Vulnerability of the Fetal Brain to Hypoxic-Ischemia

胎儿大脑对缺氧缺血的脆弱性

• 批准号: 6748613
• 负责人: JORGE Pablo FIGUEROA
• 金额: $ 22.68万
• 依托单位: WAKE FOREST UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6748613
• 关键词: Bax gene /protein; apoptosis; brain injury; brain mapping; cerebellum; cerebral ischemia /hypoxia; cysteine endopeptidases; embryo /fetus hypoxia; enzyme activity; gene expression; gestational age; hippocampus; neuroprotectants; neurotoxicology; newborn animals; nitric oxide; nitric oxide synthase; perinatal; sheep; terminal nick end labeling; western blottings

Hypoxic-ischemic insult during the perinatal period remains a significant cause of morbidity and mortality in both term and preterm newborns. Epidemiological data suggest that chronically hypoxic fetuses have a higher incidence of neurological morbidity. In this proposal we will test the following interrelated hypotheses: 1) Chronic mild hypoxemia increases the expression of Type I Nitric Oxide Synthase in fetal brain; regional differences in Type I NOS expression following hypoxia are determined by differential expression of alternative splice variants. 2) Chronic fetal hypoxia increases the vulnerability of the fetal brain to neuronal damage. 3) The mechanism by which chronic hypoxia increases fetal brain vulnerability is the augmented release of NO secondary to upregulation of Type I NOS. 4) Increased apoptosis is one of the mechanisms by which NO induces neuronal death following a hypoxic-ischemic insult. In this proposal, we will concentrate in areas of the brain prone to hypoxic-ischemic damage and known to express Type I NOS (Sensory-motor cortex, striatum, hippocampus and cerebellum). Specific Aim 1 examines the effects of chronic hypoxia on Type I NOS expression by measuring enzymatic activity and protein mass using the citrulline assay and western blotting in distinct subcellular compartments of selected brain regions. Also, the Type I NOS mRNA response to hypoxia will be evaluated studying the expression of specific alternative splice variants. In particular, variants of exons 1 and 2. Specific Aim 2 examines the effect of hypoxia on brain vulnerability to acute ischemia using cord occlusion to induced neuronal damage. Coronal sections of the fetal brain will be stained with thionin/acid fuchsin to evaluate the proportion of dead neurons 72 hours after the insult. Specific Aim 3 will determine if the increased vulnerability of the chronically hypoxic fetus can be abrogated by a selective Type I NOS inhibitor (LVNIO) administered before the cord occlusion. Specific Aim 4 examines the effect of hypoxia and cord occlusion in the activation of the apoptosis cascade by measuring cytochrome c release, BAX translocation, cyclophilin D binding to the MPT, caspase-3 activation and DNA laddering 24 hours after the insult. In addition, we will evaluate the number of neurons stained with the TUNEL method in brains obtained in Aim 2. Fetal hypoxemia is a common obstetrical complication in pregnancies in which there is placental insufficiency, i.e., IUGR and preeclampsia. Thus, the data to be obtained are important since they will provide experimental evidence to support the epidemiological association of chronic hypoxia and increased neural morbidity. Also, it will be the first work to demonstrate a differential Type I NOS gene upregulation response to hypoxia in different regions of the fetal brain and its potential role as a mechanism for increasing fetal brain vulnerability.

围产期缺氧缺血性损伤仍然是足月和早产新生儿发病和死亡的重要原因。 流行病学数据表明，长期缺氧的胎儿神经系统疾病的发生率较高。 在本提案中，我们将测试以下相互关联的假设：1）慢性轻度低氧血症会增加胎儿大脑中I型一氧化氮合酶的表达；缺氧后 I 型 NOS 表达的区域差异是由选择性剪接变体的差异表达决定的。 2）胎儿慢性缺氧会增加胎儿大脑对神经元损伤的脆弱性。 3）慢性缺氧增加胎儿大脑脆弱性的机制是I型NOS上调继发的NO释放增加。 4) 细胞凋亡增加是缺氧缺血性损伤后NO 诱导神经元死亡的机制之一。 在这项提案中，我们将重点关注容易发生缺氧缺血性损伤并已知表达 I 型 NOS（感觉运动皮层、纹状体、海马体和小脑）的大脑区域。 具体目标 1 通过使用瓜氨酸测定和蛋白质印迹法在选定大脑区域的不同亚细胞区室中测量酶活性和蛋白质质量，检查慢性缺氧对 I 型 NOS 表达的影响。 此外，还将通过研究特定选择性剪接变体的表达来评估 I 型 NOS mRNA 对缺氧的反应。 特别是外显子 1 和 2 的变体。具体目标 2 使用脊髓闭塞诱导神经元损伤来检查缺氧对大脑急性缺血脆弱性的影响。 胎儿大脑的冠状切片将用硫堇/酸性品红染色，以评估损伤后 72 小时死亡神经元的比例。 具体目标 3 将确定是否可以通过在脐带闭塞前施用选择性 I 型 NOS 抑制剂 (LVNIO) 来消除胎儿长期缺氧所增加的脆弱性。具体目标 4 通过测量损伤后 24 小时的细胞色素 c 释放、BAX 易位、亲环蛋白 D 与 MPT 的结合、caspase-3 激活和 DNA 阶梯，检查缺氧和脐带闭塞对细胞凋亡级联激活的影响。 此外，我们将评估目标 2 中获得的大脑中用 TUNEL 方法染色的神经元数量。胎儿低氧血症是胎盘功能不全妊娠中常见的产科并发症，即 IUGR 和先兆子痫。 因此，要获得的数据很重要，因为它们将提供实验证据来支持慢性缺氧和神经发病率增加的流行病学关联。 此外，这将是第一项证明 I 型 NOS 基因对胎儿大脑不同区域缺氧的差异上调反应及其作为增加胎儿大脑脆弱性的机制的潜在作用的工作。