More Frequent Electrical Stimulation Impairs Nerve Regeneration After Median Nerve Repair in a Rat Model

Peripheral nerve regeneration via electrical stimulation on nerve repair has been shown to potentiate axonal growth and functional recovery. However, it remains unknown whether increasing the frequency of electrical stimulation improves, diminishes, or has no effect on the quality of nerve regeneration after repair. Additionally, few animal studies have investigated the potential beneficial effects of serial stimulations on accelerating and improving end-organ functionality over time. We therefore sought to evaluate the additive effect of weekly versus biweekly serial electrical stimulation on median nerve regeneration after transection and repair in a rat model using a stimulated grip strength testing method.

(1) Does biweekly versus weekly electrical stimulation lead to greater grip strength forces and therefore, accelerated nerve regeneration? (2) Does frequency of electrical stimulation affect muscle morphology, compound muscle action potential amplitude, muscle weight, or axon count?

Twenty-one male Sprague Dawley rats (7 to 8 weeks old, weighing 225 to 250 g at study onset) were randomized into three groups (7 rats per group): (1) median nerve transection and repair with biweekly electrical stimulation, (2) median nerve transection and repair with weekly electrical stimulation, and (3) control group receiving weekly electrical stimulation without median nerve transection. Electrical stimulation was performed via two needle point electrodes placed percutaneously into the right axilla proximal to the pectoralis major insertion (pulsed DC current, 0.25 ms pulse duration, 3V at 50 Hz). The tested right forelimb of the anesthetized rats was placed in abduction at 90° with the elbow and wrist secured in extension. Under electrical stimulation, the maximum force generated before the loss of grip strength from a clenched paw was measured via force transducer. After administration of electrical stimulation therapy, three trials of stimulated grip strength testing were performed with 1 minute of rest between trials, and the maximum force was recorded. This was repeated at weekly or biweekly intervals depending on the experimental group. After 11 weeks, compound muscle action potentials were measured across all flexor digitorum superficialis muscles, bilaterally, and the muscles were harvested from both forearms for weight atrophy and morphology assessments by precision scale. Recovery was expressed as a ratio of compound muscle action potential amplitude of the repaired right limb amplitude to the contralateral uninjured left limb amplitude. We chose the flexor digitorum superficialis because this muscle is innervated exclusively by and its recovery is solely dependent on the median nerve. In all groups, a 1-cm segment of the ulnar nerve was excised to ensure only muscles innervated by the median nerve were contributing to the force measurements. All nerve specimens were histologically evaluated to compare extrapolated axon count among the weekly, biweekly, and control groups. Statistical analysis was performed via ANOVA testing with the Tukey post hoc test and p value set at 0.05.

By 11 weeks, weekly stimulation was associated with greater grip strength than biweekly stimulation (weekly 3.9 ± 1.0 N versus biweekly 2.6 ± 0.9 N, mean difference 1.3 N [95% CI 0.0 to 2.5]; p = 0.0499). Grip strength did not differ between the weekly and control groups (weekly 3.9 ± 1.0 N versus control 4.9 ± 1.4 N, mean difference 1 N [95% CI -0.2 to 2.3]; p = 0.11), and the control group had greater grip strength than the biweekly group (mean difference 2.3 N [95% CI 1.1 to 3.6]; p < 0.001). Weekly stimulation resulted in greater muscle preservation than biweekly stimulation (weekly 0.84 ± 0.02 versus biweekly 0.75 ± 0.05, mean difference 0.09 [95% CI 0.04 to 0.15]; p = 0.003), with the control animals demonstrating the greatest muscle weight. Compound muscle amplitude ratios were similar between groups, with no differences between weekly and biweekly, weekly and control, or control and biweekly. Axon regeneration on the repaired side differed by stimulation frequency. Model-estimated axon counts were lower in the biweekly group (1930 [95% CI 1462 to 2398]) than in the control group (p = 0.0013), whereas axon counts did not differ between the biweekly and weekly groups (3618 [95% CI 2101 to 5135]; p = 0.08). Weekly and control groups did not differ on the repaired side. Within-group comparisons showed fewer axons on the repaired than the uninjured side in the biweekly group (1930 versus 3807; p = 0.009), whereas weekly and control groups showed no side-to-side differences.

Weekly electrical stimulation was associated with greater grip strength and muscle preservation than biweekly stimulation, whereas axon counts did not differ between stimulation frequencies, suggesting that more frequent stimulation does not confer additional benefit and may impede regeneration. These findings support further investigation of stimulation frequency as a potentially modifiable factor in nerve regeneration.

Our in vivo rat study suggests that increasing the frequency of brief, repeated stimulation may hinder recovery. Clinically, these results highlight the risks of overstimulation as a potential pitfall to the regenerative process, underscoring the need to further refine stimulation parameters. Larger-animal and human studies are needed to determine translatability to patient care.