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. ? ? ?
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