Impact of methodology on estrogens' effects on cerebral ischemia in rats: an updated meta-analysis

Abstract

Background: Although most animal stroke studies have demonstrated potent neuroprotective effects of estrogens, there are a number of articles reporting the opposite. In 2009, we made the case that this dichotomy was related to administered estrogen dose. Several other suggestions for the discordant results have also been propagated, including the age of the experimental animals and the length of hypoestrogenicity prior to estrogen administration. These two suggestions have gained much popularity, probably because of their kinship with the window of opportunity hypothesis, which is commonly used to explain the analogous dichotomy among human studies. We were therefore encouraged to perform an updated meta-analysis, and to improve it by including all relevant variables in a large multiple regression model, where the impact of confounders could be controlled for.

Results: The multiple regression model revealed an indisputable impact of estrogen administration mode on the effects of estrogens in ischemic stroke. Subcutaneous slow-release pellets differed from the injection and silastic capsule treatments in terms of impact of estrogens on ischemic stroke, showing that the first mentioned were more prone to render estrogens damaging. Neither the use of elderly animals nor the adoption of longer wash-out periods influenced estrogens' effects on experimental ischemic stroke in rats.

Conclusions: We conclude that the discordant results regarding estrogens' effects in rat models of ischemic stroke are a consequence of differences in estrogen administration modes. These results are not only of importance for the ongoing debate regarding menopausal hormone therapy, but also have an important bearing on experimental stroke methodology and the apparent translational roadblock for suggested stroke interventions.

Figure 1

Three hundred and thirty-three articles were assessed for inclusion according to criteria A to I. Consensus was reached to finally include 61 articles, of which 45 were included in the previous systematic analysis, and 16 were not [12].

Figure 2

After controlling for confounding factors, it remained clear that slow-release pellets rendered significantly higher EC-ratios than the injection and silastic capsule regimens did (p < 0.001). The black diamonds mark the mean EC-ratio with 95% confidence interval for each of the three administration modes. Of note, the slow-release pellet diamond reaches over the 100% EC-ratio line, demonstrating the pellets’ potential for harm as well as protection. EC-ratio = infarct size in estrogen treated group divided by infarct size in control group.

Figure 3

By three simple linear regression analyses, it was found that higher pellet doses significantly increased EC-ratio (y = 72.8 + 0.05x; p = 0.001), while there was an opposite trend (not significant) among injection and silastic capsule regimens. The numbers of group pairs included in the slow-release pellet, injection and silastic capsule simple regression models were 39, 50 and 31, respectively. Note that the injection graph has been compressed in the upper dose range to accommodate three group pairs being administered very high doses.

Figure 4

The frequencies of the different categories in the 124 group pairs are presented as percentages. Please note that the Disease and Estrogen type variables were omitted from the statistical analysis because too few studies used diseased animals and other estrogens than 17β-estradiol, respectively.