Inflammatory responses mediate brain-heart interaction after ischemic stroke in adult mice

Abstract

Stroke induces cardiac dysfunction which increases post stroke mortality and morbidity particularly in aging population. Here, we investigated the effects of inflammatory responses as underlying mediators of cardiac dysfunction after stroke in adult mice. Adult (eight-to-nine months) male C57BL/6 mice were subjected to photothrombotic stroke. To test whether immunoresponse to stroke leads to cardiac dysfunction, splenectomy was performed with stroke. Immunohistochemistry, flow cytometry, PCR, ELISA and echocardiography were performed. We found marginal cardiac dysfunction at acute phase and significant cardiac dysfunction at chronic phase of stroke as indicated by significant decrease of left ventricular ejection fraction (LVEF) and shortening fraction (LVSF). Stroke significantly increases macrophage infiltration into the heart and increases IL-1β, IL-6, MCP-1, TGF-β and macrophage-associated inflammatory cytokine levels in the heart as well as induces cardiac-fibrosis and hypertrophy. Splenectomy with stroke significantly reduces macrophage infiltration into heart, decreases inflammatory factor expression in the heart, decreases cardiac hypertrophy and fibrosis, as well as significantly improves cardiac function compared to non-splenectomized adult stroke mice. Therefore, cerebral ischemic stroke in adult mice induces chronic cardiac dysfunction and secondary immune response may contribute to post stroke cardiac dysfunction.

Keywords: Stroke; cardiac dysfunction; immune system; macrophages; splenectomy.

Figure 1.

Splenectomy with stroke attenuates cerebral ischemic stroke-induced cardiac dysfunction in adult mice. (a) Representative images of echocardiography from sham control, splenectomy alone, stroke alone and stroke with splenectomy groups at one month after stroke in adult mice. (b) Echocardiography data at three days and one month after stroke shows that stroke induces marginal cardiac dysfunction at acute stage and significant cardiac dysfunction at one month after stroke Splenectomy with stroke in adult mice significantly improves cardiac function as indicated by increased LVEF and LVSF. The respective F values are 2.4, 2.92, 9.2, 7.96, sample size of n = 9/group was employed for all groups except 1m-spx n=6, 1m-spx-stroke n = 8, *p < 0.05, One-way ANOVA (Tukey). (c) Stroke or splenectomy with stroke does not alter blood pressure or body temperature compared to sham control mice. The respective F values were: 0.6, 0.85, 0.69. Sample size: control n = 5, 1m-spx n = 5, 1m-stroke n = 5, 1m-spx-stroke n = 4, *p < 0.05, One-way ANOVA (Tukey). (d) Splenectomy prior to stroke significantly improves neurological function (n = 9/group, ANCOVA, *p < 0.05) and decreases lesion volume (n = 6/group, *p < 0.05, One-way ANOVA (Tukey)) at one month after stroke. The respective F values are 27.59, 37.27, 0.6, 0.85, 0.64.

Figure 2.

Splenectomy with stroke decreases chronic cardiac fibrosis and hypertrophy in adult mice. (a) Representative images for PicroSirius Red immunostaining in heart tissue at one month after stroke. (b) Splenectomy with stroke in adult mice significantly attenuates stroke induced cardiomyocyte hypertrophy (MCSA), interstitial fibrosis (Sirius red), and increased heart weight at one month after stroke in adult mice. The respective F values are 31.87, 8.35, 6.65. Sample size: Control n = 9, 1m-Spx n = 5, 1m-Stroke n = 9, 1m-spx-stroke n = 8, *p < 0.05, One-way ANOVA (Tukey). (c) TGF-β immunostaining and (d) quantification data indicate that splenectomy with stroke in adult mice significantly decreases stroke-induced TGF-β expression in heart. F value = 6.023. Sample size: Control n = 5, 1m-Spx n = 5, 1m-Stroke n = 5, 1m-spx-stroke n = 4, *p < 0.05, One-way ANOVA (Tukey).

Figure 3.

Splenectomy with stroke decreases stroke-induced acute cardiac inflammatory factor expression. PCR results indicate that splenectomy with stroke significantly decreases stroke-induced inflammatory factor gene expression such as (a) IL-1β, (b) IL-6, (c) MCP-1 and (d) ED1 compared to sham-control mice. However, stroke with or without splenectomy does not significantly alter (e) IL-10 gene expression at three days after stroke compared to sham control mice. F values are 7.66, 9.07, 22.96, and 17.09, respectively. Sample size: Control n = 6, 3d-Spx n = 4, 3d-stroke n = 6, 3d-Spx stroke n = 6; *p < 0.05, One-way ANOVA (Tukey).

Figure 4.

Splenectomy with stroke decreases invasion of immune cells into heart. (a–b) Representative flow cytometry plots show the gating strategy of CD45 + leukocytes population and CD45+CD11b+F4/80 + macrophage population in heart, and activated monocytes (CD11b+F4/80+) in blood. (c) Splenectomy with stroke significantly decreases infiltration of leukocytes and macrophages into heart, compared to stroke alone mice. F values are 5.49, 11.42, respectively. (d) Splenectomy with stroke significantly decreases the leukocytes and activated monocytes in blood, compared to stroke alone mice. F values are 10.84 and 5.64, respectively. N=6/group, *p < 0.05, One-way ANOVA (Tukey).

Figure 5.

Stroke induces macrophage infiltration into heart which persists from acute to chronic phase of stroke. (a) CD45 immunostaining and quantification data indicate that stroke significantly increases CD45 + cells in heart at three days and one month after stroke compared to sham control and splenectomy only group, respectively. F value=8.61. (b) IBA-1 immunostaining and quantification data indicate that stroke significantly increases IBA-1 positive macrophages in heart at three days as well as one month after stroke compared to sham control mice. Splenectomy with stroke significantly decreases IBA-1 positive macrophages at three days after stroke. F value=6.25. Sample size: Control n = 5, 3d-spx=4, 3d-stroke n = 5, 3d-spx-stroke n = 5, 1m-spx n = 5, 1m-stroke n = 5, 1m-spx-stroke=4. *p < 0.05, One-way ANOVA (Tukey).

Figure 6.

Splenectomy with stroke decreases stroke-induced cardiac MCP-1 expression. Representative images for MCP-1 immunostaining in heart tissue at (a) three days and (b) one month after stroke and (c) quantification data. Stroke significantly increases cardiac MCP-1 expression compared to sham control mice as well as splenectomy alone mice. MCP-1 expression is significantly greater at one month after stroke compared to three days after stroke. Splenectomy with stroke significantly decreases MCP-1 expression compared to stroke alone group at three days as well as one month after stroke. F value = 28.54. Sample size: Control n = 6, 3d-spx=5, 3d-stroke n = 6, 3d-spx-stroke n = 6, 1m-spx n = 5, 1m-stroke n = 6, 1m-spx-stroke=6. *p < 0.05, One-way ANOVA (Tukey). (d) ELISA data indicate that splenectomy with stroke significantly decreases MCP-1 expression compared to stroke alone group at three days as well as one month after stroke in adult mice.