Vulnerability of the Fetal Brain to Hypoxic-Ischemia

围产期缺氧缺血性损伤仍然是足月儿和早产儿发病率和死亡率的重要原因。 流行病学数据表明，长期缺氧的胎儿有较高的神经系统发病率。 在本研究中，我们将检验以下相互关联的假设：1）慢性轻度低氧血症增加了胎儿脑中I型一氧化氮合酶的表达;低氧后I型NOS表达的区域差异由可变剪接变体的差异表达决定。 2)慢性胎儿缺氧增加了胎儿脑神经元损伤的脆弱性。3)慢性缺氧增加胎儿脑易损性的机制是继发于I型NOS上调的NO释放增加。 4)凋亡增加是NO诱导缺氧缺血性损伤后神经元死亡的机制之一。 在这个建议中，我们将集中在大脑的区域容易缺氧缺血性损伤和已知的表达I型NOS（感觉运动皮层，纹状体，海马和小脑）。 具体目标1通过使用瓜氨酸测定和蛋白质印迹法在选定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，I型NOS mRNA对缺氧的反应将通过研究特异性选择性剪接变体的表达来评估。 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脑对急性缺血的脆弱性的影响。 将胎脑的冠状切片用硫堇/酸性品红染色，以评价损伤后72小时死亡神经元的比例。 具体目标3将确定是否增加的脆弱性的慢性缺氧胎儿可以废除的选择性I型NOS抑制剂（LVNIO）的脊髓闭塞前管理。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、亲环素D与MPT的结合、半胱天冬酶-3活化和DNA梯状化来检查缺氧和脐带闭塞在细胞凋亡级联活化中的作用。 此外，我们将评价在目标2中获得的脑中用TUNEL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，获得的数据是重要的，因为它们将提供实验证据，以支持慢性缺氧和神经发病率增加的流行病学关联。 此外，这将是第一个工作，以证明一个差异的I型NOS基因上调反应，缺氧在不同地区的胎儿大脑和其潜在的作用，作为一种机制，增加胎儿大脑的脆弱性。

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.