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Focal ischemic reperfusion stroke model in rats and the role of galanin
Linköping University, Department of Clinical and Experimental Medicine, Clinical Chemistry. Linköping University, Faculty of Health Sciences.
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Stroke is the third most common cause for mortality in industrialised countries and amongst the major causes of long- time morbidity. While the mortality due to myocardial infarction has been dramatically reduced during the last 10-15 years, mortality due to stroke remains almost the same, despite the fact that the two share similar basic pathogenic mechanisms including atherosclerosis, hypertension and diabetes. Treatment modalities of reperfusion therapy for acute ischemic stroke, including the use of tissue plasminogen activator for thrombolysis and endovascular treatments, are eff ective if applied early after onset of the first symptoms. The more frequent use of reperfusion therapy, especially in the most common type of stroke aff ecting the middle cerebral artery (MCA), increase the clinical relevance and demand for experimental models of temporary and focal ischemia of the brain. The primary goal of the present work was to develop a model in rats for studying the mechanisms underlying focal and temporary ischemia in brain regions supplied by the MCA.

We have modified the intracranial method of occluding the MCA originally described by Tamura et al. in the early 1980es by introducing a microclip to occlude the artery and induce reperfusion under direct visual control through an operating microscope. The goal was to create a mild ischemia model with low morbidity and mortality, optimizing conditions for the animals postoperatively and allowing longterm (weeks) observation periods of high relevance for human stroke. Morbidity and mortality in experimental stroke models are crucial confounders. Change of anesthesia from intraperitoneally administrated chloral hydrate to isoflurane inhalation anesthesia with endotracheal intubation and controlled ventilation reduced mortality markedly from 25% to ~10%. Improved overall skills in anesthesia and surgical techniques further reduced mortality to <3%.

Hypothermia reduces brain lesions caused by ischemia not only when administered before and during the ischemic episode, but also afterwards. Several studies have shown that galanin concentrations are increased in response to various types of lesions to the nervous system, and galanin may be amongst the factors supporting neuronal survival and functions. We therefore investigated whether or not hypothermia-induced alterations in galanin concentrations could constitute a part of the established neuroprotective effect of hypothermia in our rat stroke model. 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. The galanin concentrations were lower in the ischemic hemisphere in both the normo- and hypothermic animals compared to the corresponding contralateral intact hemisphere (p = 0.049). The hypothermia and not the ischemic/reperfusion lesions explained the major part of the observed changes in galanin concentrations. Hypothermia-induced elevation in galanin concentration is therefore not likely to be amongst the major protective mechanisms of hypothermia. Our results support the notion that hypothermia-induced increase in tissue concentrations of galanin in the brain are the result of changes from optimal homeostatic conditions – the hypothermia-induced stress – rather than the ischemic/reperfusion lesion- induced changes in galanin concentrations.

Whether the lesion-induced increase in galanin concentrations is primarily a signal that a lesion has occurred, a consequence of the lesion or a mechanism for facilitating neuronal survival is an open question. We therefore infused three different concentrations of galanin intracerebroventricularly in a direct attempt to investigate whether or not galanin has neuroprotective properties in a rat model of MCA occlusion. Furthermore, we infused the GalR2/3 agonist Gal(2-11) (AR-M1896) shown to subserve neuroprotective functions. The lesion was 98% larger seven days after a 60 min transient MCA occlusion and continuous administration of the GalR2/3 agonist Gal(2-11). No differences were found after seven days in the groups treated with galanin in three different concentrations (0.24, 2.4 and 24 nmol/day; p = 0.939, 0.715 and 0.977, respectively). There was also no difference in the size of the ischemic lesion measured after three days in the galanin-treated group (2.4 nmol/d) compared to artificial cerebrospinal fl uid (p = 0.925).

The expression of the galanin, GalR1, GalR2 and GalR3 receptor genes were investigated in the female rat brain seven days after a 60-min unilateral occlusion/reperfusion of the MCA. Galanin gene expression showed a 2.5-fold increase and GalR1 a 1.5-fold increase in the locus coeruleus of the ischemic hemisphere compared to the control side, and the GalR1 mRNA levels decreased by 35% in the cortex of the ischemic hemisphere. Thus, stroke-induced forebrain lesion upregulates synthesis of galanin and GalR1 in the locus coeruleus, a noradrenergic cell group projecting to many forebrain areas, including cortex and the hippocampal formation, supporting the notion that galanin may play a role in the response of the central nervous system to injury and have trophic effects.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press , 2011. , 92 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1242
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-68086ISBN: 978-91-7393-185-4OAI: oai:DiVA.org:liu-68086DiVA: diva2:416160
Public defence
2011-05-20, Berzeliussalen, Ingång 65, Campus US, Linköpings universitet, Linköping, 09:00 (English)
Opponent
Supervisors
Available from: 2011-05-10 Created: 2011-05-10 Last updated: 2011-05-10Bibliographically approved
List of papers
1. 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: 2012-02-27Bibliographically approved
2. Hypothermia-induced increase in galanin concentrations and ischemic neuroprotection in the rat brain
Open this publication in new window or tab >>Hypothermia-induced increase in galanin concentrations and ischemic neuroprotection in the rat brain
2008 (English)In: Neuropeptides, ISSN 0143-4179, E-ISSN 1532-2785, Vol. 42, no 1, 79-87 p.Article in journal (Refereed) Published
Abstract [en]

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.

Keyword
Hypothermia; Galanin; Neuropeptides; Stroke; Cerebral ischemia; Middle cerebral artery occlusion; Neuroprotection; Rat 1. Introduction Hypothermia applied either during or soon after cerebral ischemia, has in several studies been shown to result in effect
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-43142 (URN)10.1016/j.npep.2007.09.008 (DOI)72059 (Local ID)72059 (Archive number)72059 (OAI)
Available from: 2009-10-10 Created: 2009-10-10 Last updated: 2012-02-27Bibliographically approved
3. Effects of intracerebroventricular galanin or a galanin receptor 2/3 agonist on the lesion induced by transient occlusion of the middle cerebral artery in female rats
Open this publication in new window or tab >>Effects of intracerebroventricular galanin or a galanin receptor 2/3 agonist on the lesion induced by transient occlusion of the middle cerebral artery in female rats
2011 (English)In: Neuropeptides, ISSN 0143-4179, E-ISSN 1532-2785, Vol. 45, no 1, 17-23 p.Article in journal (Refereed) Published
Abstract [en]

Several studies have shown that injury to the central and peripheral nervous system can increase expression of galanin, a 29 amino acid neuropeptide. Moreover, there is evidence that galanin, especially through its galanin receptor 2 (GalR2) receptor, plays a neuroprotective role in different injury models. However, direct studies of a possible neuroprotective effect of galanin in experimental stroke models are lacking. Galanin, a GalR2/3 agonist or artificial CSF was continuously infused intracerebroventricularly (i.c.v.) in naive female rats after a 60 min transient and focal occlusion of the middle cerebral artery. The animals were sacrificed, and the ischemic lesion was visualized using 2,3,5-triphenyltetrazolium hydrochloride (TTC) staining. The lesion was 98% larger after i.c.v, administration of the GalR2/3 agonist (2.4 nmol/day) seven days after occlusion compared to artificial CSF (p = 0.023). No statistically significant differences were found after seven days in the groups treated with galanin in three different concentrations (0.24, 2.4 and 24 nmol/day; p = 0.939, 0.715 and 0.977, respectively). There was no difference in the size of the ischemic lesions measured after three days in the galanin-treated group (2.4 nmol/d) compared to artificial CSF (p = 0.925). The present results show, surprisingly, that a GalR2/3 agonist doubled the size of the ischemic lesion. Whether this effect primarily reflects the properties of the current model, species, gender and/or the mode of galanin administration, e.g. causing desensitization, or whether galanin indeed lacks neuroprotective effect of its own, remains to be corroborated.

Place, publisher, year, edition, pages
Elsevier Science B.V., Amsterdam, 2011
Keyword
Cerebral ischemia, Neuropeptide, Neuroprotection, Stroke
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-66126 (URN)10.1016/j.npep.2010.09.002 (DOI)000286857800003 ()
Available from: 2011-03-04 Created: 2011-03-04 Last updated: 2011-05-10Bibliographically approved
4. Changes in galanin and GalR1 gene expression in discrete brain regions after transient occlusion of the middle cerebral artery in female rats
Open this publication in new window or tab >>Changes in galanin and GalR1 gene expression in discrete brain regions after transient occlusion of the middle cerebral artery in female rats
Show others...
2012 (English)In: Neuropeptides, ISSN 0143-4179, E-ISSN 1532-2785, Vol. 46, no 1, 19-27 p.Article in journal (Refereed) Published
Abstract [en]

Injury to neurons results in upregulation of galanin in some central and peripheral systems, and it has been suggested that this neuropeptide may play a protective and trophic role, primarily mediated by galanin receptor 2 (GalR2). The objective of the present study was to investigate galanin, GalR1, GalR2 and GalR3 gene expression in the female rat brain seven days after a 60-min unilateral occlusion of the middle cerebral artery followed by reperfusion. Quantitative real-time PCR was employed in punch-biopsies from the locus coeruleus, somatosensory cortex and dorsal hippocampal formation including sham-operated rats as controls. Galanin gene expression showed a ~2.5-fold increase and GalR1 a ~1.5-fold increase in the locus coeruleus of the ischemic hemisphere compared to the control side. Furthermore, the GalR1 mRNA levels decreased by 35% in the cortex of the ischemic hemisphere. The present results indicate that a stroke-induced forebrain lesion upregulates synthesis of galanin and GalR1 in the locus coeruleus, a noradrenergic cell group projecting to many forebrain areas, including cortex and the hippocampal formation. These results support the notion that galanin may play a role in the response of the central nervous system to injury and have trophic eff ects.

Place, publisher, year, edition, pages
Elsevier, 2012
Keyword
cerebral ischemia, galanin receptor, RT-PCR
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-68085 (URN)10.1016/j.npep.2011.11.001 (DOI)000300268800003 ()
Note

On the day of the defence day the status of this article was Manuscript funding agencies|County Council of Ostergotland, Sweden||Swedish Research Council||Marianne and Marcus Wallenberg Foundation||

Available from: 2011-05-10 Created: 2011-05-10 Last updated: 2013-11-26Bibliographically approved

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