Brief electrical stimulation promotes the speed and accuracy of motor axonal regeneration

Abstract

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.

Fig. 1.

Diagrammatic representations of the following.a, The femoral nerve, the branch to the quadriceps muscles, and the saphenous nerve branch containing sensory nerves to the skin. b, Application of retrograde neurotracers to count motoneurons that regenerated their axons into the muscle and cutaneous branches of the cut and surgically repaired femoral nerve (see text for details). c, Placement of bipolar electrodes to stimulate chronically the cut and regenerating nerve fibers proximal to the site of nerve transection and surgical repair.

Fig. 2.

Diagrammatic representation of the experimental method used to establish the blocking dose of TTX on the femoral nerve. Bipolar-stimulating electrodes placed on each of the two ventral roots that supply the motor fibers in the femoral nerve were stimulated supramaximally, and the evoked action potential was recorded on the femoral nerve distal to the application of TTX to the nerve. Doses of 60 mg/ml were found to be effective in completely blocking action potential conduction (see the text for further details).VR, Ventral root.

Fig. 3.

Counting the number of femoral motoneurons that regenerated their axons into the appropriate muscle branch and into the inappropriate cutaneous sensory branch and those that regenerated axons into both. a, Retrogradely labeled motoneurons that had regenerated their axons into the appropriate muscle branch (mu; fluororuby) or the inappropriate cutaneous sensory branch (cu; fluorogold) and those that regenerated axons into both (b; double labeled). b, The mean number of backlabeled motoneurons that regenerated into the appropriate muscle branch (mu; filledbars), the inappropriate cutaneous branch (cu; strippedbars), and both branches (b; openbars) 2, 3, 4, 6, 8, and 10 weeks after femoral repair.c, The mean number ± SE of total backlabeled motoneurons (All) that regenerated their axons into the appropriate muscle branch (Muscle) and into the inappropriate cutaneous branch (Cutaneous) as a function of time after femoral nerve transection and repair.

Fig. 4.

Effects of electrical stimulation on motor axonal regeneration and PMR. a, The mean number ± SE of motoneurons that regenerated into appropriate muscle (mu; filledbars) and inappropriate cutaneous (cu; strippedbars) branches and both branches (b;openbars) 2, 3, and 8 weeks after femoral nerve transection and surgical repair and 2 weeks of 20 Hz continuous electrical stimulation. b–d, Comparison of the mean number ± SE of motoneurons that regenerated after femoral nerve transection and surgical repair without (●) and with 20 Hz continuous electrical stimulation for 1 hr (▿), 1 d (■), 1 week (▵), and 2 weeks (⋄). b, All motoneurons.c, Motoneurons that regenerated into the appropriate muscle branch. d, Motoneurons that regenerated into the inappropriate cutaneous branch. The shadedhorizontalbar in b–drepresents ±SE of the mean number of regenerated motoneurons 8 and 10 weeks after femoral nerve repair with no stimulation, when there was no longer any significant difference between mean number of regenerated motoneurons (p > 0.05). stim, Stimulation.

Fig. 5.

Short-term stimulation is as effective as long-term stimulation in accelerating axonal regeneration and PMR.a, Comparison of the effects of different periods of 20 Hz continuous electrical stimulation (1 hr, 1 d, 1 week, and 2 weeks) on the mean number ± SE of motoneurons that regenerated into muscle (mu; filledbars), cutaneous (cu;strippedbars), and both (b; openbars) branches of the femoral nerve 3 weeks after nerve repair with the effects of no stimulation or sham stimulation. b, c, Data from individual rats (numbered on the x-axis) showing the effects of 1 hr of sham stimulation (b) and 1 hr of 20 Hz continuous electrical stimulation of the proximal nerve stump immediately after nerve repair (c).stim, Stimulation.

Fig. 6.

TTX block of retrograde transmission of action potentials to the cell body. TTX (60 mg/ml) completely blocked the effect of 1 hr of 20 Hz continuous electrical stimulation in accelerating regeneration and PMR 3 weeks after nerve transection and surgical repair. b (openbars), Both branches; cu(strippedbars), cutaneous branch;mu (filledbars), muscle branch; Stim, stimulation.