Department of Neurosurgery, University of Florida, Gainesville, FL, USA.
Department of Physiologic Sciences, University of Florida, Gainesville, FL, USA.
J Neuroinflammation. 2022 Sep 16;19(1):228. doi: 10.1186/s12974-022-02592-x.
Cerebral vasospasm (CV) can contribute to significant morbidity in subarachnoid hemorrhage (SAH) patients. A key unknown is how CV induction is triggered following SAH.
Human aneurysmal blood and cerebral spinal fluid were collected for evaluation. To confirm mechanism, c57/bl6 wild type and c57/bl6 IL-6 female knockout (KO) mice were utilized with groups: saline injected, SAH, SAH + IL-6 blockade, SAH IL-6 KO, SAH IL-6 KO + IL-6 administration, SAH + p-STAT3 inhibition. Dual-labeled microglia/myeloid mice were used to show myeloid diapedesis. For SAH, 50 μm blood was collected from tail puncture and administered into basal cisterns. IL-6 blockade was given at various time points. Various markers of neuroinflammation were measured with western blot and immunohistochemistry. Cerebral blood flow was also measured. Vasospasm was measured via cardiac injection of India ink/gelatin. Turning test and Garcia's modified SAH score were utilized. P < 0.05 was considered significant.
IL-6 expression peaked 3 days following SAH (p < 0.05). Human IL-6 was increased in aneurysmal blood (p < 0.05) and in cerebral spinal fluid (p < 0.01). Receptor upregulation was periventricular and perivascular. Microglia activation following SAH resulted in increased caveolin 3 and myeloid diapedesis. A significant increase in BBB markers endothelin 1 and occludin was noted following SAH, but reduced with IL-6 blockade (p < 0.01). CV occurred 5 days post-SAH, but was absent in IL-6 KO mice and mitigated with IL-6 blockade (p < 0.05). IL-6 blockade, and IL-6 KO mitigated effects of SAH on cerebral blood flow (p < 0.05). SAH mice had impaired performance on turn test and poor modified Garcia scores compared to saline and IL-6 blockade. A distinct microglia phenotype was noted day 5 in the SAH group (overlap coefficients r = 0.96 and r = 0.94) for Arg1 and iNOS, which was altered by IL-6 blockade. Day 7, a significant increase in toll-like receptor 4 and Stat3 was noted. This was mitigated by IL-6 blockade and IL-6 KO, which also reduced Caspase 3 (p < 0.05). To confirm the mechanism, we developed a p-STAT3 inhibitor that targets the IL-6 pathway and this reduced NFΚB, TLR4, and nitrotyrosine (p < 0.001). Ventricular dilation and increased Tunel positivity was noted day 9, but resolved by IL-6 blockade (p < 0.05).
Correlation between IL-6 and CV has been well documented. We show that a mechanistic connection exists via the p-STAT3 pathway, and IL-6 blockade provides benefit in reducing CV and its consequences mediated by myeloid cell origin diapedesis.
蛛网膜下腔出血（SAH）后脑血管痉挛（CV）可导致显著的发病率。一个关键的未知因素是 SAH 后如何引发 CV 诱导。
收集人动脉瘤血液和脑脊液进行评估。为了证实机制，利用 c57/bl6 野生型和 c57/bl6 IL-6 雌性敲除（KO）小鼠进行分组：盐水注射、SAH、SAH+IL-6 阻断、SAH IL-6 KO、SAH IL-6 KO+IL-6 给药、SAH+p-STAT3 抑制。使用双标记小胶质细胞/髓样细胞小鼠显示髓样细胞穿出。对于 SAH，从尾穿刺收集 50μm 的血液并注入基底池。IL-6 阻断在不同时间点给予。通过 Western blot 和免疫组织化学测量各种神经炎症标志物。还测量了脑血流。通过心脏注射印度墨水/明胶测量血管痉挛。使用转体试验和加西亚改良的 SAH 评分。p<0.05 被认为具有统计学意义。
IL-6 表达在 SAH 后 3 天达到峰值（p<0.05）。人 IL-6 在动脉瘤血液中增加（p<0.05）和脑脊液中增加（p<0.01）。受体上调位于脑室周围和血管周围。SAH 后小胶质细胞激活导致 caveolin 3 和髓样细胞穿出增加。SAH 后内皮素 1 和闭合蛋白的 BBB 标志物显著增加，但 IL-6 阻断后减少（p<0.01）。CV 发生在 SAH 后 5 天，但在 IL-6 KO 小鼠中不存在，并且 IL-6 阻断减轻了 CV（p<0.05）。IL-6 阻断和 IL-6 KO 减轻了 SAH 对脑血流的影响（p<0.05）。与盐水和 IL-6 阻断相比，SAH 小鼠在转体试验中表现出受损的性能和较差的改良加西亚评分。在 SAH 组第 5 天观察到明显的小胶质细胞表型（Arg1 和 iNOS 的重叠系数 r=0.96 和 r=0.94），IL-6 阻断改变了这种表型。第 7 天，观察到 Toll 样受体 4 和 Stat3 的显著增加。IL-6 阻断和 IL-6 KO 减轻了这种情况，还减少了 Caspase 3（p<0.05）。为了证实这一机制，我们开发了一种针对 IL-6 途径的 p-STAT3 抑制剂，该抑制剂降低了 NFΚB、TLR4 和硝基酪氨酸（p<0.001）。第 9 天观察到脑室扩张和 Tunel 阳性增加，但 IL-6 阻断后得到缓解（p<0.05）。
IL-6 与 CV 之间的相关性已得到充分证实。我们表明，存在一种通过 p-STAT3 途径的机制联系，并且 IL-6 阻断通过减少由髓样细胞起源的穿出介导的 CV 及其后果提供益处。
Zotero 插件
