Splenectomy protects aged mice from injury after experimental stroke

Abstract

Elderly stroke patients and aged animals subjected to experimental stroke have significantly worse functional recovery and higher mortality compared to younger subjects. Activation of the peripheral immune system is known to influence stroke outcome. Prior studies have shown that splenectomy reduces ischemic brain injury in young mice. As immune function changes with aging, it is unclear whether splenectomy will confer similar benefits in aged animals. We investigated the contribution of spleen to brain injury after cerebral ischemia in aged male mice. Splenic architecture and immune cell composition were altered in aged mice. Splenectomy 2 weeks before stroke resulted in improved neurobehavioral and infarct outcomes in aged male mice. In addition, there was a reduction in peripheral immune cell infiltration into the brain and decreased levels of peripheral inflammatory cytokines after stroke in aged splenectomized mice. Splenectomy immediately after reperfusion also improved behavioral and infarct outcomes. This study suggests that inhibition of the splenic immune response is a translationally relevant target to pursue for stroke treatment in aged individuals.

Keywords: Age; Immune response; Middle cerebral artery occlusion model; Splenectomy; Stroke.

Fig 1. Spleen changes with aging

(A) Increase in body weight with aging. (B) Disarrangement of white pulp with aging. Data is presented as mean ± SEM, n=8-9, and analyzed by Wilcoxon rank sum test. **p<0.01

Fig 2. Splenectomy improved functional and infarct outcomes in young and aged mice

(A) Reduction in the percentage spleen body weight after MCAo in both young and aged mice. (B) Reduced neurological deficit scores. (C) Increase in number of entries and (D) percentage alterations on Y maze (E) Decrease in corner test score was observed in the splenectomized aged mice after MCAo. (F) Decrease in percentage infarct area was seen in the splenectomized aged mice at 4 days after MCAo. Data is presented as mean± SEM, n=7-8; analyzed by two-way ANOVA or general linear model followed by post hoc Tukey test, *p<0.05, **p<0.01, ***p<0.001.

Fig 3. Decline in the infiltrating immune cells in the splenectomized aged mice after MCAo

(A) Reduced counts of infiltrating CD45hi cells (B) Decreased monocyte counts (c) decrease neutrophil counts was observed in the splenectomized aged mice after MCAo as compared to spleen intact mice. Data is presented as mean± SEM, n=7-8; analyzed by two-way ANOVA or general linear model followed by post hoc Tukey test, *p<0.05, **p<0.01, ***p<0.001.

Fig 4. Decline in the peripheral pro-inflammatory cytokines in the splenectomized aged mice after MCAo

Decline in the circulating TNF α (A) and IL-12 (B) was observed in the splenectomized mice as compared to spleen intact. Reduced plasma levels of IL-1α and IL-6 was observed in the splenectomized MCAo mice compared to splenectomized sham group. Data is presented as mean± SEM analyzed by two-way ANOVA or general linear model followed by post hoc Tukey test, n=7-10; *p<0.05, **p<0.01, ***p<0.001.

Fig 5. Splenectomy does not increase mortality and LBP levels after MCAo in aged mice

(A) Shows the Kaplan –Meier survival curves. (B) Increase in plasma LBP levels was observed after MCAo in spleen intact and splenectomized aged mice. Data is presented as mean± SEM, mortality data analyzed by log-rank test and LBP data was analyzed by two-way ANOVA or general linear model followed by post hoc Tukey test, n=7-8; p<0.05.

Fig 6. Splenectomy post stroke improves functional and infarct outcomes in aged mice

(A) Reduced neurological deficit scores. (B) Decrease in corner test score was observed in the post stroke splenectomized aged mice. (C) Kaplan –Meier survival curves for MCAo intact and splenectomy mice. (D) Decrease in percentage infarct area was seen in the post stroke splenectomized aged mice at 4 days. Data is presented as mean± SEM, and analyzed by two sample t test or Wilcoxon rank sum test. Mortality data was analyzed by log rank test. n=6; *p<0.05. **p<0.01. n=6; p<0.05.