Objective: Cerebral amyloid angiopathy (CAA) is characterized by the accumulation of amyloid β (Aβ) in the walls of cortical vessels and the accrual of microbleeds and microinfarcts over time. The relationship between CAA severity and microbleeds and microinfarcts as well as the sequence of events that lead to lesion formation remain poorly understood. Methods: We scanned intact formalin-fixed hemispheres of 12 CAA cases with magnetic resonance imaging (MRI), followed by histopathological examination in predefined areas and serial sectioning in targeted areas with multiple lesions. Results: In total, 1,168 cortical microbleeds and 472 cortical microinfarcts were observed on ex vivo MRI. Increasing CAA severity at the whole-brain or regional level was not associated with the number of microbleeds or microinfarcts. However, locally, the density of Aβ-positive cortical vessels was lower surrounding a microbleed compared to a simulated control lesion, and higher surrounding microinfarcts. Serial sectioning revealed that for (n = 28) microbleeds, both Aβ (4%) and smooth muscle cells (4%) were almost never present in the vessel wall at the site of bleeding, but Aβ was frequently observed upstream or downstream (71%), as was extensive fibrin(ogen) buildup (87%). In contrast, for (n = 22) microinfarcts, vascular Aβ was almost always observed at the core of the lesion (91%, p < 0.001) as well as upstream or downstream (82%), but few vessels associated with microinfarcts had intact smooth muscle cells (9%). Interpretation: These observations provide a model for how a single neuropathologic process such as CAA may result in hemorrhagic or ischemic brain lesions potentially through 2 different mechanistic pathways.

Cortical superficial siderosis is an established haemorrhagic neuroimaging marker of cerebral amyloid angiopathy. In fact, cortical superficial siderosis is emerging as a strong independent risk factor for future lobar intracerebral haemorrhage. However, the underlying neuropathological correlates and pathophysiological mechanisms of cortical superficial siderosis remain elusive. Here we use an in vivo MRI, ex vivo MRI, histopathology approach to assess the neuropathological correlates and vascular pathology underlying cortical superficial siderosis. Fourteen autopsy cases with cerebral amyloid angiopathy (mean age at death 73 years, nine males) and three controls (mean age at death 91 years, one male) were included in the study. Intact formalin-fixed cerebral hemispheres were scanned on a 3 T MRI scanner. Cortical superficial siderosis was assessed on ex vivo gradient echo and turbo spin echo MRI sequences and compared to findings on available in vivo MRI. Subsequently, 11 representative areas in four cases with available in vivo MRI scans were sampled for histopathological verification of MRI-defined cortical superficial siderosis. In addition, samples were taken from predefined standard areas of the brain, blinded to MRI findings. Serial sections were stained for haematoxylin and eosin and Perls’ Prussian blue, and immunohistochemistry was performed against amyloid-β and GFAP. Cortical superficial siderosis was present on ex vivo MRI in 8/14 cases (57%) and 0/3 controls (P = 0.072). Histopathologically, cortical superficial siderosis corresponded to iron-positive haemosiderin deposits in the subarachnoid space and superficial cortical layers, indicative of chronic bleeding events originating from the leptomeningeal vessels. Increased severity of cortical superficial siderosis was associated with upregulation of reactive astrocytes. Next, cortical superficial siderosis was assessed on a total of 65 Perls’-stained sections from MRI-targeted and untargeted sampling combined in cerebral amyloid angiopathy cases. Moderate-to-severe cortical superficial siderosis was associated with concentric splitting of the vessel wall (an advanced form of cerebral amyloid angiopathy-related vascular damage) in leptomeningeal vessels (P &lt; 0.0001), but reduced cerebral amyloid angiopathy severity in cortical vessels (P = 0.048). In terms of secondary tissue injury, moderate-to-severe cortical superficial siderosis was associated with the presence of microinfarcts (P = 0.025), though not microbleeds (P = 0.973). Collectively, these data suggest that cortical superficial siderosis on MRI corresponds to iron-positive deposits in the superficial cortical layers, representing the chronic manifestation of bleeding episodes from leptomeningeal vessels. Cortical superficial siderosis appears to be the result of predominantly advanced cerebral amyloid angiopathy of the leptomeningeal vessels and may trigger secondary ischaemic injury in affected areas.

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Small subclinical hyperintense lesions are frequently encountered on brain diffusion-weighted imaging (DWI) scans of patients with cerebral amyloid angiopathy (CAA). Interpretation of these DWI+ lesions, however, has been limited by absence of histopathological examination. We aimed to determine whether DWI+ lesions represent acute microinfarcts on histopathology in brains with advanced CAA, using a combined in vivo MRI-ex vivo MRI-histopathology approach. We first investigated the histopathology of a punctate cortical DWI+ lesion observed on clinical in vivo MRI 7 days prior to death in a CAA case. Subsequently, we assessed the use of ex vivo DWI to identify similar punctate cortical lesions postmortem. Intact formalin-fixed hemispheres of 12 consecutive cases with CAA and three non-CAA controls were subjected to high-resolution 3 T ex vivo DWI and T2 imaging. Small cortical lesions were classified as either DWI+/T2+ or DWI−/ T2+. A representative subset of lesions from three CAA cases was selected for detailed histopathological examination. The DWI+ lesion observed on in vivo MRI could be matched to an area with evidence of recent ischemia on histopathology. Ex vivo MRI of the intact hemispheres revealed a total of 130 DWI+/T2+ lesions in 10/12 CAA cases, but none in controls (p = 0.022). DWI+/T2+ lesions examined histopathologically proved to be acute microinfarcts (classification accuracy 100%), characterized by presence of eosinophilic neurons on hematoxylin and eosin and absence of reactive astrocytes on glial fibrillary acidic protein-stained sections. In conclusion, we suggest that small DWI+ lesions in CAA represent acute microinfarcts. Furthermore, our findings support the use of ex vivo DWI as a method to detect acute microinfarcts postmortem, which may benefit future histopathological investigations on the etiology of microinfarcts.

The pathophysiology of microhemorrhages in the context of cerebral amyloid angiopathy (CAA) remains poorly understood. Here we used in vivo two-photon microscopy in aged APP/PS1 mice with mild-to-moderate CAA to assess the formation of microhemorrhages and their spatial relationship with vascular Aβ depositions in the surrounding microvascular network. Mice with chronic cranial windows were intravenously injected with fluorescent dextran to visualize the vessels and a fluorescently labeled anti-fibrin antibody to visualize microhemorrhages. Focal vessel irradiations resulted in extravascular fibrin-positive clots at individual rupture sites that remained visible for weeks. Spontaneous extravascular fibrin-positive clots were more often observed in 19-month-old transgenic APP/PS1 mice compared to their wild-type littermate controls ( p = 0.039), after heparin administration. In the transgenic mice, these spontaneous leakage sites frequently occurred at arteriolar segments without CAA at bifurcations or vessel bends. These findings suggest that the presence of vascular Aβ per se does not directly predispose vessels to leak, but that complex flow dynamics within CAA-affected vascular networks likely play a role. Our in vivo approach for the detection of individual spontaneous leakage sites may be used in longitudinal studies aimed to assess structural and functional alterations at the single-vessel level leading up to microhemorrhage formation.

Cerebral amyloid angiopathy is a small vessel disease associated with cortical microbleeds and lobar intracerebral hemorrhage due to amyloid-β deposition in the walls of leptomeningeal and cortical arterioles. The mechanisms of cerebral amyloid angiopathy-related hemorrhage remain largely unknown. Recent work has demonstrated that ruptured blood vessels have limited (or no) amyloid-β at the site of bleeding and evidence of local vascular remodeling. We hypothesized that blood-brain barrier leakage and perivascular inflammation may be involved in this remodeling process. This study examined cortical arterioles at various stages of cerebral amyloid angiopathy-related vascular pathology (without evidence of microhemorrhage) in autopsy tissue from seven cases with definite cerebral amyloid angiopathy. We included temporo-occipital sections with microbleeds guided by ex vivo MRI from two cases with severe cerebral amyloid angiopathy and systematically sampled occipital sections from five consecutive cases with varying cerebral amyloid angiopathy severity. Hematoxylin and eosin stains and immunohistochemistry against amyloid-β, fibrin(ogen), smooth muscle actin, reactive astrocytes (glial fibrillary acidic protein), and activated microglia (cluster of differentiation 68) were performed. Arterioles were graded using a previously proposed scale of individual vessel cerebral amyloid angiopathy severity, and a blinded assessment for blood-brain barrier leakage, smooth muscle actin, and perivascular inflammation was performed. Blood-brain barrier leakage and smooth muscle actin loss were observed in significantly more vessels with mild amyloid-β deposition (Grade 1 vessels; p = 0.044 and p = 0.012 respectively) as compared to vessels with no amyloid-β (Grade 0), and blood-brain barrier leakage was observed in 100% of vessels with evidence of vessel remodeling (Grade 3-4). Perivascular inflammation in the form of reactive astrocytes and activated microglia was observed predominantly surrounding arterioles at later stages of vessel pathology (Grades 2-4), and consistently around vessels with the same morphological features as ruptured vessel segments (Grade 4). These findings suggest a role for blood-brain barrier leakage and perivascular inflammation leading to arteriolar remodeling and hemorrhage in cerebral amyloid angiopathy, with early blood-brain barrier leakage as a potential trigger for subsequent perivascular inflammation.

Traditionally, analysis of neuropathological markers in neurodegenerative diseases has relied on visual assessments of stained sections. Resulting semiquantitative scores often vary between individual raters and research centers, limiting statistical approaches. To overcome these issues, we have developed six deep learning-based models, that identify some of the most characteristic markers of Alzheimer’s disease (AD) and cerebral amyloid angiopathy (CAA). The deep learning-based models are trained to differentially detect parenchymal amyloid β (Aβ)-plaques, vascular Aβ-deposition, iron and calcium deposition, reactive astrocytes, microglia, as well as fibrin extravasation. The models were trained on digitized histopathological slides from brains of patients with AD and CAA, using a workflow that allows neuropathology experts to train convolutional neural networks (CNNs) on a cloud-based graphical interface. Validation of all models indicated a very good to excellent performance compared to three independent expert human raters. Furthermore, the Aβ and iron models were consistent with previously acquired semiquantitative scores in the same dataset and allowed the use of more complex statistical approaches. For example, linear mixed effects models could be used to confirm the previously described relationship between leptomeningeal CAA severity and cortical iron accumulation. A similar approach enabled us to explore the association between neuroinflammation and disparate Aβ pathologies. The presented workflow is easy for researchers with pathological expertise to implement and is customizable for additional histopathological markers. The implementation of deep learning-assisted analyses of histopathological slides is likely to promote standardization of the assessment of neuropathological markers across research centers, which will allow specific pathophysiological questions in neurodegenerative disease to be addressed in a harmonized way and on a larger scale.

The impact of vascular lesions on cognition is location-dependent. Here, we assessed the contribution of small vessel disease lesions in the corpus callosum (CC) to vascular cognitive impairment in cerebral amyloid angiopathy, as a model for cerebral small vessel disease. 65 patients with probable cerebral amyloid angiopathy underwent 3 T MRI, including a diffusion tensor imaging scan, and neuropsychological testing. Microstructural white matter integrity was quantified by fractional anisotropy and mean diffusivity. Z-scores on individual neuropsychological tests were averaged into five cognitive domains: information processing speed, executive functioning, memory, language, and visuospatial ability. Corpus callosum lesions were defined as hemorrhagic (microbleeds or larger bleeds) or ischemic (microinfarcts, larger infarcts, diffuse fluid-attenuated inversion recovery hyperintensities). Associations between corpus callosum lesion presence, microstructural white matter integrity, and cognitive performance were examined with multiple regression models. The prevalence of corpus callosum lesions was confirmed in an independent cohort of memory clinic patients with and without cerebral amyloid angiopathy (n = 82). In parallel, we assessed corpus callosum lesions on ex vivo MRI in cerebral amyloid angiopathy patients (n = 19) and controls (n = 5) and determined associated tissue abnormalities with histopathology. A total number of 21 corpus callosum lesions was found in 19/65 (29%) cerebral amyloid angiopathy patients. Corpus callosum lesion presence was associated with reduced microstructural white matter integrity within the corpus callosum and in the whole brain white matter. Patients with corpus callosum lesions performed significantly worse on all cognitive domains except language, compared to those without corpus callosum lesions after correcting for age, sex, education, and time between MRI and neuropsychological assessment. This association was independent of presence of intracerebral hemorrhage, whole brain fractional anisotropy and mean diffusivity, and white matter hyperintensity volume and brain volume for the domains of information processing speed and executive functioning. In the memory clinic patient cohort, corpus callosum lesions were present in 14/54 (26%) patients with probable and 2/8 (25%) patients with possible cerebral amyloid angiopathy, and in 3/20 (15%) patients without cerebral amyloid angiopathy. In the ex vivo cohort, corpus callosum lesions were present in 10/19 (53%) patients and 2/5 (40%) controls. On histopathology, ischemic corpus callosum lesions were associated with tissue loss and demyelination, which extended beyond the lesion core. Together, these data suggest that corpus callosum lesions are a frequent finding in cerebral amyloid angiopathy, and that they independently contribute to cognitive impairment through strategic microstructural disruption of white matter tracts.