Fig. 3

4-AP treatment improves the molecular organization of excitable axonal domains that are disrupted by TBI. Confocal imaging analysis of node of Ranvier complexes in individual corpus callosum axons of Thy1-YFP-16 mice at 7 days post-TBI or sham procedures. (A) Immunostaining along YFP-labeled axons (green) detected clustering of voltage-gated sodium channels (Nav1.6, white) at the node and Caspr (red) in the paranode region, where myelin attaches to the axon. B-D TBI disrupted paranode domain organization, which was normalized by 4-AP treatment. (B) “Symmetrical” Caspr paranodes exhibit paired Caspr bands of approximately symmetrical length, while “Asymmetrical” paranodes exhibit uneven Caspr domain lengths. Single Caspr paranodes with a missing domain counterpart on the opposite side of a node were classified as “Heminodes”. C TBI increased the number of Caspr heminodes while acute 4-AP treatment post-TBI resulted in heminode numbers similar to sham levels. D TBI increased the asymmetry of Caspr domains in TBI mice compared to sham. 4-AP treatment significantly improved paranode organization following TBI. E–F Kv1.2 channel (red) and Caspr (white) immunostaining along individual YFP (green) axons in single confocal optical slices. E In injured mice, Kv1.2 channels mislocalize from the juxtaparanode domain into Caspr-labeled paranode domain. F Quantification of atypical Kv1.2 domains that overlapped with Caspr domains and/or were asymmetrical in length. Acute 4-AP treatment reduced the percentage of atypical Kv1.2 domains after TBI, but the distribution patterns of Kv1.2 channels was not fully normalized to sham levels. C, D, F Bars represent mean ± SEM with an individual data point shown for each mouse. Two-way ANOVA for main effect of injury or drug with Holm-Sidak’s multiple comparisons test for significance of post-hoc pair effects. See Fig. 1 for mouse sample numbers and Table 1 for statistical details