Wood Charles E, Giroux Damian, Gridley Kelly
Department of Physiology and Functional Genomics, University of Florida College of Medicine, P.O. Box 100274, Gainesville, FL 32610-0274, USA.
Brain Res. 2003 Dec 12;993(1-2):84-9. doi: 10.1016/j.brainres.2003.09.001.
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.
我们之前已经证明，脑灌注不足会刺激多种生理和分子反应，这些反应是稳态反射的组成部分。胎儿血浆雌二醇浓度的生理性升高会调节胎儿大脑对低血压的反应性。在本研究中，我们测试了脑灌注不足和/或雌二醇对妊娠晚期胎羊中c-fos基因的蛋白质产物Fos表达的影响。我们假设，单独的雌激素和脑灌注不足会增加包括下丘脑和脑干在内的各个脑区的Fos丰度，并且雌激素会增强或改变Fos对脑灌注不足的反应。使用无菌技术，对定时妊娠母羊的单胎或双胎胎儿进行长期插管，并在头臂动脉周围安装血管外球囊阻塞器。在一半的胎儿中，我们皮下植入一个药丸，该药丸以21天5毫克的速率释放雌二醇。在手术后至少5天（妊娠124 - 128天，足月约为147天）对胎儿进行研究。一半的胎儿接受10分钟的头臂动脉阻塞（BCO）。实验开始1小时后，对母羊和胎儿实施安乐死，迅速取出胎儿大脑，进行解剖，并在丙酮/干冰浴中的聚丙烯管中冷冻。将脑组织在沸腾的裂解缓冲液中匀浆，使用Bradford法测量蛋白质浓度。提取的蛋白质在7.5%聚丙烯酰胺凝胶上进行电泳，转移到硝酸纤维素膜上，并检测Fos。在大多数脑区，雌二醇或BCO改变了Fos的表达。通过双向方差分析，在脑干、小脑和海马中，雌二醇和BCO之间存在统计学显著的相互作用（p<0.05），在下丘脑中接近显著（p = 0.07），在大脑皮层中无统计学显著意义。在这些具有统计学显著相互作用的区域，雌二醇和BCO联合处理后Fos的表达低于单独任何一种处理的反应之和。我们得出结论，雌二醇对胎儿大脑有强大作用，在脑干、小脑和海马中可观察到，并且它调节Fos对脑灌注不足的反应。使用蛋白质印迹法测量区域Fos反应揭示了雌激素和BCO之间的负性相互作用，这可能是由于脑血流或代谢的改变所致。
