Fig. 4: CSD stress leads to increased numbers of LysM⁺ myeloid cells in vascular channels connecting skull BM to meninges.

a LysM^gfp/+ mice were subjected to CSD stress and behaviorally phenotyped (Fig. S8). Before TomL intravascular (iv) injection, blood was drawn for flow cytometry (Fig. S9). Dorsal meninges and brain (Fig. S10) were prepared for imaging. A separate cohort of mice was used for tissue clearing. b Flow cytometry data from blood shows high GFP expression in neutrophils compared to other cell types. c Left: Representative image for dorsal whole-mount meningeal tissue. Scale bar = 1 mm. Right: LysM-GFP⁺ cells adjacent to a blood vessel; merge and individual channels. Scale bar = 10μm. d Representative images from HC and CSD mice showing hand-counted cells, normalized to indicated area. Inset box represents size of area shown in (c, Right). e Quantification of total meningeal LysM-GFP⁺ cells shows an increase with CSD stress (Unpaired t test, *p = 0.046, t = 2.2, df = 23, 95% CI [0.019, 0.86], n_HC = 13, n_CSD = 12. N = 2 experiments). ^‡natural log-transformed. f LysM-GFP⁺ cells from (e) were separated into three categories based on their location in the tissue: >10μm away from a blood vessel (“non-vascular”), ≤10μm away from a blood vessel (“abluminal”), and intravascular (“iv”). CSD led to an overall increase in LysM-GFP⁺ cells (RM^-ANOVA: group, *p = 0.043, F_(1,23) = 4.6, n_HC = 13, n_CSD = 12); asterisk indicates main effect of CSD, but there were no significant post-hoc comparisons. ^†log₁₀ -transformed. g Representative image from cleared skulls showing vascular channels between skull BM and the meninges. Scale bar = 100μm. Left: Individual channels. Middle: Merged image, HC mouse. Right: Merged image, CSD mouse. Quantification of LysM-GFP⁺ cells per channel (h) indicates increased egress from skull BM following CSD stress (Unpaired t test, *p = 0.049, t = 2.4, df = 7, 95% CI [0.0028, 0.70]), and (i) no differences in the number of channels counted between groups (n_HC = 3, n_CSD = 6. N = 2 experiments). Data points represent individual mice. T tests were two-tailed. BM = bone marrow, BV = blood vessel, CSD = chronic social defeat stress, HC = home cage, iv = intravascular, SSC = side scatter complexity, TomL = tomato lectin. Data shown as mean ± SEM. *p < 0.05. Source data are provided as a Source Data file. Schematic created in BioRender. Kigar, S. (2025) https://BioRender.com/p6bsp7f.