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.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
1R01HD038964-01A2
Application #
6471092
Study Section
Human Embryology and Development Subcommittee 1 (HED)
Program Officer
Hanson, James W
Project Start
2002-07-01
Project End
2007-06-30
Budget Start
2002-07-01
Budget End
2003-06-30
Support Year
1
Fiscal Year
2002
Total Cost
$247,320
Indirect Cost
Name
Wake Forest University Health Sciences
Department
Obstetrics & Gynecology
Type
Schools of Medicine
DUNS #
937727907
City
Winston-Salem
State
NC
Country
United States
Zip Code
27157
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Csordas, Attila; Pankotai, Eszter; Snipes, James A et al. (2007) Human heart mitochondria do not produce physiologically relevant quantities of nitric oxide. Life Sci 80:633-7
Lacza, Zsombor; Kozlov, Andrey V; Pankotai, Eszter et al. (2006) Mitochondria produce reactive nitrogen species via an arginine-independent pathway. Free Radic Res 40:369-78
Lacza, Zsombor; Pankotai, Eszter; Csordas, Attila et al. (2006) Mitochondrial NO and reactive nitrogen species production: does mtNOS exist? Nitric Oxide 14:162-8
Lacza, Zsombor; Horvath, Eszter M; Pankotai, Eszter et al. (2005) The novel red-fluorescent probe DAR-4M measures reactive nitrogen species rather than NO. J Pharmacol Toxicol Methods 52:335-40
Lacza, Zsombor; Snipes, James A; Zhang, Jie et al. (2003) Mitochondrial nitric oxide synthase is not eNOS, nNOS or iNOS. Free Radic Biol Med 35:1217-28