A transient decrease in spleen size following stroke corresponds to splenocyte release into systemic circulation

Abstract

The splenic response to stroke is a proinflammatory reaction to ischemic injury resulting in expanded neurodegeneration. Splenectomy reduces neural injury in rodent models of hemorrhagic and ischemic stroke, however the exact nature of this response has yet to be fully understood. This study examines the migration of splenocytes after brain ischemia utilizing carboxyfluorescein diacetate succinimidyl ester (CFSE) to label them in vivo. The spleen was found to significantly decrease in size from 24 to 48 h following middle cerebral artery occlusion (MCAO) in rats compared to sham operated controls. By 96 h post-MCAO the spleen size returned to levels not different from sham operated rats. To track splenocyte migration following MCAO, spleens were injected with CFSE to label cells. CFSE positive cell numbers were significantly reduced in the 48 h MCAO group versus 48 h sham and CFSE labeled cells were equivalent in 96 h MCAO and sham groups. A significant increase of labeled lymphocyte, monocytes, and neutrophils was detected in the blood at 48 h post-MCAO when compared to the other groups. CFSE labeled cells migrated to the brain following MCAO but appear to remain within the vasculature. These cells were identified as natural killer cells (NK) and monocytes at 48 h and at 96 h post-MCAO NK cells, T cells and monocytes. After ischemic injury, splenocytes enter into systemic circulation and migrate to the brain exacerbating neurodegeneration.

Fig 1. Splenic response to MCAO

Mean spleen weights plotted over time following MCAO. Spleen weights were significantly decreased by the 24, 48, and 51 h time points compared to sham operated controls (* p<0.05). Spleen weights were not significantly different from sham operated controls at the 3, 72, and 96 h time points (a). The total number of CFSE positive splenocytes was decreased in the 48 h MCAO group compared to the 48 h sham and the CFSE only groups. No differences were detected between the 96 h MCAO and sham groups, which were also significantly different from the 48 h sham and CFSE only groups (* p<0.0001). The CFSE only group were rats that were euthanized 5 days post injection, the same time the other groups underwent sham or MCAO procedures (n≥4) (b). Brain sections from CFSE treated rats at 48 and 96 h post-MCAO were immunostained with antibodies that recognize CD161 (NK cell), CD11b (monocytes), and CD3 (T cell). CD161 expression co-localized with CFSE labeled cells at 48 h (c) and 96 h (e). CD11b co-localized with CFSE labeled cells at 48 h (d) and 96 h (f). CD3 immunoreactivity was only detected with CFSE at 96 h (g). Arrows indicate double labeled cells. Scale bars = 50μm.