Fig. 1: Meningeal neutrophils are elevated following chronic social defeat (CSD) stress, which causes depressive-like behavioral changes.

a CSD mice were behaviorally tested on days 10-13, and tissue harvested at day 14. A fluorescently labeled CD45 antibody was retro-orbitally injected to discern blood-exposed meningeal cells. CSD causes expected sexual (b; ****p < 0.0001, U = 13, Hodges-Lehmann estimate = −24.5, n_HC = 16, n_CSD = 13. N = 5 experiments.) and social (c; *p = 0.011, U = 323, Hodges-Lehmann estimate = −4.0, n_HC = 34, n_CSD = 30. N = 10 experiments) anhedonia in USM and SI tests, respectively. In (b), pink arrowheads indicate female stimulus urine, blue arrowheads indicate representative urine scent marks from male test mice. Heatmaps (c, d) indicate animal tracking; red color indicates greater time spent in that location. CSD causes increased anxiety-like behavior in the OF (d; ****p < 0.0001, U = 65, Hodges-Lehmann estimate = −3.4, n_HC = 23, n_CSD = 21. N = 8 experiments) and LD box (e; ***p = 0.0007, U = 38, Hodges-Lehmann estimate = −11.0, n_HC = 15, n_CSD = 16. N = 7 experiments) tests. Flow cytometric analysis shows CSD causes an increase in: (f) iv^- meningeal neutrophils (CD45iv^-; CD11b⁺; Ly6G⁺; Ly6C^int), both as a percent of live CD45⁺ cells (****p < 0.0001, U = 573, Hodges-Lehmann estimate = −6.6, n_HC = 52, n_CSD = 44. N = 19 experiments), and in absolute cell counts (Unpaired t test, *p = 0.038, t = 2.2, df = 22, 95% CI [128.4, 4107], n_HC = 14, n_CSD = 10. N = 4 experiments); (g) iv⁺ meningeal neutrophils (CD45iv⁺)^, as a percentage (Unpaired t test, **p = 0.0024, t = 3.1, df = 94, 95% CI [0.24, 1.08], n_HC = 52, n_CSD = 44), but not absolute counts (n_HC=8, n_CSD = 4. N = 1 experiment); and (h) blood neutrophils (CD45iv⁺), in both percentage (****p < 0.0001, U = 146, Hodges-Lehmann estimate=34.2, n_HC = 47, n_CSD = 37. N = 17 experiments) and absolute counts (Unpaired t test, *p = 0.021, t = 2.6, df = 17, 95% CI [11.6, 122.8], n_HC = 12, n_CSD = 7. N = 3 experiments). Data points represent individual mice. Two-tailed tests were used in (b–h); all tests were Mann-Whitney tests unless otherwise indicated. No adjustments for multiple comparisons were made. Additional data shown in Figs. S1–4. Gating strategy shown in Fig. S2. CSD = chronic social defeat stress, HC = home cage, LD = light/dark, OF = open field, SI = social interaction (S = social, NS = non-social), USM = urine scent marking. Data shown as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Source data provided in Source Data file. Schematics from BioRender. Kigar, S. (2025) https://BioRender.com/v98waxl.