Abstract TP295: Temporal Characterization of Immune Responses in the Rat Brain Following Ischemic Stroke Using In-Depth Flow Cytometry Panels

Abstract

Introduction: Rat models are widely used in stroke research as they are well suited for behavior studies and brain imaging techniques. However, methods for studying immune responses and identifying distinct leukocyte subsets in rats lag behind mice in part due to lack of specific antibodies for flow cytometry. Several flow cytometry antibody panels have been used to study the immune response of the rat brain after stroke, but they did not distinguish between pro- and anti-inflammatory macrophages, which is critical for stroke-induced neuroinflammation research. Here, we developed flow cytometry panels to measure myeloid and lymphoid leukocyte subsets in rat brain, blood, and spleen, with an emphasis on macrophage subtypes, and validated them in an ischemic stroke model. Method: Adult male (8-12 wk old) Nude and Sprague Dawley (SD) rats were subjected to distal MCAO. Ipsi- and contralateral hemispheres, blood, and spleen were collected and analyzed by flow cytometry at different time points post-stroke. Cell populations were sorted using FACS and characterized using qPCR or morphological analysis to confirm our FACS gating strategy. Results: We discriminated microglia, 3 monocyte/macrophage subsets, granulocytes, dendritic cells, NK cells, B cells, and T cells in the brain and periphery. The temporal profile of leukocyte subsets after stroke resembled those observed in mice, validating the reliability of our antibody panels: granulocytes and monocytes peaked in the first 2-3 days post-stroke, B and T cells peaked around 4-5 days, followed by a gradual increase of ‘microglia’ over 14 days. Using a combination of CD43 and His48 to differentiate monocyte/macrophage subpopulations, we observed a shift in these subsets over time, akin to the shift from pro- to anti-inflammatory macrophages reported in stroke-injured mouse brains. Notably, Nude rats differed from the SD rats in terms of His48 expression; SD rat brain monocyte/macrophage population was predominantly His48lo, while Nude rats had both His48lo and His48hi subsets. Conclusion: Our flow cytometry panels for rats distinguished leukocyte subsets, including monocyte/macrophage subpopulations, providing a valuable tool to study the complex immune responses in rat models of neurological disorders.