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，慢性低氧胎儿的神经系统发病率较高。在这个方案中，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大脑中I型一氧化氮合酶的表达；低氧后I型一氧化氮合酶表达的地区差异是由选择性剪接变异体的差异表达决定的。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)慢性缺氧增加胎脑易损性的机制是I型一氧化氮合酶上调导致的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。在这项提案中，我们将集中在大脑中容易发生缺氧缺血损伤的区域，并已知表达I型一氧化氮合酶(感觉-运动皮质、纹状体、海马体和小脑)。具体目标1通过使用瓜氨酸分析和免疫印迹技术在选定脑区的不同亚细胞区段测量酶活性和蛋白质质量，研究慢性低氧对I型一氧化氮合酶表达的影响。此外，将通过研究特定的选择性剪接变异体的表达来评估I型一氧化氮合酶对低氧的反应。特别是外显子1和外显子2的变体。特殊目的2通过阻断脊髓诱导神经元损伤来研究缺氧对急性脑缺血易感性的影响。胎儿大脑的冠状切片将被硫化/酸性品红染色，以评估侮辱72小时后死亡神经元的比例。具体目标3将确定在脊髓闭塞之前给予选择性的I型一氧化氮合酶抑制剂(LVNIO)是否可以消除慢性缺氧胎儿增加的脆弱性。特异靶4通过检测损伤后24小时细胞色素c的释放、Bax转位、亲环素D与MPT的结合、caspase-3的激活和DNA梯状条带，探讨缺氧和脊髓闭塞在细胞凋亡级联反应中的作用。此外，我们还将评估在AIM 2中获得的大脑中神经元的TUNEL染色数量。胎儿低氧血症是妊娠合并胎盘功能不全的常见产科并发症，即IUGR和先兆子痫。因此，将获得的数据是重要的，因为它们将提供实验证据，支持慢性缺氧与神经发病率增加的流行病学联系。此外，这将是第一次展示胎儿大脑不同区域对缺氧的差异I型一氧化氮合酶基因上调反应，以及它作为增加胎儿大脑脆弱性的潜在机制的潜在作用。

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.