Rong Gu1, Minghua Liu2, Yonghong Wang3, Yuanda Zhou4, Haixia He4

1Department of Pharmacy, Children’s Hospital of Chongqing Medical University, Chongqing  400016, China
2Department of Emergency, Southwest Hospital of Third Military Medical University, Chongqing  400016, China
3Chongqing Key Laboratory of Neurology, Department of Neurology, First Hospital of Chongqing Medical University, Chongqing  400016, China
4Research Laboratory of Bioengineering, Basic Medical School, Department of Clinical Pharmacology, First Hospital of Chongqing Medical University, Chongqing  400016, China

the Natural Science Foundation of Chongqing, No. CSTC2006EB5030*

Abstract
BACKGROUND: Estrogen has been clinically demonstrated to attenuate ischemic brain injury. However, the precise mechanisms remain controversial.
OBJECTIVE: To investigate the effects of estradiol on angiopoietin-1 mRNA and Bcl-2 expression, as well as apoptosis and cerebral blood flow, in ovariectomized rats with focal cerebral ischemia following reperfusion.
DESIGN, TIME AND SETTING: Randomized, controlled, animal experiment. The study was per-formed at the Central Laboratory, Chongqing Medical University from September to December 2005.
MATERIALS: Estradiol benzoate was purchased from Shanghai Ninth Pharmaceutical Factory; corn oil was purchased from Walmart Supercenter; TUNEL kit, rabbit anti-rat Bcl-2 polyclonal anti-body, and biotin-labeled goat anti-rabbit antibody were purchased from Wuhan Boster, China.   
METHODS: Healthy, female, 6-month-old Wistar rats–wild-type and estrogen alpha receptor gene  knockout (ERKO)–were randomly divided into estradiol and control groups with 25 animals in each group. The rats were intramuscularly injected with estradiol benzoate (100 μg/kg per day) at 30 days following bilateral ovariectomy or corn oil (1 mL/kg per day) for seven consecutive days. Following administration, cerebral ischemia/reperfusion models were established using the right middle cere-bral artery occlusion (MCAO) method. After 30 minutes of MCAO, estradiol and control groups were separately injected with estradiol benzoate and corn oil with the above-mentioned doses.
MAIN OUTCOME MEASURES: Cell apoptosis was determined by TUNEL; angiopoietin-1 mRNA and Bcl-2 gene expression was determined, respectively, by immunohistochemical staining and RT-PCR. In addition, changes in cerebral blood flow were measured by laser Doppler flowmetry.
RESULTS: Changes in angiopoietin-1 mRNA and cerebral blood flow in estradiol-treated, wild-type, MCAO rats following ischemia/reperfusion were greater than in control rats (P < 0.01 or 0.05). However, no significant difference was observed between estradiol-treated ERKO MCAO rats and control rats. In addition, estradiol-treated wild-type and ERKO MCAO rats exhibited significantly in-creased Bcl-2 expression (P < 0.05) and decreased number of apoptotic cells in brain tissues compared with control groups (P < 0.05).
CONCLUSION: Estradiol upregulated angiopoietin-1 mRNA and Bcl-2 expression, suggesting that estradiol might be involved in protective mechanisms of cerebral ischemia/reperfusion injury.

Key Words: estradiol; angiopoietin; apoptosis; Bcl-2; cerebral ischemia/reperfusion

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