Fig. 2: Conditional knockdown of Cldn5 expression in the hippocampus exacerbates kainic acid-induced epilepsy.

a Kainic acid (KA) injection site and timeline of experiment (top). Figure Created with BioRender. Open field testing of mice post KA injection with significant decreases in the number of entries in the centre zone (bottom) at both timepoints compared to saline (****p < 0.0001 for acute and chronic). b Claudin-5 (green) and CD31 (red) staining in the hippocampus in saline injected animals and the acute and chronic stages post KA injection. Scale bars, 50 μm. c Levels of claudin-5 expression were significantly decreased following KA injection at the acute (*p = 0.0187) and chronic stage (**p = 0.0036). d Cldn5 (*p = 0.0102), e Ocln (*p = 0.0124) and f Tjp1 (*p = 0.0129) mRNA levels were decreased at 3 h and 24 h post KA injection. g S100B levels were significantly increased in serum of mice 3 h post KA injection (*p = 0.011). h Immunohistochemistry for Sulfo-NHS-Biotin and claudin-5 following intrahippocampal injection of KA. Scale bars, 50 μm. i Biotin extravasated into the brain at the acute stage post injection of KA (***p = 0.0007). Figure Created with BioRender. j AAV expressing claudin-5 shRNA injected bilaterally into the hippocampus and timeline of experiment. k Claudin-5 protein (**p = 0.0024) (green) and l mRNA (*p = 0.043) was significantly decreased in brain endothelial cells (red) following AAV-shRNA-Cldn5 injection in the hippocampus of mice compared to that in AAV-shRNA-injected mice, following doxycycline (Dox) treatment. Scale bar, 20 μm. m Reduced performance in the novel object recognition (*p = 0.0146) and n open field test (**p = 0.0023) post claudin-5 suppression in KA injected mice. o Increased sodium fluorescein (NaFl) extravasation post claudin-5 suppression in KA injected mice (***p = 0.0006). p Increased IBA1 positive microglia (**p = 0.0044) and GFAP positive astrocytes (**p = 0.0024) post claudin-5 suppression in kainic acid injected mice. Scale bar, 20 μm. Data represent means ± s.e.m.; each datapoint represents one animal. Number of animals (n) is indicated on graphs. Two-way ANOVA followed by Bonferroni’s multiple comparison test for sodium fluorescein permeability; two-sided unpaired t-test for AAV experiments; one-way ANOVA followed by Tukey’s multiple comparison test for all other graphs. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Source data are provided as a Source Data file.