Effect of Intraoperative Electrical Stimulation on Recovery after Rat Sciatic Nerve Isograft Repair

Abstract

Peripheral nerve injuries, associated with significant morbidity, can benefit from electrical stimulation (ES), as demonstrated in animal studies through improved axonal growth. This study combined the clinical gold standard of isograft repair in a rat model of sciatic nerve injury to evaluate the effects of intraoperative ES on functional tests and histology. Forty rats underwent a surgically induced gap injury to the right sciatic nerve and subsequent repair with an isograft. Half of these rats were randomly selected to receive 10 min of intraoperative ES. Functional testing, including response time to a heat stimulus and motor functional tests, were conducted. Histology of the sciatic nerves and gastrocnemius muscles were analyzed after 6 and 12 weeks of recovery. Rats that underwent ES treatment showed incremental improvements in motor function between weeks 2 and 12, with a significantly higher push-off response than the no-ES controls after 6 weeks. Although no differences were detected between groups in the sensory testing, significant improvements over time were noted in the ES group. Histology parameters, sciatic nerve measures, and gastrocnemius muscle weights demonstrated nerve recovery over time for both the ES and no-ES control groups. Although ES promoted improvements in motor function comparable to that in previous studies, the benefits of intraoperative ES were not detectable in other metrics of this rat model of peripheral nerve injury. Future work is needed to optimize sensory testing in the rodent injury model and compare electrical activity of collagen scaffolds to native tissue to detect differences.

Keywords: electrical stimulation; histology; motor function; peripheral nerve injury; sensory function.

FIG. 1.

Motor function was evaluated by EPT (A–C) and SFI (D). (A) MD%, a normalized value of injured to sham leg response of each rat, showed a decreasing trend over the 12 weeks as motor function improved. Data from 12-week animals are reported on the figure. Statistical analysis on the 12-week animals showed an MD% decrease between weeks 4 and 10 (control, p = 0.002; ES, p = 0.05), 4 and 12 (control, p = 0.003; ES, p = 0.002), and between weeks 6 and 10 (control, p = 0.013; ES, p = 0.008). Additionally, the control group showed MD% differences between weeks 4 and 8 (p = 0.009), weeks 6 and 8 (p = 0.035), and weeks 6 and 12 (p = 0.044). The ES group showed MD% differences between weeks 2 and 12 (p < 0.001). (B) Push-off force of the uninjured sham leg showed a consistency in data collection over the 12-week testing period with no differences between the control and ES groups at any of the time-points. However, some differences were found between time-points within the groups, week 2 and 6 in the control group (p = 0.039), between weeks 2 and 12 (control, p = 0.005; ES, p = 0.001), and between weeks 6 and 12 in the ES group (p = 0.021). (C) As expected, push-off force of the injured experimental leg showed a steady increase over the 12-week testing period, with differences between week 2 and 6 in the ES group (p < 0.001), between weeks 2 and 12 (control, p = 0.001; ES, p < 0.001), and between weeks 6 and 12 (control, p = 0.001; ES, p = 0.002). The ES group showed a significantly higher push-off force than the control group at the 6-week time-point (p = 0.005). Although the push-off averages were consistently higher in the ES group than in the control group (p = 0.03 at week 12), no further differences were found between the groups at any of the time-points with a conservative Bonferroni correction to adjust for multiple comparisons. (D) SFI was determined every 2 weeks for each group. All animals except one in the control group and three in the ES group developed contractures, and therefore, the paw print could not be accurately tracked. Contractures is a common problem with SFI, which is why both EPT and SFI were used to track motor function. EPT, extensor postural thrust; ES, electrical stimulation; MD%, percent motor deficit; SFI, sciatic functional index.

FIG. 2.

Sensory function. (A) For animals tested over 12 weeks, sensory response of the uninjured sham leg, the paw-lift response to a thermal stimulus, was consistent over the weeks of data collection and in the range of 5–10 sec, which was the pre-surgery latency response time. No statistical differences were found between groups or time-points. (B) Sensory response of the injured experimental leg showed a loss of function at week 2, with higher sensory response times, and a steady regaining of function with sensory response times decreasing throughout the 12 weeks of recovery. Differences were found in the ES group between weeks 2 and 6 (p = 0.037), weeks 2 and 8 (p = 0.03), weeks 2 and 10 (p = 0.016), weeks 2 and 12 (p = 0.005), weeks 4 and 8 (p = 0.002), weeks 4 and 10 (p < 0.001), and weeks 4 and 12 (p = 0.002). ES, electrical stimulation.

FIG. 3.

Muscle histology. (A) Healthy muscle fibers, as shown by a representative section with an H&E stain from the sham leg of a rat in the 6-week ES group. Muscle fibers are clearly defined, with purple nuclei. All muscle image scale bars are 100 μm. (B) Degenerated muscle fibers, as shown by a representative section from the injured leg of a rat in the 6-week ES group. Muscle fibers show signs of shrinking from muscle degeneration, with more dense nuclei per sampling area. (C) Visible regeneration, as shown by a representative section from the injured leg of a rat in the 12-week ES group. Muscle fiber size increased from week 6, with a less dense nuclei per sampling area. (D) Relative muscle mass ratio, a normalized value of the injured leg muscle weight to the sham for each rat, showed a significant increase between weeks 6 and 12 for both the control and ES group (p < 0.001). * denotes p < 0.05. ES, electrical stimulation; H&E, hematoxylin and eosin.

FIG. 4.

Nerve histology. (A) Fiber density, a measure of the total number of axons per sampling area, was calculated for all nerve sections, taken from the distal, midline, and proximal segments of the injured-leg nerve of each rat. Although there were no differences between the groups, both groups showed a significant increase in fiber density between 6 and 12 weeks in the distal, midline, and proximal segments (p ≤ 0.001). (B) Mean fiber width, a measure of the axons showing maturity of the regenerating fibers, was calculated for all nerve sections taken from the distal, midline, and proximal segments of the injured-leg nerve of each rat. Differences were found between weeks 6 and 12 in the ES group at the midline segment (p = 0.009) and in the control group at the proximal segment (p = 0.012). A difference was found between the ES and control groups in the midline sections of the 6-week time-point (p = 0.009). (C) Percent nerve, a measure of the axon area relative to the sampling area, was calculated for all nerve sections taken from the distal, midline, and proximal segments of the injured-leg nerve of each rat. Differences were found between weeks 6 and 12 in the midline and distal segments (p ≤ 0.001) and in the proximal segment (control, p < 0.001; ES, p = 0.003). (D) Representative nerve sections at both time-points, 6 weeks and 12 weeks, with samples from ES and control groups at the distal and proximal segments of the nerve, stained with osmium tetroxide. Early degeneration of the nerve can be observed at the 6-week time-point in the difference between the distal and proximal sections, where the proximal end has a healthier appearance with clearly defined black myelinated axons. Regeneration can be observed in comparing the 12-week distal segments with their 6-week counterparts, where the number of visible axons increases. This is supported by quantified data, where there are significant increases of fiber density, percent nerve, and fiber width between weeks 6 and 12. Scale bar for all images is 50 μm. ES, electrical stimulation.