The structural integrity of the white matter is required for neuronal communication within the brain which is essential for normal cognitive function. Post-mortem and clinical imaging studies of elderly individuals have demonstrated that white matter integrity is weakened with increasing age which is proposed to underlie age-related cognitive decline. Whilst the exact mechanisms are unknown it is thought that modest age-related reductions in cerebral blood flow, termed chronic cerebral hypoperfusion, may contribute to white matter disruption and impaired cognition with ageing. Investigating the effects of white matter integrity in humans is limited as it is difficult to definitively ascertain a cause and effect relationship. Indeed, elderly individuals with cerebral hypoperfusion often have co-existing disease such as hypertension thus the effects of hypoperfusion in isolation cannot be determined. This has led to the development of a mouse model of chronic cerebral hypoperfusion which provides the opportunity to directly assess whether cerebral hypoperfusion results in disruption to white matter and cognitive impairment. This is achieved by applying small wire coils around both common carotid arteries of the mouse resulting in a global reduction in cerebral blood flow. Importantly the extent of blood flow reduction is dependent on the internal diameter of the coils meaning that differing severities of hypoperfusion can be studied. Previous studies using this model have demonstrated diffuse white matter pathology in white matter tracts including the corpus callosum, internal capsule and optic tract following 1 month of hypoperfusion which is accompanied by impaired spatial working memory. This thesis sought to test the hypothesis that chronic cerebral hypoperfusion would influence the structural integrity of nodal and paranodal domains of myelinated axons of the white matter and result in decreased numbers of oligodendroglial cells. It was additionally hypothesised that treatment with the anti-inflammatory and antioxidant drug dimethyl fumarate (DMF) would ameliorate structural and functional alterations to white matter following hypoperfusion. Aim 1 – To determine the impact of chronic cerebral hypoperfusion on the structural integrity of nodal and paranodal domains of myelinated axons The first aim of this thesis was to investigate the effects of chronic cerebral hypoperfusion on the structural integrity of nodal and paranodal domains of myelinated axons. This was addressed by examining key myelin and axonal proteins found at nodal, paranodal and internodal domains. This revealed significant alterations to the distribution of voltage-gated sodium (Nav1.6) channels at nodes of Ranvier which were differentially altered in response to increasing durations of chronic cerebral hypoperfusion. Specifically an increase in the Nav1.6+ domain length was observed in the corpus callosum following 3 days (p < 0.0001) and 1 month (p < 0.001) of chronic cerebral hypoperfusion but was not significantly different from sham controls following 6 weeks of hypoperfusion (p = 0.066). A significant decrease in Nav1.6 domain length was observed following 3 months of hypoperfusion (p = 0.003). Assessment of paranodal integrity was carried out by measuring nodal gap length and by ultrastructural analysis of paranodal domains. This revealed pronounced alterations to nodal gap length, loss of paranodal septate-like junctions and abnormal morphology of paranodal loops. Furthermore this study revealed a significant loss of myelin associated glycoprotein, a key protein involved in the maintenance of axon-glial integrity, as early as 3 days following the onset of hypoperfusion. A further aim of this study was to examine potential mechanisms underlying the observed alterations to nodal and paranodal domains following cerebral hypoperfusion. It was hypothesised that increased inflammation and accumulation of mitochondria at nodes of Ranvier would be observed following hypoperfusion. The extent of inflammation was assessed by counting numbers of microglia which revealed no significant difference between groups following 3 days of hypoperfusion (p = 0.425) but a significant increase in microglial number was observed following 1 month of hypoperfusion (p = 0.001). In addition, assessment of mitochondrial distribution along myelinated axons revealed decreased numbers of nodes containing mitochondria following 6 weeks of hypoperfusion (p = 0.03) with no difference between groups observed following 3 months (p = 0.742). Taken together the results from this study provide evidence that chronic cerebral hypoperfusion results in dynamic alterations in the localisation of Nav1.6 channels which are accompanied by disruption to paranodal domains and impaired axon-glial integrity. Furthermore microglial number does not appear to mediate nodal and paranodal disruption following 3 days but may contribute to ongoing pathology following 1 month of chronic cerebral hypoperfusion. Aim 2 – To determine the effects of chronic cerebral hypoperfusion on oligodendroglial populations. The second aim of this thesis was to determine the effect of chronic cerebral hypoperfusion on numbers of mature oligodendrocytes and oligodendrocyte precursor cells (OPCs). This revealed a significant decrease in numbers of both populations following 3 days of cerebral hypoperfusion however following 1 month numbers of OPCs were restored and a significant increase in mature oligodendrocyte number was observed. Assessment of OPC proliferation demonstrated low numbers of proliferating cells but revealed that a proportion of newly generated cells had differentiated into mature oligodendrocytes. To determine a potential mechanism involved in OPC differentiation following cerebral hypoperfusion the expression of the GPR17 receptor was examined which has recently been reported to mediate OPC differentiation in response to injury. The results demonstrated decreased expression of GPR17 following 3 days of hypoperfusion (p = 0.007) with no difference between groups observed following 1 month (p = 0.362) indicating that this receptor is not involved in differentiation of OPCs following hypoperfusion. Taken together the results from this study show that mature oligodendrocytes and OPCs are lost early in response to hypoperfusion but that these cells recover over time, highlighting the regenerative capacity of the white matter following cerebral hypoperfusion.Aim 3 – To investigate whether modulation of inflammation and oxidative stress could ameliorate alterations to white matter structure and function following severe chronic cerebral hypoperfusion The third and final aim of this thesis was to determine whether treatment with the anti-inflammatory and antioxidant drug DMF could ameliorate structural and functional alterations to white matter following severe chronic cerebral hypoperfusion. This was achieved by examining myelin and axonal integrity in addition to numbers of oligodendrocytes and OPCs following 7 days of severe chronic cerebral hypoperfusion. This revealed that myelin integrity was significantly decreased in vehicle-treated hypoperfused animals as compared to shams (p = 0.005). However no differences in myelin integrity were observed between sham and hypoperfused mice treated with DMF (p = 0.312). In contrast to the previous study, numbers of oligodendrocytes and OPCs were not altered following severe hypoperfusion however DMF treatment led to significantly increased numbers of oligodendrocytes in sham animals (p = 0.003). Assessment of white matter function using electrophysiology revealed that the conduction velocity of myelinated axons was significantly increased in DMF-treated hypoperfused animals as compared to those treated with vehicle (p = 0.04). Taken together the results of this study demonstrate that modulation of inflammation and oxidative stress may improve structural and functional white matter alterations following chronic cerebral hypoperfusion. Conclusions: The results presented in this thesis demonstrate that chronic cerebral hypoperfusion results in structural alterations to myelinated axons and to oligodendroglial populations within the white matter which are accompanied by impaired spatial working memory. Whilst previous studies using the model have reported that cerebral hypoperfusion results in diffuse white matter pathology, this study has highlighted the vulnerability of nodal and paranodal domains of myelinated axons as regions which are altered early in response to hypoperfusion. Furthermore, characterisation of oligodendroglial populations has revealed that these cells are replaced over time despite ongoing hypoperfusion which demonstrates the regenerative capacity of the white matter following cerebral hypoperfusion. Critically the results presented in this thesis demonstrate that treatment with DMF improved the function of myelinated axons in response to severe reductions in cerebral blood flow and thus may represent an appropriate therapeutic strategy for chronic cerebral hypoperfusion.

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.

Recent clinical studies suggest that white matter pathology rather than grey matter abnormality is the major neurobiological substrate of age- related cognitive decline during “healthy” aging. According to this hypothesis, cerebrovascular (e.g. chronic cerebral hypoperfusion) and molecular (e.g. APOE, epigenetics) factors might contribute to age-related white matter pathology and cognitive decline. To test this, I used a new mouse model of chronic cerebral hypoperfusion and examined the following predictions: 1) hypoperfusion- induced white matter pathology might be associated with cognitive deficits, 2) APOE deficiency might be associated with white matter anomalies under normal physiological conditions and more severe hypoperfusion- induced white matter pathology, 3) chronic cerebral hypoperfusion might impact on hydroxymethylation (a newly discovered epigenetic marker) in white matter, via perturbations in associated epigenetic pathways, namely methylation and/ or TETs. I. Effects of chronic cerebral hypoperfusion on white matter integrity and cognitive abilities in mice To test the hypothesis suggesting that hypoperfusion- induced white matter pathology is associated with working memory and executive function impairment in mice, behavioural performance and neuropathology were systematically examined in two separate cohorts of sham and hypoperfused C57Bl6J mice. Spatial working memory, memory flexibility, learning capacity, short and long term memory recall were taxed using radial arm maze and water maze paradigms one month after surgery. At the completion of the behavioural testing white and grey matter integrity, inflammation were evaluated using standard immunohistochemistry with antibodies recognizing neuronal axons (APP), myelin sheath (MAG) and microglia (Iba1) as well as H&E histological staining to examine neuronal morphology and ischemic injury. In agreement with previous reports, the behavioral data indicated spatial working memory impairment in the absence of spatial memory flexibility, learning, short- and long- term memory recall deficits in hypoperfused mice However, in contrast to previous reports, a spectrum of white and grey matter abnormalities accompanied by an increased inflammation were observed in hypoperfused mice Although there was a significant association between hypoperfusion- induced inflammation in white matter and performance on a working memory radial arm maze task (p<0.05), the present pathological findings suggest that white matter abnormalities, neuronal ischemia and increased inflammation might be at the basis of hypoperfusioninduced cognitive impairment in mice. Further, chronic cerebral hypoperfusion might have affected alternative, non- examined brain processes (e.g. cerebral metabolism, neurotransmission) which might have contributed to the observed cognitive deficits in hypoperfused mice. II. Effects of APOE on white matter integrity under normal physiological and chronically hypoperfused conditions in mice To test the hypothesis suggesting that mouse APOE deficiency might be associated with white matter anomalies under normal physiological conditions and the development of more severe white matter pathology following chronic cerebral hypoperfusion, white and grey matter integrity, inflammation were examined in APOE deficient mice on a C57Bl6J background (APOEKO) and C57Bl6J wild- type (WT) counterparts one month after chronic cerebral hypoperfusion or sham surgery. A combined neuroimaging (MRI- DTI)/ immunochemical approach was attempted in these mice as an additional step towards translation of this research to human subjects. The ex vivo MRI- DTI findings demonstrated APOE genotype effects on the development of white matter abnormalities following chronic cerebral hypoperfusion in mice. Significant reductions in MRI metrics (FA and MTR) of white matter integrity were observed in examined white matter areas of APOEKO hypoperfused mice compared with WT hypoperfused counterparts (p<0.05). However, the neuroimaigng findings were not supported by the pathological analysis where no significant APOE differences were observed in hypoperfusion- induced axonal (APP), myelin (MAG, dMBP) pathology and inflammation (Iba1) (p>0.05). No significant differences in MRI parameters and pathological grades of white matter integrity were evidenced between APOEKO and WT sham mice (p>0.05). An absence of grey matter abnormalities was evidenced on T2- weighted scans and corresponding H&E stained brain sections in all experimental animals. However, significant reductions in MTR values and dMBP immunoreactivity (myelin pathology) (p<0.05) were observed in grey matter (the hippocampus) following chronic cerebral hypoperfusion in the absence of significant APOE genotype effect (p>0.05) suggesting the existence of both white and grey matter abnormalities in this animal model. Overall, the present neuroimaging data, but not pathological analysis, partially validated the main study hypothesis suggesting that APOE deficiency might be associated with the development of more severe white matter abnormalities in hypoperfused mice. III. Characterization of methylation and hydroxymethylation in white matter under normal physiological and chronically hypoperfused conditions in mice Lastly, I sought to test the hypothesis that chronic cerebral hypoperfusion might alter oxygen dependent DNA hydroxymethylation (5hmC) in white matter regions via perturbations in methylation (5mC) and/ or Ten- eleven translocation proteins (e.g. TET2) in mice. DNA methylation (5mC), hydroxymethylation (5hmC) and TET2 were immunochemically studied in white and grey matter of sham and chronically hypoperfused C57Bl6J mice a month after surgery. The immunochemical results demonstrated significant increases (p<0.05) in 5hmC in the hypoperfused corpus callosum (CC) in the absence of significant hypoperfusion- induced alterations in the distribution of 5mC and TET2 (p>0.05) in white matter. Significant hypoperfusion- induced increases were evident for TET2 in the cerebral cortex (Cx) (p<0.05). These data partially validated the main study hypothesis suggesting hypoperfusion- induced alterations in 5hmC in white matter. However, in contrast to the study hypothesis, the observed hypoperfusion- induced alterations in 5hmC occurred in the absence of changes in 5mC and TET2 in white matter. A subsequent correlation analysis between hydroxymethylation and 5mC, TET2 in the CC failed to show significant associations (p>0.05). In search of the cellular determinants of 5hmC in the CC, hydroxymethylation was examined in relation to some of the cell types in white matter- mature oligodendrocytes, oligodendrolial progenitors (OPC) and microglia both in vivo and in vitro. Specifically, a separate parametric correlation analysis between the proportion of 5hmC positive cells and the respective proportions of mature oligodendrocytes, OPC and microglia in the CC demonstrated that hydroxymethylation correlated significantly only with microglia in vivo (p<0.05). Following this, 5hmC immunochemical distribution was studied in vitro in oligodendroglia cells at different stages of maturation, and interferon γ/ lypopolisaccharide activated and nonactivated microglia. The in vitro analysis demonstrated that 5hmC is high in OPC, activated and nonactivated microglia, but it is low in mature oligodendrocytes. Taken together the in vivo and in vitro cellular analyses suggest that the processes of hydroxymethylation in white matter might be immunoregulated. However, it is possible that in vivo in addition to microglia, other cell types (e.g. astrocytes, OPC) contributed to the presently observed 5hmC upregulation in the hypoperfused CC. Conclusion The experimental work presented in this thesis further developed and characterized a new mouse model of chronic cerebral hypoperfusion by confirming previous behavioural findings (e.g. working memory deficits) and revealing previously undetected spectrum of white and grey matter pathology in this animal model. The thesis demonstrated for the first time by using a newly developed ex vivo MRI procedure that APOE might modulate hypoperfusion- induced white matter pathology in mice. Additional immunochemical analysis revealed important hypoperfusion- induced epigenetic alterations in white (5hmC) and grey (TET2) matter in this animal model. Future experiments on chronically hypoperfused mice would allow to get a better insight into the neurobiological determinants (e.g. white vs. grey matter) underlying the observed cognitive deficits in this animal model, the involved cellular and molecular pathways as well as the functional significance of genetic (APOE) and epigenetic (5hmC, TETs) alterations in the hypoperfused brain. Future experimental work on this animal model would potentially reveal new biological targets for the pre- clinical development of therapies for age- related cognitive decline. Further development and optimization of the newly developed ex vivo MRI procedure would allow its broader application in preclinical settings and would facilitate the translation of experimental findings to clinics.

Intact white matter is critical for normal cognitive function. In traumatic brain injury (TBI), chronic cerebral hypoperfusion and Alzheimer’s disease (AD) damage to white matter is associated with cognitive impairment. However, these conditions are associated with grey matter damage or with other pathological states and the contribution of white matter damage in isolation to their pathogenesis is not known. Furthermore, TBI is a risk factor for AD and cerebral hypoperfusion is an early feature of AD. It is hypothesised that white matter damage following TBI or chronic cerebral hypoperfusion will be associated with cognitive deficits and that white matter changes after injury contribute to AD pathogenesis. To investigate this, this thesis examined the contribution of white matter damage to cognitive deficits after TBI and chronic cerebral hypoperfusion and furthermore, investigated the role of white matter damage in the relationship between TBI and AD. Three studies addressed these aims. In the first, mild TBI was induced in wild-type mice and the effects on axons, myelin and neuronal cell bodies examined at time points from 4 hours to 6 weeks after injury. Spatial reference learning and memory was tested at 3 and 6 weeks after injury. Injured mice showed axonal damage in the cingulum, close to the injury site in the hours after injury and at 6 weeks, damage in the thalamus and external capsule were apparent. Injured and sham animals had comparable levels of neuronal damage and no change was observed in myelin. Injured animals showed impaired spatial reference learning at 3 weeks after injury, demonstrating that selective axonal damage is sufficient to impair cognition. In the second study mild TBI was induced in a transgenic mouse model of AD and the effects on white matter pathology and AD-related proteins examined 24 hours after injury. There was a significant increase in axonal damage in the cingulum and external capsule and parallel accumulations of amyloid were observed in these regions. There were no changes in tau or in overall levels of AD-related proteins. This suggests that axonal damage may have a role in mediating the link between TBI and AD. The third study used a model of chronic cerebral hypoperfusion in wild type mice and investigated white matter changes after one and two months of hypoperfusion as well as a comprehensive assessment of learning and memory. Chronic cerebral hypoperfusion resulted in diffuse myelin damage in the absence of ischaemic neuronal damage at both 1 and 2 months after induction of hypoperfusion. Hypoperfused animals also showed minimal axonal damage and microglial activation. Cognitive testing revealed a selective impairment in spatial working memory but not spatial reference or episodic memory in hypoperfused animals, showing that modest reductions in blood flow have effects on white matter sufficient to cause cognitive impairment. These results demonstrate that selective damage to white matter components can have a long-term impact on cognitive function as well as on the development of AD. This suggests that minimisation of axonal damage after TBI is a target for reducing subsequent risk of AD and that repair or prevention of white matter damage is a promising strategy for rescuing cognitive function in individuals who have experienced mild TBI or chronic cerebral hypoperfusion.

Vascular cognitive impairment describes a heterogeneous condition in which cognitive decline is precipitated by underlying cerebrovascular dysfunction. Ageing, as well as vascular diseases such as hypertension, stroke, cerebral small vessel disease and cerebral amyloid angiopathy, are risk factors for vascular cognitive impairment. The precise mechanisms by which these conditions impact the cerebral vasculature to drive cognitive decline, however, are unknown. Previous research has indicated that vascular risk factors can lead to microvascular oxidative stress, inflammation and endothelial dysfunction that can lead to tissue hypoperfusion, the development of white and grey matter vascular lesions (microinfarcts and microbleeds) and cognitive impairment. It was hypothesised that ageing, a prominent risk factor for cognitive decline, would induce impairments on neurovascular coupling resulting from neurovascular unit disruption. It was further hypothesised that induction of chronic cerebral hypoperfusion would mediate neurovascular dysfunction and vascular lesion development through increased oxidative stress, resulting in cognitive decline. Finally, it was also hypothesised that neurovascular impairments resulting from ageing and chronic cerebral hypoperfusion would be exacerbated in the presence of amyloid deposition. Four studies were performed in order to test these hypotheses. Vascular risk factors can be reproduced using experimental mouse models and provide a valuable basis in which to test hypotheses and therapeutic interventions. As such, a primary aim of this thesis was to develop and validate sensitive MRI approaches that would allow the detection of vascular alterations in vivo. In the first series of studies, MRI techniques to assess resting cerebral blood flow, vessel number, vascular lesions and inflammation in experimental mice were validated using established in vivo and ex vivo techniques, so that these techniques could be used in subsequent studies for vascular assessments in vivo. Arterial spin labelling was developed to assess resting cerebral blood flow, and was able to detect reductions in blood flow following cerebral hypoperfusion that correlated well with those obtained from laser speckle imaging. Q-map imaging was able to detect reductions in vessel number in acute lesions, and in non-lesioned mice measures of vessel number correlated well with histopathological measures. Structural T2 imaging was performed in order to detect ischaemic and haemorrhagic lesions in chronically hypoperfused mice, and was validated using H&E and Perls’ staining. Finally, contrast-enhanced T2* imaging was used to detect iron oxide uptake by macrophages in the brains of hypoperfused mice, which was further validated by the identification of iron-containing macrophages in immunostained brain sections. The second study was conducted to test the hypothesis that ageing would impair neurovascular unit function and structure, and that these impairments would be exacerbated in the presence of amyloid pathology. The aim of the study was to incorporate previously developed in vivo imaging approaches in the assessment of vascular function and alterations in neurovascular unit structure in both wild type and TgSwDI mice. As predicted, ageing caused a pronounced deficit on measures of neurovascular coupling, however this was not exacerbated by accumulation of amyloid in TgSwDI mice and was not associated with alterations in baseline blood flow measured by arterial spin labelling. Structural assessment of the neurovascular unit revealed a loss of contact between astrocytic endfeet and vasculature, which was significantly associated with the impairment on neurovascular coupling, in addition to other markers of breakdown of the neurovascular unit such as loss of pericyte coverage and microglial activation. Age and thalamic vascular amyloid accumulation were also associated with an increase in the NADPH oxidase (NOX) subunit p47, indicative of increased oxidative stress. Data from this experiment indicate that ageing can profoundly impair neurovascular coupling, mediated by gliosis and loss of astrocytic contacts with vasculature. The third study aimed to test the hypothesis that chronic cerebral hypoperfusion (a prominent early feature of vascular cognitive impairment) would impair vascular function and induce the development of vascular lesions and cognitive decline. The impact of hypoperfusion on neurovascular coupling, ischaemic and haemorrhagic lesion burden and cognition was investigated in wild type and TgSwDI mice. Hypoperfusion induced deficits on neurovascular coupling, increased lesion burden and inflammation assessed with T2 and contrast-enhanced T2* imaging, and caused impairment on measures of learning and memory. Hypoperfusion was also associated with an increase in the levels of NOX2, NOX4 and 3-NT at 3 months following surgery, indicating persistent reactive oxygen species production and oxidative damage in hypoperfused mice. The findings from this study indicate that vascular dysfunction and cognitive impairment following hypoperfusion may be mediated by increased NADPH oxidase activity and resulting oxidative stress. The previous studies indicated that markers of oxidative stress were induced in response to ageing, vascular amyloid accumulation and cerebral hypoperfusion. The final study sought to determine whether increased NOX activity mediates downstream pathological effects on vascular function, vascular lesion development and cognitive decline following hypoperfusion. NOX activity was inhibited pharmacologically by administration of apocynin to hypoperfused TgSwDI mice for 3 months following surgery. Treatment with apocynin significantly restored neurovascular coupling to a level similar to sham-operated mice, and there was a trend toward reduction of ischaemic vascular lesions. However, it was unable to rescue the prominent inflammatory response or decline in cognitive ability, as apocynin-treated mice were no different on these measures to non-treated hypoperfused mice. The data indicate that whilst inhibiting NOX may have potential therapeutic value in improving vascular function, additional interventions, for example to reduce inflammation, may also be required in order to prevent cognitive decline. Overall, the work outlined within the thesis indicate that vascular risk factors of ageing, cerebral amyloid angiopathy and cerebral hypoperfusion may converge on common pathways involving oxidative stress and increased inflammation in order to drive vascular dysfunction and lead to cognitive decline. Inhibition of NOX activity was able to rescue vascular function, however the results indicate that this was not sufficient to protect against cognitive impairment, suggesting additional therapeutic targets may need to be sought in order to fully preserve vascular health and prevent cognitive decline.

A hipoperfusão cerebral crônica (HCC) é um importante fator de risco para o declínio cognitivo e outras disfunções cerebrais, tais como Doença de Alzheimer e Demência Vascular. O objetivo deste estudo foi investigar o efeito neuroprotetor do Resveratrol (RSV), avaliando parâmetros comportamentais, bioquímicos e morfológicos, em um modelo experimental de Demência Vascular. Ratos Wistar adultos foram submetidos ao modelo modificado da HCC através da oclusão permanente de 2 vasos (2VO) e tratamento diário, iniciado uma hora após oclusão permanente dos vasos, com injeções intraperitoneais (20 mg/kg) de RSV durante 7 dias. Os testes comportamentais foram realizados entre o 35° e 45° dias após a cirurgia - 2VO, através da tarefa do labirinto aquático de Morris, na qual os animais foram avaliados quanto ao desempenho da memória espacial. Ao final dos testes comportamentais, um grupo dos animais foi perfundido transcardiacamente para análise histológica, outro grupo foi submetido à eutanásia em 3 tempos (3, 14 e 45 dias após a lesão isquêmica) para avaliação da expressão de NGF (fator de crescimento do nervo). Os resultados demonstraram que o tratamento com RSV atenuou significativamente a morte das células piramidais na região CA1 do hipocampo e preveniu o déficit da memória espacial. O aumento da expressão de NGF foi evidenciado no 3° dia após indução da HCC em todos os animais isquêmicos e no 45° dia após indução da HCC nos animais tratados com RSV. Com base nesses dados, hipotetizamos que o aumento, em longo prazo, na expressão de NGF no hipocampo após a HCC pode caracterizar uma das vias envolvidas nos mecanismos neuroprotetores do RSV. / Chronic cerebral hypoperfusion (CCH) is an important risk factor for cognitive decline and other brain dysfunctions, such as Alzheimer’s Disease and Vascular Dementia. The aim of the present study was to investigate the neuroprotective effect of Resveratrol (RSV) on behavioral, biochemical and morphological parameters in an experimental model of Vascular Dementia. Adult Wistar rats were submitted to the CCH modified model by means of permanent 2-vessel occlusion (2VO) and daily treatment, initiated one hour after permanent vessel occlusion, with intraperitoneal injections (20 mg/kg) of RSV for 7 days. Behavioral testing was performed between the 35th and the 45th day after 2VO surgery in the Morris Water Maze task, allowing for the evaluation of spatial memory function. At the end of the behavioral assessment, half of the animals were transcardially perfused for histological analysis and the remaining were euthanized in 3 times (3. 14 and 45 days after ischemic injury) for NGF expression evaluation (neural growth factor). Results demonstrate that the treatment with RSV significantly attenuated pyramidal cell death in CA1 hippocampal field and prevented spatial memory impairment. The increase of NGF expression was evidenced on the 3rd day after CCH induction in all ischemic animals and on the 45th day after CCH induction in animals treated with RSV. On the basis of these data, we hypothesize that the long term increase in NGF expression in the hippocampus after CCH may characterize one pathway involved in the neuroprotective mechanisms of RSV.

A hipoperfusão cerebral crônica (HCC) é um importante fator de risco para o declínio cognitivo e outras disfunções cerebrais, tais como Doença de Alzheimer e Demência Vascular. O objetivo deste estudo foi investigar o efeito neuroprotetor do Resveratrol (RSV), avaliando parâmetros comportamentais, bioquímicos e morfológicos, em um modelo experimental de Demência Vascular. Ratos Wistar adultos foram submetidos ao modelo modificado da HCC através da oclusão permanente de 2 vasos (2VO) e tratamento diário, iniciado uma hora após oclusão permanente dos vasos, com injeções intraperitoneais (20 mg/kg) de RSV durante 7 dias. Os testes comportamentais foram realizados entre o 35° e 45° dias após a cirurgia - 2VO, através da tarefa do labirinto aquático de Morris, na qual os animais foram avaliados quanto ao desempenho da memória espacial. Ao final dos testes comportamentais, um grupo dos animais foi perfundido transcardiacamente para análise histológica, outro grupo foi submetido à eutanásia em 3 tempos (3, 14 e 45 dias após a lesão isquêmica) para avaliação da expressão de NGF (fator de crescimento do nervo). Os resultados demonstraram que o tratamento com RSV atenuou significativamente a morte das células piramidais na região CA1 do hipocampo e preveniu o déficit da memória espacial. O aumento da expressão de NGF foi evidenciado no 3° dia após indução da HCC em todos os animais isquêmicos e no 45° dia após indução da HCC nos animais tratados com RSV. Com base nesses dados, hipotetizamos que o aumento, em longo prazo, na expressão de NGF no hipocampo após a HCC pode caracterizar uma das vias envolvidas nos mecanismos neuroprotetores do RSV. / Chronic cerebral hypoperfusion (CCH) is an important risk factor for cognitive decline and other brain dysfunctions, such as Alzheimer’s Disease and Vascular Dementia. The aim of the present study was to investigate the neuroprotective effect of Resveratrol (RSV) on behavioral, biochemical and morphological parameters in an experimental model of Vascular Dementia. Adult Wistar rats were submitted to the CCH modified model by means of permanent 2-vessel occlusion (2VO) and daily treatment, initiated one hour after permanent vessel occlusion, with intraperitoneal injections (20 mg/kg) of RSV for 7 days. Behavioral testing was performed between the 35th and the 45th day after 2VO surgery in the Morris Water Maze task, allowing for the evaluation of spatial memory function. At the end of the behavioral assessment, half of the animals were transcardially perfused for histological analysis and the remaining were euthanized in 3 times (3. 14 and 45 days after ischemic injury) for NGF expression evaluation (neural growth factor). Results demonstrate that the treatment with RSV significantly attenuated pyramidal cell death in CA1 hippocampal field and prevented spatial memory impairment. The increase of NGF expression was evidenced on the 3rd day after CCH induction in all ischemic animals and on the 45th day after CCH induction in animals treated with RSV. On the basis of these data, we hypothesize that the long term increase in NGF expression in the hippocampus after CCH may characterize one pathway involved in the neuroprotective mechanisms of RSV.

A hipoperfusão cerebral crônica (HCC) é um importante fator de risco para o declínio cognitivo e outras disfunções cerebrais, tais como Doença de Alzheimer e Demência Vascular. O objetivo deste estudo foi investigar o efeito neuroprotetor do Resveratrol (RSV), avaliando parâmetros comportamentais, bioquímicos e morfológicos, em um modelo experimental de Demência Vascular. Ratos Wistar adultos foram submetidos ao modelo modificado da HCC através da oclusão permanente de 2 vasos (2VO) e tratamento diário, iniciado uma hora após oclusão permanente dos vasos, com injeções intraperitoneais (20 mg/kg) de RSV durante 7 dias. Os testes comportamentais foram realizados entre o 35° e 45° dias após a cirurgia - 2VO, através da tarefa do labirinto aquático de Morris, na qual os animais foram avaliados quanto ao desempenho da memória espacial. Ao final dos testes comportamentais, um grupo dos animais foi perfundido transcardiacamente para análise histológica, outro grupo foi submetido à eutanásia em 3 tempos (3, 14 e 45 dias após a lesão isquêmica) para avaliação da expressão de NGF (fator de crescimento do nervo). Os resultados demonstraram que o tratamento com RSV atenuou significativamente a morte das células piramidais na região CA1 do hipocampo e preveniu o déficit da memória espacial. O aumento da expressão de NGF foi evidenciado no 3° dia após indução da HCC em todos os animais isquêmicos e no 45° dia após indução da HCC nos animais tratados com RSV. Com base nesses dados, hipotetizamos que o aumento, em longo prazo, na expressão de NGF no hipocampo após a HCC pode caracterizar uma das vias envolvidas nos mecanismos neuroprotetores do RSV. / Chronic cerebral hypoperfusion (CCH) is an important risk factor for cognitive decline and other brain dysfunctions, such as Alzheimer’s Disease and Vascular Dementia. The aim of the present study was to investigate the neuroprotective effect of Resveratrol (RSV) on behavioral, biochemical and morphological parameters in an experimental model of Vascular Dementia. Adult Wistar rats were submitted to the CCH modified model by means of permanent 2-vessel occlusion (2VO) and daily treatment, initiated one hour after permanent vessel occlusion, with intraperitoneal injections (20 mg/kg) of RSV for 7 days. Behavioral testing was performed between the 35th and the 45th day after 2VO surgery in the Morris Water Maze task, allowing for the evaluation of spatial memory function. At the end of the behavioral assessment, half of the animals were transcardially perfused for histological analysis and the remaining were euthanized in 3 times (3. 14 and 45 days after ischemic injury) for NGF expression evaluation (neural growth factor). Results demonstrate that the treatment with RSV significantly attenuated pyramidal cell death in CA1 hippocampal field and prevented spatial memory impairment. The increase of NGF expression was evidenced on the 3rd day after CCH induction in all ischemic animals and on the 45th day after CCH induction in animals treated with RSV. On the basis of these data, we hypothesize that the long term increase in NGF expression in the hippocampus after CCH may characterize one pathway involved in the neuroprotective mechanisms of RSV.

Cerebral small vessel pathology is now known to be associated with the development of cognitive impairment and mild motor impairments such as gait disturbance in a variety of neurodegenerative diseases. This dissertation explores the hypothesis that blood brain barrier dysfunction is an early event in cerebral ischemia and contributes to the development of cerebral small vessel disease (CSVD). A common rodent model of CSVD is permanent bilateral common carotid artery occlusion in the rat. This model was used to study several aspects of the progression of CSVD including the timecourse of blood brain barrier permeability changes following the onset of ischemia, gait disturbance, the expression of tight junction proteins and cytokine expression. It was determined that BBB permeability was elevated for 2 weeks following BCCAO and ischemic rats displayed lower gait velocity. There was no change in expression of TJ proteins. However, ischemic rats had higher levels of some proinflammatory cytokines and chemokines in brain tissue with no obvious changes in plasma levels. The mechanisms underlying the increase in BBB permeability were studied in vitro using artificial barriers made of confluent rat brain microvascular endothelial cells. Cerebral ischemia has been reported to cause an increase in plasma toxicity, likely by elevating the numbers of circulating microparticles (MPs). MPs isolated from the plasma of ischemic rats were applied to artificial barriers where it was found that they act mainly as vectors of TNF-α signaling. MPs induce activation of caspase-3 and the Rho/Rho kinase pathways. It is concluded that most of the increase in barrier permeability is due to apoptosis and disassembly of actin cytoskeleton and disruption of adherens junctions IV and not an increase in transcellular transport. The effects of treatment with the type III phosphodiesterase inhibitor cilostazol on dye extravasation in the brain, glial activation, white matter damage and motor performance were evaluated. It was determined that cilostazol could improve the increased BBB permeability and gait disturbance and microglial activation in optic tract following BCCAO. Also, the effects of treatment with cilostazol on plasma toxicity in vivo (24h and 14d following BCCAO) and artificial barriers (in vitro) were assessed. It was found that cilostazol could reduce plasma toxicity at 24h and improve increased endothelial barrier permeability that is induced by MP treatment respectively. In summary BBB dysfunction occurs in the rat model of chronic cerebral hypoperfusion with no differences in expression of TJ proteins. There is a mild motor disturbance in the form of lower gait velocity following BCCAO. Cytokines released in brain tissue may be associated with pathological consequences following BCCAO while there is no significant difference in plasma levels and circulating MPs may play a role in BBB dysfunction.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22336号 / 医博第4577号 / 新制||医||1041(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 高橋 淳, 教授 渡邉 大, 教授 伊佐 正 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM