WHAT IS NORMAL? NEUROMUSCULAR JUNCTION REINNERVATION AFTER NERVE INJURY

Abstract

Introduction:

We present a reproducible technique to assess motor recovery after nerve injury via neuromuscular junction (NMJ) immunostaining and electrodiagnostic testing.

Methods:

Wildtype mice underwent sciatic nerve transection with repair. Hindlimb muscles were collected for microscopy up to 30 weeks after injury. Immunostaining assessed axons (NF200), Schwann cells (S100), and motor endplates (α-bungarotoxin). Compound motor action potential (CMAP) amplitude assessed tibialis anterior (TA) function.

Results:

One week after injury, nearly all (98.0%) endplates were denervated. At 8 weeks, endplates were either partially (28.3%) or fully (71.7%) reinnervated. At 16 weeks, NMJ reinnervation reached 87.3%. CMAP amplitude was 83% of naïve mice at 16 weeks and correlated with percentage of fully reinnervated NMJs. Morphological differences were noted between injured and non-injured NMJs.

Discussion:

We present a reproducible method for evaluating NMJ reinnervation. Electrodiagnostic data summarize NMJ recovery. Characterization of wildtype reinnervation provides important data for consideration in experimental design and interpretation.

Introduction

Peripheral nerve injuries are debilitating conditions that affect more than twenty million Americans.¹ Despite our current understanding of the mechanisms of nerve regeneration and advances made in nerve reconstruction, a majority of patients with nerve injury do not regain satisfactory function after surgical intervention.¹ The extent to which motor function can be regained after nerve repair depends on several factors, including injury type and location, denervation duration, and patient age.^2,3 If reinnervation does not occur within 12–18 months, neuromuscular junctions (NMJs) degenerate, precluding reinnervation.^1,4 Considering the suboptimal outcomes for many patients with nerve injuries, more rigorous investigation is needed to elucidate the mechanisms promoting motor recovery after injury, including evaluation at the end-target muscle.

Functional motor recovery after peripheral nerve injury depends on two key factors: (1) nerve regeneration at the injury site and (2) NMJ reinnervation within target muscle. The NMJ represents the interface of nerve and muscle and is composed of three main structures: the nerve terminal, which contains acetylcholine vesicles to be released across the synaptic cleft, the motor endplate covered in acetylcholine receptors (AChRs), and 3 to 5 non-myelinating terminal Schwann cells (tSCs), or peri-synaptic Schwann cells, that encase the nerve terminal and synapse.^5,6 The NMJ is not a static structure as it undergoes continuous remodeling throughout the lifetime of the animal and after injury.^6,7 Whereas nerve regeneration at the injury site has been the primary subject of many investigations, the process of NMJ reinnervation after nerve injury is still incompletely characterized.^8,9

Transgenic mice are used for various experimental applications. In experimental conditions, however, appropriate controls may not be available for comparison. For example, transgenic models may differ in background strain. As a result, wildtype (WT) mice may be needed for experimental controls. Morphological differences may exist in structures in transgenic mice compared to the same structures visualized with immunostaining in WT mice. Transgenic mice may have more intense fluorescence of structures of interest compared to immunostaining of the same structures in WT mice. Because of less intense fluorescence after immunostaining in WT mice, however, fine details of structures may be better seen. Each model may offer advantages and disadvantages, depending on the planned analysis. As such, the two should not be compared as they differ at baseline. In this study, we characterize NMJ reinnervation and motor recovery in WT mice after injury.

The primary goals of this study were to comprehensively evaluate NMJ reinnervation patterns and NMJ morphology at different time points following sciatic nerve injury in a murine model.

Methods

Mice

All surgeries were performed in 3-month-old adult WT C57BL/6J mice. Sexes were mixed across all experimental groups (t=0, 1, 2, 3, 4, 8, 12, 16, and 30 weeks after nerve injury). NMJ morphology was also compared with 3-month-old transgenic mice: S100-GFP and Thy1-YFP (Supplemental Fig. 1). Pre- and postoperative animals were housed in a central animal housing facility and were maintained in strict accordance with the National Institutes of Health guidelines and protocols approved by the Institutional Animal Care and Use Committee at Washington University School of Medicine.

Sciatic nerve injury and repair

The sciatic nerve of the right hindlimb was exposed via a muscle-splitting technique. The sciatic nerve was sharply transected 3 mm proximal to the trifurcation. Immediately following nerve transection, the nerve was repaired using microsuture and fibrin glue (Tisseel, Baxter, Deerfield, IL, USA). After coaptation, the muscle and skin were closed appropriately. Animals were recovered on a heating pad, anesthesia was reversed with atipamezole hydrochloride (1 mg/kg), and animals were returned to the central animal housing facility within 12 hours for close monitoring and postoperative care.

Muscle harvest

At the assigned time point (t=0, 1, 2, 3, 4, 8, 12, 16, and 30 weeks after nerve injury), animals were anesthetized, and an incision was made over the anterior right leg extending from the dorsal foot to the knee. After identification, the distal tendon of the tibialis anterior (TA) muscle was transected to expose the extensor hallucis longus (EHL) tendon, immediately posterior to the TA tendon. The TA was then retracted and cut at the insertion at the knee to provide better exposure of the EHL and extensor digitorum longus (EDL) tendons and muscle bellies. The EHL was then carefully dissected proximally to avoid tearing where the branch of the peroneal nerve inserts. The EDL tendons were transected distally below the ankle and proximally at the insertion at the knee. After dissection, the EHL and EDL muscles were immediately placed in cold 0.1 M phosphate buffered saline (PBS, pH 7.4) solution to remove any debris or hair. Animals underwent cervical dislocation under deep anesthesia.

Whole mount immunofluorescent NMJ staining

After harvest, EHL and EDL muscles were placed in 2% paraformaldehyde solution (Electron Microscopy Science, Hatfield, PA, USA) in PBS for 15 minutes for EHL and 30 minutes for EDL muscles at room temperature. Following fixation of whole muscles, the four component tendons of the EDL were dissected out in cold PBS solution. Immunofluorescent staining was performed as previously described.¹⁰ All muscles were washed in PBS and incubated in blocking buffer (5% normal goat serum, 2% Triton X-100, 5% bovine serum albumin in PBS) for 1 hour at room temperature. Tissues were then stained with primary antibodies, rabbit anti-neurofilament 200 (NF200,1:500; Millipore Sigma, St. Louis, MO, USA) or rabbit anti-S100b (1:1000, DAKO North America, Via Real, Carpinteria, CA), overnight at 4°C. After rinsing in washing buffer (PBS with 0.2% Triton X-100) for 45 minutes, muscles were incubated with goat anti-rabbit IgG-Alexa Fluor 488 (1:1000; Invitrogen-Molecular Probes, Carlsbad, CA, USA) for 1 hour at room temperature. Muscles were further rinsed in washing buffer for 45 minutes prior to incubating with Alexa Fluor 555-α-BTX (1:1000; Molecular Probes, Eugene, OR, USA), to label AChRs, for 1 hour at room temperature. Muscles were mounted using Vectashield mounting medium with DAPI (Vector Laboratories, Inc. Burlingame, CA, USA).

NMJ Imaging

Whole mount muscles were imaged using an Axio Imager M2 fluorescent microscope (Zeiss, Thornwood, NY, USA). Sequential capture was used to separate the green and red channels in order to prevent crosstalk between dyes. Z-serial images were collected with 20x, 40x, and 63x objectives to allow analysis of three-dimensional structures. Images were viewed and analyzed using NIH ImageJ (http://rsb.info.nih.gov/iJ/). Figures were prepared using GraphPad Prism 8 (GraphPad Software Inc., La Jolla, CA, USA), Adobe Photoshop CC 2015, and Adobe Illustrator CC 2015 system (Adobe Systems, San Jose, CA, USA).

NMJ evaluation and reinnervation quantification

NMJ reinnervation in EDL and EHL muscles was evaluated at 0, 1, 2, 3, 4, 8, 12, and 16 weeks after sciatic nerve transection with immediate repair. Endplate reinnervation was visualized and quantified with a compound confocal microscope by multiple, independent, blinded reviewers. Whenever possible, muscles were imaged immediately after mounting to minimize potential for staining deterioration over time. Each slide was assessed for staining quality and adequate antibody penetration. Reinnervation quantification was exclusively based on the most superficial layer of the muscle as previously described.^10,11 NMJ reinnervation was then defined and graded based on colocalization of αBTX and NF200 staining. For each NMJ, motor endplates (αBTX staining) lying en face were visualized and overlap with NF200 staining was assessed. Reinnervation assessment required multiple sequential images for each NMJ. This method ensured that all motor endplates were evaluated. After each endplate was assessed, a reference image was chosen for record keeping, and a running endplate tally was recorded, classifying each endplate as: 1) denervated, 2) partially innervated, or 3) fully innervated (Supplemental Fig. 2). Denervation was defined as the absence of αBTX and NF200 colocalization, despite insurance of adequate staining (Supplemental Fig. 2A, A′). Partial reinnervation was defined as partial and/or interrupted colocalization of αBTX and NF200 stains (Supplemental Fig. 2B, B′). Full reinnervation was defined as the complete and continuous colocalization of αBTX and NF200 stains in a superficially located endplate (Supplemental Fig. 2C, C′).

Evaluation of polyinnervation and axonal sprouting

Polyaxonal innervation is characteristic of NMJ development, but may occur after reinnervation. Axonal sprouting is a characteristic feature of nerve regeneration after injury. Rates of polyaxonal reinnervation and axonal sprouting were assessed in naïve EDL and EHL muscles and in EDL and EHL muscles 2, 3, 4, 8, 12 and 16 weeks after sciatic nerve transection with immediate repair. We defined polyaxonal reinnervation as the presence of more than one axon reaching the endplate and axonal sprouting as an axon branch extending away from an endplate.

Evaluation of motor endplate fragmentation

Motor endplate morphology was assessed in EDL and EHL muscles at 2, 3, 8, 12, 16, and 30 weeks after sciatic nerve transection with immediate repair, and in contralateral uninjured muscles. Endplates stained with αBTX were imaged by blinded reviewers. Each endplate lying en face was analyzed at high magnification with image editing software (Windows Photo Viewer, Redmond, WA, USA) and assessed for the presence of gray-black background interposed between islands of αBTX staining within the same endplate. The reviewers then outlined these fragments with a bright color to facilitate counting. The numbers of fragments per endplate in each group were then tallied.

Electrodiagnostic testing

Functional capacity of naïve TA muscles and functional recovery of TA muscles at 2, 3, 4, 6, and 16 weeks after sciatic nerve transection and immediate repair were assessed using compound motor action potential (CMAP) amplitudes. Measurement of TA muscle activation (mV), evoked by electrical stimulation of the sciatic nerve at 50 Hz, was utilized to assess the functional capacity. The experimental technique and data acquisition were performed as previously described.^12,13

Statistical analysis

Data are reported as mean ± SEM. Quantifications were performed in a blinded fashion from at least three experimental groups, unless otherwise noted. Statistical analyses were performed in either Microsoft Excel (Microsoft, Redmond, WA, USA) or GraphPad Prism 8 (GraphPad Software, Inc., La Jolla, CA, USA). Two-tailed, unpaired Student’s t-tests (parametric data) or Mann-Whitney U tests (nonparametric data) were utilized to assess statistical differences between data sets. Correlation was calculated using the Pearson Correlation coefficient. Statistical significance was set at p < 0.05.

Results

A total of 31 mice were used for analysis, including 19 females and 12 males. A total of 1513 NMJs were evaluated for reinnervation, 1350 NMJs for motor endplate fragmentation, and 978 NMJs for polyaxonal innervation and sprouting.

Reinnervation

Reinnervation results are displayed in Figure 1. One week after sciatic nerve transection, 98% of EHL and EDL muscles were denervated (Fig. 1A, H). By 2 weeks, the majority of endplates were at least partially reinnervated (Fig. 1B, H). In addition, early evidence of axonal branch pattern reorganization was present (Fig. 1B). By 8 weeks, all NMJs were partially (28.3%) or fully (71.7%) reinnervated, and the normal tree-like axonal branching pattern was reconstituted (Fig. 1E, H). Beyond the 8-week time point, the percentage of endplates classified as fully reinnervated continued to increase (Fig. 1F–H), and by 16 weeks, the vast majority (87.3%) of endplates were fully reinnervated (Fig. 1G, H).

Figure 1.

Neuromuscular junction (NMJ) reinnervation increases and axonal branch pattern morphology reconstitutes with time after motor nerve injury. (A-G) Confocal images of adult WT extensor digitorum longus (EDL) muscles at 1, 2, 3, 4, 8, 12, and 16 weeks after sciatic nerve transection and immediate repair. (E-G) Inset images are representative higher magnification images of reinnervated NMJs at 8, 12, and 16 weeks after injury. (H) Quantification of NMJ reinnervation is summarized (n=3 mice per time point, except n=2 mice at 16 weeks; average 266 NMJs evaluated per time point). Reinnervation was classified as full, partial, or denervated. Graph shows the proportions of NMJs with the three categories of innervation at the various experimental time points. NF200 Ab = anti-neurofilament antibody (for axons, green), BTX = α‐bungarotoxin (for acetylcholine receptors, red), and DAPI (nuclear staining, blue). Scale bar = 20 μm.

Although the percentage of reinnervated NMJs increased over time, we noticed changes in regenerating axon configuration, including polyaxonal reinnervation and nerve terminal sprouting from the endplate (Fig. 2). The percentage of NMJs with axonal sprouting increased over time, with no sprouting observed 2 weeks after injury compared to a maximum of 39% sprouting observed 16 weeks after injury (Fig. 2D). Polyaxonal reinnervation was less prevalent than axonal sprouting—a maximum of ~10% of endplates exhibited polyaxonal reinnervation across all time points (Fig. 2D). Polyaxonal innervation and/or axonal sprouting were rarely noted in the absence of nerve injury.

Figure 2.

Polyaxonal innervation and axonal sprouting increase in frequency with time after motor nerve injury in adult WT extensor digitorum longus (EDL) and extensor hallucis longus (EHL) muscles. Representative confocal images of (A) normal monoaxonal innervation (asterisk), (B) polyaxonal innervation (arrowheads), and (C) axonal sprouting (arrow) in EDL muscles. (D) Quantification of axonal patterns present in naïve WT muscles (n=3 mice) and in WT muscles 2, 3, 4, 8, 12, and 16 weeks after nerve injury (n=3 mice per time point, except n=2 mice at 16 weeks; average 140 neuromuscular junctions (NMJs) evaluated per time point) are shown. Graph summarizes the proportions of NMJs with each of the three axonal patterns. NF200 Ab = anti neurofilament antibody (for axons, green), BTX = α‐bungarotoxin (for acetylcholine receptors, red), and DAPI (nuclear staining, blue). Scale bar = 20 μm.

Motor endplate fragmentation is evident, despite NMJ reinnervation

Although reinnervation rates increased steadily after injury, reinnervation did not represent a return to naïve axon-endplate interactions. Differences in endplate morphology and axonal configuration were apparent between injured and uninjured groups (Fig. 3). After nerve injury, motor endplates were increasingly fragmented over time (Fig. 3B), as opposed to the more typical “pretzel-like” conformation (Fig. 3A). At 2 and 3 weeks, both injured and uninjured conditions had an average of 3–4 fragments per endplate. At 8 weeks, we observed 6.8 ± 3.8 fragments per endplate in the injured condition, compared to 3.4 ± 2.2 in the uninjured group. By 12 weeks, the number of fragments per endplate in the injured group remained significantly different, more than twice those of the uninjured group. At 16 weeks, we observed 8.4 ± 4.7 fragments per endplate in the injured condition, significantly more than 3.1 ± 1.5 in the uninjured condition. At our final time point of 30 weeks, there were 12.1 ± 6.3 fragments per endplate in the injured group, compared to 3.8 ± 3.4 in the uninjured group (p < 0.0001). Notably, endplate fragmentation was not accompanied by a loss of innervation, nor the loss of the one-to-one axon to endplate relationship. Rather, we observed axons splitting into smaller branches thereby covering each fragment constituting an endplate.

Figure 3.

Motor endplate fragmentation increases significantly with time after motor nerve injury compared to controls. (A) Representative confocal image of a naïve WT extensor digitorum longus (EDL) motor endplate showing the “normal” pretzel-like configuration, with 7 α‐bungarotoxin (BTX) fragments. (B) Representative image of a “fragmented” WT EDL motor endplate 30 weeks after sciatic nerve transection and immediate repair, with 17 BTX fragments. (C) Graph shows the average number of fragments per endplate in WT EDL muscles at 2, 3, 8, 12, 16, and 30 weeks after sciatic nerve transection with immediate repair (injured side), compared to contralateral control muscles (uninjured side) (n=2 mice per time point, except n=1 mouse at 30 weeks; average 225 endplates evaluated per time point). At 2 and 3 weeks after injury, endplate fragmentation does not differ between the injured and uninjured groups. At 8 weeks and beyond, injured groups display significantly more endplate fragmentation than uninjured controls. Note that average number of endplate fragments in controls remains stable over time (3–4 fragments). BTX = α‐bungarotoxin (for acetylcholine receptors, grey). Scale bar = 20 μm. Data ± SEM; **** p < 0.0001.

Functional recovery progresses over time and correlates with full NMJ reinnervation

At 2 weeks after injury, TA CMAP amplitude was significantly reduced to 21% of naïve function (Fig. 4). TA CMAP amplitude demonstrated progressive functional recovery over 16 weeks post-operatively. By 4 weeks, CMAP amplitude measured 40% of naïve function and by 6 weeks, 54% of naïve TA. At 16 weeks after injury, TA CMAP amplitude reached 83% of naïve function; however, TA function was still significantly reduced compared to naïve (p < 0.01).

Figure 4.

Tibialis anterior (TA) muscle function steadily recovers with time after motor nerve injury, but remains significantly less at 16 weeks compared to naïve mice. Evoked compound motor action potential (CMAP) amplitudes were recorded from naïve WT TA muscles (n=3 mice) and from WT TA muscles 2, 3, 4, 6, and 16 weeks after sciatic nerve transection with immediate repair (n=3 mice per time point). CMAP amplitude in naïve wildtype TA measured 8.55 ± 0.76 mV. At 2 weeks after injury, TA CMAP amplitude was 1.77 ± 2.08 mV (21% of naïve function). By 3 weeks, CMAP amplitude measured 1.98 ± 0.11 mV (23% of naïve); by 4 weeks, 3.44 ± 0.63 mV (40% of naïve); and by 6 weeks, 4.63 ± 1.17 mV (54% of naïve). At 16 weeks after injury, TA CMAP amplitude reached 83% of naïve function (7.13 ± 1.75 mV); however, TA function was still significantly reduced compared to naïve. Data ± SEM; **** p < 0.0001, ** p<0.01.

We observed a strong correlation between the percentage of naïve CMAP amplitude and percentage of fully reinnervated NMJs after nerve injury and repair, with Pearson correlation coefficient = 0.835 (Fig. 5). At 2 weeks after nerve injury and repair, 12.9% of NMJs were fully reinnervated, and average CMAP amplitude was 21% that of naïve muscle. At 3 weeks, 28.5% of NMJs were fully reinnervated, and CMAP amplitude was 23% of naïve muscle. By 4 weeks, full reinnervation was seen in 40.2% of NMJs, and average CMAP amplitude was 40% of naïve. By 16 weeks, 87.3% of NMJs were fully reinnervated, and average CMAP amplitude was 83% of naïve.

Figure 5.

There is a strong relationship between the percentage of fully reinnervated endplates and compound motor action potential (CMAP) amplitude after nerve injury. Line graph displays the percentage of fully reinnervated endplates in extensor digitorum longus (EDL) muscles (presented in Fig. 1) and the percentage CMAP amplitude recovery relative to naïve tibialis anterior (TA) muscle CMAP amplitude (derived from Fig. 4) at 2, 3, 4, and 16 weeks after sciatic nerve transection and immediate repair. The data at each time point are highly correlated, with Pearson coefficient = 0.835, p < 0.001. Data ± SEM.

Discussion

Investigation of nerve regeneration and recovery after injury is important to better understand reinnervation and to identify innovative therapeutic targets for clinical translation. Most scientific investigations employ comparison of experimental conditions to controls; the mouse provides an excellent model for nerve research, and WT mice are commonly used as a control group. In this study, NMJ reinnervation was partially or fully complete in all studied NMJs by 8 weeks after nerve repair, and full reinnervation was present in 87% of NMJs by 16 weeks after repair. Polyaxonal NMJ reinnervation was present throughout the study, axonal sprouting was noted at higher rates at later time points, and endplate fragmentation increased with time. Interestingly, we found that functional recovery, as measured by CMAP amplitude, correlated closely with the percentage of fully, but not partially, reinnervated NMJs throughout all assessed time points. Functional recovery did not, however, achieve that of naïve muscle by 16 weeks after nerve injury and repair. This study provides an important reference for both morphological and functional comparison for interpretation of experimental results.

NMJ reinnervation rates

The reinnervation rates noted in this study are consistent with those from previous studies. We found no NMJs remained denervated beyond 8 weeks following nerve repair. Similarly, no remaining denervated NMJs were reported in wildtype (C57BL/6J) mice 8 weeks following facial nerve cut and repair.¹⁴ In the neck, sternomastoid reinnervation after spinal accessory nerve cut and repair was reported to occur between postoperative days 14 to 30¹⁵ and after one month for nerve cut and no repair.¹⁶ Sternomastoid reinnervation is slightly faster given the proximity of nerve injury to the muscle. Reinnervation of the biceps brachii was noted 3 weeks after nerve cut and repair in C57BL/6J mice.¹⁷

Advantages of systematic assessment of NMJ reinnervation

Closely assessing NMJ reinnervation histologically is a surrogate of functional recovery after injury. Reinnervation is commonly reported as an all-or-none phenomenon. In this study, reinnervation is specifically characterized as the degree of co-localization of the nerve terminal with motor endplates (AChRs). Our assessment method of NMJ reinnervation has several advantages. First, it allows for visualization of two major NMJ components: the nerve terminal and motor endplates. Second, it is reproducible with simple, defined parameters. Third, our protocol makes use of the EHL and EDL muscles as opposed to thicker muscles, such as TA. Both the EHL and EDL muscles are located in the anterior compartment of the lower leg, are fast-twitch muscles¹⁸, and are innervated by the deep peroneal branch of the sciatic nerve. Additionally, both muscles are ideally suited for imaging using whole mount confocal microscopy due to their thin muscle profile and EDL’s ability to be split into its four tendinous components. Thus, the use of EHL and EDL muscles facilitates better endplate visualization such that reinnervation can be comprehensively and systematically assessed.

Polyinnervation, axonal sprouting, and endplate fragmentation occur with reinnervation

Despite a high percentage of NMJ reinnervation via immunohistochemistry by week 16 and similar functional recovery by CMAP in our study, morphological NMJ differences persisted after injury. Polyinnervation and axonal sprouting progressed with time from injury and repair. These phenomena are frequently noted during NMJ development^19–21 and early in reinnervation^16,22–24, rather than at later time points. Similar changes, however, including polyinnervation and axonal sprouting, have been reported in aged rats beyond 8 weeks after nerve crush.²⁵ The presence of increasing motor endplate fragmentation was unexpected, particularly in the setting of progressive NMJ reinnervation. Previous studies from our lab and others have shown endplate fragmentation occurs with aging.^10,26–28 In the present study, age did not contribute to endplate fragment number in uninjured mice; the average number of fragments in uninjured mice was 3–4 at all time points, and all mice were under 10 months of age. Motor endplates, however, constantly turnover in homeostatic conditions.¹⁵ After NMJ denervation, endplate turnover rates accelerate due to synthesis of more rapidly degrading AChRs in denervated muscle. In the sternomastoid muscle, motor endplate half-lives decreased from 8–12 days to 3 days when nerves were prevented from regenerating, but half-lives return to baseline levels after reinnervation.^15,29,30 After denervation, then, two AChR populations exist—the original population that degrades more slowly and the more rapidly degenerating population. Once several half-lives pass following reinnervation, the more rapidly degrading AChRs become sequentially fewer in number, and homeostatic turnover rates are restored.²⁹ The fragmented endplates noted in our study may reflect this shift in endplate population to the more rapidly degrading AChRs. The reason for persistence of this morphological difference as well as the functional implications, however, are unknown. Importantly, fragmented endplates have been shown to have no decline in synaptic transmission in aged mice.²⁸ Any of these morphological changes could easily be interpreted as abnormal in different experimental conditions or as a result of treatment effects. Here, we see these morphological differences in WT mice after nerve cut and repair.

CMAP amplitude provides a functional correlate of NMJ recovery

Functional TA muscle recovery increased throughout the study period, but surprisingly, even at 16 weeks after nerve cut and repair, CMAP amplitude had not achieved levels of naïve mice. This timeline is important when planning and interpreting experiments. English et al³¹ described similar results of low evoked gastrocnemius muscle maximal response amplitudes, at 10–30% of baseline, in C57BL/6J mice at 8 weeks after sciatic nerve cut and repair.³¹ After tibial nerve cut and repair in the NMRI mouse, in vitro evoked tetanic force in the soleus was noted to be low (54% of the uninjured side) at 4 weeks post-injury, but with nearly complete recovery (98% of the uninjured side) by 2 months post-injury.³² These differing results suggest variability in motor recovery, which may be related to muscle fiber type. The TA is 59% Type IIB fibers and the gastrocnemius is 54% Type IIB fibers in the C57BL/6J mouse, compared to the soleus composition of 37% Type I fibers and 38% Type IIA fibers.³³ Interestingly, we found that functional recovery of the TA correlated with the percentage of fully reinnervated NMJs noted histologically. Although nearly all endplates were occupied by nerve by 3 weeks after nerve injury and repair, functional recovery was poor at that time point (23% of naïve) and only correlated with full NMJ reinnervation rather than partial reinnervation. This observation highlights the importance of fully restored synaptic transmission for overall muscle function. These data also suggest that CMAP amplitude provides a sensitive assessment of complete NMJ recovery. Measurement of functional recovery in rodent nerve injury models can be challenging and fraught with limitations. Challenges associated with commonly used functional metrics, such as walking track analysis, include: (1) low sensitivity, (2) difficulty discriminating between the effects of the specific nerve injury compared to the general effects of surgery, (3) the training time required for animals to learn motor tasks and (4) the potential need for expensive and special equipment.^24,34 This study supports the use of electrodiagnostic testing as a functional correlate of NMJ recovery.

While the use of specific transgenic mice facilitates visualization and quantification of reinnervation, those mice are often ill-suited to serve as controls for studies in which experimental mice are not of the same transgenic line or background strain. For this reason, we believe a comprehensive description of WT NMJ reinnervation offers an important reference to the literature.

Conclusions

These data highlight the importance of complete NMJ reinnervation for functional recovery and provide timelines for expected reinnervation in a murine model. Interestingly, functional recovery did not reach levels of uninjured mice even 16 weeks post-injury, but CMAP amplitude correlated closely with the percentage of fully reinnervated NMJs. We propose a reproducible method of reinnervation assessment and provide a reference for reinnervation in WT mice.

Abbreviations

AChRs

acetylcholine receptors

CMAP

compound motor action potential

EDL

extensor digitorum longus

EHL

extensor hallucis longus

NMJ

neuromuscular junction

TA

tibialis anterior

tSCs

terminal Schwann cells

WT

wildtype