China-America Institute of Neuroscience, Beijing Luhe Hospital, Capital Medical University, Beijing, 101100, China.
Department of Pathology and Pathophysiology, School of Basic Medical Sciences, Capital Medical University, Beijing, China.
J Neuroinflammation. 2018 May 28;15(1):167. doi: 10.1186/s12974-018-1190-9.
Remote 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.
Noninvasive 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.
Flow cytometry analysis demonstrated that the RIPC promptly increased the percentages of CD3CD8 cytotoxic T (Tc) cells in the spleen with a relatively delayed elevation in CD3CD161 natural killer T (NKT) and CD3CD45RA 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.
These results reveal an immunomodulatory effect of the spleen, effecting mainly the spleen-derived lymphocytes, during RIPC-afforded neuroprotection against cerebral ischemia.
肢体的远程缺血预处理（RIPC）已被报道可预防缺血性中风。我们之前的研究结果表明，RIPC 介导的神经保护与循环免疫细胞群的改变有关。在这里，我们评估了脾脏作为最大的免疫细胞库对 RIPC 介导的中风神经保护作用的影响。
通过使用止血带对后肢进行 4 个 5 分钟的血流阻断循环，实现非侵入性的 RIPC。在预处理前、预处理后 1 小时和 3 天采集血液和脾脏，以分析 RIPC 对脾脏的影响以及脾脏和外周淋巴细胞之间的相关性。此外，比较了有或没有 RIPC 的中风大鼠的脾脏重量和脾淋巴细胞。最后，在 RIPC 前 1 天或 2 周进行脾切除术，并进行 90 分钟大脑中动脉闭塞（MCAO）。评估梗死面积和缺损。在 RIPC 后 1 小时和 MCAO 后 3 天采集血液，以探讨脾切除术对 RIPC 诱导的神经保护和免疫变化的影响。在 MCAO 后 3 天采集对侧和同侧半球，以检测 RIPC 和脾切除术后免疫细胞的浸润。
流式细胞术分析表明，RIPC 可迅速增加脾脏中 CD3CD8 细胞毒性 T（Tc）细胞的百分比，而 CD3CD161 自然杀伤 T（NKT）和 CD3CD45RA B 淋巴细胞的升高相对较晚。正常大鼠中循环淋巴细胞的百分比与脾脏淋巴细胞的百分比呈正相关。有趣的是，RIPC 导致脾脏和循环 T 淋巴细胞之间的百分比呈负相关，而脾脏和循环 B 淋巴细胞之间的相关性仍为正相关。对于接受 RIPC 后再进行 MCAO 的动物，RIPC 增加了脾脏体积，并在 MCAO 后 3 天扩大了脾脏淋巴细胞。此外，在 RIPC 和 MCAO 前 1 天或 2 周进行脾切除术会降低 RIPC 对缺血性脑损伤的保护作用，并逆转 RIPC 对循环免疫细胞组成的影响。RIPC 可显著减少 Tc 和 NKT 细胞在大脑中的浸润。脾切除术对 RIPC 和中风后免疫细胞浸润无影响。
这些结果揭示了脾脏的免疫调节作用，主要影响 RIPC 介导的脑缺血神经保护作用期间的脾脏衍生淋巴细胞。
