Chronic Hypoxia Mediated Fetal Brain Injury is Associated with a Rho/Rho-kinase

描述(申请人提供)：缺氧是胎儿脑损伤的主要原因，包括脑性瘫痪(CP)。最近的报道表明Rho/Rho-Kinase在神经元退行性变中起作用，我们的实验室已经证明慢性缺氧所致的胎儿脑损伤与Rho家族基因的激活有关。这些胎儿特有的缺氧触发过程背后的机制还知之甚少。我们假设，从早产开始的慢性低氧通过一氧化氮(NO)-丝裂原激活蛋白激酶(MAPKs)信号通路激活Rho/Rho-Kinase导致胎儿脑损伤，这是一种适应性反应的例子，在常见的宫内/分娩条件下，这种适应性反应变得不适应。我们使用了一个慢性低氧胎鼠模型和低氧原代细胞培养加摇石敲除小鼠模型，在三个特定目标下的实验中验证了这一假说：特定目的1：验证慢性低氧所致脑损伤与Rho/Rho激酶激活以细胞特异性方式相关的假说。在两个时间点(10.5%O2-HPX-5d和10.5%O2-HPX-NEURE)对慢性缺氧信号级联基因(Rho家族基因、Rho蛋白、NOSS、MAPKs、ICAM-1、NFAT)进行研究。Rho基因和细胞类型(神经元或神经胶质细胞)的共定位将通过双荧光免疫染色进行研究。体内研究将通过低氧原代细胞模型(神经元和神经胶质细胞)(1%O2 12小时)来证实。特定目的2：在原代细胞培养中验证慢性低氧通过NO-MAPK通路激活Rho/Rho Kinase通路，以细胞特异性方式引起神经胶质细胞和神经元损伤的假说。原代小鼠神经元和神经胶质细胞将在1%O2中培养12小时。通过抑制/激活Rho家族基因、Rho-激酶、一氧化氮合酶亚型、MAPKs等信号元件来研究Rho/Rho激酶及其在低氧诱导下的信号级联调控的机制。Rho介导的替代介质(ICAM-1、NFAT)将在低氧细胞模型中进行初步研究。具体目的3：利用ROCK基因敲除模型验证Rho是慢性低氧信号级联反应的关键启动者的假设。我们将研究慢性缺氧信号介质(Rho家族基因、Rho激酶、总NO、NOSS、NO活性、MAPKs、细胞黏附标记(ICAM-1)、细胞骨架调节因子(NFAT)和胎脑损伤指数)，利用基因组工程小鼠确认Rho/Rho-Kinase在胎脑损伤中的重要作用。慢性胎儿缺氧是脑损伤的主要风险，其机制仍鲜为人知。这些实验将增加对与慢性低氧相关的胎儿脑损伤的介质和相互作用的了解，将扩大核心低氧动物模型的应用，并可能导致改善胎盘植入不良后遗症的治疗干预。 公共卫生相关性：在初步研究中，我们发现慢性缺氧会在几种哺乳动物中触发Rho/Rho-Kinase。我们建议在小鼠身上验证这一假说，即开始早产的慢性缺氧通过上调MAPKs通过Rho/Rho-Kinase激活介导的NO释放来触发胎儿脑损伤。这项研究将增加对新的介质及其在胎儿慢性缺氧过程中的相互作用的认识。

DESCRIPTION (provided by applicant): Hypoxia is a major cause of fetal brain injury, including cerebral palsy (CP). Recent reports suggest a role for Rho/Rho-kinase in neuronal degeneration, and our laboratory has shown that chronic hypoxia-induced fetal brain injury is associated with Rho family gene activation. The mechanisms underlying these fetal specific hypoxia-triggered processes are poorly understood. We hypothesize that chronic hypoxia, beginning preterm, causes fetal brain injury via activation of Rho/Rho-kinase through the nitric oxide (NO)-mitogen activated protein kinase (MAPKs) signaling pathway, and is an example of an adaptive response that, under common intrauterine/ intrapartum conditions, becomes maladaptive. We use a chronic hypoxia fetal mouse model and hypoxic primary cell cultures plus a ROCK Knock-out mice model to test the hypothesis in experiments performed under 3 specific aims: SPECIFIC AIM 1: Test the hypothesis that chronic hypoxia-induced brain injury is associated with Rho/Rho- kinase activation in a cell-specific manner. A fetal chronic hypoxia mouse will be generated, chronic hypoxia signaling cascade genes (Rho family genes, Rho kinases, NOSs, MAPKs, ICAM-1, NFAT) will be studied at two time points(10.5%O2-HPX-5d, and 10.5%O2-HPX-newborn). Co-localization of Rho genes and cell types (neuron or glial) will be studied by double-fluorescent immunostaining. In vivo studies will be confirmed by hypoxia primary cell models (neuron and glial) (1%O2 for 12 hours). SPECIFIC AIM 2: Test the hypothesis in primary cell cultures that chronic hypoxia activates Rho/Rho Kinase pathway through the NO-MAPK pathway, causing glial and neuronal injury in a cell-specific manner. Primary mouse neuron and glial cells will be cultured at 1%O2 for 12 hours. The mechanism of Rho/Rho kinase and its signaling cascade regulation mediated by hypoxia will be studied by inhibition/activation of these signaling elements (Rho family genes, Rho-kinase, NOS isoforms, MAPKs), respectively. Alternative mediators (ICAM-1, NFAT) mediated by Rho will be studied initially in hypoxia cell models. SPECIFIC AIM 3: Test the hypothesis that Rho is the key initiator in the chronic hypoxia signaling cascade by using a ROCK knock-out model. We will study chronic hypoxia-signaling mediators (Rho family genes, Rho kinase, total NO, NOSs, NO activity, MAPKs, cellular adhesion marker (ICAM-1), cytoskeletal regulator (NFAT) and fetal brain injury indices, using genome engineered mice to confirm the important role Rho/Rho-kinase plays in fetal brain injury. Chronic fetal hypoxia is a major risk for brain injury by mechanisms that are still poorly understood. These experiments will increase knowledge of mediators and interactions which contribute to fetal brain injury associated with chronic hypoxia, will expand applications of the core hypoxia animal model, and potentially lead to therapeutic interventions that ameliorate the sequelae of poor placental implantation. PUBLIC HEALTH RELEVANCE: In preliminary study, we find that chronic hypoxia triggers Rho/Rho-kinase in several mammals. We propose to test the hypothesis in the mouse that chronic hypoxia, beginning preterm, triggers fetal brain injury by up- regulating MAPKs-mediated release of NO via Rho/Rho-kinase activation. The study will increase knowledge of novel mediators and their interactions during fetal chronic hypoxia.