Thesis (M. Phil.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

<p>Stroke is the major cause of serious long-term disability with a sufficient acute treatment for only a very limited number of patients. Limited recovery of neurological functions occurs and can be elevated by a permissive post-stroke milieu. Housing animals in an enriched environment modulates regenerative mechanisms in the nonischemic peri-infarct area which might be an attractive target for pharmacological treatments to promote recovery.</p><p>Upon ischemia, cellular lipids are released due to massive cell damage and free lipids significantly contribute to the progression of acute and delayed cell death. The aim of this study was to evalute the effect of enriched environment on lipid metabolism. In particular we characterize the activation of the transcription factor liver X receptor (LXR) in glial scar formation and regulation of cholesterol balance of relevance for functional recovery following stroke.                                      Brain tissues from animals subjected to permanent occlusion of middle cerebral artery (pMCAo) were analysed for LXRα and β protein expression. We found an upregulation and an increased transcriptional activity of LXRβ in the peri-infarct area of rats housing in an enriched environment following pMCAO. Our data anticipate that enriched environment may have positive effects on lipid recycling in the ischemic hemisphere following experimental stroke.<strong></strong></p>

<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>

<p>Even if popliteal artery aneurysm (PAA) is the most common peripheral aneurysm, no single surgeon or institution has enough patients to study this disease with appropriate scientific methods, and no population-based investigation exists. </p><p>PAA epidemiology, treatment, management, and outcome were studied in a population-based study of 571 patients (717 legs) primarily operated on for PAAs and 100 episodes of preoperative thrombolysis in Sweden between 1987 and 2002. Patients were identified in the Swedish Vascular Registry and case-records were reviewed. Information on amputation and survival was obtained for all patients, and 190 patients were re-examined with ultrasound, after mean 7.2 years (range 2-18)</p><p>Median age was 71 years; 5.8% were women. Patients with unilateral PAA had AAA in 28%, increasing to 38% when PAAs were bilateral. Crude survival was 91.4% at one and 70% at five years, significantly lower than among age and sex matched controls. The cumulative incidence for operation of PAA in Sweden was estimated to 8.3/million person year. One-year amputation-rate was 8.8 %, increasing to 11% after follow-up (7.2 years). Independent risk factors for amputation within one year were poor run-off, age, emergency procedure, and prosthetic graft. Run-off was improved by preoperative thrombolysis among 87% of legs, when acute ischemia. After surgical repair with a medial approach the risk of late expansion of the aneurysm was 33%, with a posterior approach 8% , p=0.014. Among 190 re-examined patients, 108 (57%) had at least one additional aneurysm at index-operation, increasing to 131 (68%) at re-examination, the total number of aneurysms increasing by 42% (from 244 to 346).</p><p>Conclusions: Multiple aneurysms are common among patients operated on for PAA. Preoperative thrombolysis improves run-off and decreases the amputation-rate in PAAs with acute ischemia. Vein grafts do better than prosthetic grafts, especially when a long bypass is needed. Posterior approach, when possible, reduces the risk of late expansion. A complete examination of the aorto-iliac and femoro-popliteal arteries is warranted at the time of surgery. All patients should be kept under life-long surveillance in order to detect and treat newly developed aneurysms timely. Normal arterial segments should be re-examined after three years.</p>

Cardiovascular and metabolic diseases are still the primary cause of death and disability in modern society. Although genetic factors play a fundamental role in the development of these chronic conditions, the remarkable variability in an individual’s susceptibility to develop these pathologies cannot be completely explained by genetics. The early programming of adult diseases theory became established in the 1980’s, and is now supported by a growing body of evidence demonstrating that exposure to suboptimal environmental conditions during crucial periods of time can predispose an individual to the development of chronic conditions (including cardiovascular and metabolic diseases) later in life. Among the multitude of factors that can cause early programming, we have focused on the study of pregnancy complications leading to fetal hypoxia and causing intrauterine growth restriction (IUGR). To this end, we have used an animal model in which pregnant Sprague Dawley rats were exposed to either normal (∼21% O2) or hypoxic (∼11.5% O2) conditions during the last third of pregnancy. We then followed and studied the cardiovascular and metabolic characteristics of the offspring later in life. The studies presented in this thesis demonstrate that hypoxic prenatal insults have long-term consequences on cardiac structure, function and susceptibility to ischemia. We also demonstrated that programmed susceptibility to myocardial ischemia was associated with changes in cardiac energy metabolism and increased levels of myocardial oxidative stress. Moreover, we described the interaction between prenatal hypoxic insults, aging and sex differences in the later development of cardiovascular conditions. Additional studies presented in this thesis demonstrate that offspring born IUGR are more susceptible to develop most components of the metabolic syndrome when exposed to a high-fat (HF) diet. Furthermore, we also demonstrated that the exacerbated deleterious response to a HF diet described in offspring born IUGR can be prevented by postnatal administration of Resveratrol, which is a natural compound with anti-oxidant and anti-aging properties. In conclusion, the results presented in this thesis are an important contribution to the understanding of the long-term cardiovascular and metabolic effects of prenatal hypoxic insults causing IUGR and provide evidence regarding possible mechanisms and treatment alternatives that could be considered.

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.

Even if popliteal artery aneurysm (PAA) is the most common peripheral aneurysm, no single surgeon or institution has enough patients to study this disease with appropriate scientific methods, and no population-based investigation exists. PAA epidemiology, treatment, management, and outcome were studied in a population-based study of 571 patients (717 legs) primarily operated on for PAAs and 100 episodes of preoperative thrombolysis in Sweden between 1987 and 2002. Patients were identified in the Swedish Vascular Registry and case-records were reviewed. Information on amputation and survival was obtained for all patients, and 190 patients were re-examined with ultrasound, after mean 7.2 years (range 2-18) Median age was 71 years; 5.8% were women. Patients with unilateral PAA had AAA in 28%, increasing to 38% when PAAs were bilateral. Crude survival was 91.4% at one and 70% at five years, significantly lower than among age and sex matched controls. The cumulative incidence for operation of PAA in Sweden was estimated to 8.3/million person year. One-year amputation-rate was 8.8 %, increasing to 11% after follow-up (7.2 years). Independent risk factors for amputation within one year were poor run-off, age, emergency procedure, and prosthetic graft. Run-off was improved by preoperative thrombolysis among 87% of legs, when acute ischemia. After surgical repair with a medial approach the risk of late expansion of the aneurysm was 33%, with a posterior approach 8% , p=0.014. Among 190 re-examined patients, 108 (57%) had at least one additional aneurysm at index-operation, increasing to 131 (68%) at re-examination, the total number of aneurysms increasing by 42% (from 244 to 346). Conclusions: Multiple aneurysms are common among patients operated on for PAA. Preoperative thrombolysis improves run-off and decreases the amputation-rate in PAAs with acute ischemia. Vein grafts do better than prosthetic grafts, especially when a long bypass is needed. Posterior approach, when possible, reduces the risk of late expansion. A complete examination of the aorto-iliac and femoro-popliteal arteries is warranted at the time of surgery. All patients should be kept under life-long surveillance in order to detect and treat newly developed aneurysms timely. Normal arterial segments should be re-examined after three years.

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.

Stroke is the major cause of serious long-term disability with a sufficient acute treatment for only a very limited number of patients. Limited recovery of neurological functions occurs and can be elevated by a permissive post-stroke milieu. Housing animals in an enriched environment modulates regenerative mechanisms in the nonischemic peri-infarct area which might be an attractive target for pharmacological treatments to promote recovery. Upon ischemia, cellular lipids are released due to massive cell damage and free lipids significantly contribute to the progression of acute and delayed cell death. The aim of this study was to evalute the effect of enriched environment on lipid metabolism. In particular we characterize the activation of the transcription factor liver X receptor (LXR) in glial scar formation and regulation of cholesterol balance of relevance for functional recovery following stroke.                                      Brain tissues from animals subjected to permanent occlusion of middle cerebral artery (pMCAo) were analysed for LXRα and β protein expression. We found an upregulation and an increased transcriptional activity of LXRβ in the peri-infarct area of rats housing in an enriched environment following pMCAO. Our data anticipate that enriched environment may have positive effects on lipid recycling in the ischemic hemisphere following experimental stroke.

Traumatic brain injury (TBI) is a major cause of death and disability. TBI is frequently followed by cerebral ischemia which is a great contributor to secondary brain damage. The main causes of cerebral ischemia are pathophysiological changes in cerebral blood flow and metabolism. Treatment of TBI patients is currently based on intracranial pressure (ICP) and cerebral perfusion pressure (CPP) targeted treatment protocols. However, ICP and CPP alone do not provide information of the oxygen availability in the brain. Monitoring of brain tissue oxygenation (BtipO2) may give additional and valuable information about the risk for development of ischemia in TBI patients. The aims of this thesis were to study BtipO2 monitoring devices in-vitro regarding accuracy and stability, to detect threshold level of cerebral ischemia in-vivo and finally to examine the cerebral oxygen levels and cerebral metabolism in TBI patients. The BtipO2 probes performed with high accuracy and stability at different clinically relevant oxygen concentrations. A pig TBI model was developed by step-wise intracranial volume/pressure increase. Volume increase resulted in a gradual increased ICP, decreased CPP, intracranial compliance and BtipO2, respectively. Brain death (BD) was confirmed by negative CPP and negligible amount of previously injected microspheres in the brain tissue. The model simulated the clinical development of BD in humans with a classical pressure-volume response and systemic cardiovascular reactions. The model should be suitable for studies of brain injury mechanisms. From the same in-vivo model it was also possible to detect the threshold level of cerebral ischemia in the pig, where BtipO2 below 10 mmHg and CPP below 30 mmHg was associated with an impaired cerebral metabolism (microdialysis lactate to pyruvate ratio &gt;30). BtipO2 together with cerebral microdialysis were studied in 23 severe TBI patients. We observed different patterns of changes in BtipO2 and cerebral microdialysis biomarkers in focal and diffuse TBI.  Increased cerebral microdialysis levels of glutamate, glycerol or the lactate/pyruvate ratio were observed at BtipO2 &lt; 5 mmHg, indicating increased vulnerability of the brain at this critical level of tissue oxygenation in TBI patients.