Selective knockout of astrocytic Na(+) /H(+) exchanger isoform 1 reduces astrogliosis, BBB damage, infarction, and improves neurological function after ischemic stroke.
Selective knockout of astrocytic Na(+) /H(+) exchanger isoform 1 reduces astrogliosis, BBB damage, infarction, and improves neurological function after ischemic stroke.
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作者：
Begum G;Song S;Wang S;Zhao H;Bhuiyan MIH;Li E;Nepomuceno R;Ye Q;Sun M;Calderon MJ;Stolz DB;St Croix C;Watkins SC;Chen Y;He P;Shull GE;Sun D
星形胶质细胞中Na+/H+交换体亚型1（NHE 1）的刺激导致缺血条件下的离子失调。在这项研究中，我们创建了一个Nhe 1flox/flox（Nhe 1f/f）小鼠系，Nhe 1的外显子5两侧有两个loxP位点，通过将Nhe 1f/f小鼠与Gfap-CreERT 2 Cre重组酶小鼠杂交，实现了星形胶质细胞中Nhe 1的选择性消融。将出生后第60-90天的Gfap-CreERT 2 +/-; Nhe 1f/f小鼠用玉米油或他莫昔芬（Tam，75 mg/kg/天，i. p.）连续5d注射后30天，小鼠经历短暂的大脑中动脉闭塞（tMCAO）以诱导缺血性中风。与油-溶剂组（对照）相比，TAM处理的Gfap-CreERT 2 +/-; Nhe 1f/f（Nhe 1 KO）小鼠在tMCAO后1-5天发生的缺血性梗死显著更小，水肿更少，神经功能缺损更少。免疫细胞化学分析显示Nhe 1基因敲除小鼠脑内星形胶质细胞增殖、细胞肥大和病变周围神经胶质增生较少。选择性缺失星形胶质细胞中的Nhe 1也减少了缺血脑中的脑微血管损伤和血脑屏障（BBB）损伤。对照脑的BBB微血管显示肿胀的内皮细胞、开放的紧密连接、促炎蛋白酶MMP-9的表达增加和紧密连接蛋白occludin的显著损失。相比之下，Nhe 1 KO小鼠表现出减少的BBB破坏和正常的紧密连接结构，具有增加的occludin表达和减少的MMP-9。最重要的是，星形胶质细胞Nhe 1基因的缺失显著增加了MCAO后24小时缺血半球的局部脑血流量。总之，我们的研究提供了星形胶质细胞NHE 1蛋白在反应性星形胶质细胞增生和缺血性神经血管损伤中的致病作用的第一条证据。
Stimulation of Na+/H+ exchanger isoform 1 (NHE1) in astrocytes causes ionic dysregulation under ischemic conditions. In this study, we created a Nhe1flox/flox (Nhe1f/f) mouse line with exon 5 of Nhe1 flanked with two loxP sites and selective ablation of Nhe1 in astrocytes was achieved by crossing Nhe1f/f mice with Gfap-CreERT2 Cre-recombinase mice. Gfap-CreERT2+/-;Nhe1f/f mice at postnatal day 60-90 were treated with either corn oil or tamoxifen (Tam, 75mg/kg/day, i.p.) for 5 days. After 30-day post injection, mice underwent transient middle cerebral artery occlusion (tMCAO) to induce ischemic stroke. Compared to the oil-vehicle group (control), Tam-treated Gfap-CreERT2+/-;Nhe1f/f (Nhe1 KO) mice developed significantly smaller ischemic infarction, less edema, and less neurological function deficits at 1-5 days after tMCAO. Immunocytochemical analysis revealed less astrocytic proliferation, less cellular hypertrophy, and less peri-lesion gliosis in Nhe1 KO mouse brains. Selective deletion of Nhe1 in astrocytes also reduced cerebral micro-vessel damage and blood-brain barrier (BBB) injury in ischemic brains. The BBB micro-vessels of the control brains show swollen endothelial cells, opened tight junctions, increased expression of pro-inflammatory protease MMP-9 and significant loss of tight junction protein occludin. In contrast, the Nhe1 KO mice exhibited reduced BBB breakdown and normal tight junction structure, with increased expression of occludin and reduced MMP-9. Most importantly, deletion of astrocytic Nhe1 gene significantly increased regional cerebral blood flow in the ischemic hemisphere at 24 hours post-MCAO. Taken together, our study provides the first line of evidence for a causative role of astrocytic NHE1 protein in reactive astrogliosis and ischemic neurovascular damage.