Volume loss during muscle reinnervation surgery is correlated with reduced CMAP amplitude but not reduced force output in a rat hindlimb model

Abstract

Introduction: Muscle reinnervation (MR) surgery offers rehabilitative benefits to amputees by taking severely damaged nerves and providing them with new denervated muscle targets (DMTs). However, the influence of physical changes to muscle tissue during MR surgery on long-term functional outcomes remains understudied. Methods: Our rat hindlimb model of MR surgery utilizes vascularized, directly neurotized DMTs made from the lateral gastrocnemius (LG), which we employed to assess the impact of muscle tissue size on reinnervation outcomes, specifically pairing the DMT with the transected peroneal nerve. We conducted MR surgery with both DMTs at full volume and DMTs with partial volume loss of 500 mg at the time of surgery (n = 6 per group) and measured functional outcomes after 100 days of reinnervation. Compound motor action potentials (CMAPs) and isometric tetanic force production was recorded from reinnervated DMTs and compared to contralateral naïve LG muscles as positive controls. Results: Reinnervated DMTs consistently exhibited lower mass than positive controls, while DMTs with partial volume loss showed no significant mass reduction compared to full volume DMTs (p = 0.872). CMAP amplitudes were lower on average in reinnervated DMTs, but a broad linear correlation also exists between muscle mass and maximum CMAP amplitude irrespective of surgical group (R² = 0.495). Surprisingly, neither MR group, with or without volume loss, demonstrated decreased force compared to positive controls. The average force output of reinnervated DMTs, as a fraction of the contralateral LG's force output, approached 100% for both MR groups, a notable deviation from the 9.6% (±6.3%) force output observed in our negative control group at 7 days post-surgery. Tissue histology analysis revealed few significant differences except for a marked decrease in average muscle fiber area of reinnervated DMTs with volume loss compared to positive controls (p = 0.001). Discussion: The results from our rat model of MR suggests that tissue electrophysiology (CMAPs) and kinesiology (force production) may recover on different time scales, with volumetric muscle loss at the time of MR surgery not significantly reducing functional outcome measurements for the DMTs after 100 days of reinnervation.

Keywords: compound motor action potential amplitude; isometric tetanic force testing; muscle histology; muscle reinnervation; peripheral nerve.

FIGURE 1

Surgical variations of the MR procedure. (A) MR surgery is often performed in amputees to prevent the development of neuropathic pain and amplify motor nerve signals. Over time, the reinnervated muscle will produce EMG signals that reflect activity in the connected neural tissue. (B) In surgery, the nerve that has lost its original target post-amputation can be transferred to a nearby area with the available muscle tissue. Suturing the transected nerve end-to-end with the distal nerve stump of a DMT in that area is referred to as nerve coaptation. (C) Rather than nerve coaptation, which does not disturb the muscle tissue itself, a free-floating muscle graft taken from another area of the body can be directly wrapped around the cut nerve ending. This is direct nerve-to-muscle neurotization, which represents a different surgical strategy that is used regularly in human subjects. Directly neurotized DMTs are often devascularized, but they can be partially tethered to the vascular system by a “leash” or flap. This enhances tissue survival but reduces spatial freedom for tissue reconstruction.

FIGURE 2

Rat hindlimb model of MR surgery in vascularized, directly neurotized DMTs, with and without volume loss. In this rat hindlimb model of MR, the LG muscle is used as the source for the DMT, and the peroneal nerve acts as the transected nerve. First, the LG must be relieved of its endogenous innervation by cutting the lateral branch of the tibial nerve. This creates the DMT. Then, reinnervation is performed by suturing the cut peroneal nerve directly to the DMT’s epimysium, either with or without volume loss. To model volumetric muscle loss, 500 mg of muscle tissue was removed before neurotization. Both MR groups were given 100 days to reinnervate before functional outcomes were measured. The MR-negative group was given 7 days of denervation time before being measured.

FIGURE 3

Functional outcomes of muscle reinnervation. (A) After reinnervation, CMAPs were recorded using needle electrodes and a clinical EMG recording system. ITFT was performed by suturing the distal end of the DMT to the lever arm of a force sensor. The sciatic nerve was stimulated upstream to generate the muscle force. (B) The raw values for each animal (n = 6 per group) are reported, in which triangles represent the left DMT measurement, squares represent the right naïve LG, and bold lines represent the muscle group average. One-way ANOVA (df = 5) for muscle mass (F = 18.2), maximum CMAP amplitude (F = 10.4), and maximum force production (F = 8.3) resulted in statistical differences (p < 0.001 for all ANOVA tests). Within all three surgical groups, DMTs had significantly lower mass than naïve LG muscles. For CMAP amplitudes, there was a general trend that DMTs produced lower values than naïve muscles. As for the results of ITFT, only the negative group (7 days of denervation) had significantly lower force production in DMTs than the naïve LG muscles of the same group. (C) For each individual animal, measurement ratios were calculated, with left leg DMT values being normalized by the same animal’s right leg naïve LG values. The averaged ratio values are reported for animals in each surgical group (n = 6 per group), plus/minus one standard deviation. One-way ANOVA (df = 2) showed only statistical differences in the maximum force ratios (F = 4.88 and p = 0.023). Both MR surgical groups had force ratios close to 1, but only MR in the full-volume group was significantly larger than that in the MR-negative group (p = 0.019).

FIGURE 4

Histology of reinnervated muscle fibers. (A) Muscle tissue cross-sections were stained with Masson’s trichrome, which differentially stains muscle fibers and connective tissues. Scanned tissue images were classified by pixel using ilastik segmentation software. (B) Individual muscle fibers were identified and measured using FIJI open-source software. These are the raw values for each animal’s left and right limbs (n = 6 per group). The muscle fiber area was defined as any continuous area labeled “muscle fiber” that falls within the size range of 10²–10⁴ μm (Hsu and Cohen, 2013). The total muscle fiber area is the summed area of all the muscle fibers detected in a sample; df = 5, F = 4.42, and p = 0.004. The average muscle fiber area is the mean of all the discrete muscle fibers in a sample; df = 5, F = 8.73, and p < 0.001.

FIGURE 5

Muscle fiber type analysis. (A) Muscle tissue cross-sections were fluorescently labeled for Type 1, Type 2a, and Type 2b muscle fibers (n = 3 samples in the MR group and n = 9 samples in the Naïve LG group). The MR-negative DMTs being analyzed for fiber type were collected 100 days post-denervation. One hallmark of the reinnervated tissue that is present is an increased clumping of fiber types, especially Type 2a (white arrows). (B) The only significant difference seen between fiber type groups is a slight increase in Type 2a fibers for the MR-negative (100 days) and the MR with volume loss groups; df = 3, F = 6.05, and p = 0.007.

FIGURE 6

Multivariate relationships in functional outcomes All data points represent one leg of a single animal subject that was measured, where triangles represent the left leg DMT and squares represent the right leg naïve LG muscle (36 total data points). The axes for the 3D plot are muscle mass (mg) versus maximum CMAP amplitude (mV) versus maximum tetanic force production (mN). Linear regression was performed on the data (regardless of the reinnervation status) to produce best-fit equations as a function of muscle mass. The correlation observed between muscle mass and CMAP amplitude is stronger than the relationship seen between muscle mass and maximum force production (R ² = 0.495 and R ² = 0.118, respectively).