Effects of chronic and acute oestrogen replacement therapy in aged animals after experimental stroke.

Stroke is a disease that mainly affects the elderly. Stroke rates more than double every 10 years after the age of 55 years in both men and women (1). However, functional outcomes after stroke are worse in women than in men, with higher associated disability and mortality (2). Most international databases consistently demonstrate that women enjoy a lower incidence of stroke relative to men until well after menopause, after which stroke incidence in women climbs dramatically (3, 4). Although the age-related risk of stroke is evident, very few studies have examined the neurochemical changes that occur in aged brains after stroke because most of studies have been exclusively performed in young animals. Oestrogens, primarily 17β-oestradiol (E2), are potent neuroprotective agents in various animal models, including experimental ischaemic stroke (5). Numerous observational and retrospective clinical studies have also suggested that oestrogen replacement therapy (ERT) protects postmenopausal women against age-related diseases, including cardiovascular disease and stroke (6). However, two important clinical ERT trials, the Women Estrogen Stroke Trial (WEST) and the Women Health Initiative (WHI) trial, reported an unexpected increase in stroke incidence in E2-treated women (7, 8). The explanation for these findings has been debated extensively in the literature (9). One major factor is the timing of E2 replacement. Participants recruited in these trials were well beyond menopause when they received ERT; and it has been hypothesised that ERT should be initiated immediately at menopause to achieve its protective effects (10, 11), which is an effect recently modelled in preclinical studies. Prolonged periods of hypo-oestrogenicity led to an enhanced inflammatory response when ERT was administered, which exacerbated stroke damage; however, animals in that study were not aged. Intriguingly, the latest study (12) reporting on post-intervention outcomes in women involved in the WHI trial who were taken off ERT found that, after 10.7 years of follow-up, there was no significant difference in stroke risk between E2 and placebo-treated groups. Recent studies (13, 14) have revealed that brain ischaemia is a powerful stimulus that triggers a series of events leading to the activation of several transcription factors involved in the inflammatory responses, including nuclear factor kappa B (NF-κB) (15). After ischaemia NF-κB translocates from the cytoplasm to the nucleus and up-regulates the expression of many pro-inflammatory genes, such as tumour necrosis factor (TNF)-α, monocyte chemotactic protein (MCP)-1 and interlukin (IL)-6, etc. (15, 16). A growing body of evidence (17, 18) has shown that E2 interacts with NF-κB through oestrogen receptor (ER)α to down-regulate the expression of inflammatory cytokines. However, whether E2 continues to have these beneficial effects in the aged brain is not yet known. The present study aimed to evaluate the effects of either chronic or acute ERT on aged mice subjected to ischaemic stroke and to explore the possible mechanism underlying the effects of E2 in the aged brain. The mice utilised in the present study were 17 months old, which is an age equivalent to a 50–55-year-old woman (19) (i.e. the approximate age of menopause). We hypothesised that only chronic ERT initiated early after reproductive senescence would exert neuroprotective effects on mice after stroke, and predicted that the effect is related to the down-regulation of NF-κB activation as a result of continuous exposure to E2 after menopause. We also performed studies in aged males, aiming to determine whether there were sex differences in the response to ERT and to control for other factors associated with ageing.