Functional recovery is often poor despite the capacity for axonal regeneration in the peripheral nervous system and advances in microsurgical technique. Regeneration of axons in mixed nerve into inappropriate pathways is a major contributing factor to this failure. In this study, we use the rat femoral nerve model of transection and surgical repair to evaluate (1) the effect of nerve transection on the speed of regeneration and the generation of motor-sensory specificity, (2) the efficacy of electrical stimulation in accelerating axonal regeneration and promoting the reinnervation of appropriate muscle pathways by femoral motor nerves, and (3) the mechanism of action of electrical stimulation. Using the retrograde neurotracers fluorogold and fluororuby to backlabel motoneurons that regenerate axons into muscle and cutaneous pathways, we found the following. (1) There is a very protracted period (10 weeks) of axonal outgrowth that adds substantially to the delay in axonal regeneration (staggered regeneration). This process of staggered regeneration is associated with preferential motor reinnervation (PMR). (2) One hour to 2 weeks of 20 Hz continuous electrical stimulation of the parent axons proximal to the repair site dramatically reduces this period (to 3 weeks) and accelerates PMR. (3) The positive effect of short-term electrical stimulation is mediated via the cell body, implicating an enhanced growth program. The effectiveness of such a short-period low-frequency electrical stimulation suggests a new therapeutic approach to accelerate nerve regeneration after injury and, in turn, improve functional recovery.

Schwann cell phenotype is classified as either myelinating or nonmyelinating. Additional phenotypic specialization is suggested, however, by the preferential reinnervation of muscle pathways by motoneurons. To explore potential differences in growth factor expression between sensory and motor nerve, grafts of cutaneous nerve or ventral root were denervated, reinnervated with cutaneous axons, or reinnervated with motor axons. Competitive reverse transcription-PCR was performed on normal cutaneous nerve and ventral root and on graft preparations 5, 15, and 30 d after surgery. mRNA for nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), vascular endothelial growth factor, hepatocyte growth factor, and insulin-like growth factor-1 was expressed vigorously by denervated and reinnervated cutaneous nerve but minimally by ventral root. In contrast, mRNA for pleiotrophin (PTN) and glial cell line-derived neurotrophic factor was upregulated to a greater degree in ventral root. ELISA confirmed that NGF and BDNF protein were significantly more abundant in denervated cutaneous nerve than in denervated ventral root, but that PTN protein was more abundant in denervated ventral root. The motor phenotype was not immutable and could be modified toward the sensory phenotype by prolonged reinnervation of ventral root by cutaneous axons. Retrograde labeling to quantify regenerating neurons demonstrated that cutaneous nerve preferentially supported cutaneous axon regeneration, whereas ventral root preferentially supported motor axon regeneration. Schwann cells thus express distinct sensory and motor phenotypes that are associated with the support of regeneration in a phenotype-specific manner. These findings suggest that current techniques of bridging gaps in motor and mixed nerve with cutaneous graft could be improved by matching axon and Schwann cell properties.

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Electrical stimulation promotes the speed and accuracy of motor axonal regeneration. The positive effects of stimulation are mediated at the cell body. Here we characterize the effect of electrical stimulation on motoneuronal expression of BDNF and its receptor, trkB, two genes whose expression levels in motoneurons correlate with regeneration and are regulated by electrical activity in a variety of neurons. We used semiquantitative in situ hybridization to measure expression of mRNA encoding BDNF and the full-length trkB receptor at intervals of 8 h, 2 days and 7 days after unilateral femoral nerve cut, suture, and stimulation. Expression in regenerating motoneurons was compared to that of contralateral intact motoneurons. BDNF and trkB signals were not significantly upregulated 8 h and 2 days after femoral nerve suture and sham stimulation. By 7 days, there was a 2-fold increase in both BDNF and trkB mRNA expression. In contrast, stimulation of cut and repaired nerves for only 1 h led to rapid upregulation of BDNF and trkB mRNA by 3-fold and 2-fold, respectively, within the first 8 h. The stimulation effect peaked at 2 days with 6-fold and 4-fold increases in the signals, respectively. Thereafter, the levels of BDNF and trkB mRNA expression declined to equal the 2-fold increase seen at 7 days after nerve repair and sham-stimulation. We conclude that brief electrical stimulation stimulates BDNF and trkB expression in regenerating motoneurons. Because electrical stimulation is known to accelerate axonal regeneration, we suggest that changes in the expression of BDNF and trkB correlate with acceleration of axonal regeneration.