Cognitive impairment in amyotrophic lateral sclerosis (ALS) is characterized by deficits on tests of executive functions however the contribution of processing speed is unknown. By contrast, multiple sclerosis (MS) is a disorder in which slowed processing speed is regarded as the core deficit, however, methodology is often confounded by tasks which depend on motor speed. MRI studies have revealed multi-system cerebral involvement in ALS, with evidence of reduced white matter volume and integrity in predominantly frontotemporal regions. The current study had two aims. Firstly, to investigate whether cognitive impairments in ALS and MS are due to executive dysfunction or slowed processing speed, independent of motor dysfunction. Secondly, to investigate the relationship between specific cognitive impairments and the integrity of distinct white matter tracts in ALS. Twenty-nine ALS patients, twenty-five MS patients, and matched healthy control groups were administered a dual task paradigm and processing speed tasks in which stimulus presentation times were manipulated. In addition background measures of executive functioning, working memory, verbal memory, and language were administered. White matter integrity was investigated using region-of-interest (ROI) and tract based spatial statistics (TBSS) analyses of diffusion MRI data. ALS patients did not show impairments in tests of processing speed, but deficits were revealed in the dual task, as well as background tests of executive functioning, working memory, and verbal memory. MS patients also exhibited deficits in the dual task as well as background tests of executive functioning, working memory, and verbal memory. However, in contrast to ALS patients, a processing speed deficit was also observed in MS. ROI analyses revealed significant differences in fractional anisotropy (FA) and mean diffusivity (<D>) between ALS patients and healthy controls. Reduced integrity was observed in the corticospinal tracts and prefrontal and temporal white matter tracts including uncinate fasciculus, inferior longitudinal fasciculus, and regions of the cingulum. Significant differences also emerged in the white matter underlying the superior, medial and inferior frontal gyri, and the temporal gyri. Similar group differences were found in the TBSS analyses; ALS patients displayed prominent changes in the corticospinal tract and corpus callosum as well as extensive changes in prefrontal and temporal tracts and association fibres. Correlations between task performance and ROI parameters revealed that dual task performance was associated with FA in the middle frontal gyrus white matter while letter fluency indices correlated with FA in the corpus callosum and corticospinal tracts. Furthermore, verbal memory performance correlated with FA in the inferior longitudinal fasciculus and working memory performance correlated with <D> in uncinate fasciculus and hippocampal portion of the cingulum. Correlations with TBSS revealed significant associations between letter fluency indices and FA in the corticospinal tracts and anterior corpus callosum. The current study demonstrates that cognitive impairment in ALS is not due to slowed processing speed. Moreover dual task deficits are related to distinct prefrontal tract involvement in ALS, whilst fluency deficits may reflect decreasing callosal integrity. Deficits in working memory and verbal memory are related to white matter changes in fibre bundles connecting prefrontal, temporal, and limbic structures.

Adult survivors of childhood posterior fossa tumors can experience reading difficulties related to white matter integrity. Previously, reading was shown to be related to cortical white matter tracts, however information transfer across the corpus callosum (CC) may also play a role in reading. The current study used both macro- and microstructural measures of the WM structure of the corpus callosum. The current study examined how white matter volume and fractional anisotropy (FA) in five divisions of the CC was related to degree of neurological risk and reading skill, and tested two mediation models predicting reading. Participants included 20 adult survivors of childhood posterior fossa tumor and 23 healthy controls. Volume and FA were measured in five divisions of the mid-sagittal corpus callosum. Total intracranial vault was used as a covariate in volume analyses. FA was reduced in CC1 and volume was reduced in each subregion in survivors. Volume but not FA was related to degree of neurological risk. Results identified that reduced volume in CC1 and CC5, and FA in CC5 appear to be specifically related to reading skill in line with the cortical reading regions that connect in these subregions of the CC. Mediation models indicate that processing speed is the mechanism by which volume is related to reading skill. These findings have implications for addressing processing speed in reading interventions in survivors and provide insight into the interhemispheric connections in the reading network.

Magnetic resonance imaging (MRI) methods thought to assess myelin and axon integrity are improving the understanding of white matter diseases like multiple sclerosis (MS). This thesis improved the understanding of how microstructural tissue changes caused by various pathologies influence MRI metrics by developing and applying MRI methods in a longitudinal study using the cuprizone mouse model of MS. In vivo and ex vivo MRI measurments (T1 and T2 relaxometry, diffusion tensor imaging, and quantitative magnetization transfer imaging) were correlated with tissue measurements taken from electron microscopy images of control and cuprizone fed mice at weeks 2 and 3 of cuprizone feeding. Significant Spearman correlations included mean diffusivity vs. myelinated axon fraction (ρ=0.84), ex vivo T2 vs. myelinated axon fraction (ρ=0.68), and normalized T2-weighted signal vs. myelinated axon fraction (ρ =-0.80). Multiparametric MRI studies show promise in bridging the gap between damage detected in images and clinical status associated with MS. / May 2016

Aging is associated with a loss of cognitive function related to learning, memory, and executive function with varying severity. Although there is no age-related loss of neurons in healthy aging, myelin damage accumulates and is associated with cognitive decline. The brain’s resident macrophages, microglia, are responsible for clearing damaged myelin and promoting subsequent oligodendrocyte-mediated remyelination. To test the hypothesis that age-related dysfunction of microglial phagocytosis and oligodendrocyte remyelination capacity contributes to myelin pathology and cognitive impairment. To test this, rhesus monkeys from across the lifespan (7-30 years of age) were tested in three specific aims. 1) To characterize gene expression of myelin basic protein (MBP) in the brain and clearance of MBP to the cerebrospinal fluid (CSF) in relation to age-related myelin pathology. The density of myelinated axons visualized using label-free spectral confocal reflectance imaging did not correlate with age, but was significantly lower in aged animals with cognitive impairment. Next, MBP gene expression was measured using qPCR in the dorsal prefrontal cortex along with quantification of MBP protein levels in the CSF using ELISA. Age-dependent increases of MBP gene expression in the brain and MBP protein levels in the CSF were observed. Interestingly, MBP levels in the CSF were lower in animals with cognitive impairment. 2) To test the hypothesis that microglia would become increasingly primed for phagocytosis with age-related myelin pathology. The number of microglia immunostained with galectin-3, a marker for phagocytic activation, was quantified in the frontal white matter and increases in both aging and cognitive decline were detected. 3) To evaluate the hypothesis that lipofuscin, an age-related accumulation indicative of autophagic dysfunction, would accumulate and impair glial cells of the white matter in aged animals. Lipofuscin accumulation was increased with age in the frontal white matter and the size of lipofuscin clusters was associated with cognitive impairment. Lipofuscin was found primarily in microglia and oligodendrocytes, but not in astrocytes. These data suggest that lipofuscin burden in microglia and oligodendrocytes inhibits their homeostatic functions resulting in improper myelin clearance and turnover, leading to a devastating feed-forward cycle of myelin damage that contributes to age-related cognitive impairment.

Introduction: Lacunar strokes are both common and disabling; they cause up to 52 strokes per 100,000 people per year and 29-46% of survivors are disabled. Lacunar stroke is part of the spectrum of small vessel disease (SVD) which also causes cognitive impairment and gait disturbance; together these lead to dementia, falls and disability. Current evidence suggests that SVD is caused by a separate aetiology from large vessel stroke, which may be mediated by blood brain barrier (BBB) permeability and may affect organs other than the brain. We set out to establish whether SVD is a multi-system disorder of primary endothelial function, with leakage of blood-brain barrier leading to lacunar stroke, disability, and cognitive impairment. Methods: We recruited 264 patients with a lacunar or cortical stroke (118 lacunar, 146 cortical). All patients received baseline assessment of clinical features, magnetic resonance imaging (MRI), renal function, and assessment of dietary salt. At 1-3 months post-stroke we carried out cognitive testing and contrast MRI to assess blood-brain barrier integrity. We followed patients up at 12 months post-stroke with repeat cognitive testing, MRI, and assessment of disability and recurrent stroke. Results: We established that lacunar stroke has a different risk factor profile to cortical stroke, confirming findings from previous cohorts, but adding dietary salt as a risk factor for lacunar stroke and other SVD features. We confirmed that patients with a clinical stroke who did not have a lesion on diffusion-weighted MRI had the same clinical outcomes at 1 year post-stroke as those patients who did have a lesion. We established that patients who have a lacunar stroke are at as high a risk of post-stroke cognitive impairment as those with a cortical stroke. We found that blood brain barrier leakage predicted cognitive impairment at one year after lacunar and cortical stroke. We established the rates of disability and cognitive impairment at one-year post-lacunar stroke to estimate the required sample size for future trials. Conclusions: Taken together these findings confirm that lacunar stroke is part of a syndrome separate to large vessel stroke and may be mediated through blood brain barrier leakage. Dietary salt is an additional risk factor. The findings support further randomised controlled trials of treatments aimed specifically at lacunar stroke and lifestyle interventions including dietary salt reduction.

The aim of this study was to further investigate the role of pro-inflammatory cytokines in the pathogenesis of fetal cererbral white matter injury associated with chorioamnionitis by charaterizing the time course of the cytokine response in the pregnant guinea pig following a maternal inflammatory insult. Chorioamnionitis increases the risk for fetal brain injury. In the guinea pig, a threshold maternal inflammatory response must be reached for significant fetal brain injury to occur. However, a previous study demonstrated that, by seven days after an acute maternal inflammatory insult, cytokine levels in both maternal and fetal compartments are not different from controls. The purpose of this study, therefore, was to test the hypothesis that a significant cytokine response occurs within the first seven days following an acute maternal inflammatory response. Pregnant guinea pigs (n=34) were injected intraperitoneally with 100µg/kg lipopolysaccharide (LPS) at 70% gestation and euthanized at 24 hours, 48 hours or 5 days following endotoxin exposure. Control animals were euthanized at 70% gestation without exposure. Concentrations of interleukin-6, interleukin 1-β and tumour necrosis factor-α (IL-6, IL-1β, TNF-α) were quantified in the maternal serum and amniotic fluid by enzyme-linked immunosorbent assay. IL-6 and IL-1β concentrations were elevated in the maternal serum at 24 hours and returned to control levels by five days. In the amniotic fluid, IL-6 peaked at 48 hours and IL-1β at 24 hours. TNF-α levels were not significantly increased. A single maternal LPS injection produces transient increases in cytokine concentrations in the maternal serum and amniotic fluid. This further implicates the cytokines as potential mediators of fetal white matter damage. Although this response might not be sufficient to produce the brain injury itself, it may initiate harmful pro-inflammatory cytokine cascades, which could even continue to harm the fetus following delivery. A human diagnostic protocol was developed to assess the use of serial serum biomarkers, including IL-6 and TNF-α, in the prediction of histological chorioamnionitis. Preliminary analysis of the pilot study suggests that certain biomarkers might be worthy of further investigation in a larger-scale study. / Thesis (Master, Anatomy & Cell Biology) -- Queen's University, 2007-11-28 08:23:04.327

The integrity of brain white matter is vital for the interneuronal signalling between distinct brain regions required for normal cognitive function. White matter integrity is compromised with ageing and could contribute to age-related cognitive decline. Chronic cerebral hypoperfusion is thought to underlie the development of white matter pathology and cognitive changes, often seen in the elderly. Additionally, the development of regional hypoperfusion and white matter damage are thought to be early events in Alzheimer’s disease (AD) pathogenesis. This thesis set out to test the hypothesis that chronic cerebral hypoperfusion underlies the development of white matter pathology and cognitive decline and also that chronic cerebral hypoperfusion causes the development of Ab pathology in AD. The first aim was to investigate the impact of hypoperfusion on the development of white matter damage and different aspects of cognition in a mouse model of chronic cerebral hypoperfusion. Two studies were undertaken to address this. The first study examined the temporal development of pathology following hypoperfusion induced by bilateral carotid artery stenosis (BCAS) using microcoils Hypoperfusion was induced in wild type (WT) mice and the pathological changes examined at one week, two weeks, one month and two months. Hypoperfused animals developed a diffuse and widespread white matter pathology, present from one week, which occurred predominantly in the myelin component of white matter; this was accompanied by minimal axonal damage. A second study examined the impact of hypoperfusion on different aspects of spatial memory and further investigated pathological changes in the model at one and two months. Behavioural testing revealed a significant impairment in spatial working memory but not episodic memory or spatial reference memory in hypoperfused animals. In the same mice, pathological assessment indicated that there was a significant increase in levels of myelin damage and elevated levels of microglial activation as compared to shams. These results demonstrate that modest reductions in cerebral blood flow are sufficient to cause the development of white matter damage and the development of cognitive deficits. The second aim was to investigate the impact of hypoperfusion on the development of white matter and amyloid pathology in a mouse model (3xTg-AD) of AD. To address this, using 2 different sizes of microcoils (0.18mm and 0.16mm internal diameter) BCAS of varying severities was induced in 3xTg-AD mice and white matter and Ab pathology were assessed at one month. Circle of Willis (CoW) architecture was also compared between WT and 3xTg-AD mice. Overall white matter pathology was not exacerbated in experimental 3xTg-AD mice with BCAS induced by 0.18mm coils. However with a greater level of stenosis (0.16mm coil) ischaemic damage to neuronal perikarya was present in most experimental animals. In addition to ischaemic damage, localised areas of severe white matter pathology were also observed in conjunction with subtle changes to white matter Ab levels. Hypoperfusion did not impact on the development of intraneuronal Ab pathology, other than in the presence of ischaemic damage when levels were reduced. Comparison of CoW architecture between WT and 3xTg-AD mice revealed strain specific differences in the presence and morphology of the posterior communicating artery which may explain the lack of pathology in 3xTg-AD mice as compared to WT following BCAS induced using 0.18mm dia. microcoils. The third aim was to investigate whether white matter protein composition changed with age and also whether ageing conferred increased vulnerability to hypoperfusion. To address this, white matter protein levels were compared between young (3-4 months) and old (12-13 months) 3xTg-AD mice. White matter pathology was compared between sham and hypoperfused animals in the aged cohort. Levels of myelin basic protein and 2', 3'-cyclic nucleotide 3'- phosphodiesterase were found to be significantly increased whilst levels of myelin associated glycoprotein were significantly reduced with ageing. These results suggest that changes in myelin protein composition may contribute to the development of age related white matter pathology. White matter pathology was not exacerbated in aged hypoperfused animals following one month of hypoperfusion as compared to shams. The results presented within the thesis demonstrate that chronic cerebral hypoperfusion precipitates the development of selective white matter damage and impacts on cognition. Also it has been shown that where hypoperfusion is severe enough to cause ischaemic damage to neuronal perikarya and localised areas of severe white matter pathology, alterations in white matter Ab levels can occur. Hypoperfusion does not impact on APP processing or on intraneuronal levels of APP or Ab, other than in the presence of ischaemic damage to neuronal perikarya, when levels are reduced. These findings highlight the importance of early intervention strategies in the treatment of vascular risk factors which can lead to hypoperfusion and the development of white matter damage and a decline in cognitive function in later life. These findings also suggest that repair or prevention of white matter damage may be an appropriate strategy for the attenuation of cognitive decline following onset of hypoperfusion. This thesis also highlights some of the limitations of animal models of human disease.

Intracerebral hemorrhage (ICH) results in secondary brain injury caused partially by blood and its metabolites. Survivors of ICH are often left with severe disabilities, therefore, decreasing the extent of this secondary injury may improve functional outcome of patients. Incubation of mouse brain slices with partially oxidized bilirubin, a neurotoxic blood breakdown product, caused a dose- and time-dependent decrease in axonal function, suggesting a reduced number of conducting myelinated axons. These effects did not occur when tissue was incubated with non-oxidized bilirubin. Injection of bilirubin into the corpus callosum of mice caused functional impairment of unmeylinated axons; however, immunohistochemical staining of the tissue showed evidence of structural damage to both oligodendrocytes and axons. This data provides evidence for functional and structural damage to white matter in the presence of partially oxidized bilirubin Therefore, diminishing the duration of presence of bilirubin and its oxidation in the brain warrants study as a means of decreasing secondary brain injury after ICH.

Intracerebral hemorrhage (ICH) results in secondary brain injury caused partially by blood and its metabolites. Survivors of ICH are often left with severe disabilities, therefore, decreasing the extent of this secondary injury may improve functional outcome of patients. Incubation of mouse brain slices with partially oxidized bilirubin, a neurotoxic blood breakdown product, caused a dose- and time-dependent decrease in axonal function, suggesting a reduced number of conducting myelinated axons. These effects did not occur when tissue was incubated with non-oxidized bilirubin. Injection of bilirubin into the corpus callosum of mice caused functional impairment of unmeylinated axons; however, immunohistochemical staining of the tissue showed evidence of structural damage to both oligodendrocytes and axons. This data provides evidence for functional and structural damage to white matter in the presence of partially oxidized bilirubin Therefore, diminishing the duration of presence of bilirubin and its oxidation in the brain warrants study as a means of decreasing secondary brain injury after ICH.

Calorie restriction (CR) is one of the few treatments that has been observed to significantly extend life in a wide variety of species. While its life-extending properties are still being investigated in primates, there is general agreement that it reduces oxidative stress and inflammation. Interestingly, there is some evidence that it may ameliorate or delay the onset of a number of neurodegenerative diseases, including age-related white matter degeneration. The processes underlying its neuroprotective effects in non-human primates are unknown, but oxidative stress and inflammation are potential contributors to age-related white matter pathologies that characterize aging in the monkey brain and correlate with cognitive decline. To determine if CR reduces damage due to oxidative stress and inflammation in the monkey brain, brains from four calorie restricted monkeys and four matched controls brains were processed for immunohistochemical analysis using an antibody against the pro-inflammatory protein S100b. S100b is a widely expressed calcium-binding cytoplasmic protein associated with neurological insults like ischemia, atrophy, and neurofibrillary tangles and plaques in Alzheimer's disease. It is primarily expressed in astrocytes, but is also expressed to a lesser extent in microglia, oligodendrocytes, and some neuronal populations. Stereology was used to estimate density of S100b labeling in the cingulum, corpus callosum and visual cortex. No significant difference between calorie restricted animals and controls was found. More specific markers of oxidative stress and inflammation may be more effective in revealing any significant differences between CR and control brains. Potential alternatives include antibodies against 4-hydroxynonenal, a lipid peroxidation product, and encephalitogenic peptides of myelin basic protein, which are only exposed to the extracellular environment when myelin is damaged.