Intact female stroke-prone hypertensive rats lack responsiveness to mineralocorticoid receptor antagonists

Abstract

Data from the Framingham Heart Study suggest that women may be more sensitive to the deleterious cardiovascular remodeling effects of aldosterone. Previous studies from our laboratory have shown that chronic treatment with spironolactone, a mineralocorticoid receptor (MR) antagonist, decreases ischemic cerebral infarct size and prevents remodeling of the middle cerebral artery (MCA) in male spontaneously hypertensive stroke-prone rats (SHRSP). Therefore, we hypothesized that MR antagonism would reduce ischemic infarct size and prevent MCA remodeling in female SHRSP. Six-week-old female SHRSP were treated for 6 wk with spironolactone (25 or 50 mg.kg(-1).day(-1)) or eplerenone (100 mg.kg(-1).day(-1)) and compared with untreated controls. At 12 wk, cerebral ischemia was induced for 18 h using the intraluminal suture occlusion technique, or the MCA was isolated for analysis of passive structure using a pressurized arteriograph. MR antagonism had no effect on infarct size or passive MCA structure in female SHRSP. To study the potential effects of estrogen, the above experiments were repeated in bilaterally ovariectomized (OVX) female SHRSP treated with spironolactone (25 mg.kg(-1).day(-1)). Infarct size and vessel structure in OVX SHRSP were not different from control SHRSP. Spironolactone had no effect on infarct size in OVX SHRSP. However, MCA lumen and outer diameters were increased in spironolactone-treated OVX SHRSP, suggesting an effect of estrogen. Cerebral artery MR expression, assessed by Western blotting, was increased in female, compared with male, SHRSP. These studies highlight an apparent sexual dimorphism of MR expression and activity in the cerebral vasculature from hypertensive rats.

Fig. 1

Effect of mineralocorticoid receptor (MR) antagonists on cerebral infarct size after 18 h of permanent ischemia in female spontaneously hypertensive stroke-prone rats (SHRSP) and Wistar Kyoto (WKY) rats. Treatments were started at 6 wk of age in female SHRSP. Permanent cerebral ischemia was induced using middle cerebral artery occlusion (MCAO) at 12 wk of age. Brains were sliced in 2-mm-thick sections and stained to visualize the infarcted regions, which are indicated by the white areas on the brain slice. Representative brain slices are shown for each group. The percent hemisphere infarcted (%HI) was calculated using the Swanson equation after digital analysis of 3 brain slices from each animal. Spironolactone (Spir; 25 and 50 mg/kg) and eplerenone treatment had no effect on ischemic infarct size in female SHRSP. Infarct sizes were significantly smaller in control WKY rats compared with all SHRSP groups (*P <0.05, ANOVA).

Fig. 2

Effect of MR antagonists on middle cerebral artery (MCA) remodeling in female SHRSP. Treatments were started at 6 wk of age in female SHRSP. MCAs from 12-wk-old female SHRSP and WKY rats were mounted in a pressurized arteriograph, and measurements of passive lumen (A) and outer (B) diameters were acquired over a range of pressures using video microscopy. MCA luminal diameters were larger in control WKY rats compared with all SHRSP groups (*P < 0.05, ANOVA). In eplerenone-treated SHRSP, MCA luminal diameters were increased compared with control and spironolactone-treated (25 mg/kg) SHRSP (†P < 0.05, ANOVA). MR antagonists had no effect on MCA outer diameters in SHRSP. MCA outer diameters were larger in control WKY rats compared with all SHRSP groups (*P < 0.05, ANOVA).

Fig. 3

Effect of MR antagonists on MCA compliance in female SHRSP. Circumferential wall stress and strain were calculated from passive MCA luminal diameter and wall thickness measurements obtained over a range of pressures using a pressurized arteriograph system. A rightward shift in the stress-strain curve indicates increased compliance, and a leftward shift indicates decreased compliance. There was a rightward shift in the MCA stress-strain curve from spironolactone-treated (25 mg/kg) SHRSP.

Fig. 4

Effect of spironolactone (25 mg/kg) treatment on cerebral infarct size after 18 h of permanent ischemia in ovariectomized (OVX) SHRSP. Bilateral ovariectomy was performed, and spironolactone treatment was started, at 6 wk of age in female SHRSP. Permanent cerebral ischemia was induced using MCAO at 12 wk of age. Brains were sliced in 2-mm-thick sections and stained to visualize the infarcted regions, which are indicated by the white areas on the brain slice. Representative brain slices are shown for each group. The %HI was calculated using the Swanson equation after digital analysis of 3 brain slices from each animal. Spironolactone (25 mg/kg) treatment had no effect on ischemic infarct size in OVX SHRSP. Infarct sizes from control female WKY rats were included on the graph to highlight the severity of cerebral damage in SHRSP.

Fig. 5

Effect of spironolactone (25 mg/kg) treatment on MCA remodeling in OVX SHRSP. Bilateral ovariectomy was performed, and spironolactone treatment was started, at 6 wk of age in female SHRSP. MCAs from 12-wk-old OVX SHRSP were mounted in a pressurized arteriograph, and measurements of passive luminal (A) and outer (B) diameters were acquired over a range of pressures using video microscopy. Spironolactone (25 mg/kg) treatment increased MCA luminal and outer diameters in OVX SHRSP (*P <0.05, ANOVA). MCA luminal and outer diameters from control WKY rats were included to show remodeling of MCAs from SHRSP.

Fig. 6

Effect of spironolactone (25 mg/kg) treatment on MCA compliance in OVX SHRSP. Circumferential wall stress and strain were calculated from passive MCA luminal diameter and wall thickness measurements obtained over a range of pressures using a pressurized arteriograph system. A rightward shift in the stress-strain curve indicates increased compliance, and a leftward shift indicates decreased compliance. Spironolactone treatment did not cause a shift in the MCA stress-strain curve from OVX SHRSP.

Fig. 7

MR expression in cerebral blood vessels. Cerebral arteries were collected from nontreated 12-wk-old male and female SHRSP and OVX and intact female SHRSP. Western blotting was performed on protein extracts (25 μg for male/female and 30 μg for intact/OVX) using an antibody specific for the MR. To control for equal loading, blots were stripped and reprobed using an antibody specific for β-tubulin. Representative Western blots are shown. A: MR expression was increased in cerebral arteries collected from female SHRSP compared with male SHRSP (*P < 0.05, t-test). B: MR expression was not different in cerebral arteries collected from OVX compared with intact, female SHRSP (P > 0.05, t-test).