Hippocampal capillary pericytes in post-stroke and vascular dementias and Alzheimer's disease and experimental chronic cerebral hypoperfusion.

Neurovascular unit mural cells called 'pericytes' maintain the blood-brain barrier and local cerebral blood flow. Pathological changes in the hippocampus predispose to cognitive impairment and dementia. The role of hippocampal pericytes in dementia is largely unknown. We investigated hippocampal pericytes in 90 post-mortem brains from post-stroke dementia (PSD), vascular dementia (VaD), Alzheimer's disease (AD), and AD-VaD (Mixed) subjects, and post-stroke non-demented survivors as well as similar age controls. We used collagen IV immunohistochemistry to determine pericyte densities and a mouse model of VaD to validate the effects of chronic cerebral hypoperfusion. Despite increased trends in hippocampal microvascular densities across all dementias, mean pericyte densities were reduced by ~25-40% in PSD, VaD and AD subjects compared to those in controls, which calculated to 14.1 ± 0.7 per mm capillary length, specifically in the cornu ammonis (CA) 1 region (P = 0.01). In mice with chronic bilateral carotid artery occlusion, hippocampal pericyte loss was ~60% relative to controls (P < 0.001). Pericyte densities were correlated with CA1 volumes (r = 0.54, P = 0.006) but not in any other sub-region. However, mice subjected to the full-time environmental enrichment (EE) paradigm showed remarkable attenuation of hippocampal CA1 pericyte loss in tandem with CA1 atrophy. Our results suggest loss of hippocampal microvascular pericytes across common dementias is explained by a vascular aetiology, whilst the EE paradigm offers significant protection.

ER stress induced immunopathology involving complement in CADASIL: implications for therapeutics.

Cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is caused by NOTCH3 mutations. Typical CADASIL is characterised by subcortical ischemic strokes due to severe arteriopathy and fibrotic thickening of small arteries. Arteriolar vascular smooth muscle cells (VSMCs) are the key target in CADASIL, but the potential mechanisms involved in their degeneration are still unclear. Focusing on cerebral microvessels in the frontal and anterior temporal lobes and the basal ganglia, we used advanced proteomic and immunohistochemical methods to explore the extent of inflammatory and immune responses in CADASIL subjects compared to similar age normal and other disease controls. There was variable loss of VSMC in medial layers of arteries in white matter as well as the cortex, that could not be distinguished whether NOTCH3 mutations were in the epidermal growth factor (EGFr) domains 1-6 or EGFr7-34. Proteomics of isolated cerebral microvessels showed alterations in several proteins, many associated with endoplasmic reticulum (ER) stress including heat shock proteins. Cerebral vessels with sparsely populated VSMCs also attracted robust accrual of perivascular microglia/macrophages in order CD45+ > CD163+ > CD68+cells, with > 60% of vessel walls exhibiting intercellular adhesion molecule-1 (ICAM-1) immunoreactivity. Functional VSMC cultures bearing the NOTCH3 Arg133Cys mutation showed increased gene expression of the pro-inflammatory cytokine interleukin 6 and ICAM-1 by 16- and 50-fold, respectively. We further found evidence for activation of the alternative pathway of complement. Immunolocalisation of complement Factor B, C3d and C5-9 terminal complex but not C1q was apparent in ~ 70% of cerebral vessels. Increased complement expression was corroborated in > 70% of cultured VSMCs bearing the Arg133Cys mutation independent of N3ECD immunoreactivity. Our observations suggest that ER stress and other cellular features associated with arteriolar VSMC damage instigate robust localized inflammatory and immune responses in CADASIL. Our study has important implications for immunomodulation approaches to counter the characteristic arteriopathy of CADASIL.

Transcriptomic Profiling Reveals Discrete Poststroke Dementia Neuronal and Gliovascular Signatures.

Poststroke dementia (PSD) is associated with pathology in frontal brain regions, in particular dorsolateral prefrontal cortex (DLPFC) neurons and white matter, remote from the infarct. We hypothesised that PSD results from progressive DLPFC neuronal damage, associated with frontal white matter gliovascular unit (GVU) alterations. We investigated the transcriptomic profile of the neurons and white matter GVU cells previously implicated in pathology. Laser-capture microdissected neurons, astrocytes and endothelial cells were obtained from the Cognitive Function After Stroke cohort of control, PSD and poststroke non-dementia (PSND) human subjects. Gene expression was assessed using microarrays and pathway analysis to compare changes in PSD with controls and PSND. Neuronal findings were validated using NanoString technology and compared with those in the bilateral common carotid artery stenosis (BCAS) mouse model. Comparing changes in PSD compared to controls with changes in PSND compared to controls identified transcriptomic changes associated specifically with dementia. DLPFC neurons showed defects in energy production (tricarboxylic acid (TCA) cycle, adenosine triphosphate (ATP) binding and mitochondria), signalling and communication (MAPK signalling, Toll-like receptor signalling, endocytosis). Similar changes were identified in neurons isolated from BCAS mice. Neuronal findings accompanied by altered astrocyte communication and endothelium immune changes in the frontal white matter, suggesting GVU dysfunction. We propose a pathogenic model in PSD whereby neuronal changes are associated with frontal white matter GVU dysfunction leading to astrocyte failure in supporting neuronal circuits resulting in delayed cognitive decline associated with PSD. Therefore, targeting these processes could potentially ameliorate the dementia seen in PSD.

Trajectories of cognitive change following stroke: stepwise decline towards dementia in the elderly.

Stroke events increase the risk of developing dementia, 10% for a first-ever stroke and 30% for recurrent strokes. However, the effects of stroke on global cognition, leading up to dementia, remain poorly understood. We investigated: (i) post-stroke trajectories of cognitive change, (ii) trajectories of cognitive decline in those who develop dementia over periods of follow-up length and (iii) risk factors precipitating the onset of dementia. Prospective cohort of hospital-based stroke survivors in North-East England was followed for up to 12 years. In this study, we included 355 stroke survivors of ≥75 years of age, not demented 3 months post-stroke, who had had annual assessments during follow-up. Global cognition was measured annually and characterized using standardized tests: Cambridge Cognition Examination-Revised and Mini-Mental State Examination. Demographic data and risk factors were recorded at baseline. Mixed-effects models were used to study trajectories in global cognition, and logistic models to test associations between the onset of dementia and key risk factors, adjusted for age and sex. Of the 355 participants, 91 (25.6%) developed dementia during follow-up. The dementia group had a sharper decline in Cambridge Cognition Examination-Revised (coeff. = -1.91, 95% confidence interval = -2.23 to -1.59, P < 0.01) and Mini-Mental State Examination (coeff. = -0.46, 95% confidence interval = -0.58 to -0.34, P < 0.01) scores during follow-up. Stroke survivors who developed dementia within 3 years after stroke showed a steep decline in global cognition. However, a period of cognitive stability after stroke lasting 3 years was identified for individuals diagnosed with dementia in 4-6 years (coeff. = 0.28, 95% confidence interval = -3.28 to 3.8, P = 0.88) of 4 years when diagnosed at 7-9 years (coeff. = -3.00, 95% confidence interval = -6.45 to 0.45, P = 0.09); and of 6 years when diagnosed at 10-12 years (coeff. = -6.50, 95% confidence interval = -13.27 to 0.27, P = 0.06). These groups then showed a steep decline in Cambridge Cognition Examination-Revised in the 3 years prior to diagnosis of dementia. Risk factors for dementia within 3 years include recurrent stroke (odds ratio = 3.99, 95% confidence interval = 1.30-12.25, P = 0.016) and previous disabling stroke, total number of risk factors for dementia (odds ratio = 2.02, 95% confidence interval = 1.26-3.25, P = 0.004) and a Cambridge Cognition Examination-Revised score below 80 at baseline (odds ratio = 3.50, 95% confidence interval = 1.29-9.49, P = 0.014). Our unique longitudinal study showed cognitive decline following stroke occurs in two stages, a period of cognitive stability followed by rapid decline before a diagnosis of dementia. This pattern suggests stroke may predispose survivors for dementia by diminishing cognitive reserve but with a smaller impact on cognitive function, where cognitive decline may be precipitated by subsequent events, e.g. another cerebrovascular event. This supports the assertion that the development of vascular dementia can be stepwise even when patients have small stroke lesions.

Differential perivascular microglial activation in the deep white matter in vascular dementia developed post-stroke.

With the hypothesis that perivascular microglia are involved as neuroinflammatory components of the gliovascular unit contributing to white matter hyperintensities on MRI and pathophysiology, we assessed their status in stroke survivors who develop dementia. Immunohistochemical and immunofluorescent methods were used to assess the distribution and quantification of total and perivascular microglial cell densities in 68 brains focusing on the frontal lobe WM and overlying neocortex in post-stroke dementia (PSD), post-stroke non-dementia (PSND) and similar age control subjects. We primarily used CD68 as a marker of phagocytic microglia, as well as other markers of microglia including Iba-1 and TMEM119, and the myeloid cell marker TREM2 to assess dementia-specific changes. We first noted greater total densities of CD68+ and TREM2+ cells per mm2 in the frontal WM compared to the overlying cortex across the stroke cases and controls (p = 0.001). PSD subjects showed increased percentage of activated perivascular CD68+ cells distinct from ramified or primed microglia in the WM (p < 0.05). However, there was no apparent change in perivascular TREM2+ cells. Total densities of TREM2+ cells were only ~10% of CD68+ cells but there was high degree of overlap (>70%) between them in both the WM and the cortex. CD68 and Iba-1 or CD68 and TMEM119 markers were colocalised by ~55%. Within the deep WM, ~30% of CD68+ cells were co-localised with fragments of degraded myelin basic protein. Among fragmented CD68+ cells in adjacent WM of PSD subjects, >80% of the cells expressed cleaved caspase-3. Our observations suggest although the overall repertoire of perivascular microglial cells is not changed in the parenchyma, PSD subjects accrue more perivascular-activated CD68+ microglia rather than TREM2+ cells. This implies there is a subset of CD68+ cells, which are responsible for the differential response in perivascular inflammation within the gliovascular unit of the deep WM.

Evidence of beta amyloid independent small vessel disease in familial Alzheimer's disease.

We studied small vessel disease (SVD) pathology in Familial Alzheimer's disease (FAD) subjects carrying the presenilin 1 (PSEN1) p.Glu280Ala mutation in comparison to those with sporadic Alzheimer's disease (SAD) as a positive control for Alzheimer's pathology and Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) bearing different NOTCH3 mutations, as positive controls for SVD pathology. Upon magnetic resonance imaging (MRI) in life, some FAD showed mild white matter hyperintensities and no further radiologic evidence of SVD. In post-mortem studies, total SVD pathology in cortical areas and basal ganglia was similar in PSEN1 FAD and CADASIL subjects, except for the feature of arteriosclerosis which was higher in CADASIL subjects than in PSEN1 FAD subjects. Further only a few SAD subjects showed a similar degree of SVD pathology as observed in CADASIL. Furthermore, we found significantly enlarged perivascular spaces in vessels devoid of cerebral amyloid angiopathy in FAD compared with SAD and CADASIL subjects. As expected, there was greater fibrinogen-positive perivascular reactivity in CADASIL but similar reactivity in PSEN1 FAD and SAD groups. Fibrinogen immunoreactivity correlated with onset age in the PSEN1 FAD cases, suggesting increased vascular permeability may contribute to cognitive decline. Additionally, we found reduced perivascular expression of PDGFRß AQP4 in microvessels with enlarged PVS in PSEN1 FAD cases. We demonstrate that there is Aß-independent SVD pathology in PSEN1 FAD, that was marginally lower than that in CADASIL subjects although not evident by MRI. These observations suggest presence of covert SVD even in PSEN1, contributing to disease progression. As is the case in SAD, these consequences may be preventable by early recognition and actively controlling vascular disease risk, even in familial forms of dementia.

Loss with ageing but preservation of frontal cortical capillary pericytes in post-stroke dementia, vascular dementia and Alzheimer's disease.

Cerebral pericytes are an integral component of the neurovascular unit, which governs the blood-brain barrier. There is paucity of knowledge on cortical pericytes across different dementias. We quantified cortical pericytes in capillaries in 124 post-mortem brains from subjects with post-stroke dementia (PSD), vascular dementia (VaD), Alzheimer's disease (AD) and AD-VaD (Mixed) and, post-stroke non-demented (PSND) stroke survivors as well as normal ageing controls. Collagen 4 (COL4)-positive nucleated pericyte soma were identified as protrusions on capillaries of the frontal cortex. The COL4-positive somata or nodule-like cell bodies were also verified by platelet derived growth factor receptor-ß (PDGFR-ß) immunohistochemistry. The mean (± SEM) pericyte somata in frontal cortical capillaries in normal young controls (46-65 years of age) was estimated as 5.2 ± 0.2 per mm capillary length. This number was reduced by 45% in older controls (> 78 years) to 2.9 ± 0.1 per mm capillary length (P < 0.001). We further found that the numbers of pericyte cell bodies per COL4 mm2 area or per mm capillary length were not decreased but rather preserved or increased in PSD, AD and Mixed dementia groups compared to similar age older controls (P < 0.01). Consistent with this, we noted that capillary length densities identified by the endothelial marker glucose transporter 1 or COL4 were not different across the dementias compared to older controls. There was a negative correlation with age (P < 0.001) suggesting fewer pericyte somata in older age, although the % COL4 immunoreactive capillary area was increased in older controls compared to young controls. Using a proven reliable method to quantify COL4-positive nucleated pericytes, our observations demonstrate ageing related loss but mostly preserved pericytes in the frontal cortex of vascular and AD dementias. We suggest there is differential regulation of capillary pericytes in the frontal lobe between the cortex and white matter in ageing-related dementias.

The role of the medial prefrontal cortex in cognition, ageing and dementia.

Humans require a plethora of higher cognitive skills to perform executive functions, such as reasoning, planning, language and social interactions, which are regulated predominantly by the prefrontal cortex. The prefrontal cortex comprises the lateral, medial and orbitofrontal regions. In higher primates, the lateral prefrontal cortex is further separated into the respective dorsal and ventral subregions. However, all these regions have variably been implicated in several fronto-subcortical circuits. Dysfunction of these circuits has been highlighted in vascular and other neurocognitive disorders. Recent advances suggest the medial prefrontal cortex plays an important regulatory role in numerous cognitive functions, including attention, inhibitory control, habit formation and working, spatial or long-term memory. The medial prefrontal cortex appears highly interconnected with subcortical regions (thalamus, amygdala and hippocampus) and exerts top-down executive control over various cognitive domains and stimuli. Much of our knowledge comes from rodent models using precise lesions and electrophysiology readouts from specific medial prefrontal cortex locations. Although, anatomical disparities of the rodent medial prefrontal cortex compared to the primate homologue are apparent, current rodent models have effectively implicated the medial prefrontal cortex as a neural substrate of cognitive decline within ageing and dementia. Human brain connectivity-based neuroimaging has demonstrated that large-scale medial prefrontal cortex networks, such as the default mode network, are equally important for cognition. However, there is little consensus on how medial prefrontal cortex functional connectivity specifically changes during brain pathological states. In context with previous work in rodents and non-human primates, we attempt to convey a consensus on the current understanding of the role of predominantly the medial prefrontal cortex and its functional connectivity measured by resting-state functional MRI in ageing associated disorders, including prodromal dementia states, Alzheimer's disease, post-ischaemic stroke, Parkinsonism and frontotemporal dementia. Previous cross-sectional studies suggest that medial prefrontal cortex functional connectivity abnormalities are consistently found in the default mode network across both ageing and neurocognitive disorders such as Alzheimer's disease and vascular cognitive impairment. Distinct disease-specific patterns of medial prefrontal cortex functional connectivity alterations within specific large-scale networks appear to consistently feature in the default mode network, whilst detrimental connectivity alterations are associated with cognitive impairments independently from structural pathological aberrations, such as grey matter atrophy. These disease-specific patterns of medial prefrontal cortex functional connectivity also precede structural pathological changes and may be driven by ageing-related vascular mechanisms. The default mode network supports utility as a potential biomarker and therapeutic target for dementia-associated conditions. Yet, these associations still require validation in longitudinal studies using larger sample sizes.

Neuronal densities and vascular pathology in the hippocampal formation in CADASIL.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most common form of hereditary cerebral small vessel disease. Previous neuroimaging studies have suggested loss of hippocampal volume is a pathway for cognitive impairment in CADASIL. We used unbiased stereological methods to estimate SMI32-positive and total numbers and volumes of neurons in the hippocampal formation of 12 patients with CADASIL and similar age controls (young controls) and older controls. We found densities of SMI32-positive neurons in the entorhinal cortex, layer V, and cornu ammonis CA2 regions were reduced by 26%-50% in patients with CADASIL compared with young controls (p < 0.01), with a decreasing trend observed in older controls in the order of young controls> older controls ≥ CADASIL. These changes were not explained by any hippocampal infarct or vascular pathology or glial changes. Our results suggest notable loss of subsets of projection neurons within the hippocampal formation that may contribute to certain memory deficits in CADASIL, which is purely a vascular disease. It is likely that the severe arteriopathy leads to white matter damage which disconnects cortico-cortical and subcortical-cortical networks including the hippocampal formation.

Insulin-Independent and Dependent Glucose Transporters in Brain Mural Cells in CADASIL.

Typical cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is caused by mutations in the human NOTCH3 gene. Cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy is characterized by subcortical ischemic strokes due to severe arteriopathy and fibrotic thickening of small vessels. Blood regulating vascular smooth muscle cells (VSMCs) appear as the key target in CADASIL but the pathogenic mechanisms remain unclear. With the hypothesis that brain glucose metabolism is disrupted in VSMCs in CADASIL, we investigated post-mortem tissues and VSMCs derived from CADASIL patients to explore gene expression and protein immunoreactivity of glucose transporters (GLUTs), particularly GLUT4 and GLUT2 using quantitative RT-PCR and immunohistochemical techniques. In vitro cell model analysis indicated that both GLUT4 and -2 gene expression levels were down-regulated in VSMCs derived from CADASIL patients, compared to controls. In vitro studies further indicated that the down regulation of GLUT4 coincided with impaired glucose uptake in VSMCs, which could be partially rescued by insulin treatment. Our observations on reduction in GLUTs in VSMCs are consistent with previous findings of decreased cerebral blood flow and glucose uptake in CADASIL patients. That impaired ability of glucose uptake is rescued by insulin is also consistent with previously reported lower proliferation rates of VSMCs derived from CADASIL subjects. Overall, these observations are consistent with the development of severe cerebral arteriopathy in CADASIL, in which VSMCs are replaced by widespread fibrosis.

Loss of capillary pericytes and the blood-brain barrier in white matter in poststroke and vascular dementias and Alzheimer's disease.

White matter (WM) disease is associated with disruption of the gliovascular unit, which involves breach of the blood-brain barrier (BBB). We quantified pericytes as components of the gliovascular unit and assessed their status in vascular and other common dementias. Immunohistochemical and immunofluorescent methods were developed to assess the distribution and quantification of pericytes connected to the frontal lobe WM capillaries. Pericytes with a nucleus were identified by collagen 4 (COL4) and platelet-derived growth factor receptor-ß (PDGFR-ß) antibodies with further verification using PDGFR-ß-specific ELISA. We evaluated a total of 124 post-mortem brains from subjects with post-stroke dementia (PSD), vascular dementia (VaD), Alzheimer's disease (AD), AD-VaD (Mixed) and post-stroke non-demented (PSND) stroke survivors as well as normal aging controls. COL4 and PDGFR-ß reactive pericytes adopted the characteristic "crescent" or nodule-like shapes around capillary walls. We estimated densities of pericyte somata to be 225 ±38 and 200 ±13 (SEM) per COL4 mm2 area or 2.0 ± 0.1 and 1.7 ± 0.1 per mm capillary length in young and older aging controls. Remarkably, WM pericytes were reduced by ~35%-45% in the frontal lobe of PSD, VaD, Mixed and AD subjects compared to PSND and controls subjects (P < 0.001). We also found pericyte numbers were correlated with PDGFR-ß reactivity in the WM. Our results first demonstrate a reliable method to quantify COL4-positive pericytes and then, indicate that deep WM pericytes are decreased across different dementias including PSD, VaD, Mixed and AD. Our findings suggest that downregulation of pericytes is associated with the disruption of the BBB in the deep WM in several aging-related dementias.

Long-term effects of experimental carotid stenosis on hippocampal infarct pathology, neurons and glia and amelioration by environmental enrichment.

Hippocampal atrophy and pathology are common in ageing-related disorders and associated with cognitive impairment and dementia. We explored whether environmental enrichment (EE) ameliorated the pathological sequelae in the hippocampus subsequent to chronic cerebral hypoperfusion induced by bilateral common carotid artery stenosis (BCAS). Seventy-four male C57BL/6 J mice underwent BCAS or sham surgery. One-week after surgery, mice were exposed to three different degrees of EE; either standard housing conditions (std), limited 3 -h exposure to EE per day (3 h) or full-time exposure to EE (full) for 3 months. Four months after surgery, the hippocampus was examined for the extent of vascular brain injury and neuronal and glial changes. Results showed that long-term BCAS induced strokes, most often in CA1 subfield, reduced 40-50 % CA1 neurons (P < 0.01) and increased microglia/macrophage in CA1-CA3 subfields (P < 0.02). Remarkably, both 3 h and full-time EE regimes attenuated hippocampal neuronal death and repressed recurrent strokes with complete prevention of larger infarcts in mice on full-time EE (P < 0.01). Full-time EE also reduced astrocytic clasmatodendrosis and microglial/macrophage activation in all CA subfields. Our results suggest that exposure to EE differentially reduces long-term hypoperfusive hippocampal damage. The implementation of even limited EE may be beneficial for patients diagnosed with vascular cognitive impairment.

Small vessel disease pathological changes in neurodegenerative and vascular dementias concomitant with autonomic dysfunction.

We performed a clinicopathological study to assess the burden of small vessel disease (SVD) type of pathological changes in elderly demented subjects, who had clinical evidence of autonomic dysfunction, either carotid sinus hypersensitivity or orthostatic hypotension or both or had exhibited unexpected repeated falls. Clinical and neuropathological diagnoses in 112 demented subjects comprised dementia with Lewy bodies (DLB), Parkinson's disease with dementia (PDD), Alzheimer's disease (AD), Mixed dementia (mostly AD-DLB) and vascular dementia (VaD). Of these, 12 DLB subjects had no recorded unexpected falls in life and therefore no evidence of concomitant autonomic dysfunction. A further 17 subjects were assessed as aging controls without significant pathology or signs of autonomic dysfunction. We quantified brain vascular pathological changes and determined severities of neurodegenerative lesions including α-synuclein pathology. We found moderate-severe vascular changes and high-vascular pathology scores (P < 0.01) in all neurodegenerative dementias and as expected in VaD compared to similar age controls. Arteriolosclerosis, perivascular spacing and microinfarcts were frequent in the basal ganglia and frontal white matter (WM) across all dementias, whereas small infarcts (<5 mm) were restricted to VaD. In a sub-set of demented subjects, we found that vascular pathology scores were correlated with WM hyperintensity volumes determined by MRI in life (P < 0.02). Sclerotic index values were increased by ~50% in both the WM and neocortex in all dementias compared to similar age controls. We found no evidence for increased α-synuclein deposition in subjects with autonomic dysfunction. Our findings suggest greater SVD pathological changes occur in the elderly diagnosed with neurodegenerative dementias including DLB and who develop autonomic dysfunction. SVD changes may not necessarily manifest in clinically overt symptoms but they likely confound motor or cognitive dysfunction. We propose dysautonomia promotes chronic cerebral hypoperfusion to impact upon aging-related neurodegenerative disorders and characterize their end-stage clinical syndromes.

Blood brain barrier leakage is not a consistent feature of white matter lesions in CADASIL.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a genetic paradigm of small vessel disease (SVD) caused by NOTCH3 mutations that stereotypically lead to the vascular accumulation of NOTCH3 around smooth muscle cells and pericytes. White matter (WM) lesions (WMLs) are the earliest and most frequent abnormalities, and can be associated with lacunar infarcts and enlarged perivascular spaces (ePVS). The prevailing view is that blood brain barrier (BBB) leakage, possibly mediated by pericyte deficiency, plays a pivotal role in the formation of WMLs. Herein, we investigated the involvement of BBB leakage and pericyte loss in CADASIL WMLs. Using post-mortem brain tissue from 12 CADASIL patients and 10 age-matched controls, we found that WMLs are heterogeneous, and that BBB leakage reflects the heterogeneity. Specifically, while fibrinogen extravasation was significantly increased in WMLs surrounding ePVS and lacunes, levels of fibrinogen leakage were comparable in WMLs without other pathology ("pure" WMLs) to those seen in the normal appearing WM of patients and controls. In a mouse model of CADASIL, which develops WMLs but no lacunes or ePVS, we detected no extravasation of endogenous fibrinogen, nor of injected small or large tracers in WMLs. Moreover, there was no evidence of pericyte coverage modification in any type of WML in either CADASIL patients or mice. These data together indicate that WMLs in CADASIL encompass distinct classes of WM changes and argue against the prevailing hypothesis that pericyte coverage loss and BBB leakage are the primary drivers of WMLs. Our results also have important implications for the interpretation of studies on the BBB in living patients, which may misinterpret evidence of BBB leakage within WM hyperintensities as suggesting a BBB related mechanism for all WMLs, when in fact this may only apply to a subset of these lesions.

Neurovascular Ageing and Age-Related Diseases.

Proper functioning of the brain is dependent on integrity of the cerebral vasculature. During ageing, a number of factors including aortic or arterial stiffness, autonomic dysregulation, neurovascular uncoupling and blood-brain barrier (BBB) damage will define the dynamics of brain blood flow and local perfusion. The nature and extent of ageing-related cerebrovascular changes, the degree of involvement of the heart and extracranial vessels and the consequent location of tissue pathology may vary considerably. Atheromatous disease retarding flow is a common vascular insult, which increases exponentially with increasing age. Arteriolosclerosis characterized as a prominent feature of small vessel disease is one of the first changes to occur during the natural history of cerebrovascular pathology. At the capillary level, the cerebral endothelium, which forms the BBB undergoes changes including reduced cytoplasm, fewer mitochondria, loss of tight junctions and thickened basement membranes with collagenosis. Astrocyte end-feet protecting the BBB retract as part of the clasmatodendrotic response whereas pericyte coverage is altered. The consequences of these microvascular changes are lacunar infarcts, cortical and subcortical microinfarcts, microbleeds and diffuse white matter disease, which involves myelin loss and axonal abnormalities. The deeper structures are particularly vulnerable because of the relatively reduced density of the microvascular network formed by perforating and penetrating end arteries. Ultimately, the integrity of both the neurovascular and gliovascular units is compromised such that there is an overall synergistic effect reflecting on ageing associated cerebral perfusion and permeability. More than one protagonist appears to be involved in ageing-related cognitive dysfunction characteristically associated with the neurocognitive disorders.

White matter capillaries in vascular and neurodegenerative dementias.

Previous studies suggest white matter (WM) integrity is vulnerable to chronic hypoperfusion during brain ageing. We assessed ~ 0.7 million capillary profiles in the frontal lobe WM across several dementias comprising Alzheimer's disease, dementia with Lewy bodies, Parkinson's disease with dementia, vascular dementia, mixed dementias, post-stroke dementia as well as post-stroke no dementia and similar age ageing and young controls without significant brain pathology. Standard histopathological methods were used to determine microvascular pathology and capillary width and densities in 153 subjects using markers of the basement membrane (collagen IV; COL4) and endothelium (glucose transporter-1; GLUT-1). Variable microvascular pathology including coiled, tortuous, collapsed and degenerated capillaries as well as occasional microaneurysms was present in all dementias. As expected, WM microvascular densities were 20-49% lower than in the overlying cortex. This differential in density between WM and cortex was clearly demonstrated by COL4, which was highly correlated with GLUT-1 densities (Spearman's rho = 0.79, P = 0.000). WM COL4 immunopositive microvascular densities were decreased by ~ 18% across the neurodegenerative dementias. However, we found WM COL4 densities were increased by ~ 57% in post-stroke dementia versus ageing and young controls and other dementias. Using three different methods to measure capillary diameters, we found WM capillaries to be significantly wider by 19-45% compared to those in overlying neocortex apparent with both COL4 and GLUT-1. Remarkably, WM capillary widths were increased by ~ 20% across all dementias compared to ageing and young controls (P < 0.01). We also noted mean WM pathology scores incorporating myelin loss, arteriolosclerosis and perivascular spacing were correlated with COL4 immunopositive capillary widths (Pearson's r = 0.71, P = 0.032). Our key finding indicates that WM capillaries are wider compared to those in the overlying neocortex in controls but they dilate further during dementia pathogenesis. We suggest capillaries undergo restructuring in the deep WM in different dementias. This reflects compensatory changes to retain WM perfusion and integrity during hypoperfusive states in ageing-related dementias.

Brain-derived and circulating vesicle profiles indicate neurovascular unit dysfunction in early Alzheimer's disease.

Vascular factors that reduce blood flow to the brain are involved in apparition and progression of dementia. We hypothesized that cerebral hypoperfusion (CH) might alter the molecular compositions of brain intercellular communication mechanisms while affecting the neurovascular unit in preclinical and clinical human dementias. To test that hypothesis, mice were subjected to bilateral common carotid stenosis (BCAS) and the molecular compositions of brain-derived and circulating extracellular vesicles (EVs) were assessed. Murine brain vesicle profiles were then analyzed in parallel with brain EVs from post-mortem subjects affected by preclinical Alzheimer's Disease (AD) and mixed dementias. Brain EVs were identified with molecular mediators of hypoxia responses, neuroprotection and neurotoxicity in BCAS mice, patterns also partially resembled by subjects with preclinical AD and mixed dementias. Together these findings indicate that brain EVs represent a promising source of therapeutic targets and circulating markers of neurovascular insult in idiopathic dementias. Furthermore, the results obtained generate novel and compelling hypotheses about the molecular involvement of the vascular component in the etiology of human dementias.

Montreal Cognitive Assessment score correlates with regional cerebral blood flow in post-stroke patients.

OBJECTIVE: Valid and reliable measures are needed to assess post-stroke cognitive impairment. The Montreal Cognitive Assessment (MoCA) has been considered a superior screening test to the Mini-Mental State Examination (MMSE) for patients with post-stroke cognitive impairment, particularly in executive function, which may be related to reduction in regional cerebral blood flow (rCBF). In this study, we determined whether MoCA and MMSE scores correlate with rCBF assessed with SPECT in the subacute phase after ischemic stroke. PATIENTS AND METHODS: We retrospectively enrolled 28 patients who were admitted to the Red Cross Otsu Hospital with acute cerebral infarction, which was confirmed by magnetic resonance imaging (MRI), if they underwent cognitive assessment (MoCA/MMSE) and 123I-IMP SPECT imaging within 3 weeks post-stroke during a study period of 5 months. Correlation analyses between rCBF and MoCA or MMSE scores were performed by statistical parametric mapping (SPM) and volume-of-interest (VOI) analyses. RESULTS: Total MoCA score correlated with the rCBF in the prefrontal cortex, cingulate cortex, caudate nucleus and thalamus by SPM analysis (uncorrected p < 0.001; cluster-level corrected p < 0.05). Among the subtest scores of MoCA, visuoexecutive function, attention, language and delayed recall scores were positively correlated with rCBF in the prefrontal cortex by VOI analysis (p < 0.05). However, total MMSE score did not correlate significantly with any of the rCBF measures. CONCLUSIONS: Post-stroke cognitive performance assessed with MoCA positively correlated with rCBF in brain regions mainly comprising the prefrontal-subcortical circuits. The findings of this hypothesis-generating study support the notion that MoCA is useful for assessing post-stroke cognitive status.

Severe white matter astrocytopathy in CADASIL.

OBJECTIVES: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is characterized by strategic white matter (WM) hyperintensities on MRI. Pathological features include WM degeneration, arteriolosclerosis, lacunar infarcts, and the deposition of granular osmiophilic material. Based on the hypothesis that the gliovascular unit is compromised, we assessed the nature of astrocyte damage in the deep WM of CADASIL subjects. METHODS: We evaluated post-mortem brains from CADASIL, cerebral small vessel disease, similar age cognitively normal and older control subjects. Standard immunohistochemical, immunofluorescent, and unbiased stereological methods were used to evaluate the distribution of astrocytes, microvessels, and autophagy markers in five different brain regions. RESULTS: Compared to the controls, the deep WM of CADASIL subjects overall showed increased numbers of glial fibrillary acidic protein (GFAP)-positive clasmatodendritic astrocytes (P=0.037) and a decrease in the percentage of normal appearing astrocytes (P=0.025). In accord with confluent WM hyperintensities, the anterior temporal pole contained abundant clasmatodendritic astrocytes with displaced aquaporin 4 immunoreactivity. Remarkably, we also found strong evidence for the immunolocalization of autophagy markers including microtubule-associated protein 1, light chain 3 (LC3), and sequestosome 1/p62 and Caspase-3 in GFAP-positive clasmatodendritic cells, particularly within perivascular regions of the deep WM. LC3 was co-localized in more than 90% of the GFAP-positive clasmatodendrocytes. CONCLUSIONS: Our novel findings show astrocytes undergo autophagy-like cell death in CADASIL, with the anterior temporal pole being highly vulnerable. We propose astrocytes transform from normal appearing type A to hypertrophic type B and eventually to clasmatodendritic type C cells. These observations also suggest the gliovascular unit of the deep WM is severely impaired in CADASIL.