Axonal regeneration and functional recovery after complete spinal cord transection in rats by delayed treatment with transplants and neurotrophins

Abstract

Little axonal regeneration occurs after spinal cord injury in adult mammals. Regrowth of mature CNS axons can be induced, however, by altering the intrinsic capacity of the neurons for growth or by providing a permissive environment at the injury site. Fetal spinal cord transplants and neurotrophins were used to influence axonal regeneration in the adult rat after complete spinal cord transection at a midthoracic level. Transplants were placed into the lesion cavity either immediately after transection (acute injury) or after a 2-4 week delay (delayed or chronic transplants), and either vehicle or neurotrophic factors were administered exogenously via an implanted minipump. Host axons grew into the transplant in all groups. Surprisingly, regeneration from supraspinal pathways and recovery of motor function were dramatically increased when transplants and neurotrophins were delayed until 2-4 weeks after transection rather than applied acutely. Axonal growth back into the spinal cord below the lesion and transplants was seen only in the presence of neurotrophic factors. Furthermore, the restoration of anatomical connections across the injury site was associated with recovery of function with animals exhibiting plantar foot placement and weight-supported stepping. These findings suggest that the opportunity for intervention after spinal cord injury may be greater than originally envisioned and that CNS neurons with long-standing injuries can reinitiate growth, leading to improvement in motor function.

Fig. 1.

Transplants of fetal spinal cord tissue survived and bridged the gap at the transection site. A, Montage of longitudinal cresyl violet-stained sections through the lesion and transplant site. Rostral and caudal host cord are to thetop and bottom, respectively, and transplant is in the middle. Animals were killed 5 weeks after transplantation. Scale bar, 1 mm. The spinal cord containing lesion and transplant and the caudal segment with an additional 10 mm of spinal cord was examined. B–D, Raphe–spinal axons in longitudinal sections through the transplant were stained for 5-HT. Fibers were detected 7–8 mm from the caudal border of the transplant in the ventral horn gray matter and white matter. B, Acute transection and transplant. C, Acute transection and transplant with neurotrophins. D, Transection with delayed transplant and neurotrophins. Scale bar, 50 μm.

Fig. 2.

Raphe–spinal axons were able to reenter the host spinal cord caudal to the transplant and enter both gray and white matter. Longitudinal sections through the host spinal cord caudal to the transplant were stained for 5-HT. Serotonergic axons that regrew in host spinal cord caudal to transection and transplant in the acute transplant group were scattered, thin, and relatively unbranched.A shows fibers in the gray matter. Delayed transplant and neurotrophins resulted in denser axonal growth with more branching and larger cluster sizes. B shows fibers in the gray matter, and C shows fibers in the white matter. InA–C, axons are marked with arrowheads. Scale bar, 50 μm.

Fig. 3.

A, Total length of serotonergic fibers per animal was measured over a distance of 10 mm caudal to the lesion (length is mean ± SEM micrometers). A greater total fiber length was observed in the gray matter compared with the white matter (p = 0.004 for acute; p< 0.001 for delayed; Mann–Whitney U test).B, The average number of branch points was significantly higher in the gray matter in the delayed transplant groups compared with the acute transplant group (*p < 0.01; **p < 0.001; Mann–Whitney U test). C, The average size of serotonergic axon clusters was significantly larger in the gray matter for the delayed transplant group (**p < 0.001) and in the white matter in animals that received BDNF (*p < 0.01; Mann–WhitneyU test).

Fig. 4.

Fluoro-Gold retrograde labeling of regenerated neurons. Neuronal cytoplasm is labeled (arrows) by Fluoro-Gold in transverse sections of the caudal one-third of the red nucleus (A), locus Ceruleus (B), raphe nucleus (C), lateral vestibular nucleus (D), and reticular formation (E). Fluoro-Gold staining is also present in the cytoplasm of spinal neurons rostral to transplant in host cervical cord (longitudinal section) (F) and in the cell bodies of cortical neurons (data not shown). Scale bars, 100 μm.

Fig. 5.

Fluoro-Ruby anterograde labeling of corticospinal neurons. Fluoro-Ruby was injected into the motor cortex 8 weeks after transplantation of fetal spinal cord tissue and neurotrophin administration. Animals survived for 10 d and were killed. Fluorescent micrographs show fibers labeled caudal to the transection site in longitudinal sections from thoracic cord (A) and transverse sections from lumbar cord (B). Scale bar, 100 μm.Arrows indicate labeled corticospinal axons traveling toward the gray matter.

Fig. 6.

A, Histogram comparing number of weight-supported plantar steps (±SD) on a treadmill in animals with a transection (TX) only, with a transplant (Acute TP), with a delayed transplant (Chronic TP), with acute administration of transplant and neurotrophins (Acute TP + NTF), or with delayed administration of transplant and NTF (Chronic TP + NTF). Animals with delayed administration of transplant and NTF had significantly more steps than those with acute administration of transplant and NTF (★p < 0.001; ANOVA; Tukey's multiple comparison test). B, Kinematic analysis of hindlimb step cycles using the Peak Performance System for transection only (TX ONLY), normal, and delayed transplant with NT-3 (TX + TP + NT-3). Bony landmarks for pelvis, hip, knee, ankle, and fifth metatarsal were digitized directly from videotapes. BS, Beginning of stance; MS, middle stance; ES, end of stance; MSW, mid-swing. The horizontal line below each stick figure represents the surface of the treadmill.

Fig. 7.

Stair ascension. A, Sequential video frames (interval, 27 frames) of a transection only animal (left panels) and transection with delayed transplant and neurotrophins (right panels) during stair ascension 2 months after injury. Animals are represented during best performances. Note that the transection only animals show no weight support on the stairs; hindlimbs are dragged passively behind the body. Animals that receive a delayed transplant with neurotrophins demonstrate bilateral weight support, plantar foot placement (seearrows), and coordinated forelimb–hindlimb stepping during stair climbing. B, Histogram comparing mean ± SD total number of weight-supported steps during stair ascension on best day motor performance. Transection only (TX Only) animals showed no weight-supported stepping during stair climbing. Animals that received delayed transplants with neurotrophins (Chronic TP + NTF) had significantly more weight-supported steps (p < 0.001; ANOVA; Tukey's multiple comparison test).