Vulnerability of the Fetal Brain to Hypoxic-Ischemia

围产期缺氧缺血性损伤仍然是足月新生儿和早产儿发病和死亡的重要原因。流行病学资料显示，慢性缺氧胎儿有较高的神经系统发病率。在本研究中，我们将检验以下相关假设：1)慢性轻度低氧血症增加胎儿大脑中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异的差异表达决定的。2)胎儿慢性缺氧增加了胎儿大脑对神经元损伤的易感性。3)慢性缺氧增加胎儿脑易损的机制是继发于I型NOS上调的NO释放增加。4)细胞凋亡增加是NO诱导缺氧缺血性损伤后神经元死亡的机制之一。在本研究中，我们将集中研究易发生缺氧缺血性损伤且已知表达I型NOS的大脑区域（感觉-运动皮层、纹状体、海马和小脑）。特异性目的1研究慢性缺氧对I型NOS表达的影响，方法是使用瓜氨酸测定法和western blotting在选定的脑区域的不同亚细胞区室中测量酶活性和蛋白质质量。此外，将通过研究特定备选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的变异。特异性目的2探讨缺氧对脑对急性缺血易损的影响，通过脊髓闭塞诱导神经元损伤。在损伤72小时后，用硫蛋白/酸性品红染色胎儿脑冠状面，评估死亡神经元的比例。特异性目的3将确定是否可以通过在脐带阻断前给予选择性I型NOS抑制剂（LVNIO）来消除慢性缺氧胎儿的脆弱性增加。特异性目的4通过测量损伤后24小时细胞色素c释放、BAX易位、亲环蛋白D与MPT结合、caspase-3激活和DNA阶梯，检测缺氧和脊髓闭塞对细胞凋亡级联激活的影响。此外，我们将评估Aim 2中获得的TUNEL方法染色的脑内神经元数量。胎儿低氧血症是胎盘功能不全妊娠的常见产科并发症，即IUGR和先兆子痫。因此，获得的数据很重要，因为它们将为支持慢性缺氧与神经发病率增加之间的流行病学关联提供实验证据。此外，这将是首次证明I型NOS基因在胎儿大脑不同区域对缺氧的差异上调反应及其作为胎儿大脑易感性增加机制的潜在作用。

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.