Contributions of pathway and neuron to preferential motor reinnervation

Abstract

Motor axons regenerating after transection of mixed nerve preferentially reinnervate distal muscle branches, a process termed preferential motor reinnervation (PMR). Motor axon collaterals appear to enter both cutaneous and muscle Schwann cell tubes on a random basis. Double-labeling studies suggest that PMR is generated by pruning collaterals from cutaneous pathways while maintaining those in motor pathways (the "pruning hypothesis"). If all collaterals projecting to muscle are saved, then stimulation of regenerative sprouting should increase specificity by increasing the number of motoneurons with at least one collateral in a muscle pathway. In the current experiments, collateral sprouting is stimulated by crushing the nerve proximal to the repair site before suture, a maneuver that also conditions the neuron and predegenerates the distal pathway. Control experiments are performed to separate these effects from those of collateral generation. Experiments were performed on the rat femoral nerve and evaluated by exposing its terminal cutaneous and muscle branches to HRP or Fluoro-Gold. Crush proximal to the repair site increased motor axon collaterals at least fivefold and significantly increased the percentage of correctly projecting motoneurons, consistent with the pruning hypothesis. Conditioning the nerve with distal crushes before repair had no effect on specificity. A graft model was used to separate the effects of collateral generation and distal stump predegeneration. Previous crush of the proximal femoral nerve significantly increased the specificity of fresh graft reinnervation. Stimulation of regenerative collateral sprouting thus increased PMR, confirming the pruning hypothesis. However, this effect was overshadowed by the dramatic specificity with which predegenerated grafts were reinnervated by fresh uncrushed proximal axons. These unexpected effects of predegeneration on specificity could involve a variety of possible mechanisms and warrant further study because of their mechanistic and clinical implications.

Fig. 1.

Preparation of nerve repairs. Surgery was performed on the proximal femoral nerves of 250 gm female Sprague Dawley rats. In the repair group, the nerve was sharply transected and sutured under 20–40× with two 11–0 sutures. In the conditioned repair group, the distal motor and sensory branches were each crushed for 5 sec with #5 jeweler’s forceps. The crushes were administered 4 and 2 weeks before transection and suture of the proximal nerve. This preparation conditioned the neuron without stimulating collateral formation at the repair site. In the crush repair group, the proximal nerve was crushed, and after 2 weeks a second crush was delivered 2 mm distal to the first. After an additional 2 weeks, the nerve was repaired 2 mm distal to the second crush. This preparation maximized the number of axon collaterals at the suture site. M, Muscle projections; C, cutaneous projections.

Fig. 2.

Results of nerve repair. Each triad of vertical bars represents the mean motoneuron count obtained in 20 nerve preparations. The white bars represent the mean number of motoneurons projecting correctly to the muscle branch, theblack bars represent the mean number projecting incorrectly to the cutaneous branch, and the stippled bars represent the mean number of double-labeled neurons, which project collaterals simultaneously to both cutaneous and muscle branches. In repair and conditioned repair groups, motoneuron projections were random at 3 weeks; PMR was clearly evident by 3 months. After crush repair, however, specificity was already apparent at 3 weeks and was dramatic by 3 months. This specificity was achieved by lowering the number of incorrect projections rather than by increasing the number of motoneurons projecting correctly to quadriceps muscle.

Fig. 3.

Sequential double labeling. Repair and crush repair animals were prepared as in Figure 2. After 3 months of regeneration, the cutaneous nerve was exposed to tracer, in this case FG, to label all motoneurons misdirected to skin. Forty-eight hours later, the proximal nerve was labeled with a different tracer, HRP, to label all motoneurons regenerating past the repair to the level of the iliacus nerve. Motoneurons projecting to skin will be double-labeled, and those projecting to muscle will be labeled with only the second tracer.

Fig. 4.

Results of sequential double labeling. Each triad of vertical bars represents the mean counts from 20 nerve preparations. The black bars represent the mean number of double-labeled motoneurons, which project incorrectly to skin, thewhite bars represent motoneurons projecting correctly to muscle, and the hatched bars represent the total number of motoneurons regenerating. Crush repair shunted motor axons from cutaneous to muscle nerve without decreasing the total volume of regeneration.

Fig. 5.

Preparation of grafting experiments. In the graft group, the femoral nerve trunk and branches were excised and sewn into the bed of the opposite femoral nerve, correctly aligning cutaneous, muscle, and iliacus branches. In the predegenerated graft group, the donor segment was predegenerated 4 and 2 weeks before transfer and then transposed to the unoperated limb to be reinnervated by fresh, previously uninjured axons. This sequence was reversed in the crush graft group; crushes were delivered to the recipient nerve to stimulate collateral sprouting, after which a fresh graft was transferred to receive these sprouts. The iliacus branch (not shown) was repaired in all experiments.

Fig. 6.

Results of grafting experiments. Each triad of vertical bars represents the means of correct, incorrect, and double-labeled projections from 20 experimental animals. Proximal crush to stimulate regenerative sprouting increased the specificity with which fresh graft was reinnervated, confirming the pruning hypothesis. When predegenerated graft was reinnervated with fresh axons, both specificity (%M) and the absolute number of motoneurons reinnervating the quadriceps muscle were significantly increased. The results in the predegenerated graft group equal those of crush repair (Fig. 2).