| Background and Purpose—cerebral ischemic stroke is one of the leading cause of death and disability in the world. Post-stroke, immune depression occurs with a corresponding reduction in spleen size and spleen wight, while robust inflammation appears in the injured brain. Stroke also results in multiple organ injuries including the lung, liver, and heart. We used a focal ischemic rat model to investigate brain inflammation and immunosuppression caused by stroke. We also investigate the protective effects of moderate hypothermia (30℃), a robust inhibitor of ischemic infarction, on inflammatory response in the brain, immunosuppression and peripheral organ injury.Methods—focal ischemia in the cerebral cortex was induced by permanent occlusion of the left distal MCA and60min occlusion of the bilateral CCA under intra-ischemic normothermia (37℃) and brief moderate hypothermia (30℃) in SD rats. Firstly, we evaluated rats'neurological scores and measured infarct size by TTC staining. Secondly, to investigate the brain inflammation caused by stroke and the effect of hypothermia on this inflammatory response, we harvested mononuclear cells from brain72h after stroke onset. We evaluated total mononuclear cells number in the brain and used flow cytometry to characterize lymphocyte subpopulations and SIRP+ monocytes/macrophages/macroglias. We then detected CD3+T lymphocytes, CD68+activated microglia/macrophages and MPO+neutrophils infiltration and distribution in the ischemic area using immunostaining. In addition, we used real time RT-PCR to quantify gene expression levels of various cytokines (IL-1β, TNF-α. IL-4, IL-10, IL-2, INF-y), chemokines (MIP-2) and other inflammatory factors (INOS, COX-2) in the brain5,24,72h after stroke. Next, to investigate immunosuppression and peripheral organ injuries induced by stroke, as well as the role of hypothermia, we harvested and counted mononuclear leukocytes from blood, lung and splenocytes72h after stroke onset. We then evaluated lymphocyte subpopulations and absolute number of SERP+monocytes/macrophages in these organs. Moreover, we still used real time RT-PCR to quantify gene expression levels of above9inflammatory factors in the spleen, liver, and lung5,24,72h after stroke onset. At last, to further demonstrated stroke induced immunosuppression and clarified the effect of hypothermia on immunesuppression caused by stroke. We isolated splenocytes from SD rats and cultured at37℃,5%CO2for66h with0μg/ml,1μg/ml,2.5μg/ml and5μg/ml ConA, then detected the proliferative response of splenocytes with a [3H]-thymidine incorporation assay. At the same time, subcutaneous injection of Ovalbumin (OVA) was used to measure a delayed-type hypersensitivity (DTH) reaction in the ears, which represents cell mediated immune function in vivo.Results—after permanent occlusion of the distal middle cerebral artery, rats' neurological severity scores were significantly increased to more than3, while hypothermia significantly improved neurological score, which was no more than2. All animals studied in the nomothermic group had large infarct areas. The average was47.36±3.195%of cortex, while hypothermia significantly reduced the infarct volume to20.33±3.362%of cortex. After72h stroke onset, the total number of mononuclear leukocytes was (0.483±0.0953)×106in the ipsilateral side, which significantly increased compared with (0.173±0.0177)×106of cells in contralateral side in ischemic brain. Among them, the absolute numbers of T lymphocytes, CD4+T lymphocytes and SIRP+monocytes/macrophages/microglias significantly increased. Though rose insignificantly, B lymphocytes, CD4+T lymphocytes, NK cells and NKT cells showed increase trends in absolute numbers. Also, the expression of CD3, CD68and MPO increased in ischemic area, which represents T lymphocytes, activated microglias/macrophages and neutrophils, respectively. Hypothermia treatment decreased the total number of mononuclear leukocytes to (0.245±0.0185)×106. Hypothermia also decreased absolute numbers of T lymphocytes, B lymphocytes, CD4+T lymphocytes and SIRP+monocytes/macrophages/microglias significantly. In addition, in ischemic area, the expression of CD3, CD68and MPO decreased obviously. Stroke increased gene expression of EL-1β, TNF-α, IL-2, INF-γ, IL-10, MIP-2, iNOS and tended to increase IL-4and COX-2in the brain at least at one or more time points. Hypothermia inhibited expression of the former group, but had no significant effect on the latter two genes. No significant changes in gene expression were seen in the spleen, liver and lung after stroke. Also, hypothermia had no effect on these changes. In the blood and spleen, total mononuclear leukocytes number were (5.95±0.449)×10and (245.91±22.595) x106in normothermic sham group, respectively, while the numbers were reduced by stroke, which were (3.56±0.427) x105and (140.94±18.270)×106, respectively. However, this reduction was attenuated by hypothermia, the total mononuclear leukocytes number were (4.55±0.488) x106and (206.94±32.699)×106in blood and spleen, respectively. In the blood, the absolute number of T cells, B cells, CD4+and CD8+T cells decreased after stroke, and hypothermia significantly attenuated this effect. In the spleen, except T cells, B cells, CD4+and CD8+T cells, NK, NKT and SIRP+monocytes/macrophages also decreased after stroke, hypothermia attenuated the decrease. However, in the lung, both stroke and hypothermia didn't affect the total number and all cell subpopulations. Furthermore, stroke significantly inhibited splenocytes proliferation in vitro. The CPM of splenocytes proliferative activity stimulated by1μg/ml,2.5μg/ml and5μg/ml ConA in normothermic sham group and normothermic stroke group were63850±7185vs18886±5360,48577±8552vs22591±5853and31366±5252vs10137±1824, respectively. The inhibitory effect of stroke on splenocytes proliferation was significantly reversed by hypothermia. The CPM of hypothermic stroke group were40017±8418,40594±3131and21582±3435, respectively. At last, the DTH reaction was also inhibited by normothermic stroke and hypothermia attenuated the inhibition. The thicknesses of ear pinna were1.391±0.0799mm vs0.996±0.0051mm in normothermic sham group and normothermic stroke group, respectively, while in hypothermic stroke group, it was1.077±0.0356mm.Conclusions—we further demonstrated that stroke results in robust brain inflammation and induces peripheral immunosuppression comprehensively and systematically. We found that hypothermia protected against acute inflammation in the ischemic brain. We reported, for the first time, that brief, intra-ischemic moderate hypothermia did not result in immune suppression directly and worsen immune suppression induced by stroke. Instead, it attenuated stroke-induced immune dysfunction and did not damage peripheral organs directly or by cooperating with stroke. Our study offers strong evidence to support clinical translation of intra-ischemic hypothermia. However, we did not find peripheral organs injuries after stroke due to our dMCAo model. |
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