Department of Neurological Surgery, Washington University School of Medicine, 660 S, Euclid Ave,, Box 8057, St, Louis, MO 63110, USA.
J Neuroinflammation. 2012 Feb 16;9:33. doi: 10.1186/1742-2094-9-33.
A brief exposure to systemic hypoxia (i.e., hypoxic preconditioning; HPC) prior to transient middle cerebral artery occlusion (tMCAo) reduces infarct volume, blood-brain barrier disruption, and leukocyte migration. CCL2 (MCP-1), typically regarded as a leukocyte-derived pro-inflammatory chemokine, can also be directly upregulated by hypoxia-induced transcription. We hypothesized that such a hypoxia-induced upregulation of CCL2 is required for HPC-induced ischemic tolerance.
Adult male SW/ND4, CCL2-null, and wild-type mice were used in these studies. Cortical CCL2/CCR2 message, protein, and cell-type specific immunoreactivity were determined following HPC (4 h, 8% O2) or room air control (21% O2) from 6 h through 2 weeks following HPC. Circulating leukocyte subsets were determined by multi-parameter flow cytometry in naïve mice and 12 h after HPC. CCL2-null and wild-type mice were exposed to HPC 2 days prior to tMCAo, with immunoneutralization of CCL2 during HPC achieved by a monoclonal CCL2 antibody.
Cortical CCL2 mRNA and protein expression peaked at 12 h after HPC (both p < 0.01), predominantly in cortical neurons, and returned to baseline by 2 days. A delayed cerebral endothelial CCL2 message expression (p < 0.05) occurred 2 days after HPC. The levels of circulating monocytes (p < 0.0001), T lymphocytes (p < 0.0001), and granulocytes were decreased 12 h after HPC, and those of B lymphocytes were increased (p < 0.0001), but the magnitude of these respective changes did not differ between wild-type and CCL2-null mice. HPC did decrease the number of circulating CCR2+ monocytes (p < 0.0001) in a CCL2-dependent manner, but immunohistochemical analyses at this 12 h timepoint indicated that this leukocyte subpopulation did not move into the CNS. While HPC reduced infarct volumes by 27% (p < 0.01) in wild-type mice, CCL2-null mice subjected to tMCAo were not protected by HPC. Moreover, administration of a CCL2 immunoneutralizing antibody prior to HPC completely blocked (p < 0.0001 vs. HPC-treated mice) the development of ischemic tolerance.
The early expression of CCL2 in neurons, the delayed expression of CCL2 in cerebral endothelial cells, and CCL2-mediated actions on circulating CCR2+ monocytes, appear to be required to establish ischemic tolerance to focal stroke in response to HPC, and thus represent a novel role for this chemokine in endogenous neurovascular protection.
短暂性大脑中动脉闭塞（tMCAo）前短暂暴露于全身缺氧（即缺氧预处理；HPC）可减少梗死体积、血脑屏障破坏和白细胞迁移。CCL2（单核细胞趋化蛋白-1；MCP-1）通常被认为是一种白细胞衍生的促炎趋化因子，也可以被缺氧诱导的转录直接上调。我们假设这种缺氧诱导的 CCL2 上调对于 HPC 诱导的缺血耐受是必需的。
本研究使用成年雄性 SW/ND4、CCL2 基因敲除和野生型小鼠。在 HPC（4 小时，8%O2）或空气对照（21%O2）后，通过实时定量 RT-PCR 检测皮质 CCL2/CCR2 信使、蛋白质和细胞类型特异性免疫反应，从 HPC 后 6 小时到 2 周。在 HPC 前 12 小时，通过多参数流式细胞术检测未处理的小鼠和 HPC 后的循环白细胞亚群。在 tMCAo 前 2 天对 CCL2 基因敲除和野生型小鼠进行 HPC，在 HPC 期间通过单克隆 CCL2 抗体实现 CCL2 的免疫中和。
皮质 CCL2 mRNA 和蛋白表达在 HPC 后 12 小时达到峰值（均 p < 0.01），主要在皮质神经元中，并在 2 天内恢复到基线。在 HPC 后 2 天出现延迟的脑内皮细胞 CCL2 信使表达（p < 0.05）。HPC 后 12 小时，循环单核细胞（p < 0.0001）、T 淋巴细胞（p < 0.0001）和嗜中性粒细胞的水平下降，B 淋巴细胞的水平升高（p < 0.0001），但这些变化的幅度在野生型和 CCL2 基因敲除小鼠之间没有差异。HPC 确实以 CCL2 依赖性方式减少了循环 CCR2+单核细胞的数量（p < 0.0001），但在这个 12 小时的时间点的免疫组织化学分析表明，这个白细胞亚群并没有进入中枢神经系统。虽然 HPC 使野生型小鼠的梗死体积减少了 27%（p < 0.01），但 HPC 处理的 CCL2 基因敲除小鼠并未得到保护。此外，在 HPC 前给予 CCL2 免疫中和抗体完全阻断（p < 0.0001 与 HPC 处理的小鼠相比）缺血耐受的发展。
神经元中 CCL2 的早期表达、脑内皮细胞中 CCL2 的延迟表达以及 CCL2 对循环 CCR2+单核细胞的作用，似乎是对 HPC 引起的局灶性卒中建立缺血耐受所必需的，因此，这种趋化因子在中枢神经血管保护中具有新的作用。
