Centre for Research in Neuroscience, The Research Institute of the McGill University Health Center, 1650 Cedar Ave., Montreal, Quebec H3G 1A4, Canada.
Centre for Research in Neuroscience, The Research Institute of the McGill University Health Center, 1650 Cedar Ave., Montreal, Quebec H3G 1A4, Canada.
Exp Neurol. 2018 Mar;301(Pt B):120-132. doi: 10.1016/j.expneurol.2017.08.011. Epub 2017 Aug 24.
We studied the expression of pro- and anti-inflammatory molecules in microglia and infiltrating monocyte-derived macrophages after permanent Middle Cerebral Artery Occlusion (pMCAO). LysM-EGFP knock-in mice were used to distinguish between these two cell types, as peripheral myeloid cells are LysM-EGFP, while microglia are not. This was confirmed with P2ry12 (a microglial specific marker), Iba-1 and EGFP immunostaining. The peak of LysM-EGFP myeloid cell infiltration was 72h post-ischemia, and were distributed evenly in the lesion core, surrounded by a dense region of microglia. Flow cytometry showed that a higher percentage of microglia expressed TNF-α at 3 (24.3% vs 1.4%) and 7 (18.8% vs 3.4%) days post-pMCAO as compared to infiltrating macrophages. Microglia and macrophages were purified by fluorescence activated cell sorting 72h post-ischemia to assess the mRNA expression of inflammatory markers. Macrophages upregulated expression of mRNA for arginase-1 (Arg-1) by 1000-fold, and IL-1β by 90-fold as compared to microglia. At the protein level, a significantly number of macrophages expressed Arg-1, while few if any microglia expressed Arg-1. However, IL-1β protein was not detected in macrophages by flow cytometry or immunofluorescence labeling of tissue sections. It was, however, detected in astrocytes along the lesion border. A PCR-array screen of 84 inflammatory genes revealed that pro-inflammatory chemokines and cytokines were predominantly upregulated in macrophages but down-regulated in microglia in the ischemic brain. Our results show clear differences in the inflammatory expression profiles between microglia and macrophages 72h post-ischemia which may shape repair and pro-regenerative mechanisms after stroke.
我们研究了永久性大脑中动脉闭塞（pMCAO）后小胶质细胞和浸润的单核细胞衍生的巨噬细胞中促炎和抗炎分子的表达。LysM-EGFP 敲入小鼠被用于区分这两种细胞类型，因为外周髓样细胞是 LysM-EGFP，而小胶质细胞不是。这一点通过 P2ry12（一种小胶质细胞特异性标记物）、Iba-1 和 EGFP 免疫染色得到了证实。LysM-EGFP 髓样细胞浸润的峰值出现在缺血后 72 小时，均匀分布在病变核心，被密集的小胶质细胞区包围。流式细胞术显示，与浸润的巨噬细胞相比，在 pMCAO 后 3 天（24.3%比 1.4%）和 7 天（18.8%比 3.4%），小胶质细胞中 TNF-α的表达比例更高。缺血后 72 小时通过荧光激活细胞分选分离小胶质细胞和巨噬细胞，以评估炎症标志物的 mRNA 表达。巨噬细胞上调了 Arg-1（Arg-1）的 mRNA 表达 1000 倍，上调了 IL-1β的 mRNA 表达 90 倍，而与小胶质细胞相比。在蛋白质水平上，大量巨噬细胞表达 Arg-1，而很少有小胶质细胞表达 Arg-1。然而，通过流式细胞术或组织切片免疫荧光标记，在巨噬细胞中没有检测到 IL-1β 蛋白。然而，在病变边界的星形胶质细胞中检测到了 IL-1β。对 84 个炎症基因的 PCR 阵列筛选显示，促炎趋化因子和细胞因子主要在上皮细胞中上调，但在缺血大脑中的小胶质细胞中下调。我们的研究结果表明，在缺血后 72 小时，小胶质细胞和巨噬细胞之间的炎症表达谱存在明显差异，这可能会影响中风后的修复和促再生机制。
