Fig. 4: Assessment of spatially-confined conduit treatment efficacy through histopathological analysis and pain-related behaviors.

a Toluidine blue staining (TBS) images of regenerated nerves at 12 weeks postoperatively. b Myelin sheath quantifications on regenerated nerves displaying (i) average myelin thickness (n = 12) and (ii) number of regenerated axons per square millimeter (n = 3). c TEM images of nerve regeneration at 12 weeks postoperatively. d G-ratio analyses of regenerated axons, categorized by differing diameters. e Immunohistochemical (IHC) staining for C-fos, a protein expressed following noxious stimulation. f IHC staining for substance-P, a neuropeptide that enhances pain signal transmission and triggers inflammatory responses. g Quantification of activated cells per 10,000 μm² in C-fos IHC staining (n = 10). h Quantitative analysis of the positive area ratio per 10,000 μm² in substance-P IHC staining (n = 6). i. Diagram for pain behavior assessment across different time frames. j Result of autotomy scoring across different time frames (n = 6). k Footprints of rat limb gait over a 3 s interval and the intensity of the left hind limb footprints at 12 weeks after surgery. l The ratio of standing time during various time frames within a 5 s interval. m The ratio of maximum contact area during various time frames within a 5 s interval. Mean values are shown and error bars represent ± s.d., as analyzed by one-way ANOVA with Tukey’s post hoc tests in (b–j). Mean values are shown and error bars represent ± SEM, as analyzed by one-way ANOVA with Tukey’s post hoc tests in (l) and (m). The experiments in (c) were independently repeated at least three times with similar results.