Cryab^−/− mice have larger lesions and worse neurological scores. (A) Representative images of TTC-stained brain sections of wild-type and Cryab^−/− mice (Left) and quantification of lesion sizes 2 d after stroke (Right, n = 10 per group). (B) Representative images of silver-stained brain sections (Left) and quantification of lesion sizes 7 d after stroke (Right). (C) Quantification of 28-point neurological scoring in wild-type and Cryab^−/− mice at 12 h, 2 d, and 7 d after stroke. For B and C, n = 10 and 7 for wild-type and Cryab^−/− groups, respectively. *P < 0.05, Student t test. Data represent means ± SEM.

There is more immune cell infiltration in the brains of Cryab^−/− mice, and Cryab deficiency in the immune system causes larger lesion sizes after stroke. (A) Representative CD45 vs. CD11b flow cytometry plots of brain immune cells in wild-type and Cryab^−/− mice 2 d and 7 d after stroke. (B) Quantification of total cell numbers of microglia (CD11b⁺CD45^low), granulocytes and macrophages (CD11b^highCD45^high), subpopulation of monocytes (CD11b^lowCD45^high), and lymphoid cells (CD11b⁻CD45^high) in wild-type and Cryab^−/− mice brains at 2 d and 7 d after stroke. (C) Schematic plots showing the analysis method with F4/80 and Gr1 markers of CD11b^highCD45^high population (F4/80⁻Gr1⁺: granulocytes, F4/80⁺Gr1⁺: activated macrophages, F4/80⁺Gr1⁻: macrophages). (D) Quantification of granulocytes, activated (act.) macrophages, and macrophages in wild-type and Cryab^−/− mice brains 2 d and 7 d after stroke. (E) Representative CD45 vs. CD3 flow cytometry plots of brain immune cells in wild-type and Cryab^−/− mice 2 d and 7 d after stroke. (F) Quantification of number of CD3⁺, CD3⁺CD4⁺, CD3⁺CD8⁺, and CD3⁺γδTCR⁺ T cells. n = 6 per group for each time point. *P < 0.05, Mann–Whitney analysis. (G) Representative γδ-TCR vs. IL-17a flow cytometry plots of brain immune cells in wild-type and Cryab^−/− mice 7 d after stroke. (H) Scheme of timetable for the bone marrow-chimeric mice experiments. (I) Quantification of lesion sizes 7 d after stroke in the bone marrow-chimeric mice, wild-type cells into wild-type host: WT→WT; Cryab^−/− cells into wild-type host: KO→WT; wild-type cells into Cryab^−/− host: WT→KO; and Cryab^−/− cells into Cryab^−/− host: KO→KO (n = 10 for all groups except KO→KO, n = 6). *P < 0.05, **P < 0.01, ^#P = 0.063, Student t test. Data represent means ± SEM.

Plasma Cryab increases after stroke, and restoration of plasma Cryab in Cryab^−/− mice confers neuroprotection and modulates the peripheral immune response. (A) Quantification of plasma Cryab levels in naïve mice and in mice at 12 h, 2 d, and 7 d after stroke (n = 8, 11, 10, and 8 for naïve, 12-h, 2-d, and 7-d groups, respectively). *P < 0.05, Kruskal–Wallis analysis. (B) Quantification of the levels of Cryab in human plasma and (C) correlation of lesion volume with the levels of Cryab in human plasma at presentation (<4 h), 24 h, and 72 h after the symptom onset (n = 5, 7, and 6 for healthy controls, younger and older patients, respectively). (D) Quantification of lesion sizes in PBS-treated wild-type, PBS-treated Cryab^−/−, and Cryab-treated Cryab^−/− mice 7 d after stroke as assessed by silver stain (Upper) and timetable of Cryab injections as indicated by vertical black arrows (Lower; n = 15, 12, and 13 for PBS-treated wild-type, PBS-treated Cryab^−/−, and Cryab-treated Cryab^−/− groups, respectively). *P < 0.05, one-way ANOVA. (E) Quantification of cytokines after stimulation with anti-CD3/anti-CD28, Concanavalin-A (ConA), or LPS; representative graphs from one of the two experiments with similar results. *P < 0.05, **P < 0.01, ***P < 0.005, ^#P = 0.053, ^##P = 0.056, ^§P = 0.06, Student t test.

Cryab administration to wild-type mice reduces the lesion size after stroke and modulates the peripheral immune response. (A) Quantification of lesion sizes in PBS- and Cryab-treated wild-type mice 2 d after stroke as assessed by TTC staining (Upper) and timetable of injections (Lower; n = 7 per group). (B) Quantification of lesion sizes in PBS- and Cryab-treated (starting the treatment 1 h before stroke) wild-type mice 7 d after stroke as assessed by silver stain (Upper) and timetable of injections (Lower; n = 7 per group). (C) Quantification of lesion sizes in PBS- and Cryab-treated (starting the treatment 12 h after stroke) wild-type mice 7 d after stroke as assessed by silver stain (Upper) and timetable of injections (Lower; n = 14 and 15 for PBS- and Cryab-treated groups, respectively). *P < 0.05, **P < 0.005, Student t test. (D–K) Quantification of IL-2, IL-17, IFN-γ, IL-12p40, IL-6, IL-10, and TNF levels after ConA and LPS stimulation of splenocytes from PBS- and Cryab-treated mice 7 d after stroke; representative graphs from one of the four experiments with similar results. *P < 0.05, **P < 0.01, ***P < 0.005, ^#P = 0.053, Student t test.

The effect of Cryab treatment on brain T cells after stroke. (A) Representative flow cytometry plots of CD3+ cells from PBS- and Cryab-treated mice brains at 7 d after stroke. (B–E) Quantification of CD3⁺, CD4⁺, CD8⁺, and γδ-T cells in brains of PBS- and Cryab-treated mice 7 d after stroke (n = 6 and 7, respectively); pooled analysis of two experiments with similar results. (F) Representative histogram analysis of IL-17⁺ γδ-T cells in brains of PBS- and Cryab-treated mice 7 d after stroke. (G) Quantitative analysis of number of IL-17-PE molecules per cell in brains of PBS- and Cryab-treated mice 7 d after stroke (n = 10 and 11, respectively); pooled analysis of three experiments with similar results. *P < 0.05, #P = 0.053, Student t test. (H) Quantitative analysis of number of IFN-γ-PE molecules per cell in brains of PBS- and Cryab-treated mice 7 d after stroke (n = 4 and 6, respectively); pooled analysis of two experiments with similar results.