■GPR65(G蛋白偶联受体65)可以感知细胞外酸性环境以调节病理生理过程。GPR65激动剂BTB09089的预处理已被证明在急性缺血性中风中产生神经保护作用。然而,延迟BTB09089治疗和神经元GPR65激活是否促进神经恢复尚不清楚.
■在野生型(WT)或GPR65敲除(GPR65-/-)小鼠中通过光血栓性缺血诱导缺血性中风。在中风后第3、7或14天,每隔一天对雄性小鼠腹膜内注射BTB09089。AAV-Syn-GPR65(腺相关病毒-突触素-GPR65)用于在GPR65-/-和WT小鼠的梗死周围皮质神经元中过表达GPR65。通过网格行走和气缸测试监测电机功能。通过免疫组织化学观察BTB09089的神经修复作用。高尔基-考克斯染色,和西方印迹。
■BTB09089显着促进WT的运动结果,但在GPR65-/-小鼠中没有,即使BTB09089延迟3至7天。BTB09089在WT中抑制小胶质细胞的活化和神经胶质瘢痕进展,但在GPR65-/-小鼠中不抑制。同时,BTB09089减少WT小鼠的神经元密度下降,但这种益处在GPR65-/-小鼠中被取消,并通过在梗死周围皮质神经元中过度表达GPR65而重新出现.此外,BTB09089增加了GAP43(生长相关蛋白-43)和突触素斑点密度,树突棘密度,树突状分支长度,通过在GPR65-/-小鼠的梗死周围皮质神经元中过度表达GPR65和树突复杂性,伴随着p-CREB(磷酸化cAMP反应元件结合蛋白)水平的升高。此外,通过在WT小鼠梗死周围皮质神经元中过度表达GPR65,BTB09089的治疗窗口延长至第14天.
■我们的研究结果表明,延迟BTB09089治疗可通过激活神经元GRP65改善卒中后神经功能恢复和脑组织修复。GPR65过表达可能是扩大GPR65激动剂用于缺血性卒中后神经康复的治疗时间窗的潜在策略。
UNASSIGNED: GPR65 (G protein-coupled receptor 65) can sense extracellular acidic environment to regulate pathophysiological processes. Pretreatment with the GPR65 agonist BTB09089 has been proven to produce neuroprotection in acute ischemic stroke. However, whether delayed BTB09089 treatment and neuronal GPR65 activation promote neurorestoration remains unknown.
UNASSIGNED: Ischemic stroke was induced in wild-type (WT) or GPR65 knockout (GPR65-/-) mice by photothrombotic ischemia. Male mice were injected intraperitoneally with BTB09089 every other day at days 3, 7, or 14 poststroke. AAV-Syn-GPR65 (adenoassociated virus-synapsin-GPR65) was utilized to overexpress GPR65 in the peri-infarct cortical neurons of GPR65-/- and WT mice. Motor function was monitored by grid-walk and cylinder tests. The neurorestorative effects of BTB09089 were observed by immunohistochemistry, Golgi-Cox staining, and Western blotting.
UNASSIGNED: BTB09089 significantly promoted motor outcomes in WT but not in GPR65-/- mice, even when BTB09089 was delayed for 3 to 7 days. BTB09089 inhibited the activation of microglia and glial scar progression in WT but not in GPR65-/- mice. Meanwhile, BTB09089 reduced the decrease in neuronal density in WT mice, but this benefit was abolished in GPR65-/- mice and reemerged by overexpressing GPR65 in peri-infarct cortical neurons. Furthermore, BTB09089 increased the GAP43 (growth-associated protein-43) and synaptophysin puncta density, dendritic spine density, dendritic branch length, and dendritic complexity by overexpressing GPR65 in the peri-infarct cortical neurons of GPR65-/- mice, which was accompanied by increased levels of p-CREB (phosphorylated cAMP-responsive element-binding protein). In addition, the therapeutic window of BTB09089 was extended to day 14 by overexpressing GPR65 in the peri-infarct cortical neurons of WT mice.
UNASSIGNED: Our findings indicated that delayed BTB09089 treatment improved neurological functional recovery and brain tissue repair poststroke through activating neuronal GRP65. GPR65 overexpression may be a potential strategy to expand the therapeutic time window of GPR65 agonists for neurorehabilitation after ischemic stroke.