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Challenges in experimental stroke research: The 17β-estradiol example
Linköping University, Department of Clinical and Experimental Medicine, Division of Neuro and Inflammation Science. Linköping University, Faculty of Medicine and Health Sciences.ORCID iD: 0000-0001-8813-0384
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Ischemic stroke causes millions of deaths around the world each year, and surviving patients often suffer from long-term disability. Hundreds of promising drug candidates have been identified in animal models, but the clinical trials have repeatedly failed. Lack of methodological quality in the animal studies, e.g. low statistical power as a result of small group sizes in combination with high outcome variability and high mortality, has been suggested to in part explain the lack of translational success. In the meta-analytical Papers II and Paper V, we therefore investigated how method parameters impact infarct size variation and mortality in rodent stroke studies. These findings can help researchers to optimize their animal models or to more exactly predict variability and mortality given a certain experimental setup.

The relation between ischemic stroke and estrogens is complex. Premenopausal women have a lower risk of stroke than men of the same age, suggesting that female sex hormones provide protection against cerebrovascular events. The idea of a beneficial effect on the brain of estrogens was also supported by epidemiological studies showing that estrogens given as postmenopausal hormone replacement therapy decreased the risk of stroke. However, subsequent clinical trials reported the opposite, an increased risk. Interestingly, discrepancies exist also in the animal stroke literature. The majority of the rodent studies on the effects of estrogens have shown protection, but there are also several examples of increased damage. Based on experimental results and a meta-analysis, it was hypothesized that differences in hormone administration methods and their resulting plasma concentrations of estrogens might explain the previous discordant animal findings. Paper I investigated the commonly used methods for 17β-estradiol administration and found that the popular slow-release pellets produced high and unpredictable serum concentrations. A novel method with 17β-estradiol administered orally in Nutella® was also evaluated with promising results. Paper III extracted data regarding methodological choices from all previously published estrogen-stroke studies, and showed through metaanalysis that slow-release pellets are more prone to render estrogens damaging. Finally, Paper IV tested whether estrogens could both exert neuroprotection and promote detrimental effects merely depending on dose and irrespective of the administration route. Surprisingly, and in contrast to the hypothesis, a significant negative correlation was found between 17β-estradiol dose group and infarct size meaning that the higher the dose, the smaller the infarcts.

In summary, this thesis does not confirm the hypothesis of dose-related neuroprotective vs neurodamaging effects of estrogens on ischemic stroke. If high estrogen doses/plasma concentrations per se can cause increased stroke damage, such a phenomenon is not very robust, and seems to depend on tight dose ranges and/or other experimental circumstances. Although not directly applicable to the clinical situation, hopefully in a long-term perspective these findings may contribute in elucidating when estrogens are beneficial and when they are harmful. Further, it adds to the growing literature on how the quality of experimental stroke research can be increased to try to overcome translational difficulties.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2016. , 70 p.
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1504
National Category
Basic Medicine Neurosciences
URN: urn:nbn:se:liu:diva-123893DOI: 10.3384/diss.diva-123893ISBN: 978-91-7685-852-3 (print)OAI: diva2:893625
Public defence
2016-02-05, Berzeliussalen, Ingång 65, Campus US, Linköping, 09:00 (Swedish)
Available from: 2016-01-13 Created: 2016-01-13 Last updated: 2016-01-15Bibliographically approved
List of papers
1. Methods for long-term 17β-estradiol administration to mice
Open this publication in new window or tab >>Methods for long-term 17β-estradiol administration to mice
2012 (English)In: General and Comparative Endocrinology, ISSN 0016-6480, E-ISSN 1095-6840, Vol. 175, no 1, 188-193 p.Article in journal (Refereed) Published
Abstract [en]

Rodent models constitute a cornerstone in the elucidation of the effects and biological mechanisms of 17β-estradiol. However, a thorough assessment of the methods for long-term administration of 17β-estradiol to mice is lacking. The fact that 17β-estradiol has been demonstrated to exert different effects depending on dose emphasizes the need for validated administration regimens. Therefore, 169 female C57BL/6 mice were ovariectomized and administered 17β-estradiol using one of the two commonly used subcutaneous methods; slow-release pellets (0.18 mg, 60-day release pellets; 0.72 mg, 90-day release pellets) and silastic capsules (with/without convalescence period, silastic laboratory tubing, inner/outer diameter: 1.575/3.175 mm, filled with a 14 mm column of 36 μg 17β-estradiol/mL sesame oil), or a novel peroral method (56 μg 17β-estradiol/day/kg body weight in the hazelnut cream Nutella). Forty animals were used as ovariectomized and intact controls. Serum samples were obtained weekly for five weeks and 17β-estradiol concentrations were measured using radioimmunoassay. The peroral method resulted in steady concentrations within – except on one occasion – the physiological range and the silastic capsules produced predominantly physiological concentrations, although exceeding the range by maximum a factor three during the first three weeks. The 0.18 mg pellet yielded initial concentrations an order of magnitude higher than the physiological range, which then decreased drastically, and the 0.72 mg pellet produced between 18 and 40 times higher concentrations than the physiological range during the entire experiment. The peroral method and silastic capsules described in this article constitute reliable modes of administration of 17β-estradiol, superior to the widely used commercial pellets.

Place, publisher, year, edition, pages
Elsevier, 2012
Estradiol; Slow-release pellets; Silastic capsule; Per os; Uterine weight
National Category
Pharmaceutical Sciences
urn:nbn:se:liu:diva-107100 (URN)10.1016/j.ygcen.2011.11.014 (DOI)000299065800022 ()22137913 (PubMedID)2-s2.0-84855192470 (ScopusID)
Available from: 2014-06-05 Created: 2014-06-05 Last updated: 2016-01-13Bibliographically approved
2. Method parameters’ impact on mortality and variability in rat stroke experiments: a meta-analysis
Open this publication in new window or tab >>Method parameters’ impact on mortality and variability in rat stroke experiments: a meta-analysis
2013 (English)In: BMC neuroscience (Online), ISSN 1471-2202, E-ISSN 1471-2202, Vol. 14, no 41Article in journal (Refereed) Published
Abstract [en]


Even though more than 600 stroke treatments have been shown effective in preclinical studies, clinically proven treatment alternatives for cerebral infarction remain scarce. Amongst the reasons for the discrepancy may be methodological shortcomings, such as high mortality and outcome variability, in the preclinical studies. A common approach in animal stroke experiments is that A) focal cerebral ischemia is inflicted, B) some type of treatment is administered and C) the infarct sizes are assessed. However, within this paradigm, the researcher has to make numerous methodological decisions, including choosing rat strain and type of surgical procedure. Even though a few studies have attempted to address the questions experimentally, a lack of consensus regarding the optimal methodology remains.


We therefore meta-analyzed data from 502 control groups described in 346 articles to find out how rat strain, procedure for causing focal cerebral ischemia and the type of filament coating affected mortality and infarct size variability.


The Wistar strain and intraluminal filament procedure using a silicone coated filament was found optimal in lowering infarct size variability. The direct and endothelin methods rendered lower mortality rate, whereas the embolus method increased it compared to the filament method.


The current article provides means for researchers to adjust their middle cerebral artery occlusion (MCAo) protocols to minimize infarct size variability and mortality.

Place, publisher, year, edition, pages
BioMed Central, 2013
Brain infarction, Middle cerebral artery occlusion, Rats, Methods, Mortality, Variability
National Category
Medical and Health Sciences
urn:nbn:se:liu:diva-93981 (URN)10.1186/1471-2202-14-41 (DOI)000318616000001 ()23548160 (PubMedID)

Funding Agencies|County Council of Ostergotland, Sweden||

Available from: 2013-06-13 Created: 2013-06-13 Last updated: 2016-02-29Bibliographically approved
3. Impact of methodology on estrogens’ effects on cerebral ischemia in rats: an updated meta-analysis
Open this publication in new window or tab >>Impact of methodology on estrogens’ effects on cerebral ischemia in rats: an updated meta-analysis
2014 (English)In: BMC neuroscience (Online), ISSN 1471-2202, E-ISSN 1471-2202, Vol. 15, no 22Article in journal (Refereed) Published
Abstract [en]


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.


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.


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.

Place, publisher, year, edition, pages
BioMed Central, 2014
Cerebral ischemia; Estradiol; Estrogens; Meta-analysis; Rats; Stroke
National Category
Basic Medicine
urn:nbn:se:liu:diva-106875 (URN)10.1186/1471-2202-15-22 (DOI)000334885400001 ()24495535 (PubMedID)
Available from: 2014-05-28 Created: 2014-05-23 Last updated: 2016-02-29Bibliographically approved
4. Effects of high and low 17β-estradiol doses on focal cerebral ischemia in rats
Open this publication in new window or tab >>Effects of high and low 17β-estradiol doses on focal cerebral ischemia in rats
2016 (English)Manuscript (preprint) (Other academic)
Abstract [en]

The majority of the numerous animal studies of the effects of estrogens on cerebral ischemia have reported neuroprotective results, but a few have shown increased damage. Differences in hormone administration methods, resulting in highly different 17β-estradiol levels, may explain the discrepancies in previously reported effects. The objective of the present study was to test the hypothesis that it is the delivered dose per se, and not the route and method of administration, that determines the effect, and that high doses are damaging while lower doses are protective. One hundred and twenty ovariectomized female Wistar rats (n=40 per group) were randomized into three groups, subcutaneously administered different doses of 17β-estradiol and subjected to transient middle cerebral artery occlusion. The modifi ed sticky tape test was performed after 24 h and the rats were subsequently sacrifi ced for infarct size measurements. In contrast to our hypothesis, a signifi cant negative correlation between 17β-estradiol dose and infarct size was found (p=0.018). Thus, no support was found for the hypothesis that 17β-estradiol can be both neuroprotective and neurotoxic merely depending on dose. In fact, on the contrary, the fi ndings indicate that the higher the dose of 17β-estradiol, the smaller the infarct.

National Category
Clinical Medicine Microbiology in the medical area
urn:nbn:se:liu:diva-123890 (URN)
Available from: 2016-01-13 Created: 2016-01-13 Last updated: 2016-01-13Bibliographically approved
5. Method parameters’ impact on mortality and variability in mouse stroke experiments: a meta-analysis
Open this publication in new window or tab >>Method parameters’ impact on mortality and variability in mouse stroke experiments: a meta-analysis
Show others...
2016 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6Article in journal (Refereed) Published
Abstract [en]

Although hundreds of promising substances have been tested in clinical trials, thrombolysis currently remains the only specifi c pharmacological treatment for ischemic stroke. Poor quality, e.g. low statistical power, in the preclinical studies has been suggested to play an important role in these failures. Therefore, it would be attractive to use animal models optimized to minimize unnecessary mortality and outcome variability, or at least to be able to power studies more exactly by predicting variability and mortality given a certain experimental setup. The possible combinations of methodological parameters are innumerous, and an experimental comparison of them all is therefore not feasible. As an alternative approach, we extracted data from 334 experimental mouse stroke articles and, using a hypothesis-driven meta-analysis, investigated the method parameters’ impact on infarct size variability and mortality. The use of Swiss and C57BL6 mice as well as permanent occlusion of the middle cerebral artery rendered the lowest variability of the infarct size while the emboli methods increased variability. The use of Swiss mice increased mortality. Our study offers guidance for researchers striving to optimize mouse stroke models.

Place, publisher, year, edition, pages
Nature Publishing Group, 2016
National Category
Clinical Medicine Microbiology in the medical area
urn:nbn:se:liu:diva-123892 (URN)10.1038/srep21086 (DOI)000370034300001 ()

Funding agencies:  County Council of Ostergotland, Sweden

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Available from: 2016-01-13 Created: 2016-01-13 Last updated: 2016-03-09Bibliographically approved

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