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Estrogen-inducible neuropeptides in the rat brain: role in focal ischemic lesions
Linköping University, Department of Clinical and Experimental Medicine, Clinical Chemistry. Linköping University, Faculty of Health Sciences.
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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).

Place, publisher, year, edition, pages
Institutionen för biomedicin och kirurgi , 2005.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 923
Keyword [en]
Estrogen, neuropeptides, galanin, cerebral ischemia, middle cerebral artery occlusion
National Category
Neurosciences
Identifiers
URN: urn:nbn:se:liu:diva-4716ISBN: 91-85497-58-4 (print)OAI: oai:DiVA.org:liu-4716DiVA: diva2:20739
Public defence
2005-12-09, Berzeliussalen, Ingång 65, Campus US, Linköpings universitet, Linköping, 09:00 (English)
Opponent
Supervisors
Note
On the day of the public defence of the doctoral thesis, the status of article V was: Available on line since 24th of May 2005.Available from: 2005-11-11 Created: 2005-11-11 Last updated: 2009-08-23
List of papers
1. Sex differences in neuropeptide distribution in the rat brain
Open this publication in new window or tab >>Sex differences in neuropeptide distribution in the rat brain
Show others...
1999 (English)In: Peptides, ISSN 0196-9781, E-ISSN 1873-5169, Vol. 20, no 1, 81-86 p.Article in journal (Refereed) Published
Abstract [en]

We have investigated possible sex differences in the regional concentrations of neuropeptides in the rat brain. Immunoreactive neurotensin (NT), neurokinin A (NKA), galanin (GAL), calcitonin gene-related peptide (CGRP), substance P (SP) and neuropeptide Y (NPY) were measured by radioimmunoassay in frontal cortex, occipital cortex, hippocampus, striatum, hypothalamus and pituitary in male and female pre- and postpubertal rats. Sex differences were found for NPY (p < 0.001), NT (p < 0.01) and GAL (p < 0.05), in particular in hippocampus, striatum, hypothalamus and pituitary, but not for CGRP, SP and NKA. Results from analysis of neuropeptides in one sex may not be entirely applicable to the other.

Keyword
Sex differences, Neuropeptide Y, Neurotensin, Galanin, Hippocampus, Pituitary, Frontal cortex, Puberty
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-13441 (URN)10.1016/S0196-9781(98)00139-9 (DOI)
Available from: 2005-11-11 Created: 2005-11-11 Last updated: 2017-12-13Bibliographically approved
2. Effects of estradiol, progesterone, and norethisterone on regional concentrations of galanin in the rat brain
Open this publication in new window or tab >>Effects of estradiol, progesterone, and norethisterone on regional concentrations of galanin in the rat brain
1999 (English)In: Peptides, ISSN 0196-9781, E-ISSN 1873-5169, Vol. 20, no 6, 743-748 p.Article in journal (Refereed) Published
Abstract [en]

Concentrations of immunoreactive galanin were compared in eight gross brain regions of ovariectomized female rats treated with either estradiol, estradiol + progesterone, estradiol + norethisterone, or placebo. Higher concentrations with estradiol treatment compared with placebo were found in the pituitary (357%), frontal cortex (162%), occipital cortex (174%), hippocampus (170%), and median eminence (202%). A more profound difference with addition of progesterone or norethisterone was seen in the pituitary (529% and 467%, respectively). Sex steroids, particularly estradiol, modulate galanin concentrations not only in reproductive, but also in nonreproductive, brain regions.

Keyword
Galanin, Estradiol, Progesterone, Norethisterone, Hippocampus, Pituitary, Frontal cortex
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-13442 (URN)10.1016/S0196-9781(99)00057-1 (DOI)
Available from: 2005-11-11 Created: 2005-11-11 Last updated: 2017-12-13Bibliographically approved
3. Modern anesthesia and peroperative monitoring methods reduce per- and postoperative mortality during transient occlusion of the middle cerebral artery in rats
Open this publication in new window or tab >>Modern anesthesia and peroperative monitoring methods reduce per- and postoperative mortality during transient occlusion of the middle cerebral artery in rats
2005 (English)In: Brain Research Protocols, ISSN 1385-299X, E-ISSN 1872-809X, Vol. 14, no 3, 181-190 p.Article in journal (Refereed) Published
Abstract [en]

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.

Keyword
Cerebral ischemia; Focal; Middle cerebral artery occlusion; Anesthetic method; Isoflurane; Rat
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-13443 (URN)10.1016/j.brainresprot.2005.01.002 (DOI)
Available from: 2005-11-11 Created: 2005-11-11 Last updated: 2017-12-13Bibliographically approved
4. Serum concentrations of 17β-estradiol in ovariectomized rats during 2 times 6 weeks crossover treatment by daily injections in comparison with slow-release pellets
Open this publication in new window or tab >>Serum concentrations of 17β-estradiol in ovariectomized rats during 2 times 6 weeks crossover treatment by daily injections in comparison with slow-release pellets
Show others...
2005 (English)In: Scandinavian Journal of Clinical and Laboratory Investigation, ISSN 1502-7686 (electronic) 0036-5513 (paper), Vol. 65, no 8, 699-706 p.Article in journal (Refereed) Published
Abstract [en]

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.

Keyword
Estrogen; 17β-estradiol; rat; slow-release pellet
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-13444 (URN)10.1080/00365510500375206 (DOI)
Available from: 2005-11-11 Created: 2005-11-11 Last updated: 2009-06-05
5. 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
Open this publication in new window or tab >>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 (English)In: Peptides, ISSN 0196-9781, Vol. 26, no 11, 2257-2264 p.Article in journal (Refereed) Published
Abstract [en]

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.

Keyword
Galanin; Neuropeptide Y; Neuropeptides; Stroke; Cerebral ischemia; Estrogen; Neuroprotection; Middle cerebral artery occlusion; Rat
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-13445 (URN)10.1016/j.peptides.2005.04.013 (DOI)
Available from: 2005-11-11 Created: 2005-11-11 Last updated: 2009-06-05
6. Galanin administered intracerebroventricularly increases ischemic lesions measured 7 days after focal and transient occlusion of the middle cerebral artery in female rats
Open this publication in new window or tab >>Galanin administered intracerebroventricularly increases ischemic lesions measured 7 days after focal and transient occlusion of the middle cerebral artery in female rats
2005 (English)Article in journal (Refereed) Submitted
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-13446 (URN)
Available from: 2005-11-11 Created: 2005-11-11

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