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，女性激素替代疗法可能不是有益的，甚至是有害的。本项目是基于这样的概念，即临床相关的情况下，可能会发生雌激素替代治疗（ERT）不再是神经保护，但神经毒性。一种这样的情况是糖尿病/慢性高血糖症（CH）。最近的结果表明，而慢性“生理”ERT，在非糖尿病，卵巢切除（OVX）大鼠保护脑缺血性损伤，ERT在糖尿病OVX女性加重损害。本项目旨在阐明这种“转变”背后的机制。基本原理来自于平行观察结果，即CH促进晚期糖基化终产物（AGEs）水平增加，而ERT促进脑血管内皮细胞上AGE受体（AGEs）表达增加。由此产生的TNF-α激活的增加释放级联反应，导致促炎转录调节因子NF κ B的活性持续增加，以及缺血性损伤后脑炎症活性的可能性增加。本提案的4个具体目标由以下假设指导：1）糖尿病（链脲佐菌素治疗，慢性[>1个月]高血糖）OVX女性患者的ERT将加重，而非糖尿病患者的ERT将减轻缺血后（短暂性前脑缺血）脑血管炎症，如白细胞粘附于软脑膜小静脉的程度所反映。2)阻断腺苷酸、其配体（例如，AGE）或其下游效应物NFkB通过反义或药理学抑制剂治疗将阻止ERT在糖尿病患者中的促粘附作用。3)预防白细胞 粘附（通过白细胞减少症），除了AGE、NFkB或NFkB阻断之外，将防止缺血性脑损伤的ERT相关恶化。4)长期孕酮（P）替代，当与ERT联合使用时，将防止在糖尿病OVX女性中单独使用ERT时观察到的促炎和神经毒性作用。将根据雌性动物是否为完整、OVX或OVX +17 B-雌二醇（E2）给药（或E2+ P给药）以及大鼠是否为糖尿病或非糖尿病大鼠评价结果。要监测的主要变量将是缺血后（再灌注0- 10小时）罗丹明-6G标记的白细胞的软脑膜小静脉粘附;神经元细胞损失（再灌注72小时）;和关键蛋白质的表达。用于定量白细胞粘附的既定技术包括长期放置闭合的颅窗、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“黑暗面”背后的一些机制，并应导致激素替代治疗的临床疗效的改善。

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.