Timing of estrogen therapy after ovariectomy dictates the efficacy of its neuroprotective and antiinflammatory actions

Abstract

Recent studies describing the seemingly contradictory actions of estrogens in ischemic stroke injury have led us to reevaluate the circumstances under which estrogen therapy (ET) provides benefits against cerebral stroke and decipher its mechanisms of action. One prominent feature that follows stroke injury is massive central and peripheral inflammatory responses. Evidence now suggests that postischemic inflammatory responses strongly contribute to the extent of brain injury, and 17beta-estradiol (E(2)) may protect the ischemic brain by exerting antiinflammatory actions. In an attempt to explain recently reported dichotomous effects of E(2) in stroke injury, we tested the hypothesis that an extended period of hypoestrogenicity both prevents E(2) from protecting the brain against ischemia and simultaneously suppresses its antiinflammatory actions. We report that E(2) exerts profound neuroprotective action when administered immediately upon ovariectomy, but not when administered after 10 weeks of hypoestrogenicity. Consistently, E(2) treatment given immediately at the time of ovariectomy attenuated central and peripheral production of proinflammatory cytokines after ischemic stroke. In contrast, E(2) did not suppress production of proinflammatory molecules when it was administered after 10 weeks postovariectomy. These results demonstrate that a prolonged period of hypoestrogenicity disrupts both neuroprotective and antiinflammatory actions of E(2). Our findings may help to explain the results of the Women's Health initiative that reported no beneficial effect of ET against stroke because the majority of the subjects initiated ET after an extended period of hypoestrogenicity.

Fig. 1.

Low physiological levels of E₂ protect the brain against ischemic injury. (A) C57BL/6J mice (19-week-old) were ovariectomized and immediately implanted with capsules containing either oil (n = 8) or E₂ (n = 11) for 1 week. Subsequently, animals underwent experimental ischemia by middle cerebral artery occlusion (MCAO) at 20 weeks of age and were killed 24 h after the onset of injury. (B and C) Infarct volumes were measured by using TTC staining in oil- (B) or E₂- (C) treated mice. (D–F) Immediate E₂ treatment significantly reduced the total infarct volume (D; ∗, P < 0.02) and the extent of injury in both the cortex (E; ∗, P < 0.05) and striatum (F; ∗, P < 0.03) of 20-week-old mice (unpaired two-tailed t test, mean ± SEM, n = 8–11).

Fig. 2.

Prolonged hypoestrogenicity disrupts the ability of E₂ to protect the ischemic brain. (A) Nine-week-old C57BL/6J mice were ovariectomized and 10 weeks later implanted with oil (n = 12) or E₂ (n = 10) capsules for 1 week before they underwent experimental ischemia. This paradigm of E₂ treatment leads to a prolonged period of hypoestrogenicity before stable serum levels of E₂ are restored through a delayed implantation of a Silastic capsule. (B and C) In contrast to animals that received capsules immediately at the time of ovariectomy (Fig. 1), mice in this group exhibited extensive ischemic brain injury regardless of E₂ treatment revealed by TTC staining. (D–F) A delayed E₂ treatment did not reduce the total infarct volume (D; P = 0.697). Specifically, after a prolonged period of hypoestrogenicity, E₂ failed to reduce brain infarction in the cortex (E; P = 0.447) as well as in the striatum (F; P = 0.581), compared with oil-treated counterparts (unpaired two-tailed t test, mean ± SEM, n = 10–12).

Fig. 3.

Immediate E₂ treatment attenuates proinflammatory responses in the brain. In the first paradigm, mice were ovariectomized at 19 weeks of age and immediately implanted with capsules containing either oil (n = 5) or E₂ (n = 8) for 1 week. (A and B) A mouse cytokine multiplex proteomic array revealed that ischemic injury increased the expression of monocyte chemoattractant protein-1 (MCP-1; #, P < 0.0001 for oil-treated mice; #, P = 0.0153 for E₂-treated mice) and IL-6 (IL-6; #, P < 0.0001 for oil-treated mice; #, P = 0.0043 for E₂-treated mice) on the injured side of the brain compared with the contralateral side. An immediate E₂ treatment attenuated ischemia-induced up-regulation of MCP-1 (∗, P = 0.0093) and IL-6 (∗, P = 0.0271) on the ipsilateral side of the ischemic brain. (C) E₂ prevented injury-induced down-regulation (#, P = 0.0004) of the neuroprotective VEGF (∗, P = 0.0024). In the second paradigm, mice at 9 weeks of age were ovariectomized for 10 weeks and subsequently implanted with an oil (n = 5) or E₂ (n = 6) capsule for 1 week. (D) When administered 10 weeks after ovariectomy, E₂ did not suppress (P = 0.285) the injury-induced production of MCP-1 (#, P = 0.0052). (E) Similarly, E₂ did not attenuate (P = 0.541) the ischemia-induced up-regulation of IL-6 production (#, P = 0.0261). (F) A delayed E₂ treatment caused no changes in the expression of VEGF (P = 0.326). In both paradigms, data represent the mean ± SEM of five to eight animals per experimental group.

Fig. 4.

E₂ exerted antiinflammatory actions only when administered immediately after ovariectomy. (A–C) Immediate E₂ treatment (n = 9) suppressed plasma levels of IL-6 (A; ∗, P = 0.004), TNFα (B; ∗, P < 0.05), and GM-CSF (C; ∗, P = 0.0002), compared with oil-treated controls (n = 8). (D–F) When E₂ was administered 10 weeks postovariectomy (n = 11), it did not suppress the production of IL-6 (D; P = 0.737), TNFα (E; P = 0.351), and GM-CSF (F; P = 0.663), compared with oil-treated counterparts (n = 14). In both series, data represent the mean ± SEM of 8–14 animals per experimental group.