Jacalin immunoreactive or NaV immunoreactive nodes and MAG immunoreactive incisures were counted in a similar way as for JAM-C. The results were expressed as densities (e.g., the number of JAM-C immunoreactive paranodes/mm2). Regions were analyzed from at least six sections per animal. Statistical analysis All results are expressed as mean ± standard errors of the means (SEM). One-way, Akt inhibitor two-way, or repeated Inhibitors,research,lifescience,medical measures of analysis of variance (ANOVA)

were used when appropriate. The Tukey–Kramer multicomparison adjustment was used as the post-hoc test to calculate the significance levels. P < 0.05 was considered statistically significant. Results JAM-C localization in normal sciatic nerve Immunohistochemistry Inhibitors,research,lifescience,medical on longitudinal sections of sciatic nerves of sham surgery control adult rats demonstrated JAM-C localization in peripheral nerves (Fig. 1a). Double labeling with two markers of nodes of Ranvier (jacalin and pan-NaV) and with a marker of Schmidt–Lantermann incisures (MAG) confirmed that JAM-C is concentrated in paranodal regions of nerves (Fig. 1b and c) and in Schmidt–Lantermann incisures (Fig. 1d). Double labeling Inhibitors,research,lifescience,medical with antibodies to neurofilament and to P0 confirmed that JAM-C immunoreactive structures are associated with axons and

with regions that lack compact myelin (Fig. 1e and f). Figure 1 JAM-C localization in sham surgery control rat sciatic nerve. (a) JAM-C immunostaining in the peripheral nerve with labeling of paranodes Inhibitors,research,lifescience,medical (arrows) and incisures (double-arrows). Rows (b–f) show a sequence of double labeling with JAM-C to illustrate … Sciatic nerve crush induces changes in JAM-C localization In order to examine the localization of JAM-C after peripheral nerve injury (PNI), immunolabeling followed by quantitative analysis of paranodes and incisures was performed spatially in the near, mid-, and far-most distal regions (1.4, 4.0, and 6.6 mm, respectively, from the crush site) along the distal nerve. Additionally, Inhibitors,research,lifescience,medical this localization

was examined temporally at various time points; namely three, Bay 11-7085 14, 28, and 56 days after nerve crush. These time points were selected as covering both the degeneration stage (three days) and the remyelination process, which is known to begin within two weeks of the onset of axonal regeneration in rats (Burnett and Zager 2004). The spatiotemporal localization of JAM-C immunoreactive paranodes in the regenerating nerve At three (not illustrated) and 14 days (Fig. 2a, c, e, and g) after injury, JAM-C immunoreactive paranodes appeared to be decreased distal to the crush site, and this decrease was confirmed by quantitative analysis (Fig. 3a). In the distal region closest to the crush site (1.4 mm distal), the density of JAM-C immunoreactive paranodes was decreased at three days, but this decrease was not statistically significant.