Angiopoietin-1 mRNA expression in estradiol-treated ovariectomized rats with focal cerebral ischemia after reperfusion

摘要

BACKGROUND: Epidemiologic studies have indicated that the incidence of stroke in premenopausal females is lower than in males at the same age, but it significantly rises in postmenopausal females. Estrogen is used clinically to alleviate injury caused by cerebral ischemia. It has been hypothesized that the neuroprotective role of estrogen relates to angiopoietin (Angpt), which plays an important role in vascularization, vascular remodeling and maturation. OBJECTIVE: To observe and validate the effect of estradiol on angiopoietin-1 (Angpt1) mRNA expression in ovariectomized rats with focal cerebral ischemia after reperfusion, so as to explore the molecular mechanisms of estradiol-mediated protection from cerebral ischemic damage. DESIGN, TIME AND SETTING: Randomized, controlled, molecular biology, prospective animal study. The experiment was performed at the Central Laboratory of Chongqing Medical University from September to December 2005. MATERIALS: Fifty healthy female wild type (WT) rats aged 6 months and fifty female rats aged 6 months with knockout of the estrogen-alpha receptor gene (ERKO). METHODS: WT rats and ERKO rats were divided into estradiol and control groups (n = 25), and injected in- tramuscularly with estradiol benzoate (100 μg/kg per day) or corn oil (1 mL/kg per day) for 7 days, 30 days after bilateral ovariectomy. Rat models of cerebral ischemia/reperfusion were established with the middle cerebral artery occlusion method. After 30 minutes of middle cerebral artery occlusion, rats from the estradiol and control groups were injected intramuscularly with estradiol benzoate or corn oil at the above dose. MAIN OUTCOME MEASURES: We used radio-immunity analysis and laser-Doppler flowmetry to measure plasma estradiol levels and changes in cerebral blood flow. We used immunohistochemical staining of CD34 epitopes to measure changes in the capillary density in brain following cerebral ischemia/reperfusion, and quantitative RT-PCR analysis to assess mRNA expression levels of Angpt1, Angpt2, Tie2, vascular endothelial growth factor (Vegf), VegfR1, and VegfR2. RESULTS: In WT ovariectomized rats treated with estradiol, the change in cerebral blood flow following cerebral ischemia/reperfusion, capillary density in the basal nuclei and parietal lobe cortex and Angpt1 mRNA level were significantly higher than in the control group (P < 0.01). We did not identify any such changes in ERKO rats treated with estradiol. In addition, the plasma estradiol levels in WT and ERKO ovariectomized rats treated with estradiol were remarkably higher than in their corresponding control groups (P < 0.01). CONCLUSION: Angpt1 is a critical factor in many processes during the repair of cerebral ischemia/reperfusion injury. For example, it confers estrogen-mediated protection, restoration of cerebral blood flow and increases in brain capillary density. It is emerging as an important molecule for estradiol-mediated neuroprotection.