Fig. 3
(A-H): Influence of circadian rhythm after SAH in vivo
Analysis of Hmox1fl/fl and HO-1 deficient LyzM-Cre-Hmox1fl/fl mice. After blood injection, mice were air or carbon monoxide (CO) exposed daily (1 h); blood injection at ZT2 = 2nd hour and ZT12 = 12th hour of the 12-hour light cycle of the mice. (A) Survival 7 days post-SAH in Hmox1fl/fl and LyzM-Cre-Hmox1fl/fl air or CO exposed (ZT2 n = 6–7 and ZT12 n = 5–8, p = 0.0449). (B) Relative latency times ZT2 vs. ZT12 in the Barnes maze spatial memory paradigm of Hmox1fl/fl and LyzM-Cre-Hmox1fl/fl air or CO exposed on day 14 (7 days post-SAH) n = 5–7. (C) Hematoxylin/Eosin staining of the middle cerebral artery (MCA) 7 days post-SAH of Hmox1fl/fl and LyzM-Cre-Hmox1fl/fl controls and treated with CO at ZT2 and ZT12. (D) Vasospasm index (lumen radius [LR]/wall thickness [WT] normalized to Hmox1fl/fl control ZT2, representative images in 4 C, n = 3–4 mice, 2–3 images per mouse, Hmox1fl/fl control p = 0.035, LyzM-Cre-Hmox1fl/fl control p = 0.038). (E) Representative microglia in the hippocampus; measurement of soma area; scale bars 20 μm, 7 days post-SAH. (F) Hippocampus (HC) microglia soma size (representative images in 4E, n = 3–4 mice; 2–4 images with microglia per mouse, Hmox1fl/fl control p = 0.0046 and, LyzM-Cre-Hmox1fl/fl control p = 0.0032). (G) For phagocytosis analysis, colocalization of immunofluorescent stained Ter-119 positive erythrocytes and Iba-1 stained microglia were analyzed; representative images of the analyzed brain region (base of the brain with proximity to the hypothalamic nuclei). White arrows show erythrocytes associated with microglia (colocalization of Ter-119 and Iba-1 signals) and red arrows show unassociated erythrocytes (Ter-119 signal alone). Scale bars 100 μm. (H) Erythrocytes associated with Hmox1fl/fl or LyzM-Cre-Hmox1fl/fl microglia normalized to Hmox1fl/fl control ZT2. Analysis of phagocytosis (representative images in 4G; n = 3–4 mice with 2–4 images per mouse, Hmox1fl/fl control p = 0.0062). Used statistical analysis: Log-rank (Mantel-Cox) test (Fig. 4A), two-way ANOVA with multiple comparison test; results presented as box blot with median, minimum and maximum. Statistically significant values were defined as p ≤ 0.05 (* p ≤ 0.05; ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001)