Tetrahydrobiopterin in Hypoxia-Ischemia Induced Fetal Brain Dysfunction

描述(由申请人提供)：本提案的主要目标是确定早产儿脑内缺氧-缺血(H-I)和四氢生物蝶呤(BH4)丢失与神经功能之间的联系。尽管有证据表明氧化应激起了一定作用，但缺氧对发育中的大脑造成损害的机制尚不清楚。因此，可能会发生多巴胺能神经元的丢失。先天性BH4缺乏会导致运动障碍，在某些情况下，可以用BH4治疗。我们最近建立了一种早产胎兔脑H-I损伤的模型，导致新生儿高张和运动障碍，并表明早产胎兔脑中BH4的水平比报道的HPH-1小鼠低得多，HPH-1是一种遗传性BH4缺乏症和L多巴反应障碍的模型。因此，低水平的BH4可能是H-I所致胎儿脑功能障碍的关键发育因素。我们还表明，有限的BH4水平支持nNOS解偶联，以增加超氧化物的形成。因此，早产胎兔脑为研究人员提供了一个以前没有的机会来确定H-I和BH4依赖机制(一氧化氮、超氧化物、L-多巴)在运动障碍病因中的联系。假设：缺氧缺血引起的高氧化应激破坏早产儿脑内BH4的稳态，导致运动障碍。目的：1)研究是否可以通过BH4调节超氧化物和nNOS产生NO来解释早产儿脑发育易感性；2)研究导致缺氧后BH4和nNOS解偶联的急性和慢性机制；以及单独补充BH4或与维生素C(一种BH4稳定抗氧化剂)联合使用的效果。方法：用高效液相色谱法测定脑组织BH4、7，8-BH2和新喋呤水平及BH4依赖酶的活性。二氢乙锭转化为2-羟乙锭后的超氧化物定量将用高效液相色谱法进行。NNOS活性之后会有14C-瓜氨酸和NO衍生的化学发光。意义：围产期缺氧是导致儿童残疾的重要危险因素，通常会导致认知和运动障碍(脑瘫)。目前，还没有有效的治疗方法来预防缺氧诱导的脑损伤对发育中的脑的影响。从这项提案中获得的信息将为确定BH4在预防胎儿大脑功能障碍中的使用提供基础。缺氧缺血(H-I)或低氧供应被认为是未成熟胎儿脑损伤的主要危险因素。目前，还没有干预措施来预防或减少任何临床情况下的脑损伤，怀疑是缺氧。H-I的并发症和预后与年龄有关，提示发育因素在该病中的重要性。四氢生物蝶呤是大脑中的一种关键代谢物，调节多种功能和氧化剂的产生。它可能参与了H-I后的脑功能障碍。我们期望为四氢生物蝶呤的发育生物学及其在胎脑氧化损伤机制中的作用提供重要的新信息。这些信息可能会定义四氢生物蝶呤在预防和/或改善缺氧诱导的胎儿脑功能障碍方面的使用，这仍然是一个主要的医学挑战。

DESCRIPTION (provided by applicant): The broad objective of this proposal is to determine the link between hypoxia-ischemia (H-I) and loss of tetrahydrobiopterin (BH4) and neurological function in the preterm fetal brain. The mechanism by which H-I causes damage to the developing brain remains unknown, although evidence indicates that oxidative stress plays a role. Loss of dopaminergic neurons may occur as a consequence. Congenital BH4 deficiency causes motor deficits that, in some cases, can be treated with BH4. We recently presented a model of global H-I injury in preterm fetal rabbit brain that causes hypertonia and motor deficits in the neonates and shows that BH4 levels in the preterm fetal rabbit brain are much lower compared to those reported in the hph-1 mice, a model for genetic BH4 deficiency and L-dopa-responsive-dystonia. Thus, low BH4 levels may be a critical developmental factor associated with H-I induced fetal brain dysfunction. We also showed that limited BH4 levels supports nNOS uncoupling to increase superoxide formation. Thus, preterm fetal rabbit brain gives investigators an opportunity that was not available before to ascertain the connection between H-I and BH4- dependent mechanisms (NO, superoxide, L-dopa) in the etiology of motor impairments. Hypothesis: High oxidative stress induced by hypoxia-ischemia disrupts BH4-homeostasis in the preterm fetal brain leading to motor deficits. Aims: 1) Examine whether the developmental susceptibility of the premature brain can be explained by BH4 modulation of superoxide and NO production from nNOS; 2) Examine the acute and chronic mechanisms causing loss of BH4 and nNOS uncoupling after H-I; and the effects of BH4 supplementation alone or in combination with ascorbate, a BH4 stabilizing antioxidant. Methods: Brain BH4, 7,8-BH2 and neopterin levels and activity of BH4-dependent enzymes will be measured by HPLC. Superoxide quantification following conversion of dihydroethidium to 2-hydroxyethidium will be performed by HPLC. nNOS activity will be followed by 14 C-citrulline and NO-derived chemiluminescence. Significance: Perinatal H-I is an important risk factor for disability in children often resulting in cognitive and motor impairment (cerebral palsy). Currently, there is no effective treatment to prevent the consequences of H-I mediated injury to the developing brain. The information obtained from this proposal will provide the basis to define the use of BH4 in preventing fetal brain dysfunction. Hypoxia-ischemia (H-I) or low oxygen supply is considered a major risk factor for brain damage in the immature fetal brain. Currently, there are no interventional measures to prevent or decrease brain damage after any clinical situation where deficient oxygen supply is suspected. The complications and prognosis of H-I are age related, indicating the importance of developmental factors in the disease. Tetrahydrobiopterin is a key metabolite in the brain regulating several functions and oxidant production. It is possibly involved in brain dysfunction, following H-I. We expect to provide important new information on the developmental biology of tetrahydrobiopterin and its involvement in the mechanisms of oxidative damage in fetal brain. This information will likely define the use of tetrahydrobiopterin in the prevention and/or amelioration of H-I induced fetal brain dysfunction, which remains a major medical challenge.