Spinal Cord Transplants Support the Regeneration of Axotomized Neurons after Spinal Cord Lesions at Birth: A Quantitative Double-Labeling Study

Abstract

After spinal cord lesions in newborn rats, transplants of fetal spinal cord tissue rescue immature axotomized neurons, support the growth of axons into and through the site of injury and prolong the critical period for developmental plasticity. Both late-developing (uninjured) and regenerating axons contribute to this transplant-induced anatomical plasticity. After lesions in the mature CNS, transplant-induced axonal elongation is spatially restricted. The current study was designed (1) to determine the magnitude of transplant-induced regeneration, (2) to test the hypothesis that the long distance growth beyond the site of injury is mediated by late-developing axonal pathways, whereas axonal elongation by regenerating pathways is spatially restricted as it is in the adult, and (3) to determine if particular nuclei have a greater inherent capacity for regeneration than others. We used temporally spaced retrograde tracing with the fluorescent dyes fast blue and diamidino yellow to address this issue. Fast blue was placed into the site of a spinal cord over hemisection in rat pups <48 h old to label those neurons which were axotomized by a neonatal lesion. The tracer was removed and a transplant of Embryonic Day 14 fetal spinal cord tissue was placed into the lesion site. Three to six weeks later a second tracer (diamidino yellow) was injected bilaterally into the host spinal cord caudal to the transplant. We counted the number of double-labeled (regenerated), single diamidino yellow-labeled (late-growing), and single fast blue-labeled (nonbridging) neurons in the cortex, red nucleus, raphe nuclei, and locus coeruleus. By systematically varying the distance of the diamidino yellow injection site caudal to the transplant, we were able to compare the distance which injured axons regenerate with the distance that late-growing axons extend. When the diamidino yellow in injection was placed within 5 mm caudal to the transplant 28% of the axotomized neurons in the red nucleus, 32% of the axotomized neurons in the locus coeruleus, and 37% of the axotomized neurons in the raphe nuclei were double-labeled (regenerating). Although the percentage of double-labeled neurons decreased as the distance beyond the transplant increased, a substantial population of regenerating neurons was identified in each of the brain stem nuclei examined following diamidino yellow injections up to 15 mm caudal to the transplant. Thus, after spinal cord lesions and transplants at birth, both regenerating neurons and late-developing neurons extended axons long distances (up to 15 mm) caudal to the lesion site. The capacity for regenerative growth was similar in each of the nuclei examined.