Fig 2: Microglia activation and increase in proinflammatory cytokine levels after AQP4 immunization.(A) Immunostaining for Iba-1 in the spinal cord of naive, pEmpty(C/P+), pAQP4(C/P–), and pAQP4(C/P+) mice. Scale bar: 50 μm. Original magnification, ×400 (inset). (B) Quantification of Iba-1 immunofluorescence intensity. (C) Coimmunostaining for Iba-1 and GFAP. (D–F) ELISA of TNF-α, IL-1β, and IL-6 levels in the spinal cord homogenate of naive, pEmpty(C/P+), pAQP4(C/P–), and pAQP4(C/P+) mice. Images are representative photomicrographs showing the ventrolateral white matter of cervical spinal cord cross sections from 5 mice per group. Nuclei were counterstained with DAPI. Data are mean ± SEM; n = 5 per group (B), n = 3 per group (D–F). ***P < 0.001, 1-way ANOVA with post hoc Tukey’s test. Scale bar: 50 μm.

Fig 3: Expression of neuroinflammatory markers following LPS challenge is reduced in Orai1 cKO mice.a A schematic of the protocol used for the experiment. Orai1fl/fl (WT) and Orai1fl/fl Aldh1l1-Cre/ERT2 mice were injected with tamoxifen for five days to induce the deletion of Orai1. LPS or saline was delivered as indicated on day 10 following the last tamoxifen injection. Animals were sacrificed 2 days later and analyzed by immunohistochemistry and ELISA. b, c Immunohistochemistry of hippocampal GFAP (to label astrocytes) and IBA1 (to label microglia) in the dentate gyrus and the CA1 regions of the hippocampus. The images shown were obtained from male mice; female mice showed a similar pattern (Supplementary Fig. 8). Scale bar = 50 µm. d Quantification of GFAP and IBA1 expression assessed by measuring the % area of the region of interest occupied by fluorescent signal. Data are given as means +/- SEM for n = 14–18 images from 3 WT saline mice, n = 14–20 images from 5 WT LPS mice, n = 11–13 images from 3 KO saline mice, n = 10–20 images from 4 KO LPS mice. e Levels of IL-1α and IL-6 measured in homogenized hippocampal tissue lysates by ELISA. (IL-1α - WT saline group: n = 5 male, 2 female mice; WT LPS group: n = 6 male, 2 female mice; KO saline group: n = 2 male, 2 female mice; KO + LPS group: n = 3 male, 3 female mice. IL-6 - WT saline group: n = 5 male, 2 female mice; WT LPS group: n = 5 male, 2 female mice; KO saline group: n = 1 male, 3 female mice; KO LPS group: n = 5 male, 2 female mice. f Peripheral LPS administration increases thrombin levels in the brain. Thrombin (A) and SDF-1α (B) were measured via ELISA in homogenized hippocampal tissue lysates in saline (white bars) and LPS-injected (black bars) mice 48 h after injection. Thrombin is significantly elevated in LPS-treated WT and cKO mice, while SDF1α is modestly elevated. (WT saline group: n = 3 female and 5 male mice. WT LPS group, n = 3 female and 5 male mice. Orai1 cKO saline group: n = 3 female, 1 male mice, Orai1 cKO LPS group: n = 4 male, 2 female KO mice). All data are given as mean +/- SEM. Statistical tests were conducted for the pooled data by two-way ANOVA followed by Tukey posthoc tests in each graph. Source data are provided as a Source Data file.