基于eNOS功能障碍创建可逆性脑血管收缩综合征(RCVS)小鼠模型的研究

Reversible cerebral vasoconstriction syndrome (RCVS) is characterized by recurrent headaches and reversible segmental cerebral vasoconstriction. Its etiology and pathogenesis remain unknown. RCVS is clinically and radiologically complex and volatile and there are no animal models to study so far. There are also no effective treatments for RCVS and severe cases are fatal. Recently, we have demonstrated in mice that targeted deletion of endothelial nitric oxide synthase (eNOS) gene results in multifocal cerebral ischemia/infarctions, blood-brain barrier (BBB) breakdown, cerebral/subarachnoid hemorrhages, and abnormal segmental narrowing or dilatation of cerebral arteries. These findings are consistent with the typical features of head computed tomography (CT)/magnetic resonance imaging (MRI) and cerebral angiography in patients with RCVS. We propose that decreased endothelial nitric oxide (NO) due to eNOS dysfunctions plays a key role in the development of RCVS. Accordingly, in this project, the homozygous and heterozygous eNOS knockout mice and different dose eNOS inhibitor-treated wild-type mice will be used to make the experimental models. Using fluorescent angiography of the whole brain, the location and severity of cerebral vasoconstriction in the mouse models will be scored and correlated with cerebral perfusion and BBB. Angiopathies of the brain, if any, will be determined by pathohistology. Combined with serial magnetic resonance angiography (MRA) of the brain, headache behavior evaluation, and the intervention with NO donor and eNOS activator, the RCVS mouse model established on the basis of decreased expression/activity of eNOS will be validated and standardized. Starting from endothelial NO and focusing on the neurovascular unit that regulates and maintains cerebrovascular tone and BBB, we will be able to elucidate the cellular and the underlying molecular mechanisms in eNOS dysfunction-induced RCVS. From this project, it is expected to provide new theory, potential targets, and ideal animal model for innovative researches on the prevention and treatment of RCVS.

可逆性脑血管收缩综合征（RCVS）是一组以反复头痛和可逆性节段性脑血管收缩为特征的综合征。因其病因和发病机制不明，临床和影像学表现复杂多变，迄今无实验动物模型，亦无有效治疗方法，重者可致死。我们最近的研究发现，敲除小鼠内皮一氧化氮合酶（eNOS）基因可导致多灶性脑缺血或梗死、血-脑屏障破坏、脑或蛛网膜下腔出血及多发性节段性脑动脉异常狭窄或扩张，其表现与RCVS患者典型的头部CT/MRI和脑血管造影所见一致。我们推测，eNOS功能障碍使内皮一氧化氮（NO）减少在RCVS的发生发展中起关键作用。据此本项目拟用eNOS基因敲除纯合子及杂合子小鼠和不同剂量eNOS抑制剂处理野生型小鼠制备实验模型，荧光全脑血管造影和神经病理分析脑血管狭窄或扩张的部位、性质和严重程度及其对脑灌注和血-脑屏障的影响，结合动态磁共振血管造影（MRA）、头痛行为学评估、NO供体及eNOS增强剂等的干预效应，鉴定和标准化基于eNOS表达或活性降低创建的RCVS小鼠模型，并以内皮NO为切入点，围绕调控和维持脑血管张力及血-脑屏障的神经血管单元（NVU），探讨eNOS功能障碍介导RCVS的细胞及分子机制，为RCVS的防治研究提供新理论、新靶点和理想的动物模型。

可逆性脑血管收缩综合征(RCVS)以反复头痛和血管壁组织病理学正常的脑血管可逆性节段性收缩为其临床病理特征，其病因和发病机制不明，临床和影像学表现复杂多变，迄今无实验动物模型，亦无有效治疗方法，重者可致死。. 本研究采用内皮一氧化氮合酶(eNOS)基因敲除纯合子(eNOS-KO)和杂合子(eNOS-Het)小鼠制备实验模型，以野生型(eNOS-WT)小鼠为对照，结合荧光全脑血管造影、神经病理、免疫组化和Western blot等方法，鉴定和筛选基于eNOS表达和活性降低创建的RCVS小鼠模型，并探讨其病理生理机制。. 主要结果显示：(1)小鼠eNOS表达降低可导致脑血管节段性狭窄和/或扩张，脑血管造影典型表现为“串珠样”，异常血管主要见于颈内动脉和椎-基底动脉系统远心段和终末段,其严重程度与eNOS表达水平负相关，但与年龄和性别无显著相关性；(2) eNOS表达降低小鼠脑部节段性异常血管常并发局灶性脑缺血/梗死和血-脑屏障破坏/脑或蛛网膜下腔出血，典型表现为微梗死和微出血，脑梗死和微量出血灶的数量与eNOS表达水平负相关，与年龄呈正相关；(3) 3月龄eNOS表达降低小鼠脑部节段性异常血管管壁的病理组织学无明显异常，内弹力膜完整，血管平滑肌无明显增厚或变薄，无炎症细胞浸润、动脉粥样硬化和淀粉样蛋白沉积等改变，但6月龄和12月龄eNOS表达降低小鼠脑部血管可见局灶性动脉内膜增厚/毛糙、动脉粥样硬化斑块形成和β-淀粉样蛋白沉积，年龄越大越明显,且与eNOS表达水平负相关。研究结果提示：eNOS表达和活性降低与RCVS的发生密切相关，3月龄eNOS-KO小鼠可高度模拟人类RCVS的神经影像和病理组织学改变。我们用eNOS增强剂治疗1例经脑活检确诊的重症RCVS患者取得满意效果提示，该小鼠模型对阐明RCVS的发病机制、发现药物新靶点及临床前药效评价具有重要的科学意义和广阔的应用前景。

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