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- Chaireti, Roza, 1979-
(author)
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Thrombin generation in different cohorts : Evaluation of the haemostatic potential
- 2013
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Doctoral thesis (other academic/artistic)abstract
- The aim of this thesis is to evaluate thrombin generation in patients with thrombophilia (Paper I), in patients with venous thromboembolism (Paper II), in healthy women during the menstrual cycle (Paper III), in patients with liver disease (Paper IV) and in patients with mild deficiency of factor VII (Paper V).For this purpose, thrombin generation was measured in platelet poor plasma by the calibrated automated thrombogram (CAT®) assay. Thrombin generation expresses the overall haemostatic potential, in contrast to the more traditional coagulation tests, which concentrate on individual factors or coagulation pathways. The thrombin generation markers that were measured and studied were: lagtime (clotting time), endogenous thrombin potential (ETP, total thrombin concentration), peak (maximum thrombin concentration) and time to peak (ttpeak).The cohorts for Papers I and II are part of a larger cohort (The LInköping Study on Thrombosis, LIST), which included 516 consecutive patients who presented at the Emergency Department of Linköping University Hospital, Sweden with the clinical suspicion of venous thrombosis. In Paper I thrombin generation was measured in the absence of thrombomodulin in patients with thrombophilia (factor V Leiden, n=98 and prothrombin G20210A mutation, n=15) and in an equal number of age- and gendermatched controls. The results were associated with the presence of thrombosis, as well as gender and age. It was shown that thrombin generation did not differ significantly among patients and controls. Patients with and patients without thrombophilia who had suffered a thrombosis upon inclusion had longer lagtime compared with their counterparts without thrombosis. Neither age nor gender had any effect on the results.In Paper II, thrombin generation at the time of an acute thromboembolic episode was studied as a potential early marker for recurrence during a 7-year follow-up in 115 patients with venous thrombosis upon inclusion. It was shown that patients with recurrences during follow-up had longer lagtime and ttpeak at the time of the acute thrombosis, whereas those without recurrences had higher ETP and peak. Those results were particularly evident in the group of patients with an unprovoked thrombosis upon inclusion.In Paper III, thrombin generation was measured in the follicular and luteal phase of a normal menstrual cycle in 102 healthy women not taking oral contraceptives. The results were associated with haemostatic parameters (fibrinogen, antithrombin, D-dimer, plasminogen activator inhibitor-1, factors VII, VIII, X and von Willebrand) as well as the physiological concentrations of oestradiol, progesterone, antimüllerian hormone and sex hormone-binding globulin and the number of pregnancies and deliveries for these women. ETP was significantly higher during the luteal phase. However, this could not be explained by the elevation of other procoagulant factors during the same phase. Progesterone was found to exert a more significant effect on haemostasis than oestradiol during both phases (multiple regression analysis).In Paper IV, thrombin generation was measured in the presence and absence of thrombomodulin in 47 patients with portal vein thrombosis, PVT (11 with cirrhotic PVT and 36 with non-cirrhotic PVT), 15 patients with Budd-Chiari syndrome and 24 patients with cirrhosis, as well as 21 healthy controls. Since 15 patients with PVT (2 with cirrhotic PVT and 13 with non-cirrhotic PVT) and 10 patients with Budd-Chiari syndrome were treated with warfarin at the time of the blood sampling, an equal number of patients matched for age, gender and prothrombin time-international normalized ratio with atrial fibrillation and no hepatic diseases were used as controls. It was shown that hypercoagulability, expressed as total and maximum concentration of generated thrombin as well as thrombomodulin resistance [thrombin generation markers measured in the presence]/[thrombin generation markers measured in the absence of thrombomodulin] was pronounced in the groups of patients with cirrhosis, regardless of the presence of splanchnic thrombosis.In Paper V, thrombin generation in the presence of human and different concentrations of rabbit thromboplastin was measured in 10 patients with mild deficiency of factor VII and in 12 controls. In these patients, the levels of factor VII varied slightly depending on the origin of the thromboplastin used in the reagent. Nine out of 10 patients had a mutation in common (Arg353Gln), which was, however, not associated with the diversity in the factor VII measurements due to the origin of thromboplastin. ETP in patients with mild factor VII deficiency was about 86% of the ETP in the control group. The expected thrombin generation patterns with increasing concentrations of thromboplastin did not differ depending on the origin of thromboplastin in the patient group.
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| 2. |
- Holm, Lovisa, 1962-
(author)
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Focal ischemic reperfusion stroke model in rats and the role of galanin
- 2011
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Doctoral thesis (other academic/artistic)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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| 3. |
- Ström, Jakob
(author)
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The dose-dependent effects of estrogens on ischemic stroke
- 2012
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Doctoral thesis (other academic/artistic)abstract
- 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.
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