Fig. 2

Pretreatment with NOX2-blocking peptides or shRNA attenuates SNI-induced mechanical allodynia and hyperexcitability in DRG neurons. a Images of the L5 DRG (a(a, b)) and proximal spinal cord (a(c, d)) after the local application of CFSE (100 μM in 2 μl) by gelatin sponge. b, c Local application of gp91-tat (26 μg or 52 μg) to L4-L6 DRGs before (b) but not after (c) SNI surgery significantly attenuated mechanical allodynia at 3–14 days after SNI. The dotted arrow indicates the time of gp91-tat or vehicle application. Kruskal-Wallis test followed by Dunn’s multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001 versus the sham group; #p < 0.05, ##p < 0.01 versus the vehicle-treated SNI group. d Western blotting showed that pretreatment with gp91-tat decreased NOX2 expression in L4-L6 DRGs at 7 days after SNI (n = 7). One-way ANOVA followed by Tukey’s test. *p < 0.05 versus the sham group, #p < 0.05 versus SNI group. e Representative traces illustrating the action potentials and their rheobase currents recorded in L4–L6 DRG neurons. f Representative traces showing the action potentials elicited by 1×rheobase in L4-L6 DRG neurons. g Quantification analysis of rheobase currents (a), one-way ANOVA followed by Tukey’s test. **p < 0.01 versus the sham group, #p < 0.05 versus the SNI group. Numbers of action potentials (b), Kruskal-Wallis test followed by Dunn’s multiple comparison test. **p < 0.01 versus the sham group. Membrane capacitances (c). Peak amplitudes (d) and half-widths (e) of action potentials, as well as input resistance (f) in each group (n = 9–11/group). h Experimental diagram showing the timeline of AAV-shRNA injection and behavioral tests. i(a-b) Representative immunofluorescence staining showing the expression of cybb-shRNA-mCherry or scramble-shRNA-mCherry in transduced cells. j Western blotting analysis showed that cybb-shRNA injected into the DRGs diminished the protein expression of NOX2 (n = 4). Two-tailed unpaired Student’s t test. ***p < 0.001 versus scrambled shRNA. k NOX2 cybb-shRNA but not scrambled shRNA significantly abolished the initiation of mechanical allodynia after SNI (n = 5–6/group). Kruskal-Wallis test followed by Dunn’s multiple comparison test. *p < 0.05, **p < 0.01 versus the sham group; #p < 0.05 versus the cybb-shRNA group