Abstract 2494: A Transient Decrease in Spleen Size Following Stoke Corresponds to Splenocyte Release into Systemic Circulation

Abstract

Objective: Splenectomy is protective in ischemic injuries to renal, hepatic, and gastrointestinal systems, showing that the splenic response promotes cellular degeneration. Removal of the spleen is also protective in ischemic and hemorrhagic stroke. Therefore, we investigated the cellular response of the spleen to brain ischemia. Hypothesis: Immune cells are released from the spleen following cerebral ischemia contributing to neural injury. Methods: A time course was conducted to investigate splenic size changes in rats over time following permanent middle cerebral artery occlusion (MCAO) and sham-MCAO (n≥3). In a second experiment, splenocytes were labeled in situ with carboxyfluorescein diacetate, succinimidyl ester (CFSE). Five evenly spaced splenic injections, 4 mg/ml in DMSO, were given to all groups five days prior to MCAO or sham-MCAO. The experimental groups were: 48 hr post-MCAO (n=4), 48 hr post-sham-MCAO (n=4), 96 hr post-MCAO (n=6), 96 hr post-sham-MCAO (n=4), and a CFSE only group that was euthanized five days following injection of CFSE without undergoing MCAO (n=4). Spleen, brain, thymus, and blood smears were collected for all groups. Results: The spleen was found to decrease in size at 48 hrs following MCAO in rats compared to controls (p<0.01). However, by 96 hrs post-MCAO the spleen size returned to levels not different from sham operated rats. CFSE was found to be non-toxic at the 4 mg/ml concentration and five days following injection 15% of the splenocytes were labeled. In the spleen, there was a significant increase (p<0.0001) in CFSE positive cells in the 48 hr sham group versus the 96 hr sham and both 48 and 96 hr MCAO groups. Blood smears showed a significant increase in total CFSE positive cells (p<0.0007) at 48 hrs post-MCAO. CFSE positive cells in the blood were identified by Giemsa staining. A significant increase of lymphocytes (p<0.005), monocytes (p<0.02), and neutrophils (p<0.0005) was found at 48 hrs post-MCAO when compared to the other groups. Conclusion: These results demonstrate that CFSE is a viable and safe way to track immune cells in situ in an animal model of stroke. The spleen transiently decreases in size post-MCAO in rats and releases splenocytes into systemic circulation at 48 hrs following MCAO. These cells are migrating to the brain or other lymphoid organs, affecting the overall immune response to ischemia. Brains and thymi from the CFSE injected animals will be analyzed for the presence of these cells.