| 1. |
- Dock, Hua, et al.
(författare)
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DNA Methylation Inhibitor Zebularine Confers Stroke Protection in Ischemic Rats
- 2015
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Ingår i: TRANSLATIONAL STROKE RESEARCH. - : Springer Verlag (Germany). - 1868-4483 .- 1868-601X. ; 6:4, s. 296-300
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Tidskriftsartikel (refereegranskat)abstract
- 5-Aza-deoxycytidine (5-aza-dC) confers neuroprotection in ischemic mice by inhibiting DNA methylation. Zebularine is another DNA methylation inhibitor, less toxic and more stable in aqueous solutions and, therefore more biologically suitable. We investigated Zebularines effects on brain ischemia in a rat middle cerebral artery occlusion (MCAo) model in order to elucidate its therapeutic potential. Male Wistar wild-type (WT) rats were randomly allocated to three treatment groups, vehicle, Zebularine 100 mu g, and Zebularine 500 mu g. Saline (10 mu L) or Zebularine (10 mu L) was administered intracerebroventricularly 20 min before 45-min occlusion of the middle cerebral artery. Reperfusion was allowed after 45-min occlusion, and the rats were sacrificed at 24-h reperfusion. The brains were removed, sliced, and stained with 2 % 2,3,5-triphenyltetrazolium chloride (TTC) before measuring infarct size. Zebularine (500 mu g) reduced infarct volumes significantly (p less than 0.05) by 61 % from 20.7 +/- 4.2 % in the vehicle treated to 8.1 +/- 1.6 % in the Zebularine treated. Zebularine (100 mu g) also reduced infarct volumes dramatically by 55 to 9.4 +/- 1.2 %. The mechanisms behind this neuroprotection is not yet known, but the results agree with previous studies and support the notion that Zebularine-induced inhibition of DNA methyltransferase ameliorates ischemic brain injury in rats.
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| 2. |
- Holm, Lovisa, et al.
(författare)
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Changes in galanin and GalR1 gene expression in discrete brain regions after transient occlusion of the middle cerebral artery in female rats
- 2012
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Ingår i: Neuropeptides. - : Elsevier. - 0143-4179 .- 1532-2785. ; 46:1, s. 19-27
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 3. |
- Holm, Lovisa, et al.
(författare)
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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
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Ingår i: Neuropeptides. - : Elsevier Science B.V., Amsterdam. - 0143-4179 .- 1532-2785. ; 45:1, s. 17-23
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 4. |
- Holm, Lovisa, 1962-
(författare)
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Focal ischemic reperfusion stroke model in rats and the role of galanin
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 5. |
- Ingberg, Edvin, 1988-
(författare)
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Challenges in experimental stroke research : The 17β-estradiol example
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 6. |
- Ingberg, Edvin, 1988-, et al.
(författare)
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Effect of laser Doppler flowmetry and occlusion time on outcome variability and mortality in rat middle cerebral artery occlusion : inconclusive results
- 2018
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Ingår i: BMC Neuroscience. - London : BioMed Central. - 1471-2202. ; 19:1
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Stroke is among the leading causes of death and disability. Although intense research efforts have provided promising treatment options in animals, most clinical trials in humans have failed and the therapeutic options are few. Several factors have been suggested to explain this translational difficulty, particularly concerning methodology and study design. Consistent infarcts and low mortality might be desirable in some, but not all, studies. Here, we aimed to investigate whether the use of laser Doppler flowmetry (LDF) and the occlusion time (60 vs. 45 min) affected outcome variability and mortality in a rat stroke model. Eighty ovariectomized female Wistar rats were subjected to ischemic stroke using intraluminal filament middle cerebral artery occlusion with or without LDF and with occlusion times of 45 or 60 min. Outcome was evaluated by triphenyl tetrazolium chloride staining of brain slices to measure infarct size and a modified sticky tape test.RESULTS: Neither LDF nor occlusion times of 45 versus 60 min significantly affected mortality, outcome variability or outcome severity.CONCLUSIONS: Due to the unexpectedly high mortality and variability the statistical power was very low and thus the results were inconclusive.
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| 7. |
- Ingberg, Edvin, et al.
(författare)
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Effects of high and low 17 beta-estradiol doses on focal cerebral ischemia in rats
- 2016
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Ingår i: Scientific Reports. - : NATURE PUBLISHING GROUP. - 2045-2322. ; 6:20228
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Tidskriftsartikel (refereegranskat)abstract
- 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 17 beta-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 17 beta-estradiol and subjected to transient middle cerebral artery occlusion. The modified sticky tape test was performed after 24 h and the rats were subsequently sacrificed for infarct size measurements. In contrast to our hypothesis, a significant negative correlation between 17 beta-estradiol dose and infarct size was found (p = 0.018). Thus, no support was found for the hypothesis that 17 beta-estradiol can be both neuroprotective and neurotoxic merely depending on dose. In fact, on the contrary, the findings indicate that the higher the dose of 17 beta-estradiol, the smaller the infarct.
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| 8. |
- Ingberg, Edvin, 1988-, et al.
(författare)
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Effects of high and low 17β-estradiol doses on focal cerebral ischemia in rats
- 2016
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Ingår i: Scientific Reports. - London, United Kingdom : Nature Publishing Group. - 2045-2322. ; 6
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Tidskriftsartikel (refereegranskat)abstract
- 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 modified sticky tape test was performed after 24 h and the rats were subsequently sacrificed for infarct size measurements. In contrast to our hypothesis, a significant 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 findings indicate that the higher the dose of 17β-estradiol, the smaller the infarct.
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| 9. |
- Ingberg, Edvin, 1988-, et al.
(författare)
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Elevated body swing test after focal cerebral ischemia in rodents : methodological considerations
- 2015
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Ingår i: BMC Neuroscience. - : Springer Science and Business Media LLC. - 1471-2202. ; 16
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Tidskriftsartikel (refereegranskat)abstract
- Background: The elevated body swing test (EBST) is a behavioral test used to evaluate experimental stroke in rodents. The basic idea is that when the animal is suspended vertically by the tail, it will swing its head laterally to the left or right depending on lesion side. In a previous study from our lab using the EBST after middle cerebral artery occlusion (MCAo), rats swung contralateral to the infarct day 1 post-MCAo, but ipsilateral day 3 post-MCAo. This shift was unexpected and prompted us to perform the present study. First, the literature was systematically reviewed to elucidate whether a similar shift had been noticed before, and if consensus existed regarding swing direction. Secondly, an experiment was conducted to systematically investigate the suggested behavior. Eighty-three adult male and female Sprague-Dawley rats were subjected to MCAo or sham surgery and the EBST was performed up to 7 days after the lesion.Results: Both experimentally and through systematic literature review, the present study shows that the direction of biased swing activity in the EBST for rodents after cerebral ischemia can differ and even shift over time in some situations. The EBST curve for females was significantly different from that of males after the same occlusion time (p = 0.023).Conclusions: This study highlights the importance of adequate reporting of behavioral tests for lateralization and it is concluded that the EBST cannot be recommended as a test for motor asymmetry after MCAo in rats.
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| 10. |
- Ingberg, Edvin, 1988-, et al.
(författare)
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Method parameters' impact on mortality and variability in mouse stroke experiments : a meta-analysis
- 2016
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Ingår i: Scientific Reports. - London, England : Nature Publishing Group. - 2045-2322. ; 11:SUPP 3, s. 108-108
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Tidskriftsartikel (refereegranskat)abstract
- Although hundreds of promising substances have been tested in clinical trials, thrombolysis currently remains the only specific 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.
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