Abstract：
Cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL) is a life-threatening, inherited, nonhypertensive arteriole disease of the brain. Therapeutic strategy for CARASIL is limited because its pathogenesis is not clear. We previously reported the first family with CARASIL in China, which involves a high-temperature requirement serine protease gene mutation (HtrA1L364P ). Based on this previous study, we constructed a CARASIL mouse model (Mut-hHtrA1L364P mouse, hereinafter referred to as Mut). This paper aimed to systematically study the behavior, pathology, and molecular biology of Mut mice and explore the pathogenesis and possible therapeutic strategies of CARASIL.
Food maze and water maze experiments were used in the behavioral studies. Pathological studies were carried out by arteriole labeling staining and electron microscopy. The mRNA and protein expression levels of the key factors of TGF-β/Smad signaling pathway (TGF-β, Smad2, Smad3, and Smad4) in the brain of the model mice were detected by immunohistochemistry, real-time quantitative polymerase chain reaction (RT-PCR), and Western blot assay.
The food maze and water maze experiment data showed significant differences between the Mut and wild-type (WT) mice in the first time to find food, the time to contact the escape table for the first time, and the number of times to travel in the escape table quadrant (p < 0.001). The results of vascular labeling staining showed that some small arteries in the brain of Mut mice lost normal structure. The results of electron microscopy showed that the cell morphologies in the cortex and hippocampus of Mut mice were abnormal; the number of synapses was reduced; the walls of capillaries, venules, and arterioles thickened; lumen stenosis and other abnormal phenomenon occurred; and lipofuscin deposition and autophagosomes were found in the hippocampus. Immunohistochemistry, RT-PCR, and Western Blot results showed that the mRNA and protein expression levels of TGF-β, Smad2, and Smad3 in the brain of Mut mice increased to different degrees.
The most significant innovation of this study is the first study on the pathogenesis of CARASIL disease using model animals. The Mut mice can well simulate the pathogenesis of CARASIL in behavioral and pathological aspects. The TGF-β/Smad signaling pathway, which is involved in the pathogenesis of CARASIL, is abnormally upregulated in the brain of Mut mice.
Food maze and water maze experiments were used in the behavioral studies. Pathological studies were carried out by arteriole labeling staining and electron microscopy. The mRNA and protein expression levels of the key factors of TGF-β/Smad signaling pathway (TGF-β, Smad2, Smad3, and Smad4) in the brain of the model mice were detected by immunohistochemistry, real-time quantitative polymerase chain reaction (RT-PCR), and Western blot assay.
The food maze and water maze experiment data showed significant differences between the Mut and wild-type (WT) mice in the first time to find food, the time to contact the escape table for the first time, and the number of times to travel in the escape table quadrant (p < 0.001). The results of vascular labeling staining showed that some small arteries in the brain of Mut mice lost normal structure. The results of electron microscopy showed that the cell morphologies in the cortex and hippocampus of Mut mice were abnormal; the number of synapses was reduced; the walls of capillaries, venules, and arterioles thickened; lumen stenosis and other abnormal phenomenon occurred; and lipofuscin deposition and autophagosomes were found in the hippocampus. Immunohistochemistry, RT-PCR, and Western Blot results showed that the mRNA and protein expression levels of TGF-β, Smad2, and Smad3 in the brain of Mut mice increased to different degrees.
The most significant innovation of this study is the first study on the pathogenesis of CARASIL disease using model animals. The Mut mice can well simulate the pathogenesis of CARASIL in behavioral and pathological aspects. The TGF-β/Smad signaling pathway, which is involved in the pathogenesis of CARASIL, is abnormally upregulated in the brain of Mut mice.
摘要：
伴有皮质下梗死和白质脑病的常染色体隐性遗传性脑动脉病(CARASIL)是一种危及生命的,继承,非高血压性小动脉疾病。CARASIL的治疗策略是有限的，因为其发病机制尚不清楚。我们之前报道了中国第一个患有CARASIL的家庭，其中涉及高温要求丝氨酸蛋白酶基因突变（HtrA1L364P）。根据以前的研究，我们构建了CARASIL小鼠模型（Mut-hHtrA1L364P小鼠，以下简称Mut)。本文旨在系统地研究行为，病理学,并探讨CARASIL的发病机制和可能的治疗策略。
在行为研究中使用了食物迷宫和水迷宫实验。通过小动脉标记染色和电子显微镜进行病理研究。TGF-β/Smad信号通路关键因子的mRNA和蛋白表达水平(TGF-β,通过免疫组织化学检测模型小鼠脑中的Smad2，Smad3和Smad4），实时定量聚合酶链反应(RT-PCR),和蛋白质印迹分析。
食物迷宫和水迷宫实验数据显示，首次发现食物的Mut和野生型（WT）小鼠之间存在显着差异，第一次联系转义表的时间，以及在逃生表象限中旅行的次数(p<0.001)。血管标记染色结果显示Mut小鼠脑内部分小动脉失去正常结构。电镜结果显示,Mut小鼠大脑皮层和海马的细胞形态异常;突触数量减少;毛细血管壁,小静脉,小动脉增厚；管腔狭窄等异常现象；海马中发现脂褐素沉积和自噬体。免疫组织化学,RT-PCR,WesternBlot结果显示TGF-β的mRNA和蛋白表达水平,Smad2、Smad3在Mut小鼠脑内有不同程度的升高。
本研究最重大的创新是首次使用模型动物对CARASIL病的发病机理进行研究。Mut小鼠可以很好地模拟CARASIL在行为和病理方面的发病机理。TGF-β/Smad信号通路,这与CARASIL的发病机理有关，在Mut小鼠的大脑中异常上调。
在行为研究中使用了食物迷宫和水迷宫实验。通过小动脉标记染色和电子显微镜进行病理研究。TGF-β/Smad信号通路关键因子的mRNA和蛋白表达水平(TGF-β,通过免疫组织化学检测模型小鼠脑中的Smad2，Smad3和Smad4），实时定量聚合酶链反应(RT-PCR),和蛋白质印迹分析。
食物迷宫和水迷宫实验数据显示，首次发现食物的Mut和野生型（WT）小鼠之间存在显着差异，第一次联系转义表的时间，以及在逃生表象限中旅行的次数(p<0.001)。血管标记染色结果显示Mut小鼠脑内部分小动脉失去正常结构。电镜结果显示,Mut小鼠大脑皮层和海马的细胞形态异常;突触数量减少;毛细血管壁,小静脉,小动脉增厚；管腔狭窄等异常现象；海马中发现脂褐素沉积和自噬体。免疫组织化学,RT-PCR,WesternBlot结果显示TGF-β的mRNA和蛋白表达水平,Smad2、Smad3在Mut小鼠脑内有不同程度的升高。
本研究最重大的创新是首次使用模型动物对CARASIL病的发病机理进行研究。Mut小鼠可以很好地模拟CARASIL在行为和病理方面的发病机理。TGF-β/Smad信号通路,这与CARASIL的发病机理有关，在Mut小鼠的大脑中异常上调。
