Secondary denervation is a chronic pathophysiologic sequela of volumetric muscle loss

Abstract

Volumetric muscle loss (VML) is the traumatic loss of muscle tissue that results in long-term functional impairments. Despite the loss of myofibers, there remains an unexplained significant decline in muscle function. VML injury likely extends beyond the defect area, causing negative secondary outcomes to the neuromuscular system, including the neuromuscular junctions (NMJs), yet the extent to which VML induces denervation is unclear. This study systematically examined NMJs surrounding the VML injury, hypothesizing that the sequela of VML includes denervation. The VML injury removed ∼20% of the tibialis anterior (TA) muscle in adult male inbred Lewis rats (n = 43), the noninjured leg served as an intra-animal control. Muscles were harvested up to 48 days post-VML. Synaptic terminals were identified immunohistochemically, and quantitative confocal microscopy evaluated 2,613 individual NMJ. Significant denervation was apparent by 21 and 48 days post-VML. Initially, denervation increased ∼10% within 3 days of injury; with time, denervation further increased to ∼22% and 32% by 21 and 48 days post-VML, respectively, suggesting significant secondary denervation. The appearance of terminal axon sprouting and polyinnervation were observed as early as 7 days post-VML, increasing in number and complexity throughout 48 days. There was no evidence of VML-induced NMJ size alteration, which may be beneficial for interventions aimed at restoring muscle function. This work recognizes VML-induced secondary denervation and poor remodeling of the NMJ as part of the sequela of VML injury; moreover, secondary denervation is a possible contributing factor to the chronic functional impairments and potentially an overlooked treatment target.NEW & NOTEWORTHY This work advances our understanding of the pathophysiologic complexity of volumetric muscle loss injury. Specifically, we identified secondary denervation in the muscle remaining after volumetric muscle loss injuries as a novel aspect of the injury sequela. Denervation increased chronically, in parallel with the appearance of irregular morphological characteristics and destabilization of the neuromuscular junction, which is expected to further confound chronic functional impairments.

Keywords: innervation; neuromuscular junction; polyinnervation; skeletal muscle; sprouting.

Figure 1.

Individual presynaptic (red) and postsynaptic (green) NMJ structures were stained, imaged, binarized, and used to quantitatively measure two-dimensional (2-D) planar area, three-dimensional (3-D) volume, colocalization, and complexity. Images were also used for visual analysis of NMJ denervation and the appearance of irregular morphological characteristics. Scale bar 10 µm and 3-D color depth scale range 0–30 µm. NMJ, neuromuscular junctions.

Figure 2.

A: representative images showing both individual and merged presynaptic (red) and postsynaptic (green) terminals along with the overlap area (colocalization; white) for an innervated and a partially denervated NMJ. B: the percentage of colocalization was determined by comparing the colocalization area to the postsynaptic area. Colocalization data were analyzed as a mixed model where individual animals were used as a random effect (P < 0.001). Data are presented as overall group means ± SD; additionally each dot represents an individual NMJ and each color represents an individual muscle. C: evidence of denervation was significantly greater in VML injured muscle, particularly at later time points. The numbers within the bars represent the total number of NMJs analyzed. Data are analyzed by one-way-ANOVA and presented as individual animal means ± SD. Significant difference (P < 0.05) compared to *controls; §day 3; ◊day 7; ‡day 14. Representative images of innervated (D) and denervated (E) NMJs. For visualization, confocal z-stacks were rendered into 3-D stacks using Volume View in Nikon NIS-Elements and displayed as binary reconstructions. Denervation was defined as the partial or complete absence of the presynaptic terminal based on maximum intensity projections of en face NMJs. NMJ, neuromuscular junctions; VML, volumetric muscle loss.

Figure 3.

A: representative maximum projection of confocal images of control and VML-injured NMJs, labeled with synaptic vesicle/neurofilament (red; presynaptic terminal and axon) and α-bungarotoxin (green; postsynaptic motor end-plate). The merged image displays overlap of pre- and postsynaptic structures. B: two-dimensional (2-D) planar area of the presynaptic and postsynaptic terminals were similar in control and VML-injured NMJs. Data were analyzed using a mixed linear model where the random effect of animal was significant (presynaptic P = 0.004; postsynaptic P = 0.007). Data are presented as overall group means ± SD; additionally each dot represents an individual NMJ and each color represents an individual muscle. There was a significant shift in NMJ size distributions for both pre- (C) and post-synaptic (D) area as determined by chi-square analysis (P < 0.05) compared with control. Red bars represent controls, blue bars indicate significantly different from controls, and gray bars indicate no difference from control; individual P values are noted at each group. NMJ, neuromuscular junctions; VML, volumetric muscle loss.

Figure 4.

A: the volume of the presynaptic terminal was similar in control and VML-injured NMJs (P = 0.667). The postsynaptic 3-D volume was significantly larger at 21 days post-VML compared with control (noted by *P = 0.005). Data were analyzed using a mixed linear model where the random effect of animal was significant (presynaptic P < 0.001; postsynaptic P < 0.001). Data are presented as overall group means ± SD; additionally each dot represents an individual NMJ and each color represents an individual muscle. B: the presynaptic volume distribution was similar for all groups, and there was a shift toward larger (C) postsynaptic volume distributions at day 21 as determined by chi-square analysis compared with control (P < 0.05). Red bars represent controls, blue bars indicate significantly different from controls, and gray bars indicate no difference from control; individual P values are noted at each group. NMJ, neuromuscular junctions; VML, volumetric muscle loss.

Figure 5.

A: representative images of a single innervated NMJ compared with NMJs with irregular morphology, including sprouting (the motor axon terminal forms extensions that do not correspond with the postsynapse), polyinnervation (a single postsynaptic site is innervated by two or more motor axons), or a combination of sprouting and polyinnervation. B: irregular presynaptic morphological alterations appeared more frequently in VML-injured NMJs. The numbers within the bars represent the number of innervated NMJs analyzed. Data are analyzed by one-way-ANOVA and presented as individual muscle means ± SD. Significant (P < 0.05) differences compared with *controls; §day 3; ‡day 14. NMJ, neuromuscular junctions; VML, volumetric muscle loss.

Figure 6.

A: complexity of NMJs was measured by comparing the two-dimensional area of the postsynaptic terminal to the total orthogonal area of the image. Data were analyzed as a mixed linear model where individual animals were used as a random effect (P < 0.001). Complexity was similar between groups (P = 0.103). Data are presented as means ± SD; additionally each dot represents an individual NMJ and each color represents an individual muscle. B: fragmentation was qualitatively analyzed and significantly greater by 48 days compared with controls (noted by *P = 0.013). Data are analyzed by one-way-ANOVA and presented as individual animal means ± SD; total NMJ n = 2,606. C: representative images of fragmented NMJs displaying the postsynaptic terminal separating into a series of islands. NMJ, neuromuscular junction; VML, volumetric muscle loss.