgical neurology 1976;6(1) 35.

Exp Neurol 1987;95(3) 548-70.

agents access. Acta Ortopédica Brasileira 2007;15(3) 155-7.

acute cord injury model in the rat. Surg Neurol 197810(1) 38-43.

#### **9. Conclusion and future perspectives**

Animal models of SCI have confirmed to be helpful for the development of experimental therapies, and certainly will continue to play an essential role in the studies related to SCI. They give researchers an opportunity to discover the characteristic pattern of cell death and sparing, and measurement of any neuroprotection, regeneration, collateral sprouting, demye‐ lination, and recovery of locomotor or other deficits. All injury paradigms are useful, but differ in the information that can be gained. The contusion models better simulate the biomechanics and neuropathology of human injury. The transection models, either completely or partially, are valuable for investigating the anatomic regeneration. The conclusions of rodent studies should examine in other animal models to survey their biological responses. In parallel, controlling and monitoring the injury mechanism within the surgical field, and evaluation of behavioral and histological outcomes have to be enhanced by applying technological im‐ provements. Finally, more experimental studies should be designed to quantify neuronal damage after ischemic SCI.

#### **Author details**

Mahdi Sharif-Alhoseini1 and Vafa Rahimi-Movaghar1,2\*

\*Address all correspondence to: v\_rahimi@sina.tums.ac.ir, v\_rahimi@yahoo.com

1 Sina Trauma and Surgery Research Center, Tehran University of Medical Sciences, Tehran, Iran

2 Research Centre for Neural Repair, University of Tehran, Tehran, Iran

#### **References**

animal experiments and investigate what animals are appropriate for each area of study in their models. Reproducible experimental SCI requires suitable training, animal care, experi‐ ence with animal spine surgery, and proper surgical equipment. A standard housing envi‐ ronment with ad libitum access to food and water is a necessity for animal experiments. Pretraining and habituation of animals are important. When a behavioral testing is planned, animals must be trained inadequate sessions pre-operatively. Anesthetizing, surgery, and/or sacrificing have to been performed based on confirmed methods, attentively. All animals should be inspected regularly for wound healing, weight loss, dehydration, infection, auto‐ phagia and any discomfort [77]. Animal models, particularly complete SCI ones, need to serious care including preparation of supportive fluids, analgesia, and antibiotics, and also continuous bladder and bowel care. Appropriate veterinary care was provided as needed. All behavioral, histological, etc. analysis should be precisely selected before beginning a study and

Animal models of SCI have confirmed to be helpful for the development of experimental therapies, and certainly will continue to play an essential role in the studies related to SCI. They give researchers an opportunity to discover the characteristic pattern of cell death and sparing, and measurement of any neuroprotection, regeneration, collateral sprouting, demye‐ lination, and recovery of locomotor or other deficits. All injury paradigms are useful, but differ in the information that can be gained. The contusion models better simulate the biomechanics and neuropathology of human injury. The transection models, either completely or partially, are valuable for investigating the anatomic regeneration. The conclusions of rodent studies should examine in other animal models to survey their biological responses. In parallel, controlling and monitoring the injury mechanism within the surgical field, and evaluation of behavioral and histological outcomes have to be enhanced by applying technological im‐ provements. Finally, more experimental studies should be designed to quantify neuronal

and Vafa Rahimi-Movaghar1,2\*

\*Address all correspondence to: v\_rahimi@sina.tums.ac.ir, v\_rahimi@yahoo.com

2 Research Centre for Neural Repair, University of Tehran, Tehran, Iran

1 Sina Trauma and Surgery Research Center, Tehran University of Medical Sciences, Tehran,

conducted by personnel blind to groups of study.

**9. Conclusion and future perspectives**

damage after ischemic SCI.

**Author details**

222 Topics in Paraplegia

Iran

Mahdi Sharif-Alhoseini1


[14] Kwon BK, Oxland TR, Tetzlaff W. Animal models used in spinal cord regeneration research. Spine (Phila Pa 1976) 2002;27(14) 1504-10.

[27] Ohta K, Fujimura Y, Nakamura M, Watanabe M, Yato Y. Experimental study on MRI evaluation of the course of cervical spinal cord injury. Spinal Cord 1999;37(8) 580-4.

Animal Models in Traumatic Spinal Cord Injury

http://dx.doi.org/10.5772/57189

225

[28] Nobunaga AI, Go BK, Karunas RB. Recent demographic and injury trends in people served by the model spinal cord injury care systems. Archives of Physical Medicine

[29] Jakeman LB, Guan Z, Wei P, Ponnappan R, Dzwonczyk R, Popovich PG, et al. Trau‐ matic spinal cord injury produced by controlled contusion in mouse. J Neurotrauma

[30] Stokes BT, Jakeman LB. Experimental modelling of human spinal cord injury: a mod‐ el that crosses the species barrier and mimics the spectrum of human cytopathology.

[31] Scheff SW, Rabchevsky AG, Fugaccia I, Main JA, Lumpp JE, Jr. Experimental model‐ ing of spinal cord injury: characterization of a force-defined injury device. J Neuro‐

[32] Brösamle C, Huber AB. Cracking the black box–and putting it back together again: Animal models of spinal cord injury. Drug Discovery Today: Disease Models

[33] Rahimi-Movaghar V, Yazdi A, Karimi M, Mohammadi M, Firouzi M, Zanjani LO, et al. Effect of decompression on complete spinal cord injury in rats. Int J Neurosci

[34] Jazayeri SB, Firouzi M, Abdollah Zadegan S, Saeedi N, Pirouz E, Nategh M, et al. The effect of timing of decompression on neurologic recovery and histopathologic find‐ ings after spinal cord compression in a rat model. Acta Med Iran 2013;51(7) 431-7. [35] Lim JH, Jung CS, Byeon YE, Kim WH, Yoon JH, Kang KS, et al. Establishment of a canine spinal cord injury model induced by epidural balloon compression. J Vet Sci

[36] Dimar JRI, Glassman SD, Raque GH, Zhang YP, Shields CB. The Influence of Spinal Canal Narrowing and Timing of Decompression on Neurologic Recovery After Spi‐

[37] Talac R, Friedman JA, Moore MJ, Lu L, Jabbari E, Windebank AJ, et al. Animal mod‐ els of spinal cord injury for evaluation of tissue engineering treatment strategies. Bio‐

[38] Nakae A, Nakai K, Yano K, Hosokawa K, Shibata M, Mashimo T. The animal model of spinal cord injury as an experimental pain model. J Biomed Biotechnol 2011; doi:

nal Cord Contusion in a Rat Model. Spine 1999;24(16) 1623.

and Rehabilitation 1999;80(11) 1372-82.

2000;17(4) 299-319.

Spinal Cord 2002;40(3) 101-9.

trauma 2003;20(2) 179-93.

2007;3(4) 341-7.

2007;8(1) 89-94.

materials 2004;25(9) 1505-10.

10.1155/2011/939023

2008;118(10) 1359-73.


[27] Ohta K, Fujimura Y, Nakamura M, Watanabe M, Yato Y. Experimental study on MRI evaluation of the course of cervical spinal cord injury. Spinal Cord 1999;37(8) 580-4.

[14] Kwon BK, Oxland TR, Tetzlaff W. Animal models used in spinal cord regeneration

[15] Agrawal G, Kerr C, Thakor NV, All AH. Characterization of Graded MASCIS Contu‐ sion Spinal Cord Injury using Somatosensory Evoked Potentials. Spine 2010;35(11)

[16] Lang-Lazdunski L, Matsushita K, Hirt L, Waeber C, Vonsattel J-PG, Moskowitz MA. Spinal Cord Ischemia: Development of a Model in the Mouse. Stroke 2000;31(1)

[17] Gaviria M, Haton H, Sandillon F, Privat A. A mouse model of acute ischemic spinal

[18] Nouri M, Rasouli M, Shafiei S, Tavasoly A, Dehpour AR, Rahimi-Movaghar V. Does abdominal aorta clamping, as a method of spinal ischemia in rats, really work? Surg

[19] Rasouli MR, Rahimi-Movaghar V, Vaccaro AR. Re: Usul H, Arslan E, Cansever T, et al. Effects of clotrimazole on experimental spinal cord ischemia/reperfusion injury in

[20] Rahimi-Movaghar V, Sayyah MK, Akbari H, Khorramirouz R, Rasouli MR, Moradi-Lakeh M, et al. Epidemiology of Traumatic Spinal Cord Injury in Developing Coun‐

[21] Gensel JC, Tovar CA, Hamers FP, Deibert RJ, Beattie MS, Bresnahan JC. Behavioral and histological characterization of unilateral cervical spinal cord contusion injury in

[22] Lane MA, Fuller DD, White TE, Reier PJ. Respiratory neuroplasticity and cervical spinal cord injury: translational perspectives. Trends in Neurosciences 2008;31(10)

[23] Goshgarian HG, Koistinen JM, Schmidt ER. Cell death and changes in the retrograde transport of horseradish peroxidase in rubrospinal neurons following spinal cord

[24] Soblosky JS, Song J-H, Dinh DH. Graded unilateral cervical spinal cord injury in the rat: evaluation of forelimb recovery and histological effects. Behavioural Brain Re‐

[25] Rahimi-Movaghar V. Clinical trials for the treatment of spinal cord injury: cervical

[26] Pearse DD, Lo TP, Jr., Cho KS, Lynch MP, Garg MS, Marcillo AE, et al. Histopatho‐ logical and behavioral characterization of a novel cervical spinal cord displacement

and lumbar enlargements versus thoracic area. Brain 2009;132(7) e115.

tries: A Systematic Review. Neuroepidemiology 2013;41(2) 65-85.

hemisection in the adult rat. J Comp Neurol 1983;214(3) 251-7.

contusion injury in the rat. J Neurotrauma 2005;22(6) 680-702.

research. Spine (Phila Pa 1976) 2002;27(14) 1504-10.

cord injury. J Neurotrauma 2002;19(2) 205-21.

rats. Spine (Phila Pa 1976) 2009;34(17) 1884.

rats. J Neurotrauma 2006 Jan;23(1) 36-54.

1122.

224 Topics in Paraplegia

208-13.

538-47.

search 2001;119(1) 1-13.

Neurol 2006;66(3) 332-3.


[39] Steward O, Zheng B, Tessier-Lavigne M. False resurrections: Distinguishing regener‐ ated from spared axons in the injured central nervous system. The Journal of Compa‐ rative Neurology 2003;459(1) 1-8.

[52] Khan T, Havey RM, Sayers ST, Patwardhan A, King WW. Animal models of spinal

Animal Models in Traumatic Spinal Cord Injury

http://dx.doi.org/10.5772/57189

227

[53] Metz GA, Curt A, van de Meent H, Klusman I, Schwab ME, Dietz V. Validation of the weight-drop contusion model in rats: a comparative study of human spinal cord

[54] Jakeman L, Ma M, Stokes B. Considering the Use of Transgenic Mice in Spinal Cord

[55] Jakeman L, Ma M, Stokes B. Considering the use of transgenic animals in spinal cord injury research. In: Marwah J, Dixon E, Banik NL. (ed.) Traumatic CNS Injury. Prom‐

[56] Rossignol S, Bouyer L, Langlet C, Barthélemy D, Chau C, Giroux N, et al. Determi‐ nants of locomotor recovery after spinal injury in the cat. In: Shigemi Mori DGS,

[57] Basso DM. Behavioral testing after spinal cord injury: congruities, complexities, and

[58] Šedý J, Urdzíková L, Jendelová P, Syková E. Methods for behavioral testing of spinal cord injured rats. Neuroscience & Biobehavioral Reviews 2008;32(3) 550-80.

[59] Rahimi-Movaghar V, Yazdi A, Mohammadi M. Usefulness of the tail-flick reflex in the prognosis of functional recovery in paraplegic rats. Surgical neurology 2008;70(3)

[60] Onifer SM, Rabchevsky AG, Scheff SW. Rat Models of Traumatic Spinal Cord Injury

[61] Onifer SM, Zhang YP, Burke DA, Brooks DL, Decker JA, McClure NJ, et al. Adult rat forelimb dysfunction after dorsal cervical spinal cord injury. Experimental Neurolo‐

[62] Beaumont E, Onifer SM, Reed WR, Magnuson DSK. Magnetically evoked inter-en‐ largement response: An assessment of ascending propriospinal fibers following spi‐

[63] Linden RD, Zhang YP, Burke DA, Hunt MA, Harpring JE, Shields CB. Magnetic mo‐ tor evoked potential monitoring in the rat. J Neurosurg 1999;91(2 Suppl) 205-10. [64] Blight AR. Motor evoked potentials in CNS trauma. Cent Nerv Syst Trauma 1986;3(3)

[65] Skinner SA, Transfeldt EE. Electromyography in the detection of mechanically in‐ duced spinal motor tract injury: observations in diverse porcine models. Journal of

Mario W. (ed.) Progress in Brain Research. Elsevier; 2004. p163-72.

cord contusion injuries. Comparative Medicine 1999;49(2) 161-72.

injury. J Neurotrauma 2000;17(1) 1-17.

inent Press; 2001. p180–201.

323-5.

207-14.

gy 2005;192(1) 25-38.

Injury Research. New York: Prominent Press; 2001.

controversies. J Neurotrauma 2004;21(4) 395-404.

to Assess Motor Recovery. ILAR Journal 2007;48(4) 385-95.

nal cord injury. Experimental Neurology 2006;201(2) 428-40.

Neurosurgery: Spine 2009;11(3) 369-74.


[52] Khan T, Havey RM, Sayers ST, Patwardhan A, King WW. Animal models of spinal cord contusion injuries. Comparative Medicine 1999;49(2) 161-72.

[39] Steward O, Zheng B, Tessier-Lavigne M. False resurrections: Distinguishing regener‐ ated from spared axons in the injured central nervous system. The Journal of Compa‐

[40] Watson BD, Prado R, Dietrich WD, Ginsberg MD, Green BA. Photochemically in‐

[41] Piao MS, Lee JK, Jang JW, Kim SH, Kim HS. A mouse model of photochemically in‐

[42] Awad H, Ankeny DP, Guan Z, Wei P, McTigue DM, Popovich PG. A mouse model of ischemic spinal cord injury with delayed paralysis caused by aortic cross-clamp‐

[43] Yezierski RP, Liu S, Ruenes GL, Kajander KJ, Brewer KL. Excitotoxic spinal cord in‐ jury: behavioral and morphological characteristics of a central pain model. PAIN

[44] Zheng YH, Fang Z, Cao P, Zheng T, Sun CH, Lu J, et al. A model of acute compres‐ sion spinal cord injury by a mini-invasive expandable balloon in goats. Zhonghua Yi

[45] Jeffery N, Smith P, Lakatos A, Ibanez C, Ito D, Franklin R. Clinical canine spinal cord injury provides an opportunity to examine the issues in translating laboratory techni‐

[46] Fukuda S, Nakamura T, Kishigami Y, Endo K, Azuma T, Fujikawa T, et al. New ca‐ nine spinal cord injury model free from laminectomy. Brain Res Protoc 2005;14

[47] Moon L, Bunge MB. From animal models to humans: strategies for promoting CNS axon regeneration and recovery of limb function after spinal cord injury. Journal of

[48] Lemon RN, Griffiths J. Comparing the function of the corticospinal system in differ‐ ent species: organizational differences for motor specialization? Muscle & nerve

[49] Courtine G, Bunge MB, Fawcett JW, Grossman RG, Kaas JH, Lemon R, et al. Can ex‐ periments in nonhuman primates expedite the translation of treatments for spinal

[50] Doyle LMF, Stafford PP, Roberts BL. Recovery of locomotion correlated with axonal regeneration after a complete spinal transection in the eel. Neuroscience 2001;107(1)

[51] Lee D-H, Lee J. Animal models of axon regeneration after spinal cord injury. Neuro‐

duced spinal cord injury in the rat. Brain Res 1986;367(1-2) 296-300.

duced spinal cord injury. J Korean Neurosurg Soc 2009;46(5) 479-83.

rative Neurology 2003;459(1) 1-8.

ing. Anesthesiology 2010;113(4) 880-91.

Xue Za Zhi 2012;92(23) 1591-5. [In Chinese]

Neurologic Physical Therapy 2005;29(2) 55-69.

cord injury in humans? Nat Med 2007;13(5) 561-6.

ques into practical therapy. Spinal Cord 2006;44(10) 584-93.

1998;75(1) 141-55.

226 Topics in Paraplegia

171-80.

169-79.

2005;32(3) 261-79.

sci Bull 2013;29(4) 436-44.


[66] Rivas DA, Chancellor MB, Huang B, Salzman SK. Autonomic dysreflexia in a rat model spinal cord injury and the effect of pharmacologic agents. Neurourol Urodyn 1995;14(2) 141-52.

**Chapter 11**

**Mesenchymal Stem Cells in Spinal Cord Injury**

Spinal cord injury is the most devastating neural injury associated with road traffic accidents or fall from height. Due to the compact arrangement of nerve fibers injury often leads to significant deficits. In addition the cellular components of the spinal cord are highly susceptible to injury. Together with the brain the ability of self-repair in comparison to other tissues of the body is poor.[1]. Recently it is noted that the tissue response of the spinal cord to injury is distinctly different from that of brain. The structure, cellular arrangement, vascularity, blood spinal cord barrier, and lack of exposure to inflammatory cells are some of the limiting factors for repair. Added to it receptor and membrane specializations that allow chemical and electrical neuro transmission is prone to major ionic shifts. Though regeneration of spinal cord in teleost fishes and urodele amphibians is established, no adult mammal is able to regenerate. Hence, any insult can result in permanent and significant loss of body function. The therapies currently practiced (surgery, drugs, rehabilitation), are grossly inadequate. The available surgical treatment could only achieve prevention of further injury, maintain and support blood flow, relieve the compression and secure stabilization of spine for early mobilization and rehabilitation. Thus any new treatment for spinal cord injury that enables recovery of function is the need of the hour and could be a significant advancement in clinical care. Biological therapies are now being developed to augment the endogenous repair capabilities. They are aimed at preservation of tissue, promotion of cell survival, activation of neuronal regrowth, reduction in growth inhibition, scarring and cavitation, promotion of myelin repair thus

We have studied and evaluated such applications to attempt spinal cord regeneration. Adult human mesenchymal stem cells were the obvious choice due to their self-renewal property, ease of availability, hypo-immunogenic property, non-teratogenicity, multi-potentiality with

> © 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

N.K. Venkataramanaa and Rakhi Pal

http://dx.doi.org/10.5772/58323

enhancing neuronal circuits.

high genetic stability.

**1. Introduction**

Additional information is available at the end of the chapter


### **Mesenchymal Stem Cells in Spinal Cord Injury**

N.K. Venkataramanaa and Rakhi Pal

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/58323

#### **1. Introduction**

[66] Rivas DA, Chancellor MB, Huang B, Salzman SK. Autonomic dysreflexia in a rat model spinal cord injury and the effect of pharmacologic agents. Neurourol Urodyn

[67] Lammertse D, Dungan D, Dreisbach J, Falci S, Flanders A, Marino R, et al. Neuroi‐ maging in traumatic spinal cord injury: an evidence-based review for clinical practice

[68] Hofstetter CP, Schweinhardt P, Klason T, Olson L, Spenger C. Numb rats walk - a behavioural and fMRI comparison of mild and moderate spinal cord injury. Eur J

[69] Schwartz ED, Hackney DB. Diffusion-weighted MRI and the evaluation of spinal cord axonal integrity following injury and treatment. Experimental Neurology

[70] Voor MJ, Brown EH, Xu Q, Waddell SW, Burden RL, Jr., Burke DA, et al. Bone loss following spinal cord injury in a rat model. J Neurotrauma 2012;29(8) 1676-82.

[71] Wu Y, Satkunendrarajah K, Teng Y, Chow DS, Buttigieg J, Fehlings MG. Delayed post-injury administration of riluzole is neuroprotective in a preclinical rodent model

[72] Seitz A, Aglow E, Heber-Katz E. Recovery from spinal cord injury: a new transection

[73] Pal R, Gopinath C, Rao NM, Banerjee P, Krishnamoorthy V, Venkataramana NK, et al. Functional recovery after transplantation of bone marrow-derived human mesen‐ chymal stromal cells in a rat model of spinal cord injury. Cytotherapy 2010;12(6)

[74] Martin D, Robe P, Franzen R, Delree P, Schoenen J, Stevenaert A, et al. Effects of Schwann cell transplantation in a contusion model of rat spinal cord injury. J Neuro‐

[75] Hodgetts SI, Simmons PJ, Plant GW. Human mesenchymal precursor cells (Stro-1(+)) from spinal cord injury patients improve functional recovery and tissue sparing in an

[76] Abematsu M, Tsujimura K, Yamano M, Saito M, Kohno K, Kohyama J, et al. Neurons derived from transplanted neural stem cells restore disrupted neuronal circuitry in a

[77] Rahimi-Movaghar V, Yazdi A, Saadat S. Saturated Picric Acid Prevents Autophagia and Self-Mutilation in Laboratory Rats. Acta Medica Iranica 2008;46(4) 283-6.

acute spinal cord injury rat model. Cell Transplant 2013;22(3) 393-412.

mouse model of spinal cord injury. J Clin Invest 2010;120(9) 3255-66.

of cervical spinal cord injury. J Neurotrauma 2013;30(6) 441-52.

model in the C57Bl/6 mouse. J Neurosci Res 2002;67(3) 337-45.

and research. J Spinal Cord Med 2007;30(3) 205-14.

1995;14(2) 141-52.

228 Topics in Paraplegia

Neurosci 2003;18(11) 3061-8.

2003;184(2) 570-89.

792-806.

sci Res 1996;45(5) 588-97.

Spinal cord injury is the most devastating neural injury associated with road traffic accidents or fall from height. Due to the compact arrangement of nerve fibers injury often leads to significant deficits. In addition the cellular components of the spinal cord are highly susceptible to injury. Together with the brain the ability of self-repair in comparison to other tissues of the body is poor.[1]. Recently it is noted that the tissue response of the spinal cord to injury is distinctly different from that of brain. The structure, cellular arrangement, vascularity, blood spinal cord barrier, and lack of exposure to inflammatory cells are some of the limiting factors for repair. Added to it receptor and membrane specializations that allow chemical and electrical neuro transmission is prone to major ionic shifts. Though regeneration of spinal cord in teleost fishes and urodele amphibians is established, no adult mammal is able to regenerate. Hence, any insult can result in permanent and significant loss of body function. The therapies currently practiced (surgery, drugs, rehabilitation), are grossly inadequate. The available surgical treatment could only achieve prevention of further injury, maintain and support blood flow, relieve the compression and secure stabilization of spine for early mobilization and rehabilitation. Thus any new treatment for spinal cord injury that enables recovery of function is the need of the hour and could be a significant advancement in clinical care. Biological therapies are now being developed to augment the endogenous repair capabilities. They are aimed at preservation of tissue, promotion of cell survival, activation of neuronal regrowth, reduction in growth inhibition, scarring and cavitation, promotion of myelin repair thus enhancing neuronal circuits.

We have studied and evaluated such applications to attempt spinal cord regeneration. Adult human mesenchymal stem cells were the obvious choice due to their self-renewal property, ease of availability, hypo-immunogenic property, non-teratogenicity, multi-potentiality with high genetic stability.

© 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

#### **2. Incidence**

More than half of all spinal cord injuries occur in the cervical area; and a third of them affect thoracic region. And the rest afflicts lumbar region. Most of the affected ones are young, in their teens or twenties.. The leading causes of acute spinal cord injury include vehicular accidents-41%, violence-22%, falls-21% and sports-8% [2]. Population studies shows the incidence that vary between 2-20%. The official figure is 12% majority being, due to trauma. The total number of people suffering a spinal cord injury in the US alone is 200,000; and 11,000 being added annually. The United Kingdom had over 700 new spinal cord injuries in 2004 (according to the International Campaign for Cures of Spinal Cord Injury).

Spinal cord injury (SCI) is the third most prevalent disease in our country after diabetes and myocardial infarction. More than 12% of the Indian population suffers from the complications associated with spinal cord injury and at least 10,000 are being affected annually [3]. Majority are in the age group 21-36 years, the most productive years of life and 10-12% of severe head injuries are associated with spinal injury. Awareness of this fact is important to protect spine during pre hospital care. The clinical dictum is to suspect spinal injury in all high speed injuries.. Penetrating injuries are relatively rare.

#### **3. Pathophysiology**

The biological response to spinal cord injury is customarily categorized into 3 phases that follows a distinct but somewhat overlapping temporal sequence: acute or primary (seconds to minutes after the injury), secondary or sub-acute (minutes to weeks after the injury), and chronic (months to years after the injury) [4] Table 1. Primary injury is due to direct impact, damaging the neurons, cell membranes, disrupting blood supply, and destabilizing the spinal column. Secondary damage soon follows causing oedema, inflammation and free radical production. A series of molecular changes then produce a cascading effect with liberation of toxins compounding the primary injury. This can continue for few days to even up to six months. [5,6,7] Diverse type of cells and molecules from nervous, immune and vascular system are known to be involved in each phase. Most of the involved cells reside within the spinal cord; also some other cells are recruited through the circulatory system [8]. Hypotension and hypoxia can induce secondary permanent damage.

revealed accumulation of intra-and extracellular fluids in the intercellular space. Ischemia or local anemia has been reported within first few hours after severe trauma using angiographic methods. A major reduction in spinal cord blood flow and lack of perfusion has been observed. This ischemic zone encompasses a large portion of gray matter and surrounding white matter. The main reasons postulated for ischemia are vasospasm (due to vasoconstrictors and vasoactive amines), thrombosis, and platelet aggregation and hemorrhage [9]. By 4th hour, axonal degeneration followed by vesicular disruption in myelin sheaths and ischemia becomes evident. In other patients who do survive the initial injury, hyperaemia and other vascular changes become prominent in 12 to 24 hours. These reactions are mediated through prosta‐ glandins, catecholamines and other agents [10]. At the end of 24 hours, necrosis starts and remains active for another 24 hours which triggers the inflammatory response and disruption of cell membranes resulting in release of intracellular contents of neurons and endothelial cells lining them. This progressive, coagulative and patchy necrosis generally occupies the previous hemorrhagic region and develops infarcts. Increased intracellular calcium influences enzymes, such as phospholipases and phosphatases, to promote the breakdown of the cell membrane. This results in liberation of free fatty acids, which are converted to prostaglandins which

**Table 1.** Illustrates the different phases of spinal cord injury and the cascade of events associated with it.

**Spinal shock (12 hrs –3 weeks)**

functions. 4. Quadriplegia. 5 Anesthesia below affected level. 6. Delayed gastric emptying. 7. Paralytic ileus

1. Neurogenic shock. 2. Respiratory distress. 3. Impaired autonomic **Post spinal shock Reflexes reappear**

http://dx.doi.org/10.5772/58323

231

Mesenchymal Stem Cells in Spinal Cord Injury

reflex

reflex

1. Superficial abdominal

2. Criemasteric reflex 3. Bulbocavernous

4. Withdrawal reflex 5. Beevar sign etc

**Primary events (0-2 hrs)**

Mechanical compression of neural elements by bone fragments, disc material, and ligaments, laceration, shear and distraction

**Secondary events**

1. Toxic metabolites 2. Electrolyte loss

microcirculation

hemorrhage.

cell integrity

peroxidation 10. Demyelination 11. Edema

3. Hypoperfusion of gray matter 4. Loss of autoregulation and

5. Vasospasm, thrombosis and

7. Elevated calcium levels

6. Accumulation of neurotransmitters : like Glutamate leading to excitotoxicity

8. Cell membrane disruption and loss of

9. Cytotoxicity/free radicals/apoptosis/ prostaglandin release and lipid

12. Invasion of glial cells and activation of resident microglial cell population.

**(1-6 hrs)**

The onset of acute phase begins within seconds after an insult to spinal cord injury and is marked by both local and systemic events. Cascade of sequential pathological changes can occur during this phase. Local events such as cord compression, release and accumulation of various neurotransmitters such as catecholamines and excitotoxic amino acids to a toxic level enough to kill neural cells have been postulated to occur within seconds of injury [8]. Soon after trauma, hypotension, shock, low cardiac output and respiratory failure and hypoxia occur due to autonomic system failure. Between 15 to 30 minutes of post trauma, edema in white matter and hemorrhage in gray matter have been reported. Electron microscopic studies


**2. Incidence**

230 Topics in Paraplegia

More than half of all spinal cord injuries occur in the cervical area; and a third of them affect thoracic region. And the rest afflicts lumbar region. Most of the affected ones are young, in their teens or twenties.. The leading causes of acute spinal cord injury include vehicular accidents-41%, violence-22%, falls-21% and sports-8% [2]. Population studies shows the incidence that vary between 2-20%. The official figure is 12% majority being, due to trauma. The total number of people suffering a spinal cord injury in the US alone is 200,000; and 11,000 being added annually. The United Kingdom had over 700 new spinal cord injuries in 2004

Spinal cord injury (SCI) is the third most prevalent disease in our country after diabetes and myocardial infarction. More than 12% of the Indian population suffers from the complications associated with spinal cord injury and at least 10,000 are being affected annually [3]. Majority are in the age group 21-36 years, the most productive years of life and 10-12% of severe head injuries are associated with spinal injury. Awareness of this fact is important to protect spine during pre hospital care. The clinical dictum is to suspect spinal injury in all high speed

The biological response to spinal cord injury is customarily categorized into 3 phases that follows a distinct but somewhat overlapping temporal sequence: acute or primary (seconds to minutes after the injury), secondary or sub-acute (minutes to weeks after the injury), and chronic (months to years after the injury) [4] Table 1. Primary injury is due to direct impact, damaging the neurons, cell membranes, disrupting blood supply, and destabilizing the spinal column. Secondary damage soon follows causing oedema, inflammation and free radical production. A series of molecular changes then produce a cascading effect with liberation of toxins compounding the primary injury. This can continue for few days to even up to six months. [5,6,7] Diverse type of cells and molecules from nervous, immune and vascular system are known to be involved in each phase. Most of the involved cells reside within the spinal cord; also some other cells are recruited through the circulatory system [8]. Hypotension and

The onset of acute phase begins within seconds after an insult to spinal cord injury and is marked by both local and systemic events. Cascade of sequential pathological changes can occur during this phase. Local events such as cord compression, release and accumulation of various neurotransmitters such as catecholamines and excitotoxic amino acids to a toxic level enough to kill neural cells have been postulated to occur within seconds of injury [8]. Soon after trauma, hypotension, shock, low cardiac output and respiratory failure and hypoxia occur due to autonomic system failure. Between 15 to 30 minutes of post trauma, edema in white matter and hemorrhage in gray matter have been reported. Electron microscopic studies

(according to the International Campaign for Cures of Spinal Cord Injury).

injuries.. Penetrating injuries are relatively rare.

hypoxia can induce secondary permanent damage.

**3. Pathophysiology**

**Table 1.** Illustrates the different phases of spinal cord injury and the cascade of events associated with it.

revealed accumulation of intra-and extracellular fluids in the intercellular space. Ischemia or local anemia has been reported within first few hours after severe trauma using angiographic methods. A major reduction in spinal cord blood flow and lack of perfusion has been observed. This ischemic zone encompasses a large portion of gray matter and surrounding white matter. The main reasons postulated for ischemia are vasospasm (due to vasoconstrictors and vasoactive amines), thrombosis, and platelet aggregation and hemorrhage [9]. By 4th hour, axonal degeneration followed by vesicular disruption in myelin sheaths and ischemia becomes evident. In other patients who do survive the initial injury, hyperaemia and other vascular changes become prominent in 12 to 24 hours. These reactions are mediated through prosta‐ glandins, catecholamines and other agents [10]. At the end of 24 hours, necrosis starts and remains active for another 24 hours which triggers the inflammatory response and disruption of cell membranes resulting in release of intracellular contents of neurons and endothelial cells lining them. This progressive, coagulative and patchy necrosis generally occupies the previous hemorrhagic region and develops infarcts. Increased intracellular calcium influences enzymes, such as phospholipases and phosphatases, to promote the breakdown of the cell membrane. This results in liberation of free fatty acids, which are converted to prostaglandins which further increases the constriction of the blood vessels (vasospasm), which in turn contributes to final cell death [11].

Finally the reactive astrogliosis itself hinders the axonal regrowth and the functional recovery

Mesenchymal Stem Cells in Spinal Cord Injury

http://dx.doi.org/10.5772/58323

233

Cord tissue comprises of several components with variable sensitivity to injury.[1].Injury often causes cavitation of epicenter due to cell death, ischemia, mechanical injury, exitotoxicity and neuro inflammation. This cavity can enlarge extending the injury up and down. In addition it becomes a physical barrier for regeneration and cell transmission and cell migration. Body attempts to contain the injury and promote healing resulting in gliosis. This astrocytic gliosis becomes an impediment to the growth of axon described by Raymon Y Cajal in 1928 [1]. These gliotic cells also secrete inhibitory molecules for the axonal growth and connectivity. Inflam‐ mation slows down the initial angiogenesis response and oligodendrocytes (secrete Nogo molecules), glycoproteins Semaphoring 4D and Epherin B3,also have been shown to have

Techniques now have been developed that allow researchers to isolate and grow popula‐ tions of neurons to investigate the effects of specific proteins and molecules on neuronal injury and repair. Neurons can be grown in isolation or with glial cells such as oligodendro‐ cytes or Schwann cells to study the processes of axonal outgrowth and myelination using DNA or protein analysis. Furthermore, the elucidation of the signaling pathways responsi‐ ble for this switch in response may lead to the discovery of a strategy for enhancing axon

Often, in vitro assays are tested along with animal models which allow better understanding of the effects detected in vitro and to be validated in a more complex system. The best studied example includes chondroitin sulfate proteoglycans, a potent inhibitor of neurite outgrowth in *in vitro* experiments. Analysis with animal models demonstrated that the levels of these proteoglycans are enhanced, or up-regulated, during central nervous system (CNS) injury and led to the development of a strategy to break down these substances and promote the regrowth

No single animal model has dominated for research in this area. Two broad classes of models have accounted for the great majority of studies. Both involve surgical exposure of the cord. Most commonly used models are transection or partial injuries for detailed studies of regeneration and experimental contusion and compression. Allen's weight drop model, the oldest method in use and produced by dropping a known weight onto the dorsal side of the exposed spinal cord. This is mainly to address the early processes of

of the injured spinal cord [22].

inhibitory role.[21,22,23]

regeneration.

**4.1. Molecular, genetic, and in vitro tools**

**4. Tools for assessing spinal cord injury and repair**

of axons in the intact rat spinal cord after an injury [21].

**4.2. Animal models for spinal cord injury**

**3.1. Impediments for regeneration**

The secondary mechanisms are still ill understood. In literature( shows) there are approxi‐ mately 25 well established secondary injury mechanisms are described [12, 13]. Secon‐ dary phase sets in minutes and lasts from days to months. Some classical examples of secondary injury mechanisms are continuation of events from the acute phase as outlined by Charles. They are vasospasm, cell death from direct insult, ischemia, edema, derange‐ ments in ionic homeostasis and accumulation of neurotransmitters. In addition some novel features, marks the secondary phase such as free-radical production, lipid peroxidation, nitrous oxide excess, conduction block, excess noradrenaline, energy failure and de‐ creased ATP, immune cells invasion and release of cytokines, inflammatory mediated cell death, neurite growth-inhibitory factors (Nogo-A, Rho-A, oligodendrocyte myelin glycopro‐ tein (OMgp) myelin-associated, glycoprotein (MAG), and chondroitin sulfate proteogly‐ cans, central chromatolysis,). vertebral compression / column instability, demyelination of surviving axons, initiation of central cavitation, astroglial scar launch, plasma membrane compromise /permeability, mitochondrial malfunctions and activation of death signals causing apoptosis [8] are the remaining..

The third phase (chronic phase), along with the events in secondary phase, such as demyeli‐ nation, apoptosis, central cavitation, glial scar formation, is marked by the emergence of new types of pathologies both at micro and macro level [8]. At microlevel, death of oligodendro‐ cytes, susceptible to Reactive Oxygen Species (ROS), loss of electrical impulse conduction by axons due to demyelination and altered neurocircuits and alteration of ion channels and receptors occur [9]. At macrolevel, formation of the glial scar represents an attempt by Glial cells to contain the injury site and promote healing. In addition to reactive astrocytes, scar formation also involves oligodendrocyte precursor cells, microglia, and macrophages. The pathobiology of glial scar is due to reactive gliosis and extra cellular matrix (ECM) remodeling [8]. These changes during reactive astrogliosis have the potential to alter astrocyte activities both through gain and loss of functions that could be beneficial as well as detrimental to surrounding neural and non-neural cells [16, 17, and 18].

More than a quarter of spinal cord injured patients develop cavities which eventually lead to Syringomyelia [19]. Pathogenesis of post traumatic syrinx is not clear. Widely accepted theory recognized two steps in the pathogenesis, namely formation of cavity followed by its enlarge‐ ment and extension. Microscopic examination demonstrated gliosis, which is an astrocytic response to adjacent tissue damage, appears as high MRI signals around the syrinx. [20]. The intial cystic lesion results from multiple factors like mechanical damage, local ischemia [19], arterial and venous obstruction, liquifaction of hematoma, by lysosomal and other intracellular enzymes [21].

Beside, chronic phase also initiates number of neuroprotective and regenerative responses.But they are insufficient for regeneration of the nerve root by Schwann cells or oligodendrocytes. Some compensation by spared neurons (sprouting) often with inappropriate connectivity. Finally the reactive astrogliosis itself hinders the axonal regrowth and the functional recovery of the injured spinal cord [22].

#### **3.1. Impediments for regeneration**

further increases the constriction of the blood vessels (vasospasm), which in turn contributes

The secondary mechanisms are still ill understood. In literature( shows) there are approxi‐ mately 25 well established secondary injury mechanisms are described [12, 13]. Secon‐ dary phase sets in minutes and lasts from days to months. Some classical examples of secondary injury mechanisms are continuation of events from the acute phase as outlined by Charles. They are vasospasm, cell death from direct insult, ischemia, edema, derange‐ ments in ionic homeostasis and accumulation of neurotransmitters. In addition some novel features, marks the secondary phase such as free-radical production, lipid peroxidation, nitrous oxide excess, conduction block, excess noradrenaline, energy failure and de‐ creased ATP, immune cells invasion and release of cytokines, inflammatory mediated cell death, neurite growth-inhibitory factors (Nogo-A, Rho-A, oligodendrocyte myelin glycopro‐ tein (OMgp) myelin-associated, glycoprotein (MAG), and chondroitin sulfate proteogly‐ cans, central chromatolysis,). vertebral compression / column instability, demyelination of surviving axons, initiation of central cavitation, astroglial scar launch, plasma membrane compromise /permeability, mitochondrial malfunctions and activation of death signals

The third phase (chronic phase), along with the events in secondary phase, such as demyeli‐ nation, apoptosis, central cavitation, glial scar formation, is marked by the emergence of new types of pathologies both at micro and macro level [8]. At microlevel, death of oligodendro‐ cytes, susceptible to Reactive Oxygen Species (ROS), loss of electrical impulse conduction by axons due to demyelination and altered neurocircuits and alteration of ion channels and receptors occur [9]. At macrolevel, formation of the glial scar represents an attempt by Glial cells to contain the injury site and promote healing. In addition to reactive astrocytes, scar formation also involves oligodendrocyte precursor cells, microglia, and macrophages. The pathobiology of glial scar is due to reactive gliosis and extra cellular matrix (ECM) remodeling [8]. These changes during reactive astrogliosis have the potential to alter astrocyte activities both through gain and loss of functions that could be beneficial as well as detrimental to

More than a quarter of spinal cord injured patients develop cavities which eventually lead to Syringomyelia [19]. Pathogenesis of post traumatic syrinx is not clear. Widely accepted theory recognized two steps in the pathogenesis, namely formation of cavity followed by its enlarge‐ ment and extension. Microscopic examination demonstrated gliosis, which is an astrocytic response to adjacent tissue damage, appears as high MRI signals around the syrinx. [20]. The intial cystic lesion results from multiple factors like mechanical damage, local ischemia [19], arterial and venous obstruction, liquifaction of hematoma, by lysosomal and other intracellular

Beside, chronic phase also initiates number of neuroprotective and regenerative responses.But they are insufficient for regeneration of the nerve root by Schwann cells or oligodendrocytes. Some compensation by spared neurons (sprouting) often with inappropriate connectivity.

to final cell death [11].

232 Topics in Paraplegia

causing apoptosis [8] are the remaining..

surrounding neural and non-neural cells [16, 17, and 18].

enzymes [21].

Cord tissue comprises of several components with variable sensitivity to injury.[1].Injury often causes cavitation of epicenter due to cell death, ischemia, mechanical injury, exitotoxicity and neuro inflammation. This cavity can enlarge extending the injury up and down. In addition it becomes a physical barrier for regeneration and cell transmission and cell migration. Body attempts to contain the injury and promote healing resulting in gliosis. This astrocytic gliosis becomes an impediment to the growth of axon described by Raymon Y Cajal in 1928 [1]. These gliotic cells also secrete inhibitory molecules for the axonal growth and connectivity. Inflam‐ mation slows down the initial angiogenesis response and oligodendrocytes (secrete Nogo molecules), glycoproteins Semaphoring 4D and Epherin B3,also have been shown to have inhibitory role.[21,22,23]

#### **4. Tools for assessing spinal cord injury and repair**

#### **4.1. Molecular, genetic, and in vitro tools**

Techniques now have been developed that allow researchers to isolate and grow popula‐ tions of neurons to investigate the effects of specific proteins and molecules on neuronal injury and repair. Neurons can be grown in isolation or with glial cells such as oligodendro‐ cytes or Schwann cells to study the processes of axonal outgrowth and myelination using DNA or protein analysis. Furthermore, the elucidation of the signaling pathways responsi‐ ble for this switch in response may lead to the discovery of a strategy for enhancing axon regeneration.

Often, in vitro assays are tested along with animal models which allow better understanding of the effects detected in vitro and to be validated in a more complex system. The best studied example includes chondroitin sulfate proteoglycans, a potent inhibitor of neurite outgrowth in *in vitro* experiments. Analysis with animal models demonstrated that the levels of these proteoglycans are enhanced, or up-regulated, during central nervous system (CNS) injury and led to the development of a strategy to break down these substances and promote the regrowth of axons in the intact rat spinal cord after an injury [21].

#### **4.2. Animal models for spinal cord injury**

No single animal model has dominated for research in this area. Two broad classes of models have accounted for the great majority of studies. Both involve surgical exposure of the cord. Most commonly used models are transection or partial injuries for detailed studies of regeneration and experimental contusion and compression. Allen's weight drop model, the oldest method in use and produced by dropping a known weight onto the dorsal side of the exposed spinal cord. This is mainly to address the early processes of injury. Besides the above mentioned category, microlesion formation and transgenic models, [25] Photochemical SCI model, excitotoxic spinal cord injury have also been developed in recent years [26].

categories: neuroprotection, repair/regeneration, enhancing the plasticity and replace/assist

Mesenchymal Stem Cells in Spinal Cord Injury

http://dx.doi.org/10.5772/58323

235

Clinical trials should augment the neurological recovery data with outcome measures designed to assess the functional significance of the neurological recovery. To date more than 70 clinical trials have been done on functional recovery of Spinal Cord Injury with drugs and other therapeutic intervention (http://clinicaltrials.gov/). Of those, drugs which have direct application in treatment regimen for SCI and the reason for their pitfalls are discussed here.

The first and extensive studied drug is Methyl prednisolone sodium succinate (MPSS), an anti inflammatory corticosteroid exerting its function as antioxidant, enhancer of spinal cord blood flow, by reducing calcium influx, posttraumatic axonal die back and attenuating lipid perox‐ idation. The drawback of this drug is that it did not rescue neurons from cell death and [16] its high rates of adverse events such as the occurrence of pulmonary and gastrointestinal

A noncompetitive N-methyl-d-aspartate receptor antagonist, gacyclidine (GK-11), showed promise as a neuroprotective agent as evidenced by walking recovery, motor performance, attenuation of spinal cord damage, reducing apoptosis of oligodendrocytes via inhibition of proNGF production in microglia [18] etc, in rat model. However, this agent is no longer being pursued for SCI [11] and the use of minocycline following contusion of cord requires further

Minocycline, an antibiotic and anti-inflammatory substance facilitated overall motor recovery and attenuated mechanical hyperalgesia in a rat model [8], but did not increase the survival

**2.** GM-1(Sygen), a ganglioside found in the neuronal cell membranes, was found to promote recovery in a number of animal models. In human trials it resulted in statistically signif‐ icant improvement in ASIA motor score but failed to demonstrate a significant difference in its primary outcome measure, a 2-point improvement on the modified Benzel walking

**3.** Erythropoietin, a potent cytokine [25], and its analogues have been thoroughly investi‐ gated [26] and shown to protect neuronal cell in vitro from apoptosis and also suppress the up-regulated expression of TGF-β [27, 25] reduces the inflammation, and restores the

Inflammatory processes that occur at the injury site of the spinal cord are both beneficial and harmful. Phagocytic macrophages have been indicated in secondary destruction of neural tissue post SCI [28; 29] but are not sufficient as compared with peripheral nerve injury. Rapalino *et al.,* [30], has demonstrated that implantation of activated macrophages in the site

function. The details of all can be found at ICORD website (http://icord.org/).

**5.3. Neuroprotection (randomized clinical trials)**

**1.** Pharmacological therapy

complications and others. [8, 9, 15].

scale [8, 9].

vascular integrity [21].

**4.** Immunomodulatory treatment

investigation before clinical trials are implemented [17].

of the preganglionic parasympathetic neurons (PPNs) [20].

During the last two decades, various researchers have shown interest in developing variety of animal models based on the above two categories, that mimic different attributes associated with spinal cord injuries. Depending upon the purpose of the study and the specific aspect of the injury to be investigated, researchers determine which animal model most closely repli‐ cates the injury in humans. Commonly used animal models for the investigation includes [8] Primates – to test the safety and efficacy of the therapy, [9] Cat – to examine and define spinal cord circuitry, [10] Mouse and rat – mainly used for the investigations of molecular, genetic and anatomical response to injury and to modify genes to test the effect of restoration or loss of function.

The kind of inquiries currently in focus can be addressed with rodent models, for which the maximum number of biological reagents and tools are available. In time, there may be a need to examine the conclusions of rodent studies in other models, to deal with questions of species differences (biological responses, chromosomal arrangements, genetic variability and the spatial arrangement of the nerve tracts) and mechanical scale (animal size, limited number of animals for experimentation) and ethics [9].

#### **5. Assessing SCI and repair mechanisms**

#### **5.1. Conventional treatment strategies**

Treatment for Spinal cord injury starts at the site of accident or trauma. Manual spine immobilization or using cervical collar and spine board, followed by administration of analgesics to reduce pain is an established practice to achieve comfort to the patient. Careful monitoring of airway, respiration, and arterial pressure is essential. Hypotension, hypoxia are deleterious and should be avoided at all cost. From the scene of trauma, the patient is moved to the medical center and assessed further with neurological status and clinical level of injury. Base line clinical status is established and documented. In parallel other system‐ ic injuries were also evaluated. ASIA impairment scale modified BENZEL scale and FRANKEL scales are commonly used to evaluate progress. MRI is the gold standard in imaging to delineate the anatomy of injury. In addition, size and extent of cord contu‐ sion, hemorrhage and edema have prognostic significance. Throughout its mandatory to avoid secondary insults to spinal cord. Several drugs have been tried with no demonstra‐ ble benefit. There is no role for steroids and Methylprednisolone. All attempts of direct surgical repair of spinal cord have failed.

#### **5.2. Experimental strategies**

Almost every aspect of the management of SCI is controversial, due to lack of good-quality evidence. Currently all the modes of the experimental therapy falls into any of the following categories: neuroprotection, repair/regeneration, enhancing the plasticity and replace/assist function. The details of all can be found at ICORD website (http://icord.org/).

#### **5.3. Neuroprotection (randomized clinical trials)**

Clinical trials should augment the neurological recovery data with outcome measures designed to assess the functional significance of the neurological recovery. To date more than 70 clinical trials have been done on functional recovery of Spinal Cord Injury with drugs and other therapeutic intervention (http://clinicaltrials.gov/). Of those, drugs which have direct application in treatment regimen for SCI and the reason for their pitfalls are discussed here.

**1.** Pharmacological therapy

injury. Besides the above mentioned category, microlesion formation and transgenic models, [25] Photochemical SCI model, excitotoxic spinal cord injury have also been

During the last two decades, various researchers have shown interest in developing variety of animal models based on the above two categories, that mimic different attributes associated with spinal cord injuries. Depending upon the purpose of the study and the specific aspect of the injury to be investigated, researchers determine which animal model most closely repli‐ cates the injury in humans. Commonly used animal models for the investigation includes [8] Primates – to test the safety and efficacy of the therapy, [9] Cat – to examine and define spinal cord circuitry, [10] Mouse and rat – mainly used for the investigations of molecular, genetic and anatomical response to injury and to modify genes to test the effect of restoration or loss

The kind of inquiries currently in focus can be addressed with rodent models, for which the maximum number of biological reagents and tools are available. In time, there may be a need to examine the conclusions of rodent studies in other models, to deal with questions of species differences (biological responses, chromosomal arrangements, genetic variability and the spatial arrangement of the nerve tracts) and mechanical scale (animal size, limited number of

Treatment for Spinal cord injury starts at the site of accident or trauma. Manual spine immobilization or using cervical collar and spine board, followed by administration of analgesics to reduce pain is an established practice to achieve comfort to the patient. Careful monitoring of airway, respiration, and arterial pressure is essential. Hypotension, hypoxia are deleterious and should be avoided at all cost. From the scene of trauma, the patient is moved to the medical center and assessed further with neurological status and clinical level of injury. Base line clinical status is established and documented. In parallel other system‐ ic injuries were also evaluated. ASIA impairment scale modified BENZEL scale and FRANKEL scales are commonly used to evaluate progress. MRI is the gold standard in imaging to delineate the anatomy of injury. In addition, size and extent of cord contu‐ sion, hemorrhage and edema have prognostic significance. Throughout its mandatory to avoid secondary insults to spinal cord. Several drugs have been tried with no demonstra‐ ble benefit. There is no role for steroids and Methylprednisolone. All attempts of direct

Almost every aspect of the management of SCI is controversial, due to lack of good-quality evidence. Currently all the modes of the experimental therapy falls into any of the following

developed in recent years [26].

animals for experimentation) and ethics [9].

**5.1. Conventional treatment strategies**

surgical repair of spinal cord have failed.

**5.2. Experimental strategies**

**5. Assessing SCI and repair mechanisms**

of function.

234 Topics in Paraplegia

The first and extensive studied drug is Methyl prednisolone sodium succinate (MPSS), an anti inflammatory corticosteroid exerting its function as antioxidant, enhancer of spinal cord blood flow, by reducing calcium influx, posttraumatic axonal die back and attenuating lipid perox‐ idation. The drawback of this drug is that it did not rescue neurons from cell death and [16] its high rates of adverse events such as the occurrence of pulmonary and gastrointestinal complications and others. [8, 9, 15].

A noncompetitive N-methyl-d-aspartate receptor antagonist, gacyclidine (GK-11), showed promise as a neuroprotective agent as evidenced by walking recovery, motor performance, attenuation of spinal cord damage, reducing apoptosis of oligodendrocytes via inhibition of proNGF production in microglia [18] etc, in rat model. However, this agent is no longer being pursued for SCI [11] and the use of minocycline following contusion of cord requires further investigation before clinical trials are implemented [17].

Minocycline, an antibiotic and anti-inflammatory substance facilitated overall motor recovery and attenuated mechanical hyperalgesia in a rat model [8], but did not increase the survival of the preganglionic parasympathetic neurons (PPNs) [20].


Inflammatory processes that occur at the injury site of the spinal cord are both beneficial and harmful. Phagocytic macrophages have been indicated in secondary destruction of neural tissue post SCI [28; 29] but are not sufficient as compared with peripheral nerve injury. Rapalino *et al.,* [30], has demonstrated that implantation of activated macrophages in the site of injury in adult injured rats results in partial recovery. On the contrary, Popovich *et al.,* [31] suggested that depletion of macrophages may result in preservation of myelinated axons and functional recovery following injury. A phase I clinical trial demonstrated the safety of autologous macrophage transplantation into the damaged spinal cord within 14 days of injury.

patient move their upper limb and lower limb and support their body weight after a spinal trauma. Upon discharge from a hospital setting, family members, other caregivers, or both share the burden of care. Medical insurance programs have required reliable data on which to determine benefits, including coverage of durable medical equipment, treatment, and care giving assistance. Task specific training i.e., activities of daily living (ADL) which include self feeding, bathing, bowel and bladder maintenance, dressing, hygiene maintenance, computer usage etc., plays a central role for the patient to be independent. Other techniques such as body weight support and treadmill training using upper and lower limb orthosis and knee orthosis, have shown recovery in maintaining the body gait and postures. Tilt table standing, robotaided gait training, electric stimulated wheel chairs are also used in recent days for posture maintenance. Recreation and leisure skill development such as reading, writing, painting, exercises, All-Terrain Vehicles (ATVs) (cycling, fishing, horseback riding, climbing, diving, etc) arm ergometry and Nautilus-type machines. Although these techniques are considered to be promising, less is known about their mechanism and efficacy on the functional recovery. Hence a deeper understanding of the underlying mechanism for adaptation and plasticity after

Mesenchymal Stem Cells in Spinal Cord Injury

http://dx.doi.org/10.5772/58323

237

Functional electrical stimulation (FES) is the technique of applying safe levels of electric current to activate the damaged or disabled nervous system. Although no absolute contraindications exist for the use of externally applied FES, a patient with a cardiac demand pacemaker or an automatic implanted defibrillator should be approached with extreme caution. Some of the relative contraindications for FES include patients with cardiac arrhythmias, congestive heart failure, pregnancy, electrode sensitivity, and patients with healing wound(s) that could be stressed during stimulation (i.e., muscle stimulation would adversely move healing tissues). As with any implant in the body, individuals with implanted FES systems need to obtain antibiotic prophylaxis when undergoing invasive procedures such as oral surgery. Functional uses for FES after SCI include applications in standing, walking, hand grasp (and release), bladder, bowel, and sexual function, respiratory assist, and electro ejaculation for fertility. [82-88]. Functional magnetic stimulation (FMS) can be defined as a technology that applies a time varying magnetic field to produce useful bodily function. There were no significant side effects of magnetic stimulation that were reported. However safety consideration such as magnetic effect, electric effect and power dissipation should be kept in mind during stimula‐ tion. A few reports have shown that repetitive transcranial magnetic stimulation may result

Hypothermia, CSF Drainage, durotomy and subarachnoid perfusion, Functional electrical stimulation, Electromagnetic stimulation, hyperbaric oxygen were tried with some success.

Over the past 2 decades, advances in understanding the pathophysiology of spinal cord injury (SCI) have stimulated the recent emergence of therapeutic strategies. Functional repair of the

But none of them reached to the level of functional therapeutic options.

spinal cord injury is needed to improve rehabilitation regimes [65-81].

**6.2. Non pharmacological intervention for the treatment of SCI**

in increased seizure activities [89-94].

**6.3. Replace or assist function**


#### **6. Repair and regeneration**

A variety of promising substances have been tested in animal models, but few have had potential application to human spinal cord injury (SCI) patients. This category of treat‐ ment includes both the pharmacological intervention using FDA approved drugs and cell transplantation. (The latter will be discussed in detail in the forthcoming titles). Several drugs were tested for their efficacy in restoring spinal cord function as evidenced by multiple preclinical studies. Some of the Drugs such as Cethrin [47-50], rolipram [41-45], ATI-355 [45-51], chondroitinase [51-56] and riluzole [57-64] were thoroughly reviewed which are not limited to neuroprotection, axonal regeneration, motor neuron recovery, reduction in muscle spasms, enhanced sprouting of corticospinal axons, improved behavio‐ ral outcome and corticospinal plasticity, recovery of forelimb function, inhibition of apoptosis and suppression of glial scar formation with varying degree of success. The major drawback of the pharmacological intervention is their side effects and direct application in human trials.

#### **6.1. Plasticity enhancement and rehabilitation**

An inability to perform self-care activities is considered a "burden of care" by the medical community. The individual with acute SCI faces many challenges with the resumption of selfcare tasks. Hence considerable efforts have been taken by the therapist in order to guide the patient move their upper limb and lower limb and support their body weight after a spinal trauma. Upon discharge from a hospital setting, family members, other caregivers, or both share the burden of care. Medical insurance programs have required reliable data on which to determine benefits, including coverage of durable medical equipment, treatment, and care giving assistance. Task specific training i.e., activities of daily living (ADL) which include self feeding, bathing, bowel and bladder maintenance, dressing, hygiene maintenance, computer usage etc., plays a central role for the patient to be independent. Other techniques such as body weight support and treadmill training using upper and lower limb orthosis and knee orthosis, have shown recovery in maintaining the body gait and postures. Tilt table standing, robotaided gait training, electric stimulated wheel chairs are also used in recent days for posture maintenance. Recreation and leisure skill development such as reading, writing, painting, exercises, All-Terrain Vehicles (ATVs) (cycling, fishing, horseback riding, climbing, diving, etc) arm ergometry and Nautilus-type machines. Although these techniques are considered to be promising, less is known about their mechanism and efficacy on the functional recovery. Hence a deeper understanding of the underlying mechanism for adaptation and plasticity after spinal cord injury is needed to improve rehabilitation regimes [65-81].

#### **6.2. Non pharmacological intervention for the treatment of SCI**

Functional electrical stimulation (FES) is the technique of applying safe levels of electric current to activate the damaged or disabled nervous system. Although no absolute contraindications exist for the use of externally applied FES, a patient with a cardiac demand pacemaker or an automatic implanted defibrillator should be approached with extreme caution. Some of the relative contraindications for FES include patients with cardiac arrhythmias, congestive heart failure, pregnancy, electrode sensitivity, and patients with healing wound(s) that could be stressed during stimulation (i.e., muscle stimulation would adversely move healing tissues). As with any implant in the body, individuals with implanted FES systems need to obtain antibiotic prophylaxis when undergoing invasive procedures such as oral surgery. Functional uses for FES after SCI include applications in standing, walking, hand grasp (and release), bladder, bowel, and sexual function, respiratory assist, and electro ejaculation for fertility. [82-88]. Functional magnetic stimulation (FMS) can be defined as a technology that applies a time varying magnetic field to produce useful bodily function. There were no significant side effects of magnetic stimulation that were reported. However safety consideration such as magnetic effect, electric effect and power dissipation should be kept in mind during stimula‐ tion. A few reports have shown that repetitive transcranial magnetic stimulation may result in increased seizure activities [89-94].

Hypothermia, CSF Drainage, durotomy and subarachnoid perfusion, Functional electrical stimulation, Electromagnetic stimulation, hyperbaric oxygen were tried with some success. But none of them reached to the level of functional therapeutic options.

#### **6.3. Replace or assist function**

of injury in adult injured rats results in partial recovery. On the contrary, Popovich *et al.,* [31] suggested that depletion of macrophages may result in preservation of myelinated axons and functional recovery following injury. A phase I clinical trial demonstrated the safety of autologous macrophage transplantation into the damaged spinal cord within 14 days of injury. **5.** Neurotrophic factors: Neurotrophic factors have been documented to improve cell survival and axonal regeneration and various approaches have been developed to deliver these factors to the site of injury. Stem cells from different sources like bone marrow [32, 33, adipose tissue, dental pulp [34], Wharton's jelly, olfactory ensheathing cells [35], neural stem cells [36] and embryonic stem cells [37] when transplanted *in vivo* have shown

**6.** In a controlled double-blinded study, 20 patients receiving thyrotrophin releasing hormone treatment showed significantly higher motor, sensory, and Sunnybrook scores than placebo treatment. But because of patients lost to subsequent follow-up, data were not highly informative [18]. Another study in rats treated with thyrotropin-releasing hormone showed significant improvement in Neural Scores 14 days post-injury, but there were no significant differences in morphometric parameters between saline-and TRHtreated rats [19]. TRH has disadvantages, including its analeptic, endocrine, and auto‐ nomic effects, but a new generation of TRH analogs has been developed that have the

A variety of promising substances have been tested in animal models, but few have had potential application to human spinal cord injury (SCI) patients. This category of treat‐ ment includes both the pharmacological intervention using FDA approved drugs and cell transplantation. (The latter will be discussed in detail in the forthcoming titles). Several drugs were tested for their efficacy in restoring spinal cord function as evidenced by multiple preclinical studies. Some of the Drugs such as Cethrin [47-50], rolipram [41-45], ATI-355 [45-51], chondroitinase [51-56] and riluzole [57-64] were thoroughly reviewed which are not limited to neuroprotection, axonal regeneration, motor neuron recovery, reduction in muscle spasms, enhanced sprouting of corticospinal axons, improved behavio‐ ral outcome and corticospinal plasticity, recovery of forelimb function, inhibition of apoptosis and suppression of glial scar formation with varying degree of success. The major drawback of the pharmacological intervention is their side effects and direct application in

An inability to perform self-care activities is considered a "burden of care" by the medical community. The individual with acute SCI faces many challenges with the resumption of selfcare tasks. Hence considerable efforts have been taken by the therapist in order to guide the

protective effects of TRH without its adverse effects [20].

significant recovery.

236 Topics in Paraplegia

**6. Repair and regeneration**

**6.1. Plasticity enhancement and rehabilitation**

human trials.

Over the past 2 decades, advances in understanding the pathophysiology of spinal cord injury (SCI) have stimulated the recent emergence of therapeutic strategies. Functional repair of the injured central nervous system (CNS) is one of the greatest challenges addressed by neurobi‐ ologists. The rapidly growing field of stem cell biology offers a promising future for cell replacement and neural regeneration therapies. Stem cells have seen its good days with success in Parkinson disease and Huntington's disease and hold a long history of research on the possible use of progenitor cells in the treatment of SCI. The application of cell-based therapies to SCI is a natural expansion of research in other fields, such as cancer, diabetes, and heart diseases.

**6.4. Proposed scope of using stem cells / regenerative medicine**

Partial cervical hemisection injury

Compression lesion

**Site of injection**

Intravenously, Intraventricula Intravenous route – least efficient. Intrathecally and intraventricular administration shows enhanced cell migration and

grafting

Higher BBB scores and better recovery of hind limb sensitivity

Improved neurological signs in pelvic limbs Significant high Olby scores

Intrathecally and Lumbar puncture

Intravenously (BMSCs) and subcutaneousl y (GCSF)

Intrathecal

rly,

**Results Salient features Reference**

Axonal growth was observed around the transplanted cells. Grafted LRNP cells

Mesenchymal Stem Cells in Spinal Cord Injury

http://dx.doi.org/10.5772/58323

239

differentiation into mature neurons, their survival and integration within the host

Ajay bakshi et al

Lucia Urdzíková et

Dong-In Jung et al

al

spinal

Cord might lead to functional recovery.

Recovery may be due to the synchronized action of various factors as evidenced by high rate of mRNA expression for neurotrophic

factors.

Significant increases in the spared volume of white matter due to the synchronized action of various factors released by MSCs

**Type of stem cells Species Injury type**

Inbred Fisher-344 rats

Male Wistar rats

Adult Beagle dogs

BM MSC

Transgenically labeled BMMSC (autologous) and lineage restricted neural precursor

(LRNP) (2x106 cells)

BMSCs and GCSF

Bone-marrowderived

mesenchymal stem cell (autologous and allogenic)

Spinal Cord Injury and Stem cells: Some Cellular transplantation strategies

Spinal cord injury though uncommon leads to profound lifelong disability and systemic effects. So far no single therapy have proved its efficacy,.therefore combination therapies might hold the future design. In order to repair the injured spinal cord, it is essential to reduce secondary damage and promote regeneration. Several biological agents such as proteins, antibodies, enzymes and cells were used to achieve this goal..

The adult spinal cord has an endogenous progenitor cell pool said to have been located, in the ependymal region around the central canal. [95-97]. While others believe their presence throughout the spinal cord [98]. The response of these endogenous cells post injury is insuffi‐ cient and does not bring about adequate recovery following SCI [95-97]; probably due to the insufficient cell numbers, microenvironment at the injury site, and presence of tissue debris. Neural inflammation, immune mediated destruction and loss of vascularity also becomes major hindrance. There could be an imbalance between the degree of repair vs damage. Cell transplant strategies have the potential of reducing such secondary damage and promoting regeneration by replacement of dead cells and production of Neurotrophic factors promoting regeneration [99-101].

Oligodendrocytes and astrocytes are the major supportive cells within the central nervous system and are responsible for myelination of axons, so it is believed that replacement of this cell population will support the frame work in regenerative processes.

Immature glial cells have been shown to reduce the inhibitory properties of the lesion epicentre and promote axonal growth [102]. Immature oligodendrocytes provide remyelination after injury [103], whereas immature astrocytes promote axonal growth and survival after injury [104]. A recent study supports the idea of ensuring both of these cell types, astroglial, are replaced, since oligodendrocytes precursors failed to remyelinate the spinal cord in the absence of astrocytes.

Cao *et al.,* [99] has demonstrated that after the transplantation of stem cells into lesioned adult rat spinal cord most of these transplanted cells have differentiated into astrocytes and no neurons or oligodendrocytes were observed. This indicates that it would be essential to transplant a progenitor cell population capable of trans-differentiating into a mixed lineage *in vivo* or should be able to secrete neurotrophic factors *in vivo*. Studies have elucidated that MSCs do have the capacity to Trans-differentiate into the astroglial lineage and also secrete cytokines which may be essential for regeneration in spinal cord injury.


#### **6.4. Proposed scope of using stem cells / regenerative medicine**

injured central nervous system (CNS) is one of the greatest challenges addressed by neurobi‐ ologists. The rapidly growing field of stem cell biology offers a promising future for cell replacement and neural regeneration therapies. Stem cells have seen its good days with success in Parkinson disease and Huntington's disease and hold a long history of research on the possible use of progenitor cells in the treatment of SCI. The application of cell-based therapies to SCI is a natural expansion of research in other fields, such as cancer, diabetes, and heart

Spinal cord injury though uncommon leads to profound lifelong disability and systemic effects. So far no single therapy have proved its efficacy,.therefore combination therapies might hold the future design. In order to repair the injured spinal cord, it is essential to reduce secondary damage and promote regeneration. Several biological agents such as proteins,

The adult spinal cord has an endogenous progenitor cell pool said to have been located, in the ependymal region around the central canal. [95-97]. While others believe their presence throughout the spinal cord [98]. The response of these endogenous cells post injury is insuffi‐ cient and does not bring about adequate recovery following SCI [95-97]; probably due to the insufficient cell numbers, microenvironment at the injury site, and presence of tissue debris. Neural inflammation, immune mediated destruction and loss of vascularity also becomes major hindrance. There could be an imbalance between the degree of repair vs damage. Cell transplant strategies have the potential of reducing such secondary damage and promoting regeneration by replacement of dead cells and production of Neurotrophic factors promoting

Oligodendrocytes and astrocytes are the major supportive cells within the central nervous system and are responsible for myelination of axons, so it is believed that replacement of this

Immature glial cells have been shown to reduce the inhibitory properties of the lesion epicentre and promote axonal growth [102]. Immature oligodendrocytes provide remyelination after injury [103], whereas immature astrocytes promote axonal growth and survival after injury [104]. A recent study supports the idea of ensuring both of these cell types, astroglial, are replaced, since oligodendrocytes precursors failed to remyelinate the spinal cord in the absence

Cao *et al.,* [99] has demonstrated that after the transplantation of stem cells into lesioned adult rat spinal cord most of these transplanted cells have differentiated into astrocytes and no neurons or oligodendrocytes were observed. This indicates that it would be essential to transplant a progenitor cell population capable of trans-differentiating into a mixed lineage *in vivo* or should be able to secrete neurotrophic factors *in vivo*. Studies have elucidated that MSCs do have the capacity to Trans-differentiate into the astroglial lineage and also secrete cytokines

cell population will support the frame work in regenerative processes.

which may be essential for regeneration in spinal cord injury.

Spinal Cord Injury and Stem cells: Some Cellular transplantation strategies

antibodies, enzymes and cells were used to achieve this goal..

diseases.

238 Topics in Paraplegia

regeneration [99-101].

of astrocytes.


**Type of stem cells Species Injury type**

Adult Female Sprague-Dawley rats

Adult Female Fischer 344 rats

African green monkey

Human umbilical cord mesenchymal stem cells

GLIAL CELLS

NSC/ NSPC

stem cells

poly(lactide-coglycolide) (PLGA) polymer seeded with human neural

Multineurotrophin-Expressing Glial-Restricted Precursor Cells

**Site of injection**

Transection Lesion site

Contusion Lesion site

Hemisection Lesion site

**Results Salient features Reference**

Mesenchymal Stem Cells in Spinal Cord Injury

http://dx.doi.org/10.5772/58323

241

factor and neurotrophin-3.

Production of large amounts of

human neutrophilactivating protein-2, NT-3,

bFGF, glucocorticoid induced TNFreceptor, and VEGFR 3 in host spinal cord, may have helped in spinal cord repair.

Grafted GRPs formed normalappearing myelin sheaths around the axons in the ventrolateral funiculus (VLF) of spinal cord and restores the conduction.

Major mechanism of action of implanted cells may be due to trophic support rather than

Chang-Ching Yang et al

Qilin Cao et

Pritchard et

al

al

Fewer reactive astrocytes were observed

Promotion of regrowth of injured corticospinal fibers change in the distribution of astrocytes in spinal

cords

Improved transcranial magnetic motorevoked potential responses Improved

Electrophysiologica l and locomotor functional recovery

Enhanced hindlimb motor neuron performance

reduction in the activation of microglia


**Type of stem cells Species Injury type**

Adult mixed-breed dogs

Adult Male Sprague-Dawley rats

Adult Female SD rats

Female Sprague-Dawley rats Epidural balloon compression

Clip compression

LA MSC

240 Topics in Paraplegia

Adipose-derived stem cells (allogenic)

Cotransplantation of Mouse Neural Stem Cells (mNSCs) With Adipose Tissue-Derived Mesenchymal Stem

Cells

WJ MSC

Human Umbilical Cord-Derived Schwann-Like Cell Combined with Neurotrophin-3

Human umbilical cord mesenchymal stem cells

**Site of injection**

Injured site

Epicenter of the injured spinal cord

Transection Lesion site

At the dorsal spinal cord 2 mm rostrally and 2 mm caudally to the injury site

Weight drop method (contusion)

**Results Salient features Reference**

Neuronal transdifferentiati

Survived MSCs produces large amounts of bFGF and VEGFR 3 which aid the recovery

Biomolecular substances secreted by ATMSCs improve mNSC survival and inhibit mNSC apoptosis, mainly

Jin Soo Oh et al

Guo Yan-Wu et al

Hu SL et al

VEGF

GDNF, BDNF, NT-3 and bFGF provided neurotrophic support

Transplanted cells survived, migrated over short distances, and produced large amounts of glial cell linederived neurotrophic

on

Nerve conduction velocity was significantly improved GFAP, Tuj-1 and NF160 were observed

AT-MSCs inhibited the apoptosis of mNSCs mNSCs transplanted with AT-MSCs showed better survival rates

NT-3

administration significantly promoted the survival

of the grafted cells Improved motor function and promotes neurite outgrowth

Significant Recovery of hindlimb

structures

locomotor function Increased length of neurofilamentpositive fibers and increased numbers of growth cone-like


**Type of stem cells Species Injury type**

Adult Sprague– Dawley female rats

Contusion

Adult dog Hemisection Lesion site

transection Site of injury

Contusion Lesion site

**Table 2.** Provides a list of preclinical animal studies conducted for spinal cord injury

Adult rats Complete

Female Sprague Dawley adult rats

mesenchymal stromal cells (BMSCS)

Immortalized human NSC line over expressing VEGF (F3.VEGF

Poly(lactic-coglycolic acid) (PLGA) seeded with neural stem cell (NSC)

hESC-derived oligodendrocyte progenitors (OPC)

hESC derived Oligodendrocyte Progenitor Cells

and/or motoneuron progenitors (MP)

hESC derived PROGENITOR CELLS

cells)

**Site of injection**

2 mm rostral and 2 mm caudal from the lesion epicenter

Elevated the amount of VEGF in the injured spinal cord tissue and increased

phosphorylation of VEGFR flk-1 Enhanced cellular proliferation and tissue sparing

Grafted NSC survived the implantation procedure and showed migratory

behavior

Locomotor function was significantly enhanced OPC and MP survived, migrated, and differentiated into mature oligodendrocytes and neurons

Transplanted cells survived, redistributed over short distances, and differentiated

oligodendrocytes

into

**Results Salient features Reference**

space of the spinal cord

Mesenchymal Stem Cells in Spinal Cord Injury

http://dx.doi.org/10.5772/58323

243

VEGF increased the number of

proliferating cells

Kim et al

Kim et al

SLAVEN ERCEG et al (

Keirstead et

al

differentiated into mature oligodendrocytes

early

that

Ectopic expression of a therapeutic neurotrophin-3 gene was observed

The recoveries can be attributed

reconnection of the axons above and below the lesion site

Widespread oligodendrocyte remyelination throughout the white matter

to the


**Table 2.** Provides a list of preclinical animal studies conducted for spinal cord injury

**Type of stem cells Species Injury type**

Adult male rat

Neural stem cells Marmoset Contusion Lesion site

Wild type C57BL6/J and C57BL/6- TgN (ACTbEGFP) 1Osb

Rat Compression

Contusion Intraspinal

Lumbar puncture

Clip compression

Spinal cord-derived NSPCs and BMSCs

242 Topics in Paraplegia

Epidermal Neural Crest Stem Cell (EPI-NCSC)

Spinal cord-derived neural stem/ progenitor cells (NSPCS) and Bone Marrow-derived

**Site of injection**

Lesion site

**Results Salient features Reference**

neuronal replacement

Differentiation of NSPCs into astrocytes and oligodendrocytes promoting remyelination, and potential axonal guidance

Grafted human NSCs survived

differentiated into neurons astrocytes and oligodendrocytes

restores motor function

combination of pertinent functions including cell replacement, neuroprotection, angiogenesis and modulation of

and

and

scar formation

wide

dissemination of cells in the subarachnoid

Parr et al

Iwanami et

al

Sieber

Mothe et al

No functional improvement was seen in either transplant group. But significant inverse corelation between the functional scores and the number of transplanted astrocytes was observed

Recovery of motor

Significantly higher spontaneous movement

Differentiated into gabaergic neurons and myelinating oligodendrocytes

Expression of oligodendrocyte markers

function was observed mainly in the hindlimbs

#### *6.4.1. Current status of cell replacement therapy*

During the last 2 decades, the search for new therapies has been revolutionized by the discovery of stem cells, which has inspired scientists and clinicians to search for stem cell– based reparative approaches to many diseases. The adult spinal cord harbors endogenous stem/progenitor cells, collectively referred to as neural progenitor cells (NPCs) that might be responsible for normal turnover of the cells. However, the proliferative activity of endogenous NPCs is too limited to support significant self-repair after SCI. Thus, various cellular trans‐ plantation strategies have been adopted in models of SCI.

cant increase in nerve conduction, neuronal transdifferentiation and production of bFGF and VEGFR3 in large quantity. In yet another study [2] using rat as animal model of SCI, trans‐ planted ADMSC and Mouse Neural Stem Cells (mNSCs) and observed that ADMSC protect mNSCs from apoptosis and increases the survival rates by secreting biomolecular substances, preferably VEGF in various conditions like hypoxia, oxidative stress and combined injury.

Mesenchymal Stem Cells in Spinal Cord Injury

http://dx.doi.org/10.5772/58323

245

In two trans-section and one contusion injury model of rats studied using Human umbili‐ cal cord mesenchymal stem cells had revealed that the grafted cells survived, migrated and produced large amount of GDNF, BDNF, NT-3, bFGF [9] glial cell line-derived neurotro‐ phic factor, [13] neutrophil-activating protein-2, glucocorticoid induced TNF-receptor, and

Improved transcranial magnetic motor-evoked potential responses and improved electrophy‐ siological and locomotor functional recovery was observed in rat contusion model of spinal trauma using Multi-neurotrophin-expressing glial-restricted precursor cells. The reason behind the functional recovery in restoring conduction was proposed to be formation of myelin

Various animal injury models were studied for the transplantation of NSC/NPC. This include primates and rodents model. NSC in PLGA scaffold was tested in African Green Monkey using hemisection and Pritchard concluded the regulatory mechanism as the signaling by various factors released by NSCs [10]. In a contusion model of injury using marmoset, Iwanami et al [18] reported the differentiation of NSPCs into astrocytes and oligodendrocytes which promotes remyelination and promotes functional recovery. In another contusion injury model, the efficacy of EPI-NSC in restoring function was due to differentiation of grafted cells into Gaba-ergic neurons and myelinating oligodendrocytes resulting in neuroprotection, angio‐

Expression of a therapeutic neurotrophin-3 gene, which leads to the recovery, was observed when PLGA coated with NSC was grafted in a canine hemisection model [20]. While others [25] observed no functional improvements in either groups transplanted with spinal cordderived NSPCs and BMSCs on rats at the lesion site, Mothe et al observed recovery by injecting the cells via LP. Differentiation of SC derived NSPC into astrocytes and oligodendrocytes were observed by both the teams. Elevated amount of VEGF in the injured spinal cord tissue and increased phosphorylation of VEGFR flk-1 enhanced cellular proliferation and tissue sparing and increase in the density of blood vessels was the result reported by Kim et al using immortalized human NSC line over expressing VEGF (F3.VEGF cells) in a contusion model of

*Human umbilical cord mesenchymal stem cells*

sheath around the axons by the grafted cells [12].

*Neural stem cells and Neural progenitor cells*

genesis and scar modulation [18].

injury in rats [15].

VEGFR 3 [16].

*Glial precursor cells*

Current goals of cell replacement approach are broadly classified into two broad types: 1) regeneration and 2) repair. Alternatively the cell transplanted may promote protection to the endogenous cells from further damage.

A summary of cell therapy approaches has been listed in Table 2 mentioned above.

#### *6.4.2. Different cell types proposed to have therapeutic potential*

#### *Human Embryonic Stem Cell derived progenitor cells*

Cocultures of hESC derived ologodendrocytes with or without motor neuron progenitors have been used for the treatment of SCI by different researchers with different injury models [14, 17]. The functional recovery concluded by both study is in vivo differentiation of the trans‐ planted cells into oligodendrocytes and neurons promoting remyelination and axonal regrowth.

#### *Adult derived stem cells*

#### *Bone marrow derived stem cells*

In a hemisection model of rats, Bakshi et al has shown that BMSC co-transplanted with that of neural progenitors shows better cell migration and grafting when injected intraventricularly or intrathecally. However, intravenous route shows the least cell migration to the site of injury. Alternatively, Urdzíková and his team reported that when BMSCs were transplanted intra‐ venously with GCSF in subcutaneous region, spared white matter increases in size and enhanced recovery of hind limb sensitivity was observed. A canine model of injury using both auto and allogeneic BMSCs transplanted intrathecally shows improvement in neurological signs. But the mechanism of recovery observed was the synchronized action of the growth factors released by the grafted cells [11].

Strangely no functional recovery was observed in rat model of SCI wherein a co culture of Spinal cord-derived NSPCs and BMSCs were transplanted at the lesion site. Alternatively a reverse correlation was observed between the functional scores and number of astrocytes transplanted [25]. But the same group of cells when injected via LP shows potent oligoden‐ drocyte marker [21].

#### *Adipose tissue derived stem cells*

In 2009, Hak-Hyun Ryu and his colleagues reported the use of adipose derived stem cells on a canine model of SCI using compression method. ADMSCs show better recovery by signifi‐ cant increase in nerve conduction, neuronal transdifferentiation and production of bFGF and VEGFR3 in large quantity. In yet another study [2] using rat as animal model of SCI, trans‐ planted ADMSC and Mouse Neural Stem Cells (mNSCs) and observed that ADMSC protect mNSCs from apoptosis and increases the survival rates by secreting biomolecular substances, preferably VEGF in various conditions like hypoxia, oxidative stress and combined injury.

#### *Human umbilical cord mesenchymal stem cells*

In two trans-section and one contusion injury model of rats studied using Human umbili‐ cal cord mesenchymal stem cells had revealed that the grafted cells survived, migrated and produced large amount of GDNF, BDNF, NT-3, bFGF [9] glial cell line-derived neurotro‐ phic factor, [13] neutrophil-activating protein-2, glucocorticoid induced TNF-receptor, and VEGFR 3 [16].

#### *Glial precursor cells*

*6.4.1. Current status of cell replacement therapy*

endogenous cells from further damage.

growth.

244 Topics in Paraplegia

*Adult derived stem cells*

drocyte marker [21].

*Adipose tissue derived stem cells*

*Bone marrow derived stem cells*

factors released by the grafted cells [11].

plantation strategies have been adopted in models of SCI.

*6.4.2. Different cell types proposed to have therapeutic potential*

*Human Embryonic Stem Cell derived progenitor cells*

During the last 2 decades, the search for new therapies has been revolutionized by the discovery of stem cells, which has inspired scientists and clinicians to search for stem cell– based reparative approaches to many diseases. The adult spinal cord harbors endogenous stem/progenitor cells, collectively referred to as neural progenitor cells (NPCs) that might be responsible for normal turnover of the cells. However, the proliferative activity of endogenous NPCs is too limited to support significant self-repair after SCI. Thus, various cellular trans‐

Current goals of cell replacement approach are broadly classified into two broad types: 1) regeneration and 2) repair. Alternatively the cell transplanted may promote protection to the

Cocultures of hESC derived ologodendrocytes with or without motor neuron progenitors have been used for the treatment of SCI by different researchers with different injury models [14, 17]. The functional recovery concluded by both study is in vivo differentiation of the trans‐ planted cells into oligodendrocytes and neurons promoting remyelination and axonal re-

In a hemisection model of rats, Bakshi et al has shown that BMSC co-transplanted with that of neural progenitors shows better cell migration and grafting when injected intraventricularly or intrathecally. However, intravenous route shows the least cell migration to the site of injury. Alternatively, Urdzíková and his team reported that when BMSCs were transplanted intra‐ venously with GCSF in subcutaneous region, spared white matter increases in size and enhanced recovery of hind limb sensitivity was observed. A canine model of injury using both auto and allogeneic BMSCs transplanted intrathecally shows improvement in neurological signs. But the mechanism of recovery observed was the synchronized action of the growth

Strangely no functional recovery was observed in rat model of SCI wherein a co culture of Spinal cord-derived NSPCs and BMSCs were transplanted at the lesion site. Alternatively a reverse correlation was observed between the functional scores and number of astrocytes transplanted [25]. But the same group of cells when injected via LP shows potent oligoden‐

In 2009, Hak-Hyun Ryu and his colleagues reported the use of adipose derived stem cells on a canine model of SCI using compression method. ADMSCs show better recovery by signifi‐

A summary of cell therapy approaches has been listed in Table 2 mentioned above.

Improved transcranial magnetic motor-evoked potential responses and improved electrophy‐ siological and locomotor functional recovery was observed in rat contusion model of spinal trauma using Multi-neurotrophin-expressing glial-restricted precursor cells. The reason behind the functional recovery in restoring conduction was proposed to be formation of myelin sheath around the axons by the grafted cells [12].

#### *Neural stem cells and Neural progenitor cells*

Various animal injury models were studied for the transplantation of NSC/NPC. This include primates and rodents model. NSC in PLGA scaffold was tested in African Green Monkey using hemisection and Pritchard concluded the regulatory mechanism as the signaling by various factors released by NSCs [10]. In a contusion model of injury using marmoset, Iwanami et al [18] reported the differentiation of NSPCs into astrocytes and oligodendrocytes which promotes remyelination and promotes functional recovery. In another contusion injury model, the efficacy of EPI-NSC in restoring function was due to differentiation of grafted cells into Gaba-ergic neurons and myelinating oligodendrocytes resulting in neuroprotection, angio‐ genesis and scar modulation [18].

Expression of a therapeutic neurotrophin-3 gene, which leads to the recovery, was observed when PLGA coated with NSC was grafted in a canine hemisection model [20]. While others [25] observed no functional improvements in either groups transplanted with spinal cordderived NSPCs and BMSCs on rats at the lesion site, Mothe et al observed recovery by injecting the cells via LP. Differentiation of SC derived NSPC into astrocytes and oligodendrocytes were observed by both the teams. Elevated amount of VEGF in the injured spinal cord tissue and increased phosphorylation of VEGFR flk-1 enhanced cellular proliferation and tissue sparing and increase in the density of blood vessels was the result reported by Kim et al using immortalized human NSC line over expressing VEGF (F3.VEGF cells) in a contusion model of injury in rats [15].

#### **6.5. Clinical trials for SCI**

Cell transplantation therapies have become a major focus in pre-clinical research as a prom‐ ising strategy for the treatment of spinal cord injury. Various types of stem cells such as bone marrow stromal cells (BMSCs), adipose tissue Mesenchymal stem cells (ADMSCs), Schwann cells, olfactory ensheathing cells (OECs), neural stem cells or progenitor cells have been reported for their potential to form myelin, promote axonal regrowth and guidance, bridging the site of injury.

**S. No NCT study**

8 NCT01186679

9 NCT01274975

10 NCT00816803

11 NCT01217008

12 NCT01231893

subacute stage [8].

trials of cell therapies for SCI [9].

as a therapeutic agent.

**number Title/ Brief summary Study type/**

(HuCNS-SC®) in Subjects With Thoracic (T2-T11) Spinal

Surgical Transplantation of Autologous Bone Marrow Stem Cells With Glial Scar Resection for Patients of Chronic Spinal Cord Injury and Intra-thecal Injection for

To assess the safety of intravenous autologous adipose derived mesenchymal stem cells transplant in spinal

To assess the safety of autologous bone marrow derived cell transplant in chronic spinal cord injury

To evaluate the safety of GRNOPC1 administered at a single time-point between 7 and 14 days post spinal

transplantation of autologous olfactory ensheathing glia and olfactory fibroblasts obtained from the olfactory mucosa in patients with complete spinal cord

In an article published, Wolfram Tetzlaff et al has reviewed in detail, all the types of cells being used in the treatment of spinal cord injury from the available pre-clinical literature. Their review shows that rodent stem cells have been most extensively studied for SCI. Limited studies have been done on human stem cells. Majority of trials are with bone marrow stromal cells. Also reported was, while chronic treatments were rare and often failed to yield functional benefits, all the preclinical studies conducted, was in acute and

Also Fehlings et al [9] in his recently reviewed article has shown the efficacies and limitations of every type of cells, either alone or in various combinations as registered for trial studies, in use and has demonstrated the potential use of other promising candidate stem cells evaluated in pre-clinical studies but are not yet in Clinical Trials. Also they have made recommendations for the conduct and evaluation of pre-clinical studies and clinical

However no clinical intervention is risk free and we require understanding more on the pathophysiology of SCI and the clinical potential of stem cells to translate the use of the same

Assessment of the safety and feasibility of

Cord Trauma

Acute and Subacute Injury

cord injury patients.

patients.

cord injury

injury.

**phase Study status**

Mesenchymal Stem Cells in Spinal Cord Injury

http://dx.doi.org/10.5772/58323

247

Phase I / II Completed

Phase I Completed

Phase I / II Completed

Phase I Recruiting

Active, not recruiting

Interventional,

Interventional,

Interventional,

Interventional, Phase I

Interventional,

More than a dozen of clinical trials have been registered in the official website of clinical trials (http://www.clinicaltrials.gov). A brief listing of the selected trials is given below.


The results obtained are as follows:


**6.5. Clinical trials for SCI**

The results obtained are as follows:

the site of injury.

246 Topics in Paraplegia

**S. No NCT study**

1 NCT01325103

2 NCT01490242

3 NCT01393977

4 NCT01328860

5 NCT01446640

6 NCT01162915

7 NCT01321333

Cell transplantation therapies have become a major focus in pre-clinical research as a prom‐ ising strategy for the treatment of spinal cord injury. Various types of stem cells such as bone marrow stromal cells (BMSCs), adipose tissue Mesenchymal stem cells (ADMSCs), Schwann cells, olfactory ensheathing cells (OECs), neural stem cells or progenitor cells have been reported for their potential to form myelin, promote axonal regrowth and guidance, bridging

More than a dozen of clinical trials have been registered in the official website of clinical trials

**phase Study status**

Phase I / II Recruiting

Phase II Recruiting

Phase I Recruiting

Phase I / II Recruiting

Phase I / II Recruiting

Active, not recruiting

Active, not recruiting

Interventional, Phase I

Interventional,

Interventional,

Interventional,

Interventional,

Interventional, Phase I

Interventional,

(http://www.clinicaltrials.gov). A brief listing of the selected trials is given below.

**number Title/ Brief summary Study type/**

Phase I/II, multicenter, prospective, non-randomized, open label study to evaluate the safety/efficacy of autologous bone marrow-derived stem cell transplantation in spinal cord injury patients.

To study the efficacy difference between Rehabilitation Therapy and Umbilical Cord Derived Mesenchymal

2. To determine if late functional outcome is improved

A phase I/II trial designed to establish the safety and efficacy of intravenous combined with intrathecal administration of autologous bone marrow derived

A Phase I, single-center trial to assess the safety and tolerability of an intrathecal infusion (lumbar puncture) of autologous, ex vivo expanded bone marrow-derived

A Phase I/II Study of the Safety and Preliminary Efficacy of Intramedullary Spinal Cord Transplantation of Human Central Nervous System (CNS) Stem Cells

Stem Cells transplantation

mesenchymal stem cells

mesenchymal stem cells

1. To see if Bone Marrow Cell harvest and transplantation are safe in children and

following Bone Marrow Cell transplantation.

To evaluate autologous bone marrow stem cells transplantation as a safe and potentially beneficial treatment for patients with spinal cord injury

> In an article published, Wolfram Tetzlaff et al has reviewed in detail, all the types of cells being used in the treatment of spinal cord injury from the available pre-clinical literature. Their review shows that rodent stem cells have been most extensively studied for SCI. Limited studies have been done on human stem cells. Majority of trials are with bone marrow stromal cells. Also reported was, while chronic treatments were rare and often failed to yield functional benefits, all the preclinical studies conducted, was in acute and subacute stage [8].

> Also Fehlings et al [9] in his recently reviewed article has shown the efficacies and limitations of every type of cells, either alone or in various combinations as registered for trial studies, in use and has demonstrated the potential use of other promising candidate stem cells evaluated in pre-clinical studies but are not yet in Clinical Trials. Also they have made recommendations for the conduct and evaluation of pre-clinical studies and clinical trials of cell therapies for SCI [9].

> However no clinical intervention is risk free and we require understanding more on the pathophysiology of SCI and the clinical potential of stem cells to translate the use of the same as a therapeutic agent.

#### NPCs/OPCs:

Geron conducted a Phase 1 clinical trial in the United States in October 2010, to evaluate the safety of human embryonic stem cell-based product candidate, GRNOPC1, in patients with thoracic spinal cord injuries. Accordingly, GRNOPC1, an investigational product for treatment of Spinal Cord Injury, is a population of living cells containing oligodendrocyte progenitor cells (OPC).

neuro-rehabilitation. The authors conclude that *in vitro* cultures of MSCs and anti CNS T cells

Mesenchymal Stem Cells in Spinal Cord Injury

http://dx.doi.org/10.5772/58323

249

Kang *et al.,* 2005 [109] transplanted human umbilical cord cells into a 37 year old female with T11/T12 complete injury and have observed recovery but have not ruled out the fact that the laminectomy itself may have released compressed areas of the spinal cord and brought about

Zhou *et al.,* 2004 differentiated BM-MSCs into neural stem cells and transplanted them into SCI patients. 3 patients reported adverse events of intracranial infection requirement treat‐ ment. This study has not mentioned the baseline status of the patients, neither the details of

Deda *et al.,* 2009 [110], reported that autologous hematopoietic progenitor stem cells are an effective and safe method for treatment of chronic SCI. In this study autologous hematopoietic progenitor stem cells were injected at the site of injury and three weeks post transplantation

Realizing the unmet medical need in producing reasonable clinical recovery in spinal cord injury we have designed a preclinical experimental animal study. We developed a rodent model of spinal cord contusion injury and transplanted bone marrow derieved mesenchymal stem cells both at the site of injury and into the CSF using lumbar puncture technique. The results were very encouraging [111]. Motivated by this an initial pilot study was conducted on 10 patients with chronic spinal cord injury. The initial results showed only partial sensory improvement. Only 2 patients showed minimal motor improvement but not clinically useful. Surprisingly 4 of them showed reasonable improvement in bladder function. This fact has triggered further interest in us to pursue this and try different methods to improvise the clinical results. Though an attempt was made to quantify the recovery, none of the existing methods

But this study has raised several questions like a)Timing of intervention) Route of cell administration c)Dosage of cells D)Type of cell e) Number and interval of doses f)Autologous vs allogenic MSC g) problems of chronic injury h)Method of monitoring, evaluation and

In our further study we attempted to address some of these questions: 1) route-Intrathecal, direct at the site of injury, Direct delivery into the cord during surgery 2) excision of scar 3) scaffold to bridge the damaged ends of the cord 4) number of injections 5) number of cells 6) Cell type – mesenchymal autologous, allogenic & mononuclear 7) source-bone marrow,adi‐ pose and Wharton jelly 8) additional systemic injections. 100 volunteers with clinically complete cord injury were recruited. Clinical, MRI and tractography were done at baseline

and at periodic intervals to monitor the course of events post stem cell infusion.

can induce transdifferentiation of MSCs into neural stem cells.

the follow-up study conducted nor the details of the intervention.

the patients have demonstrated improved sensory and motor functions.

recovery.

**7. Our experience**

were satisfactory.

quantifying the results.

#### HUMSCs:

WJCs can undergo repeated freeze–thaw cycles without a significant loss of viability, meso‐ dermal differentiation potential, and without accumulating karyotypic abnormalities and thus represent a potential for the treatment of the neurodegenerative disorders including SCI. Two studies so far have examined the use of WJCs in SCI models, but were poorly conceived and designed.

#### **6.6. Ongoing clinical trials for spinal cord injury using stem cells**

Based on the encouraging preclinical animal results, Sarel et al, has conducted a phase II clinical trial of a cell therapy for patients with acute spinal cord injury using monocytes isolated from peripheral blood of human donors. They were able to stimulate by co-incubation with skin tissue, producing a distinct cellular phenotype which is said to be associated with wound healing. These features of skin-co incubated macrophages suggest possible mechanisms by which they may support an immune response that promotes neuronal cell survival and repair.

Jones *et al.,* 2004 [105] observed the long-term outcomes after complete spinal cord injury followed by subsequent treatment with a therapy consisting of autologous incubated macro‐ phages that have been pre-incubated with autologous skin and injected into the lesion site. The study so far has been conducted on 14 patients. Recovery of clinically significant neuro‐ logical function has been observed in several subjects after treatment, whereas untreated patients with complete SCI rarely recover significant function.

Auerbach *et al.,* 2004 [106], has conducted open-label, non-randomized trials to assess the safety of autologous macrophages in 16 patients with acute complete spinal cord injury. The macrophages were prepared from monocytes isolated from patient blood and co-incubated with autologous skin tissue. The cells were then injected into the spinal cord parenchyma within 14 days of injury. The study shows that administration of autologous macrophages has a favorable benefit to risk ratio for the treatment of patients with acute, complete spinal cord injury.

Keirstead *et al.,* 2005 [107] have shown human embryonic stem cells differentiate into oligo‐ dendrocytes in high purity and showed regeneration of the spinal cord in rat. On the basis of this study Geron Inc is currently conducting a FDA approved phase-I clinical trial.

Moviglia *et al.,* 2006, [108] demonstrated a case report of two patients who were administered BM-MSCs co cultured with an autologous pure population of T cells, intravenously 48 hours prior to transplantation of trans-differentiated NCS. This was followed up with 6 months of neuro-rehabilitation. The authors conclude that *in vitro* cultures of MSCs and anti CNS T cells can induce transdifferentiation of MSCs into neural stem cells.

Kang *et al.,* 2005 [109] transplanted human umbilical cord cells into a 37 year old female with T11/T12 complete injury and have observed recovery but have not ruled out the fact that the laminectomy itself may have released compressed areas of the spinal cord and brought about recovery.

Zhou *et al.,* 2004 differentiated BM-MSCs into neural stem cells and transplanted them into SCI patients. 3 patients reported adverse events of intracranial infection requirement treat‐ ment. This study has not mentioned the baseline status of the patients, neither the details of the follow-up study conducted nor the details of the intervention.

Deda *et al.,* 2009 [110], reported that autologous hematopoietic progenitor stem cells are an effective and safe method for treatment of chronic SCI. In this study autologous hematopoietic progenitor stem cells were injected at the site of injury and three weeks post transplantation the patients have demonstrated improved sensory and motor functions.

#### **7. Our experience**

NPCs/OPCs:

248 Topics in Paraplegia

cells (OPC).

HUMSCs:

designed.

injury.

Geron conducted a Phase 1 clinical trial in the United States in October 2010, to evaluate the safety of human embryonic stem cell-based product candidate, GRNOPC1, in patients with thoracic spinal cord injuries. Accordingly, GRNOPC1, an investigational product for treatment of Spinal Cord Injury, is a population of living cells containing oligodendrocyte progenitor

WJCs can undergo repeated freeze–thaw cycles without a significant loss of viability, meso‐ dermal differentiation potential, and without accumulating karyotypic abnormalities and thus represent a potential for the treatment of the neurodegenerative disorders including SCI. Two studies so far have examined the use of WJCs in SCI models, but were poorly conceived and

Based on the encouraging preclinical animal results, Sarel et al, has conducted a phase II clinical trial of a cell therapy for patients with acute spinal cord injury using monocytes isolated from peripheral blood of human donors. They were able to stimulate by co-incubation with skin tissue, producing a distinct cellular phenotype which is said to be associated with wound healing. These features of skin-co incubated macrophages suggest possible mechanisms by which they may support an immune response that promotes neuronal cell survival and repair.

Jones *et al.,* 2004 [105] observed the long-term outcomes after complete spinal cord injury followed by subsequent treatment with a therapy consisting of autologous incubated macro‐ phages that have been pre-incubated with autologous skin and injected into the lesion site. The study so far has been conducted on 14 patients. Recovery of clinically significant neuro‐ logical function has been observed in several subjects after treatment, whereas untreated

Auerbach *et al.,* 2004 [106], has conducted open-label, non-randomized trials to assess the safety of autologous macrophages in 16 patients with acute complete spinal cord injury. The macrophages were prepared from monocytes isolated from patient blood and co-incubated with autologous skin tissue. The cells were then injected into the spinal cord parenchyma within 14 days of injury. The study shows that administration of autologous macrophages has a favorable benefit to risk ratio for the treatment of patients with acute, complete spinal cord

Keirstead *et al.,* 2005 [107] have shown human embryonic stem cells differentiate into oligo‐ dendrocytes in high purity and showed regeneration of the spinal cord in rat. On the basis of

Moviglia *et al.,* 2006, [108] demonstrated a case report of two patients who were administered BM-MSCs co cultured with an autologous pure population of T cells, intravenously 48 hours prior to transplantation of trans-differentiated NCS. This was followed up with 6 months of

this study Geron Inc is currently conducting a FDA approved phase-I clinical trial.

**6.6. Ongoing clinical trials for spinal cord injury using stem cells**

patients with complete SCI rarely recover significant function.

Realizing the unmet medical need in producing reasonable clinical recovery in spinal cord injury we have designed a preclinical experimental animal study. We developed a rodent model of spinal cord contusion injury and transplanted bone marrow derieved mesenchymal stem cells both at the site of injury and into the CSF using lumbar puncture technique. The results were very encouraging [111]. Motivated by this an initial pilot study was conducted on 10 patients with chronic spinal cord injury. The initial results showed only partial sensory improvement. Only 2 patients showed minimal motor improvement but not clinically useful. Surprisingly 4 of them showed reasonable improvement in bladder function. This fact has triggered further interest in us to pursue this and try different methods to improvise the clinical results. Though an attempt was made to quantify the recovery, none of the existing methods were satisfactory.

But this study has raised several questions like a)Timing of intervention) Route of cell administration c)Dosage of cells D)Type of cell e) Number and interval of doses f)Autologous vs allogenic MSC g) problems of chronic injury h)Method of monitoring, evaluation and quantifying the results.

In our further study we attempted to address some of these questions: 1) route-Intrathecal, direct at the site of injury, Direct delivery into the cord during surgery 2) excision of scar 3) scaffold to bridge the damaged ends of the cord 4) number of injections 5) number of cells 6) Cell type – mesenchymal autologous, allogenic & mononuclear 7) source-bone marrow,adi‐ pose and Wharton jelly 8) additional systemic injections. 100 volunteers with clinically complete cord injury were recruited. Clinical, MRI and tractography were done at baseline and at periodic intervals to monitor the course of events post stem cell infusion.

#### **8. Study plan**

The objective of this study was to demonstrate the safety and feasibility of various stem cells as a possible therapeutic strategy for Spinal cord injury. For this, 52 volunteers were recruited and grouped into 4, on the basis of stem cells they received for the treatment. Group 1 received autologous bone marrow derived mononuclear cells (BMMNCs) for transplantation, group 2 were infused with autologous bone marrow derived Mesenchymal stem cells (BMMSCs), while group 3 were transplanted with different allogeneic stem cells (subgroup 1: Bone Marrow derived Mesenchymal cells, subgroup 2: Wharton's Jelly derived Mesenchymal stem cells (WJMSCs) and subgroup 3: Adipose Tissue derived Mesenchymal cells (ADMSCs). Also, in this study, we demonstrated, delivery of stem cells via 3 different routes (laminectomy, lumbar puncture, site of injury guided by CT scan and intravenous delivery) were safe and feasible and do not cause any infections and adverse reactions post transplantation.

**c.** Screening of the patients:

procedure.

**d.** Isolation and propagation of stem cells:

and expanded using a method reported previously [1].

Before enrollment each patient was screened for HIV: Human Immunodeficiency Virus; HBV: Hepatitis B Virus; HCV: Hepatitis C Virus; CMV: Cytomegalovirus; and VDRL: Venereal

Mesenchymal Stem Cells in Spinal Cord Injury

http://dx.doi.org/10.5772/58323

251

Patients willing to undergo autologous cell transplantation were screened 7 days before the aspiration for infectious disease mentioned above. Thereafter, BM-derived MSC were isolated

Briefly, 60 ml BM was aspirated aseptically from the iliac crest of each patient under aseptic conditions. The BM was diluted (1:1) with Knockout Dulbecco's modified Eagle's medium (KO-DMEM) and centrifuged at 1800 r.p.m. for 10 min to remove anticoagulants. The super‐ natant was discarded and the BM washed once with culture medium. Mononuclear cells (MNC) were isolated by layering onto a lymphoprep (Axis Shield, Norway) density gradient. The MNC present in the buffy coat were washed again with culture medium. The mononuclear fractions containing MSC were plated at a density of 1000 cells/cm2 onto T-75cm2 flasks and cultured in KO-DMEM. The media were supplemented with 10% fetal bovine serum (FBS), 200 mM Glutamax and Pen-Strep. The cultures were maintained at 370C in a humidified 5% CO2 atmosphere for 2 days. The non-adherent cells were removed after 48 h of culture and replenished with fresh medium. Subsequently, the medium was replenished every 5th day until the required number of cells obtained. Once confluent, the culture flasks were washed with Dulbecco`s Phosphate Buffered Saline (DPBS) and harvested using 0.25% Trypsin-EDTA solution and re-plated in 5 cell stacks (Corning, USA) for further expansion till the required number of cells obtained. On the day of transplantation the cells were harvested and sus‐ pended in saline solution, packed in sterile container and given for the transplantation

Disease Research Laboratory, by a nationally certified testing laboratory.

**i. Autologous Bone Marrow derived Mesenchymal Stem Cells:**

**ii. Autologous Bone Marrow derived Mononuclear cells (MNCs):**

resuspended in the same and given for infusion.

All the patients were examined by a designated medically qualified staff member to establish their eligibility for bone marrow aspiration. Briefly, 60 ml BM was aspirated aseptically from the iliac crest of each patient under aseptic conditions. The BM was diluted (1:1) with Knockout Dulbecco's modified Eagle's medium (KO-DMEM) and centrifuged at 1800 r.p.m. for 10 min to remove anticoagulants. The supernatant was discarded and the BM washed once with culture medium. Mononuclear cells (MNC) were isolated by layering the bone marrow samples onto a lymphoprep (Axis Shield, Norway) density gradient. The MNC present in the buffy coat were washed again with culture medium and then with saline for 2-3 times,

**a.** Regulatory approval, Informed consent:

As per national guidelines, approval from institutional ethics committee (IEC) was taken and informed consent was obtained from every patient who participated in the study. Any deviations, drop-outs and adverse events were documented and the IEC informed.

**b.** Patient selection:

Patients were enrolled for this study as per the inclusion and exclusion criteria designed by and adapted in a pilot clinical study [1]. The inclusion Criteria in the study was as follows


Exclusion criteria for the study was


#### **c.** Screening of the patients:

**8. Study plan**

250 Topics in Paraplegia

**b.** Patient selection:

The objective of this study was to demonstrate the safety and feasibility of various stem cells as a possible therapeutic strategy for Spinal cord injury. For this, 52 volunteers were recruited and grouped into 4, on the basis of stem cells they received for the treatment. Group 1 received autologous bone marrow derived mononuclear cells (BMMNCs) for transplantation, group 2 were infused with autologous bone marrow derived Mesenchymal stem cells (BMMSCs), while group 3 were transplanted with different allogeneic stem cells (subgroup 1: Bone Marrow derived Mesenchymal cells, subgroup 2: Wharton's Jelly derived Mesenchymal stem cells (WJMSCs) and subgroup 3: Adipose Tissue derived Mesenchymal cells (ADMSCs). Also, in this study, we demonstrated, delivery of stem cells via 3 different routes (laminectomy, lumbar puncture, site of injury guided by CT scan and intravenous delivery) were safe and

feasible and do not cause any infections and adverse reactions post transplantation.

deviations, drop-outs and adverse events were documented and the IEC informed.

**iii.** the level of spinal injury between C4 and T10 level (neurologic),

As per national guidelines, approval from institutional ethics committee (IEC) was taken and informed consent was obtained from every patient who participated in the study. Any

Patients were enrolled for this study as per the inclusion and exclusion criteria designed by and adapted in a pilot clinical study [1]. The inclusion Criteria in the study was as follows

**iv.** (SCI was clinically complete and categorized as per the American Spinal Injury

**i.** Difficulty in assessing the size and location of the injury multiple sites of injury,

**iii.** serious pre-existing medical conditions, disease or impairment that precluded

**a.** Regulatory approval, Informed consent:

**i.** the patients could be of either sex,

Exclusion criteria for the study was

**viii.** Fixed deformities.

**ii.** gun shot or penetrating injuries,

**ii.** must be between the ages 18 and 55 years,

Association (ASIA) impairment scale.

adequate neurologic examination **iv.** Respiratory insufficiency requiring support. **v.** if he/she is enrolled in any other clinical trial

**vi.** Not able to understand and comply with follow up

**vii.** Diagnosed with infections like HIV, HCV,CMV and VDRL.

Before enrollment each patient was screened for HIV: Human Immunodeficiency Virus; HBV: Hepatitis B Virus; HCV: Hepatitis C Virus; CMV: Cytomegalovirus; and VDRL: Venereal Disease Research Laboratory, by a nationally certified testing laboratory.

**d.** Isolation and propagation of stem cells:

#### **i. Autologous Bone Marrow derived Mesenchymal Stem Cells:**

Patients willing to undergo autologous cell transplantation were screened 7 days before the aspiration for infectious disease mentioned above. Thereafter, BM-derived MSC were isolated and expanded using a method reported previously [1].

Briefly, 60 ml BM was aspirated aseptically from the iliac crest of each patient under aseptic conditions. The BM was diluted (1:1) with Knockout Dulbecco's modified Eagle's medium (KO-DMEM) and centrifuged at 1800 r.p.m. for 10 min to remove anticoagulants. The super‐ natant was discarded and the BM washed once with culture medium. Mononuclear cells (MNC) were isolated by layering onto a lymphoprep (Axis Shield, Norway) density gradient. The MNC present in the buffy coat were washed again with culture medium. The mononuclear fractions containing MSC were plated at a density of 1000 cells/cm2 onto T-75cm2 flasks and cultured in KO-DMEM. The media were supplemented with 10% fetal bovine serum (FBS), 200 mM Glutamax and Pen-Strep. The cultures were maintained at 370C in a humidified 5% CO2 atmosphere for 2 days. The non-adherent cells were removed after 48 h of culture and replenished with fresh medium. Subsequently, the medium was replenished every 5th day until the required number of cells obtained. Once confluent, the culture flasks were washed with Dulbecco`s Phosphate Buffered Saline (DPBS) and harvested using 0.25% Trypsin-EDTA solution and re-plated in 5 cell stacks (Corning, USA) for further expansion till the required number of cells obtained. On the day of transplantation the cells were harvested and sus‐ pended in saline solution, packed in sterile container and given for the transplantation procedure.

#### **ii. Autologous Bone Marrow derived Mononuclear cells (MNCs):**

All the patients were examined by a designated medically qualified staff member to establish their eligibility for bone marrow aspiration. Briefly, 60 ml BM was aspirated aseptically from the iliac crest of each patient under aseptic conditions. The BM was diluted (1:1) with Knockout Dulbecco's modified Eagle's medium (KO-DMEM) and centrifuged at 1800 r.p.m. for 10 min to remove anticoagulants. The supernatant was discarded and the BM washed once with culture medium. Mononuclear cells (MNC) were isolated by layering the bone marrow samples onto a lymphoprep (Axis Shield, Norway) density gradient. The MNC present in the buffy coat were washed again with culture medium and then with saline for 2-3 times, resuspended in the same and given for infusion.

#### **9. Scaffolds**

Scaffolds are basically structures to support and connect the cut ends of spinal cord. They are used after scar excision or otherwise in chronic injuries to bridge the healthy ends. The stem cells are deposited over the membrane. It helps to hold the cells in place, and grows along using this as support.We have used Gelfoam as well as a special biological membrane, which is inert and biocompatible made out of Chitosin. It is a thin and transparent glucosamine polymer. Stem cells have grown in sheets over this membrane in our invitro studies.

for additional 4 or 5 days till it reached 80-85% confluency. On confluency, confluence, adherent cells were detached by treatment with a Trypsin-EDTA solution and re-plated at a density of 1000 cells/cm2 in 5 cell stacks and cultured in the same condition for 14-16. The cell stacks were checked regularly and replenished with medium on every 5th day. The cells were then harvested at 80-90% confluency and cryopreserved in 10% Dimethyl Sulfoxide (DMSO, Sigma-Aldrich) and 85% Plasmalyte (Baxter, USA) and 5% Human Serum Albumin (HSA,

The use of lipoaspirate as a source for stem cells with multipotent differentiation poten‐ tial offers a far less invasive procedure for cell sampling than the aspiration of bone marrow (BM), and numbers of stem cells obtained are reportedly higher in lipoaspirate than its BM counterpart. Lipoaspirate, an otherwise disposable byproduct of cosmetic surgery, has been shown to contain a putative population of stem cells, termed adipose-derived stem cells (ADSCs) that share many similarities to marrow stromal cells (MSCs) from BM, including multilineage differentiation capacity. Furthermore, these cells also show high colonyforming unit frequencies as well as an apparent pluripotent ability to differentiate to cells

This protocol describes the preparation of MSCs from human lipoaspirate obtained from cosmetic surgery. Briefly, the liposuctioned fat first washed thoroughly in phosphate-buffered saline (PBS) with antibiotic solution (Penstrep, 2X), until the bottom layer containing blood cells contaminant was clear, before being subjected to enzymatic digestion using collagenase type I (0.2%, diluted in KO-DMEM) for 45-60 minutes at 37°C in shaking condition, in order to obtain a soupy single-cell suspension. After digestion, the action of collagenase was neutralized by the addition of FBS. The suspension was then mixed well and passed through 40 μm cell strainer before being subjected to centrifugation at 1400 rpm for 10 minutes. After centrifugation, cell pellet, termed as stromal vascular fraction (SVF) is resuspended in KO-

flask at a density of 1,000 cells/cm2

were removed after 48 h of culture and replenished with fresh medium. Subsequently, the medium was replenished every 4th day and the cells were harvested at 80% confluency and replated in 5 cell stacks to obtain the sufficient number of cells required for the infusion. The plates were checked for confluence every day and the cells are fed with fresh medium. After the cell stacks were confluent enough, the cells were harvested using Trypsin-EDTA solution

Studies have demonstrated the multipotent properties of mesenchymal stromal cells isolated from the inner matrix of the Wharton's Jelly derived from the umbilical cord. These cells have

. The non-adherent cells

Mesenchymal Stem Cells in Spinal Cord Injury

http://dx.doi.org/10.5772/58323

253

Baxter) in liquid nitrogen till further use.

of a neuronal phenotype [9, 10].

DMEM and seeded in a T-75cm2

and cryopreserved in liquid nitrogen till further use.

**13. Wharton's Jelly derived mesenchymal stem cells**

**12. Adipose tissue derived mesenchymal stem cells**

In acute phase, the chemical changes resulted out of injury presumably attracts stem cells even after remote injection whereas in chronic injuries there are additional problems.

1. Scar intervenes ends of normal cords 2.In severe injuries there is thinning and atrophy causing anatomical discontinuity. 3. Due to ongoing degeneration there is a functional void between the two ends, with or without an abnormal cord intervening. The main purpose of scaffolds is to bridge this gap and create continuity for the cells to reach both ends.

#### **10. Screening of potential donors for bone marrow aspiration**

Potential voluntary donors were interviewed, counseled and examined by the investigator or a designated medically qualified staff member to establish their eligibility for bone marrow aspiration. Donors were informed with full description about the nature and purpose of the aspiration and written consent were obtained from them before proceeding with study. Some of the inclusion criteria include (i) the donor must be healthy (ii) may be of either sex (iii) must be between 18-30 years of age (iv) able to understand the voluntary donation program, and ready to provide voluntary written informed consent. The donors were excluded if (i) diagnosed with a past history of illness such as autoimmune disorders, tuberculosis, malaria and any other infection, any illness which precludes the use of general anesthesia, history of malignancy, diabetes, hypertension, significant heart disease, genetic or chromosomal disorders, history of any inherited disorders, hemoglobin less than 10, and pregnant women. Also, at the time of obtaining informed consent they were screened for infection with human immunodeficiency virus (HIV), hepatitis B (HBV), hepatitis C (HCV), cytomegalovirus (CMV), and syphilis (VDRL) and excluded, if found positive.

#### **11. Allogeneic BM-MSCs**

As per the donor selection criteria, donors were recruited and bone marrow samples were aspirated from the iliac crest of the donors and further processed for the isolation of mono‐ nuclear fraction using Lymphoprep (Axis Shield, Norway) density gradient. Thus obtained fraction was seeded in T-75cm2 and cultured at 37°C in 5% CO2 atmosphere. The non-adherent cells were removed after 48 hours by replacing the medium and the adherent cells were grown for additional 4 or 5 days till it reached 80-85% confluency. On confluency, confluence, adherent cells were detached by treatment with a Trypsin-EDTA solution and re-plated at a density of 1000 cells/cm2 in 5 cell stacks and cultured in the same condition for 14-16. The cell stacks were checked regularly and replenished with medium on every 5th day. The cells were then harvested at 80-90% confluency and cryopreserved in 10% Dimethyl Sulfoxide (DMSO, Sigma-Aldrich) and 85% Plasmalyte (Baxter, USA) and 5% Human Serum Albumin (HSA, Baxter) in liquid nitrogen till further use.

#### **12. Adipose tissue derived mesenchymal stem cells**

**9. Scaffolds**

252 Topics in Paraplegia

Scaffolds are basically structures to support and connect the cut ends of spinal cord. They are used after scar excision or otherwise in chronic injuries to bridge the healthy ends. The stem cells are deposited over the membrane. It helps to hold the cells in place, and grows along using this as support.We have used Gelfoam as well as a special biological membrane, which is inert and biocompatible made out of Chitosin. It is a thin and transparent glucosamine

In acute phase, the chemical changes resulted out of injury presumably attracts stem cells even

1. Scar intervenes ends of normal cords 2.In severe injuries there is thinning and atrophy causing anatomical discontinuity. 3. Due to ongoing degeneration there is a functional void between the two ends, with or without an abnormal cord intervening. The main purpose of

Potential voluntary donors were interviewed, counseled and examined by the investigator or a designated medically qualified staff member to establish their eligibility for bone marrow aspiration. Donors were informed with full description about the nature and purpose of the aspiration and written consent were obtained from them before proceeding with study. Some of the inclusion criteria include (i) the donor must be healthy (ii) may be of either sex (iii) must be between 18-30 years of age (iv) able to understand the voluntary donation program, and ready to provide voluntary written informed consent. The donors were excluded if (i) diagnosed with a past history of illness such as autoimmune disorders, tuberculosis, malaria and any other infection, any illness which precludes the use of general anesthesia, history of malignancy, diabetes, hypertension, significant heart disease, genetic or chromosomal disorders, history of any inherited disorders, hemoglobin less than 10, and pregnant women. Also, at the time of obtaining informed consent they were screened for infection with human immunodeficiency virus (HIV), hepatitis B (HBV), hepatitis C (HCV), cytomegalovirus (CMV),

As per the donor selection criteria, donors were recruited and bone marrow samples were aspirated from the iliac crest of the donors and further processed for the isolation of mono‐ nuclear fraction using Lymphoprep (Axis Shield, Norway) density gradient. Thus obtained

cells were removed after 48 hours by replacing the medium and the adherent cells were grown

and cultured at 37°C in 5% CO2 atmosphere. The non-adherent

polymer. Stem cells have grown in sheets over this membrane in our invitro studies.

after remote injection whereas in chronic injuries there are additional problems.

scaffolds is to bridge this gap and create continuity for the cells to reach both ends.

**10. Screening of potential donors for bone marrow aspiration**

and syphilis (VDRL) and excluded, if found positive.

**11. Allogeneic BM-MSCs**

fraction was seeded in T-75cm2

The use of lipoaspirate as a source for stem cells with multipotent differentiation poten‐ tial offers a far less invasive procedure for cell sampling than the aspiration of bone marrow (BM), and numbers of stem cells obtained are reportedly higher in lipoaspirate than its BM counterpart. Lipoaspirate, an otherwise disposable byproduct of cosmetic surgery, has been shown to contain a putative population of stem cells, termed adipose-derived stem cells (ADSCs) that share many similarities to marrow stromal cells (MSCs) from BM, including multilineage differentiation capacity. Furthermore, these cells also show high colonyforming unit frequencies as well as an apparent pluripotent ability to differentiate to cells of a neuronal phenotype [9, 10].

This protocol describes the preparation of MSCs from human lipoaspirate obtained from cosmetic surgery. Briefly, the liposuctioned fat first washed thoroughly in phosphate-buffered saline (PBS) with antibiotic solution (Penstrep, 2X), until the bottom layer containing blood cells contaminant was clear, before being subjected to enzymatic digestion using collagenase type I (0.2%, diluted in KO-DMEM) for 45-60 minutes at 37°C in shaking condition, in order to obtain a soupy single-cell suspension. After digestion, the action of collagenase was neutralized by the addition of FBS. The suspension was then mixed well and passed through 40 μm cell strainer before being subjected to centrifugation at 1400 rpm for 10 minutes. After centrifugation, cell pellet, termed as stromal vascular fraction (SVF) is resuspended in KO-DMEM and seeded in a T-75cm2 flask at a density of 1,000 cells/cm2 . The non-adherent cells were removed after 48 h of culture and replenished with fresh medium. Subsequently, the medium was replenished every 4th day and the cells were harvested at 80% confluency and replated in 5 cell stacks to obtain the sufficient number of cells required for the infusion. The plates were checked for confluence every day and the cells are fed with fresh medium. After the cell stacks were confluent enough, the cells were harvested using Trypsin-EDTA solution and cryopreserved in liquid nitrogen till further use.

#### **13. Wharton's Jelly derived mesenchymal stem cells**

Studies have demonstrated the multipotent properties of mesenchymal stromal cells isolated from the inner matrix of the Wharton's Jelly derived from the umbilical cord. These cells have also been demonstrated to differentiate into neuronal lineage and supporting glia [11, 12]. Based on these studies, in the current trial we have attempted to understand the therapeutic potential of WJ-MSCs in spinal cord injury.

phosphate (Sigma-Aldrich Chemical Private Limited, Bangalore, Karnataka, India) for 3 weeks. Fresh medium was replenished every 3 days. Calcium accumulation was assessed by von Kossa staining. The differentiated cells were washed with PBS and fixed with 10% formalin for 30 min. The fixed cells were incubated with 5% AgNO3 for 60 min under ultraviolet (UV) light and then treated with 2.5% sodium thiosulphate for 5 min. Images were captured using an Nikon Eclipse 90i microscope (Nikon Corporation, Towa Optics, New Delhi, India; www.nikon.com) and Image-Pro Express soft ware (Media Cybernetics Inc., Silver Spring,

Mesenchymal Stem Cells in Spinal Cord Injury

http://dx.doi.org/10.5772/58323

255

To induce *adipogenic differentiation*, human MSCs were cultured for 21 days in KO-DMEM supplemented with 10% FBS, 200 mM Glutamax, 1 μm dexamethasone, 0.5 mM isobutylme‐ thylxanthine, 1 μg/ml insulin and 100 μm indomethacin (from Sigma-Aldrich). Inducing factors were fixed in 10% formalin for 20 min and 200 μl Oil Red O staining solution added and incubated for 10 min at room temperature. The cells were rinsed five times with distilled water. The images were captured using Nikon Eclipse 90i microscope (Nikon) and Image-Pro

For *chondrogenic differentiation*, human MSCs were cultured for 21 days using Chondrogenesis differentiation kit (Life Technologies, USA) as per the manufacturer's recommendations and stained with Safranin O as specified. The images were captured using Nikon Eclipse 90i

A standard G-banding protocol was performed by analyzing more than 200 cells per sample and reported according to the International System for Human Cytogenetic Nomenclature (ISCN). If the cells did not fall under the set standard of the above mentioned tests, they would

Based on the ISCT guidelines, certain quality control tests were performed on the end product before transplantation. These include Mycoplasma (using RT-PCR based method), Endotoxin testing by Limulus Amebocyte Lysate (LAL) method and cell surface markers like CD73, CD90, CD105, CD166, CD34, and CD45 via flow cytometry. The positive markers (CD73, CD90, CD105 and CD166) should be greater than 95% positive, while the negative markers (CD34 and CD45) must be less than 2% positive. 7-AAD (7-amino actinomycin D) was also analyzed

As described above, the cells were harvested and processed for transplantation. Briefly, the total cell count was calculated using a standard hemocytometer. The cells were washed several times with normal saline solution and finally resuspended in saline containing 0.2% human serum albumin. All the syringes and bottles were appropriately labeled. These were packaged

not be released to the patient for transplantation was discarded appropriately.

microscope (Nikon Corporation, Towa Optics, New Delhi, India).

MD, USA; www. mediacy.com).

Express software (Media Cybernetics).

**14.1. Karyotyping**

**14.2. Quality control testing**

via flow cytometry to determine the cell viability.

**14.3. Processing of cells for transplantation**

After appropriate informed consent, a clean, healthy, straight clamped umbilical cord approx‐ imately 10 cms in length was collected in sterile normal saline bottle and transported to the laboratory. Briefly, the umbilical cord was washed with normal saline followed by DPBS (with 0.2% of Penstrep solution) wash for 3-4 times. This was followed by quick dip in 100% ethanol and was cleared off in DPBS. The tissue was washed free of contaminating blood with normal saline throughout the process and cut into 2-5 mm3 pieces. Using sterile scalpel and forceps the cord was dissected, unfolded and the exposed arteries and vein were removed and discarded. The cord was then scrapped gently with scalpel to obtain the viscous, jelly like substance. The obtained suspension was passed through a 100 mm cell strainer to obtain singlecell suspension. The resultant suspension was then diluted with saline to reduce the viscosity of the suspension. Cells were centrifuged at 1400 rpm for 10 minutes at 37°C and the pellet was resuspended in KO-DMEM supplemented with FBS (10%), Glutamax (1%) , Penstrep (0.5%), FGF-2 (1ng/ml) and cultured in T-75cm2 flasks at 370 C in 5% CO2 until confluent (80-85%). Upon confluency, the cells were harvested from the flasks and transferred to 5 cell stacks at a seeding density of 1000 cells/cm2 for 10-12 days in order to obtain the required number of cells for the transplantation. The harvested cells were processed and frozen in cryobags in liquid nitrogen till use.

#### **14. Characterization**

**1.** Immunophenotype:

This is a technique used to study the expression of cell surface antigens on the MSCs using flow cytometry. Briefly, the cells were dissociated with 0.25% Trypsin-EDTA and resuspended in wash buffer at a concentration of 1 × 10^6 cells/ml. 200 μL cell suspensions were incubated in the dark for 15 min at 4°C with saturating concentrations of phycoerythrin (PE) conjugated antibodies. The following markers were analyzed: CD34-PE, CD45-PE, CD73-PE, CD105-PE, CD166-PE, and CD90-PE (BD Pharmingen, San Diego, CA, USA). Flow cytometry was performed on a 5HT Guava instrument. Appropriate isotype-matched controls were used to set the instrument parameters. Cell viability was measured using 7-amino actinomycin D (7- AAD). Cells were identified by light scatter for 10,000 gated events and analyzed.

**2.** Multipotent differentiation assay

The mesenchymal properties of human stem cells isolated from various sources as described above, were investigated using specific differentiation kits for the three different lineages i.e., osteogenic, adipogenic and chondrogenic (as per ISCT criteria).

Briefly, *Osteoblast differentiation* was induced by culturing human MSCs in KO-DMEM supplemented with 10% FBS (Hyclone), 200 26mM Glutamax (Invitrogen), 10-8 M dexa methasone (Sigma-Aldrich), 30 μgm/ml ascorbic acid (Sigma-Aldrich) and 10 mM β-glycero‐ phosphate (Sigma-Aldrich Chemical Private Limited, Bangalore, Karnataka, India) for 3 weeks. Fresh medium was replenished every 3 days. Calcium accumulation was assessed by von Kossa staining. The differentiated cells were washed with PBS and fixed with 10% formalin for 30 min. The fixed cells were incubated with 5% AgNO3 for 60 min under ultraviolet (UV) light and then treated with 2.5% sodium thiosulphate for 5 min. Images were captured using an Nikon Eclipse 90i microscope (Nikon Corporation, Towa Optics, New Delhi, India; www.nikon.com) and Image-Pro Express soft ware (Media Cybernetics Inc., Silver Spring, MD, USA; www. mediacy.com).

To induce *adipogenic differentiation*, human MSCs were cultured for 21 days in KO-DMEM supplemented with 10% FBS, 200 mM Glutamax, 1 μm dexamethasone, 0.5 mM isobutylme‐ thylxanthine, 1 μg/ml insulin and 100 μm indomethacin (from Sigma-Aldrich). Inducing factors were fixed in 10% formalin for 20 min and 200 μl Oil Red O staining solution added and incubated for 10 min at room temperature. The cells were rinsed five times with distilled water. The images were captured using Nikon Eclipse 90i microscope (Nikon) and Image-Pro Express software (Media Cybernetics).

For *chondrogenic differentiation*, human MSCs were cultured for 21 days using Chondrogenesis differentiation kit (Life Technologies, USA) as per the manufacturer's recommendations and stained with Safranin O as specified. The images were captured using Nikon Eclipse 90i microscope (Nikon Corporation, Towa Optics, New Delhi, India).

#### **14.1. Karyotyping**

also been demonstrated to differentiate into neuronal lineage and supporting glia [11, 12]. Based on these studies, in the current trial we have attempted to understand the therapeutic

After appropriate informed consent, a clean, healthy, straight clamped umbilical cord approx‐ imately 10 cms in length was collected in sterile normal saline bottle and transported to the laboratory. Briefly, the umbilical cord was washed with normal saline followed by DPBS (with 0.2% of Penstrep solution) wash for 3-4 times. This was followed by quick dip in 100% ethanol and was cleared off in DPBS. The tissue was washed free of contaminating blood with normal

the cord was dissected, unfolded and the exposed arteries and vein were removed and discarded. The cord was then scrapped gently with scalpel to obtain the viscous, jelly like substance. The obtained suspension was passed through a 100 mm cell strainer to obtain singlecell suspension. The resultant suspension was then diluted with saline to reduce the viscosity of the suspension. Cells were centrifuged at 1400 rpm for 10 minutes at 37°C and the pellet was resuspended in KO-DMEM supplemented with FBS (10%), Glutamax (1%) , Penstrep

(80-85%). Upon confluency, the cells were harvested from the flasks and transferred to 5 cell

number of cells for the transplantation. The harvested cells were processed and frozen in

This is a technique used to study the expression of cell surface antigens on the MSCs using flow cytometry. Briefly, the cells were dissociated with 0.25% Trypsin-EDTA and resuspended in wash buffer at a concentration of 1 × 10^6 cells/ml. 200 μL cell suspensions were incubated in the dark for 15 min at 4°C with saturating concentrations of phycoerythrin (PE) conjugated antibodies. The following markers were analyzed: CD34-PE, CD45-PE, CD73-PE, CD105-PE, CD166-PE, and CD90-PE (BD Pharmingen, San Diego, CA, USA). Flow cytometry was performed on a 5HT Guava instrument. Appropriate isotype-matched controls were used to set the instrument parameters. Cell viability was measured using 7-amino actinomycin D (7-

The mesenchymal properties of human stem cells isolated from various sources as described above, were investigated using specific differentiation kits for the three different lineages i.e.,

Briefly, *Osteoblast differentiation* was induced by culturing human MSCs in KO-DMEM supplemented with 10% FBS (Hyclone), 200 26mM Glutamax (Invitrogen), 10-8 M dexa methasone (Sigma-Aldrich), 30 μgm/ml ascorbic acid (Sigma-Aldrich) and 10 mM β-glycero‐

AAD). Cells were identified by light scatter for 10,000 gated events and analyzed.

osteogenic, adipogenic and chondrogenic (as per ISCT criteria).

pieces. Using sterile scalpel and forceps

flasks at 370 C in 5% CO2 until confluent

for 10-12 days in order to obtain the required

potential of WJ-MSCs in spinal cord injury.

254 Topics in Paraplegia

saline throughout the process and cut into 2-5 mm3

(0.5%), FGF-2 (1ng/ml) and cultured in T-75cm2

stacks at a seeding density of 1000 cells/cm2

cryobags in liquid nitrogen till use.

**2.** Multipotent differentiation assay

**14. Characterization**

**1.** Immunophenotype:

A standard G-banding protocol was performed by analyzing more than 200 cells per sample and reported according to the International System for Human Cytogenetic Nomenclature (ISCN). If the cells did not fall under the set standard of the above mentioned tests, they would not be released to the patient for transplantation was discarded appropriately.

#### **14.2. Quality control testing**

Based on the ISCT guidelines, certain quality control tests were performed on the end product before transplantation. These include Mycoplasma (using RT-PCR based method), Endotoxin testing by Limulus Amebocyte Lysate (LAL) method and cell surface markers like CD73, CD90, CD105, CD166, CD34, and CD45 via flow cytometry. The positive markers (CD73, CD90, CD105 and CD166) should be greater than 95% positive, while the negative markers (CD34 and CD45) must be less than 2% positive. 7-AAD (7-amino actinomycin D) was also analyzed via flow cytometry to determine the cell viability.

#### **14.3. Processing of cells for transplantation**

As described above, the cells were harvested and processed for transplantation. Briefly, the total cell count was calculated using a standard hemocytometer. The cells were washed several times with normal saline solution and finally resuspended in saline containing 0.2% human serum albumin. All the syringes and bottles were appropriately labeled. These were packaged in a sterile container and dispatched in a transportation container maintained at 22°C to the hospital for transplantation via the shortest route.

**14.5. Follow up schedule**

**stem cells**

transplantation.

performed to observe structural changes, if any.

**15.2. Autologous bone marrow derived MSCs**

**15.1. Autologous BM derived MNCs**

**15.3. Allogenic BM derived MSCs**

At every follow-up, the patients were assessed clinically using the ASIA scale rating system and with the Barthel's index (BI) for degree of independence and patient rating. MRI was

Mesenchymal Stem Cells in Spinal Cord Injury

http://dx.doi.org/10.5772/58323

257

**15. Isolation and identification of mononuclear cells and mesenchymal**

BM samples were aspirated from the patients (n=9) after getting proper consent and the samples were processed in cGMP compliant clean room facility for the isolation of the MNCs following the standardized protocol as described above. CD34 expression was analyzed using PE conjugated CD34 antibody in flow cytometer and cell count was performed prior to

BM samples obtained from the patients (n=11) after getting proper consent were processed in cGMP compliant clean room facility for the isolation propagation and expansion following the standardized protocol as described above. Flow cytometry analysis revealed that cell samples with positive markers are >95% and <2% of negative markers with >90% viability with 7AAD staining indicating the cells were mesenchymal in nature. Multipotent characteristics, as determined by Oil Red O stain, Von Kossa stain and Safranin O stain, respectively indicates the cell samples undergo adipogenic, osteogenic and chondrogenic differentiation. Karyotypes of all the cell samples were normal and no abnormalities/aberrations were found after ex vivo propagation figure. Endotoxin test using LAL method and Mycoplasma test using RT-PCR

BM samples obtained from the donors after appropriate informed consent and the samples were processed in cGMP compliant clean room facility for the isolation, propagation and expansion. Stem cells thus extracted are cryopreserved as master cell bank (MCB) in liquid nitrogen. From MCB, working cell banks (WCB) were raised in tissue culture plates until required number of cells obtained for the infusion. The cells were then harvested and frozen as investigational product (IP) until use. Prior to transplantation the cells were thawed and processed further. The cell samples were found to express positive markers >95% and <2% for negative markers, with >95% viability, when stained with 7AAD as determined by flow cytometry indicating the Mesenchymal nature of the processed cells. Multipotent character‐ istics, as determined by Oil Red O stain, Von Kossa stain and Safranin O stain, respectively, indicating the cell samples undergo adipogenic, osteogenic and chondrogenic differentiation. Karyotype of all the cell samples was normal and no abnormalities/aberrations were found

were found to be negative indicating the cells were safe for transplantation.

#### **14.4. Route of administration**


Laminectomy-was performed where ever decompression was indicated with or without stabilization. Surgical technique includes prone position and exposure of lamina at appropriate level under general anesthesia. Dura was opened and injured cord was inspected.

Scar excision-In chronic injuries with glial scar or neuroma the intervening tissue was removed gently till healthy appearing tissue was seen under high magnification. Cavity was decompressed. The cells were injected into the ends of the cord tissue through an insulin syringe. If the edges are apart a scaffold or gelfoam was used to bridge the gap. Dura was closed water tight. If the cord was oedematous (acute injury) doroplasty was performed. Additional cells were delivered into intra thecal space and Laminectomy was closed using standard technique.

Image guidance method-In chronic complete injuries CT guided technique was used to deliver the cells directly at the site of injury.

Clinical assessment was performed on all patients based on the parameters of the ASIA impairment scale (American Spinal Injury Association). This was considered as the primary measurable outcome of the clinical study.


**Table 3.** ASIA impairment scale

#### **14.5. Follow up schedule**

in a sterile container and dispatched in a transportation container maintained at 22°C to the

**1.** Intrathecal administration through Lumbar Puncture (LP) method:. The pateinet was positioned in lateral decubitous position and the part prepared. Under aseptic conditions lumbar puncture was performed at lowest possible level usually L4-5 or L5-S1 levels. Once clear CSF was obtained the cells were delivered into the intra thecal space gently. The

**2.** Intra Venous – Regular intravenous infusion of cells in 50 ml saline administered into the

Laminectomy-was performed where ever decompression was indicated with or without stabilization. Surgical technique includes prone position and exposure of lamina at appropriate

Scar excision-In chronic injuries with glial scar or neuroma the intervening tissue was removed gently till healthy appearing tissue was seen under high magnification. Cavity was decompressed. The cells were injected into the ends of the cord tissue through an insulin syringe. If the edges are apart a scaffold or gelfoam was used to bridge the gap. Dura was closed water tight. If the cord was oedematous (acute injury) doroplasty was performed. Additional cells were delivered into intra thecal space and Laminectomy was

Image guidance method-In chronic complete injuries CT guided technique was used to deliver

Clinical assessment was performed on all patients based on the parameters of the ASIA impairment scale (American Spinal Injury Association). This was considered as the primary

C Incomplete: Motor function is preserved below the neurological level, and more than half of key muscles below

D Incomplete: Motor function is preserved below the neurological level, and at least half of key muscles below the

level under general anesthesia. Dura was opened and injured cord was inspected.

hospital for transplantation via the shortest route.

procedure was repeated as per the protocol.

**3.** At the site of injury-either by laminectomy or image guidance

A Complete: No motor or sensory function is preserved in the sacral segments S4-S5. B Incomplete: Sensory but not motor function is preserved below the neurological level and

peripheral veins of the hand.

closed using standard technique.

the cells directly at the site of injury.

Includes the sacral segments S4-S5.

**Table 3.** ASIA impairment scale

measurable outcome of the clinical study.

the neurological level have a muscle grade less than 3.

neurological level have a muscle grade of 3 or more. E Normal: Motor and sensory functions are normal.

**14.4. Route of administration**

256 Topics in Paraplegia

At every follow-up, the patients were assessed clinically using the ASIA scale rating system and with the Barthel's index (BI) for degree of independence and patient rating. MRI was performed to observe structural changes, if any.

#### **15. Isolation and identification of mononuclear cells and mesenchymal stem cells**

#### **15.1. Autologous BM derived MNCs**

BM samples were aspirated from the patients (n=9) after getting proper consent and the samples were processed in cGMP compliant clean room facility for the isolation of the MNCs following the standardized protocol as described above. CD34 expression was analyzed using PE conjugated CD34 antibody in flow cytometer and cell count was performed prior to transplantation.

#### **15.2. Autologous bone marrow derived MSCs**

BM samples obtained from the patients (n=11) after getting proper consent were processed in cGMP compliant clean room facility for the isolation propagation and expansion following the standardized protocol as described above. Flow cytometry analysis revealed that cell samples with positive markers are >95% and <2% of negative markers with >90% viability with 7AAD staining indicating the cells were mesenchymal in nature. Multipotent characteristics, as determined by Oil Red O stain, Von Kossa stain and Safranin O stain, respectively indicates the cell samples undergo adipogenic, osteogenic and chondrogenic differentiation. Karyotypes of all the cell samples were normal and no abnormalities/aberrations were found after ex vivo propagation figure. Endotoxin test using LAL method and Mycoplasma test using RT-PCR were found to be negative indicating the cells were safe for transplantation.

#### **15.3. Allogenic BM derived MSCs**

BM samples obtained from the donors after appropriate informed consent and the samples were processed in cGMP compliant clean room facility for the isolation, propagation and expansion. Stem cells thus extracted are cryopreserved as master cell bank (MCB) in liquid nitrogen. From MCB, working cell banks (WCB) were raised in tissue culture plates until required number of cells obtained for the infusion. The cells were then harvested and frozen as investigational product (IP) until use. Prior to transplantation the cells were thawed and processed further. The cell samples were found to express positive markers >95% and <2% for negative markers, with >95% viability, when stained with 7AAD as determined by flow cytometry indicating the Mesenchymal nature of the processed cells. Multipotent character‐ istics, as determined by Oil Red O stain, Von Kossa stain and Safranin O stain, respectively, indicating the cell samples undergo adipogenic, osteogenic and chondrogenic differentiation. Karyotype of all the cell samples was normal and no abnormalities/aberrations were found after ex vivo propagation figure. End product testing such as endotoxin test using LAL method and Mycoplasma test using RT-PCR were found to be negative indicating the cells were safe.

**16.2. Results**

study are given in Tables below.

**Case No. Age Sex Level of**

**Case No. Age Sex**

**injury**

As per the inclusion and exclusion criteria mentioned above, 52 volunteers were recruited for this study. This includes 8 females and 44 males between the age group 17 and 66 years. Duration of injury varied between 15 days after injury to 20 years. All the patients were divided into 4 groups based on the type of cells received. The details of the patients recruited for this

> **Duration of injury**

> **Duration of injury**

 23 M D11-D12 0 month 1 Laminectomy + IV 31 M D4, C6-7 7 months 1 Laminectomy + IV 23 M D11 4 months 1 Laminectomy + IV 26 F C5-C6 1 year 1 Laminectomy + IV 21 M C4-C5 1 year 1 Laminectomy + IV 26 F C5-C6 1 year 1 Laminectomy + IV 53 M C6-C7 3 years 1 Laminectomy + IV 23 M C5-C6 3 years 1 Laminectomy + IV 31 M C6-C7 4 years 1 Laminectomy + IV

**Table 4.** Group 1-Autologous Bone Marrow derived mononuclear cells (BMMNCs; n=9).

 59 F D3-D5 7 years 1 CT Guided 34 M C7 6 years 1 CT Guided 56 M D4 14 years 1 CT Guided 54 M D5-D6 2 years 1 CT Guided 49 M D6 4 years 1 CT Guided 26 M D12 2 years 1 CT Guided 23 M C4-C6 1 years 1 CT Guided 31 M L1 4 years 1 CT Guided 42 M D12 3 years 1 CT Guided 28 M C5-C6 3 years 1 CT Guided 28 M D5-D6 5 years 1 CT Guided

**Table 5.** Group 2-Autologous Bone Marrow derived Mesenchymal stem cells (BMMSCs; n=11)

**Level of injury**

**No. of injection**

**No. of injection** **Route of infusion**

Mesenchymal Stem Cells in Spinal Cord Injury

http://dx.doi.org/10.5772/58323

259

**Route of infusion**

#### **15.4. Adipose tissue derived MSCs**

Mesenchymal stem cells isolated from fat samples received in a sterile container after liposuction were expanded in above mentioned conditions until the cells were confluent. Post confluent, the cells were harvested and stored frozen in liquid nitrogen as MCB, from which WCB were raised. IP were cultured on appropriate tissue culture plates on re‐ quest. Prior to transplantation, in process test and end process test were done. Flow cytometric analysis showed that the cells express the surface markers with >95% for positive markers (fig) and <2% for negative markers (fig) with >90%% viabilty. The cells were found to undergo adipogenic, osteogenic and chondrogenic differentiation as determined by Oil Red O stain, Von Kossa stain and Safranin O stain, respectively. All the samples showed normal karyotypes and no abnormalities/aberrations were noted after ex vivo propaga‐ tion. A representative ideogram is illustrated in Figure. The cell samples tested for endotoxin using LAL method and Mycoplasma test using RT-PCR were found to be negative indicating the cells were safe to be infused.

#### **15.5. Wharton's Jelly derived MSCs**

Umbilical cords obtained postpartum in a sterile container were processed according to the standard protocol described earlier. The cells were further up-scaled and expanded in order to provide the required number of cells for the patient. The cultured cells were found to show normal spindle shaped phenotype when observed (fig). Flow cytometric analysis showed that the cells were positive with >95% for positive markers and <2% for negative markers (fig) with >90% viability. The cells were found to undergo adipogenic, osteogenic and chondrogenic differentiation as determined by Oil Red O stain, Von Kossa stain and Safranin O stain, respectively. All the samples showed normal karyotypes and no abnormalities/aberrations were noted after ex vivo propagation by standard G banding method. A representative ideogram is illustrated in Figure. The cell samples tested for endotoxin using LAL method and Mycoplasma test using RT-PCR were found to be negative indicating the cells were safe for the transplantation.

#### **16. Clinical assessment**

#### **16.1. Clinical examination and ASIA scale scoring**

Clinical assessment was performed on all patients based on the parameters of the ASIA impairment scale. This was considered as the primary measurable outcome of the clinical study.

#### **16.2. Results**

after ex vivo propagation figure. End product testing such as endotoxin test using LAL method and Mycoplasma test using RT-PCR were found to be negative indicating the cells were safe.

Mesenchymal stem cells isolated from fat samples received in a sterile container after liposuction were expanded in above mentioned conditions until the cells were confluent. Post confluent, the cells were harvested and stored frozen in liquid nitrogen as MCB, from which WCB were raised. IP were cultured on appropriate tissue culture plates on re‐ quest. Prior to transplantation, in process test and end process test were done. Flow cytometric analysis showed that the cells express the surface markers with >95% for positive markers (fig) and <2% for negative markers (fig) with >90%% viabilty. The cells were found to undergo adipogenic, osteogenic and chondrogenic differentiation as determined by Oil Red O stain, Von Kossa stain and Safranin O stain, respectively. All the samples showed normal karyotypes and no abnormalities/aberrations were noted after ex vivo propaga‐ tion. A representative ideogram is illustrated in Figure. The cell samples tested for endotoxin using LAL method and Mycoplasma test using RT-PCR were found to be

Umbilical cords obtained postpartum in a sterile container were processed according to the standard protocol described earlier. The cells were further up-scaled and expanded in order to provide the required number of cells for the patient. The cultured cells were found to show normal spindle shaped phenotype when observed (fig). Flow cytometric analysis showed that the cells were positive with >95% for positive markers and <2% for negative markers (fig) with >90% viability. The cells were found to undergo adipogenic, osteogenic and chondrogenic differentiation as determined by Oil Red O stain, Von Kossa stain and Safranin O stain, respectively. All the samples showed normal karyotypes and no abnormalities/aberrations were noted after ex vivo propagation by standard G banding method. A representative ideogram is illustrated in Figure. The cell samples tested for endotoxin using LAL method and Mycoplasma test using RT-PCR were found to be negative indicating the cells were safe for

Clinical assessment was performed on all patients based on the parameters of the ASIA impairment scale. This was considered as the primary measurable outcome of the clinical

**15.4. Adipose tissue derived MSCs**

258 Topics in Paraplegia

negative indicating the cells were safe to be infused.

**15.5. Wharton's Jelly derived MSCs**

the transplantation.

study.

**16. Clinical assessment**

**16.1. Clinical examination and ASIA scale scoring**

As per the inclusion and exclusion criteria mentioned above, 52 volunteers were recruited for this study. This includes 8 females and 44 males between the age group 17 and 66 years. Duration of injury varied between 15 days after injury to 20 years. All the patients were divided into 4 groups based on the type of cells received. The details of the patients recruited for this study are given in Tables below.


**Table 4.** Group 1-Autologous Bone Marrow derived mononuclear cells (BMMNCs; n=9).


**Table 5.** Group 2-Autologous Bone Marrow derived Mesenchymal stem cells (BMMSCs; n=11)


time from the hospital, indicating that there were no immediate cytotoxic effects due to implantation of various cell types (as mentioned above) and the procedures were safe.

Mesenchymal Stem Cells in Spinal Cord Injury

http://dx.doi.org/10.5772/58323

261

The ASIA rating scale did not reveal any significant changes or further worsening or deterio‐

Of the total patient recruited for the study, 9 patients have shown notable clinical and func‐ tional recovery. While follow-up, one patient (G3C2; Table 4a) whose baseline report was as follows: Motor – Upper limb-5/5, Lower limb-2/5, Sensory – Loss of sensation at D 10 and below for all modalities, reported to be able to stand and walk with support and does swimming.

Another patient (G4C3; Table 5], at baseline with power at shoulder-grade 3/5, Power at elbow joint-3/5 in flexion and extension and hand grip-2/5, lower limbs-grade 0/5 with generalized wasting in all limbs and spasticity in both the lower limbs, anesthesia below C6 dermatomes

Slight improvement in Upper Limb sensation after the first dose, was reported by one patient

Post therapy one patient (G4C4; Table 5) was able to feel the bladder fullness from 3rd month of transplantation. In subsequent follow-up, the imbalance while sitting on the wheel chair has partially improved. Also, improvement in touch and pain sensation up-to the right knee on the right side and up to the upper thigh in the left side were noted. Bladder sensations have

Additionally two patients (G4C5; Table 5 and G3C4; Table 4c) has shown improvement in

Two patients (G3C1 and G3C3; Table 4c &4b) were able to walk with the aid of walker post

One patient (G3C5; Table 4c) has regained some sensation in abdomen and lower back area and below feet. The patient can now feel stretching sensation in toes when performing exercises

Out of the 52 patients treated, only 3 patients reported pain after infusion. And two patients

Barthel's index (BI) was performed on all patients, pre-and post-transplantation of the cells. No significant improvement or appreciable changes were observed in the patients with long history of injury. However, patients with less than 6 months of injury have shown improve‐

Magnetic resonance imaging (MRI) of the spinal cord before and after stem cell infusion:

No change was observed in MRI findings at baseline and post-stem cell transplantation. Also, no adverse effects of transplantation were detected on the MRI post transplantation. Further, no changes in cystic regions or syringomyelia, and no further external compression of the cord

therapy. But however, the latter patient had a fall and is now back to baseline.

and exaggerated deep tendon reflexes in lower limbs, has shown minimal recovery.

(G1C5; Table 2). However at times, the patient had painful sensations.

improved to some extent.

were lost to follow-up.

Barthel's Index Score

ment in the scores.

sensation and able to sit with support.

and becoming more aware of bowel movements.

ration in neurological or functional level pre and post stem cells therapy.


**Table 6.** (a): Subgroup 1: BMMSCs, (b): Subgroup 2: WJMSCs, (c): Sub group 3: ADMSCs

On an average, 2 million cells /kg bodyweight were transplanted via 3 different routes i.e., laminectomy, lumbar puncture, and intravenous injections. All the patients stood the proce‐ dure well, there were no postoperative complications and were discharged within a week's time from the hospital, indicating that there were no immediate cytotoxic effects due to implantation of various cell types (as mentioned above) and the procedures were safe.

The ASIA rating scale did not reveal any significant changes or further worsening or deterio‐ ration in neurological or functional level pre and post stem cells therapy.

Of the total patient recruited for the study, 9 patients have shown notable clinical and func‐ tional recovery. While follow-up, one patient (G3C2; Table 4a) whose baseline report was as follows: Motor – Upper limb-5/5, Lower limb-2/5, Sensory – Loss of sensation at D 10 and below for all modalities, reported to be able to stand and walk with support and does swimming.

Another patient (G4C3; Table 5], at baseline with power at shoulder-grade 3/5, Power at elbow joint-3/5 in flexion and extension and hand grip-2/5, lower limbs-grade 0/5 with generalized wasting in all limbs and spasticity in both the lower limbs, anesthesia below C6 dermatomes and exaggerated deep tendon reflexes in lower limbs, has shown minimal recovery.

Slight improvement in Upper Limb sensation after the first dose, was reported by one patient (G1C5; Table 2). However at times, the patient had painful sensations.

Post therapy one patient (G4C4; Table 5) was able to feel the bladder fullness from 3rd month of transplantation. In subsequent follow-up, the imbalance while sitting on the wheel chair has partially improved. Also, improvement in touch and pain sensation up-to the right knee on the right side and up to the upper thigh in the left side were noted. Bladder sensations have improved to some extent.

Additionally two patients (G4C5; Table 5 and G3C4; Table 4c) has shown improvement in sensation and able to sit with support.

Two patients (G3C1 and G3C3; Table 4c &4b) were able to walk with the aid of walker post therapy. But however, the latter patient had a fall and is now back to baseline.

One patient (G3C5; Table 4c) has regained some sensation in abdomen and lower back area and below feet. The patient can now feel stretching sensation in toes when performing exercises and becoming more aware of bowel movements.

Out of the 52 patients treated, only 3 patients reported pain after infusion. And two patients were lost to follow-up.

#### Barthel's Index Score

Table 6: Group 3-Allogeneic BMMSCs or Adipose tissue derived MSCs (ADMSCs) or Whar‐

**Duration of injury**

**(a)**

**(b)**

**(c)**

On an average, 2 million cells /kg bodyweight were transplanted via 3 different routes i.e., laminectomy, lumbar puncture, and intravenous injections. All the patients stood the proce‐ dure well, there were no postoperative complications and were discharged within a week's

Case No. Age Sex Level of injury Duration of injury No. of injection Route Of infusion 23 M Cervical 2 years 3 Intrathecal 54 M Thoracic 1 year 3 Intrathecal 54 M C4-C5 6 months 3 Intrathecal 31 M C2-D4 1 year 3 Intrathecal

Case No. Age Sex Level of injury Duration of injury No. of injection Route Of infusion 47 M Cervical 8 months 3 Intrathecal 37 M D12 4 years 3 Intrathecal 42 M Thoracic 3 Intrathecal 35 M C3-C4 0 month 3 Intrathecal 27 M C5-C6 6 years 3 Intrathecal 37 M Thoracic 13 years 3 Intrathecal

**Table 6.** (a): Subgroup 1: BMMSCs, (b): Subgroup 2: WJMSCs, (c): Sub group 3: ADMSCs

 19 F D4-D6 1 year 1 CT Guided 36 F D10 1 month 3 Intrathecal 27 M T12-L1 1 year 1 CT Guided 36 M C6-C7 2 months 1 Laminectomy + IV 26 M D3 3 months 1 Laminectomy + IV 46 M C3-C4 0 month 1 Laminectomy + IV 29 M Partial 1 year 3 Intrathecal 45 M C1-L1 1 year 1 Laminectomy + IV 50 F C2 1 year 1 Laminectomy + IV 46 F Dorsal SCI 1 year 3 Intrathecal 25 M D9-D10 1 year 3 Intrathecal 29 F D4 1 year 3 Intrathecal 27 M SCI 10 months 1 Laminectomy + IV 26 M C7-T1 3 years 3 Intrathecal 27 M Cervical 6 months 3 Intrathecal 51 M D4-D6 20 years 3 Intrathecal

**No. of injection** **Route of infusion**

ton's jelly derived MSCs (WJMSCs) (n=26)

**Level of injury**

**Case No. Age Sex**

260 Topics in Paraplegia

Barthel's index (BI) was performed on all patients, pre-and post-transplantation of the cells. No significant improvement or appreciable changes were observed in the patients with long history of injury. However, patients with less than 6 months of injury have shown improve‐ ment in the scores.

Magnetic resonance imaging (MRI) of the spinal cord before and after stem cell infusion:

No change was observed in MRI findings at baseline and post-stem cell transplantation. Also, no adverse effects of transplantation were detected on the MRI post transplantation. Further, no changes in cystic regions or syringomyelia, and no further external compression of the cord or formation of tumor-like masses in and around the injection site or along the cord, were visualized.

The advantages of allogenic cells over autologous cells for transplantation may be that, they are readily available with defined cell quality and quantity. This makes allogenic stem cells, a good choice to make extensive research on the feasibility in other therapeutic interventions. In addition it offers an opportunity to use the cells as early as possible. It has been the obser‐ vation that early intervention within few days has yielded marginally better results suggesting an optimal temporal window for cell mediated therapy. [121]. several studies have indicated better results with early intervention and acute injury. [122, 123, 121]. The preclinical literature also has suggested that there is an earlier window for the optimization of cell therapies [125, 124]. Now its well known that these cells do HOME at the required site. Homing could be mediated by the ongoing cell reactions, products of cell death or inflammation or some chemo attractants. We believe timing of delivery of cells is crucial for these cells to impregnate in large numbers.In delayed or chronic injuries cell reach may be poor and once gliosis sets in cell penetration may be difficult. In addition spinalcord –csf barrier doesn't allow cell migration

Mesenchymal Stem Cells in Spinal Cord Injury

http://dx.doi.org/10.5772/58323

263

The strategy to cord injury is twofold-Initial control of damage and minimizing secondary deleterious effects, and later promotion of recovery. Cell therapy can play a role in both provided they were given at the right time.. However there is no sufficient data to indicate the exact time of maximizing the benefit. In general those who are likely to be benefited must be treated before the molecular mechanisms cause the irreparable damage. [5] The drawback of our study is timing could not be controlled since they were inducted as and when they came

A canine study from South Korea 2009 used autologous and allogenic cells. Though autologous BM-MSCs had better results than allogenic both showed better results com‐

Literature shows several routes of administration like intra arterial, intravenous, intra thecal and direct injections to the injured cord. Intra thecal injection was most frequently used method. [121]. Our study also demonstrates that administration of MSCs via multiple routes such as laminectomy, lumbar puncture and intravenous delivery, are feasible and safe. Though direct injection into the cord appears logical,the apprehension of enhancing the injury always exists. In our opinion it is invasive and should be reserved for those where decompression or stabilization is indicated and most suitable in chronic complete inuries.. Saberi et al [119] reported no serious adverse effect after intraparenchymal injections. They also reported transient low grade fever with nausea, vomiting and headache. But we did not encounter such complications in our series. The use of scaffold is complimentary and may have positive influence. [110]. In chronic injuries widening of anatomical gap between the functional tissues of the cord is a challenge and a possible reason for poor outcomes. Degeneration makes this anatomical and functional gap wider with time. Often this gap gets replaced by glial scar which becomes a physical barrier preventing cellular penetration,regeneration and migration. Scaffolds can act as an anatomical substrate on which these cells can grow and connect the

It appears that the cytokines and bio active molecules secreted by these cells play a significant role in acute as well as sub acute phases. Therefore it is essential to retain the cells at the required

to our clinical service. In addition its difficult to have clinical controls.

into the parenchyma.

pared to controls [126]

physiological ends.

#### **17. Discussion**

Spinal cord essentially is a conduit integrating relay and transmission of signals and the functions of the body (motor, sensory and autonomic) with the higher centers (brain brainstem & cerebellum). SCI can be devastating with lifelong disability due to its complex architecture and compounding consequences that follows an injury. Disruption of such local integrative networks interrupts ascending and descending input and outputs resulting in dysfunction of motor, sensory, autonomic and dysregulation of various reflexes in the body. Majority are in the age range of 16-35. Damage to the spinal cord progresses rapidly in stages. In the last two decades, researchers have made their efforts to understand this complex pathobiology from several animal studies [6]. Ischemia,Scarring, cavitation, wallarian degeneration, axonal die back, excitotoxins, inflammation and several complex cellular and molecular changes are known to influence recovery of such injury. Several medical (pharmacological and others) and surgical attempts did not influence any substantial positive outcomes. Hence the attention was turned towards neurotrophic factors and cell based treatments. As a result spontaneous neurological recovery has been reported only in 6-13% of patients with only 2% gaining any functional recovery. [113-116].

Cell death is often rapid after SCI. The adult spinal cord harbors endogenous stem/progenitor cells, collectively referred to as Neural Progenitor Cells (NPCs) that might be responsible for normal turnover of the cells and repair process. Several studies have confirmed that new cells are born around the central canal from the ependymal precursors [1].However, the prolifera‐ tive activity of endogenous NPCs is too limited and grossly inadequate to support spontaneous repair after SCI. Hence various cell transplantation strategies have been adopted in models of SCI such as embryonic stem cells, Wharton's jelly, adult neural stem cells, bone marrow and adipose tissue derived Mesenchymal stem cells [13]. They are currently being studied as potential sources of neurons, glial cells or neurotrophic factors. Transplantation of these cells to create or regenerate spinal cord as an alternative therapy has generated lot of interest. This study clearly documents the feasibility of such cell replacement strategies [14].

Several studies have reported several protocols different timings and type of cells [117-120]. We have studied autologous BMMNCs, BMMSCs (autologous and allogenic), WJMSCs and ADMSCs (allogenic) have been used to study their therapeutic potential in spinal cord injury. Those who received autologous BMMNCs showed only minimal improvement. The reason may be due to variations in age; extent, duration of injury; and variance in cell quantity and quality.

Nevertheless, autologous BMMSCs had shown good improvement, the concern with the transplantation is the availability of cells in time and other problems as mentioned above.

Recently, allogenic MSCs from sources like Bone marrow, Adipose tissue and Wharton's jelly shown to have attracted many, to use it as source for treatment and conducting trials on them. The advantages of allogenic cells over autologous cells for transplantation may be that, they are readily available with defined cell quality and quantity. This makes allogenic stem cells, a good choice to make extensive research on the feasibility in other therapeutic interventions. In addition it offers an opportunity to use the cells as early as possible. It has been the obser‐ vation that early intervention within few days has yielded marginally better results suggesting an optimal temporal window for cell mediated therapy. [121]. several studies have indicated better results with early intervention and acute injury. [122, 123, 121]. The preclinical literature also has suggested that there is an earlier window for the optimization of cell therapies [125, 124]. Now its well known that these cells do HOME at the required site. Homing could be mediated by the ongoing cell reactions, products of cell death or inflammation or some chemo attractants. We believe timing of delivery of cells is crucial for these cells to impregnate in large numbers.In delayed or chronic injuries cell reach may be poor and once gliosis sets in cell penetration may be difficult. In addition spinalcord –csf barrier doesn't allow cell migration into the parenchyma.

or formation of tumor-like masses in and around the injection site or along the cord, were

Spinal cord essentially is a conduit integrating relay and transmission of signals and the functions of the body (motor, sensory and autonomic) with the higher centers (brain brainstem & cerebellum). SCI can be devastating with lifelong disability due to its complex architecture and compounding consequences that follows an injury. Disruption of such local integrative networks interrupts ascending and descending input and outputs resulting in dysfunction of motor, sensory, autonomic and dysregulation of various reflexes in the body. Majority are in the age range of 16-35. Damage to the spinal cord progresses rapidly in stages. In the last two decades, researchers have made their efforts to understand this complex pathobiology from several animal studies [6]. Ischemia,Scarring, cavitation, wallarian degeneration, axonal die back, excitotoxins, inflammation and several complex cellular and molecular changes are known to influence recovery of such injury. Several medical (pharmacological and others) and surgical attempts did not influence any substantial positive outcomes. Hence the attention was turned towards neurotrophic factors and cell based treatments. As a result spontaneous neurological recovery has been reported only in 6-13% of patients with only 2% gaining any

Cell death is often rapid after SCI. The adult spinal cord harbors endogenous stem/progenitor cells, collectively referred to as Neural Progenitor Cells (NPCs) that might be responsible for normal turnover of the cells and repair process. Several studies have confirmed that new cells are born around the central canal from the ependymal precursors [1].However, the prolifera‐ tive activity of endogenous NPCs is too limited and grossly inadequate to support spontaneous repair after SCI. Hence various cell transplantation strategies have been adopted in models of SCI such as embryonic stem cells, Wharton's jelly, adult neural stem cells, bone marrow and adipose tissue derived Mesenchymal stem cells [13]. They are currently being studied as potential sources of neurons, glial cells or neurotrophic factors. Transplantation of these cells to create or regenerate spinal cord as an alternative therapy has generated lot of interest. This

Several studies have reported several protocols different timings and type of cells [117-120]. We have studied autologous BMMNCs, BMMSCs (autologous and allogenic), WJMSCs and ADMSCs (allogenic) have been used to study their therapeutic potential in spinal cord injury. Those who received autologous BMMNCs showed only minimal improvement. The reason may be due to variations in age; extent, duration of injury; and variance in cell quantity and

Nevertheless, autologous BMMSCs had shown good improvement, the concern with the transplantation is the availability of cells in time and other problems as mentioned above. Recently, allogenic MSCs from sources like Bone marrow, Adipose tissue and Wharton's jelly shown to have attracted many, to use it as source for treatment and conducting trials on them.

study clearly documents the feasibility of such cell replacement strategies [14].

visualized.

262 Topics in Paraplegia

**17. Discussion**

functional recovery. [113-116].

quality.

The strategy to cord injury is twofold-Initial control of damage and minimizing secondary deleterious effects, and later promotion of recovery. Cell therapy can play a role in both provided they were given at the right time.. However there is no sufficient data to indicate the exact time of maximizing the benefit. In general those who are likely to be benefited must be treated before the molecular mechanisms cause the irreparable damage. [5] The drawback of our study is timing could not be controlled since they were inducted as and when they came to our clinical service. In addition its difficult to have clinical controls.

A canine study from South Korea 2009 used autologous and allogenic cells. Though autologous BM-MSCs had better results than allogenic both showed better results com‐ pared to controls [126]

Literature shows several routes of administration like intra arterial, intravenous, intra thecal and direct injections to the injured cord. Intra thecal injection was most frequently used method. [121]. Our study also demonstrates that administration of MSCs via multiple routes such as laminectomy, lumbar puncture and intravenous delivery, are feasible and safe. Though direct injection into the cord appears logical,the apprehension of enhancing the injury always exists. In our opinion it is invasive and should be reserved for those where decompression or stabilization is indicated and most suitable in chronic complete inuries.. Saberi et al [119] reported no serious adverse effect after intraparenchymal injections. They also reported transient low grade fever with nausea, vomiting and headache. But we did not encounter such complications in our series. The use of scaffold is complimentary and may have positive influence. [110]. In chronic injuries widening of anatomical gap between the functional tissues of the cord is a challenge and a possible reason for poor outcomes. Degeneration makes this anatomical and functional gap wider with time. Often this gap gets replaced by glial scar which becomes a physical barrier preventing cellular penetration,regeneration and migration. Scaffolds can act as an anatomical substrate on which these cells can grow and connect the physiological ends.

It appears that the cytokines and bio active molecules secreted by these cells play a significant role in acute as well as sub acute phases. Therefore it is essential to retain the cells at the required area in sufficient numbers. We have included only complete injuries so as to remove the bias of spontaneous recovery. Based on our results and the positive role of anatomical continuity we feel partial injuries shall definitely benefit more. Cell therapy can augment the spontaneous recovery either by promotion or reducing the derogatory inhibitory influences.

methods and mechanisms alone or in combination need to put in place.Rehabilitation does play a significant role in those with clinical recovery. We speculate that combination of rehab and regeneration may be better. The local neuronal circuits within the segments of the cord must be sustained to retain the integrity of the reflex arc. This appears complex and needs

Mesenchymal Stem Cells in Spinal Cord Injury

http://dx.doi.org/10.5772/58323

265

In conclusion, the surge of research activities in the cell therapies for SCI has yielded only mixed results. While the pre clinical studies are quite promising it is difficult to reproduce similar results in the clinical scenario.. Knowing the mechanisms involved stem cells seems to have specific role and prospects for future studies. Future direction should focus on enhancing the benefits of cell therapy by combination of methods systematically addressing the chal‐ lenges involved. Our study documents safety and influence on recovery to some extent paves the way for further preclinical and clinical studies with proper design. Such larger clinical

The author acknowledges the assistance provided by the neurosurgery team, clinical research

[1] James Guest: Principles of Translation of Biologic Therapies in Spinal Cord Injury Youmans Neurological Surgery, 6th Edition, Chapter 267, Volume 3; 2011.

further experimental and clinical data to understand the underlying mechanisms.

studies only can overcome the present diversity in methods and outcomes.

team at BGS Global Hospital and by the research team from ANSA.

and Rakhi Pal2

2 Advanced Neuroscience Allies Research Foundation, India

The authors state that there is no conflict of interest.

1 Department of Neurosurgery. BGS Global Hospital, Kengeri, Bangalore, India

**18. Conclusion**

**Acknowledgements**

**Author details**

**References**

N.K. Venkataramanaa1

In our findings delivery through lumbar puncture is simple and equally effective [111-112]. But cell survival in CSF and their functionality need to be enhanced. Retaining large number of cells at the site of injury is also a challenge. We did not encounter any adverse reaction or infection. After lumbar puncture majority had low pressure headaches which were treated with fluid therapy and analgesics effectively. Kishk et al reported neuropathic pain in 56% of their patients following intrathecal injection which was not noticed in our series.

Though the results of animal experiments are enticing the overall translation into clinical benefit is minimal and quite disappointing. Irrespective of site of injury, route of administra‐ tion and type of cell the clinical recovery is very minimal and only less than 1/3rd showed signs of recovery. Useful functional recovery was seen only in 7-9%. This is rather disappointing. Therefore it appears that even cell therapy has its limitations. But there has been definite evidence of clinical recovery in few and are useful to understand the role of cell therapy. It appears we are somewhere closer to some success yet needs understanding to augment these benefits. Young age, focal segmental injury and early intervention seem to benefit or compli‐ ment recovery. Though all our patients expressed subjective well being, ability to sit for longer periods and actively participation in physical excercises following cell therapy could be mediated by cytokines and growth factors. In addition trunk muscles just above the site of injury showed definite clinical improvement. This could explain the need of anatomical integrity for recovery and also their enhanced ability to sit longer. In the distal segment sensory, long tracts and bladder have shown signs of recovery in many, but few had clinically useful benefit. Motor recovery is the most difficult to achieve. Possibly due to loss of trophic influence from higher centers, vascularity which leads to loss of anterior horn cells. Presently available imaging and electro physiological methods are not sensitive enough to detect or monitor regeneration in spinal cord. Those who recovered could be potential partial injuries (anatom‐ ical continuity) although behaved as complete injuries clinically.. This could be the possible reason of useful clinical recovery observed in our study. Presently we feel role of cell therapy is only complimentary. MSCs are known for immunomodulation and once administered in the right time may help in minimiging neural inflammation and immune mediated damage. Early intervention might reduce gliosis and promote recovery through secretion of cytokines, bioactive molecules and growth factors. These cells also known for angiogenesis hence benefit by revascularization of spinal cord. Lastly the role of effective activation native progenitor cells to come the rescue of adequate repair needs further exploration. Preservation and promotion of recovery of ant horn cells and reestablishing neuronal functional circuits should be the focus.

Going forward, SCI appears to be the most difficult clinical challenge today. Our understand‐ ing of its pathobiology is not complete. The challenges are local (site of injury), peripheral (body below the site of injury) and central (higher centers). The future strategy need to target all the three. Augmenting central influences; sustaining muscles with proper neurotization, rehabilitation, promoting recovery and regeneration at the site seems to be the goal. Several methods and mechanisms alone or in combination need to put in place.Rehabilitation does play a significant role in those with clinical recovery. We speculate that combination of rehab and regeneration may be better. The local neuronal circuits within the segments of the cord must be sustained to retain the integrity of the reflex arc. This appears complex and needs further experimental and clinical data to understand the underlying mechanisms.

#### **18. Conclusion**

area in sufficient numbers. We have included only complete injuries so as to remove the bias of spontaneous recovery. Based on our results and the positive role of anatomical continuity we feel partial injuries shall definitely benefit more. Cell therapy can augment the spontaneous

In our findings delivery through lumbar puncture is simple and equally effective [111-112]. But cell survival in CSF and their functionality need to be enhanced. Retaining large number of cells at the site of injury is also a challenge. We did not encounter any adverse reaction or infection. After lumbar puncture majority had low pressure headaches which were treated with fluid therapy and analgesics effectively. Kishk et al reported neuropathic pain in 56% of

Though the results of animal experiments are enticing the overall translation into clinical benefit is minimal and quite disappointing. Irrespective of site of injury, route of administra‐ tion and type of cell the clinical recovery is very minimal and only less than 1/3rd showed signs of recovery. Useful functional recovery was seen only in 7-9%. This is rather disappointing. Therefore it appears that even cell therapy has its limitations. But there has been definite evidence of clinical recovery in few and are useful to understand the role of cell therapy. It appears we are somewhere closer to some success yet needs understanding to augment these benefits. Young age, focal segmental injury and early intervention seem to benefit or compli‐ ment recovery. Though all our patients expressed subjective well being, ability to sit for longer periods and actively participation in physical excercises following cell therapy could be mediated by cytokines and growth factors. In addition trunk muscles just above the site of injury showed definite clinical improvement. This could explain the need of anatomical integrity for recovery and also their enhanced ability to sit longer. In the distal segment sensory, long tracts and bladder have shown signs of recovery in many, but few had clinically useful benefit. Motor recovery is the most difficult to achieve. Possibly due to loss of trophic influence from higher centers, vascularity which leads to loss of anterior horn cells. Presently available imaging and electro physiological methods are not sensitive enough to detect or monitor regeneration in spinal cord. Those who recovered could be potential partial injuries (anatom‐ ical continuity) although behaved as complete injuries clinically.. This could be the possible reason of useful clinical recovery observed in our study. Presently we feel role of cell therapy is only complimentary. MSCs are known for immunomodulation and once administered in the right time may help in minimiging neural inflammation and immune mediated damage. Early intervention might reduce gliosis and promote recovery through secretion of cytokines, bioactive molecules and growth factors. These cells also known for angiogenesis hence benefit by revascularization of spinal cord. Lastly the role of effective activation native progenitor cells to come the rescue of adequate repair needs further exploration. Preservation and promotion of recovery of ant horn cells and reestablishing neuronal functional circuits should be the focus.

Going forward, SCI appears to be the most difficult clinical challenge today. Our understand‐ ing of its pathobiology is not complete. The challenges are local (site of injury), peripheral (body below the site of injury) and central (higher centers). The future strategy need to target all the three. Augmenting central influences; sustaining muscles with proper neurotization, rehabilitation, promoting recovery and regeneration at the site seems to be the goal. Several

recovery either by promotion or reducing the derogatory inhibitory influences.

264 Topics in Paraplegia

their patients following intrathecal injection which was not noticed in our series.

In conclusion, the surge of research activities in the cell therapies for SCI has yielded only mixed results. While the pre clinical studies are quite promising it is difficult to reproduce similar results in the clinical scenario.. Knowing the mechanisms involved stem cells seems to have specific role and prospects for future studies. Future direction should focus on enhancing the benefits of cell therapy by combination of methods systematically addressing the chal‐ lenges involved. Our study documents safety and influence on recovery to some extent paves the way for further preclinical and clinical studies with proper design. Such larger clinical studies only can overcome the present diversity in methods and outcomes.

#### **Acknowledgements**

The author acknowledges the assistance provided by the neurosurgery team, clinical research team at BGS Global Hospital and by the research team from ANSA.

#### **Author details**


The authors state that there is no conflict of interest.

#### **References**

[1] James Guest: Principles of Translation of Biologic Therapies in Spinal Cord Injury Youmans Neurological Surgery, 6th Edition, Chapter 267, Volume 3; 2011.

[2] Barnett SC, Riddell JS. Olfactory ensheathing cells (OECs) and the treatment of CNS injury: advantages and possible caveats. J Anat. 2004 Jan;204(1):57-67. Review.

[14] Cho DC, Cheong JH, Yang MS, Hwang SJ, Kim JM, Kim CH. (2011). The effect of minocycline on motor neuron recovery and neuropathic pain in rat model of spinal

Mesenchymal Stem Cells in Spinal Cord Injury

http://dx.doi.org/10.5772/58323

267

[15] Ricardo Vieira Botelho, Jefferson Walter Daniel, José Luis Rom eo Boulosa, Benedicto Oscar Colli, Ronald de Lucena Farias, Osmar José Santos Mor aes, Wilson Eloy Pi‐ menta Jr., Carlos Henrique Ribeiro, Francisco Ricardo Bor ges Ribeiro, Mario Augus‐ to Taricco, Marcio Vinhal de Carvalho, Wanderley Marques Bernardo (2010) Effectiveness of methylprednisolone in acute spinal cord injury – a systematic review

[16] Jin-Moo Lee, Ping Yan, Qingli Xiao, Shawei Chen, Kuang-Yung Lee, Chung Y. Hsu, and Jan Xu. (2008) Methylprednisolone protects oligodendrocytes but not neurons

[17] Alberto Pinzon, Alexander Marcillo, Ada Quintana, Sarah Stamler, Mary Bartlett Bunge, Helen M. Bramlett, and W. Dalton Dietrich (2008) A Re-assessment of Mino‐ cycline as a Neuroprotective Agent in a Rat Spinal Cord Contusion Model. Brain Res.

[18] Tae Y. Yune, Jee Y. Lee, Gil Y. Jung, Sun J. Kim, Mei H. Jiang, Young C. Kim, Young J. Oh, George J. Markelonis, and Tae H. Oh. (2007) Minocycline Alleviates Death of oligodendrocytes by Inhibiting Pro-Nerve Growth Factor Production in Microglia af‐

[19] FANG Xiang-qian, FANG Mei, FAN Shun-wu and GU Chuan-long. (2009) Protection of erythropoietin on experimental spinal cord injury by reducing the expression of thrombospondin-1 and transforming growth factor-β. Chinese Medical Journal.

[20] Thao X. Hoang, Mahnaz Akhavan, Jun Wu, and Leif A. Havton. (2008) Minocycline Protects Motor but not Autonomic Neurons after Cauda Equina Injury. Exp Brain

[21] Georgios K. Matis Theodossios A. Birbilis. (2009) Erythropoietin in spinal cord in‐

[24] Liu BP, Fournier A, GrandPre T, Strittmatter SM, Myeliin –associated Glycoprotein

[25] Murat Celik, Necati G kmen, Serhat Erbayraktar, Mustafa Akhisaroglu, Selman Ko‐ nakc, Cagnur Ulukus, Sermin Genc, Kursad Genc, Emel Sagiroglu, Anthony Cerami and Michael Brines. (2002) Erythropoietin prevents motor neuron apoptosis and neu‐ rologic disability in experimental spinal cord ischemic injury. PNAS vol. 99 no.

[22] Schwab NE. Nogo and axon regeneration. Curr Opin Nerobiol:1118-24,2004.

as a functional ligand for the Nogo-66 receptor.Science;297:1190-03; 2002.

[23] Schwab ME, Repairing the Injured Spinal Cord. Science:295:1029-31,2002

of randomized controlled trials. Rev Assoc Med Bras 56(6): 729-37.

ter Spinal Cord Injury. Journal of Neuroscience, 27(29):7751–7761.

cord injury. J Korean Neurosurg Soc. 49:83-91.

following spinal cord. J Neurosci. 28(12): 3141–3149.

3; 1243: 146–151

122(14):1631-1635

Res. 189(1): 71–77.

42258–2263.

jury. Eur Spine J 18:314–323.


[14] Cho DC, Cheong JH, Yang MS, Hwang SJ, Kim JM, Kim CH. (2011). The effect of minocycline on motor neuron recovery and neuropathic pain in rat model of spinal cord injury. J Korean Neurosurg Soc. 49:83-91.

[2] Barnett SC, Riddell JS. Olfactory ensheathing cells (OECs) and the treatment of CNS injury: advantages and possible caveats. J Anat. 2004 Jan;204(1):57-67. Review.

[3] Singh R, Sharma SC, Mittal R, Sharma A. Traumatic spinal cord injuries in Haryana: An epidemiological study. Indian journal of community medicine. 2003; XXV111(4):

[4] Gregory W. J. Hawryluk, James Rowland, Brian K. Kwon and Michael G. Fehlings. (2008). Protection and repair of the injured spinal cord: a review of completed, ongo‐ ing, and planned clinical trials for acute spinal cord injury. Neurosurg Focus 25

[5] Anjan Kumar Das, Renjitha Gopurappilly & Ishwar Parhar: Current Status & Pro‐ spective Application of Stemcell – Based Therapies for Spinal Cord Injury. Current

[6] Schultz SS: Adult stemcell application in Spinal cord injury. Curr Drug Targets

[7] Totoiu MO, Kierstead HS. Spinal Cord Injury is accompanied by chronic progressive

[8] Gerlinde A.S. Metz, Armin Curt, Henk Van De Meent, Isabel Klusman, Martin E. Schwab, Volker Dietz (2000). Validation of the Weight-Drop Contusion Model in Rats: A Comparative Study of Human Spinal Cord Injury. Journal of Neurotrauma.

[9] Fehlings, Michael G.; Rao, Sanjay C.; Tator, Charles H.; Skaf, Ghassan; Arnold, Paul; Benzel, Edward; Dickman, Curtis; Cuddy, Brian; Green, Barth; Hitchon, Patrick; Northrup, Bruce; Sonntag, Volker; Wagner, Frank; Wilberger, Jack (1999) The Opti‐ mal Radiologic Method for Assessing Spinal Canal Compromise and Cord Compres‐ sion in Patients With Cervical Spinal Cord Injury: Part II: Results of a Multicenter

[10] Manuel Gaviria, Alain Privat, Pierre D'arbigny, Jean-Marc Kamenka, Henri Haton, and Freddy Ohanna (2000) Neuroprotective Effects of Gacyclidine After Experimen‐ tal Photochemical Spinal Cord Lesion in Adult Rats: Dose-Window and Time-Win‐

[11] Pitts LH, Ross A, Chase GA, Faden AI. (1995) Treatment with thyrotropin-releasing hormone (TRH) in patients with traumatic spinal cord injuries. J Neurotrauma. 12(3):

[12] Takami K, Hashimoto T, Shino A, Fukuda N. (1991). Effect of thyrotropin-releasing hormone (TRH) in experimental spinal cord injury: a quantitative histopathologic

[13] Natasha Olby (1999). Current Concepts in the Management of Acute Spinal Cord In‐

dow Effects. Journal of Neurotrauma. Vol. 17, No. 1: 19-30

Stem Cell Research & Therapy, Vol.6,No.1,6,00,2011.

demyelination. J Comp Neurol;486;373-83, 2005.

184-86.

266 Topics in Paraplegia

(5):E14.

6:63-73; 2006.

17(1): 1-17.

235-43.

Study. Spine, 24(6):605-613.

study. Jpn J Pharmacol. 57(3):405-17.

jury. J Vet Intern Med 13:399–407


[26] Carelli S, Marfia G, Di Giulio AM, Ghilardi G, Gorio A. (2011) Erythropoietin: recent developments in the treatment of spinal cord injury. Neurol Res Int.

[40] Fehlings MG, Theodore N, Harrop J, Maurais G, Kuntz C, Shaffrey CI, Kwon BK, Chapman J, Yee A, Tighe A, McKerracher L. (2011) A phase I/IIa clinical trial of a re‐ combinant Rho protein antagonist in acute spinal cord injury. J Neurotrauma. 28(5):

Mesenchymal Stem Cells in Spinal Cord Injury

http://dx.doi.org/10.5772/58323

269

[41] Beaumont E, Whitaker CM, Burke DA, Hetman M, Onifer SM. (2009) Effects of roli‐ pram on adult rat oligodendrocytes and functional recovery after contusive cervical

[42] Satkunendrarajah Kajana, and Harry G Goshgarian. (2009) Systemic administration of rolipram increases medullary and spinal cAMP and activates a latent respiratory motor pathway after high cervical spinal cord injury. Spinal Cord Med. 32(2):175-82.

[43] Whitaker CM, Beaumont E, Wells MJ, Magnuson DS, Hetman M, Onifer SM. (2008) Rolipram attenuates acute oligodendrocyte death in the adult rat ventrolateral funi‐ culus following contusive cervical spinal cord injury. Neurosci Lett. 438(2):200-4. [44] Nikulina E, Tidwell JL, Dai HN, Bregman BS, Filbin MT. (2004) The phosphodiester‐ ase inhibitor rolipram delivered after a spinal cord lesion promotes axonal regenera‐

[45] Gonzenbach RR, Zoerner B, Schnell L, Weinmann O, Mir AK, Schwab ME. (2011) De‐ layed Anti-Nogo-A Antibody Application after Spinal Cord Injury Shows Progres‐

[46] Maier IC, Ichiyama RM, Courtine G, Schnell L, Lavrov I, Edgerton VR, Schwab ME. (2009) Differential effects of anti-Nogo-A antibody treatment and treadmill training

[47] Gonzenbach RR, Gasser P, Zörner B, Hochreutener E, Dietz V, Schwab ME. (2010) Nogo-A antibodies and training reduce muscle spasms in spinal cord-injured rats.

[48] Freund P, Wannier T, Schmidlin E, Bloch J, Mir A, Schwab ME, Rouiller EM. (2007) Anti-Nogo-A antibody treatment enhances sprouting of corticospinal axons rostral to a unilateral cervical spinal cord lesion in adult macaque monkey. J Comp Neurol.

[49] Liebscher T, Schnell L, Schnell D, Scholl J, Schneider R, Gullo M, Fouad K, Mir A, Rausch M, Kindler D, Hamers FP, Schwab ME. (2005) Nogo-A antibody improves re‐ generation and locomotion of spinal cord-injured rats. Ann Neurol. 58(5):706-19. [50] Wiessner C, Bareyre FM, Allegrini PR, Mir AK, Frentzel S, Zurini M, Schnell L, Oer‐ tle T, Schwab ME. Anti-Nogo-A antibody infusion 24 hours after experimental stroke improved behavioral outcome and corticospinal plasticity in normotensive and spon‐

taneously hypertensive rats. J Cereb Blood Flow Metab. 23(2):154-65.

tion and functional recovery. Proc Natl Acad Sci 101(23):8786-90.

in rats with incomplete spinal cord injury. Brain. 132(Pt 6):1426-40.

spinal cord injury. Neuroscience. 163(4):985-90.

sive Loss of Responsiveness. J Neurotrauma.

Ann Neurol. 68(1):48-57.

502(4): 644-59.

787-96.


[40] Fehlings MG, Theodore N, Harrop J, Maurais G, Kuntz C, Shaffrey CI, Kwon BK, Chapman J, Yee A, Tighe A, McKerracher L. (2011) A phase I/IIa clinical trial of a re‐ combinant Rho protein antagonist in acute spinal cord injury. J Neurotrauma. 28(5): 787-96.

[26] Carelli S, Marfia G, Di Giulio AM, Ghilardi G, Gorio A. (2011) Erythropoietin: recent

[27] Dergham P, Ellezam B, Essagian C, Avedissian H, Lubell WD, McKerracher L. (2002) Rho signaling pathway targeted to promote spinal cord repair. J Neurosci. 22(15):

[28] Blight AR. Computer simulation of action potentials and afterpotentials in mammali‐ an myelinated axons: the case for a lower resistance myelin sheath. Neuroscience.

[29] Blight AR. Macrophages and inflammatory damage in spinal cord injury. J Neuro‐

[30] Rapalino O, Lazarov-Spiegler O, Agranov E, Velan GJ, Yoles E, Fraidakis M, Solo‐ mon A, Gepstein R, Katz A, Belkin M, Hadani M, Schwartz M. Implantation of stimulated homologous macrophages results in partial recovery of paraplegic rats.

[31] Popovich PG, Guan Z, Wei P, Huitinga I, van Rooijen N, Stokes BT. Depletion of hematogenous macrophages promotes partial hindlimb recovery and neuroanatomi‐ cal repair after experimental spinal cord injury. Exp Neurol. 1999 Aug;158(2):351-65.

[32] Chopp M, Zhang XH, Li Y, Wang L, Chen J, Lu D, Lu M, Rosenblum M. Spinal cord injury in rat: treatment with bone marrow stromal cell transplantation. Neuroreport.

[33] Ankeny DP, McTigue DM, Jakeman LB. One marrow transplants provide tissue pro‐ tection and directional guidance for axons after contusive spinal cord injury in rats.

[34] Nosrat IV, Widenfalk J, Olson L, Nosrat CA. Dental pulp cells produce neurotrophic factors, interact with trigeminal neurons *in vitro*, and rescue motoneurons after spinal

[35] Lipson AC, Widenfalk J, Lindqvist E, Ebendal T, Olson Neurotrophic properties of

[36] Umezawa A, Ogawa S. Cardiomyocytes can be generated from marrow stromal cells

[37] McDonald JW, Belegu V. Demyelination and remyelination after spinal cord injury. J

[38] Fournier AE, Takizawa BT, Strittmatter SM. (2003) Rho kinase inhibition enhances

[39] Sung JK, Miao L, Calvert JW, Huang L, Louis Harkey H, Zhang JH. (2003) A possible role of RhoA/Rho-kinase in experimental spinal cord injury in rat. Brain Res. 959(1):

olfactory ensheathing glia.LExp Neurol. 2003 Apr;180(2):167-71.

axonal regeneration in the injured CNS. J Neurosci. 23(4):1416-23.

developments in the treatment of spinal cord injury. Neurol Res Int.

6570-7.

268 Topics in Paraplegia

1985 May;15(1):13-31.

Nat Med. 1998 Jul;4(7):814-21.

2000 Sep 11;11(13):3001-5.

Exp Neurol. 2004 Nov;190(1):17-31.

*in vitro*. J Clin Invest 1999 103: 697-705.

29-38.

cord injury. Dev Biol. 2001 Oct 1;238(1):120-32.

Neurotrauma. 2006 Mar-Apr;23(3-4):345-59. Review.

trauma. 1992 Mar;9 Suppl 1:S83-91. Review.


[51] Bukhari N, Torres L, Robinson JK, Tsirka SE. (2011) Axonal regrowth after spinal cord injury via chondroitinase and the tissue plasminogen activator (tPA)/plasmin system. J Neurosci. 31(42):14931-43.

[62] Pintér S, Gloviczki B, Szabó A, Márton G, Nógrádi A. J Neurotrauma. (2010) In‐ creased survival and reinnervation of cervical motoneurons by riluzole after avulsion

Mesenchymal Stem Cells in Spinal Cord Injury

http://dx.doi.org/10.5772/58323

271

[63] Kitzman PH. (2009) Effectiveness of riluzole in suppressing spasticity in the spinal

[64] Nógrádi A, Szabó A, Pintér S, Vrbová G. (2007) Delayed riluzole treatment is able to

[65] Faghri PD, Glaser RM, Figoni SF. (1992) Functional electrical stimulation leg cycle er‐ gometer exercise: training effects on cardiorespiratory responses of spinal cord in‐ jured subjects at rest and during submaximal exercise. Arch Phys Med Rehabil.

[66] Hicks AL, Martin KA, Ditor DS, et al. (2003) Long-term exercise training in persons with spinal cord injury: effects on strength, arm ergometry performance, and psycho‐

[67] Astorino TA, Tyerman N, Wong K, Harness E. (2008) Efficacy of a new rehabilitative device for individuals with spinal cord injury. J Spinal Cord Med. 31(5):586-91. [68] Nash MS. (2005) Exercise as a health-promoting activity following spinal cord injury.

[69] Hagglund KJ, Clark MJ, Mokelke EK, Stout BJ. (2004) The current state of personal assistance services: implications for policy and future research. NeuroRehabilitation.

[70] Adams MM, Hicks AL. (2011) Comparison of the effects of body-weight-supported treadmill training and tilt-table standing on spasticity in individuals with chronic

[71] Stevens S, Morgan DW. (2010) Underwater treadmill training in adults with incom‐

[72] Epifanov VA. (1980). Effect of physical exercises on the hemodynamic state in pa‐ tients with an injury of the cervical spine and spinal cord. Vopr Kurortol Fizioter

[73] Rayegani SM, Shojaee H, Sedighipour L, Soroush MR, Baghbani M, Amirani OB. (2010) The effect of electrical passive cycling on spasticity in war veterans with spinal

[74] Galvez JA, Budovitch A, Harkema SJ, Reinkensmeyer DJ. (2011)Trainer variability during step training after spinal cord injury: Implications for robotic gait-training de‐

rescue injured rat spinal motoneurons. Neuroscience. 144(2):431-8.

of the C7 ventral root. 27(12):2273-82.

73:1085–1093.

19(2):115-20.

Lech Fiz Kult. (3):54-7.

cord injury. Front Neurol. 2:39.

vice design. J Rehabil Res Dev 48(2):147-60

cord injured rat. Neurosci Lett. 455(2):150-3.

logical well-being. Spinal Cord. 41:34–43.

J Neurol Phys Ther. 29(2):87-103, 106.

spinal cord injury. J Spinal Cord Med. 34(5):488-94

plete spinal cord injuries. J Rehabil Res Dev. 47(7): vii-x.


[62] Pintér S, Gloviczki B, Szabó A, Márton G, Nógrádi A. J Neurotrauma. (2010) In‐ creased survival and reinnervation of cervical motoneurons by riluzole after avulsion of the C7 ventral root. 27(12):2273-82.

[51] Bukhari N, Torres L, Robinson JK, Tsirka SE. (2011) Axonal regrowth after spinal cord injury via chondroitinase and the tissue plasminogen activator (tPA)/plasmin

[52] Zhao RR, Muir EM, Alves JN, Rickman H, Allan AY, Kwok JC, Roet KC, Verhaagen J, Schneider BL, Bensadoun JC, Ahmed SG, Yáñez-Muñoz RJ, Keynes RJ, Fawcett JW, Rogers JH. (2011) Lentiviral vectors express chondroitinase ABC in cortical projec‐ tions and promote sprouting of injured corticospinal axons. J Neurosci Methods.

[53] Wang D, Ichiyama RM, Zhao R, Andrews MR, Fawcett JW. (2011) Chondroitinase combined with rehabilitation promotes recovery of forelimb function in rats with

[54] Zhang C, Yao C, He XJ, Li HP. (2010). Repair of subacute spinal cord crush injury by bone marrow stromal cell transplantation and chondroitinase ABC microinjection in

[55] Karimi-Abdolrezaee S, Eftekharpour E, Wang J, Schut D, Fehlings MG. (2010) Syner‐ gistic effects of transplanted adult neural stem/progenitor cells, chondroitinase, and growth factors promote functional repair and plasticity of the chronically injured spi‐

[56] Tom VJ, Kadakia R, Santi L, Houlé JD. (2009) Administration of chondroitinase ABC rostral or caudal to a spinal cord injury site promotes anatomical but not functional

[57] Duschau-Wicke A, Caprez A, Riener R. (2011) Patient-cooperative control increases active participation of individuals with SCI during robot-aided gait training. J Neu‐

[58] Gregory W. J. Hawryluk, James Rowland, Brian K. Kwon, and Michael G. Fehlings. (2008) Protection and repair of the injured spinal cord: a review of completed, ongo‐ ing, and planned clinical trials for acute spinal cord injury. Neurosurg Focus 25

[59] Lang-Lazdunski L, Heurteaux C, Mignon A, Mantz J, Widmann C, Desmonts J, Laz‐ dunski M. (2000) Ischemic spinal cord injury induced by aortic cross-clamping: pre‐

[60] Schuster JE, Fu R, Siddique T, Heckman CJ. (2012) Effect of prolonged riluzole expo‐ sure on cultured motoneurons in a mouse model of ALS. J Neurophysiol. 107(1):

[61] Simard JM, Tsymbalyuk O, Keledjian K, Ivanov A, Ivanova S, Gerzanich V. (2011) Comparative effects of glibenclamide and riluzole in a rat model of severe cervical

vention by riluzole. Eur J Cardiothorac Surg. 18(2):174-81.

system. J Neurosci. 31(42):14931-43.

nal cord. J Neurosci. 30(5):1657-76.

roeng Rehabil. 10;7:43.

spinal cord injury. Exp Neurol.

(5):E14.

484-92.

plasticity. J Neurotrauma. 26(12):2323-33.

chronic spinal cord injury. J Neurosci. 31(25):9332-44.

adult rats. Nan Fang Yi Ke Da Xue Xue Bao. 30(9):2030-5

201(1):228-38.

270 Topics in Paraplegia


[75] Liu G, Keeler BE, Zhukareva V, Houlé JD. (2010) Cycling exercise affects the expres‐ sion of apoptosis-associated micro RNAs after spinal cord injury in rats. Exp Neurol. 226(1): 200-6

[87] Hamid S, Hayek R. (2008) Role of electrical stimulation for rehabilitation and regen‐

Mesenchymal Stem Cells in Spinal Cord Injury

http://dx.doi.org/10.5772/58323

273

[88] Thrasher TA, Popovic MR. (2008) Functional electrical stimulation of walking: func‐

[89] Ahmed Z, Wieraszko A. (2008) Combined effects of acrobatic exercise and magnetic stimulation on the functional recovery after spinal cord lesions. J Neurotrauma.

[90] Tsai PY, Wang CP, Chiu FY, Tsai YA, Chang YC, Chuang TY. (2009) Efficacy of func‐ tional magnetic stimulation in neurogenic bowel dysfunction after spinal cord injury.

[91] Krause P, Straube A. (2003) Repetitive magnetic and functional electrical stimulation reduce spastic tone increase in patients with spinal cord injury. Suppl Clin Neuro‐

[92] Lin VW, Nino-Murcia M, Frost F, Wolfe V, Hsiao I, Perkash I. (2001) Functional mag‐ netic stimulation of the colon in persons with spinal cord injury. Arch Phys Med Re‐

[93] Lin VW, Hsiao IN, Zhu E, Perkash I. (2001) Functional magnetic stimulation for con‐ ditioning of expiratory muscles in patients with spinal cord injury. Arch Phys Med

[94] Classen J, Witte OW. (1995) Epileptic seizures triggered directly by focal transcranial magnetic stimulation. Electroencephalography Clin Neurophysiology; 94:19–25.

[95] Beattie MS, Bresnahan JC, Komon J, Tovar CA, Van Meter M, Anderson DK, Faden AI, Hsu CY, Noble LJ, Salzman S, Young W.Endogenous repair after spinal cord con‐

[96] Mothe AJ, Tator CH. Proliferation, migration, and differentiation of endogenous ependymal region stem/progenitor cells following minimal spinal cord injury in the

[97] Namiki J, Tator CH.Cell proliferation and nestin expression in the ependyma of the adult rat spinal cord after injury. J Neuropathol Exp Neurol. 1999 May;58(5):489-98

[98] Horner PJ, Gage FH. Regenerating the damaged central nervous system. Nature.

[99] Cao X, Tang C, Luo Y. Effects of nerve growth factor on N-methyl-D-asparate recep‐ tor 1 after spinal cord injury in rats. Chin J Traumatol. 2002 Aug;5(4):228-31.

[100] Koike M, Uchiyama Y, Toyama Y, Okano H. Transplantation of *in vitro*-expanded fe‐ tal neural progenitor cells results in neurogenesis and functional recovery after spi‐ nal cord contusion injury in adult rats. J Neurosci Res. 2002 Sep 15;69(6):925-33.

tusion injuries in the rat. Exp Neurol. 1997 Dec;148(2):453-63.

adult rat. Neuroscience. 2005;131(1):177-87.

2000 Oct 26;407(6807):963-70.

eration after spinal cord injury: an overview. Eur Spine J. 17(9):1256-69.

tion, exercise and rehabilitation. Ann Readapt Med Phys. 51(6):452-60.

25(10): 1257-69.

physiol; 56:220-5.

habil; 82(2):167-73

Rehabil. 82(2):162-6.

J Rehabil Med; 41(1):41-7


[87] Hamid S, Hayek R. (2008) Role of electrical stimulation for rehabilitation and regen‐ eration after spinal cord injury: an overview. Eur Spine J. 17(9):1256-69.

[75] Liu G, Keeler BE, Zhukareva V, Houlé JD. (2010) Cycling exercise affects the expres‐ sion of apoptosis-associated micro RNAs after spinal cord injury in rats. Exp Neurol.

[76] Johnston TE, Smith BT, Oladeji O, Betz RR, Lauer RT. (2008) Outcomes of a home cy‐ cling program using functional electrical stimulation or passive motion for children

[77] Hetz SP, Latimer AE, Buchholz AC, Martin Ginis KA; SHAPE-SCI Research Group. (2009) Increased participation in activities of daily living is associated with lower cholesterol levels in people with spinal cord injury. Arch Phys Med Rehabil. 90(10):

[78] Chafetz RS, Mulcahey MJ, Betz RR, Anderson C, Vogel LC, Gaughan JP, Odel MA, Flanagan A, McDonald CM. (2007) Impact of prophylactic thoraco-lumbosacral or‐ thosis bracing on functional activities and activities of daily living in the pediatric

[79] Liem NR, McColl MA, King W, Smith KM. (2004) Aging with a spinal cord injury: factors associated with the need for more help with activities of daily living. Arch

[80] Watson AH, Kanny EM, White DM, Anson DK. (1995) Use of standardized activities of daily living rating scales in spinal cord injury and disease services. Am J Occup

[81] Vernon W Lin, Diana D Cardenas, Nancy C Cutter, Frederick S Frost, Margaret C Hammond, Laurie B Lindblom, Inder Perkash, FACS, Robert Waters, and Robert M Woolsey. (2003) Spinal Cord Medicine: Principles and Practice. New York: Demos

[82] Chen D, Philip M, Philip PA, Monga TN. (1990) Cardiac pacemaker inhibition by transcutaneous electrical nerve stimulation. Arch Phys Med Rehabil 71:27–30.

[83] Segreti J. (1999) Is antibiotic prophylaxis necessary for preventing prosthetic device

[84] Kahn NN, Feldman SP, Bauman WA. (2010) Lower-extremity functional electrical stimulation decreases platelet aggregation and blood coagulation in persons with

[85] Do AH, Wang PT, King CE, Abiri A, Nenadic Z. (2011) Brain-computer interface con‐ trolled functional electrical stimulation system for ankle movement. J Neuroeng Re‐

[86] Gater DR Jr, Dolbow D, Tsui B, Gorgey AS. (2011) Functional electrical stimulation

chronic spinal cord injury: a pilot study. J Spinal Cord Med. 33(2):150-8

therapies after spinal cord injury. NeuroRehabilitation 28(3):231-48

with spinal cord injury: a case series. J Spinal Cord Med 31(2):215-21.

spinal cord injury population. J Spinal Cord Med. 30 Suppl 1:S178-83

226(1): 200-6

272 Topics in Paraplegia

1755-9.

Phys Med Rehabil. 85(10):1567-77

Medical Publishing; Pg 635-731.

infection? Infect Dis Clin North Am 13(4): 871–877.

Ther. 49(3):229-34.

habil. 8:49.


[101] Yan Y, Yang D, Zarnowska ED, Du Z, Werbel B, Valliere C, Pearce RA, Thomson JA, Zhang SC. Directed differentiation of dopaminergic neuronal subtypes from human embryonic stem cells. Stem Cells. 2005 Jun-Jul;23(6):781-90

[112] Neuhuber B, Brashinger AL, Paul C, et al.Stem cell delivery by lumbar puncture as a therapeutic alternative to direct injection into injured, spinal cord.J Neurosurg Spine:

Mesenchymal Stem Cells in Spinal Cord Injury

http://dx.doi.org/10.5772/58323

275

[113] Mai Q, Yu Y, Li T, Wang L, Chen MJ, Huang SZ, Zhou C, Zhou Q. Derivation of hu‐ man embryonic stem cell lines from parthenogenetic blastocysts. Cell Res. 2007 Dec;

[114] Pal R, Gopinath C, Rao NM, Banerjee P, Krishnamoorthy V, Venkataramana NK, To‐ tey S. Functional recovery after transplantation of bone marrow-derived human mes‐

[115] Harrop JS, Naroji S. Maltenfort MG, Rathiff JK, Tjoumakaris SI, Frank B, etal: Neuro‐ logic improvement after thoracic, thoracolumbar and lumbar spinal cord (conus me‐

[116] Burns AS, Lee BS, Ditunno JF Jr, Tessler A: Patient selection for clinical trials: the reli‐ ability of the early spinal cord injury examination. J Neurotrauma 20:477-482,2013.

[117] Kirshblum S, Millis S, McKinley W, Tulsky D: Late neurologic recovery after trau‐

[118] Marino RJ, Ditunno JF Jr. Donovan WH. Maynard F Jr: Neurologica recovery after traumatic spinal cord injury: data from the Model Spinal Cord Injury:data from the Model Spinal Cord Injury Systems. Arch Phys Med Rehabil 80:1391-1396.1999.

[119] Mehta T, Feroz A, Thakkar U, Vanikar A, Shah V, Trivedi H: Subarachnoid place‐ ment of stemcells in neurological disorders. Transplatn Proc 40:1145-1147, 2008

[120] Cristante AF, Barros – Filho TE, Tatsui N. Mendrone A, Caldas JG, Camargo A, et al: Stemcells in the treatment of chronic spinal cord injury: evaluation of somatosensi‐

[121] Saberi H, Firouzi M, Habibi Z, Moshayedi P, Aghayan HR, Arjmand B, et al: Safety of intramedullary Schwann cell transplantation for post rehabilitation spinal cord in‐ juries: 2 year follow up of 33 cases. Clinical article. J Neurosurg Spine

[122] Yoon SH, Shim YS, Park YH, Chung JK, Nam JH, Kim MO, et al: Complete spinal cord injury treatment using autologous bone marrow cell transplantation and bone marrow stimulation with granulocyte macrophage-colony stimulating factor: Phase

[123] Kumar AA, Kumar SR,, Narayanan R, Arul K, Baskaran M: Autologous bone mar‐ row derived monomuclear cell therapy for spinal cord injury: a pahse I/II clinical

[124] Yoon SH, Shim YS, Park YH, Chung JK, Nam JH, Kim MO, et al: Complete spinal cord injury treatment using autologous bone marrow cell transplantation and bone

safety and primary efficacy data. Exp. Clin Transplant 7:241-248,2009.

matic spinal cord injury. Arch Phys Med Rehabil 85:1811-1817,2004.

tive evoked potentials in 39 patients. Spinal Cord 47:733-738, 2009.

I/II clinical trial. Stem Cells 25:2066-2073, 2007.

enchymal stromal cells in a rat model of spinal cord injury.

dullaris)injuries. Spine (Phila Pa 1976)36:21-25,2011.

9:390-9,2008.

17(12):1008-19.

15:515-525,2011.


[112] Neuhuber B, Brashinger AL, Paul C, et al.Stem cell delivery by lumbar puncture as a therapeutic alternative to direct injection into injured, spinal cord.J Neurosurg Spine: 9:390-9,2008.

[101] Yan Y, Yang D, Zarnowska ED, Du Z, Werbel B, Valliere C, Pearce RA, Thomson JA, Zhang SC. Directed differentiation of dopaminergic neuronal subtypes from human

[102] Houlé JD, Reier PJ. Transplantation of fetal spinal cord tissue into the chronically in‐

[103] Ankeny DP, McTigue DM, Jakeman LB. one marrow transplants provide tissue pro‐ tection and directional guidance for axons after contusive spinal cord injury in rats.

[104] Fitch MT, Doller, C, Combs CK, Landreth GE, Silver J. Cellular and molecular mech‐ anism of glial scarring and progressive cavitation: *in vivo* and *in vitro* analysis of in‐ flammation-induced secondary injury after CNS trauma. 1999 J. Neurosci., 19(19),

[105] Jones DG, Anderson ER, Galvin KA. Spinal cord regeneration: moving tentatively to‐

[106] Knoller N, Auerbach G, Fulga V, Zelig G, Attias J, Bakimer R, et al. Clinical experi‐ ence using incubated autologous macrophages as a treatment for complete spinal

[107] Nistor GI, Totoiu MO, Haque N, Carpenter MK, Keirstead HS. Human embryonic stem cells differentiate into oligodendrocytes in high purity and myelinate after spi‐

[108] Moviglia GA, Fernandez Viña R, Brizuela JA, Saslavsky J, Vrsalovic F, Varela G, Bas‐ tos F, Farina P, Etchegaray G, Barbieri M, Martinez G, Picasso F, Schmidt Y, Brizuela P, Gaeta CA, Costanzo H, Moviglia Brandolino MT, Merino S, Pes ME, Veloso MJ, Rugilo C, Tamer I, Shuster GS. Combined protocol of cell therapy for chronic spinal cord injury. Report on the electrical and functional recovery of two patients. Cyto‐

[109] Kang KS, Kim SW, Oh YH, Yu JW, Kim KY, Park HK, Song CH, Han H. A 37-yearold spinal cord-injured female patient transplanted of multipotent stem cells from human UC blood, with improved sensory perception and mobility, both functionally

[110] Deda H, Inci MC, Kureckei A, et al. Treatment of chronic spinal cord injured patients with Autologous bone marrow –derived hematopoietic stemcell transplantation. Cy‐

[111] Jung DI, Ha J, Kang BTm et. A comparison of autologous and allogenic bone mar‐ row-derived mesenchymal stem cell transplantation in canine spinal cord injury. J

and morphologically: a case study. Cytotherapy 2005 7(4):368-73.

cord injury: phase I study results. 2005 J Neurosurg Spine. Sep; 3(3):173-81.

nal cord transplantation. Glia. 2005 Feb;49(3):385-96. Wolfram Tetzlaff et al

wards new perspectives. NeuroRehabilitation. 2003 18(4):339-51.

jured adult rat spinal cord. J Comp Neurol. 1988 Mar 22;269(4):535-47.

embryonic stem cells. Stem Cells. 2005 Jun-Jul;23(6):781-90

Exp Neurol. 2004 Nov;190(1):17-31.

8182-8198.

274 Topics in Paraplegia

therapy. 2006; 8(3):202-9.

thotherapy:6:551-64,2008.

Neurol Sci ;285:67-77, 2009


marrow stimulation with granulocyte macrophage-colony stimulating factor: Phase I/II clinical trial. Stem Cells 25:2066-2073, 2007.

**Chapter 12**

**35 Years in Research on Spinal Cord Lesions and Repair**

Paraplegia by spinal cord lesion is a severe condition which cannot be cured.

It is known since the ancient times as figured figured in the Ninive basrelief (fig 1) and described in the Smith papirus (fig 2). Since those times it is known that the spinal cord, after

> © 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Giorgio Brunelli

**1. Introduction**

http://dx.doi.org/10.5772/56988

severance does not allow healing.

**Figure 1.** Dying lioness with paraplegia (Ninive bas relief)

Additional information is available at the end of the chapter


### **35 Years in Research on Spinal Cord Lesions and Repair**

#### Giorgio Brunelli

marrow stimulation with granulocyte macrophage-colony stimulating factor: Phase

[125] Tetzlaff W, Okon EB, Karmi-Abdolrezaee S, Hill CE, Sparling JS, Plemel JR, et al; A systemic review of cellular transplantation therapies for spinal cord injury. J Neuro‐

[126] Park SS, Byeon YE, Ryu HH, Kang BJ, Kim Y, Kim WH, et al: Comparison of canine umbilical cord blood-derived emsenchymal stemcell transplantation times: Involve‐ ment of astrogliosis, inflammation, intracellular actin cytoskeleton pathways and

[127] Jung DI, Ha J, Kang BTm et. A comparison of autologous and allogenic bone mar‐ row-derived mesenchymal stem cell transplantation in canine spinal cord injury. J

[128] Kishk N A, Gabr H, Hamdy S, Afifi L, Abokresha N. mahmoud H, eta l: case control series of intrathecal autologous bone marrow mesenchymal stem cell therapy for

chronic spinal cord injury. Neuro rehabil Neural Repair 24:702-708, 2010.

I/II clinical trial. Stem Cells 25:2066-2073, 2007.

neurotrophin. Cell Transplant (epub ahead of print), 2011.

trauma 28:1611-1682,2011.

276 Topics in Paraplegia

Neurol Sci ;285:67-77, 2009.

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/56988

#### **1. Introduction**

Paraplegia by spinal cord lesion is a severe condition which cannot be cured.

It is known since the ancient times as figured figured in the Ninive basrelief (fig 1) and described in the Smith papirus (fig 2). Since those times it is known that the spinal cord, after severance does not allow healing.

**Figure 1.** Dying lioness with paraplegia (Ninive bas relief)

© 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Paraplegia by S.pinal Cord Injury is a very heavy burden:

Non permissiveness and incurability of paraplegia are a DOGMA.

Various explanations of this dogma have been tried without solution.

cells which support the regeneration of the nerve fibres in peripheral nerves.

psycological point of view and

was able to help regeneration of the spinal cord.

and architectural facilities.

**Figure 4.** Removal of 1 cm of spinal cord in a rat

the world),

**a.** for patients who at the age of their most productive capacity are put in a weelchair for life

35 Years in Research on Spinal Cord Lesions and Repair

http://dx.doi.org/10.5772/56988

279

(More than 2000 new cases a year occur in Italy and 18/20 cases every milion of inhabitants in

**2.** for the families which must assist their relative from the economical, physical and

**3.** for society and the country which have to supply medical assistance, economical support

My research on spinal cord started in 1978 when, thinking that microsurgery had solved almost all the problems related to peripheral nerves lesions, I wondered why no surgical treatment

One of the hypotetical reason (besides the scar formation) was the lack (in the cord) of Schwann

Thissemplicisticideapushedmetoresectonecentimeterofcordinratsandtoputintheresulting

As a result I found that the grafts were reinhabited by the axons descending from the brain

but that at the very end of the grafts the axons stopped progressing. (fig 5 and 6)

gap several grafts of peripheral nerves (sciatic n.) which contain Schwann cells. (fig.4)

and undergo severe physical, economical and psycological problems.

**Figure 2.** Smith papirus describing the symptoms of cord lesion

It is due to the *nonpermissiveness* of the cord for the advancement of the axons that regrow from the brain neurons.

Not only walking is hindered but there is compromission of all the vegetative function and pression sores (fig 3) not to say the psicological damage of the patient and of his/her relatives and the social heavy burden for assistance and architectural modifications.

**Figure 3.** Ptressure sores in a paraplegic

Paraplegia by S.pinal Cord Injury is a very heavy burden:

**a.** for patients who at the age of their most productive capacity are put in a weelchair for life and undergo severe physical, economical and psycological problems.

(More than 2000 new cases a year occur in Italy and 18/20 cases every milion of inhabitants in the world),


Non permissiveness and incurability of paraplegia are a DOGMA.

Various explanations of this dogma have been tried without solution.

My research on spinal cord started in 1978 when, thinking that microsurgery had solved almost all the problems related to peripheral nerves lesions, I wondered why no surgical treatment was able to help regeneration of the spinal cord.

One of the hypotetical reason (besides the scar formation) was the lack (in the cord) of Schwann cells which support the regeneration of the nerve fibres in peripheral nerves.

Thissemplicisticideapushedmetoresectonecentimeterofcordinratsandtoputintheresulting gap several grafts of peripheral nerves (sciatic n.) which contain Schwann cells. (fig.4)

**Figure 4.** Removal of 1 cm of spinal cord in a rat

**Figure 2.** Smith papirus describing the symptoms of cord lesion

the brain neurons.

278 Topics in Paraplegia

**Figure 3.** Ptressure sores in a paraplegic

It is due to the *nonpermissiveness* of the cord for the advancement of the axons that regrow from

Not only walking is hindered but there is compromission of all the vegetative function and pression sores (fig 3) not to say the psicological damage of the patient and of his/her relatives

and the social heavy burden for assistance and architectural modifications.

As a result I found that the grafts were reinhabited by the axons descending from the brain but that at the very end of the grafts the axons stopped progressing. (fig 5 and 6)

**Figure 5.** Grafts of sciatic nerve put in the cord of a rat after removal of 1 cm of cord.

**Figure 7.** The team of the C.A.L.I.E.S program in Montpellier: VW: Von Wild, R: Rabishong, B:Brunelli

35 Years in Research on Spinal Cord Lesions and Repair

http://dx.doi.org/10.5772/56988

281

**Figure 8.** The multichannel control unit of the CALIES, to be implanted under the skin of the abdomen, connected with 8 muscles of the legs and stimulated transcutaneously by an antenna connected with the computer for stimula‐

These experimental operations had to be abandoned due to the high cost of the electronic devices (not to say to the necessity for the paraplegics to pull a trolley with batteries and the computerized program that had been studied by means of a meticulous gait analysis that could not anyway change the recorded program of the computer in front of unanticipated obstacles).

tion of the muscles.

**Figure 6.** The graft has been reinhabited by the axons regrowing from the upper neurons but axons stopped pro‐ gressing as soon as in contact again with the C.N.S. (cord). Only a few micron of progession was observed due to the exit of Schwann cells.

This fact confirmed the idea that the central nervous system (C.N.S. of which the cord is part) is "non permissive" for the regrowing axons progression inside it (perhaphs due to specific "no-go" molecules).

Also I took part in the C.A.L.I.E.S. (Computer Assisted Locomotion by means of Implanted Electrical Stimulation) and S.U.A.W (Stand Up And Walk) (European programs intended to obtain walking by means of implanted electrical stimulation of muscles). (fig. 7) The electrical stimulation of the muscles was given by electrodes implanted in 8 muscles of each inferior limb by means of a control-unit implanted under the abdominal skin that received impulses from a computer through a transcutaneous antenna. (fig 8)

**Figure 7.** The team of the C.A.L.I.E.S program in Montpellier: VW: Von Wild, R: Rabishong, B:Brunelli

**Figure 5.** Grafts of sciatic nerve put in the cord of a rat after removal of 1 cm of cord.

exit of Schwann cells.

280 Topics in Paraplegia

"no-go" molecules).

**Figure 6.** The graft has been reinhabited by the axons regrowing from the upper neurons but axons stopped pro‐ gressing as soon as in contact again with the C.N.S. (cord). Only a few micron of progession was observed due to the

This fact confirmed the idea that the central nervous system (C.N.S. of which the cord is part) is "non permissive" for the regrowing axons progression inside it (perhaphs due to specific

Also I took part in the C.A.L.I.E.S. (Computer Assisted Locomotion by means of Implanted Electrical Stimulation) and S.U.A.W (Stand Up And Walk) (European programs intended to obtain walking by means of implanted electrical stimulation of muscles). (fig. 7) The electrical stimulation of the muscles was given by electrodes implanted in 8 muscles of each inferior limb by means of a control-unit implanted under the abdominal skin that received impulses

from a computer through a transcutaneous antenna. (fig 8)

**Figure 8.** The multichannel control unit of the CALIES, to be implanted under the skin of the abdomen, connected with 8 muscles of the legs and stimulated transcutaneously by an antenna connected with the computer for stimula‐ tion of the muscles.

These experimental operations had to be abandoned due to the high cost of the electronic devices (not to say to the necessity for the paraplegics to pull a trolley with batteries and the computerized program that had been studied by means of a meticulous gait analysis that could not anyway change the recorded program of the computer in front of unanticipated obstacles). My second step (1981) was the connection of the above the lesion cord (by means of a graft of peripheral nerve) to peripheral nerves and muscles. (fig 9 and 10).

The advise was: go to primates. At that time I had not the facilities nor the permission for operating monkeys in Italy. Therefore I was compelled to go abroad and I found the friendy help of dr Carlstedt who hosted me in the primatology institute of the Karolinska Institutet in Solna (Stockolm). I and my staff operated on, over there, 20 "macaca fascicularis" connecting the above the lesion cord with peripheral nerves by means of autologous grafts and checking the results by means of clinical observation, electromyography, magnetic stimulation of both the nerve going to muscles and of the brain (after craniotomy) and histology of the cord and of the graft. (Fig 12,13, 14). Magnetic stimulation of the brain, external and after craniotomy as well as E.M.G. of the grafts and the muscles showed good muscle responses with different latency times according to the distance from the recording electrode (fig 15) The monkeys were

35 Years in Research on Spinal Cord Lesions and Repair

http://dx.doi.org/10.5772/56988

283

**Figure 11.** Motor-end plates newly formed at the contact of the graft with the muscle.

able to move the reinnervated muscles.

**Figure 12.** Sketch of the place where the graft is inserted.

**Figure 9.** Connection of the C.S,T, to peripheral nerves

**Figure 10.** Sketch of the connection.

The regrowing axons elongated up to the muscle forming new motor end-plates and functional connections. (fig 11 ).

By this connection the result was effective and rats could walk, even if, of course, with some limitation. The presentation of theses results at an international meeting (in San Antonio) stired up a lot of skepticism: *"rats could walk even without any attempt of repairing the cord" (*whic is not true).

**Figure 11.** Motor-end plates newly formed at the contact of the graft with the muscle.

My second step (1981) was the connection of the above the lesion cord (by means of a graft of

The regrowing axons elongated up to the muscle forming new motor end-plates and functional

By this connection the result was effective and rats could walk, even if, of course, with some limitation. The presentation of theses results at an international meeting (in San Antonio) stired up a lot of skepticism: *"rats could walk even without any attempt of repairing*

peripheral nerve) to peripheral nerves and muscles. (fig 9 and 10).

282 Topics in Paraplegia

**Figure 9.** Connection of the C.S,T, to peripheral nerves

**Figure 10.** Sketch of the connection.

*the cord" (*whic is not true).

connections. (fig 11 ).

The advise was: go to primates. At that time I had not the facilities nor the permission for operating monkeys in Italy. Therefore I was compelled to go abroad and I found the friendy help of dr Carlstedt who hosted me in the primatology institute of the Karolinska Institutet in Solna (Stockolm). I and my staff operated on, over there, 20 "macaca fascicularis" connecting the above the lesion cord with peripheral nerves by means of autologous grafts and checking the results by means of clinical observation, electromyography, magnetic stimulation of both the nerve going to muscles and of the brain (after craniotomy) and histology of the cord and of the graft. (Fig 12,13, 14). Magnetic stimulation of the brain, external and after craniotomy as well as E.M.G. of the grafts and the muscles showed good muscle responses with different latency times according to the distance from the recording electrode (fig 15) The monkeys were able to move the reinnervated muscles.

**Figure 12.** Sketch of the place where the graft is inserted.

**Figure 13.** An operated monkey grasping the bars of its cage with its foot

**Figure 14.** Craniotomy done for magnetography of the graft and muscles.

As at that time we had no effective treatment for spinal cord injury (S.C.I.) I thought, (and the Ethical Committee of the Italian Health Organisation agreed) that it could be ethically justified to operate on *fully informed volunteer patients* by means of surgical procedures already in current use, tried and checked in animals and human beeings as, for instance, the transfer of nerves (ulnar nerves from upper to lower limbs, fig 16 and 16 bis) or the grafting from the corticospinal tract of the cord to peripheral nerves of the lower limbs by means of autologous nerve-grafts. **Figure 16.** Sketch of the transfer of the ulnar nerve from the upper to the lower limb.

**Figure 15.** Emg: different latencies according to the distance of the recording electrode.

35 Years in Research on Spinal Cord Lesions and Repair

http://dx.doi.org/10.5772/56988

285

resuscitation, I got better survival rate and functional results.

palsyes of the hand).

(The damage at the donor hands due to ulnar nerve removal, was repaired by the S.Bunnell and D.Smith procedures. (fig 17) which are well-known procedures of tendon transfers for

Later on in my own laboratories (in the private hospital "saint Rocco of Franciacorta" in Ome), in the following four groups of monkeys operated on with immobilisation in plaster and

**Figure 15.** Emg: different latencies according to the distance of the recording electrode.

**Figure 13.** An operated monkey grasping the bars of its cage with its foot

284 Topics in Paraplegia

**Figure 14.** Craniotomy done for magnetography of the graft and muscles.

As at that time we had no effective treatment for spinal cord injury (S.C.I.) I thought, (and the Ethical Committee of the Italian Health Organisation agreed) that it could be ethically justified to operate on *fully informed volunteer patients* by means of surgical procedures already in current use, tried and checked in animals and human beeings as, for instance, the transfer of nerves (ulnar nerves from upper to lower limbs, fig 16 and 16 bis) or the grafting from the corticospinal tract of the cord to peripheral nerves of the lower limbs by means of autologous nerve-grafts.

**Figure 16.** Sketch of the transfer of the ulnar nerve from the upper to the lower limb.

(The damage at the donor hands due to ulnar nerve removal, was repaired by the S.Bunnell and D.Smith procedures. (fig 17) which are well-known procedures of tendon transfers for palsyes of the hand).

Later on in my own laboratories (in the private hospital "saint Rocco of Franciacorta" in Ome), in the following four groups of monkeys operated on with immobilisation in plaster and resuscitation, I got better survival rate and functional results.

In order to understand how this was possible a multidisciplinary research with various institutes of the University of Brescia has been done, in rats, to check which neurotransmitter

35 Years in Research on Spinal Cord Lesions and Repair

http://dx.doi.org/10.5772/56988

287

Vecuronium (nicotinic receptors antagonist) was injected i.v. in the saphen vein (after trache‐ ostomy for artificial ventilation) obtaining the palsy of all the muscles except the reinnervated one. Then GYKY 52466 (antagonist of the glutamate receptors) was injected intraperitoneally

Immunoblot analysis of Choline acetyltransferase, Vesicular acetylcholine transporter and vesicular glutamate transporter, demonstrated that the markers for Acetilcholine were present

**Figure 19.** Immunoblot demonstrating that in control muscles are present the markers for ACh whereas in the rein‐

This research was able to demonstrate that the receptors of the motor end plates, stimulated by the presynaptic nerve fibres (glutamatergic) were able to change one of the molecules of theyr jonic canals so accepting the glutamate transmitter (Proceeding of the National Academy

Going back to the operation of connecting the muscles to the cortico spinal tract by means of autologous nerve grafts, the peroneal component of the sciatic nerve (bilaterally) was used to

The operated patients stayed 4 to 6 days in the intensive care unit and then started a long

Two teams of microsurgeons, aneasthesiologists and nurses were at work for 12 hours.

CTB retrograde tracing of reticular formation and red nucleus neurons was positive.

graft from the C.S.T. of the cord to the muscles (glutei and quadriceps) (fig).

in controls whereas those for Glut were present in the reinnervated muscle. (fig 19).

obtaining the disappearance of the response of the reinnervated muscle.

was really rensponsible for the muscle response:

nervated muscles the Glut markers are present.

of Science - P.N.S.A).

program of re-education.

**Figure 17.** Repair of the damage done at the hands by means of Bunnel and Smith operations

Muscles were first completely disconnected from central nervous system (C.N.S.) and then reconnected to it by means of an autologous nerve graft inserted into the cortico spinal tract (C.S.T.), with exclusion of the lower motoneuron (fig 18).

**Figure 18.** Through this operation the muscle receives innervation by the upper motoneurons and by glutamate in‐ stead of acetilcholine

A question arose: how could the muscles (the receptors of which normally respond to acetilcholine) respond to a different neurotransmitter, i.e. glutamate?

In order to understand how this was possible a multidisciplinary research with various institutes of the University of Brescia has been done, in rats, to check which neurotransmitter was really rensponsible for the muscle response:

Vecuronium (nicotinic receptors antagonist) was injected i.v. in the saphen vein (after trache‐ ostomy for artificial ventilation) obtaining the palsy of all the muscles except the reinnervated one. Then GYKY 52466 (antagonist of the glutamate receptors) was injected intraperitoneally obtaining the disappearance of the response of the reinnervated muscle.

Immunoblot analysis of Choline acetyltransferase, Vesicular acetylcholine transporter and vesicular glutamate transporter, demonstrated that the markers for Acetilcholine were present in controls whereas those for Glut were present in the reinnervated muscle. (fig 19).

Muscles were first completely disconnected from central nervous system (C.N.S.) and then reconnected to it by means of an autologous nerve graft inserted into the cortico spinal tract

**Figure 17.** Repair of the damage done at the hands by means of Bunnel and Smith operations

**Figure 18.** Through this operation the muscle receives innervation by the upper motoneurons and by glutamate in‐

A question arose: how could the muscles (the receptors of which normally respond to

acetilcholine) respond to a different neurotransmitter, i.e. glutamate?

(C.S.T.), with exclusion of the lower motoneuron (fig 18).

stead of acetilcholine

286 Topics in Paraplegia

**Figure 19.** Immunoblot demonstrating that in control muscles are present the markers for ACh whereas in the rein‐ nervated muscles the Glut markers are present.

CTB retrograde tracing of reticular formation and red nucleus neurons was positive.

This research was able to demonstrate that the receptors of the motor end plates, stimulated by the presynaptic nerve fibres (glutamatergic) were able to change one of the molecules of theyr jonic canals so accepting the glutamate transmitter (Proceeding of the National Academy of Science - P.N.S.A).

Going back to the operation of connecting the muscles to the cortico spinal tract by means of autologous nerve grafts, the peroneal component of the sciatic nerve (bilaterally) was used to graft from the C.S.T. of the cord to the muscles (glutei and quadriceps) (fig).

Two teams of microsurgeons, aneasthesiologists and nurses were at work for 12 hours.

The operated patients stayed 4 to 6 days in the intensive care unit and then started a long program of re-education.

After 5 months the first voluntary movements appeared which became functional after one year (fig.20 and 21).

**Figure 20.** Active abduction

This occurs probably due to a partial remodelling of the molecules of the trans-membrane channels of the motor end-plates which go back to an embrionic type of channels changing

**Figure 22.** The patient walking with the help of an ambulator. (She had undergone a guillottine severance of the cord

35 Years in Research on Spinal Cord Lesions and Repair

http://dx.doi.org/10.5772/56988

289

**1.** the upper motoneuron can build up a cytoskeleton longer than that of the lower moto‐

**4.** alteration of the motor end-plates from cholinergic to glutamatergic takes place

**5.** brain plasticity by multiple single neurons, (not only by cortical areas) is demonstrated

The connection of the graft was inevitably random with the descending axons of the lateral

In the C.S.T. thousans and thousands of motor axons run coming from neurons of different areas of the brain cortex having different functions, destined to different muscles having

The least we could expect should have been co-contractions with severe hindrance of function. All the muscles connected with the C.S.T. should contract contemporarily with no useful

one of their 5 molecular constituents. Our research demonstrated 5 novelties:

neuron (up to the muscles)

**2.** functional connection with muscles occurs,

at T8 and grafts from the C.S.T. to the glutei and quadriceps).

bundle of the cord: the cortico-spinal-tract.

different and even contrasting movements.

function.

**3.** also selective voluntary activation of the muscles occurs,

But once explained this mistery, one more mistery became evident:

**Figure 21.** Active extension of the knee without co-contractions

Soon after the patient was able to walk with the help of an ambulator or of quadripode sticks. (fig.22) (cortico spinal tract) by means of nerve grafts, the peroneal nerve (bilaterally) was used to graft from the cortico spinal tract of the cord to the muscles.

Through this operation the muscles receive the innervation by the presynaptic neurons by means of glutamate transmitter (fig 18).

This result has been published in 2006 in the P.N.A.S.

**Figure 22.** The patient walking with the help of an ambulator. (She had undergone a guillottine severance of the cord at T8 and grafts from the C.S.T. to the glutei and quadriceps).

This occurs probably due to a partial remodelling of the molecules of the trans-membrane channels of the motor end-plates which go back to an embrionic type of channels changing one of their 5 molecular constituents.

Our research demonstrated 5 novelties:

After 5 months the first voluntary movements appeared which became functional after one

year (fig.20 and 21).

288 Topics in Paraplegia

**Figure 20.** Active abduction

**Figure 21.** Active extension of the knee without co-contractions

This result has been published in 2006 in the P.N.A.S.

means of glutamate transmitter (fig 18).

to graft from the cortico spinal tract of the cord to the muscles.

Soon after the patient was able to walk with the help of an ambulator or of quadripode sticks. (fig.22) (cortico spinal tract) by means of nerve grafts, the peroneal nerve (bilaterally) was used

Through this operation the muscles receive the innervation by the presynaptic neurons by


But once explained this mistery, one more mistery became evident:

The connection of the graft was inevitably random with the descending axons of the lateral bundle of the cord: the cortico-spinal-tract.

In the C.S.T. thousans and thousands of motor axons run coming from neurons of different areas of the brain cortex having different functions, destined to different muscles having different and even contrasting movements.

The least we could expect should have been co-contractions with severe hindrance of function. All the muscles connected with the C.S.T. should contract contemporarily with no useful function.

On the contrary only the muscles that the patient wanted to move were active. The explanation may be that there must be some until now unknown mechanism of feed back by which the mental command coming from the frontal lobe is able to acticate only those neurons that at the periphery have been connected to the wanted muscles.

This means that the single and selective movements depend on the activation of milion of single motorneurons scattered in various areas of the cortex and not on neurons of a given cortical area.

This means that there is a brain plasticity by multiple (milions) single neurons scattered in different places of the brain cortex that fire simultaneously under the mind command for the desired movement due to theyr peripheral connection even if theyr previous function was different. (fig 22)

Connections of the cord with peripheral nerves have been tried also with other different surgical protocols with the aim of correcting other types of palsies as, for instance, those due to total brachial plexus avulsions in paraplegics.

Total avulsions of the roots of the brachial plexus cannot be repaired by sutures or nerve grafts but can only be treated by extraplexual neurotisations i.e. transfers of extraplexual donor nerves.

**Figure 24.** Connection of the brachial plexus of a rat to the c.s.t.

**Figure 25.** Extension of the elbow,wrist and fingers after connection ocf the radial nerve to the C.S.T.

In human beings with paraplegia no additional damage at the lower limbs can occur.

35 Years in Research on Spinal Cord Lesions and Repair

http://dx.doi.org/10.5772/56988

291

After 5 months the extension of the wrist and of the digits was recovered.

In rare cases these lesions occur simultaneously with traumatic paraplegia (or in patients with previous paraplegia) (3 to 5 %).

With the aim of restoring function to the paralized arm in paraplegics I have set up a research in rats by cutting the radial nerve at the armpit and connecting it by means of an autologous graft to the cortico-spinal tract of the spinal cord at level of T3 – T4. (fig 23, 24 and 25)

**Figure 23.** Sketch of the experimental connection of the C.S.T. of the cord below T3 with the radial nerve at the arm‐ pit to re innervate the brachial plexus in paraplegics.

**Figure 24.** Connection of the brachial plexus of a rat to the c.s.t.

On the contrary only the muscles that the patient wanted to move were active. The explanation may be that there must be some until now unknown mechanism of feed back by which the mental command coming from the frontal lobe is able to acticate only those neurons that at

This means that the single and selective movements depend on the activation of milion of single motorneurons scattered in various areas of the cortex and not on neurons of a given

This means that there is a brain plasticity by multiple (milions) single neurons scattered in different places of the brain cortex that fire simultaneously under the mind command for the desired movement due to theyr peripheral connection even if theyr previous function was

Connections of the cord with peripheral nerves have been tried also with other different surgical protocols with the aim of correcting other types of palsies as, for instance, those due

Total avulsions of the roots of the brachial plexus cannot be repaired by sutures or nerve grafts but can only be treated by extraplexual neurotisations i.e. transfers of extraplexual donor

In rare cases these lesions occur simultaneously with traumatic paraplegia (or in patients with

With the aim of restoring function to the paralized arm in paraplegics I have set up a research in rats by cutting the radial nerve at the armpit and connecting it by means of an autologous

**Figure 23.** Sketch of the experimental connection of the C.S.T. of the cord below T3 with the radial nerve at the arm‐

graft to the cortico-spinal tract of the spinal cord at level of T3 – T4. (fig 23, 24 and 25)

the periphery have been connected to the wanted muscles.

to total brachial plexus avulsions in paraplegics.

previous paraplegia) (3 to 5 %).

pit to re innervate the brachial plexus in paraplegics.

cortical area.

290 Topics in Paraplegia

different. (fig 22)

nerves.

**Figure 25.** Extension of the elbow,wrist and fingers after connection ocf the radial nerve to the C.S.T.

After 5 months the extension of the wrist and of the digits was recovered.

In human beings with paraplegia no additional damage at the lower limbs can occur.

#### **Author details**

Giorgio Brunelli\*

Address all correspondence to: giorgio.brunelli@midollospinale.com

Medical School of the University of Brescia, Italy

#### **References**


[14] Brunelli et al: 2005: Glutamatergic reinnervation through peripheral nerve grafts dic‐ tates assembly of glutamatergic synapses at rat skeletal muscle. P.N.A.S.

35 Years in Research on Spinal Cord Lesions and Repair

http://dx.doi.org/10.5772/56988

293

[15] Brunelli: 2005: Les reparations chirugicales des lesions de la moelle epiniere. Bull,

[16] Pizzi M., Brunelli G.A. et al: 2006: Glutamatergic innervation of rat skeletal muscles

[17] von Wild K.R., Brunelli G.A.: 2003: restoration of locomotion in paraplegics with aid of autologous bypass grafts for direct neurotisation of muscles by upper motoneur‐

[18] Brunelli G and von Wild K: Unsuspected plasicity of single neurons after connection of the C.S.T.with peripheral nerves in spinal cord lesion. J.Korean neurosurg.society,

on. The future surgery of the spinal cord?. Acta Neurochir suppl. 87, 107-112.

by supraspinal neurons. Current Opinion neurobiol 16,323-328

102,8752-8757.

46, 1, 1-4.

Acad.Natle med, Paris, 189.n.6.1135-1149.


[14] Brunelli et al: 2005: Glutamatergic reinnervation through peripheral nerve grafts dic‐ tates assembly of glutamatergic synapses at rat skeletal muscle. P.N.A.S. 102,8752-8757.

**Author details**

292 Topics in Paraplegia

Giorgio Brunelli\*

**References**

Address all correspondence to: giorgio.brunelli@midollospinale.com

sioni midollari.ATTI II° SIMP.intern. pietra Ligure

P.N.S. jour, recstr. microsurg.4, 2455-250

[1] Brunelli: 1983, Innesti sperimentali per lesioni del midollo spinale:G.I.O.T, 9, 53-57.

[2] Aguayo A, 1981.Mc Gill, Montreal. Non permissiveness for regrowing axons in the

[3] Brunelli: 1983, Studio sperimentale sulla rigenerazione assonale nel midollo.Policlini‐

[4] Chiasserini: 1944, Compendio di traumatologia del sistema nervoso. Ed Humanitas

[5] Brunelli: 1984, Acquisizioni recenti sulle possibilità di riparazione chirurgica delle le‐

[6] Brunelli: 1988, Experimental spinal cord repair. In Textbook of microsurgery. Ed Bru‐

[7] Brunelli: 1988, Experimental repair of spinal cord lesions by grafting from C.N.S. to

[8] Brunelli: 1996 Results in experimental spinal cord repair. Proc 12th Symposium

[9] Brunelli: 1996, Experimental surgery in spinal cord lesions by connecting upper mo‐

[10] Brunelli et al: 1996, Sperimentazione sulla possibile riparazione delle lesioni del mid‐ ollo spinale: gli innesti dal SNC al SNP producono connessioni funzionali. Mono‐

[12] Brunelli: 1999: Restoration of walking in paraplegia by transfering the ulnar nerve to

toneurons directly to peripheral targets. J.periph.nerv.system 1, 111-118.

[11] Brunelli: 1997: Experimental spinal cord repair.Surgical tecnology 391-395.

[13] Brunelli: 2002: The future: surgery of the spinal cord?. Hand clinic: 18,541-546.

Medical School of the University of Brescia, Italy

central nervous sistem.

nelli, Masson ed, 547-554.

I.S.R.M., Singapore, 3-6.

graph, Delfo,Brescia 1-56.

the hip. Microsurgery,223-226

co, 90,1334-1336.

Nova, Roma.


### *Edited by Yannis Dionyssiotis*

Spinal cord injury related paraplegia changes a person's life in a sudden way. The most important issue for physicians, therapists and caregivers is to manage the complications that arise, and help paraplegic subjects return to a productive integrated life within society. The book Topics in Paraplegia provides modern knowledge in this direction. Addressing hot topics related to paraplegia, ranging from surgical management to research therapies with mesenchymal stem cells, this book could be a valued reference for physiatrists, neurosurgeons, orthopaedic surgeons, neurologists and physical therapists. The book is organized into four sections. The first covers the epidemiology and psychological conditions associated with paraplegia, the second discusses surgical management and common rehabilitation interventions; the third medical complications and special musculoskeletal issues, while the last outlines current research in animals and humans.

Topics in Paraplegia

Topics in Paraplegia

*Edited by Yannis Dionyssiotis*

Photo by Svetlanais / iStock