What is Normal? Neuromuscular junction reinnervation after nerve injury

Abstract

Introduction: In this study we present a reproducible technique to assess motor recovery after nerve injury via neuromuscular junction (NMJ) immunostaining and electrodiagnostic testing.

Methods: Wild-type mice underwent sciatic nerve transection with repair. Hindlimb muscles were collected for microscopy up to 30 weeks after injury. Immunostaining was used to assess axons (NF200), Schwann cells (S100), and motor endplates (α-bungarotoxin). Compound motor action potential (CMAP) amplitude was used to assess 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 naive mice at 16 weeks and correlated with percentage of fully reinnervated NMJs. Morphological differences were noted between injured and noninjured NMJs.

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

Keywords: motor endplate; motor recovery; nerve injury; neuromuscular junction; reinnervation.

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.

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.

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.

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.

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.