Vascular, especially cerebrovascular, dysfunction may be a critical factor in ageing and dementia. Cerebrovascular impairment due to risk factors such as ageing, stroke, smoking, diabetes and cerebral hypoperfusion has a deterious impact on the normal supply of basic nutrients such as oxygen and glucose to the brain; their absence leads inevitably to neuronal death. The cerebral white matter lesions found in most forms of dementia are reportedly the result of chronic cerebral hypoperfusion. However the temporal and spatial evolution of damage remains unclear. Furthermore, any decrease in the integrity of the blood-brain barrier (BBB) has been hypothesised to be a precocious attack on white matter. The “milieu interieure” the most protected in the body, namely the extracellular fluid of the brain, is no longer maintained homeostatically. The cumulation of these various pathophysiological processes alters cerebral function and it has been postulated that, in the most extreme instances, the outcome of this cascade of nefarious events leads to dementia. This thesis examines the supposition that chronic cerebral hypoperfusion could be responsible for the time-related development of white and grey matter pathology and investigates the relationships between the disturbances in the integrity of the BBB and white matter pathology. Three studies addressed these aims. In the first, chronic cerebral hypoperfusion, induced in male Wistar rats by bilateral common carotid artery occlusion (BCCAo), was chosen as the model to study changes in axons, myelin, perikarya as well as microglial activation. The groups of rats that underwent BCCAo were examined at three hours as well as three, seven, 14 and 28 days after the induction of chronic cerebral hypoperfusion. The microscopic examination revealed that, after three hours post BCCAo, damage was detected only in axons and myelin. In contrast, no visible pathology to the neuronal perikarya or enhancement of activated microglia (compared to the sham group) was observable. Injury in both white and grey matter and enhancement of activated microglia was observed from three days post BCCAo and increased with time post BCCAo. The most severe damage to the white and grey matter and enhancement of microglial activation was detected at seven days post BCCAo. These results would indicate that white matter damage precedes grey matter pathology and the enhancement of activated microglia. In the second study, the integrity of the BBB at three hours (when only white matter pathology was found according to the results of the first study) and seven days post BCCAo (when more severe damage to the white and grey matter was shown) was assessed by the use of MRI on T1-weighted image acquisitions with gadolinium as a tracer for BBB permeability. White matter integrity was measured by MTR maps from MTI acquisitions in four brain structures (corpus callosum, caudatoputamen, the external and internal capsules). No differences in white matter integrity were detected between the BCCAo and sham group at three hours and seven days. No differences in signal enhancement of gadolinium were detected three hours post BCCAo. However, a significant signal enhancement of gadolinium was detected at seven days post BCCAo in the caudatoputamen and in the external capsule. Furthermore, immunohistochemistry revealed a significant enhancement of activated microglia seven days post BCCAo compared to the sham group. This functional and immunohistochemical finding, when taken together, might indicate that chronic cerebral hypoperfusion is not in itself responsible for BBB permeability. Rather, the damage to the white matter caused by cerebral hypoperfusion may be responsible for the dysfunction of the BBB over time. Another point of interest was the evidence that the enhancement of activated microglia may play a critical role in the increased permeability of the BBB. The final study in this thesis aimed to investigate the possible pathway and proteins potentially implicated in white matter damage and BBB permeability. To address this question, protein levels and the expression of genes involved in the apoptotic and nonapoptotic hypoxic pathways were compared to the sham groups (at three hours and seven days after BCCAo), in three brain structures (cortex, corpus callosum and caudatoputamen). The levels of HIF-1α, MMP-2, Caspase-3 and VEGF were unchanged compared to the sham group after BCCAo. However, VEGF mRNA expression was found to be significantly different to the sham group seven days post BCCAo in all the three structures examined. An overexpression of HIF-1α and a significant level of Caspase-3 would indicate the activation of the apoptotic pathway. However, neither of these criteria were met and these negative results suggest that the apoptotic pathway is not implicated in the mechanisms that lead to white matter pathology after cerebral hypoperfusion. Finally, the significant expression of VEGF mRNA, compared to the sham group seven days post BCCAo, may contribute to the time-relate increased permeability of the BBB. The results presented within this thesis provide a body of evidence to support the hypothesis that chronic cerebral hypoperfusion is - at least – causal to the damage to different components of the white matter which precedes either early ischaemic changes to the perikarya or enhancement of activated microglia following BCCAo. The increased permeability of the BBB, which can be related to the significant over-expression of VEGF mRNA (compared to the sham group seven days post BCCAo), does not appear to be primarily responsible for white matter pathology, because the MRI investigations indicated that BBB integrity was not affected after three hours of BCCAo. The increased permeability of the BBB, observed seven days post BCCAo with MRI, seems to be the consequence of increased brain damage; thereafter, there is a time-dependent relationship between increasing BBB permeability and increasing brain pathology. Overall, the studies reported herein, strengthen the initial working hypothesis. The conclusion – and direction for future studies – would be that minimising white matter pathology and protecting components of the BBB represent potential targets to decrease then incidence of neuropsychological function or to obtund the cerebral dysfunction in patients who suffer from chronic cerebral hypoperfusion.

Lacunar strokes account for approximately a quarter of all ischaemic strokes and traditionally are thought to result from occlusion of a small deep perforating arteriole in the brain. Lacunar infarcts can be up to 2cm in diameter and are found in deep brain structures such as the thalamus and internal capsule. Despite their prevalence and specific accompanying clinical syndromes, the cause of lacunar stroke and its associated vascular pathology remain unclear. Many hypotheses as to the cause exist, which fall broadly into two categories; firstly, a direct occlusion via emboli or thrombus usually from a cardiac or large artery source, microatheroma (intrinsic lenticulostriate occlusion) or macroatheroma (parent artery occlusion) all operating primarily via ischaemia. Secondly, there could be an indirect occlusion resulting from vasospasm, endothelial dysfunction or other forms of endovascular damage (e.g. inflammation). Therefore the question of whether the resulting lesions are truly “ischaemic” or actually arise secondary to an alternative process is still under debate. To clarify the chain of pathological events ultimately resulting in lacunar stroke, in this thesis I firstly undertook a systematic assessment of human lacunar stroke pathology literature to determine the information currently available and the quality of these studies (including terminology). The majority of these studies were performed in patients who had died long after their stroke making it difficult to determine the early changes, and there were few patients with a clinically verified lacunar syndrome. Therefore I adopted alternative approaches. In this thesis, I systematically looked for all potential experimental models of lacunar stroke and identified what appears at present to be the most pertinent - the spontaneous pathology of the stroke-prone spontaneously hypertensive rat (SHRSP). However, the cerebral pathology described in this model to date is biased towards end stage pathology, with little information concerning the microvasculature (as opposed to the brain parenchyma) and confounding by use of salt to exacerbate pathology. Therefore, the aim of the experimental work in this thesis was to assess pathological changes within the cerebral vasculature and brain parenchyma of the SHRSP across a variety of ages (particularly young pre-hypertensive animals) and to look at the effects of salt loading on both the SHRSP and its parent strain (the Wistar Kyoto rat - WKY). Three related studies (qualitative and quantitative histology, immunohistochemistry and a microarray study of gene expression confirmed by quantitative PCR), revealed that the presence of inflammation (via significant changes in gene expression in the acute phase response pathway and increased immunostaining of activated microglia and astrocytes) plus alterations in vascular tone regulation, (via genetic alteration of the nitric oxide signaling pathway probably secondary to abnormal oxidative state), impaired structural integrity of the blood brain barrier (histological evidence of endothelial dysfunction and significantly decreased Claudin-5 staining) and reduced plasma oncotic potential (reduced albumin gene expression) are all present in the native SHRSP at 5 weeks of age, i.e. well before the onset of hypertension and without exposure to high levels of salt. We also confirmed previous findings of vessel remodelling at older ages likely as a secondary response to hypertension (thickened arteriolar smooth muscle, increased smooth muscle actin immunostaining). Furthermore, we found not only that salt exacerbated the changes see in the SHRSP at 21 weeks, but also that the control animals (WKY) exposed to a high salt intake developed features of cerebral microvascular pathology independently of hypertension (e.g. white matter vacuolation and significant changes in myelin basic protein expression). In conclusion, via the assessment of the most pertinent experimental model of lacunar stroke currently available, this thesis has provided two very important pieces of evidence: firstly that cerebral small vessel disease is primarily caused by a non-ischaemic mechanism and that any thrombotic vessel lesions occur as secondary end stage pathology; secondly that these features are not simply the consequence of exposure to raised blood pressure but occur secondary to abnormal endothelial integrity, inflammation, abnormal oxidative pathways influencing regulation of vascular tone and low plasma oncotic pressure. Patients with an innate susceptibility to increased blood brain barrier permeability and/or chronic inflammation could therefore have a higher risk of developing small vessel disease pathology and ultimately lacunar stroke and other features of small vessel disease. Research, addressing whether lacunar stroke patients should be treated differently to those with atherothromboembolic stroke is urgently needed.

The trace elements zinc and copper are essential components of many enzymes, some of which are of importance for the development and function of the central nervous system. Deficiency of the metals has been shown to lead to malformations and to the loss of myelin in animals. Earlier reports of zinc concentrations in the cerebrospinal fluid are few and the results variable. In multiple sclerosis and in epilepsy therapy with phenytoin there are varying reports of changes in serum concentrations of zinc and copper. A method was developed for the determination of zinc in cerebrospinal fluid by flame atomic absorption spectrophotometry utilizing a pulse nebulizer technique. Zinc and copper in serum were determined by flame atomic absorption spectrophotometry with conti nous aspiration. The normal concentrations of zinc in cerebrospi nal fluid was 0.16_+0.03 micromoles per litre (mean +_ S.D.). The zinc concentrations were correlated with protein and albumin concentrations in the cerebrospinal fluid but not with the serum zinc levels. In the patients with increased protein concentrations in the cerebrospinal fluid or with subarachnoid haemorrhage increased zinc levels were found. In 50 patients with multiple sclerosis lower serum concentrations of zinc were found compared to age and sex matched controls. In younger patients low serum levels of copper were also observed. There was no correlation between zinc and protein parameters in the cerebrospinal fluid of multiple sclerosis patients. In untreated epileptic males low serum zinc concentrations were observed. During the first 72 hours of phenytoin therapy increased serum concentrations of zinc and copper were found. during long-term therapy with phenytoin alone or in combination with other antiepileptic drugs there was an increased serum concentration of copper and ceruloplasmin but no change in zinc concentration compared with controls. / <p>Diss. Umeå, Umeå universitet, 1982, härtill 4 uppsatser</p> / digitalisering@umu

Cronobacter species cause serious infections such as meningitis and enteritis in newborns and neonates, with the major vehicle being contaminated powdered infant formula. The main objectives of this study were i) to identify potential virulence factors, such as enterotoxin production; ii) characterize the gene(s) involved in adhesion and invasion of the human brain microvascular endothelial cells (HBMEC); and iii) determine whether strains from clinical, food, and environmental sources differ in their ability to produce surface-attached bacterial aggregates, known as biofilms. Random transposon mutagenesis was used on strains demonstrating the best adherence and invasion to blood- brain barrier cell lines (BBB). Isogenic mutants were then screened for increased or decreased adherence and invasion. Screening of the transposon library identified one isogenic mutant of a clinical strain which lost the ability to adhere to BBB cells. The transposon rescue revealed the insertion site to be within a diguanylate cyclase (DGC) gene. The major function of DGC in many Gram-negative bacteria is to synthesize cyclic diguanylate (c-di-GMP), a secondary bacterial metabolite known for regulating biofilm formation, motility, and virulence or aspects of microbial pathogenicity. Based on the findings of this study, DGC appears to play an important role in Cronobacter species’ ability to produce biofilms and may also have a role of the pathogenicity in the microorganism.

Capacity to perform prolonged exercise is reduced in high ambient temperatures, but this premature fatigue is not adequately explained by peripheral mechanisms. The aim of this thesis was to examine some possible underlying mechanisms of central fatigue operating during prolonged exercise in a warm environment. The first series of experiments investigated the effect of nutritional manipulation of central serotonergic activity through alterations to the plasma concentration ratio of free-tryptophan to branched-chain amino acids (f-TRP:BCAA). In contrast to previous reports, acute BCAA supplementation failed to alter perceived exertion and delay the onset of fatigue (Chapter 3). This response was similar when exercise was preceded by an exercise and diet regimen designed to reduce glycogen availability (Chapter 4). The ingestion of meals containing added carbohydrate and fat did not alter f-TRP:BCAA at rest (Chapter 5). Acute dopaminergic / noradrenergic reuptake inhibition with bupropion increased exercise perfonnance by 9 % in warm conditions (30C), but this effect was not apparent at 18C (Chapter 6). This response was accompanied by attainment of a higher core temperature and heart rate towards the end of the bupropion trial in the heat despite no detectable difference in perceived exertion and thermal stress. These data suggested that maintenance of catecholaminergic activity may dampen inhibitory signals from the CNS due to the attainment of a high core temperature, allowing power output to be maintained. The blood-brain barrier (BBB) regulates the exchange of substances between the cerebral interstitial fluid and the blood to maintain a stable environment for the CNS. If the BBB is compromised this may adversely influence nonnal brain function. Serum S1OOb, a proposed peripheral marker of BBB penneability, was increased following exercise in a warm environment (Chapter 7). These data indicate that exposure to combined exercise and heat stress may result in a loss of BBB integrity.

Background: Strategies to maintain BBB integrity in diseases with a hypoxia/reoxygenation (H/R) component involve preventing glutathione (GSH) loss from endothelial cells. GSH efflux transporters include multidrug resistance proteins (Mrps). Therefore, characterization of Mrp regulation at the BBB during H/R is required to advance these transporters as therapeutic targets. Our goal was to investigate, in vivo, regulation of Abcc1, Abcc2, and Abcc4 mRNA expression (i.e., genes encoding Mrp isoforms that transport GSH) by nuclear factor E2-related factor (Nrf2) using a well-established H/R model. Methods: Female Sprague-Dawley rats (200-250 g) were subjected to normoxia (Nx, 21% O-2, 60 min), hypoxia (Hx, 6% O-2, 60 min) or H/R (6% O-2, 60 min followed by 21% O-2, 10 min, 30 min, or 1 h) or were treated with the Nrf2 activator sulforaphane (25 mg/kg, i.p.) for 3 h. Abcc mRNA expression in brain microvessels was determined using quantitative real-time PCR. Nrf2 signaling activation was examined using an electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) respectively. Data were expressed as mean +/- SD and analyzed via ANOVA followed by the post hoc Bonferroni t test. Results: We observed increased microvascular expression of Abcc1, Abcc2, and Abcc4 mRNA following H/R treatment with reoxygenation times of 10 min, 30 min, and 1 h and in animals treated with sulforaphane. Using a biotinylated Nrf2 probe, we observed an upward band shift in brain microvessels isolated from H/R animals or animals administered sulforaphane. ChIP studies showed increased Nrf2 binding to antioxidant response elements on Abcc1, Abcc2, and Abcc4 promoters following H/R or sulforaphane treatment, suggesting a role for Nrf2 signaling in Abcc gene regulation. Conclusions: Our data show increased Abcc1, Abcc2, and Abcc4 mRNA expression at the BBB in response to H/R stress and that Abcc gene expression is regulated by Nrf2 signaling. Since these Mrp isoforms transport GSH, these results may point to endogenous transporters that can be targeted for BBB protection during H/R stress. Experiments are ongoing to examine functional implications of Nrf2-mediated increases in Abcc transcript expression. Such studies will determine utility of targeting Mrp isoforms for BBB protection in diseases with an H/R component.

The rates of opioid prescription and use have continued to increase over the last few decades. In turn, a greater number of patients suffer from opioid tolerance. Treatment of acute pain is a clinical challenge for these patients. Acute pain can arise from common occurrences like surgical pain and pain resulting from the injury. P-glycoprotein (p-gp) is a transporter at the blood-brain barrier (BBB) associated with a decrease in the analgesic efficacy of morphine. Peripheral inflammatory pain (PIP) is a pain state known to cause a change in p-gp trafficking at the BBB. P-gp traffics from the nucleus to the luminal surface of endothelial cells making up the BBB. This surface where circulating blood interfaces with the endothelial cell is where p-gp will efflux morphine back into circulation. Osmotic minipumps were used as a long-term delivery method in this model of opioid tolerance in female rats. PIP induced p-gp trafficking away from nuclear stores showed a 2-fold increase when animals were exposed to opioids for 6 days. This observation presents a possible relationship between p-gp trafficking and the challenges of treating post-surgical pain in opioid tolerant patients. This could reveal potential strategies for improving pain management in these patients.

Glioblastoma multiforme is the most aggressive form of malignant brain tumor with poor prognosis. The efficacy of brain cancer treatment by chemotherapeutics is limited by the blood-brain barrier (BBB) which allows less than 2% of the small molecules and blocks almost all the macromolecules to transport into the brain. Delivery of the large molecules such as proteins and nucleic acids across the BBB is a great challenge for brain-targeted drug delivery. To overcome this obstacle, cell-penetrating peptides (CPPs) were used as vectors for delivery of nucleic acids across the BBB targeting glioblastomas. The CPPs have shown such promising carriers to deliver various cargoes ranging from small molecules to large molecules into the cells. This thesis is focused on the development of glioblastoma-targeting vectors based on modifications of the CPPs and the targeting peptides. The peptide-based vectors were developed to improve the transport of the nucleic acids across the BBB and specifically target glioblastomas. In this thesis, a series of peptide-based vectors targeting glioblastomas were synthesized and modified with targeting peptides by either covalent conjugation or non-covalent complex formation. The delivery of plasmid DNA (pDNA) in the complex with the peptide-based vectors was studied in the in vitro model of the BBB. The role of receptors expressed on the BBB was investigated. Scavenger receptors class A and B were found to be expressed on the BBB, and they were involved in the delivery of the pDNA across the BBB model. Moreover, various targeting peptides were modified with hexaglutamate to form non-covalent complexes with the CPPs for small interfering RNA (siRNA) delivery to glioblastoma cells. The non-covalent complex of the CPP and the targeting peptide showed greater gene-silencing efficiency than the consecutively covalent conjugation of the CPP and the targeting peptide for siRNA delivery to glioblastoma cells. Lastly, a number of novel, amphipathic peptides were developed based on the model amphipathic peptide. The prediction of the biological effect of the designed peptides using quantitative structure-activity relationship model showed a correlation with the experimental data. Finally, the CPP-based nucleic acid delivery vectors with homing peptide strategy have a potential for the BBB shuttle and the future use as a glioblastoma-targeted drug carrier in the in vivo studies and the clinical applications. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p>

Targeted drug delivery to the brain is difficult to achieve using conventional techniques, largely due to the blood-brain barrier’s (BBB) impediment to drug diffusion into the brain parenchyma. In response, development of convection-enhanced delivery (CED) offers the ability to circumvent the BBB and target specific areas of the brain. Predictability of infusate movement in pathological brain states during CED will maximize the effectiveness of this treatment, and therefore modeling of infusate movement must be characterized. Previous work from our lab effectively modeled CED in rats using the middle carotid artery occlusion model of cytotoxic edema. However, previous models examined for vasogenic edema study did not show pure vasogenic edema. The purpose of this study was to develop a model of pure vasogenic edema in the rat brain. In this study, we show that stereotactic 9 µL infusion of 1.0 mM DCA over 45 minutes into the rat corpus callosum reproducibly creates pure vasogenic edema, as observed in the peritumoral white matter surrounding gliomas.

Title: Acute neuroinflammatory reaction after focal cerebral ischemia Aim: The aim of this thesis is to evaluate neuroinflammatory response after focal cerebral cortical ischemia. Also, familiarizing with the method of displaying damage of blood brain barriers, neurons and the possibility of detection of microglia cells as a marker of acute neuroinflammatory processes. Methodology: This is an experimental study. We brought about cortical cerebral ischemia in rats using an application of photosensitive dye "bengal red," and a green laser. Two animals were were given the additional application of "Evans blue" in order to visualize the defects of the blood brain barrier. The animals were returned to their cage for the time needed before they were induced terminal anesthesia. This was followed by the process of brain perfusion, slicing the brain in sections 50 µm thick and then applied these sections onto slides. Sections with applied EB were immediately analyzed under the microscope. Sections to illustrate neuronal death were immunohistochemically stained via the Nissl method. Sections visualizing microglial activity were stained using CD11b antibody. Results: Following the induction of focal ischemia there occured brain tissue damage. In the vicinity of lesion there is degeneration of neurons and...