Estradiol modulates post-ischemic cerebral vascular remodeling and improves long-term functional outcome in a rat model of stroke

Abstract

We previously observed that 17β-estradiol (E2) augments ischemic borderzone vascular density 10 days after focal cerebral ischemia-reperfusion in rats. We now evaluated the effect of E2 on vascular remodeling, lesional characteristics, and motor recovery up to 30 days after injury. Peri-lesional vascular density in tissue sections from rats treated with 0.72 mg E2 pellets was higher compared to 0.18 mg E2 pellets or placebo (P) pellets: vascular density index, 1.9 ± 0.2 (0.72 mg E2) vs. 1.4 ± 0.2 (0.18 mg E2) vs. 1.5 ± 0.4 (P), p=0.01. This was consistent with perfusion magnetic resonance imaging (MRI) measurements of lesional relative cerebral blood flow (rCBF): 1.89 ± 0.32 (0.72 mg E2) vs. 1.32 ± 0.19 (P), p=0.04. Post-ischemic angiogenesis occurred in P-treated as well as E2-treated rats. There was no treatment-related effect on lesional size, but lesional tissue was better preserved in E2-treated rats: cystic component as a % of total lesion, 30 ± 12 (0.72 mg E2) vs. 29 ± 17 (0.18 mg E2) vs. 61 ± 29 (P), p=0.008. Three weeks after right middle cerebral artery territory injury, rats treated with 0.72 mg E2 pellets used the left forelimb more than P-treated or 0.18 mg E2-treated rats: limb use asymmetry score, 0.09 ± 0.43 (0.72 mg E2) vs. 0.54 ± 0.12 (0.18 mg E2) vs. 0.54 ± 0.40 (P), p=0.05. We conclude that treatment with 0.72 mg E2 pellets beginning one week prior to ischemia/reperfusion and continuing through the one-month recovery period results in augmentation of lesional vascularity and perfusion, as well as improved motor recovery.

Copyright © 2012 Elsevier B.V. All rights reserved.

Figures

Figure 1

Lesion size correlations. (A) Lesion size expressed as % contralateral volume was obtained in eight histologic sections spanning bregma 3.24 to -6.24 (Osborne et al., 1987) and correlated with lesion size expressed as % contralateral area in a histologic section at bregma 1.1. The correlation coefficient was 0.9, p=0.002, n=9. (B) Lesion size expressed as % contralateral volume was obtained using magnetic resonance imaging (MRI) and correlated with lesion size expressed as % contralateral area in a histologic section at bregma 1.1. The correlation coefficient was 0.8, p=0.005, n=11.

Figure 2

Magnetic resonance images and histology. (A) and (B): T2 maps were obtained from placebo (P)-treated (A) and 17β-estradiol (E2, 0.72) - treated (B) rats. Regions of interest (ROIs) derived from T2 threshold analyses were used to define the total ischemic lesion (red outlines) and cystic regions of the lesion (green outlines). Lesions in 0.72 mg E2-treated animals were characterized by smaller cystic components and more preserved tissue compared to P-treated animals. (C) and (D): Low magnification composites of immunolabeled brain sections corresponding to the MR T2 maps were generated to confirm lesion characteristics in P-treated (C) and E2-treated (D) rats. Tissue sections were stained with anti-GFAP antibody (green), anti-CD31 antibody (red), and Hoechst (blue). Asterisks indicate cystic regions; scale bar (C), (D), 1 mm; magnification 4X. (E) and (F): Tissue sections were stained with hematoxylin and eosin (H&E) in order to calculate lesional areas in representative coronal sections of P-treated and E2-treated (F) rats. Asterisks indicate cystic regions; scale bar (E), (F), 5 mm; magnification 0.625X. Panels (A), (C), (E) are from the same animal; (B), (D), (F) are from the same animal.

Figure 3

Lesional characteristics. Lesional endothelial cells in both placebo (P)-treated and 0.72 mg 17β-estradiol (E2) pellet -treated (E2, 0.72) rats incorporated and retained 5-bromo-2′-deoxyuridine (BrdU) administered during the first four days after cerebral ischemia-reperfusion and expressed vascular markers. (A) CD31-labeled blood vessel (red) with a light-blue nucleus (arrow) due to a overlay of blue Hoechst stain and green BrdU immunofluorescence, and an example of a Hoechst-stained, BrdU-negative endothelial cell nucleus (arrow-head) in tissue from a P-treated rat; scale bar, 20 μm; magnification 40X. (B) BrdU-labeled endothelial cells immunoreactive for zona occludens (arrow) in tissue from an E2-treated rat; scale bar, 10 μm; magnification 60X. Lesional tissue from P-treated and E2-treated rats also contained CD11b/c-positive (C) / Iba-1-negative (D) macrophages; scale bar (C), (D), 100 μm; magnification 20X. Lesional tissue (double asterisk) in contrast to uninjured tissue (single asterisk) did not express the neuronal marker microtubule-associated protein-2 (Map2), as shown in a tissue section from a P-treated rat (E), or NeuN, as shown in a tissue section from an E2-treated rat (F); scale bar (E), (F), 200 μm; magnification 10X.

Figure 4

Lesional vasculature. (A) and (B): Cerebral blood flow (CBF) maps were derived from DSC-MRI in placebo (P)-treated (A) and 17β-estradiol (E2, 0.72)-treated (B) rats; color scales are relative, with contralateral hues approximately set to unity. CBF was higher in lesional regions of interest (ROIs, delineated white overlay) specifically in remnant tissue around cysts (asterisks) of E2-treated animals compared to P-treated animals. To avoid contamination from striatal and thalamic arterial flow, analysis of CBF was performed within the ROI above the horizontal midline. Arrows indicate examples of CBF signal thought to correspond to basal ganglionic arteries. (C) and (D): Peri-lesional striatal ROIs were evaluated in tissue sections labeled with antibodies against CD31 (red) and GFAP (green) in P-treated (P) (C) and E2-treated (E2, 0.72) (D) rats. GFAP expression was enriched in borderzone, and was absent from lesional, tissue. Single asterisks indicate cysts; double asterisks in (D) indicate vascular lesional tissue in the E2-treated rat; brackets delineate glial scars; scale bar (C), (D), 50 μm; magnification 20X. (E): Vascular density in peri-lesional ROIs was quantified and found to be higher in 0.72 mg E2-treated rats compared to 0.18 mg E2 or P-treated rats, *p=0.013, ANOVA. The diagram (not to scale) shows the locations of ischemic ROIs (purple box on the right-hand side) and non-ischemic ROIs (purple box on the left-hand side) used for determination of vascular density indices.

Figure 5

Left forelimb function. Rats were evaluated using the cylinder test before (Pre-MCAO) and weekly for three weeks (Week 1, 2, 3) after transient right middle cerebral artery occlusion (MCAO). The ischemic lesion causes decreased use of the left forelimb, i.e., a positive limb use asymmetry score, derived from counting limb placements, [(right - left) / (right + left + both)]. 0.72 mg 17β-estradiol (E2)-treated, compared to placebo (P)-treated and 0.18 mg E2-treated rats exhibited increased left forelimb use three weeks after experimental ischemic stroke, *p=0.05, ANOVA.