Epidural Stimulation Combined with Triple Gene Therapy for Spinal Cord Injury Treatment.

The translation of new therapies for spinal cord injury to clinical trials can be facilitated with large animal models close in morpho-physiological scale to humans. Here, we report functional restoration and morphological reorganization after spinal contusion in pigs, following a combined treatment of locomotor training facilitated with epidural electrical stimulation (EES) and cell-mediated triple gene therapy with umbilical cord blood mononuclear cells overexpressing recombinant vascular endothelial growth factor, glial-derived neurotrophic factor, and neural cell adhesion molecule. Preliminary results obtained on a small sample of pigs 2 months after spinal contusion revealed the difference in post-traumatic spinal cord outcomes in control and treated animals. In treated pigs, motor performance was enabled by EES and the corresponding morpho-functional changes in hind limb skeletal muscles were accompanied by the reorganization of the glial cell, the reaction of stress cell, and synaptic proteins. Our data demonstrate effects of combined EES-facilitated motor training and cell-mediated triple gene therapy after spinal contusion in large animals, informing a background for further animal studies and clinical translation.

Evidence of axon connectivity across a spinal cord transection in rats treated with epidural stimulation and motor training combined with olfactory ensheathing cell transplantation.

Olfactory ensheathing cells (OECs) are unique glia that support axon outgrowth in the olfactory system, and when used as cellular therapy after spinal cord injury, improve recovery and axon regeneration. Here we assessed the effects of combining OEC transplantation with another promising therapy, epidural electrical stimulation during a rehabilitative motor task. Sprague-Dawley rats received a mid-thoracic transection and transplantation of OECs or fibroblasts (FBs) followed by lumbar stimulation while climbing an inclined grid. We injected pseudorabies virus (PRV) into hindlimb muscles 7 months post-injury to assess connectivity across the transection. Analyses showed that the number of serotonergic (5-HT) axons that crossed the rostral scar border and the area of neurofilament-positive axons in the injury site were both greater in OEC- than FB-treated rats. We detected PRV-labeled cells rostral to the transection and remarkable evidence of 5-HT and PRV axons crossing the injury site in 1 OEC- and 1 FB-treated rat. The axons that crossed suggested either axon regeneration (OEC) or small areas of probable tissue sparing (FB). Most PRV-labeled thoracic neurons were detected in laminae VII or X, and ~25% expressed Chx10, a marker for V2a interneurons. These findings suggest potential regeneration or sparing of circuits that connect thoracic interneurons to lumbar somatic motor neurons. Despite evidence of axonal connectivity, no behavioral changes were detected in this small-scale study. Together these data suggest that when supplemented with epidural stimulation and climbing, OEC transplantation can increase axonal growth across the injury site and may promote recovery of propriospinal circuitry.

Tail nerve electrical stimulation combined with scar ablation and neural transplantation promotes locomotor recovery in rats with chronically contused spinal cord.

To date, few treatment strategies applying cellular transplantation to the chronically injured spinal cord have yielded significant functional improvement in animal experiments. Here we report that significant improvement of locomotor function was achieved in rats with chronic spinal cord injury (SCI) by the application of combination treatments with tail nerve electrical stimulation (TANES), which can activate the central pattern generator, inducing active weight-supported stepping. Contusion injury (25 mm) to spinal cord T10 was produced by using the NYU impactor device in female, adult Long-Evans rats. Rats in 2 of 4 groups with SCI received basic treatments (scar ablation followed by transplantation of lamina propria of olfactory mucosa and cultured olfactory ensheathing cells into the lesion cavity) 6 weeks after SCI. Rats both with and without basic treatments were subjected to TANES one week after secondary surgery or 7 weeks after SCI. Sixteen weeks after secondary surgery or 22 weeks after SCI rats in two groups receiving TANES significantly improved their functional recovery compared with those without TANES, when evaluated with BBB open field rating scale (p<0.01). Among them, however, rats with basic treatments performed better than those without basic treatments. TANES may contribute to the activity-dependent plasticity below the injury level, which is critical for functional recovery. Additionally, TANES may promote axonal regeneration, including those from supraspinal level. Since TANES demonstrated considerable potential for achieving improvement of functional recovery in rat model, it would suggest a new strategy for chronic SCI.

Long-term functional restoration by neural progenitor cell transplantation in rat model of cognitive dysfunction: co-transplantation with olfactory ensheathing cells for neurotrophic factor support.

Neural progenitor cell transplantation has emerged as a promising approach for cell replacement therapy in the brain of neurodegenerative diseases. These are multipotent stem cells with self-renewal capabilities and can give rise to cells of all the three lineages of nervous system and can be maintained and differentiated to desirable neuronal subtypes in vitro with known trophic factors. However, like fetal cells, neural progenitor cells after differentiating to specific neuronal type also require continuous neurotrophic factor support for their long-term survival following transplantation. Recent reports suggest that olfactory ensheathing cells are capable of providing continuous neurotrophic factor to the transplanted neural progenitor cells for their long-term survival. In the present investigation, an attempt has been made to validate functional restoration in kainic acid lesioned rat model of cognitive dysfunction following co-transplantation of neural progenitor cells with olfactory ensheathing cells. Animals lesioned with kainic acid in CA3 subfield of hippocampal region were transplanted with neural progenitor cells, olfactory ensheathing cells or neural progenitor cells+olfactory ensheathing cells together. Twelve weeks post-transplantation functional restoration was assessed using neurobehavioral, neurochemical, and immunohistochemical approaches. Significant recovery in learning and memory (89%) was observed in co-transplanted group when compared to lesioned group. This was accompanied by significantly higher expression of choline acetyltransferase and restoration in cholinergic receptor binding in co-transplanted group (61%) over the animals transplanted either olfactory ensheathing cells or neural progenitor cells alone. Role of olfactory ensheathing cells in supplementing neurotrophic factors was further substantiated in vitro by pronounced differentiation of neural progenitor cells to choline acetyltransferase/acetylcholine esterase immunoreactive cells when co-cultured with olfactory ensheathing cells as compared to neural progenitor cells alone. The results strengthened the hypothesis that co-transplantation of olfactory ensheathing cells and neural progenitor cells may be a better approach for functional restoration in kainic acid induced rat model of cognitive dysfunction.

Region-specific migration of embryonic glia grafted to the neonatal brain.

Cell fate determination and region-specific migration among neurons from the developing brain have been widely studied. Because similar attributes have been mostly unexplored in reference to glia, the present study has characterized the migratory responses of glia from diverse regions of the embryonic mouse brain after their transplantation to the brains of early postnatal (still developing) rats. Through the use of the mouse-specific, glial-specific marker M2, immunocytochemical processing of host tissues three to four weeks after transplantation revealed notable difference in the migratory patterns of phylogenetically diverse populations of glia. While glia from the ventral mesencephalon, cerebral cortex, and cerebellar neuroepithelium all showed a similar affinity for the nigropallidal tract after grafting to the internal capsule, only ventral mesencephalon-derived glia showed restricted migration toward and into the substantia nigra after transplantation to the thalamus or pontine tegmentum. These results suggest the presence of a highly favourable substrate for glial migration along developing fibre tracts, but, more importantly, indicates the potential for certain glia to respond to particular (region-specific) distal cues within the developing brain.