Aim Brain clusterin is known to be associated with the amyloid‐β deposits in A lzheimer's disease ( AD ). We assessed the distribution of clusterin immunoreactivity in cerebrovascular disorders, particularly focusing on white matter changes in small vessel diseases. Methods Post‐mortem brain tissues from the frontal or temporal lobes of a total of 70 subjects with various disorders including cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy ( CADASIL ), cerebral amyloid angiopathy ( CAA ) and AD were examined using immunohistochemistry and immunofluorescence. We further used immunogold electron microscopy to study clusterin immunoreactivity in extracellular deposits in CADASIL . Results Immunostaining with clusterin antibodies revealed strong localization in arterioles and capillaries, besides cortical neurones. We found that clusterin immunostaining was significantly increased in the frontal white matter of CADASIL and pontine autosomal dominant microangiopathy and leukoencephalopathy subjects. In addition, clusterin immunostaining correlated with white matter pathology severity scores. Immunostaining in axons ranged from fine punctate deposits in single axons to larger confluent areas with numerous swollen axon bulbs, similar to that observed with known axon damage markers such as non‐phosphorylated neurofilament H and the amyloid precursor protein. Immunofluorescence and immunogold electron microscopy experiments showed that whereas clusterin immunoreactivity was closely associated with vascular amyloid‐β in CAA , it was lacking within the granular osmiophilic material immunolabelled by NOTCH 3 extracelluar domain aggregates found in CADASIL . Conclusions Our results suggest a wider role for clusterin associated with white matter damage in addition to its ability to chaperone proteins for clearance via the perivascular drainage pathways in several disease states.

AimsCerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is identified by aggregates of NOTCH3 extracellular domain (N3ECD) along capillaries and the deposition of granular osmiophilic material (GOM). We assessed the pattern of distribution of pericytes in relation to N3ECD deposits in cerebral microvessels of CADASIL subjects.MethodsWe assessed post mortem brains from (n = 50) subjects with CADASIL, cerebral small vessel disease, and similar‐age cognitively normal and older controls. Immunohistochemical and immunofluorescent staining methods were used to study the distribution and quantify immunoreactivities of the platelet‐derived growth factor receptor‐β (PDGFR‐β) (for pericytes) and microvascular markers in the frontal cortex and white matter.ResultsPDGFR‐β antibody stained cells typical of pericytes in capillaries and small arterioles in both the grey and white matter. PDGFR‐β reactive pericytes adopted ‘crescent’ morphology wrapped closely around capillary walls readily evident in cross‐sections. We noted considerable overlap between PDGFR‐β and N3ECD imunoreactivities in capillaries. Quantitative analysis of PDGFR‐β immunoreactivity revealed significant differences in PDGFR‐β %A in CADASIL compared with young controls (P < 0.05). PDGFR‐β %A was further positively correlated with the basement membrane marker collagen IV (r = 0.529, P = 0.009), but was not associated with GLUT‐1, the marker for endothelial cells.ConclusionsOur results suggest increased expression of PDGFR‐β immunoreactive pericytes in cerebral microvessels in CADASIL compared with similar age controls. While we cannot confirm whether PDGFR‐β‐expressing pericytes produce N3ECD and hence GOM, our findings demonstrate that up‐regulation of pericyte‐like cells is associated with microvascular changes, including loss of vascular smooth muscle cells in CADASIL.

BackgroundOptimal vascular function is vital for prevention of dementia. We hypothesized that elderly post‐stroke survivors who preserve cognitive function show unperturbed cerebral microvasculature compared with those who develop dementia.MethodsUsing stereological spherical probe software, we compared the length density (Lv, cumulative vessel length per unit tissue volume) of hippocampal microvessels in post mortem brain tissue from post‐stroke survivors, Alzheimer's disease (AD), vascular dementia (VaD) and normal ageing control subjects. We also assessed microvessel diameters in the same subjects. Microvessels were identified by markers of endothelial cells (glucose transporter 1; GLUT1), basement membrane (collagen IV; COL4) and smooth muscle cell α‐actin (SMA).ResultsWe found increased Lv of both GLUT1 and COL4 immunostained microvessels (P < 0.05) in the hippocampal CA1 region of post‐stroke demented (PSD) and AD cases compared with post‐stroke nondemented (PSND), control and VaD subjects. However, no changes were apparent in the CA2 region. We also noted significant increase in Lv in the entorhinal cortex of AD compared with PSND and PSD subjects. The mean diameter of microvessels was decreased in PSD, compared with PSND, as well as in AD and VaD compared with controls. Cumulative frequency analysis showed PSND subjects to have significantly greater proportion of microvessels with diameters, ranging from 7 to 12 μm.ConclusionsAn increase in microvascular Lv in AD and PSD suggests either an increase in angiogenesis or the formation of newer microvessel loops in response to cerebral hypoperfusion. The decreased vessel diameters found in AD and VaD suggests increased vasoconstriction in dementia.

Background: Magnetic resonance imaging indicates diffuse white matter (WM) changes are associated with cognitive impairment in cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). We examined whether the distribution of axonal abnormalities is related to microvascular pathology in the underlying WM. Methods: We used post-mortem brains from CADASIL subjects and similar age cognitively normal controls to examine WM axonal changes, microvascular pathology, and glial reaction in up to 16 different regions extending rostro-caudally through the cerebrum. Using unbiased stereological methods, we estimated length densities of affected axons immunostained with neurofilament antibody SMI32. Standard immunohistochemistry was used to assess amyloid precursor protein immunoreactivity per WM area. To relate WM changes to microvascular pathology, we also determined the sclerotic index (SI) in WM arterioles. Results: The degree of WM pathology consistently scored higher across all brain regions in CADASIL subjects (P < 0.01) with the WM underlying the primary motor cortex exhibiting the most severe change. SMI32 immunoreactive axons in CADASIL were invariably increased compared with controls (P < 0.01), with most prominent axonal abnormalities observed in the frontal WM (P < 0.05). The SIs of arterioles in CADASIL were increased by 25-45% throughout the regions assessed, with the highest change in the mid-frontal region (P = 0.000). Conclusions: Our results suggest disruption of either cortico-cortical or subcortical-cortical networks in the WM of the frontal lobe that may explain motor deficits and executive dysfunction in CADASIL. Widespread WM axonal changes arise from differential stenosis and sclerosis of arterioles in the WM of CADASIL subjects, possibly affecting some axons of projection neurones connecting to targets in the subcortical structures.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), the most common form of familial brain arteriopathy, is associated with deposition of granular osmiophilic material (GOM). We used immunohistochemistry and immunogold electron microscopy (EM) to examine the distribution of GOM and NOTCH3 ectodomain (N3ECD) protein in microvasculature of brain gray matter and white matter in patients with CADASIL, non-CADASIL hereditary small-vessel disease and sporadic age-related degenerative disease, and comparable-age controls. We observed intense immunostaining patterns with 2 different anti-N3ECD antibodies in CADASIL but not in young and older controls or other small-vessel disease patients. In addition, CADASIL samples exhibited immunoreactivity in arterial walls and in most capillaries. Electron microscopy revealed profound and widespread extracellular distribution of 0.2- to 2-rho m GOM deposits associated with meningeal vessels and perforating arteries and arterioles. Granular osmiophilic material was adjacent to capillaries even within white matter. Immunogold EM with antibody A1-1 to N3ECD revealed abundant particles in GOM within microvessels, vascular smooth muscle cell membranes, and perivascular cells. Granular osmiophilic material did not exhibit immunogold labeling for smooth muscle alpha-actin or collagen IV. These results showed the specificity of the antibodies and confirm the predominant localization of N3ECD within GOM deposits. The extensive distribution of N3ECD-GOM complexes within meninges, arteries, arterioles, and particularly capillaries in the brains of CADASIL patients suggests that NOTCH3 fragments are major components of GOM deposits, which may be eliminated via perivascular routes.

Objective: Most pathologic studies indicate that significant vascular changes are found in the majority of elderly persons, either alone or in association with neurodegenerative processes such as Alzheimer disease (AD) or dementia with Lewy bodies (DLB). Cumulative burden of cerebrovascular lesions can explain cognitive decline described as vascular cognitive impairment, but because there is a lack of consensus in the best way to quantify vascular pathology, the relationship between cognitive decline and cerebrovascular disease remains uncertain. We developed a rating scheme for cerebrovascular lesions using postmortem brains from patients with dementia from 2 European tertiary care memory clinics.Methods: A total of 135 brains with a neuropathologic diagnosis of vascular dementia (VaD) (n = 26), AD + VaD (n = 39), DLB + VaD (n = 21), AD + DLB + VaD (n = 9), AD (n = 19), and DLB (n = 21) were investigated in this study. Cerebrovascular lesions were rated on large sections from the hippocampus, the temporal lobe, the frontal lobe, and basal ganglia.Results: In patients with dementia, vessel wall modifications such as arteriolosclerosis or amyloid angiopathy are the most common and presumably the earliest changes. Modifications in perivascular spaces and myelin loss are the next most common. Lacunar or regional infarcts may occur as a consequence of an independent process or in the final phase of small vessel diseases.Conclusion: A staging system based on this conceptual model of cerebrovascular pathology could enable the neuropathologic quantification of the cerebrovascular burden in dementia. Further studies are needed to determine whether this system can be used in large-scale studies to understand clinical-cerebrovascular pathologic correlations. Neurology (R) 2012;78:1043-1050

Neuropathology and Applied NeurobiologyVolume 38, Issue s1 p. 7-26 Oral Presentations First published: 18 January 2012 https://doi.org/10.1111/j.1365-2990.2011.01241.xCitations: 1AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Citing Literature Volume38, Issues1Special Issue: Proceedings of the 113th Meeting of the British Neuropathological Society, 11-13 January 2012, Institute of Child Health, London, UKJanuary 2012Pages 7-26 RelatedInformation

AIMS:Carotid sinus hypersensitivity (CSH) is an ageing-related autonomic disorder, rarely occurring before the age of 50 years but increasing in incidence thereafter. Clinical symptoms of CSH include falls and dizziness, thought to be precipitated by dysfunctional baroreflex responses. CSH is highly prevalent in Alzheimer's disease (AD), Parkinson's disease (PD) and dementia with Lewy bodies (DLB); diseases that are associated with variable degeneration of medullary autonomic nuclei which regulate baroreflex responses. Currently, there are no descriptions of the integrity of medullary autonomic nuclei in CSH. We hypothesized medullary autonomic degeneration is found in elderly patients with CSH. METHODS:Using in vitro digital imaging, we quantified the burden of tau, amyloid beta and alpha-synuclein in autonomic nuclei of 12 patients prospectively assessed with CSH (age 83 years) compared with 14 (80 years) control subjects. RESULTS:We found increased tau (P < 0.000) accumulation in baroreflex associated nuclei, but not the hypoglossal or raphe in the CSH patients. Medullary tau accumulation was not related to the development of AD in the CSH patients. Tau was colocalized to catecholaminergic neurones and occurred in the absence of neuronal loss. We found no difference in alpha-synuclein, amyloid beta or microglial numbers between the CSH cases and controls. CONCLUSIONS:We suggest that hyperphosphorylated tau accumulation particularly in tyrosine hydroxylase containing neurones may impair central regulation of baroreflex responses in patients with CSH. Future clinic-pathological investigations should reveal whether medullary degeneration is the cause of CSH symptoms.

Multiple system atrophy (MSA) is a rare and fatal early-onset autonomic disorder which is characterised by Parkinsonism and orthostatic hypotension (OH). The pathophysiology of MSA is not fully understood but key features include the depletion of medullary autonomic neurons and presence of glial cellular inclusions. We hypothesise that the degeneration of medullary autonomic microvessels is an additional finding in MSA. Using digital pathology we quantified basement membrane collagen (Coll IV), smooth muscle actin (α-actin) and endothelial glucose transporter (Glut 1) expression in medullary autonomic nuclei of 8 MSA and 8 OH cases, compared with 12 controls with no autonomic dysfunction. We found decreased Coll IV (p=0.000) and Glut 1 (p=0.000) but not α-actin expression, in medullary autonomic nuclei of MSA, but not OH cases compared with control subjects. Medullary microvessel degeneration in MSA may be secondary to the primary neuro-glial pathogenesis of the disorder, and could accelerate its ageing-related progression.

OBJECTIVE:Evidence suggests that abnormal iron metabolism is associated with Parkinson disease (PD), with raised iron levels found in pathologically affected areas in PD. It is unknown if this elevated iron is actually associated with neurons or reactive glia, and we therefore addressed this issue by determining if raised iron was present in single dopaminergic neurons.METHODS:We used unfixed frozen sections from postmortem tissue of PD patients and elderly normal individuals to avoid metal contamination and translocation. Levels of iron and other elements were measured using sensitive and specific wavelength dispersive electron probe x-ray microanalysis coupled with cathodoluminescence spectroscopy in individual substantia nigra dopaminergic neurons.RESULTS:We identified raised intraneuronal iron in single defined substantia nigra neurons in PD (mean neuronal iron 2,838 vs 1,611, p < 0.0001) but not in other movement disorders such as Huntington disease. These findings were unrelated to the density of remaining neurons.CONCLUSIONS:Primary changes in neuronal iron could lead to neurodegeneration in Parkinson disease.

We previously demonstrated that rats subjected to intermittent hypoxia (IH) by exposure to 10% O2 for 4 h daily for 56 days in a normobaric chamber, developed pulmonary hypertension, right ventricular hypertrophy and wall-thickening in pulmonary arterioles, compared with normoxic (N) controls. These changes were greater in rats subjected to continuous hypoxia (CH breathing 10% O2 for 56 days). Cerebral angiogenesis was demonstrated by immunostaining with glucose transporter 1 (GLUT1) antibody, in viable vessels, in CH and to a lesser degree in IH. In this study, adult Wistar rats were subjected to the same hypoxic regimes and given the nitric oxide synthase (NOS) inhibitor N6-nitro-l-arginine methyl ester (l-NAME) in drinking water (NLN, IHLN and CHLN regimes) to induce hypertension. There was significant systemic hypertension in NLN and IHLN rats, compared with N and IH, but surprisingly not in CHLN compared with CH. Hematocrit rose in all hypoxic groups (up to 79% in CHLN). There was no significant pulmonary hypertension in IHLN versus NLN rats, although there was asymmetric wall thickening in pulmonary arterioles. Cerebral GLUT1 immunoreactivity increased with l-NAME, with or without hypoxia, especially in CHLN rats, but conspicuously there was no evidence of angiogenesis in brains of IHLN compared with NLN rats. NOS blockade may attenuate the cerebral and pulmonary vascular changes of IH while augmenting cerebral angiogenesis in continuous hypoxia. However, whether cerebral effects are due to systemic hypertension or changes in cerebral nitric oxide production needs to be evaluated.