Fig. 1. Construction and evaluation of a unilateral stroke model. (a) Workflow for inducing ischemic stroke; (b) white-light images of the skull before and after skull optical clearing; (c) fluorescence imaging of the brain in the coronal sections from Sham, PT-30 s, and PT-5 min group; the red area on the left shows the motor cortex. (d) Quantification of the infarct volume of the brain (n = 3 at each time point). (e) Schematic diagrams of the rotarod test and the grid-walking test; (f), (g) scores for the rotarod test and grid walking at each time point after stroke in the sham group, PT-30 s group, and PT-5 min group (n = 4 for each behavior test).

Fig. 2. 3D visualization and quantification of NMJs in different muscles after unilateral stroke. (a) Experimental pipeline for 3D visualization of NMJs and innervating nerves in skeletal muscles. (b), (c) 3D reconstructions of NMJs and innervating nerves in FDP and EDL; the images in the first row are enlarged and present in the second row. (d), (e) Quantification of NMJ numbers at each time point after stroke in different groups (n = 3 for each muscle at each time point); all values are presented as the mean ± SEM; statistical significance in (d) and (e) (n.s. represents not significant).

Fig. 3. Quantification of fine structural features of NMJs after unilateral stroke. (a) Comparison of the fine structure of NMJ in muscle after stroke, where the arrows point to the fragments; (b), (c) quantification of the number of fragments of NMJs at each time point after stroke in different groups in FDP and EDL muscles; (d), (e) quantification of NMJ area after stroke in different groups in FDP and EDL muscles; (f), (g) proportion of NMJs with different levels of fragmentation in FDP and EDL muscles after stroke; in FDP and EDL muscles, 562 and 635 NMJs were analyzed, respectively. “≤2” indicates the number of NMJ fragments is less than or equal to 2, representing “normal state;” “>2” indicates the number of NMJ fragments is greater than 2, representing “fragmentation.” All values are presented as the mean ± SEM; n = 3 for each muscle at each time point; statistical significance in (b)–(e) (n.s. represents not significant, *P < 0.1 and **P < 0.01).

Fig. 4. 3D visualization and quantification of NMJs in different muscles after bilateral stroke. (a) Schematic diagram of the bilateral stroke model; (b), (c) scores for rotarod and grid walking at each time point (n = 4 at each time point); (d) 3D distributions of NMJs and innervating nerves in FDP muscles at different times; (e), (f) quantification of NMJ numbers in FDP and EDL muscles at each time point (n = 3 for each muscle at each time point); (g) fine structure of NMJs after bilateral stroke, where the arrows point to the fragments; (h), (i) quantification of the number of NMJ fragments in FDP and EDL muscles; (j), (k) quantification of the NMJ area in FDP and EDL muscles (n = 3 for each muscle at each time point); (l), (m) proportion of NMJs with different levels of fragmentation in FDP and EDL muscles after bilateral stroke; in the unilateral group, 190 and 218 NMJs in FDP and EDL muscles were analyzed; in the bilateral group, 175 and 158 NMJs in FDP and EDL muscles were analyzed. “≤2” indicates the number of NMJ fragments is less than or equal to 2, representing “normal state;” “>2” indicates the number of NMJ fragments is greater than 2, representing “fragmentation.” All values are presented as the mean ± SEM; statistical significance in (b), (c), (e), (f), and (h)–(k) (n.s. represents not significant).