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

描述（由申请人提供）：缺氧是胎儿脑损伤的主要原因，包括脑瘫（CP）。最近的报道表明Rho/Rho激酶在神经元变性中的作用，我们的实验室已经表明，慢性缺氧诱导的胎儿脑损伤与Rho家族基因激活有关。这些胎儿特异性缺氧触发过程的机制知之甚少。我们假设，慢性缺氧，开始早产，导致胎儿脑损伤通过激活Rho/Rho激酶通过一氧化氮（NO）-丝裂原活化蛋白激酶（MAPKs）信号通路，是一个适应性反应的例子，在常见的子宫内/产时条件下，成为适应不良。我们使用慢性缺氧胎鼠模型和缺氧原代细胞培养物加上ROCK敲除小鼠模型来测试在3个特定目标下进行的实验中的假设：特定目标1：测试慢性缺氧诱导的脑损伤与细胞特异性方式的Rho/Rho激酶激活相关的假设。将产生胎儿慢性缺氧小鼠，将在两个时间点（10.5%O2-HPX-5d和10.5%O2-HPX-新生儿）研究慢性缺氧信号级联基因（Rho家族基因、Rho激酶、NOS、MAPK、ICAM-1、NFAT）。将通过双荧光免疫染色研究Rho基因和细胞类型（神经元或神经胶质）的共定位。将通过缺氧原代细胞模型（神经元和神经胶质）（1%O2，12小时）确认体内研究。具体目标2：在原代细胞培养物中检验慢性缺氧通过NO-MAPK途径激活Rho/Rho激酶途径，以细胞特异性方式引起神经胶质和神经元损伤的假设。将原代小鼠神经元和神经胶质细胞在1%O2下培养12小时。本论文将通过对Rho家族基因、Rho激酶、NOS亚型、MAPKs等信号元件的抑制和激活来研究Rho/Rho激酶及其信号级联调节的机制。Rho介导的替代介质（ICAM-1，NFAT）将在缺氧细胞模型中进行初步研究。具体目的3：通过使用ROCK敲除模型来检验Rho是慢性缺氧信号级联中的关键起始物的假设。本研究将利用基因工程小鼠研究慢性缺氧信号介质（Rho家族基因、Rho激酶、总NO、NOS、NO活性、MAPK、细胞粘附标志物（ICAM-1）、细胞骨架调节因子（NFAT）和胎脑损伤指标，以证实Rho/Rho激酶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，其机制仍然知之甚少。这些实验将增加介质和相互作用的知识，这有助于胎儿脑损伤与慢性缺氧，将扩大核心缺氧动物模型的应用，并可能导致治疗干预，改善胎盘植入不良的后遗症。 公共卫生相关性：在初步研究中，我们发现慢性缺氧在几种哺乳动物中触发Rho/Rho激酶。我们建议在小鼠中测试以下假设：从早产开始的慢性缺氧通过上调MAPK介导的经由Rho/Rho激酶激活的NO释放来触发胎儿脑损伤。该研究将增加对胎儿慢性缺氧过程中新介质及其相互作用的认识。

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.