<p>Stroke is the third leading cause of morbidity and mortality in the industrialized world. Subarachnoid hemorrhage (SAH), the least common form of stroke, is one of the most demanding diseases treated in neurointensive care units. Cerebral ischemia may develop rapidly, and has a major influence on outcome.To be able to save parts of the brain that are at risk for ischemic brain damage, there is a need for reliable monitoring techniques. Understanding the pathophysiology of cerebral ischemia is a prerequisite both for the correct treatment of these diseases and for the development of new monitoring techniques and treatment modalities. The main aim of this thesis was to gain insight into the mechanisms of cerebral ischemia by studying early hemodynamic and metabolic changes with positron emission tomography and neurochemical changes with microdialysis. A secondary aim was to evaluate the potential of these techniques for detecting ischemia and predicting the degree of reversibility of ischemic changes.</p><p>Early changes in cerebral blood flow (CBF) and metabolism (CMRO₂) were studied with repeated positron emission tomography in an experimental model (MCAO) of transient focal ischemia, and in SAH patients. CMRO₂ was superior to CBF in discriminating between tissue with irreversible damage and tissue with the potential for survival in the experimental model. A metabolic threshold of ischemia was found. Neurochemical changes in the ischemic regions were studied simultaneously with microdialysis. Extracellular concentrations of glucose, lactate, hypoxanthine, glutamate and glycerol were measured, and the lactate/pyruvate (LP) and lactate/glucose ratios were calculated. Changes in all the microdialysis parameters were related to the degree of ischemia (severe ischemia or penumbra). Especially the LP ratio and glycerol were found to be robust and specific markers of ischemia. In the patients, hemodynamic and metabolic changes were common, but diverse in the acute phase of SAH, and it was suggested that these changes may contribute to an increased vulnerability for secondary events and the development of secondary ischemic brain damage.</p>

Stroke is the third leading cause of morbidity and mortality in the industrialized world. Subarachnoid hemorrhage (SAH), the least common form of stroke, is one of the most demanding diseases treated in neurointensive care units. Cerebral ischemia may develop rapidly, and has a major influence on outcome.To be able to save parts of the brain that are at risk for ischemic brain damage, there is a need for reliable monitoring techniques. Understanding the pathophysiology of cerebral ischemia is a prerequisite both for the correct treatment of these diseases and for the development of new monitoring techniques and treatment modalities. The main aim of this thesis was to gain insight into the mechanisms of cerebral ischemia by studying early hemodynamic and metabolic changes with positron emission tomography and neurochemical changes with microdialysis. A secondary aim was to evaluate the potential of these techniques for detecting ischemia and predicting the degree of reversibility of ischemic changes. Early changes in cerebral blood flow (CBF) and metabolism (CMRO2) were studied with repeated positron emission tomography in an experimental model (MCAO) of transient focal ischemia, and in SAH patients. CMRO2 was superior to CBF in discriminating between tissue with irreversible damage and tissue with the potential for survival in the experimental model. A metabolic threshold of ischemia was found. Neurochemical changes in the ischemic regions were studied simultaneously with microdialysis. Extracellular concentrations of glucose, lactate, hypoxanthine, glutamate and glycerol were measured, and the lactate/pyruvate (LP) and lactate/glucose ratios were calculated. Changes in all the microdialysis parameters were related to the degree of ischemia (severe ischemia or penumbra). Especially the LP ratio and glycerol were found to be robust and specific markers of ischemia. In the patients, hemodynamic and metabolic changes were common, but diverse in the acute phase of SAH, and it was suggested that these changes may contribute to an increased vulnerability for secondary events and the development of secondary ischemic brain damage.

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). / On the day of the public defence of the doctoral thesis, the status of article V was: Available on line since 24th of May 2005.

Background Even though more than 600 stroke treatments have been shown effective in preclinical studies, clinically proven treatment alternatives for cerebral infarction remain scarce. Amongst the reasons for the discrepancy may be methodological shortcomings, such as high mortality and outcome variability, in the preclinical studies. A common approach in animal stroke experiments is that A) focal cerebral ischemia is inflicted, B) some type of treatment is administered and C) the infarct sizes are assessed. However, within this paradigm, the researcher has to make numerous methodological decisions, including choosing rat strain and type of surgical procedure. Even though a few studies have attempted to address the questions experimentally, a lack of consensus regarding the optimal methodology remains. Methods We therefore meta-analyzed data from 502 control groups described in 346 articles to find out how rat strain, procedure for causing focal cerebral ischemia and the type of filament coating affected mortality and infarct size variability. Results The Wistar strain and intraluminal filament procedure using a silicone coated filament was found optimal in lowering infarct size variability. The direct and endothelin methods rendered lower mortality rate, whereas the embolus method increased it compared to the filament method. Conclusions The current article provides means for researchers to adjust their middle cerebral artery occlusion (MCAo) protocols to minimize infarct size variability and mortality. / <p>Funding Agencies|County Council of Ostergotland, Sweden||</p>

Many populations have low intakes of n-3 fatty acids, yet there is substantial evidence that the long chain n-3 fatty acid docosahexaenoic acid (DHA; 22:6n-3), found at high concentrations in the brain, is required for the proper development of the nervous system. However, less is known about requirements of long chain n-3 fatty acids for maintenance and function of the nervous system in later life. Several recent studies have reported that high amounts of long chain n-3 fatty acids reduce the extent of brain damage caused by cerebral ischemia in animals. However, whether or not a dietary deficiency of n-3 fatty acids increases the extent of injury when cerebral ischemia occurs has not been previously reported. The present studies, therefore, sought to determine if a diet deficient in n-3 fatty acids influences the extent of brain injury in the rat following cerebral ischemia. Male rats were fed an n-3 fatty acid adequate (control), an n-3 fatty acid deficient, or a high DHA diet for 5 weeks from weaning. Middle cerebral artery occlusion (MCAO) was induced and infarct volume was measured by 2,3,5,-triphenyltetrazolium chloride staining 24 hours after the procedure. Brain and platelet fatty acids were analyzed by gas liquid chromatography. DHA (22:6n-3) was 21-28% lower in brain phospholipids, and 17% lower in brain total fatty acids in the n-3 fatty acid deficient compared to control group, while 22:6n-3 was 12% higher in total brain fatty acids in the high DHA group than the control group. There was no significant difference in infarct volume (203, 220 and 218 mm³) among the control, n-3 fatty acid deficient, and high DHA groups, respectively. Platelet fatty acids and platelet aggregation were assessed to determine if these were influenced by the high DHA diet, and could possibly explain the observation of an apparent, but not statistically significant, higher number of rats with hemorrhages in the high DHA diet group. Platelet lipid arachidonic acid was not lower and platelet aggregation, assessed ex vivo using whole blood with a platelet function analyzer, was not longer in rats fed the high DHA compared to control or n-3 fatty acid deficient diets. In summary, dietary n-3 fatty acid deficiency did not increase the extent of brain injury following cerebral ischemia. The possibility that high dietary 22:6n-3 might increase susceptibility to cerebral hemorrhage will require further study.

Many populations have low intakes of n-3 fatty acids, yet there is substantial evidence that the long chain n-3 fatty acid docosahexaenoic acid (DHA; 22:6n-3), found at high concentrations in the brain, is required for the proper development of the nervous system. However, less is known about requirements of long chain n-3 fatty acids for maintenance and function of the nervous system in later life. Several recent studies have reported that high amounts of long chain n-3 fatty acids reduce the extent of brain damage caused by cerebral ischemia in animals. However, whether or not a dietary deficiency of n-3 fatty acids increases the extent of injury when cerebral ischemia occurs has not been previously reported. The present studies, therefore, sought to determine if a diet deficient in n-3 fatty acids influences the extent of brain injury in the rat following cerebral ischemia. Male rats were fed an n-3 fatty acid adequate (control), an n-3 fatty acid deficient, or a high DHA diet for 5 weeks from weaning. Middle cerebral artery occlusion (MCAO) was induced and infarct volume was measured by 2,3,5,-triphenyltetrazolium chloride staining 24 hours after the procedure. Brain and platelet fatty acids were analyzed by gas liquid chromatography. DHA (22:6n-3) was 21-28% lower in brain phospholipids, and 17% lower in brain total fatty acids in the n-3 fatty acid deficient compared to control group, while 22:6n-3 was 12% higher in total brain fatty acids in the high DHA group than the control group. There was no significant difference in infarct volume (203, 220 and 218 mm³) among the control, n-3 fatty acid deficient, and high DHA groups, respectively. Platelet fatty acids and platelet aggregation were assessed to determine if these were influenced by the high DHA diet, and could possibly explain the observation of an apparent, but not statistically significant, higher number of rats with hemorrhages in the high DHA diet group. Platelet lipid arachidonic acid was not lower and platelet aggregation, assessed ex vivo using whole blood with a platelet function analyzer, was not longer in rats fed the high DHA compared to control or n-3 fatty acid deficient diets. In summary, dietary n-3 fatty acid deficiency did not increase the extent of brain injury following cerebral ischemia. The possibility that high dietary 22:6n-3 might increase susceptibility to cerebral hemorrhage will require further study.

Many populations have low intakes of n-3 fatty acids, yet there is substantial evidence that the long chain n-3 fatty acid docosahexaenoic acid (DHA; 22:6n-3), found at high concentrations in the brain, is required for the proper development of the nervous system. However, less is known about requirements of long chain n-3 fatty acids for maintenance and function of the nervous system in later life. Several recent studies have reported that high amounts of long chain n-3 fatty acids reduce the extent of brain damage caused by cerebral ischemia in animals. However, whether or not a dietary deficiency of n-3 fatty acids increases the extent of injury when cerebral ischemia occurs has not been previously reported. The present studies, therefore, sought to determine if a diet deficient in n-3 fatty acids influences the extent of brain injury in the rat following cerebral ischemia. Male rats were fed an n-3 fatty acid adequate (control), an n-3 fatty acid deficient, or a high DHA diet for 5 weeks from weaning. Middle cerebral artery occlusion (MCAO) was induced and infarct volume was measured by 2,3,5,-triphenyltetrazolium chloride staining 24 hours after the procedure. Brain and platelet fatty acids were analyzed by gas liquid chromatography. DHA (22:6n-3) was 21-28% lower in brain phospholipids, and 17% lower in brain total fatty acids in the n-3 fatty acid deficient compared to control group, while 22:6n-3 was 12% higher in total brain fatty acids in the high DHA group than the control group. There was no significant difference in infarct volume (203, 220 and 218 mm³) among the control, n-3 fatty acid deficient, and high DHA groups, respectively. Platelet fatty acids and platelet aggregation were assessed to determine if these were influenced by the high DHA diet, and could possibly explain the observation of an apparent, but not statistically significant, higher number of rats with hemorrhages in the high DHA diet group. Platelet lipid arachidonic acid was not lower and platelet aggregation, assessed ex vivo using whole blood with a platelet function analyzer, was not longer in rats fed the high DHA compared to control or n-3 fatty acid deficient diets. In summary, dietary n-3 fatty acid deficiency did not increase the extent of brain injury following cerebral ischemia. The possibility that high dietary 22:6n-3 might increase susceptibility to cerebral hemorrhage will require further study. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

The initial hours following an ischaemic event in the brain represent a critically important window in which therapeutic interventions to reduce neuronal damage and improve patient outcome can be made. Nevertheless, the dynamics of cerebral blood flow and oxygenation, as well as the local physiological changes, in the first few hours after reperfusion following ischaemic stroke are not well understood. In the first study, a remote filament approach was used to obtain multispectral imaging data before, during and after middle cerebral artery occlusion to investigate early changes in haemodynamic concentration of oxy-/deoxy-haemoglobin and total blood volume, in anaesthetised mice. We use immunohistochemistry to establish the extent of cortical injury and correlate the severity of damage with the change of oxygen perfusion during and after the ischaemic event. Increased numbers of platelets and activated microglia, expression of interleukin-1α, evidence of BBB breakdown and neuronal stress are all seen within the stroked hemisphere of MCAo mice and correlate with the severity of oxy-haemoglobin concentration deficit at experimental but not with the change in oxy-haemoglobin concentration during the acute stroke. In the second study, we used the same remote filament and optical imaging approach to investigate the effects of acute systemic inflammation on haemodynamics pre, during, and after induced cerebral ischaemia. We found that an acute systemic inflammatory challenge exacerbates oxy-haemoglobin deficit after 3 h of reperfusion following an ischaemic event. We investigated known pathophysiological markers to elucidate potential mechanisms that may contribute to this exacerbated oxygenation deficit and found hyper-coagulated platelets within the large and microvessels of the ipsilateral cortex. Our findings demonstrate that despite initial restoration of HbO2 supply after 30 min MCAo there is a delayed compromise that coincides with inflammatory processes that could be a future target for improved stroke outcome after thrombolysis. We also show that acute systemic inflammation exacerbates this oxy-haemoglobin deficit after an ischaemic challenge and increases pathophysiology.

Previous studies have demonstrated that plasma resistin levels were increased in patients with acute ischemic stroke. However, the role of resistin after ischemic brain injury is still unclear. In this study, we investigated the protective effects of resistin on cerebral ischemia/reperfusion injury in a middle cerebral artery occlusion mouse model. We found that resistin (i.c.v.) significantly reduced infarct volume and improved neurological deficits after 45 min of ischemia and 24 h of reperfusion. Furthermore, our data demonstrate that intraperitoneal administration of resistin (10 µg/kg body weight) also had protective effects on infarct volume, indicating the crossing of resistin through the impaired BBB after ischemia injury. Resistin treatment reduced cleaved protein level of Poly(ADP-ribose)polymerase-1 (PARP-1), a marker of cellular apoptosis, showing the anti-apoptotic activity of resistin. Resistin increased the level of phosphorylated Akt after ischemic brain injury. The neuroprotective effect of resistin was partially reversed by a PI3K inhibitor wortmannin, demonstrating that the PI3K/Akt signal pathway is involved in the anti-apoptotic mechanisms of resistin. Finally, we found that resistin treatment improved neurological function recovery at 14 days after treatment, including balance ability and muscle strength. Given these findings, resistin may have therapeutic potential for the treatment of stroke.

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