Secondary denervation is a chronic pathophysiologic sequela of volumetric muscle loss.

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.