Department of Neuroscience, UConn Health, Farmington, CT 06032, USA.
Calhoun Cardiology Center, UConn Health, Farmington, CT 06032, USA.
Exp Neurol. 2020 Jul;329:113308. doi: 10.1016/j.expneurol.2020.113308. Epub 2020 Apr 11.
Stroke remains a leading cause of disability in the United States. Despite recent advances, interventions to reduce damage and enhance recovery after stroke are lacking. P2X4R, a receptor for adenosine triphosphate (ATP), regulates activation of myeloid immune cells (infiltrating monocytes/macrophages and brain-resident microglia) after stroke injury. However, over-stimulation of P2X4Rs due to excessive ATP release from dying or damaged neuronal cells can contribute to ischemic injury. Therefore, we pharmacologically inhibited P2X4R to limit the over-stimulated myeloid cell immune response and improve both acute and chronic stroke recovery. We subjected 8-12-week-old male and female wild type mice to a 60 min right middle cerebral artery occlusion (MCAo) followed by 3 or 30 days of reperfusion. We performed histological, RNA sequencing, behavioral (sensorimotor, anxiety, and depressive), and biochemical (Evans blue dye extravasation, western blot, quantitative PCR, and flow cytometry) analyses to determine the acute (3 days after MCAo) and chronic (30 days after MCAo) effects of P2X4R antagonist 5-BDBD (1 mg/kg P.O. daily x 3 days post 4 h of MCAo) treatment. 5-BDBD treatment significantly (p < .05) reduced infarct volume, neurological deficit (ND) score, levels of cytokine interleukin-1 beta (IL-1β) and blood brain barrier (BBB) permeability in the 3-day group. Chronically, 5-BDBD treatment also conferred progressive recovery (p < .05) of motor balance and coordination using a rotarod test, as well as reduced anxiety-like behavior over 30 days. Interestingly, depressive-type behavior was not observed in mice treated with 5-BDBD for 3 days. In addition, flow cytometric analysis revealed that 5-BDBD treatment decreased the total number of infiltrated leukocytes, and among those infiltrated leukocytes, pro-inflammatory cells of myeloid origin were specifically reduced. 5-BDBD treatment reduced the cell surface expression of P2X4R in flow cytometry-sorted monocytes and microglia without reducing the total P2X4R level in brain tissue. In summary, acute P2X4R inhibition protects against ischemic injury at both acute and chronic time-points after stroke. Reduced numbers of infiltrating pro-inflammatory myeloid cells, decreased surface P2X4R expression, and reduced BBB disruption are likely its mechanism of neuroprotection and neuro-rehabilitation.
中风仍然是美国导致残疾的主要原因。尽管最近取得了进展，但缺乏减少中风后损伤和促进恢复的干预措施。P2X4R 是三磷酸腺苷（ATP）的受体，可调节中风损伤后髓样免疫细胞（浸润的单核细胞/巨噬细胞和脑驻留的小胶质细胞）的激活。然而，由于死亡或受损神经元细胞中过多的 ATP 释放，过度刺激 P2X4Rs 可能导致缺血性损伤。因此，我们通过药理学抑制 P2X4R 来限制过度刺激的髓样细胞免疫反应，并改善急性和慢性中风后的恢复。我们使 8-12 周龄的雄性和雌性野生型小鼠经历 60 分钟的右侧大脑中动脉闭塞（MCAo），然后再进行 3 或 30 天的再灌注。我们进行了组织学、RNA 测序、行为（感觉运动、焦虑和抑郁）和生化（伊文思蓝染料渗出、western blot、定量 PCR 和流式细胞术）分析，以确定 P2X4R 拮抗剂 5-BDBD（1mg/kg PO 每天 x 4h MCAo 后 3 天）治疗的急性（MCAo 后 3 天）和慢性（MCAo 后 30 天）影响。5-BDBD 治疗显著（p<0.05）降低了梗塞体积、神经功能缺损（ND）评分、细胞因子白细胞介素 1β（IL-1β）水平和血脑屏障（BBB）通透性在 3 天组。慢性组中，5-BDBD 治疗还通过旋转棒测试逐渐改善了运动平衡和协调（p<0.05），并在 30 天内减轻了焦虑样行为。有趣的是，在接受 5-BDBD 治疗 3 天的小鼠中未观察到抑郁样行为。此外，流式细胞术分析显示，5-BDBD 治疗减少了浸润白细胞的总数，并且在浸润白细胞中，髓样来源的促炎细胞特异性减少。5-BDBD 治疗减少了流式细胞术分选的单核细胞和小胶质细胞中 P2X4R 的细胞表面表达，而不降低脑组织中的总 P2X4R 水平。总之，急性 P2X4R 抑制可在中风后急性和慢性时间点均起到保护作用。浸润的促炎髓样细胞数量减少、表面 P2X4R 表达减少和 BBB 破坏减少可能是其神经保护和神经康复的机制。
