1. |
- Ström, Jakob O, et al.
(author)
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Dose-related neuroprotective versus neurodamaging effects of estrogens in rat cerebral ischemia : a systematic analysis
- 2009
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In: Journal of Cerebral Blood Flow and Metabolism. - New York, USA : SAGE Publications. - 0271-678X .- 1559-7016. ; 29:8, s. 1359-1372
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Research review (peer-reviewed)abstract
- 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.
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2. |
- Ström, Jakob O., 1983-, et al.
(author)
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Order of magnitude differences between methods for maintaining physiological 17beta-oestradiol concentrations in ovariectomized rats
- 2008
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In: Scandinavian Journal of Clinical and Laboratory Investigation. - Oslo, Norway : Taylor & Francis. - 0036-5513 .- 1502-7686. ; 68:8, s. 814-822
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Journal article (peer-reviewed)abstract
- The use of animal models, especially the rat, is crucial for elucidating the biological effects and mechanisms of the widely used hormone 17beta-oestradiol. Unfortunately, there is a lack of consensus on optimal means of obtaining and maintaining physiological 17beta-oestradiol concentrations in plasma and this may be the reason for the varying results in several studies, including the disagreement on whether 17beta-oestradiol is neuroprotective or not. Very few studies have been devoted to investigating the characteristics and biological relevance of different methods of 17beta-oestradiol administration. We therefore ovariectomized 75 Sprague-Dawley rats and, following a 2-week washout period, administered 17beta-oestradiol using three different methods; daily injections (10 microg 17beta-oestradiol/kg), slow-release pellets (0.25 mg 60 day-release pellets, 0.10 mg 90 day-release pellets) and silastic capsules (with/without washout periods) (silastic laboratory tubing, inner/outer diameter: 1.575/3.175 mm, filled with 20 mm columns of 180 microg 17beta-oestradiol/mL sesame oil). A further 45 animals were used as ovariectomized and native controls studied in different parts of the oestrous cycle. Silastic capsules produced concentrations of 17beta-oestradiol within the physiological range 4-5 weeks independently of whether a prior washout period was included or not. The slow-release pellets, irrespective of dose or release period, resulted in initial concentrations an order of magnitude above physiological concentrations during the first 2 weeks followed by a substantial decrease. Daily injections resulted in increasing 17beta-oestradiol concentrations, but within physiological levels. Silastic capsules are conveniently manufactured and used and are superior to pellets and injections in reliably producing long-term 17beta-oestradiol concentrations within the physiological range.
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3. |
- Ström, Jakob O., et al.
(author)
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Order of magnitude differences between methods for maintaining physiological 17β-estradiol concentrations in ovariectomized rats
- 2008
-
In: Scandinavian Journal of Clinical and Laboratory Investigation. - : Informa UK Limited. - 0036-5513 .- 1502-7686. ; 68:8, s. 814-822
-
Journal article (peer-reviewed)abstract
- 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.
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4. |
- Ström, Jakob O., et al.
(author)
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Substantial discrepancies in 17β-estradiol concentrations obtained with three different commercial direct radioimmunoassay kits in rat sera
- 2008
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In: Scandinavian Journal of Clinical and Laboratory Investigation. - Oslo, Norway : Informa UK Limited. - 0036-5513 .- 1502-7686. ; 68:8, s. 806-813
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Journal article (peer-reviewed)abstract
- The extensive use of estrogen for contraception and amelioration of post-menopausal symptoms has made it the subject of substantial recent research efforts. Ovariectomized (ovx) rats treated with exogenous ovarial hormones constitute important tools in the investigation of the effects and mechanisms of estrogen actions. The crucial need to control and to monitor plasma levels of 17β-estradiol calls for accurate, precise and robust assay methods. The performance of direct radioimmunoassays (RIAs) for measurement of 17β-estradiol has previously been reported for human samples, but – to our knowledge – not for rat samples. In the current study, 552 serum samples from ovx, native and hormone treated rats were used to compare the performance of three commercially manufactured direct RIAs from the companies DPC (Siemens Healthcare Diagnostics Inc., formerly Diagnostic Products Corporation), DSL (Diagnostic Systems Labs) and MPB (MP Biomedicals, formerly ICN Biomedicals). Substantial differences in results between the three assay methods were found when measuring serum 17β-estradiol concentrations. The following formulas, describing the relation between the different methods’ results, were obtained using weighted Deming’s orthogonal regression (all regression formulae in the abstract are based on pg/mL): DSL= 0.43*DPC+12.3, MPB= 2.1*DPC+84.7 and DSL= 4.8*MPB+22.2. Furthermore, a preceding diethyl ether extraction step of the serum appears to impair the RIAs’ performances in the present samples: DPCex= 0.39*DPCunex+0.76, DSLex= 0.32*DSLunex-1.7 and MPBex= 0.22*MPBunex+1.4.
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5. |
- Theodorsson, Annette, et al.
(author)
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Estradiol increases brain lesions in the cortex and lateral striatum after transient occlusion of the middle cerebral artery in rats: No effect of ischemia on galanin in the stroke area but decreased levels in the hippocampus
- 2005
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In: Peptides. - : Elsevier BV. - 0196-9781. ; 26:11, s. 2257-2264
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Journal article (peer-reviewed)abstract
- A distinctive feature of galanin expression is that it is extensively increased by neuronal injury, estrogens, Alzheimer's disease and during development. Since stroke is amongst the clinically most important causes of neuronal injury we studied the tissue concentrations of galanin in a rat stroke model and the possibility of modulating this effect with estrogen. Transient focal middle cerebral artery ischemia was induced in rats that 2 weeks earlier underwent ovariectomy and received 1.5 mg 17β-estradiol slow-release or placebo pellets. The concentrations of galanin and neuropeptide Y were measured after observation periods of 3, 7 and 14 days in extracts of punch biopsies from both the lesioned and the contra lateral control hemisphere. The galanin levels were not changed in any of the brain regions studied except in the hippocampus where they were lower in the ischemic hemisphere in both the estrogen- and placebo-treated animals compared to the corresponding contra lateral intact hemisphere (p = 0.015). Estrogen treatment up-regulated galanin concentrations in both the ventral and dorsal hippocampus (p = 0.003). The effects on the galanin concentrations were similar after all observation periods: 3, 7 and 14 days (p = 0.144). No significant changes were observed in the concentration of neuropeptide Y in response to the lesions. The ischemic lesions were markedly larger in the estrogen-treated animals observed after 3 days compared to the corresponding control group. In the estrogen group the lesion was largest at bregma and the slice 2 mm anterior to the bregma, 82% and 435% larger than in the control group (p < 0.001). A similar, but much less pronounced (not statistically significant) difference was seen in the groups observed after 7 and 14 days. Earlier studies of lesions in the peripheral and central nervous systems have generally shown an up-regulation of galanin markers in response to but at a distance from the injury. Our results indicate that galanin is not involved in the response of the ischemic penumbra itself to stroke, whereas it may participate in the reactions of the neural stem-cell rich hippocampus to stroke.
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6. |
- Theodorsson, Annette, 1958-
(author)
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Estrogen-inducible neuropeptides in the rat brain: role in focal ischemic lesions
- 2005
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Doctoral thesis (other academic/artistic)abstract
- Sex steroids in general and estrogens in particular – in addition to their effects on the reproductive organs – affect a large number of crucial bodily functions, including “higher” brain functions.Neuropeptides constitute the phylogenetically oldest neurotransmitter system and are currently thought to act mainly during stress, disease or injury. The concentration of galanin is i.a. up-regulated by injury to the nervous system and by estrogen.The main focus of the present thesis was to investigate whether the reported neuroprotective effect of 17β-estradiol in experimental animal stroke models is partially mediated through its effects on galanin and if galanin per se exerts neuroprotective effects in stroke.An exploratory study of the effects of sex steroid concentrations due to gender and pubertal development showed differences in concentrations of i.a. the neuropeptides galanin and neuropeptide Y also in brain regions of female rats important for higher brain functions, including hippocampus and cortex, brain regions not directly involved in reproduction. Puberty brings about changes in several hormonal mechanisms, and our studies showed that the major effect on the concentrations of galanin in various brain regions of ovariectomized (ovx) rats, was brought about by 17β-estradiol.The pathophysiological mechanisms involved in thrombolysis – the current treatment of choice in human stroke – attempts the re-establishment of perfusion (reperfusion) to the lesioned area of the brain. This prompted us to develop a reperfusion stroke model in rats designed to be mild, focal and transient, allowing long-term observation periods of animals thriving well postoperatively. Mortality and morbidity during and after the middle cerebral artery (MCA) occlusion are important confounding factors crucial for the results. Changing anaesthesia from intraperitoneally administered chloral hydrate to isofl urane inhalation anaesthesia using endotracheal intubation and controlled ventilation markedly reduced the mortality rate from 25% to 10.6%, which was even further reduced down to 2.7 % by successively improved surgical skills.Contrary to our initial hypothesis, long-term 17β-estradiol treatment resulted in larger ischemic lesions in our stroke model compared to control treatment. After 3 days the cerebral ischemic lesion area was doubled after 17β-estradiol treatment in rats subjected to 60 min microclip occlusion of the MCA followed by reperfusion. A similar, but not statistically signifi cant difference was found after 7 and 14 days. Three groups studying different types of experimental animal stroke and different doses of 17β-estradiol treatment have recently also demonstrated lack of neuroprotection by 17β-estradiol treatment. Furthermore, large epidemiological clinical studies have recently also reported an increased risk and poorer outcome in postmenopausal women subjected to hormone replacement therapy.The concentrations of galanin-like immunoreactivity in extracts of punch biopsies from the penumbra area after transient MCA occlusion were found unchanged, but were decreased (p=0.015) in the apparently undamaged ipsilateral hippocampus. Galanin administered by continuous intracerebroventricular infusion (2.4 nmol/day) resulted in a 30% larger ischemic lesion compared to controls, measured 7 days after the MCA occlusion. Taken together, these results indicate that galanin in the brain is primarily a factor reacting to ischemic injury rather than a neuroprotective factor in its own right.Very limited information is available about the steady state serum concentrations of 17β-estradiol in response to different modes of administration to rats for days and weeks. The need for this information has become especially apparent during recent years due to the observable dichotomy of estrogens effects – neuroprotective or not – in the various animal models of brain ischemia reported in the current scientific literature. The cause of this dichotomy is likely to be found in the experimental setup, including the mode of administration of 17β-estradiol. Delayed steady state of serum 17β-estradiol concentrations were found when comparing two common modes of exogenous administration of 17β-estradiol – slow-release osmotic pumps vs. daily subcutaneously injections of 17β-estradiol solved in sesame oil – to ovx rats during 2 times 6 weeks crossover treatment. Steady state was reached at week 4 in the daily injections group compared to at week 6 in the slow release osmotic pumps group. Once steady state was reached, the concentration was the same in both groups for the reminder of the experiment (in total 12 weeks).
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7. |
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8. |
- Theodorsson, Annette, 1958-, et al.
(author)
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Hypothermia-induced increase in galanin concentrations and ischemic neuroprotection in the rat brain
- 2008
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In: Neuropeptides. - : Elsevier BV. - 0143-4179 .- 1532-2785. ; 42:1, s. 79-87
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Journal article (peer-reviewed)abstract
- The effects of hypothermia on galanin concentrations and the relation between ischemic brain lesions, hypothermia and galanin concentrations in a transient and focal rat stroke model were investigated in order to elucidate whether hypothermia-induced alterations in galanin concentrations could constitute a part of the established neuroprotective effect of hypothermia. Female rats were allocated to normothermia (37 °C) or hypothermia (33 °C) treatments during a 60 min microclip middle cerebral artery occlusion. The ischemic lesions were visualized after observation periods of 2 or 7 days and the concentration of galanin measured by radioimmunoassay in extracts of punch biopsies from both the lesioned and the contralateral control hemisphere. Hypothermia-induced an overall increase in the concentrations of immunoreactive galanin (p < 0.001). The elevated galanin levels were predominantly found in the non-ischemic control hemisphere, in the hippocampus, thalamus and the posterior part of parietal cortex. The galanin concentrations were lower in the ischemic hemisphere in both the normo- and hypothermic animals compared to the corresponding contra lateral intact hemisphere (p = 0.049). The factor of time, 2 respectively 7 days, did not show any significant difference regarding the galanin concentrations (p = 0.844). Multivariate analyses of variance revealed significant effect of ischemia on the size of the ischemic brain lesions (p = 0.001) but no overall effect of temperature when data from both 2 and 7 days observation periods were analyzed together. The ischemic lesions were generally larger at 33 degrees after 2 days (p = 0.230). Prolonged observation time of 7 days resulted in a significant reduction of the ischemic brain lesion (p = 0.011) with smaller ischemic lesions in the hypothermic group. Our data support the notion that hypothermia-induced increase in the tissue concentrations of galanin in the brain are the result of changes from optimal homeostatic conditions - the hypothermia-induced stress - rather than the ischemia/re-perfusion lesion induced changes in galanin concentrations. Hypothermia-induced elevation in galanin concentration is therefore not likely to be amongst the major protective mechanisms of hypothermia. © 2007 Elsevier Ltd. All rights reserved.
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9. |
- Theodorsson, Annette, et al.
(author)
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Modern anesthesia and peroperative monitoring methods reduce per- and postoperative mortality during transient occlusion of the middle cerebral artery in rats
- 2005
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In: Brain Research Protocols. - : Elsevier BV. - 1385-299X .- 1872-809X. ; 14:3, s. 181-190
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Journal article (peer-reviewed)abstract
- Mortality and morbidity during and after occlusion of the middle cerebral artery in rats are important confounding factors which may be minimized by improved anesthesia and peroperative monitoring techniques. We describe state of the art techniques for inducing anesthesia, endotracheal intubation, ventilation and monitoring peroperatively in this context. Introducing the subtemporal approach of Tamura et al. in our laboratory 5 years ago, we experienced 25% peroperative and 24 h postoperative rat mortality when performing temporary clipping of the middle cerebral artery. This prompted us to abandon intraperitoneal anesthesia by chloral hydrate and ventilation by tracheotomy in favor of endotracheal intubation and isoflurane anesthesia (1% isoflurane in 30%:70% O2/N2O). These anesthetic techniques in combination with improved surgical skills have reduced our initial 25% peroperative- and 24 h postoperative mortality to 2.7% (1.8% peroperatively and 0.9% 24 h postoperatively). Furthermore, the following 14 days postoperative mortality rate was 1.8%. A total number of 203 rats have been operated with this method in different studies where a focal reperfusion stroke model combined with extended periods of observations were the cornerstone.
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10. |
- Theodorsson, Annette, et al.
(author)
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Serum concentrations of 17β-estradiol in ovariectomized rats during 2 times 6 weeks crossover treatment by daily injections in comparison with slow-release pellets
- 2005
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In: Scandinavian Journal of Clinical and Laboratory Investigation. - : Informa UK Limited. - 1502-7686 .- 0036-5513. ; 65:8, s. 699-706
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Journal article (peer-reviewed)abstract
- Estrogens exert widespread biological functions that reach far beyond their well-known role in reproduction. Exogenous administration of 17β-estradiol to ovariectomized experimental animals is of the utmost importance in elucidating its mechanisms of action. In the present study, we compared two different modes of exogenous administration of 17β-estradiol to ovariectomized rats in relation to the serum 17β-estradiol concentrations over prolonged periods of time. 17β-estradiol was administered either by slow-release pellets (Innovative Research of America, Sarasota, Fl. 34236, USA, 90-day release, NHH-115, 1.5 mg) or by daily subcutaneous injections of 15 µg 17β-estradiol dissolved in sesame oil. After 6 weeks, the mode of administration of estradiol was changed to the opposite method and continued for a further 6 weeks. Blood samples for measurement of serum 17β-estradiol were taken every second week. After 2 weeks, the serum concentrations of 17β-estradiol in group A initially receiving the pellets were 73 % higher (p<0.001) compared to those of group B receiving daily injections. The difference was even more prominent, 580 % (p<0.001), after 4 weeks. Steady state was reached at week 6 in group A, but already by week 4 in group B. Once steady state was reached, the concentrations were the same in both groups for the remainder of the experiment (12 weeks in total). Our study indicates that steady-state concentrations of 17β-estradiol occur 5-6 weeks later than the 48 h the manufacturer of the slow-release pellets claims.
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