Neuromuscular junctions (NMJs) mediate skeletal muscle contractions and play an important role in several neuromuscular disorders when their morphology and function are compromised. However, due to their small size and sparse distribution throughout the comparatively large, inherently opaque muscle tissue the analysis of NMJ morphology has been limited to teased fiber preparations, longitudinal muscle sections, and flat muscles. Consequently, whole mount analyses of NMJ morphology, numbers, their distribution, and assignment to a given muscle fiber have also been impossible to determine in muscle types that are frequently used in experimental paradigms. This impossibility is exacerbated by the lack of optical tissue clearing techniques that are compatible with clear and persistent NMJ stains. Here, we present MYOCLEAR, a novel and highly reproducible muscle tissue clearing protocol. Based on hydrogel-based tissue clearing methods, this protocol permits the labeling and detection of all NMJs in adult hindleg extensor digitorum longus muscles from wildtype and diseased mice. The method is also applicable to adult mouse diaphragm muscles and can be used for different staining agents, including toxins, lectins, antibodies, and nuclear dyes. It will be useful in understanding the distribution, morphological features, and muscle tissue context of NMJs in hindleg muscle whole mounts for biomedical and basic research.

Amongst other approaches, adipose-derived stromal cells (ASCs) have recently been tested with respect to their regenerative capacity for treatment of neuromuscular disorders. While beneficial effects of ASCs on muscle recovery were observed previously, their impact on regeneration of neuromuscular junctions (NMJs) is unclear. Here, we used a murine glycerol damage model to study disruption and regeneration of NMJs and to evaluate the effects of systemic application of ASCs on muscle and NMJ recovery. In mice that were not treated with ASCs, a differential response of NMJ preand post-synapses to glycerol-induced damage was observed. While post-synapses were still present in regions that were necrotic and lacking actin and dystrophin, presynapses disappeared soon in those affected areas. Partial regeneration of NMJs occurred within 11 days after damage. ASC treatment slightly enhanced NMJ recovery and reduced the loss of presynaptic sites, but also led to a late phase of muscle necrosis and fibrosis. In summary, the results suggest a differential sensitivity of NMJ pre-and post-synapses to glycerol-induced muscle damage and that the use of ASC for the treatment of neuromuscular disorders needs further careful evaluation.

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