Department of Neurology, University of California, San Francisco and the San Francisco Veteran's Affairs Medical Center, San Francisco, California, United States of America.
PLoS One. 2013 Aug 5;8(8):e70927. doi: 10.1371/journal.pone.0070927. Print 2013.
Microglia are among the first immune cells to respond to ischemic insults. Triggering of this inflammatory response may involve the microglial purinergic GPCR, P2Y12, activation via extracellular release of nucleotides from injured cells. It is also the inhibitory target of the widely used antiplatelet drug, clopidogrel. Thus, inhibiting this GPCR in microglia should inhibit microglial mediated neurotoxicity following ischemic brain injury.
Experimental cerebral ischemia was induced, in vitro with oxygen-glucose deprivation (OGD), or in vivo via bilateral common carotid artery occlusion (BCCAO). Genetic knock-down in vitro via siRNA, or in vivo P2Y12 transgenic mice (P2Y12-/- or P2Y12+/-), or in vivo treatment with clopidogrel, were used to manipulate the receptor. Neuron death, microglial activation, and microglial migration were assessed.
The addition of microglia to neuron-astrocyte cultures increases neurotoxicity following OGD, which is mitigated by microglial P2Y12 deficiency (P<0.05). Wildtype microglia form clusters around these neurons following injury, which is also prevented in P2Y12 deficient microglia (P<0.01). P2Y12 knock-out microglia migrated less than WT controls in response to OGD-conditioned neuronal supernatant. P2Y12 (+/-) or clopidogrel treated mice subjected to global cerebral ischemia suffered less neuronal injury (P<0.01, P<0.001) compared to wild-type littermates or placebo treated controls. There were also fewer microglia surrounding areas of injury, and less activation of the pro-inflammatory transcription factor, nuclear factor Kappa B (NFkB).
P2Y12 participates in ischemia related inflammation by mediating microglial migration and potentiation of neurotoxicity. These data also suggest an additional anti-inflammatory, neuroprotective benefit of clopidogrel.
小胶质细胞是对缺血性损伤最早做出反应的免疫细胞之一。这种炎症反应的触发可能涉及到小胶质细胞嘌呤能 GPCR P2Y12，通过损伤细胞释放的细胞外核苷酸激活。它也是广泛使用的抗血小板药物氯吡格雷的抑制靶点。因此，抑制小胶质细胞中的这种 GPCR 应该可以抑制缺血性脑损伤后的小胶质细胞介导的神经毒性。
通过体外氧葡萄糖剥夺（OGD）或体内双侧颈总动脉闭塞（BCCAO）诱导实验性脑缺血。通过 siRNA 体外基因敲低，或体内 P2Y12 转基因小鼠（P2Y12-/-或 P2Y12+/-），或体内氯吡格雷治疗，来操纵该受体。评估神经元死亡、小胶质细胞活化和小胶质细胞迁移。
将小胶质细胞添加到神经元-星形胶质细胞培养物中会增加 OGD 后的神经毒性，而小胶质细胞 P2Y12 缺乏可减轻这种毒性（P<0.05）。在损伤后，野生型小胶质细胞在这些神经元周围形成簇，而在 P2Y12 缺乏的小胶质细胞中则不会（P<0.01）。在 OGD 条件下神经元上清液刺激下，P2Y12 敲除小胶质细胞的迁移能力低于 WT 对照。与野生型同窝仔或安慰剂治疗对照组相比，接受全脑缺血的 P2Y12（+/-）或氯吡格雷治疗的小鼠神经元损伤更少（P<0.01，P<0.001）。损伤周围的小胶质细胞也更少，促炎转录因子核因子 Kappa B（NFkB）的激活也更少。
P2Y12 通过介导小胶质细胞迁移和增强神经毒性参与缺血相关炎症。这些数据还表明氯吡格雷具有额外的抗炎、神经保护作用。
