Fig. 2

Morphological NMJ regeneration of the LAL muscle following distal nerve crush injury. a Experimental design for the regenerative model. The posterior auricular branch of the facial nerve was crushed for 30 seconds. b LAL muscles from control adult mice and mice 5, 15, 30, 60, and 90 days after nerve damage were dissected and processed for immunofluorescence staining with 2H3 (neurofilaments) plus SV2 (synaptic vesicles) antibodies to reveal motor axons and presynaptic terminals (magenta), with an anti S100B antibody to stain Schwann cells (yellow), and with BTX (cyan) to stain AChRs located at postsynaptic densities. Insets in 5d and 15d show only pre- and postsynaptic staining of NMJs. Bar = 100 μm. c–f Nerve terminal perimeter, nerve terminal area, AChR aggregates area, and overlap between pre and postsynaptic apparatuses were measured at different times after nerve crush injury and in control muscles. g The behavior of tSCs was analyzed by following tSCs covering the NMJ (arrows) (left panel), tSCs covering the NMJ and extending cell projections (middle panel), and tSC at the periphery (i.e., within a 50 μm perimeter from the postsynaptic region) of each NMJ (arrowheads) (right panel). Bar = 50 μm. h–j Quantification of tSC behavior after nerve crush injury. The results are represented as the mean ± SEM and each individual value (N: 3–4 mice; 2 female plus 1–2 male mice). *p < 0.05, **p < 0.01, ****p< 0.0001, one-way ANOVA test (c, d, e, f). n.s. = non-significant (h, i, j)