Abstract
BackgroundRemote ischemic preconditioning (RIPC) of a limb has been reported to protect against ischemic stroke. Our previous results demonstrated that the RIPC-mediated neuroprotection is associated with alterations in circulating immune cell populations. Here, we evaluated the effect of the spleen, the largest reservoir of immune cells, on RIPC-mediated neuroprotection against stroke.MethodsNoninvasive RIPC was achieved by four repeated cycles of 5-min blood flow constriction in the hindlimbs using a tourniquet. The blood and spleens were collected before and 1 h and 3 days after preconditioning to analyze the effect of RIPC on the spleen and the correlation between splenic and peripheral lymphocytes. Moreover, spleen weight and splenic lymphocytes were compared in stroke rats with or without RIPC. Finally, splenectomy was made 1 day or 2 weeks before RIPC and 90-min middle cerebral artery occlusion (MCAO). The infarct areas and deficits were assessed. Blood was collected 1 h after RIPC and 3 days after MCAO to explore the impact of splenectomy on RIPC-induced neuroprotection and immune changes. The contralateral and ipsilateral hemispheres were collected 3 days after MCAO to detect the infiltration of immune cells after RIPC and splenectomy.ResultsFlow cytometry analysis demonstrated that the RIPC promptly increased the percentages of CD3+CD8+ cytotoxic T (Tc) cells in the spleen with a relatively delayed elevation in CD3+CD161+ natural killer T (NKT) and CD3−CD45RA+ B lymphocytes. The percentages of circulating lymphocytes are positively correlated with the percentages of splenic lymphocytes in normal rats. Interestingly, RIPC resulted in negative correlations between the percentages of splenic and circulating T lymphocytes, while the correlation between splenic and circulating B lymphocytes remained positive. For animals subjected to RIPC followed by MCAO, RIPC increased splenic volume with an expansion of splenic lymphocytes 3 days after MCAO. Furthermore, the removal of the spleen 1 day or 2 weeks before RIPC and MCAO reduced the protective effect of RIPC on ischemic brain injury and reversed the effects of RIPC on circulating immune cell composition. RIPC significantly reduced brain infiltration of Tc and NKT cells. Prior splenectomy showed no effect on immune cell infiltration after RIPC and stroke.ConclusionThese results reveal an immunomodulatory effect of the spleen, effecting mainly the spleen-derived lymphocytes, during RIPC-afforded neuroprotection against cerebral ischemia.
Highlights
Remote ischemic preconditioning (RIPC) of a limb has been reported to protect against ischemic stroke
remote ischemic preconditioning (RIPC) increased the number of lymphocytes in the spleen In order to identify the effect of RIPC on the spleen, spleens were removed at different time points and splenic lymphocytes were analyzed by flow cytometry (Fig. 1a)
RIPC dramatically increased the B cell percentage in the spleen 3 days after RIPC. These results suggest that RIPC can increase the percentages of splenic lymphocyte populations
Summary
Remote ischemic preconditioning (RIPC) of a limb has been reported to protect against ischemic stroke. Accumulating evidence indicates that brief episodes of ischemic pre-treatment have a protective effect on subsequent cerebral ischemia-reperfusion injury [1,2,3]. Such ischemic preconditioning may vary greatly in terms of location, timing, and duration. We have reported that brief repetitive bilateral upper arm ischemic preconditioning improves cerebral perfusion and reduces recurrent strokes in patients with intracranial arterial stenosis [7]. These clinical evidences suggest a neuroprotective effect of RIPC in stroke. The mechanism underlying RIPC-afforded neuroprotection is not clear
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