Fortuitous benefits of activity-based rehabilitation in stem cell-based therapy for spinal cord repair: enhancing graft survival

Traumatic injuries to spinal cord elicit diverse signaling pathways leading to unselective and complex pathological outcomes: death of multiple classes of neural cells, formation of cystic cavities and glial scars, disruption of axonal connections, and demyelination of spared axons, all of which can contribute more or less to debilitating functional impairments found in patients with spinal cord injury. The multitude of pathobiological processes involved in spinal cord trauma may make it highly challenging to develop a clinically meaningful therapeutic approach targeting only a specific molecule or signaling pathway. A hopeful alternative might be a cell therapy, especially a transplantation approach using neural stem cells (NSC) with a clear potential to differentiate into various neural cell types. Provision of NSCs with capacity to differentiate into mature neural cells can ideally replace lost segmental neurons and dying oligodendrocytes around surviving axons. Furthermore, NSCs secrete various growth factors that provide protective or pro-regenerative effects. It has been also demonstrated that NSCs can exert powerful modulatory effects on immune cells ameliorating secondary degenerative processes. The basic premise for successful stem cell-based therapy would be a good extent of survival of NSC grafts. Survival of transplanted NSCs would be particularly critical when attempting to replace lost neural cells. In the authors’ opinion, the issue of graft survival has been underestimated partly because of a frequent use of genetically immune-compromised animals as hosts for NSC grafts (for example SCID mice or athymic rats). Complete avoidance of immunological interactions between host and grafts would be unimaginable in clinical situations, and there was a report that even induced pluripotent stem cells derived from the same host animal can evoke some degree of inflammatory reactions (Zhao et al., 2011). Therefore, designing strategies to enhance survival of NSC grafts should be considered as a prerequisite for the successful translation of stem cell-based transplantation therapy. We have recently reported that treadmill locomotor training (TMT), which is routinely prescribed for patients with paraplegia, substantially enhances survival of grafted NSCs in a rat spinal cord injury model (Hwang et al., 2014). In our model where rat NSCs are transplanted into rat spinal cord (allograft), a majority of grafted stem cells disappear within several days after transplantation. This is consistent with the previous studies showing that more than 90% transplanted NSCs die within several days in injured CNS (Okada et al., 2005; Nakagomi et al., 2009). When a group of animals with NSC grafts were subjected to intensive TMT (three sessions per day with each lasting 20 minutes and 6 days per week), they showed three times and five times higher number of surviving NSCs at 3 and 9 weeks after injury, respectively. We also found that the markers of cellular stresses related to reactive oxygen or nitrogen species were substantially attenuated in grafted NSCs by TMT. Importantly, the increase in the number of surviving NSCs was significantly correlated with the degree of behavioral improvement in the same group of animals. What is the molecular mechanism of the TMT-induced enhancement of NSC graft survival? It has been shown that exercise increases peripheral production of insulin-like growth factor-1 (IGF-1) and the IGF-1 can be delivered to the CNS through the blood-cerebrospinal fluid (CSF) barrier (Fernandez and Torres-Aleman, 2012). We found that the level of IGF-1 was increased in serum and CSF in injured animals with TMT. To establish a causative role of IGF-1, neutralizing antibodies against IGF-1 were intrathecally delivered. Neutralization of IGF-1 almost completely abrogated the pro-survival effect of TMT on grafted NSCs. Intriguingly, neutralization of either brain-derived neurotrophic factor (BDNF) or neurotrophin-3(NT-3), which have been implicated in TMT-induced neuroplasticity and whose levels were increased within the spinal cord tissue, but not in the CSF compartment, by TMT, did not affect the effect of TMT in enhancing NSC survival. Our data suggest an interesting scenario in which IGF-1 produced in peripheral organs by TMT enters into the CNS via the blood-CSF barrier and provides beneficial effects on the survival of grafted NSCs in patients with spinal cord trauma (Figure 1). Rehabilitative exercise or training mobilizes intense muscular activities accompanied by metabolic challenges in various peripheral organs. These changes in turn are probably responsible for the production of IGF-1 and other insulin-like peptide molecules. These insulin-like peptide molecules are traditionally known as important regulators of glucose and lipid metabolism in peripheral organs. More recent studies, however, have uncovered various beneficial influences of the insulin-like peptide molecules on CNS involving brain development, neural cell differentiation, and even cognitive functions (Fernandez and Torres-Aleman, 2012). Our findings of IGF-1 regulating NSC survival add to the growing evidence that insulin and IGF receptor signaling play critical roles in NSC homeostasis (Ziegler et al., 2015). Therefore, it is conceivable that intensive rehabilitative training will be able to potentiate the therapeutic effects of NSC transplantation by not only promoting NSC survival but also enhancing multiple functionalities of NSCs. Indeed, we found that TMT increased the percentage of NSCs differentiating into neurons or oligodendrocytes. A previous study reported that IGF-1 promotes migration of newly generated neuroblasts. In consistence with the report, the rostrocaudal distribution of grafted NSCs was also greatly extended in animals with