| 1. |
- Chaireti, Roza, 1979-
(författare)
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Thrombin generation in different cohorts : Evaluation of the haemostatic potential
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)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. |
- Halling Linder, Cecilia, 1975-
(författare)
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Biochemical and functional properties of mammalian bone alkaline phosphatase isoforms during osteogenesis
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The human skeleton is a living and dynamic tissue that constantly is being renewed in a process called bone remodeling. Old bone is resorbed by osteoclasts and new bone is formed by osteoblasts. Bone is a composite material made up by mineral crystals in the form of hydroxyapatite (calcium and phosphate) that provides the hardness of bone, and collagen fibrils that provides elasticity and flexibility. Alkaline phosphatase (ALP) is a family of enzymes that is present in most species and catalyzes the hydrolysis of various phosphomonoesters at alkaline pH. Despite the generalized use of ALP as a biochemical marker of bone formation, the precise function of bone ALP (BALP) is only now becoming clear. Three circulating human BALP isoforms (B1, B2, and B/I) can be distinguished in healthy individuals and a fourth isoform (B1x) has been discovered in patients with chronic kidney disease and in bone tissue.Paper I. Three endogenous phosphocompounds, (i.e., inorganic pyrophosphate (PPi), pyridoxal 5′-phosphate (PLP) and phosphoethanolamine (PEA)), have been suggested to serve as physiological substrates for BALP. The BALP isoforms display different catalytic properties towards PPi and PLP, which is attributed to their distinct N-linked glycosylation patterns. The catalytic activity, using PEA as substrate, was barely detectable for all BALP isoforms indicating that PEA is not a physiological substrate for BALP.Paper II. Mouse serum ALP is frequently measured and interpreted in mammalian bone research. However, little is known about the circulating ALPs in mice and their relation to human ALP. We characterized the circulating and tissue-derived mouse ALP isozymes and isoforms from mixed strains of wild-type and knockout mice. All four BALP isoforms (B/I, B1x, B1, and B2) were identified in mouse serum and bone tissues, in good correspondence with those found in human bones. All mouse tissues, except liver, contained significant ALP activities. This is a notable difference as human liver contains vast amounts of ALP.Paper III. The objective of this study was to investigate the binding properties of human collagen type I to human BALP, including the two BALP isoforms B1 and B2, together with ALP from human liver, human placenta and E. coli. A surface plasmon resonance-based analysis showed that BALP binds stronger to collagen type I in comparison with ALPs expressed in non-mineralizing tissues. The B2 isoform binds significantly stronger to collagen type I in comparison with the B1 isoform, indicating that glycosylation differences in human ALPs are of crucial importance for protein–protein interactions with collagen type I.Paper IV. Tartrate-resistant acid phosphatase (TRAP) is highly expressed in osteoclasts and frequently used as a marker of bone resorption. Intriguingly, recent studies show that TRAP is also expressed in osteoblasts and osteocytes. TRAP displays enzymatic activity towards the endogenous substrates for BALP, i.e., PPi and PLP. Both TRAP and BALP can alleviate the inhibitory effect of osteopontin on mineralization by dephosphorylation, which suggests a novel role for TRAP in skeletal mineralization.
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| 3. |
- Hilke, Susanne, 1965-
(författare)
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Galanin and NPY in the rodent brain: rapid effects of 17beta-estradiol and possible roles in hippocampal plasticity
- 2005
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The neuropeptides galanin and neuropeptide Y (NPY) play an important role in the reproduction of rodents, e.g. by modulating the release of gonadal hormones, the nutritional status by effects on feeding behavior and also by influencing mating behavior. There are age- and gender- differences in galanin- and NPY- like immunoreactivities (LIs) in brain areas important for higher functions including the hippocampal formation (HiFo) and cortex, that are related to the concentrations of 17β-estradiol.Neuropeptides in general are currently not considered critical in normal integrative neuronal functions but are rather thought to act as slow modulators during periods of stress or injury. In the present thesis we attempted to investigate, if the normal cyclical changes in the female sex-hormone 17β-estradiol can affect neurotransmission in brain areas important for memory, cognition and mood. We studied not only ”long term” (days and weeks) but also ”short-term” (one hour) effects on galanin and NPY concentrations in 17β-estradiol-primed ovariectomized (ovx) rats and mice.Radioimmunoassay (RIA) of galanin-LI in extracts of brain tissues from ”long-term” 17β-estradiol-treated ovx rats showed that its effects on galanin are dependent on boththe dose and on duration. Galanin - and NPY-LI in brain tissues of young ovx rats and mice increased in response to 17β-estradiol treatment in the HiFo, frontal cortex and striatum already within hours. This effect was not blocked by Tamoxifen® in rats. The mechanism of the 17β-estradiol effects on galanin levels in the rat HiFo may be related to decreased release of galanin into the extracellular fluid, since galanin-LI decreased in microdialysis samples two hours after a single injection of 17β-estradiol. Species differences were observed with regards to galanin, possibly due to tissue and species differences in the distribution of estrogen receptors.In the HiFo and caudate nucleus of mice, we found an increase in NPY-transcript after two hours by means of insitu hybridization, perhaps a compensatory up-regulation of NPY mRNA after increased 17β-estradiol-induced release in these areas. Taken together with no effects of Tamoxifen® on the levels on galanin in the HiFo of rats, the short duration, and the fact that the density of classical estrogen receptors seems to be limited in the striatum, we suggest that these effects are mediated through a membrane-related mechanism perhaps not involving the classical ER route.With an antiserum raised against the C-terminal end of the first 16 aminoacids of galanin- the sequence important for binding of intact galanin to its receptor - we found a novel compound which appears to be a homologue to galanin. Chromatographical analysis revealed that it was not galanin(1-29) or the galanin related peptide, galaninlike peptide (GALP), but appeared with immunohistochemistry in the galanin systems in the brain and was further influenced by 17β-estradiol in the HiFo and frontal cortex in a similar manner as galanin(1-29).In conclusion, tissue concentrations of galanin, a putative galanin homologue and NPY can be altered already after one hour by 17β-estradiol treatment e.i. in the HiFo. These ”short-term” effects are most likely to be due to effects on estrogen-primed peptide release which might influence mechanisms important for memory, cognition and mood.
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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. |
- Mörelius, Evalotte, 1965-
(författare)
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Stress in infants and parents : Studies of salivary cortisol, behaviour and psychometric measures
- 2006
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The life of a preterm infant admitted to a neonatal intensive care unit may be stressful from the moment of birth. Ever since Hans Selye’s initial characterisation of the biological stress response, cortisol has been frequently measured as an indicator of stress responsivity. However, research of the stress response and cortisol in infants, especially those who are preterm and/or ill, has been scarce basically because of methodological issues.The first aim with this thesis was to investigate the acute stress response, as measured by salivary cortisol and behaviour, for preterm infants, healthy infants, and infants at high psychosocial risk in response to certain defined handling procedures. The second aim was to investigate the stress response, as measured by salivary cortisol and psychometric measures, for parents present during the handling procedure of their infants. The intention was to perform all investigations in an as naturally occurring situation as possible, which means that the studied procedures would have been performed irrespectively of the research.The present thesis includes six original articles. The results of the first study demonstrate that it is feasible to collect sufficient amounts of saliva and to analyse salivary cortisol in neonates using the presented method of collection and analysis. The second study shows that preterm infants, usually cared for in incubators, show no signs of discomfort and have variable cortisol responses during skin-to-skin care with their mothers. The mothers, however, experience stress and low control before their first skin-to-skin care with their preterm infant and do not relax completely until after the session. In the third study we found that preterm infants have higher baseline salivary cortisol as compared to healthy full-term infants. Moreover, preterm infants have higher and sustained pain response during a nappy change as compared to healthy full-term infants. The results of the fourth study shows that infants younger than three months, living in psychosocial high-risk families, have increased cortisol responses during a nappy change, performed by the mother. However, support with the aim of improving mother-infant interaction, dampens the stress response. The results of the fifth study show that oral sweet-tasting solution in combination with a pacifier dampen the levels of the stress hormone cortisol in three months old infants during routine immunisation. Moreover, parents experience more self-rated emotional stress before immunisation if it is their first child who is being immunised. The sixth paper shows that the material used for saliva collection (cotton buds with wooden or plastic sticks) is of importance when saliva is collected but for practical reasons not centrifuged within 24 hours prior to cortisol analyse.The present thesis shows that it is practically feasible to collect saliva and to analyse the stress hormone cortisol in infants. The interpretation of infants’ and parents’ salivary cortisol responses to different handling procedures are discussed in relation to shortand long-term consequences, neonatal intensive care, preterm birth, attachment, mood, and pain.
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| 7. |
- Preinbergs, Julia, 1989-
(författare)
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Testosterone Analysis in Hair
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The effects of testosterone in the body are dependent on the concentrations at the target organs, as well as the susceptibility of the androgen receptor. Steroid hormones circulate in the bloodstream bound to proteins, and only a small part is unbound and free to exert its effect at the receptors. Due to the diurnal variation in testosterone levels, the measured concentrations can change rapidly. Thus, a kind of "long-term measure" of the average free testosterone concentration would facilitate the understanding of the hormones' long-term effects on target organs.Hair seems at first sight to be a suitable matrix. The current theory is that only the free, unbound fraction of hormones passively diffuse from the bloodstream into the hair matrix as the hair grows. In this thesis, we have developed an analysis capable of analysing testosterone in the hair of men and women. We have systematically explored potential confounding factors and the pattern of testosterone concentrations in different hair segments. Furthermore, we have sought to confirm biological differences such as sex, age and BMI which affect hormone concentrations in the blood. Finally, we have investigated how hair testosterone concentrations change in relation to treatment with oral contraceptives and before an acute myocardial infarction.Our conclusions are that it is possible to measure testosterone in extracts from hair in very low concentrations. The choice of measurement method needs to balance between high specificity, which mass spectrometry can offer, and adequate detection limits, which can be achieved with immunoassays. Hair testosterone concentrations correlate significantly with testosterone concentrations in saliva, which suggests that hair testosterone reflects the average hormone concentrations in the body. The significant variation in hair growth rate within and between individuals impedes the theoretical relationship between certain hair segments and a specific time in the past. Hair testosterone concentrations are affected by the frequency of hair washing, cosmetic hair treatment, natural hair colour and the biological sex. In hair samples taken shortly after a patient was admitted to hospital due to a myocardial infarction, lower concentrations of testosterone can be seen in hair compared to individuals of the same age who have not had an acute heart attack. This suggests that a reduction in testosterone concentrations occurs shortly before the heart attack, independently of other cardiovascular risk factors. Reduced testosterone concentrations could thus be an independent risk factor for developing an acute myocardial infarction.
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| 8. |
- Ström, Jakob
(författare)
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The dose-dependent effects of estrogens on ischemic stroke
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)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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| 9. |
- Theodorsson, Annette, 1958-
(författare)
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Estrogen-inducible neuropeptides in the rat brain: role in focal ischemic lesions
- 2005
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)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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