Recent work suggests denervation of surviving muscle fibers is a contributing factor to the overall pathophysiology following volumetric muscle loss (VML) injury (Sorensen et al., 2021). Defined as the traumatic or surgical loss of skeletal muscle with resultant functional impairment (Grogan and Hsu, 2011), VML is a non-recoverable injury leading to lifelong disability in the affected region. Morphological changes at the neuromuscular junction (NMJ) after VML, such as an increase in motor endplate fragmentation, also appear to be associated with progressive secondary denervation (i.e., denervation of NMJs that were not directly disrupted or removed at the time of injury) (Sorensen et al., 2021). Yet it is unclear if these changes are indicative of degenerating or regenerating NMJs, which is an important distinction for the development of therapeutic modalities for this severe injury. Regeneration of the NMJ is well described after peripheral nerve injuries(Vannucci et al., 2019; Rios et al., 2021); however, there is little on the effects of a traumatic muscle injury impacting the NMJ. Therefore, ongoing work seeks to identify temporal changes in neural signaling and cellular responses that may provide clues as to how NMJ remodeling takes place after VML injury. Although numerous cells and pathways contribute to NMJ remodeling and maintenance, this study is focused on the response of terminal Schwann cells (tSCs) and associated signaling factors, namely neuregulin-1 (NRG1) and brain derived neurotrophic factor (BDNF).

While the NMJ is a synapse providing connection of an α-motor neuron and muscle fiber, it is often thought of as a triadic synapse due to the critical role of tSCs during development and throughout the lifespan. These cells have been shown to regulate NMJs both functionally and structurally. Functionally, tSCs are capable of sensing action potentials and giving feedback to modulate the amplitude of subsequent end-plate potentials(Robitaille, 1998; Todd et al., 2010; Rousse et al., 2010; Reddy et al., 2003). Structurally, they help support acetylcholine receptor (AChR) clustering formation, as well as presynaptic connection to the clusters, as evidenced in models of tSC ablation (Reddy et al., 2003; Barik et al., 2016). In addition, they appear to be vital for NMJ repair and regeneration after various recoverable insults to the muscle and/or nerve. After injury, tSCs from denervated NMJs sprout to form bridges with healthy NMJs, which is thought to guide re-innervation (Son and Thompson, 1995; Reynolds and Woolf, 1992; Kang et al., 2019). Moreover, axonal debris is cleared by tSC phagocytosis, a process also seen in development with competing nerve terminals (Smith et al., 2013; Cunningham et al., 2020).

Factors responsible for motor neuron survival and regeneration such as BDNF, glial cell-line derived neurotrophic factor (GDNF), and nerve growth factor are released from tSCs after injury to help with regeneration of retracted axons (Li et al., 2018). Meanwhile, the proliferation and survival of tSCs themselves is largely regulated by NRG1 binding to epidermal growth factor receptors (ErbB, 1-4) on the tSC membrane. Mutant mice lacking NRG1, ErbB2, or ErbB3 have demonstrated a loss of tSCs, in addition to poor neuromuscular development and dysregulated NMJs (Lin et al., 2000; Wolpowitz et al., 2000; Riethmacher et al., 1997). Importantly, many of these neurotrophic factors, such as NRG1 and BDNF, are secreted by myofibers in addition to motor nerves or glial cells.

Terminal Schwann cell abnormalities are a prominent finding in skeletal muscle from patients with (or animal models of) various neuromuscular diseases, which can even precede clinical manifestations of the disease (Santosa et al., 2018; Carrasco et al., 2016a; Arbour et al., 2015). For example, mouse models of spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS) show a progressive reduction in the number of tSCs per NMJ (Carrasco et al., 2016a; Murray et al., 2013; Lee et al., 2011; Carrasco et al., 2016b). Additionally, studies that do not report the number of tSCs per NMJ often demonstrate similar outcomes by reporting a loss of tSC cytoplasmic coverage over the NMJ (Personius and Sawyer, 2005). In ALS patients, the tSCs that do remain may have an impaired ability to detect and respond to synaptic activity (Arbour et al., 2015; Perez-Gonzalez et al., 2022), likely exacerbating the already impaired muscle function. Furthermore, synapse elimination and debris clearance are vitally important for proper development and maintenance of adult NMJs; however, in certain conditions such as ALS and others, it can become dysregulated and lead to NMJ abnormalities (Lee et al., 2016; Bruneteau et al., 2015; Maselli et al., 2009). Invasion of tSC processes into the synaptic cleft has been reported in multiple neuromuscular diseases, and in some cases active phagocytosis of the nerve terminal is present (Lee et al., 2016). While it is difficult to assess whether these features ultimately weaken synaptic function, tSC invasion effectively reduces the area of possible nerve to muscle contact. Moreover, rodent models of muscular dystrophy (i.e., mdx mouse) display extensive tSC sprouting that appears disorganized and lacks bridge formation with innervated NMJs (Personius and Sawyer, 2005; Marques et al., 2006).

Given VML injury displays progressive denervation resembling neuromuscular diseases, at least through 7 weeks post-injury, we hypothesize there may be tSC loss or abnormalities as well. The loss of tSCs could uncover a mechanism of failed muscle recovery after VML injury and provide therapeutic targets. Conversely, no abnormalities seen may indicate that either NMJ re-innervation is still ongoing and the lack of muscle recovery is not compounded by neural impairments, or a separate mechanism (such as fibrosis(Hoffman et al., 2022; Corona et al., 2018a; Zhang et al., 2021)) is hindering NMJ re-innervation. Thus, a temporal study design was completed, with terminal timepoints of 3, 7, 14, 21, and 48 days post-VML injury to assess the tSC response in relation to NMJ changes. Upon finding the greatest alterations in tSCs at 48 days post-VML, further evaluation of tSC morphology was completed at this timepoint specifically.