Cervical spinal cord therapeutics delivery: preclinical safety validation of a stabilized microinjection platform.

OBJECTIVE: The current series represents a preclinical safety validation study for direct parenchymal microinjection of cellular grafts into the ventral horn of the porcine cervical spinal cord. METHODS: Twenty-four 30- to 40-kg female Yorkshire farm pigs immunosuppressed with cyclosporine underwent a cervical laminectomy and ventral horn human neural progenitor cell injection. Cell transplantation in groups 1 to 3 (n = 6 pigs each) was undertaken with the intent of assessing the safety of varied injection volumes: 10, 25, and 50 microL injected at 1, 2.5, and 5 microL/min, respectively. Groups 4 and 5 (n = 3 pigs each) received prolonged immunosuppressant pretreatment in an attempt to demonstrate graft viability. The latter was undertaken in an alternate species (mini-pig versus Yorkshire pig). RESULTS: Neurological morbidity was observed in 1 animal and was attributable to the presence of a resolving epidural hematoma noted at necropsy. Although instances of ventral horn targeting were achieved in all injection groups with a coordinate-based approach, opportunities exist for improvement in accuracy and precision. A relationship between injection volume and graft site cross-sectional area suggested limited reflux. Only animals from group 5 achieved graft survival at a survival end point (t = 1 week). CONCLUSION: This series demonstrated the functional safety of targeted ventral horn microinjection despite evidence for graft site immune rejection. Improvements in graft delivery may be augmented with an adapter to improve control of the cannula entry angle, intraoperative imaging, or larger graft volumes. Finally, demonstration of long-term graft viability in future preclinical toxicity studies may require tailored immunosuppressive therapies, an allograft construct, or tailored choice of host species.

Properties of medial gastrocnemius motor units and muscle fibers reinnervated by embryonic ventral spinal cord cells.

Severe muscle atrophy occurs after complete denervation. Here, Embryonic Day 14-15 ventral spinal cord cells were transplanted into the distal tibial nerve stump of adult female Fischer rats to provide a source of neurons for muscle reinnervation. Our aim was to characterize the properties of the reinnervated motor units and muscle fibers. Some reinnervated motor units contracted spontaneously. Electrical stimulation of the transplants at increasing intensity produced an average (+/- SE) of 7 +/- 1 electromyographic and force steps. Each signal increment represented the excitation of another motor unit. These reinnervated units exerted an average force of 12.0 +/- 1.5 mN, strength similar to that of control fatigue-resistant units. Repeated transplant stimulation depleted 17% of the muscle fibers of glycogen, an indication of some functional reinnervation. Reinnervated (glycogen-depleted), denervated (no cells transplanted), and control fibers were of histochemical type I, IIA, or IIB. Fibers of the same type were grouped after reinnervation. The proportion of fiber types also changed. Reinnervated fibers were primarily type IIA, whereas most fibers in denervated and control muscles were type IIB. Reinnervated fibers of each type had significantly larger cross-sectional areas than the corresponding fiber types in denervated muscles. These data suggest that neurons with different properties can reside in the unusual environment of the adult rat peripheral nerve, make functional connections with muscle, specify muscle fiber type, and reduce the amount that each type atrophies.

Intraspinal implantation of hNT neurons into SOD1 mice with apparent motor deficit.

INTRODUCTION: The aim of this study was to determine the effect of hNT neuron transplants on motor neuron function in SOD1 (G93A) mice when motor deficits were already apparent. METHOD: The hNT neurons were implanted into L(4)-L(5) segments of the ventral horn spinal cord of mice at 15-16 weeks of age: either G93A mice, transgenic mice carrying the normal allele for human SOD1 gene (hTg), or control wild type mice (wt). Behavioral tests (rotorod, beam balance, extension reflex, footprint) were performed prior to transplantation and at weekly intervals afterwards. RESULTS: HNT neuron transplantation in the SOD1 mice delayed disease progression for 3-4 weeks, although lifespan was not affected. CONCLUSION: These results suggest that hNT neuron transplantation may be a promising therapeutic strategy for ALS in the later phase of the neurodegeneration.