Fetal brain regional responses to cerebral hypoperfusion: modulation by estrogen.
We have previously demonstrated that cerebral hypoperfusion stimulates several physiological and molecular responses which are components of homeostatic reflexes. Physiological increases in fetal plasma estradiol concentration modulate fetal brain responsiveness to hypotension. In the present study, we tested the effect of cerebral hypoperfusion and/or estradiol on the expression of Fos, the protein product of the gene c-fos in late-gestation fetal sheep. We hypothesized that estrogen and cerebral hypoperfusion alone would augment Fos abundance in various brain regions, including the hypothalamus and brainstem, and that estrogen would augment or otherwise modify the Fos response to cerebral hypoperfusion. Singleton or twin fetuses of time-dated pregnant ewes were chronically catheterized and fitted with an extravascular balloon occluder around the brachiocephalic artery using aseptic techniques. In one-half of the fetuses, we implanted a pellet subcutaneously which released estradiol at a rate of 5 mg in 21 days. Fetuses were studied at least 5 days after surgery (124-128 days' gestation, term is approximately 147 days). One-half of the fetuses were subjected to a 10-min period of brachiocephalic occlusion (BCO). One hour after the start of the experiment, the ewe and fetus were euthanized and the fetal brain was rapidly recovered, dissected, and frozen in a polypropylene tube in an acetone/dry ice bath. Brain tissue was homogenized in a boiling lysis buffer, and protein concentrations measured using the Bradford method. Extracted proteins were electrophoresed on 7.5% polyacrylamide gels, transferred to nitrocellulose membranes, and probed for Fos. In most brain regions, estradiol or BCO altered the expression of Fos. Analyzed by two-way analysis of variance, there was a statistically significant (p<0.05) interaction between estradiol and BCO in brainstem, cerebellum, and hippocampus, nearly significant in hypothalamus (p=0.07) and not statistically significant in cerebral cortex. In these regions with statistically significant interactions, the expression of Fos in response to the combined treatment of estradiol and BCO was less than the sum of responses to either treatment alone. We conclude that estradiol has a potent action on the fetal brain which is identifiable in the brainstem, cerebellum, and hippocampus and that it modulates the Fos response to cerebral hypoperfusion. The measurement of regional Fos responses using Western blot reveals a negative interaction between estrogen and BCO which might result from alterations in cerebral blood flow or metabolism.
Wood
Charles E
CE
Department of Physiology and Functional Genomics, University of Florida College of Medicine, P.O. Box 100274, Gainesville, FL 32610-0274, USA. cwood@phys.med.ufl.edu
Giroux
Damian
D
Gridley
Kelly
K
D000704:Analysis of Variance; D000818:Animals; D015153:Blotting, Western / Q000379:methods; D016122:Brachiocephalic Trunk; D001923:Brain Chemistry; D002545:Brain Ischemia / Q000150:complications / Q000378:metabolism / Q000517:prevention & control*; D003327:Coronary Disease / Q000209:etiology; D004622:Embryo, Mammalian; D004967:Estrogens / Q000627:therapeutic use*; D005260:Female; D006973:Hypertension / Q000209:etiology / Q000378:metabolism*; D008297:Male; D016761:Oncogene Proteins v-fos / Q000378:metabolism; D010477:Perfusion; D011247:Pregnancy; D012756:Sheep / Q000196:embryology; D014427:Twins
D004967:Estrogens; D016761:Oncogene Proteins v-fos