Estrogen and Bcl-2: gene induction and effect of transgene in experimental stroke

Abstract

Female rodents producing endogenous estrogens are protected from stroke damage in comparison with male counterparts. This natural protection is lost after ovariectomy or reproductive senescence. The aim of this study is to determine whether estrogen reduces early neuronal injury and cell loss after ischemia by increasing the expression of Bcl-2. Male, intact female, ovariectomized, and estrogen-repleted ovariectomized rats were subjected to middle cerebral artery occlusion, and 22 hr later the level and localization of Bcl-2 mRNA and protein were determined. The levels of post-ischemic bcl-2 mRNA and protein were increased exclusively in neurons within the peri-infarct region. Intact females and estrogen-treated castrates demonstrated increased bcl-2 mRNA and protein expression compared with males and estrogen-deficient females, accompanied by a decrease in infarct size. To test the hypothesis that the neuroprotective mechanism of estrogen functions via Bcl-2, we compared ischemic outcome in male, female, and ovariectomized wild-type mice and mice overexpressing Bcl-2 exclusively in neurons. Wild-type female mice sustained smaller infarcts compared with males. Bcl-2 overexpression reduced infarct size in males, but provided no added protection in the female. Moreover, ovariectomy exacerbated infarction in wild-type females, but had no effect in Bcl-2 overexpressors. These data indicate that overexpression of Bcl-2 simulates the protection against ischemic injury conferred by endogenous female sex steroids. We concluded that estrogen rescues neurons after focal cerebral ischemia by increasing the level of Bcl-2 in peri-infarct regions and that estrogen-induced bcl-2 gene expression is an important downstream component of neuronal protection in female stroke.

Fig. 1.

Cresyl violet-stained coronal brain sections from estrogen-treated (A) and untreated (B) ovariectomized female rats after stroke. The area of pallor delineates the “core” of cerebral infarction, and the area immediately surrounding that core represents the “penumbra” of ischemic injury. Estrogen treatment resulted in 40% reduction in cerebral infarct (n = 4 per group).Circles delineate areas from which tissue micropunches were extracted for bcl-2 mRNA quantification using RNase protection assay.

Fig. 2.

Bcl-2 in situ hybridization of female rat brain after stroke. Rat brains (n = 4 in each of 4 groups) were frozen 22 hr after 2 hr MCA.Bcl-2 mRNA is visualized as dark grainsover thionin-stained cells (purple). Labeled cells are abundant in the peri-infarct region (B), but not in uninjured (contralateral) hemisphere (A). Control sections hybridized with radiolabeled sense and with excess unlabeled antisense probes were negative for bcl-2 mRNA signal. Photomicrographs were taken at 100× magnification. Scale bar, 50 μm.

Fig. 3.

Analysis of bcl-2 mRNA expression in male (M), intact female (F), ovariectomized female (O), and estrogen-treated O (E) rats at 22 hr after 2 hr MCA occlusion in rat. RNase protection assay was performed directly on micropunches that were extracted from selected areas within the striatum (A) and the cerebral cortex (B) as defined in Figure 1. Each value represents the mean of 10 pooled micropunches (∼25–30 mg) that were extracted from the same region of five animals per group. Bcl-2mRNA expression was higher in F and Ethan in M and O in the cerebral cortex and striatum.

Fig. 4.

Immunohistochemical analysis of Bcl-2 expression at 22 hr after 2 hr MCA occlusion in the rat. A is a 10× magnification of a segment of a cresyl violet-stained coronal brain section at the level of the dorsal hippocampus. The area within the cerebral cortex in A with staining hypointensity, or “pallor,” represents tissue infarction, and thesquare at the edge of the infarct depicts the location from which the photomicrographs in B andD were taken. B and Drepresent the peri-infarct region within the cerebral cortex in the presence (B) and absence (D) of the primary antibody. Crepresents the corresponding area in the contralateral, uninjured hemisphere in the presence of the primary antibody. Bcl-2 immunoreactivity was detected in cell bodies and processes of large pyramidal neurons within the peri-infarct region (B). No immunoreactivity to Bcl-2 was apparent in the uninjured hemisphere (C). Background staining in D is caused by endogenous peroxidase activity in ischemic tissue. Scale bar, 50 μm.

Fig. 5.

Expression of Bcl-2 protein at 22 hr after 2 hr MCA occlusion in rat. A, Western blot analysis of Bcl-2 expression in contralateral (C) and ischemic (I) hemispheres in male (lanes 2C and 2I, respectively), intact female (3C, 3I), ovariectomized female (4C, 4I), and estrogen-supplemented ovariectomized female rats (5C,5I). Bcl-2 protein was identified as a 26 kDa protein in the presence of mouse myeloblast cell lysate expressing a high level of Bcl-2 as positive control (lane 1). Bcl-2 protein was induced by ischemia in all groups. Estrogen treatment and female sex were associated with increased Bcl-2 protein expression in ischemic and contralateral hemispheres. B, Western blot analysis of Bcl-2 expression within the ischemic hemisphere after stroke in male (M; n = 5), intact female (F; n = 5), ovariectomized female (O; n = 5), and estrogen-supplemented O (E;n = 5) rats. Bcl-2 expression was estimated from the optical density (OD) of bcl-2 band after normalization to the amount of transferred protein (micrograms) and the area of pallor (square millimeters) in adjacent sections after staining with cresyl violet. * denotes a statistically significant difference from both M and O(p < 0.05, ANOVA).

Fig. 6.

Comparison between the infarct size of male and female wild-type (WT) mice and transgenic mice overexpressing Bcl-2 in neurons (BCL-2) 22 hr after 90 min MCA occlusion. Cerebral infarct, identified in coronal brain sections by TTC staining, was smaller in male but not female transgenic mice compared with WT mice. Values are mean ± SEM of eight animals. * denotes statistically significant difference from WT males.

Fig. 7.

Comparison between the infarct size of intact (F) and ovariectomized (O) wild-type mice (WT) and mice overexpressing Bcl-2 (BCL2) 22 hr after 90 min MCA occlusion. Infarct size was 35% larger in ovariectomized WT female mice compared with intact females but was not different between ovariectomized and intact BCL2 females. Values are mean ± SEM of 10–11 animals. * denotes statistically significant difference from other groups (p < 0.05; ANOVA).