**2. Pathology of spinal cord injury**

Spinal cord injury (SCI) is a devastating event that significantly affects the morbidity and quality of life in adults (Average age of patients at the time of injury is 38.0 years old). [1] The prevalence of traumatic SCI worldwide is approximately 750 per million with an increasing annual incidence. [2] In order to be able to develop effective treatments for SCI, it is necessary to have a detailed understanding of the pathophysiological events that happen during SCI in the body and how they interact with each other to cause the functional deficits seen in patients. These events have complicated roles post-SCI. For example, the inflammatory response and reactive astrogliosis that are seen post-SCI have damaging *and* neuroprotective and –restora‐ tive effects. [3]

© 2013 Salewski et al.; licensee InTech. This is an open access article 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. © 2013 Salewski et al.; licensee InTech. This is a paper 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.

The pathophysiology of SCI is biphasic; it consists of a "primary" and a "secondary" stage. The primary injury is the result of an initial mechanical insult, which is followed by a series of cellular and molecular events that, in turn, lead to further destruction of the spinal cord tissue. It is well known that the mechanical injury seldom results in complete transection of the spinal cord. Most fracture dislocations or burst fractures of the spine result in disc displacement into and laceration or compression of the cord resulting in blood vessel, axonal and cellular membrane disruption.

first two hours post-SCI is the immediate stage during which the body is going through processes as a result of the primary injury itself. Severing of axons, neural and glial death, and spinal shock, all instantly result in loss of function below and at the level of the injury. The spinal cord becomes swollen which is accompanied by hemorrhage into the grey matter, cellular necrosis, vascular disruption and ischemia. Vascular disruption leads to further hemorrhage into the white matter and, as a result of the combined effect of edema and hemorrhage, many segments of the spinal cord, rostral and caudal to the site of injury, become ischemic. [6] Even though gross histopathological changes may not be visible at this stage, many pathophysiological events have already started. For example, activation of microglial cells and an instantaneous increase in pro-inflammatory cytokines TNFand IL and excitotoxic

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levels of extracellular glutamate can be detected within minutes of an injury. [3, 7, 8]

During the acute stage of the SCI, the events of the secondary injury prevail. This is subdivided into early acute (2-48 hours) and subacute (2 days-2weeks). Vascular disruption, hemorrhage, and the resulting ischemia are central constituents of the early acute stage [5, 7], which result in the loss of normal autoregulatory mechanisms. The loss of ionic homeostasis immediately following SCI and excitotoxicity are closely related processes that each significantly contribute to the propagation of cellular injury after SCI. The ionic deregulation plays an important role in increasing necrosis and cell death following SCI. For instance, irregularity of Ca2+ concen‐ tration initiates activation of calpains and caspase cascades which in turn cause further cellular apoptosis. [9] Additionally, activated microglia express FAS ligand receptors and signaling through the p75 neurotrophin receptors that initiate the caspase and calpain cascade leading to proteolysis and DNA cleavage by effector caspases and trigger even further apoptotic cell death. [10] As a result of these events, a large number of cells, including oligodendrocytes, die. In addition to death of the neural cells, loss of calcium, sodium and potassium ionic channel homeostasis, and demyelination disrupt signal transduction in the spinal cord and result in

The extracellular levels of glutamate rise rapidly due to direct injury to cells and failure of energy-dependent transporters, notably the Na+K+ adenosine triphosphatase membrane transporter that normally functions to regulate extracellular concentrations of ions, glutamate, and other molecules. [11] Excessive activation of glutamate receptors leads to an increase in the influx of Na+ and Ca2+ through the NMDA and alpha-amino-3-hydroxy-5-methylisoxazoleproprionate/kainate receptors and excitotoxicity. [12] This causes further neural and

Reactive oxygen (O2) species (ROS) is at detectable levels 12 hours after SCI and remains elevated for almost one week, returning to preinjury levels 4-5 weeks post-injury. The increased amount of ROS leads to increased membrane lipid oxidation and an increase in cellular lysis, organelle damage and Ca2+ ion concentration irregularities. [13, 14] It has been

**2.1. Stages SCI progression**

sensorimotor inactivity and paralysis. [3-5]

*2.1.1. Acute stage*

glial death.

Decreased blood supply to the tissue and cells of the spinal cord results in nutrient and oxygen deficiency in the injured tissue, which in turn is followed by a cascade of biochemical or "secondary" events that eventually lead to further tissue necrosis. [3-5] Thus, the primary insult precedes sequential pathological changes such as further vascular dysfunction and hemorrhage, edema, ischemia, excitotoxicity, electrolyte shifts, free radical production, inflammation, axonal and neuronal necrosis, demyelination, cyst formation and infarction. [5] The events will be discussed in further detail in following sections and are summarized in Figure 1.

**Figure 1.** Mechanisms of Injury in SCI. SCI pathology results from several mechanisms occurring both concurrently and in sequence. Primary mechanical insult results in initial loss of axons (1) and demyelination (2). Subsequent secondary injury is characterized by further neuronal/axonal cell death (1) and myelin degradation (2), usually due to secondary inflammation from infiltrating lymphocytes and monocytes (3) and also reactive oxygen species secreted by activated astrocytes composing the glial scar (4). Both the glial scar (4) and post-traumatic cyst or syrinx formation (5) pro- vide physical impediments to regeneration, and cyst formation can further damage neurons by exerting physical pressure on their damaged axons. \* from *Ruff and Fehlings. (2010). Panminerva Med 52:125-147, with permission*

SCI is a complex set of damaging events that occur at the cellular level and it can be divided into four main stages: the immediate, acute, intermediate, and chronic phase of SCI.[3] The first two hours post-SCI is the immediate stage during which the body is going through processes as a result of the primary injury itself. Severing of axons, neural and glial death, and spinal shock, all instantly result in loss of function below and at the level of the injury. The spinal cord becomes swollen which is accompanied by hemorrhage into the grey matter, cellular necrosis, vascular disruption and ischemia. Vascular disruption leads to further hemorrhage into the white matter and, as a result of the combined effect of edema and hemorrhage, many segments of the spinal cord, rostral and caudal to the site of injury, become ischemic. [6] Even though gross histopathological changes may not be visible at this stage, many pathophysiological events have already started. For example, activation of microglial cells and an instantaneous increase in pro-inflammatory cytokines TNFand IL and excitotoxic levels of extracellular glutamate can be detected within minutes of an injury. [3, 7, 8]

#### **2.1. Stages SCI progression**

#### *2.1.1. Acute stage*

The pathophysiology of SCI is biphasic; it consists of a "primary" and a "secondary" stage. The primary injury is the result of an initial mechanical insult, which is followed by a series of cellular and molecular events that, in turn, lead to further destruction of the spinal cord tissue. It is well known that the mechanical injury seldom results in complete transection of the spinal cord. Most fracture dislocations or burst fractures of the spine result in disc displacement into and laceration or compression of the cord resulting in blood vessel, axonal and cellular

Decreased blood supply to the tissue and cells of the spinal cord results in nutrient and oxygen deficiency in the injured tissue, which in turn is followed by a cascade of biochemical or "secondary" events that eventually lead to further tissue necrosis. [3-5] Thus, the primary insult precedes sequential pathological changes such as further vascular dysfunction and hemorrhage, edema, ischemia, excitotoxicity, electrolyte shifts, free radical production, inflammation, axonal and neuronal necrosis, demyelination, cyst formation and infarction. [5] The events will be discussed in further detail in following sections and are summarized in

**Figure 1.** Mechanisms of Injury in SCI. SCI pathology results from several mechanisms occurring both concurrently and in sequence. Primary mechanical insult results in initial loss of axons (1) and demyelination (2). Subsequent secondary injury is characterized by further neuronal/axonal cell death (1) and myelin degradation (2), usually due to secondary inflammation from infiltrating lymphocytes and monocytes (3) and also reactive oxygen species secreted by activated astrocytes composing the glial scar (4). Both the glial scar (4) and post-traumatic cyst or syrinx formation (5) pro- vide physical impediments to regeneration, and cyst formation can further damage neurons by exerting physical pressure

SCI is a complex set of damaging events that occur at the cellular level and it can be divided into four main stages: the immediate, acute, intermediate, and chronic phase of SCI.[3] The

on their damaged axons. \* from *Ruff and Fehlings. (2010). Panminerva Med 52:125-147, with permission*

membrane disruption.

272 Trends in Cell Signaling Pathways in Neuronal Fate Decision

Figure 1.

During the acute stage of the SCI, the events of the secondary injury prevail. This is subdivided into early acute (2-48 hours) and subacute (2 days-2weeks). Vascular disruption, hemorrhage, and the resulting ischemia are central constituents of the early acute stage [5, 7], which result in the loss of normal autoregulatory mechanisms. The loss of ionic homeostasis immediately following SCI and excitotoxicity are closely related processes that each significantly contribute to the propagation of cellular injury after SCI. The ionic deregulation plays an important role in increasing necrosis and cell death following SCI. For instance, irregularity of Ca2+ concen‐ tration initiates activation of calpains and caspase cascades which in turn cause further cellular apoptosis. [9] Additionally, activated microglia express FAS ligand receptors and signaling through the p75 neurotrophin receptors that initiate the caspase and calpain cascade leading to proteolysis and DNA cleavage by effector caspases and trigger even further apoptotic cell death. [10] As a result of these events, a large number of cells, including oligodendrocytes, die. In addition to death of the neural cells, loss of calcium, sodium and potassium ionic channel homeostasis, and demyelination disrupt signal transduction in the spinal cord and result in sensorimotor inactivity and paralysis. [3-5]

The extracellular levels of glutamate rise rapidly due to direct injury to cells and failure of energy-dependent transporters, notably the Na+K+ adenosine triphosphatase membrane transporter that normally functions to regulate extracellular concentrations of ions, glutamate, and other molecules. [11] Excessive activation of glutamate receptors leads to an increase in the influx of Na+ and Ca2+ through the NMDA and alpha-amino-3-hydroxy-5-methylisoxazoleproprionate/kainate receptors and excitotoxicity. [12] This causes further neural and glial death.

Reactive oxygen (O2) species (ROS) is at detectable levels 12 hours after SCI and remains elevated for almost one week, returning to preinjury levels 4-5 weeks post-injury. The increased amount of ROS leads to increased membrane lipid oxidation and an increase in cellular lysis, organelle damage and Ca2+ ion concentration irregularities. [13, 14] It has been shown that Peroxynitrite, which is generated through reaction of ROS and nitric acid, is directly involved in the induction of neural apoptosis in rat SCI. [13]

important to note that in humans, there is significantly less astroglial scarring observed than is observed in rodent SCI models. [21] Other than scar formation, astrocytes also promote the reestablishment of ionic homeostasis and the integrity of the BBB, which is beneficial in

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During the subacute stage of the injury, there is a significantly higher phagocytic response observed in the CNS compared to the PNS. While this could partly account for the lack of regenerative ability seen in the CNS, the phagocytic response is believed to be efficient in cell debris removal and, to some extent, in promotion of axonal growth inside the lesion. [13]

The intermediate phase starts at two weeks and continues to 6 months post-injury. Main characteristics of this stage are continuous maturation of the gliotic scar and the start of axonal regeneration. Regenerative axonal sprouting has been observed in corticospinal tract axons in rat models of contusive SCI from 3 weeks to 3 months following injury, and reticulospinal fibers have been observed from 3 to 8 months post injury. [23] It is encouraging to observe that regenerative potential still exists in adult spinal cord, however, these regenerations have not

The chronic phase starts about 6 months after the initial SCI and lasts throughout the lifetime of the SCI patient. Further gliotic scar formation, development of cysts and syrinxes within the lesion, and continuous Wallerian degeneration of severed and injured axons are the main characteristics of this stage. [3, 23-25] Two to three years post-injury, the lesion has usually stabilized and is characterized by formation of a cyst cavity and mayelomalacia, which is the final stage of necrotic death. [26] Treatment strategies at this stage focus on rehabilitative approaches to promote plasticity, regeneration and remyelination of injured axons. Unfortu‐ nately, to date there has not been any report of the application of stem cells at this stage

As explained in the previous section, in the pathophysiology of SCI, some processes can be considered to be double-edged swords with both positive and negative effects and interations., These are not yet completely understood. One example of such an interaction is the astrocytic response which, on one hand results in the development of astrocytic scar limiting axonal regeneration and functional recovery, and on the other results in reestablishment of the BBB, ionic homeostasis, and decreased immune cell infiltration. What makes the pathophysiology of SCI and the translation of treatment strategies into the clinic even more complicated is its variance and uniqueness among different patients with different cases of injury. Experimental SCI models, which are mostly rodent models, consist of a homogeneous population, in contrast

decreasing edema and immune cell infiltration. [22]

been able to produce significant functional recovery in severe SCI.

resulting in any significant functional improvement.

to the vastly heterogeneous human SCI population.

**2.2. Cell-based approaches in SCI**

*2.1.3. Intermediate phase*

*2.1.4. Chronic phase*

The brain blood barrier (BBB) is a highly selective endothelial filter for transport of compounds in and out of the central nervous system. This becomes highly permeable as a result of the primary mechanical insult to the cord and presence of inflammatory mediators. It has been shown that peak BBB permeability occurs at 24 hours following injury in the rat and returns control levels by 2 weeks. [15] Two inflammatory mediators – TNF and IL-1 - are known to increase vascular permeability. In addition, other compounds released by glial cells or invading immune cells which are believed to play a role in increasing BBB permeability include ROS, nitric oxide, histamine, matrix metalloproteinases, and elastase. [3, 13] Additionally, disruption of the vasculature following SCI results in the breakage of the BBB, leading to an inflammatory response and infiltration of astrocytes, microglia, T cells, neutrophils, and invading monocytes. Invasion of macrophages and neutrophils to the site of injury increases cellular disruption and causes further inflammatory-triggered necrosis. Inflammation then results in activation of microglia which, together with leukocytes, cause malfunction of oxidative metabolism in demyelinated axons and proliferation of astrocytes as well as increased expression of glial fibrillary acid protein (GFAP) and the formation of a glial or astrocytic scar. [2-4] Further to this, a multitude of noncellular mediators, including TNF, interferons, and ILs play important roles.

The inflammatory response in SCI is a highly complex event with a dual nature. Some aspects of the inflammatory response further advance the secondary injury phase, while others are beneficial in removing cellular debris and starting the regenerative process [16]. For instance, TNF plays an important role in cellular death post-SCI and its inhibition has been shown to result in functional neurological recovery after SCI. However, it has also been shown to have a neuroprotective role in *in vitro* and murine models of SCI. [17, 18]

#### *2.1.2. Subacute phase*

The subacute phase lasts from 2 days to 2 weeks after the initial injury. This is the stage where it is hoped that therapeutic strategies currently in development, including cellular treatments and strategies, will be helpful. For instance, we have shown in our lab that transplantation of adult murine neural precursor cells at 2 weeks post-injury promotes remyelination and functional recovery. Such effects were not seen when the cells were transplanted at later time points or during the chronic stage of the injury. [19] Keirstead et al, have also reported the failure of transplanted human ESC-derived oligodendrocyte progenitor cells to survive, migrate and promote functional recovery when they were injected to the site of injury either at the time of injury or after the subacute phase. [20]

During the initial hours to days following SCI, astrocytes undergo cytotoxic edema and necrotic cellular death; however, a second delayed astrocytic response happens during the subacute phase. During this stage, there is a significant increase in the astrocytic intermediate filament GFAP as a result of the proliferative and hypertrophic activity of astrocytes. These reactive astrocytes form the astrocytic (gliotic) scar through their interweaving cytoplasmic processes, which becomes a physical and chemical barrier to axonal regeneration. It is important to note that in humans, there is significantly less astroglial scarring observed than is observed in rodent SCI models. [21] Other than scar formation, astrocytes also promote the reestablishment of ionic homeostasis and the integrity of the BBB, which is beneficial in decreasing edema and immune cell infiltration. [22]

During the subacute stage of the injury, there is a significantly higher phagocytic response observed in the CNS compared to the PNS. While this could partly account for the lack of regenerative ability seen in the CNS, the phagocytic response is believed to be efficient in cell debris removal and, to some extent, in promotion of axonal growth inside the lesion. [13]

#### *2.1.3. Intermediate phase*

shown that Peroxynitrite, which is generated through reaction of ROS and nitric acid, is directly

The brain blood barrier (BBB) is a highly selective endothelial filter for transport of compounds in and out of the central nervous system. This becomes highly permeable as a result of the primary mechanical insult to the cord and presence of inflammatory mediators. It has been shown that peak BBB permeability occurs at 24 hours following injury in the rat and returns control levels by 2 weeks. [15] Two inflammatory mediators – TNF and IL-1 - are known to increase vascular permeability. In addition, other compounds released by glial cells or invading immune cells which are believed to play a role in increasing BBB permeability include ROS, nitric oxide, histamine, matrix metalloproteinases, and elastase. [3, 13] Additionally, disruption of the vasculature following SCI results in the breakage of the BBB, leading to an inflammatory response and infiltration of astrocytes, microglia, T cells, neutrophils, and invading monocytes. Invasion of macrophages and neutrophils to the site of injury increases cellular disruption and causes further inflammatory-triggered necrosis. Inflammation then results in activation of microglia which, together with leukocytes, cause malfunction of oxidative metabolism in demyelinated axons and proliferation of astrocytes as well as increased expression of glial fibrillary acid protein (GFAP) and the formation of a glial or astrocytic scar. [2-4] Further to this, a multitude of noncellular mediators, including TNF,

The inflammatory response in SCI is a highly complex event with a dual nature. Some aspects of the inflammatory response further advance the secondary injury phase, while others are beneficial in removing cellular debris and starting the regenerative process [16]. For instance, TNF plays an important role in cellular death post-SCI and its inhibition has been shown to result in functional neurological recovery after SCI. However, it has also been shown to have

The subacute phase lasts from 2 days to 2 weeks after the initial injury. This is the stage where it is hoped that therapeutic strategies currently in development, including cellular treatments and strategies, will be helpful. For instance, we have shown in our lab that transplantation of adult murine neural precursor cells at 2 weeks post-injury promotes remyelination and functional recovery. Such effects were not seen when the cells were transplanted at later time points or during the chronic stage of the injury. [19] Keirstead et al, have also reported the failure of transplanted human ESC-derived oligodendrocyte progenitor cells to survive, migrate and promote functional recovery when they were injected to the site of injury either

During the initial hours to days following SCI, astrocytes undergo cytotoxic edema and necrotic cellular death; however, a second delayed astrocytic response happens during the subacute phase. During this stage, there is a significant increase in the astrocytic intermediate filament GFAP as a result of the proliferative and hypertrophic activity of astrocytes. These reactive astrocytes form the astrocytic (gliotic) scar through their interweaving cytoplasmic processes, which becomes a physical and chemical barrier to axonal regeneration. It is

a neuroprotective role in *in vitro* and murine models of SCI. [17, 18]

at the time of injury or after the subacute phase. [20]

involved in the induction of neural apoptosis in rat SCI. [13]

274 Trends in Cell Signaling Pathways in Neuronal Fate Decision

interferons, and ILs play important roles.

*2.1.2. Subacute phase*

The intermediate phase starts at two weeks and continues to 6 months post-injury. Main characteristics of this stage are continuous maturation of the gliotic scar and the start of axonal regeneration. Regenerative axonal sprouting has been observed in corticospinal tract axons in rat models of contusive SCI from 3 weeks to 3 months following injury, and reticulospinal fibers have been observed from 3 to 8 months post injury. [23] It is encouraging to observe that regenerative potential still exists in adult spinal cord, however, these regenerations have not been able to produce significant functional recovery in severe SCI.

#### *2.1.4. Chronic phase*

The chronic phase starts about 6 months after the initial SCI and lasts throughout the lifetime of the SCI patient. Further gliotic scar formation, development of cysts and syrinxes within the lesion, and continuous Wallerian degeneration of severed and injured axons are the main characteristics of this stage. [3, 23-25] Two to three years post-injury, the lesion has usually stabilized and is characterized by formation of a cyst cavity and mayelomalacia, which is the final stage of necrotic death. [26] Treatment strategies at this stage focus on rehabilitative approaches to promote plasticity, regeneration and remyelination of injured axons. Unfortu‐ nately, to date there has not been any report of the application of stem cells at this stage resulting in any significant functional improvement.

#### **2.2. Cell-based approaches in SCI**

As explained in the previous section, in the pathophysiology of SCI, some processes can be considered to be double-edged swords with both positive and negative effects and interations., These are not yet completely understood. One example of such an interaction is the astrocytic response which, on one hand results in the development of astrocytic scar limiting axonal regeneration and functional recovery, and on the other results in reestablishment of the BBB, ionic homeostasis, and decreased immune cell infiltration. What makes the pathophysiology of SCI and the translation of treatment strategies into the clinic even more complicated is its variance and uniqueness among different patients with different cases of injury. Experimental SCI models, which are mostly rodent models, consist of a homogeneous population, in contrast to the vastly heterogeneous human SCI population.

Two main aims of cell-based treatment for SCI are replacing lost or injured cells such as oligodendrocytes and neurons, or providing the cells with a microenvironment that supports or enhances the neuroprotective and regenerative ability of cells within the lesion. Stem and progenitor cells, and Schwann cells are examples of cells that are being used in studies to replace lost cells. Additionally, many studies have shown that these cells have the ability to remyelinate axons in the injury site while also providing them with supportive growth and neuroprotective factors. [1, 19, 27-29]

planted into adult rat CNS, neither rat nor human MSCs were able to differentiate into neuronal

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There are other studies in which transplanted MSCs were tracked in rat or mouse injury models through GFP or electromagnetic tagging coupled with immunochemistry and MR imaging. Even though both types of studies report functional improvements, only a fraction of cells are reported to show expression of neuronal or astrocytic markers and only a very small number of cells are reported as having differentiated into neurons. [37-39] There are differing reports on the extent of MSC transplantation benefits in functional recovery. MSCs have shown to be effective at 4 weeks up to 1 year after the injury [41], depending on the timing of transplantation from the time of injury [40] to 1 week [37, 38, 41], or 3 months post injury [42]. Other studies show lack of any functional benefit observable through the Basso Beattie Bresnahan (BBB) open field locomotor scoring system. [43] However, all of the mentioned studies report on the ability of MSC transplants to preserve the injured tissue, both gray and white matter, and to provide micro environmental cues supportive of axonal growth, rather than replacing lost cells. [42, 43] Given the inconsistency seen in the results of transplanting MSCs into SCI models in rats, mice and primates, it is necessary to have a better understanding of the type of cells in the MSC population that are being transplanted[44] and investigate all the possible mechanisms behind

Schwann cells are the myelinating cells in the peripheral nervous system. Since the first transplantation of SCs into SCI by Duncan et al. in 1981 [34], these cells have been used in many different SCI models and have been shown to be able to remyelinate axons and provide a permissive environment for them to regenerate and grow. Binge et al. [45] showed that SCs also have a small but significant effect on increasing functional recovery. However, their remyelinating and regenerating ability does not go beyond the growth-permissive surface, which explains why there is only a slight improvement in functional recovery. [3, 44-46] Additionally, it has been shown that axons in the corticospinal tract (CST) remain unaffected by SC transplants. [34] Also, they have been shown to generate a more active astrocytic response compared to other cell types such as NPCs which results in less efficient integration of these cells into the injured spinal cord. [34] As a result, the clinical application of SCs alone

To overcome this limitation, many combinatorial strategies have evolved which associate SCs with growth factors or bioengineering scaffolds and other cells types to enhance recovery. [4, 47-50] Additionally, new sources for SCs are being investigated such as skin derived precursor cells from the dermis of the skin [51-54] and MSCs. [55-58] However, it is yet to be proven whether these cells hold any advantage in treating SCI compared with SCs that are derived

OECs are glial cells derived from the olfactory bulb or lamina propria of the olfactory mucosa and are termed olfactory nerve Schwann cells due to morphological similarities to SCs. Having a lifelong capacity to proliferate and being able to facilitate passage of new axons from

cells. Mostly, they generated perivascular macrophages in the rat brain. [35-37]

the functional recovery observed in these models.

for SCI treatment appears extremely limited.

*3.1.2. Schwann cells*

from the PNS.

*3.1.3. Olfactory ensheathing cells*
