Redox Modulation of Nitric Oxide in Olfactory Dysfunction after Fetal Hypoxia

描述(申请人提供)：研究中枢神经系统的氧化应激和其他机制的最大问题之一是缺乏反映完整动物的动态和生活状态的功能模型。这一问题在围产期研究中被放大，因为胎儿相对难以接近。这项提案引入了一种创新的动物模型系统，它使用嗅觉系统作为通往大脑其余部分的窗口。它指出需要做更多的研究来研究低氧对围产期嗅觉的影响。在脑性瘫痪(CP)患者中，感觉障碍并没有像运动障碍那样受到关注，这为研究胎儿缺氧缺血(H-I)后出生后发育障碍中的感觉障碍问题提供了一个模型。我们的假设是，活性氧(ROS)与一氧化氮的相互作用介导了胎儿H-I所致的神经元损伤。我们将使用基于胎儿H-I中锰离子摄取的功能性磁共振成像MRI来确定完整动物的活体嗅觉神经元功能。一氧化氮(NO)在嗅觉中起核心作用，并被认为是通过氧化还原调节活性氮物种(RNS)的形成而起作用的。这些概念将在最近开发的动物模型中使用多学科方法进行测试，该动物模型表现为早产子宫缺血后的CP表型，模拟急性胎盘功能不全的临床病理生理学。我们先前已经证明，在这个模型中，ROS和RNS是在H-I后在胎儿脑中产生的，母亲给予抗氧化剂可以减轻胎儿脑损伤。第一个目的是描述不同胎龄的胎儿嗅束对H-I的发育易感性。通过流式细胞术和免疫组织化学方法检测嗅上皮和嗅球中的细胞损伤、死亡和凋亡，使用摄锰的功能磁共振成像将与细胞损伤、死亡和细胞凋亡相关。第二个目的是确定ROS和RNS是否介导了胎儿H-I对嗅觉系统的损伤。我们将通过测量超氧化物歧化、脂质过氧化和/或S亚硝化和酪氨酸硝化来检验发育脆弱性是否与ROS有关。我们将通过降低ROS和调节NO水平来改善缺氧缺血损伤，以检验我们的假设。嗅觉系统的功能恢复将通过连续功能磁共振成像和成熟嗅神经元和嗅球在两个出生后的单独免疫染色来评估。我们的目标是了解H-I所致神经元损伤中氧化应激和氧化还原信号的关键机制，并最终产生R-01应用的研究。这个模型系统将对研究迄今无法接近的胎儿的疾病、可塑性和恢复的其他机制，以及在不同病理中的产前嗅觉编程，如肥胖症有价值。该提案引入了一种创新的动物模型系统，将嗅觉系统用作了解大脑其余部分的窗口，解决了对围产期嗅觉等感觉缺陷进行更多研究的需要，并研究了氧化应激在嗅觉损伤中的关键机制。这个模型系统将在研究疾病、可塑性和胎儿恢复的其他机制，以及在不同病理中的嗅觉产前编程方面具有价值。

DESCRIPTION (provided by applicant): One of the biggest problems of investigating oxidative stress and other mechanisms in the central nervous system is the lack of functional models that reflect the dynamic and living status of the intact animal. This problem is magnified for perinatal studies because the fetus is relatively inaccessible. This proposal introduces an innovative animal model system that uses the olfactory system as a window to the rest of the brain. It addresses the need of doing more studies investigating the effect of hypoxia on olfaction in the perinatal period. In patients with cerebral palsy (CP), sensory disabilities have not received as much attention as motor deficits and this provides a model for studying the issue of sensory disabilities among the postnatal developmental disabilities following fetal hypoxia-ischemia (H-I). Our hypothesis is that the interaction of reactive oxygen species (ROS) with nitric oxide mediates neuronal injury caused by fetal H-I. We will determine live olfactory neuron function in the intact animal using functional magnetic resonance imaging MRI that is based on the uptake of manganese ion in fetal H-I. Nitric oxide (NO) plays a central role in olfaction and is thought to do so by redox regulation with the formation of reactive nitrogen species (RNS). These concepts will be tested using a multidisciplinary approach in a recently developed animal model manifesting a CP phenotype following preterm uterine ischemia mimicking the clinical pathophysiology of acute placental insufficiency. We have previously shown that ROS and RNS are produced in fetal brain after H-I in this model, and administration of antioxidants to the mother ameliorates fetal brain injury. The first aim characterizes the developmental vulnerability of the olfactory tract to fetal H-I at various gestational ages. Functional MRI using manganese uptake will be correlated with cell injury, death and apoptosis by flow cytometry and immunohistochemical evidence in the olfactory epithelium and olfactory bulbs. The second aim determines if ROS and RNS mediate injury to the olfactory system caused by fetal H-I. We will test whether the developmental vulnerability is due to ROS by measuring superoxide and lipid peroxidation and/or RNS by S-nitrosylation and nitration of tyrosine. We will test our hypothesis by amelioration of the hypoxic-ischemic injury by decreasing ROS and manipulating levels of NO. The functional recovery of olfactory system will be assessed by serial functional MRI and separate immunostaining of mature olfactory neurons and olfactory bulbs at two postnatal ages. Our objectives are to understand key mechanisms of oxidative stress and redox signaling in neuronal injury from H-I and to ultimately generate the studies for an R-01application. This model system will be valuable in the study of other mechanisms of disease, plasticity, and recovery in the hitherto inaccessible fetus, and prenatal programming of smell in diverse pathologies, such as in obesity. The proposal introduces an innovative animal model system that uses the olfactory system as a window to the rest of the brain, addresses the need of doing more studies investigating sensory deficits such as olfaction in the perinatal period, and investigates a key mechanism of oxidative stress in olfactory injury. This model system will be valuable in the study of other mechanisms of disease, plasticity, and recovery in the hitherto inaccessible fetus and prenatal programming of smell in diverse pathologies.