Noradrenergic innervation of the rat spinal cord caudal to a complete spinal cord transection: effects of olfactory ensheathing glia.

Transplantation of olfactory bulb-derived olfactory ensheathing glia (OEG) combined with step training improves hindlimb locomotion in adult rats with a complete spinal cord transection. Spinal cord injury studies use the presence of noradrenergic (NA) axons caudal to the injury site as evidence of axonal regeneration and we previously found more NA axons just caudal to the transection in OEG- than media-injected spinal rats. We therefore hypothesized that OEG transplantation promotes descending coeruleospinal regeneration that contributes to the recovery of hindlimb locomotion. Now we report that NA axons are present throughout the caudal stump of both media- and OEG-injected spinal rats and they enter the spinal cord from the periphery via dorsal and ventral roots and along large penetrating blood vessels. These results indicate that the presence of NA fibers in the caudal spinal cord is not a reliable indicator of coeruleospinal regeneration. We then asked if NA axons appose cholinergic neurons associated with motor functions, i.e., central canal cluster and partition cells (active during fictive locomotion) and somatic motor neurons (SMNs). We found more NA varicosities adjacent to central canal cluster cells, partition cells, and SMNs in the lumbar enlargement of OEG- than media-injected rats. As non-synaptic release of NA is common in the spinal cord, more associations between NA varicosities and motor-associated cholinergic neurons in the lumbar spinal cord may contribute to the improved treadmill stepping observed in OEG-injected spinal rats. This effect could be mediated through direct association with SMNs and/or indirectly via cholinergic interneurons.

Serotonergic innervation of the caudal spinal stump in rats after complete spinal transection: effect of olfactory ensheathing glia.

Spinal cord injury studies use the presence of serotonin (5-HT)-immunoreactive axons caudal to the injury site as evidence of axonal regeneration. As olfactory ensheathing glia (OEG) transplantation improves hindlimb locomotion in adult rats with complete spinal cord transection, we hypothesized that more 5-HT-positive axons would be found in the caudal stump of OEG- than media-injected rats. Previously we found 5-HT-immunolabeled axons that spanned the transection site only in OEG-injected rats but detected labeled axons just caudal to the lesion in both media- and OEG-injected rats. Now we report that many 5-HT-labeled axons are present throughout the caudal stump of both media- and OEG-injected rats. We found occasional 5-HT-positive interneurons that are one likely source of 5-HT-labeled axons. These results imply that the presence of 5-HT-labeled fibers in the caudal stump is not a reliable indicator of regeneration. We then asked if 5-HT-positive axons appose cholinergic neurons associated with motor functions: central canal cluster and partition cells (active during fictive locomotion) and somatic motor neurons (SMNs). We found more 5-HT-positive varicosities in lamina X adjacent to central canal cluster cells in lumbar and sacral segments of OEG- than media-injected rats. SMNs and partition cells are less frequently apposed. As nonsynaptic release of 5-HT is common in the spinal cord, an increase in 5-HT-positive varicosities along motor-associated cholinergic neurons may contribute to the locomotor improvement observed in OEG-injected spinal rats. Furthermore, serotonin located within the caudal stump may activate lumbosacral locomotor networks.

OEG implantation and step training enhance hindlimb-stepping ability in adult spinal transected rats.

Numerous treatment strategies for spinal cord injury seek to maximize recovery of function and two strategies that show substantial promise are olfactory bulb-derived olfactory ensheathing glia (OEG) transplantation and treadmill step training. In this study we re-examined the issue of the effectiveness of OEG implantation but used objective, quantitative measures of motor performance to test if there is a complementary effect of long-term step training and olfactory bulb-derived OEG implantation. We studied complete mid-thoracic spinal cord transected adult female rats and compared four experimental groups: media-untrained, media-trained, OEG-untrained and OEG-trained. To assess the extent of hindlimb locomotor recovery at 4 and 7 months post-transection we used three quantitative measures of stepping ability: plantar stepping performance until failure, joint movement shape and movement frequency compared to sham controls. OEG transplantation alone significantly increased the number of plantar steps performed at 7 months post-transection, while training alone had no effect at either time point. Only OEG-injected rats plantar placed their hindpaws for more than two steps by the 7-month endpoint of the study. OEG transplantation combined with training resulted in the highest percentage of spinal rats per group that plantar stepped, and was the only group to significantly improve its stepping abilities between the 4- and 7-month evaluations. Additionally, OEG transplantation promoted tissue sparing at the transection site, regeneration of noradrenergic axons and serotonergic axons spanning the injury site. Interestingly, the caudal stump of media- and OEG-injected rats contained a similar density of serotonergic axons and occasional serotonin-labelled interneurons. These data demonstrate that olfactory bulb-derived OEG transplantation improves hindlimb stepping in paraplegic rats and further suggest that task-specific training may enhance this OEG effect.

Developmental distribution of reelin-positive cells and their secreted product in the rodent spinal cord.

To date, only sympathetic and parasympathetic preganglionic neurons are known to migrate abnormally in reeler mutant spinal cord. Reelin, the large extracellular matrix protein absent in reeler, is found in wild-type neurons bordering both groups of preganglionic neurons. To understand better Reelin's function in the spinal cord, we studied its developmental expression in both mice and rats. A remarkable conservation was found in the spatiotemporal pattern of Reelin in both species. Numerous Reelin-expressing cells were found in the intermediate zone, except for regions containing somatic and autonomic motor neurons. A band of Reelin-positive cells filled the superficial dorsal horn, whereas only a few immunoreactive cells populated the deep dorsal horn and dorsal commissure. High levels of diffuse Reelin product were detected in the lateral marginal and ventral ventricular zones in both rodent species. This expression pattern was detected at all segmental spinal cord levels during embryonic development and remained detectable at lower levels throughout the first postnatal month. To discriminate between the cellular and secreted forms of Reelin, brefeldin A was used to block secretion in organotypic cultures. Such perturbations revealed that the high levels of secreted Reelin in the lateral marginal zone were derived from varicose axons of more medially located Reelin-positive cells. Thus, the laterally located secreted Reelin product may normally prevent the preganglionic neurons from migrating too far medially. Based on the strong evolutionary conservation of Reelin expression and its postnatal detection, Reelin may have other important functions in addition to its role in neuronal migration.

Using robotics to teach the spinal cord to walk.

We have developed a robotic device (e.g. the rat stepper) that can be used to impose programmed forces on the hindlimbs of rats during stepping. In the present paper we describe initial experiments using this robotic device to determine the feasibility of robotically assisted locomotor training in complete spinally transected adult rats. The present results show that using the robots to increase the amount of load during stance by applying a downward force on the ankle improved lift during swing. The trajectory pattern during swing was also improved when the robot arms were programmed to move the ankle in a path that approximated the normal swing trajectory. These results suggest that critical elements for successful training of hindlimb stepping in spinal cord injured rats can be implemented rigorously and evaluated using the rat stepper.