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. 2022 Jan 27;20(1):e3001526. doi: 10.1371/journal.pbio.3001526

Fig 1. Systemic and central (intracortical) blockade of NKCC1 regulate LPS-induced inflammatory cytokine production in the brain in an opposite manner.

Fig 1

(A) Mice were subjected to either ip. or cor. LPS injections, while NKCC1 was blocked by ip. Bum administration. Central LPS injection triggers high cytokine (G-CSF, IL-1α, IL-1β) and KC responses in the brain compared to ip. LPS injection, which is blocked by ip. Bum administration. (B) Central NKCC1 inhibition by cor. Bum administration significantly increases G-CSF and IL-1β levels. See also S1 Fig for effects of systemic vs. central blockade of NKCC1 on LPS-induced cytokine responses in the periphery. (C) Flow cytometric dot plots show that cortical administration of Bum does not affect the number of microglia (CD45int/P5 gate), and recruitment of leukocytes (CD45high/P4 gate), including monocytes (CD11b+, Ly6Chigh /P9 gate), and granulocytes (CD11b+, Ly6Ghigh/P7 gate) upon central LPS injection. (D) The main source of IL-1α and IL-1β in the brain are microglia cells. Confocal images of Cx3CR1+/GFP brain slices show IL-1α-CD45-P2Y12R (above, red arrowheads) and IL-1β-CD45-P2Y12R (below, red arrowheads) labeled cells after cortical LPS injection-induced inflammation. (E) NKCC1 (encoded by Slc12a2) and P2Y12R gene expression is down-regulated in microglia isolated from adult mice 24 hours after cisterna magna LPS application. (A) Kruskall–Wallis followed by Dunn’s multiple comparison test; *p < 0.05; N (veh.) = 5, N (veh. + ip. LPS) = 5, N (Bum + ip. LPS) = 5, N (veh. + cor. LPS) = 5, N (Bum + cor. LPS) = 9. (B) Unpaired t test; *p < 0.05; N = 9/group; data were pooled from two independent studies. (C) One-way ANOVA followed by Tukey’s multiple comparison test; *p < 0.05; N (veh.) = 5, N (cor. LPS) = 6, N (cor. Bum + LPS) = 6. (D) Scale: 25 μm. (E) Unpaired t test; **p < 0.01, ***p < 0.001; N (WT) = 6, N (WT + LPS) = 5. Data underlying this figure can be found in S1 Data. Bum, bumetanide; cor., cortical; ip., intraperitoneal; ns, not significant; veh., vehicle; WT, wild type.