BRAIN MACROPHAGES AND REPRODUCTIVE AGING

大脑巨噬细胞和生殖衰老

• 批准号: 2899808
• 负责人: FREDERICK NAFTOLIN
• 金额: $ 25.05万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-01 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2899808
• 关键词: aging; antioxidants; astrocytes; biomarker; estrogen receptors; estrogens; estrus; female; flow cytometry; free radical oxygen; hormone regulation /control mechanism; hypothalamic pituitary axis; hypothalamus; immunocytochemistry; laboratory rat; leukocyte activation /transformation; macrophage; microglia; neural plasticity; neurons; oxidative stress; pituitary gonadal axis; synapses; tissue /cell culture; tocopherols

The aging female rat's reproductive life ends when she can no longer produce a surge of gonadotrophin in response to the midcycle surge of estrogen (positve feedback). Normally, this hypothalamic aging is gradual, marked by peroxidase accumulation in astroglia in the hypothalamus. We have shown a relationship between estrogen-induced synaptic retraction, elaboration of glial processes and positive feedback. To explain this aging, we propose that in the cycling rat, resident brain macrophages in the arcuate nucleus are repeatedly activated by estrogen and produce free radicals that, in turn, disables hypothalamic function. In testing this hypothesis: 1) In cycling female rats, we will characterize the physiological mid cycle activation of arcuate nucleus macrophages and determine the morphological relationship between synaptic plasticity of arcuate nucleus synapses/neurons, astrocytes and brain macrophages using light and electron microscopic immunolabeling for the neuronal marker microtubule-associated protein 2 (MAP 2), the astroglia marker glial fibrillary acidic protein (GFAP), the macrophage marker OX42, and, the appearance of a cell adhesion molecule 1 (I-CAM-1) to mark activated macrophages. 2) We propose to delay the onset of reproductive senescence in normally aging rats with the administration of vitamin E, an anti-oxidant that previously has been shown to suppress free radical production in the central nervous system. The reproductive cycles of normally aging animals will be monitored by vaginal smears and blood LH measurements. At three distinct aging milestones (3 months old, 11 months old and 15 months), control and vitamin E-treated females will be studied by light- and electron microscopic immunocytochemistry for brain macrophage markers in combination with quantitative synaptology will be carried out. The failure of reproduction is normally evident by eleven months and completed by 15 months. Thus, we will characterize aging functionally and morphologically and determine the protective effect of vitamin E. 3) To determine in vitro the cellular basis of activation of brain macrophages by estrogen, we will treat primary cultures of microglia, astrocytes, and, neuronal cell lines that express alpha, beta or alpha/beta estrogen receptors, alone and in combination, assess the effects of estrogen, alone or with vitamin E or superoxide dismutase on these. We will assess production of reactive oxygen species in these cultures by in-situ chemiluminescence and histochemistry.

当年老的雌性大鼠不能再产生促性腺激素来响应中期雌激素的激增（正反馈）时，雌性大鼠的生殖生命就结束了。正常情况下，这种下丘脑衰老是渐进的，其特征是下丘脑星形胶质细胞中过氧化物酶的积累。我们已经证明了雌激素诱导的突触收缩、胶质过程的细化和正反馈之间的关系。为了解释这种衰老，我们提出在循环大鼠中，弓形核中的常驻脑巨噬细胞被雌激素反复激活并产生自由基，进而使下丘脑功能丧失功能。为了验证这个假设：1)在循环雌性大鼠中，我们将利用光镜和电镜对神经元标记物微管相关蛋白2 （MAP 2）、星形胶质细胞标记物胶质纤维酸性蛋白（GFAP）、巨噬细胞标记物OX42和巨噬细胞进行免疫标记，表征弓形核巨噬细胞生理周期中期激活，确定弓形核突触/神经元、星形胶质细胞和脑巨噬细胞突触可塑性的形态学关系；细胞粘附分子1 （I-CAM-1）的出现，标记活化的巨噬细胞。2)我们建议通过服用维生素E来延缓正常衰老大鼠的生殖衰老，维生素E是一种抗氧化剂，先前已被证明可以抑制中枢神经系统中自由基的产生。正常衰老动物的生殖周期将通过阴道涂片和血液LH测量来监测。在三个不同的衰老里程碑（3个月大，11个月大和15个月大），对照和维生素e治疗的雌性将通过光镜和电镜免疫细胞化学研究脑巨噬细胞标志物，并结合定量突触学。繁殖失败通常在11个月时明显，在15个月时完成。因此，我们将在功能和形态学上表征衰老，并确定维生素E的保护作用。3)为了确定雌激素在体外激活脑巨噬细胞的细胞基础，我们将单独或联合处理表达α、β或α / β雌激素受体的小胶质细胞、星形胶质细胞和神经细胞系的原代培养，评估雌激素单独或与维生素E或超氧化物歧化酶一起对这些细胞的影响。我们将通过原位化学发光和组织化学来评估这些培养物中活性氧的产生。