**3.3 Umbilical cord mesenchymal stem cells**

*Ryu et al.* [44] conducted a comparison between four sources of MSCs (Bone marrow, adipose-derived, umbilical cord blood, and Wharton's Jelly) to treat a canine model with spinal cord injury. Even though data revealed no significant differences in functional recovery among the MSCs groups, they identified essential properties such as the promotion of neuronal regeneration and anti-inflammatory activity. Umbilical cord stem cells group showed more nerve regeneration, neuroprotection, and less inflammation with reduced IL-6 and COX-2 levels than other MSCs. Moreover, researchers establish improvement in locomotion measured using the Olby and modified Tarlov scores eight weeks after the application of MSCs

*Use of Mesenchymal Stem Cells in Pre-Clinical Models of Spinal Cord Injury DOI: http://dx.doi.org/10.5772/intechopen.94086*

compared with the control group, suggesting that the use of MSCs promotes functional recovery after SCI. Additionally, preclinical studies such as the one carried by *Chua S. J. et al.* [45] have detected cytokines and growth factors known by its neuroprotective, angiogenic, and anti-inflammatory effects.

In a compression SCI rat model, both BMSCs and umbilical cord-derived stem cells caused similar results and improvement in allodynia, hyperalgesia, and functional recovery. However, UCSCs were more effective in decreasing wind-up levels [46]. 13 of 22 patients treated with UC-MSCs were better in daily living activities. They had a better motor function, better motor and tactile sensation [47].

#### **3.4 Amniotic fetal mesenchymal stem cells**

There are few preclinical studies in animal models that have to use amniotic fetal derived stem cells identified in the literature [48]. Nevertheless, the specific characteristics of this source of MSCs were observed. For instance, the multipotency, the low risk of immunogenic reaction, the ease of sample processing, and the high proliferative capacity, makes amniotic fetal derived stem cells an attractive alternative for regenerative medicine [49]. These properties are supported by data observed which showed promotion of angiogenesis and support of the surrounding tissue surplus the decreased inflammatory response and apoptosis [50–52].

In a rat model of SCI, the impact of two types of MSCs: Human umbilical cord blood-derived and Human amniotic epithelial cells were assessed for the treatment of SCI-induced thermal hyperalgesia and mechanical allodynia. None of them were effective in treating the thermal hyperalgesia. Though both improved the mechanical allodynia, human amniotic epithelial stem cells were more efficacious [53].

#### **4. Animal models in spinal cord injury**

Multiple studies described the administration of MSCs to treat spinal cord injury in a variety of animal models such as rodents, primates, sheep, dogs, cats, bovine, and even humans. Rodents are the most common animal model used [54], and the most appropriate model for spinal cord injury studies [55] since large animals and non-human primates are very expensive to care, demand additional managing requirements, and have ethical implications to consider when choosing. However, the experiments of the latter approximate more to SCIs [56].

The efficacy of MSCs were also observed in cats with SCI. Improvement in the cutaneous trunci (panniculus) reflex, pain sensation, bowel, and bladder function were noted. However, no significant change in proprioception and hyperreflexia of ataxic hind limbs were observed [57]. In dogs with SCI, treatment with BMSCs also caused the same clinical improvement with no significant recovery of low proprioceptive and hyperreflexic ataxic hind limbs [58]

#### **4.1 Stem cell delivery methods in SCI animal models**

There are currently 3 different methods to deliver mesenchymal stem cells (MSCs) in animal models of spinal cord injury (SCI). These are direct implantation, intravenous (IV) infusion, and intrathecal infusion. Direct implantation refers to the injection of MSCs directly in the injured area of the spinal cord. IV infusion refers to the injection of MSCs in a major vein of the animal model (e.g. the tail vein of a mouse or rat). Lastly, intrathecal infusion refers to the injection of MSCs directly into the subarachnoid space, in the cerebrospinal fluid. The delivery methods

described in the following paragraphs concern mainly Sprague-Dawley rats. For a summary of advantages and disadvantages of delivery methods, see **Table 1**.

Direct implantation of the MSCs is done using a small syringe capable if precisely injecting cells in the damaged area [59], guaranteeing delivery to the desired site [60]. This method has been largely favored due to its high cell viability and improved survival [61], observed in higher engraftment rates in both acute and chronic SCI [59, 61, 62]. However, some authors have expressed concerns regarding the translation of this technique to clinical practice. Agglomeration of cells in the injection site, needle damage to the adjacent non-injured spinal cord, and failure of cell migration to the central parenchyma are some of the most noteworthy disadvantages of this delivery method [59–64]. Additionally, if done in humans, direct implantation of MSCs would require the patient to undergo general anesthesia and an invasive surgical procedure [61].

Since direct implantation of MSCs in humans might pose substantial risks, IV and intrathecal infusion were deemed appropriate less invasive surrogates that could potentially be clinically used. Damage to the blood-brain barrier (BBB) in SCI (particularly in traumatic SCI) allows infiltration of cells and toxic mediators that promote further neurologic damage [65]. It was initially thought that this process could improve diffusion of MSCs into the spinal cord. However, cell infusion in the first 48 hours of injury has shown conflicting evidence, with some authors reporting either the presence or absence of MSCs at the lesion site [65, 66]. Nevertheless, when present, the cell's engraftment rate with IV delivery in the spinal cord was very low in comparison to other methods [59].


#### **Table 1.**

*Advantages and disadvantages of the three main MSC delivery methods.*

#### *Use of Mesenchymal Stem Cells in Pre-Clinical Models of Spinal Cord Injury DOI: http://dx.doi.org/10.5772/intechopen.94086*

The low engraftment rates in the spinal cord with IV delivery methods are considered a consequence of the cells' first pass through the systemic circulation. MSCs have been observed in the lungs and liver in the first 24-48 hours of IV infusion [66, 67], with progressive increase of cell numbers in the spleen in the following days [61, 67]. Cell engraftment in the spleen is associated with increased levels of anti-inflammatory cytokines (e.g. IL-10, TIMP-1) [67] in plasma, which is believed to decrease BBB permeability by inhibiting monocyte adhesion to the vasculature, preventing metalloproteinase release and vascular basement membrane degradation [67]. Therefore, the anti-inflammatory environment promoted by cells engrafting outside the spinal cord prevents vascular leakage at the lesion site, decreasing hemorrhage, inflammation and further damage [66, 67].

IV infusion of MSCs can be done through the femoral vein or the tail vein. In rodents, peripheral circulation is mostly accessed through the tail vein [61, 67, 68] due to its simplicity when compared to the approach required to access the femoral vein [59, 65, 66]. Additionally, inflammation around the site of injury is higher than with intrathecal administration but lower than direct injection [59]. Even though cells administered IV have low engraftment rates, animals still score better outcomes in limb function recovery scores and grip strength [67], develop less scarring [59, 68], and have higher vascularization, myelination, and axonal density than controls [68].

Intrathecal delivery of MSCs can be done via the intracisternal approach (i.e. injection into the fourth ventricle) [61, 69, 70] or by laminectomy with injection of cells through the dura [63]. When initially injected, cells occupy the whole subarachnoid space, but progressively decrease their number in this anatomical region [63]. In contrast to intralesional delivery of MSCs, intrathecally administered cells show a more extensive migration in the neural tissue, extending from the dorsal spinal cord to its center [61]. Although the number of viable cells is only second to the intralesional delivery method [61], a decrease in engrafted cells to 5% of the original cell number has been observed after 6 weeks, with some cells attaching to the pia mater [63]. Animals with intrathecally delivered MSCs obtain higher scores in limb function recovery scores when compared with intralesional and intravenous deliveries [61].

The impact of MSCs on SCI resolution can be explained by the following characteristics: immunomodulatory, anti-inflammatory, neurotrophic/neuroprotective, and angiogenetic effects. The direct impact on the regeneration of the neurons is mainly exerted by neurotrophic and neuroprotective functions. These functions are usually mediated by the secretion of neurotrophic factors.

The spinal cord injury occurs in 2 phases. The first phase occurs immediately after the trauma and is mediated by damage to the microvascular elements, cellular membrane, and the blood-spinal cord barrier. Damage to these three structures evokes series of events that give rise to axonal fragmentation, demyelination, cyst formation, and expansion and accumulation of the microglia and macrophages in and around the injury site, which leads to the secondary injury. The secondary injury is characterized by inflammation, ischemia, disruption of ion channels, free radical production, glutamatergic excitotoxicity, necrosis, axonal demyelination, and glial scar formation [30, 71].

As previously described, MSCs from different sources have been used to treat SCI. They can directly be injected to the injury site or intravenously as they have the ability to migrate to the epicenter of the injury, demonstrating their homing abilities.

Chen *et al*. reviewed 12 randomized controlled trials on rats and mice and showed that stem cell treatment improved the mechanical reflex threshold. For the mice, improvement in thermal withdrawal latency was observed. However, no improvement was seen in rat studies [72]. In a rat spinal cord hemisection model, BMSCs were noted to promote astrocyte migration to the injury epicenter. In the group treated with a combination of the BDNF with platelet-rich plasma, more astrocyte migration, and higher rates of remyelination has been documented. This group also showed remyelination and oligodendrocytes with higher activity, while only the BMSCs group showed axonal demyelination, vacuole and whirled body formation [73].

The human ADSC have been shown to be able to convert to the oligodendrocytes and to attract oligodendrocyte precursor cells, which, in turn, mediates remyelination. Unsurprisingly, the treatment caused an improvement in the motor function of the animals with focal demyelination [74]. Differentiation of neurotrophic factors secreting cells from human ADSC to oligodendrocytes was noted in a damaged spinal cord rat model. Neurotrophic factors secreting cells promoted remyelination and increased thickness of the myelin and the diameter of the axons [75].

In a rat model of spinal cord contusion, rats treated with bone marrow-derived Schwann cells experienced better functional recovery. At the same time, the size of the cystic cavity decreased, and axonal regeneration was observed [76]. Treatment with bone-derived MSCs stimulated axonal growth in the subtotal cervical hemisection rat model. An increase in the length of the axons was observed [77]. Bone marrow mesenchymal stem cells decreased the cavity volume and increased the spared white matter, the length of the neurites, the number of axons, and the neurites in a SCI rat model [78]. Rats with a moderate contusion model of spinal cord injury showed better recovery in 2 behavioral tests (Bresnahan Locomotor Rating Scale [79] and exploratory rearing [80]). All rats experienced a decrease in the size of the cyst cavity and more axons in the injury site either through an increase in spared axons or through axon regeneration [80]. In a complete spinal cord transection rat model, human umbilical mesenchymal stem cells promoted axonal regeneration, and more neurofilament-positive fibers around the epicenter of the corticospinal tract injury was observed. Additionally, proximal and dorsal to the injury site, fewer microglia and astrocytes with reactive features were found [81].

Some studies suggest that the MSCs do not have the ability to convert to neural cells. As an example, Quertainmont et al. could not detect the stem cells after grafting. The authors described tissue sparing, vascularization in the injury epicenter, but no evidence of axonal regrowth [37].
