Study On Pathological Changes Of Cerebral Microvessels Related To Agin

Part 1 Basic characteristics of age-related cerebral microvascular pathologyBackground and purpose:Changes in microvasculature pathologies with aging could adversely affect normal cerebrovascular structure,morphology,and function.They are mainly manifested as morphological damages,including arteriolosclerosis,capillary tortuosity and density changes,and venular collagenosis.At the cellular and molecular level,injuries including basement membrane(BM)thickening and deposition of extracellular matrix proteins(ECM),are damage the integrity of the blood-brain barrier,which affects the normal function of the cerebral microvasculature and plays an important role in the development of aging-related diseases.The pathological changes of cerebral microvessels and their surrounding ECM proteins,play an important role in the occurrence and development of aging-related diseases such as cerebral small vessel disease and neurodegenerative diseases.Although extensive studies about cerebral microvasculature have been conducted,its pathological changes with aging are still not understood and described.Moreover,previous studies have assessed cerebral microvessels as a whole and did not distinguish between arterioles,capillaries,and venules.Therefore,no study has comprehensively estimated the pathological changes of various microvascular morphology and related protein molecules with aging.In this study,multi-regional brain tissues were selected from donors,and a more reliable and stable staining method was used to identify microvessel types.We aim to evaluate the integrity of endothelial cells on cerebral arterioles,capillaries,and venules,collagen deposition in media tunica,and changes in extracellular matrix proteins of basement membrane.Additionally,the percentage of arteriolosclerosis and venular collagenosis was compared.The correlation between various microvascular abnormalities and age was analyzed to increase the understanding of the pathological age-related changes in cerebral microvasculature.Methods:A total of 27 donated brain tissues without a history of cerebrovascular disease or intracranial tumor were included from the National Human Brain Bank for Development and Functions in this study.Tissue blockers from the periventricular and deep white matter,cortex,hippocampus,putamen,and other seven brain regions were taken,and vascular tissues were also taken to verify the immunohistochemical staining method for anti-monocarboxylate transporter 1.Based on the identification of arterioles,capillaries,and venules by immunohistochemical staining of adjacent sections,the severity of arteriolosclerosis,venular collagenosis,and capillary density in seven brain regions was assessed.The correlation between microvascular damages with age was analyzed by the Spearman correlation analysis.In terms of cells and molecules,we further assessed the levels of CD31,collagen Ⅳ,and extracellular matrix proteins quantitatively,including laminin,fibronectin,perlecan,and agrin with aging based on immunohistochemical staining.Results:The average age of 27 enrolled subjects was 77.33±14.07 years old,of whom 48.15%were male.This study firstly validated the reliability of the vascular type identification method based on anti-monocarboxylate transporter 1 staining in large and small caliber cerebrovessels.Secondly,the severity of arteriolosclerosis,venular collagenosis,and capillary density were assessed in seven brain regions,as well as changes in levels of ECM.Arteriolosclerosis and venular collagenosis are prevalent in aging brains,especially prominent in the white matter regions(arteriolosclerosis,57.14%;venular collagenosis,59.38%),but less commonly in the hippocampus(arteriolosclerosis,10.53%;venular collagenosis,8.11%).In the cortex and putamen,arteriolosclerosis was more evident than venular collagenosis(cortex,54.55%vs 40.00%;putamen,51.16%vs 33.33%).Arteriolosclerosis increased significantly with aging across the whole brain(all β>0,p<0.05),but venular collagenosis was not significantly increased in the putamen(β>0,p<0.05 except for putamen;for putamen,β=0.34898,p=0.0744).Further exploration in this study found that the ratio of typeⅠ to type Ⅲ collagen was significantly increased in venular walls(Col I:74.28 vs 45.42;Col Ⅲ:32.45 vs 16.90),and there was no significant change in collagen Ⅳ.In contrast to previous studies,we found that capillary density increased with aging in the white matter region,and there were no significant changes in extracellular matrix proteins except for fibronectin(4.31 vs 3.67,p=0.0029).Conclusion:Arteriolosclerosis,venular collagenosis,and changes in capillary density are prevalent in aging brains.Increased levels of fibronectin also implied that the function of cerebral microvasculature was impaired with aging.Noted that,the cerebral venules,which have been overlooked in the past,are not the same as the cerebral arterioles in terms of pathological characteristics and distributions,which indicated two different roles in cerebral microvasculature.The different mechanisms of these microvascular damages involved in various diseases are worthy of further exploration and research.Part 2 Associations between cerebral microvascular pathology and parenchymal damagesBackground and purpose:Cerebrovascular parenchymal damage is prevalent in aging brains and is often described in pathological studies as changes in lacunes,microinfarcts,microbleeds or hemosiderin deposition,leukoaraiosis,and myelin loss.Although many studies based on clinical risk factors,neuroimaging,and histopathology have been conducted on vascular-related brain parenchymal injury in the past,the vascular mechanism has not been fully elucidated.Additionally,there is little research on whether the pathological changes of cerebral venules are also involved he parenchymal damages.Recently,with the findings of the cerebral lymphoid system,the important physiological functions of the cerebral venous system and its important role in the disease process have been gradually noticed.However,there is no study has compared the differences in the mediation of brain parenchymal damages by cerebral arterioles and venules.Herein,we aimed to investigate the associations between microvascular injuries,including arteriolosclerosis and venular collagenosis,and related parenchymal damages in aging brains,to investigate the underlying correlations.Methods:A total of 27 human post-mortem brains from the National Human Brain Bank for Development and Function,without a history of stroke or brain tumor history until death were included in this study.We evaluated arteriolosclerosis and venular collagenosis in seven regions including the frontal periventricular white matter,occipital periventricular white matter,deep white matter in the parietal lobe,superior frontal cortex,occipital cortex,hippocampus,and putamen.The correlations between the ratio of arteriolosclerosis,venular collagenosis,and the severity of cerebrovascular parenchymal damage,including lacunes,microinfarcts,myelin loss,and parenchymal and perivascular hemosiderin deposits,were assessed.Results:Arteriolosclerosis and venular collagenosis became more evident with aging in the white matter(arteriolosclerosis,β=0.67248,p=0.0001;venular collagenosis,β=0.73899,p<0.0001),cortex(arteriolosclerosis,β=0.50383,p=0.0074;venular collagenosis,β=0.54967,p=0.0030),and hippocampus(arteriolosclerosis,β=0.58211,p=0.0014;venular collagenosis,β=0.54875,p=0.0030),except for the severity of venular collagenosis in the putamen(arteriolosclerosis,β=47758,p=0.0118;venular collagenosis,β=0.34898,p=0.0744).Arteriolosclerosis was associated with the presence of lacunes(59.21%vs 30.00%,p=0.0038)but not with leukoaraiosis severity.Venular collagenosis was not associated with the number of lacunes or hemosiderin,while white matter generally became paler with severe venular collagenosis in the periventricular(β=-0.430,p=0.0284)and deep white matter(β=-0.437,p=0.0254).Neither arteriolosclerosis nor venular collagenosis was associated with perivascular or brain parenchymal hemosiderin deposits in any region(p>0.1 for all comparisons),except in the superior frontal cortex,where arteriolosclerosis correlated with decreased brain parenchymal hemosiderin deposits(p=0.024).Conclusion:Our findings imply different roles for arteriolosclerosis and venular collagenosis in different subtypes of microvessel-related parenchymal damage.More severe myelin loss was significantly associated with increased collagenized venules but not sclerotic arterioles,whereas lacunes were only associated with more severe arteriolosclerosis in the putamen but not with venular collagenosis in any region.Both cerebral arterioles and venules require further investigation.Part 3 Associations between changes in microvascular walls,microvascular dysfunction,and Alzheimer’s disease neuropathologic changeBackground and purpose:Alzheimer’s disease(AD)is a common finding in aging brains and its pathogenesis is mostly believed to be related to aging,amyloid deposition,immuneinflammatory response,etc.,among which microvascular factors are often considered to be involved in its process.The microvascular system plays an important role in maintaining cerebral tissue perfusion,blood-brain barrier integrity,and draining metabolic wastes and abnormal proteins out of the brain.Decreased cerebral perfusion and abnormal protein deposition are often considered to be important drivers of AD.However,the relationship between AD-related pathological changes and cerebral microvascular system pathological changes is still unclear,and the related pathogenesis mediated by microvessels has not been elucidated.It is worth noting that,as one of the important pathways for the drainage of abnormal proteins in the brain,whether the cerebral venule system is also related to AD has not been specifically mentioned in previous pathological studies.In order to solve the above problems,this study aimed to evaluate and compare the morphological changes such as arteriolosclerosis,capillary density,and venular collagenosis in brain tissues with or without AD-related pathological changes.The changes in microvascular endothelial integrity and extracellular matrix proteins were futher explored in the two groups,suggesting the possible functional changes and potential mechanisms of cerebral microvasculature in AD.Methods:All brains underwent neuropathological assessment according to the National Institute on Aging-Alzheimer’s Association criteria.Finally,our study consisted of 27 human post-mortem brains in total,including 12 brains with AD neuropathologic change and 15 brains without AD neuropathologic change.We examined samples of white matter regions,cerebral cortex,putamen,and hippocampus.The proportions of small vessels affected by arteriolosclerosis and venular collagenosis,the capillary density,the endothelium integrity,and the levels of collagen IV,laminin fibronectin,and perlecan in extracellular matrix were quantified by immunohistochemistry and compared between the two groups.Results:In AD cases compared to controls,venular collagenosis was significantly more severe across all selected brain regions(white matter,63.25%vs 38.81%,p=0.0230;cortex,49.12%vs 21.88%,p=0.0132;hippocampus,46.88%vs 0.00%,p=0.0062;putamen,59.42%vs 0.00%,p=0.0033),while arteriolosclerosis was significantly more severe only in the putamen(62.96%vs 41.67%,p=0.0400).Similar association patterns were observed between these microvasculature changes and ABC scores.CD31 levels,which could represent endothelium integrity,were lower in AD group(0.688 vs 0.919,p=0.0002).Collagen Ⅳ(1.714 vs 2.108,p=0.0025)and fibronectin(3.774 vs 4.472,p=0.0038)expression were decreased while agrin(1.775 vs 1.299,p<0.0001)increased in AD cases and showed that changes in extracellular matrix correlated with AD neuropathologic change.Conclusion:Our data indicated a much more prominent role of collagenized venules in AD neuropathologic change than arteriolosclerosis.Additionally,the findings further support the hypothesis that significant extracellular matrix changes occur during AD pathogenesis.The study implied that venular collagenosis affecting brain glymphatic efflux of endogenous metabolites,together with the underlying brain-blood barrier dysfunction by extracellular matrix changes,could therefore drive AD progression.Significant changes of venules and extracellular matrix proteins,provide potential new biomarkers and targets for investigation.