Estrogen and brain vascular inflammation in diabetics

描述（由申请人提供）：雌激素的神经和血管保护特性已在实验室研究中得到充分证明。然而，临床上有迹象表明，激素替代疗法对女性可能没有好处，甚至是有害的。目前的项目是基于这样一个概念，即临床相关情况可能发生，雌激素替代疗法（ERT）不再是神经保护，而是神经毒性。糖尿病/慢性高血糖症（CH）就是其中之一。最近的研究结果表明，在非糖尿病卵巢切除（OVX）大鼠中，慢性“生理性”ERT可以保护大脑免受缺血性损伤，而在糖尿病卵巢切除（OVX）雌性大鼠中，ERT则加剧了这种损伤。本项目旨在描述这种“转变”背后的机制。其基本原理来源于平行观察，即CH促进晚期糖基化终产物（AGEs）水平的增加，而ERT促进AGE受体RAGE在脑血管内皮细胞上的表达增加。由此产生的RAGE激活的增加释放了一个级联反应，导致促炎转录调节因子NFkB活性的持续增加，并增加了缺血性损伤后脑炎症活动的可能性。该提案的4个具体目标基于以下假设：1)糖尿病（链脲佐菌素治疗，慢性高血糖）OVX女性的ERT会加剧，而非糖尿病患者的ERT会减弱缺血后（短暂性前脑缺血）脑血管炎症，这反映在白细胞粘附在肾小静脉的程度上。2)通过反义或药物抑制剂治疗阻断RAGE、其配体（如AGE）或其下游效应物NFkB，将阻止ERT在糖尿病患者中的促粘附作用。3)除AGE、RAGE或NFkB阻断外，预防白细胞粘附（通过白细胞减少）也可预防ert相关的缺血性脑损伤加重。4)慢性黄体酮(P)替代，当联合ERT时，将阻止促炎和神经毒性作用在糖尿病OVX女性中单独使用ERT。结果将根据雌性是否完整、OVX或OVX + 17b -雌二醇（E2）处理（或E2+ p处理）以及大鼠是否患有糖尿病来评估。需要监测的主要变量是缺血后（0-10h再灌注）罗丹明- 6g标记白细胞的动脉静脉粘连；神经元细胞损失（72h再灌注时）；以及关键蛋白的表达。已建立的用于定量白细胞粘附的技术包括慢性放置封闭的颅窗，活体显微镜/视频测量和计算机辅助图像处理。这些研究的结果将使我们能够了解雌激素转化为“阴暗面”背后的一些机制，并将改善激素替代治疗的临床疗效。

DESCRIPTION (provided by applicant): The neuro-and vasculoprotective properties of estrogen have been amply demonstrated in laboratory investigations. However, clinically, there are indications that hormone replacement therapy in women may not be beneficial, and even detrimental. The present project is based upon the concept that clinically-relevant circumstances may occur where estrogen replacement therapy (ERT) is no longer neuroprotective, but neurotoxic. One such circumstance is diabetes/chronic hyperglycemia (CH). Recent results showed that, whereas chronic "physiologic" ERT, in non-diabetic, ovariectomized (OVX) rats protects the brain against ischemic damage, ERT in diabetic OVX females exacerbates the damage. This project is designed to delineate the mechanisms behind this "transformation". The rationale derives from the parallel observations that CH promotes an increase in the levels of advanced glycation end-products (AGEs), while ERT promotes an increased expression of the AGE receptor, RAGE, on cerebrovascular endothelial cells. The resultant increase in RAGE activation unleashes a cascade leading to a sustained increase in the activity of the pro-inflammatory transcriptional regulator, NFkB, and a heightened potential for cerebral inflammatory activity following an ischemic insult. The 4 specific aims of this proposal are guided by the following hypotheses: 1) ERT in diabetic (streptozotocin-treated, chronically [>1 mo] hyperglycemic) OVX females will exacerbate, while ERT in non-diabetics will attenuate post-ischemic (transient forebrain ischemia) cerebrovascular inflammation, as reflected by the extent to which leukocytes adhere to pial venules. 2) Blocking RAGE, its ligands (e.g., AGE), or its downstream effector, NFkB, either via antisense or pharmacologic inhibitor treatments, will prevent the pro-adhesive action of ERT in diabetics. 3) Preventing leukocyte adhesion (via leukopenia), in addition to AGE, RAGE or NFkB blockade, will prevent ERT-associated exacerbation of ischemic brain damage. 4) Chronic progesterone (P) replacement, when combined with ERT, will prevent the pro-inflammatory and neurotoxic actions seen with ERT alone in the diabetic OVX female. Results will be evaluated based upon whether the females are intact, OVX, or OVX + 17B-estradiol (E2)-treated (or E2+P-treated), and whether the rats are diabetic or non-diabetic. The principal variables to be monitored will be post-ischemic (0-10h reperfusion) pial venular adhesion of rhodamine-6G-labeled leukocytes; neuronal cell loss (at 72h reperfusion); and expression of key proteins. The established technique being used for quantitating leukocyte adhesion involves chronic placement of closed cranial windows, intravital microscopy/videometry, and computer-assisted image processing. The results of these studies will permit us to gain an understanding of some of the mechanisms behind estrogen's conversion to the "dark side" and should lead to improvements in the clinical efficacy of hormone replacement treatments.