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The dose-dependent effects of estrogens on ischemic stroke
Linköping University, Department of Clinical and Experimental Medicine, Clinical Chemistry. Linköping University, Faculty of Health Sciences.
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Estrogens are a group of female sex hormones that in addition to central roles in reproductive functions also have profound impact on for example brain development, blood vessels, bone tissue, metabolism and the immune system. The dominant endogenous production sites for estrogens in females are the ovaries and adipose tissue, while exogenous sources include combined contraceptive hormone treatments and menopausal hormone therapy. A few decades ago, the observation that females in comparison to men seemed to be protected against cerebral ischemia, and that this benefit was partially lost during menopause, sparked the hypothesis that estrogens protect against stroke. This was later confirmed by epidemiological studies and a large number of experimental animal studies, which motivated extensive clinical trials in which estrogens and/or progestagens were administered with the intent to prevent degenerative conditions rather than to ameliorate menopausal symptoms. However, the results were generally disappointing. The largest study, the Women’s Health Initiative (WHI), was discontinued due to the observation of an increased risk of breast cancer, cardiovascular disease and stroke. In parallel, a small number of animal studies in which estrogens were shown to increase damage from cerebral ischemia were published, one of these originating from our laboratory. This was, despite the WHI outcome, a surprising result, since the vast majority of previous animal studies had demonstrated protective effects.

Therefore, in an attempt to explain the discordant results, Paper 1, and later Paper 4, of the current thesis were planned, in which four 17β-estradiol administration methods were tested. Substantial differences in serum hormone concentrations resulted from the different methods. Most importantly, the commercially available slow-release pellets used in our earlier experiments resulted in extremely high serum concentrations of 17β-estradiol. In Paper 2, 66 published studies that had investigated the effects of estrogens on stroke were meta-analyzed to pin-point the methodological reasons for the result dichotomy. Strikingly, in all six studies in which estrogens had produced damaging effects, the same type of slow-release pellets had been used, although these were used in a minority of the total number of studies. Paper 3 substantially strengthened the hypothesis that administration methods were crucial by showing that repeating the earlier experiment from our laboratory in which pellets had been used, but using a low-dose regimen instead, switched the estrogen effects from neurodamaging to neuroprotective. In Paper 5, an effort was made to challenge the assumption that the dose, and not the administration method per se, was the key factor, however this failed due to large intra-group infarct size variability.

The current thesis adds evidence to the notion that differences in administration methods and their resulting serum concentrations of 17β-estradiol constitute a major factor responsible for the dichotomous results in studies investigating estrogens’ effects on cerebral ischemia. Even though results from animal studies are difficult to extrapolate to humans, this has a bearing on the menopausal hormone therapy debate, indicating that the risk of stroke could be reduced if serum concentrations of estrogens are minimized.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2012. , 92 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1301
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-77193ISBN: 978-91-7519-937-5 (print)OAI: oai:DiVA.org:liu-77193DiVA: diva2:525451
Public defence
2012-06-05, Berzeliussalen, Ingång 65,, Campus US, Linköpings universitet, Linköping, 09:00 (Swedish)
Opponent
Supervisors
Available from: 2012-05-08 Created: 2012-05-08 Last updated: 2016-02-29Bibliographically approved
List of papers
1. Order of magnitude differences between methods for maintaining physiological 17β-estradiol concentrations in ovariectomized rats
Open this publication in new window or tab >>Order of magnitude differences between methods for maintaining physiological 17β-estradiol concentrations in ovariectomized rats
2008 (English)In: Scandinavian Journal of Clinical and Laboratory Investigation, ISSN 0036-5513, E-ISSN 1502-7686, Vol. 68, no 8, 814-822 p.Article in journal (Refereed) Published
Abstract [en]

The use of animal, especially rat, models, is crucial for elucidating the biological effects and mechanisms of the widely used hormone 17β-estradiol. Unfortunately there is a lack of consensus on optimal means of obtaining and maintaining physiological 17β-estradiol concentrations in plasma. This may be the reason for varying results in several studies including the disagreement on whether 17β-estradiol is neuroprotective or not. Very few studies have been devoted to investigating the characteristics and biological relevance of different methods of 17β-estradiol administration. We therefore ovariectomized 75 Sprague-Dawley rats and, following a 2 weeks wash-out period, administered 17β-estradiol using three different methods; daily injections (10 µg 17β-estradiol/kg), slow-release pellets (0.25 mg 60 day-release pellets, 0.10 mg 90 day-release pellets) and silastic capsules (with/without wash-out periods) (silastic laboratory tubing, inner/outer diameter: 1.575/3.175 mm, filled with 20 mm columns of 180 µg 17β-estradiol/mL sesame oil). Further 45 animals were used as ovariectomized and native controls, studied in different parts of the estrous cycle. Silastic capsules produced concentrations of 17β-estradiol within the physiological range for 4-5 weeks independent of whether a prior wash-out period was included or not. The slow-release pellets, irrespective of dose or release period, resulted in initial concentrations which were an order of magnitude above physiological concentrations during the first two weeks followed by a substantial decrease. Daily injections resulted in increasing 17β-estradiol concentrations, however within physiological levels. Silastic capsules are conveniently manufactured and used, and are superior to pellets and injections in reliably producing long-term 17β-estradiol concentrations within the physiological range.

Keyword
Estrogens, Injections, Pharmacokinetics, Silastic capsules, Slow-release pellets, Wash-out
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-15389 (URN)10.1080/00365510802409703 (DOI)
Note
Original publication: Jakob O. Ström, Elvar Theodorsson and Annette Theodorsson, Order of magnitude differences between methods for maintaining physiological 17β-estradiol concentrations in ovariectomized rats, 2008, Scandinavian Journal of Clinical and Laboratory Investigation.http://dx.doi.org/10.1080/00365510802409703. Copyright © Taylor & Francis Group, an informa businessAvailable from: 2008-11-05 Created: 2008-11-05 Last updated: 2017-12-14Bibliographically approved
2. Dose-related neuroprotective versus neurodamaging effects of estrogens in rat cerebral ischemia: a systematic analysis
Open this publication in new window or tab >>Dose-related neuroprotective versus neurodamaging effects of estrogens in rat cerebral ischemia: a systematic analysis
2009 (English)In: Journal of Cerebral Blood Flow and Metabolism, ISSN 0271-678X, E-ISSN 1559-7016, Vol. 29, no 8, 1359-1372 p.Article, review/survey (Refereed) Published
Abstract [en]

Numerous studies of the effects of estrogens for stroke prevention have yielded conflicting results in human and animal studies alike. We present a systematical analysis of study design and methodological differences between 66 studies where estrogens impact on ischemic brain damage in rat models has been investigated, providing evidence that the differences in results may be explained by high estrogen doses produced by slow-release pellets. These pellets have been used in all studies showing increased neurologic damage because of estrogens. Our data indicate that the increased neurologic damage is related to the pellets plasma concentration profile with an early, prolonged, supraphysiological peak. Neither the method of inducing the ischemic brain lesions, the choice of variables for measuring outcome, the measured plasma concentrations of estrogens at the time of ischemia nor rat population attributes (sex, strain, age, and diseases) are factors contributing to the discrepancies in results. This suggests that the effects of estrogens for stroke prevention are concentration related with a complex dose-response curve, and underscores the importance of carefully validating the experimental methods used. Future studies of hormone-replacement therapy in women may have to take dosage and administration regimens into account.

Keyword
ischemia, estrogen, hormone administration, methodology, rat
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-19897 (URN)10.1038/jcbfm.2009.66 (DOI)
Available from: 2009-08-14 Created: 2009-08-14 Last updated: 2017-12-13Bibliographically approved
3. Different methods for administering 17 beta-estradiol to ovariectomized rats result in opposite effects on ischemic brain damage
Open this publication in new window or tab >>Different methods for administering 17 beta-estradiol to ovariectomized rats result in opposite effects on ischemic brain damage
2010 (English)In: BMC neuroscience (Online), ISSN 1471-2202, E-ISSN 1471-2202, Vol. 11, 39- p.Article in journal (Refereed) Published
Abstract [en]

Background: Numerous stroke studies have controversially shown estrogens to be either neuroprotective or neurodamaging. The discordant results observed in rat brain ischemia models may be a consequence of discrepancies in estrogen administration modes resulting in plasma concentration profiles far from those intended. To test this hypothesis we reproduced in detail and extended an earlier study from our lab using a different mode of 17 beta-estradiol administration; home-made silastic capsules instead of commercial slow-release 17 beta-estradiol pellets. Four groups of female rats (n = 12) were ovariectomized and administered 17 beta-estradiol or placebo via silastic capsules. All animals underwent MCAo fourteen days after ovariectomy and were sacrificed three days later.

Results: In contrast to our earlier results using the commercial pellets, the group receiving 17 beta-estradiol during the entire experiment had significantly smaller lesions than the group receiving placebo (mean +/- SEM: 3.85 +/- 0.70% versus 7.15 +/- 0.27% of total slice area, respectively; p = 0.015). No significant neuroprotection was found when the 17 beta-estradiol was administered only during the two weeks before or the three days immediately after MCAo.

Conclusions: The results indicate that different estrogen treatment regimens result in diametrically different effects on cerebral ischemia. Thus the effects of estrogens on ischemic damage seem to be concentration-related, with a biphasic, or even more complex, dose-response relation. These findings have implications for the design of animal experiments and also have a bearing on the estrogen doses used for peri-menopausal hormone replacement therapy.

Place, publisher, year, edition, pages
BioMed Central, 2010
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-55058 (URN)10.1186/1471-2202-11-39 (DOI)000276444500001 ()20236508 (PubMedID)
Note

Original Publication: Jakob O Ström, Elvar Theodorsson, Lovisa Holm and Annette Theodorsson, Different methods for administering 17 beta-estradiol to ovariectomized rats result in opposite effects on ischemic brain damage, 2010, BMC NEUROSCIENCE, (11), 39. http://dx.doi.org/10.1186/1471-2202-11-39 Licensee: BioMed Central http://www.biomedcentral.com/

Available from: 2010-04-28 Created: 2010-04-28 Last updated: 2017-12-12Bibliographically approved
4. Methods for 17 beta-oestradiol administration to rats
Open this publication in new window or tab >>Methods for 17 beta-oestradiol administration to rats
2011 (English)In: Scandinavian Journal of Clinical and Laboratory Investigation, ISSN 0036-5513, E-ISSN 1502-7686, Vol. 71, no 7, 583-592 p.Article in journal (Refereed) Published
Abstract [en]

Several studies indicate that the beneficial or harmful effects of oestrogens in stroke are dose-dependent. Rats are amongst the most frequently used animals in these studies, which calls for thoroughly validated methods for administering 17 beta-oestradiol to rats. In an earlier study we characterised three different administration methods for 17 beta-oestradiol over 42 days. The present study assesses the concentrations in a short time perspective, with the addition of a novel peroral method. Female Sprague-Dawley rats were ovariectomised and administered 17 beta-oestradiol by subcutaneous injections, silastic capsules, pellets and orally (in the nut-cream Nutella (R)), respectively. One group received 17 beta-oestradiol by silastic capsules without previous washout time. Blood samples were obtained after 30 minutes, 1, 2, 4, 8, 12, 24, 48 and 168 hours and serum 17 beta-oestradiol (and oestrone sulphate in some samples) was subsequently analysed. For long-term characterisation, one group treated perorally was blood sampled after 2, 7, 14, 21, 28, 35 and 42 days. At sacrifice, uterine horns were weighed and subcutaneous tissue samples were taken for histological assessment. The pellets, silastic capsule and injection groups produced serum 17 beta-oestradiol concentrations that were initially several orders of magnitude higher than physiological levels, while the peroral groups had 17 beta-oestradiol levels that were within the physiological range during the entire experiment. The peroral method is a promising option for administering 17 beta-oestradiol if physiological levels or similarity to womens oral hormone therapy are desired. Uterine weights were found to be a very crude measure of oestrogen exposure.

Place, publisher, year, edition, pages
Informa Healthcare, 2011
Keyword
Dosage, drug administration routes, injection, oestradiol, per os, rat, silastic capsule, slow-release pellet, uterine weight, washout
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-71772 (URN)10.3109/00365513.2011.596944 (DOI)000295799300009 ()
Available from: 2011-11-04 Created: 2011-11-04 Last updated: 2017-12-08Bibliographically approved
5. Effects of high and low 17β-estradiol doses on cerebral ischemia
Open this publication in new window or tab >>Effects of high and low 17β-estradiol doses on cerebral ischemia
(English)Manuscript (preprint) (Other academic)
Abstract [en]

INTRODUCTION: Estrogens’ effects on cerebral ischemia have during the last two decades been the subject of intense research efforts. Notwithstanding this, the reasons that some studies indicate that estrogens are damaging while others show estrogen-induced neuroprotection has hitherto not been fully elucidated. Recent evidence indicates that discrepancies in hormone administration paradigms, resulting in highly different serum hormone concentrations, may account for this dichotomy. The current study was designed to test this  ypothesis.

METHODS: Sixty ovariectomized female rats were randomized into three groups differing in subsequent 17β-estradiol regimen (vehicle, low dose and high dose respectively). Following two weeks of treatment, focal cerebral ischemia was induced via an intraluminal filament middle cerebral artery occlusion (MCAo) method. All animals were subjected to a small functional testing battery, and three days after MCAo they were sacrificed for infarct size assessment.

RESULTS AND DISCUSSION: The hormone administration regimens significantly affected animal weights and feeding behavior, but infarct sizes did not differ between groups. Further, random intra-group variations in infarct size were too large to allow negative conclusions to be drawn. The large variation was possibly a consequence of too large occluding filament diameter in combination with that the animals were allowed to wake up during ongoing MCAo. After correcting the large variation, the hypothesis needs to be addressed anew.

Keyword
Administration methods, Cerebral ischemia, Elevated body swing test, Estrogens, Hormesis, Silastic capsules, Slow-release pellets
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-77192 (URN)
Available from: 2012-05-08 Created: 2012-05-08 Last updated: 2016-02-29Bibliographically approved

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