Fig. 2
IT delivery of rAAV1-HGF facilitated the regeneration of the sciatic nerves and TA muscles after sciatic nerve crush. a-h Using 2-month-old C57BL/6 mice, sciatic nerve crush was induced, and 5 × 1011 GC of rAAV1-C or rAAV1-HGF were intrathecally injected into the LSC. Behavioral tests were performed once a week for 28 days (a-b). For IHC assay, the sciatic nerves and TA muscles were collected 5 days after nerve crush (c-f). a Hindlimb strength was measured 1, 7, 10, 14, 21, and 28 days after nerve crush. Value at day 0 was measured before nerve crush. b Mean latency to fall from the rotarod was measured 1, 7, 10, 14, 21, and 27 days after nerve crush. Value at day 0 was measured before nerve crush. In Fig. 2a-b, two-way ANOVA was performed, followed by Tukey’s post-hoc test. c Representative images of the sciatic nerves from mice treated with rAAV1-C or rAAV1-HGF after nerve crush. Antibodies specific to TUJ1 and SCG10 were used as markers for neurons and regenerating axons, respectively. Crush sites are indicated by dotted lines, and the tip of SCG10 signals is indicated by white arrows. Proximodistal direction is indicated by dotted lines. d After sciatic nerve crush, lengths of regenerated nerves were measured using Fiji software and represented as a bar graph. For statistical analysis, Student’s t-test was performed. e-f An antibody specific to UCHL1 was used as a marker for presynaptic terminals, whereas that of α-bungarotoxin (α-BTX) was used for postsynaptic end plates [9]. The shapes of NMJs were analyzed to determine whether they were pretzel-shaped or distorted (e). The integrity of NMJs was determined by measuring to what degree presynaptic terminals merged with postsynaptic end plates (f). g-i After sciatic nerve crush, the shape (g) and integrity (h) of NMJs and average α-BTX area (i) were measured. For graphs, values are represented as mean ± SEM. In Fig. 2g-h, two-way ANOVA was performed, followed by Tukey’s post-hoc test. In Fig. 2g, ****p < 0.0001 for modified NMJ, ####p < 0.0001 for pretzel-shaped NMJ. In Fig. 2h, **p < 0.01 and ****p < 0.0001 for fully innervated NMJ, ####p < 0.0001 and n.s. > 0.05 partially innervated NMJ. In Fig. 2i, one-way ANOVA was performed, followed by Tukey’s post-hoc test. For the remaining bar graphs, *p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001. Scale bars: c = 200 μm; d-e = 20 μm