Published Ahead of Print on February 3, 2016 as 10.1212/WNL.0000000000002419
Heterogeneous histopathology of cortical microbleeds in cerebral amyloid angiopathy Susanne J. van Veluw, PhD Geert Jan Biessels, MD, PhD Catharina J.M. Klijn, MD, PhD Annemieke J.M. Rozemuller, MD, PhD
Correspondence to Dr. van Veluw: [email protected]
ABSTRACT
Objective: To investigate the histopathologic substrate of microbleeds detected on 7T postmortem MRI in autopsy cases with severe cerebral amyloid angiopathy (CAA) and Alzheimer pathology.
Methods: Five decedents (mean age at death 79.6 6 5.7 years) with documented severe CAA and Alzheimer pathology on standard neuropathologic examination were selected from a local database. Formalin-fixed coronal brain slices were scanned at 7T MRI, including high-resolution T2and T2*-weighted sequences. Representative microbleeds from each case were sampled for histopathologic analysis, including the presence of blood, blood breakdown products, and markers of ischemic tissue injury. Results: On MRI, we identified .300 cortical and 4 subcortical microbleeds. Two out of 15 sampled cortical microbleeds corresponded histologically to erythrocytes (suggestive of recent hemorrhages), 4 to vasculopathies (fibrinoid necrosis in 3 and a cavernoma) without substantial parenchymal tissue injury, and 9 to accumulations of iron-positive siderophages without erythrocytes (suggestive of old hemorrhages) combined with mild to moderate degrees of chronic ischemic tissue injury. Conclusions: This study provides evidence for heterogeneous pathologic substrates and possibly different pathophysiologic mechanisms underlying MRI-observed cortical microbleeds in the context of advanced CAA and Alzheimer disease. Neurology® 2016;86:1–5 GLOSSARY CAA 5 cerebral amyloid angiopathy; H&E 5 hematoxylin & eosin; MB 5 microbleed; TE 5 echo time; TR 5 repetition time; VUMC 5 VU Medical Centre.
Supplemental data at Neurology.org
Cerebral microbleeds (MBs) are a manifestation of small vessel disease on MRI, attracting increasing interest. They are associated with cerebrovascular disease and dementia, in particular Alzheimer disease. MBs are recognized as small, round, well-defined foci of low signal intensity on T2*-weighted MRI.1 Deep or infratentorial MBs have been related to hypertension, whereas cortical MBs may be indicative of cerebral amyloid angiopathy (CAA).2 MBs are believed to represent focal deposits of iron-positive blood breakdown products, causing the characteristic low signal on susceptibility-weighted MRI. However, the exact histopathology of MBs remains poorly understood and few studies have linked MRI features to histology.3 The presence of blood breakdown products implies that MRI-observed MBs are indicative of hemorrhagic processes, as inferred by their name. However, it has been suggested that MBs can also be related to hemorrhagic transformation after a primary ischemic proces,4 although direct evidence for the latter is lacking. In patients with CAA, both hemorrhagic and ischemic pathology is observed, but it is unclear how these processes relate to each other.5 This study explores histopathologic substrates of MBs detected on 7T postmortem MRI in 5 autopsy cases with severe CAA. From the Department of Neurology, Brain Center Rudolf Magnus (S.J.v.V., G.J.B., C.J.M.K.), and the Department of Pathology (A.J.M.R.), University Medical Center Utrecht; Department of Neurology, Donders Institute for Brain Cognition and Behaviour, Center for Neuroscience (C.J.M.K.), Radboud University Medical Center, Nijmegen; and Department of Pathology (A.J.M.R.), VU Medical Center, Amsterdam, the Netherlands. Go to Neurology.org for full disclosures. Funding information and disclosures deemed relevant by the authors, if any, are provided at the end of the article. © 2016 American Academy of Neurology
ª 2016 American Academy of Neurology. Unauthorized reproduction of this article is prohibited.
1
Figure 1
MRI-observed microbleeds in cerebral amyloid angiopathy correspond histopathologically to different stages of hemorrhagic tissue injury
Three cortical microbleeds in the postmortem brain tissue of an 84-year-old woman with severe cerebral amyloid angiopathy. On the T2-weighted postmortem MRI, 3 microbleeds (1–3) are visible (A), and matched on the corresponding hematoxylin & eosin section (B; black ink [encircled in gray] was applied to guide the retrieval of the MRI lesions). The whole section showed amyloid b positivity in many nearby vessels (C; taken at the level of microbleed 1). Microbleed 1 corresponded to an accumulation of siderophages (i.e., hemosiderin-containing macrophages; brown deposits) and hematoidin (yellow substance) (D). The adjacent section was iron-positive (inset in D; scale bar indicates 100 mm). Microbleed 2 corresponded to a strictly delineated round area containing intact erythrocytes (E). The adjacent section was only iron-positive on the edges of the lesion (not shown). Microbleed 3 corresponded to a focal accumulation of siderophages (F). The adjacent section was iron-positive (inset in F; scale bar indicates 100 mm). Note that the size of microbleed 2 is similar on MRI compared to histology, whereas microbleeds 1 and 3 are much larger on MRI compared to histology (possibly due to greater iron content).
METHODS Cases. From the neuropathology database of the VU Medical Centre (VUMC) Amsterdam, we selected 5 consecutive cases that had come to autopsy between 2010 and 2014 with severe CAA at neuropathologic evaluation. We selected formalin-fixed 10-mm-thick coronal brain slices, including areas from the frontal, temporoparietal, and occipital lobes. For each scan session, we submerged slices in 2
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10% formalin in a Perspex container designed to fit in the MRI head coil.
Standard protocol approvals, registrations, and patient consents. The use of the tissue was in accordance with local regulations and approved by the medical ethics committee of the University Medical Center Utrecht and VUMC.
March 1, 2016
ª 2016 American Academy of Neurology. Unauthorized reproduction of this article is prohibited.
MRI. Scans were acquired on a 7T MRI system (Philips Healthcare, Cleveland, OH) with a dual transmit and 32-channel receive head coil (Nova Medical, Wilmington, MA). The protocol included a T2-weighted (resolution 400 3 400 3 400 mm3, repetition time [TR] 3,500 ms, echo time [TE] 164 ms, scan duration 1 hour 52 minutes) and a T2*-weighted sequence (resolution 180 3 180 3 180 mm3, TR 75 ms, TE 20 ms, scan duration 4 hours 59 minutes). One rater screened the acquired images for MBs, defined as round or ovoid focal ,10 mm hypointense lesions on T2 and T2*.1 From each case we sampled at least 1 MB, if present, from each lobe (frontal, temporal, parietal, occipital). If present, we also sampled subcortical and hippocampal MBs.
Histopathology. Samples were dehydrated, embedded in paraffin, and cut in 4-mm-thick sections. Standard hematoxylin & eosin (H&E) was performed on the first section, and adjacent sections were stained for Ab and Perl iron. An experienced neuropathologist (A.J.M.R.) examined all lesions on H&E sections together with a second observer (S.J.v.V.). They noted presence of intact or lysed erythrocytes, indicative of recent hemorrhages, and presence of blood breakdown products (hematoidin, siderophages [i.e., hemosiderin-containing macrophages]), indicative of old hemorrhages. They also noted degree of gemistocytic astrocytes and tissue loss/edema observed in the presence of blood breakdown products. Based on the combination of these histopathologic observations, lesions were interpreted in terms of their primary etiology (e.g., likely to be of ischemic or hemorrhagic nature). Presence of red neurons, indicative of acute ischemic tissue injury, was also noted. The adjacent sections were examined for the presence of Ab and iron.
Case characteristics and MRI findings are provided in table e-1 on the Neurology® Web site at Neurology.org. The participants had a mean age at death of 79.6 6 5.7 years. All cases also proved to have significant Alzheimer pathology, but none showed evidence of primary intracerebral hemorrhage upon gross pathology. RESULTS
MRI. In total, we identified .300 cortical MBs in 4 out of 5 patients, one of whom had 3 MBs in the basal ganglia and one a hippocampal MB. One patient had no MBs. No MBs were observed in the white matter. Three cortical MBs had an atypical MRI appearance: 2 had an irregular shape and 1 an inhomogeneous signal Figure 2
intensity. Hence, 2 of these atypical MBs were sampled for histopathologic examination. Histopathology. Samples targeting 21 MRI-observed
MBs were subjected to histopathologic analysis, including 17 cortical MBs, 3 subcortical MBs, and 1 hippocampal MB. Two cortical MBs with typical MRI appearance could not be retrieved on histopathology, probably due to sampling errors. Detailed histopathologic findings of the retrieved MBs are described in table e-2. Recent hemorrhages. One cortical MB corresponded to intact erythrocytes, indicating an acute hemorrhage (figure 1E). One cortical atypical MB corresponded to partly lysed erythrocytes, accompanied by ironpositive siderophages and hematoidin (figure 2; this MB had an inhomogeneous MRI signal intensity), indicating a (sub) acute hemorrhage. In this MB, the ruptured vessel could be identified. Vasculopathies. Four cortical MBs corresponded to vasculopathies (3 fibrinoid necrosis [figure 2] and 1 a cavernoma [measuring ;500 mm on histology]) rather than parenchymal lesions. Old hemorrhages: Hemorrhagic microinfarcts. The remaining 9 cortical MBs corresponded to ironpositive focal or dispersed accumulations of siderophages, sometimes with hematoidin (n 5 2), but in the absence of erythrocytes (figure 1). All of these 9 lesions were accompanied by varying degrees of gemistocytic astrocytes and tissue loss/edema. Some were suggestive of old hemorrhages with a mild gliotic tissue response (figure 3D), others of chronic ischemic tissue injury with hemorrhagic transformation (figure 3B; this particular MB had an irregular shape on MRI). In some cases, interpretation in terms of the primary nature of the lesion remained inconclusive (table e-2). None of the targeted lesions showed evidence of acute ischemic injury, in the form of red neurons. The hippocampal MB corresponded to a subacute hemorrhage, characterized by lysed erythrocytes,
MRI-observed microbleeds in cerebral amyloid angiopathy correspond to hemorrhages and vasculopathies
Three cortical microbleeds in the postmortem brain tissue of an 84-year-old woman with severe cerebral amyloid angiopathy. The atypical microbleed 1 in (A) corresponds to the lesion in (B), which shows a ruptured vessel with extravasation of partly lysed erythrocytes. Microbleeds 2 and 3 in (A) correspond to the lesions in (C) and (D), respectively, which show 2 severely dilated thrombotic vessels with fibrin deposition and erythrocyte accumulation in the vessel wall, suggestive of fibrinoid necrosis (confirmed by a phosphotungstic acid/hematoxylin stain on an adjacent section). Neurology 86
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3
Figure 3
Presence of chronic ischemic tissue injury underlying MRI-observed microbleeds in cerebral amyloid angiopathy
Two cortical microbleeds in the postmortem brain tissue of an 81-year-old man with severe cerebral amyloid angiopathy. The atypical microbleed in (A) corresponds to the lesion in (B), which shows an area with pronounced tissue loss, accompanied by gemistocytic astrocytes (arrows), and a few dispersed siderophages (brown dots; broken arrows), suggestive of a hemorrhagic microinfarct. The adjacent section was iron-positive (inset in B; scale bar indicates 100 mm). Interestingly, this microbleed appeared more typical on lower resolution T2* (i.e., the resolution achieved on in vivo images). The microbleed in (C) corresponds to the lesion in (D), which shows a focal accumulation of siderophages (brown dots), gemistocytic astrocytes (arrows), and mild tissue loss, more suggestive of an old hemorrhage with a general gliotic response. The adjacent section was iron-positive (inset in D; scale bar indicates 100 mm).
accompanied by iron-positive siderophages. The basal ganglia MBs corresponded to accumulations of ironpositive siderophages, without erythrocytes and none to a mild degree of gemistocytic astrocytes and tissue loss (figure e-1). DISCUSSION The main finding of this study is that the underlying histopathology of MRI-observed cortical MBs in patients with severe CAA and Alzheimer pathology is heterogeneous, both in terms of lesion stage and the nature of the lesion. MBs represented vasculopathies, (sub) acute hemorrhages, old hemorrhages with mild to moderate degrees of ischemic tissue injury, or hemorrhagic microinfarcts. The vast majority (.98%) of observed MBs were located within the cortex. This is consistent with the fact that CAA predominantly affects cortical vessels, and has recently also been demonstrated in an in vivo 7T MRI study in CAA patients.6 Despite the fact that MBs have characteristic and well-defined imaging features,1 their underlying histopathology is 4
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heterogenous.3 In our study, only a small number of MBs corresponded to recent hemorrhagic events. The majority corresponded to focal accumulations of siderophages. Although evidence of a ruptured vessel —which would be proof of bleeding as underlying event—is often lacking, these lesions are generally interpreted as old hemorrhages. Siderophages were generally accompanied by reactive astrocytes, as has been reported before.7 This could be interpreted as a gliotic response to primary hemorrhagic injury. However, when reactive astrocytes are accompanied by pronounced tissue loss, an alternative hypothesis could be that this reflects primary ischemic tissue injury with hemorrhagic transformation. Remaining siderophages would then show up as MBs on MRI. Indeed, in our study we identified lesions resembling hemorrhagic microinfarcts, which have also been reported in previous studies.8–10 It should be noted that the cross-sectional nature of a histopathology study, such as ours, does not allow assessing the order of events, where the nature of the initial injury based
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on static histopathologic observations can be subjective. This should be further investigated in experimental studies, in which primary and secondary consequences of vessel rupture or occlusion on tissue can be assessed. When translating our findings to in vivo MRI, a few issues inherently related to ex vivo MRI need to be taken into consideration. First, ex vivo MRI may contain artefacts related to postmortem conditions (e.g., postmortem thrombi in perforating vessels, mistaken for MBs on ex vivo T2- and T2*-weighted MRI). Although we did not identify any falsepositives based on our histopathologic observations, it cannot be excluded that MBs not sampled for histology in our study may have been the result of such an artefact. Second, perimortem events may cause hemorrhages, which potentially could have resulted in an overestimation of acute hemorrhages in our sample. However, this is unlikely as we only observed one hemorrhage recent enough to be explained by such events (figure 1E). Finally, sensitivity issues play a role. With ex vivo MRI, a higher resolution can be obtained compared to in vivo, which may also explain the high number of observed MBs in this study, compared to reported MB number on clinical in vivo MRI.2 Therefore, it also remains to be seen to what extent heterogeneity of MBs may be distinguished on in vivo MRI. In this context, it would be interesting to pursue acute microinfarcts, as detected with diffusion-weighted MRI in patients, and examine whether a subset may evolve into a lesion manifesting itself as an MB on follow-up imaging. A limitation of this study is the small sample size. Nevertheless, within the 5 cases, we observed many MBs. Although there was heterogeneity in clinical histories, all cases included in this study were specifically selected for presence of severe CAA. The fact that all patients also proved to have concomitant Alzheimer pathology may be due to the fact that many patients undergoing autopsy were derived from the memory clinic at the VUMC. The histopathology of CAA-related MBs in other patient populations, for example patients with intracerebral hemorrhage, might be different, and should be studied further. Another limitation is that we did not obtain whole brain coverage by scanning approximately 3 slabs per case. This may explain why we did not observe any MBs on MRI in 1 case. Our study highlights that uniform-appearing MBs in the context of severe CAA have heterogeneous pathologic substrates likely reflecting different etiologies. Further studies into the role of ischemia in MB formation may improve our understanding of the etiology of CAA-related tissue injury.
AUTHOR CONTRIBUTIONS Susanne J. van Veluw was involved in the concept and design of the study, acquisition, analysis, and interpretation of the data, drafting and revising of the manuscript. Geert Jan Biessels was involved in study supervision, obtaining funding, concept and design of the study, interpretation of the data, drafting and revising of the manuscript. Catharina J.M. Klijn was involved in concept and design of the study, interpretation of the data, revising the manuscript. Annemieke J.M. Rozemuller was involved in acquisition of the data, interpretation of the data, and revising of the manuscript.
STUDY FUNDING Supported by grants from ZonMw (Vidi), The Netherlands Organization for Health Research and Development (91711384), and the Netherlands Heart Foundation (2010 T073) to G.J.B. C.J.M.K. is supported by a clinical established investigator grant of the Netherlands Heart Foundation (2012 T077) and an ASPASIA grant from ZonMw (015008048).
DISCLOSURE The authors report no disclosures relevant to the manuscript. Go to Neurology.org for full disclosures.
Received June 29, 2015. Accepted in final form November 5, 2015. REFERENCES 1. Gregoire SM, Chaudhary UJ, Brown MM, et al. The Microbleed Anatomical Rating Scale (MARS): reliability of a tool to map brain microbleeds. Neurology 2009;73: 1759–1766. 2. Vernooij MW, van der Lugt A, Ikram MA, et al. Prevalence and risk factors of cerebral microbleeds: the Rotterdam Scan Study. Neurology 2008;70:1208–1214. 3. Shoamanesh A, Kwok CS, Benavente O. Cerebral microbleeds: histopathological correlation of neuroimaging. Cerebrovasc Dis 2011;32:528–534. 4. Fisher M. Cerebral microbleeds: where are we now? Neurology 2014;83:1304–1305. 5. Reijmer YD, van Veluw SJ, Greenberg SM. Ischemic brain injury in cerebral amyloid angiopathy. J Cereb Blood Flow Metab Epub 2015 May 6. 6. Ni J, Auriel E, Martinez-Ramirez S, et al. Cortical localization of microbleeds in cerebral amyloid angiopathy: an ultra high-field 7T MRI study. J Alzheimers Dis 2015;43: 1325–1330. 7. Schrag M, McAuley G, Pomakian J, et al. Correlation of hypointensities in susceptibility-weighted images to tissue histology in dementia patients with cerebral amyloid angiopathy: a postmortem MRI study. AJNR Am J Neuroradiol 1999;20:637–642. 8. Van Veluw SJ, Zwanenburg JJ, Rozemuller AJ, Luijten PR, Spliet WG, Biessels GJ. The spectrum of MR detectable cortical microinfarcts: a classification study with 7-tesla postmortem MRI and histopathology. J Cereb Blood Flow Metab 2015;35:676–683. 9. Transkanen M, Mäkelä M, Myllykangas L, Rastas S, Sulkava R, Paetau A. Intracerebral hemorrhage in the oldest old: a population-based study (Vantaa 851). Front Neurol 2012;3:103. 10. Janaway BM, Simpson JE, Hoggard N, et al. Brain haemosiderin in older people: pathological evidence for an ischaemic origin of magnetic resonance imaging (MRI) microbleeds. Neuropathol Appl Neurobiol 2014;40: 258–269.
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Heterogeneous histopathology of cortical microbleeds in cerebral amyloid angiopathy Susanne J. van Veluw, Geert Jan Biessels, Catharina J.M. Klijn, et al. Neurology published online February 3, 2016 DOI 10.1212/WNL.0000000000002419 This information is current as of February 3, 2016 Updated Information & Services
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Neurology ® is the official journal of the American Academy of Neurology. Published continuously since 1951, it is now a weekly with 48 issues per year. Copyright © 2016 American Academy of Neurology. All rights reserved. Print ISSN: 0028-3878. Online ISSN: 1526-632X.
Heterogeneous histopathology of cortical microbleeds in cerebral amyloid angiopathy Susanne J. van Veluw, PhD Geert Jan Biessels, MD, PhD Catharina J.M. Klijn, MD, PhD Annemieke J.M. Rozemuller, MD, PhD
Correspondence to Dr. van Veluw: [email protected]
ABSTRACT
Objective: To investigate the histopathologic substrate of microbleeds detected on 7T postmortem MRI in autopsy cases with severe cerebral amyloid angiopathy (CAA) and Alzheimer pathology.
Methods: Five decedents (mean age at death 79.6 6 5.7 years) with documented severe CAA and Alzheimer pathology on standard neuropathologic examination were selected from a local database. Formalin-fixed coronal brain slices were scanned at 7T MRI, including high-resolution T2and T2*-weighted sequences. Representative microbleeds from each case were sampled for histopathologic analysis, including the presence of blood, blood breakdown products, and markers of ischemic tissue injury. Results: On MRI, we identified .300 cortical and 4 subcortical microbleeds. Two out of 15 sampled cortical microbleeds corresponded histologically to erythrocytes (suggestive of recent hemorrhages), 4 to vasculopathies (fibrinoid necrosis in 3 and a cavernoma) without substantial parenchymal tissue injury, and 9 to accumulations of iron-positive siderophages without erythrocytes (suggestive of old hemorrhages) combined with mild to moderate degrees of chronic ischemic tissue injury. Conclusions: This study provides evidence for heterogeneous pathologic substrates and possibly different pathophysiologic mechanisms underlying MRI-observed cortical microbleeds in the context of advanced CAA and Alzheimer disease. Neurology® 2016;86:1–5 GLOSSARY CAA 5 cerebral amyloid angiopathy; H&E 5 hematoxylin & eosin; MB 5 microbleed; TE 5 echo time; TR 5 repetition time; VUMC 5 VU Medical Centre.
Supplemental data at Neurology.org
Cerebral microbleeds (MBs) are a manifestation of small vessel disease on MRI, attracting increasing interest. They are associated with cerebrovascular disease and dementia, in particular Alzheimer disease. MBs are recognized as small, round, well-defined foci of low signal intensity on T2*-weighted MRI.1 Deep or infratentorial MBs have been related to hypertension, whereas cortical MBs may be indicative of cerebral amyloid angiopathy (CAA).2 MBs are believed to represent focal deposits of iron-positive blood breakdown products, causing the characteristic low signal on susceptibility-weighted MRI. However, the exact histopathology of MBs remains poorly understood and few studies have linked MRI features to histology.3 The presence of blood breakdown products implies that MRI-observed MBs are indicative of hemorrhagic processes, as inferred by their name. However, it has been suggested that MBs can also be related to hemorrhagic transformation after a primary ischemic proces,4 although direct evidence for the latter is lacking. In patients with CAA, both hemorrhagic and ischemic pathology is observed, but it is unclear how these processes relate to each other.5 This study explores histopathologic substrates of MBs detected on 7T postmortem MRI in 5 autopsy cases with severe CAA. From the Department of Neurology, Brain Center Rudolf Magnus (S.J.v.V., G.J.B., C.J.M.K.), and the Department of Pathology (A.J.M.R.), University Medical Center Utrecht; Department of Neurology, Donders Institute for Brain Cognition and Behaviour, Center for Neuroscience (C.J.M.K.), Radboud University Medical Center, Nijmegen; and Department of Pathology (A.J.M.R.), VU Medical Center, Amsterdam, the Netherlands. Go to Neurology.org for full disclosures. Funding information and disclosures deemed relevant by the authors, if any, are provided at the end of the article. © 2016 American Academy of Neurology
ª 2016 American Academy of Neurology. Unauthorized reproduction of this article is prohibited.
1
Figure 1
MRI-observed microbleeds in cerebral amyloid angiopathy correspond histopathologically to different stages of hemorrhagic tissue injury
Three cortical microbleeds in the postmortem brain tissue of an 84-year-old woman with severe cerebral amyloid angiopathy. On the T2-weighted postmortem MRI, 3 microbleeds (1–3) are visible (A), and matched on the corresponding hematoxylin & eosin section (B; black ink [encircled in gray] was applied to guide the retrieval of the MRI lesions). The whole section showed amyloid b positivity in many nearby vessels (C; taken at the level of microbleed 1). Microbleed 1 corresponded to an accumulation of siderophages (i.e., hemosiderin-containing macrophages; brown deposits) and hematoidin (yellow substance) (D). The adjacent section was iron-positive (inset in D; scale bar indicates 100 mm). Microbleed 2 corresponded to a strictly delineated round area containing intact erythrocytes (E). The adjacent section was only iron-positive on the edges of the lesion (not shown). Microbleed 3 corresponded to a focal accumulation of siderophages (F). The adjacent section was iron-positive (inset in F; scale bar indicates 100 mm). Note that the size of microbleed 2 is similar on MRI compared to histology, whereas microbleeds 1 and 3 are much larger on MRI compared to histology (possibly due to greater iron content).
METHODS Cases. From the neuropathology database of the VU Medical Centre (VUMC) Amsterdam, we selected 5 consecutive cases that had come to autopsy between 2010 and 2014 with severe CAA at neuropathologic evaluation. We selected formalin-fixed 10-mm-thick coronal brain slices, including areas from the frontal, temporoparietal, and occipital lobes. For each scan session, we submerged slices in 2
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10% formalin in a Perspex container designed to fit in the MRI head coil.
Standard protocol approvals, registrations, and patient consents. The use of the tissue was in accordance with local regulations and approved by the medical ethics committee of the University Medical Center Utrecht and VUMC.
March 1, 2016
ª 2016 American Academy of Neurology. Unauthorized reproduction of this article is prohibited.
MRI. Scans were acquired on a 7T MRI system (Philips Healthcare, Cleveland, OH) with a dual transmit and 32-channel receive head coil (Nova Medical, Wilmington, MA). The protocol included a T2-weighted (resolution 400 3 400 3 400 mm3, repetition time [TR] 3,500 ms, echo time [TE] 164 ms, scan duration 1 hour 52 minutes) and a T2*-weighted sequence (resolution 180 3 180 3 180 mm3, TR 75 ms, TE 20 ms, scan duration 4 hours 59 minutes). One rater screened the acquired images for MBs, defined as round or ovoid focal ,10 mm hypointense lesions on T2 and T2*.1 From each case we sampled at least 1 MB, if present, from each lobe (frontal, temporal, parietal, occipital). If present, we also sampled subcortical and hippocampal MBs.
Histopathology. Samples were dehydrated, embedded in paraffin, and cut in 4-mm-thick sections. Standard hematoxylin & eosin (H&E) was performed on the first section, and adjacent sections were stained for Ab and Perl iron. An experienced neuropathologist (A.J.M.R.) examined all lesions on H&E sections together with a second observer (S.J.v.V.). They noted presence of intact or lysed erythrocytes, indicative of recent hemorrhages, and presence of blood breakdown products (hematoidin, siderophages [i.e., hemosiderin-containing macrophages]), indicative of old hemorrhages. They also noted degree of gemistocytic astrocytes and tissue loss/edema observed in the presence of blood breakdown products. Based on the combination of these histopathologic observations, lesions were interpreted in terms of their primary etiology (e.g., likely to be of ischemic or hemorrhagic nature). Presence of red neurons, indicative of acute ischemic tissue injury, was also noted. The adjacent sections were examined for the presence of Ab and iron.
Case characteristics and MRI findings are provided in table e-1 on the Neurology® Web site at Neurology.org. The participants had a mean age at death of 79.6 6 5.7 years. All cases also proved to have significant Alzheimer pathology, but none showed evidence of primary intracerebral hemorrhage upon gross pathology. RESULTS
MRI. In total, we identified .300 cortical MBs in 4 out of 5 patients, one of whom had 3 MBs in the basal ganglia and one a hippocampal MB. One patient had no MBs. No MBs were observed in the white matter. Three cortical MBs had an atypical MRI appearance: 2 had an irregular shape and 1 an inhomogeneous signal Figure 2
intensity. Hence, 2 of these atypical MBs were sampled for histopathologic examination. Histopathology. Samples targeting 21 MRI-observed
MBs were subjected to histopathologic analysis, including 17 cortical MBs, 3 subcortical MBs, and 1 hippocampal MB. Two cortical MBs with typical MRI appearance could not be retrieved on histopathology, probably due to sampling errors. Detailed histopathologic findings of the retrieved MBs are described in table e-2. Recent hemorrhages. One cortical MB corresponded to intact erythrocytes, indicating an acute hemorrhage (figure 1E). One cortical atypical MB corresponded to partly lysed erythrocytes, accompanied by ironpositive siderophages and hematoidin (figure 2; this MB had an inhomogeneous MRI signal intensity), indicating a (sub) acute hemorrhage. In this MB, the ruptured vessel could be identified. Vasculopathies. Four cortical MBs corresponded to vasculopathies (3 fibrinoid necrosis [figure 2] and 1 a cavernoma [measuring ;500 mm on histology]) rather than parenchymal lesions. Old hemorrhages: Hemorrhagic microinfarcts. The remaining 9 cortical MBs corresponded to ironpositive focal or dispersed accumulations of siderophages, sometimes with hematoidin (n 5 2), but in the absence of erythrocytes (figure 1). All of these 9 lesions were accompanied by varying degrees of gemistocytic astrocytes and tissue loss/edema. Some were suggestive of old hemorrhages with a mild gliotic tissue response (figure 3D), others of chronic ischemic tissue injury with hemorrhagic transformation (figure 3B; this particular MB had an irregular shape on MRI). In some cases, interpretation in terms of the primary nature of the lesion remained inconclusive (table e-2). None of the targeted lesions showed evidence of acute ischemic injury, in the form of red neurons. The hippocampal MB corresponded to a subacute hemorrhage, characterized by lysed erythrocytes,
MRI-observed microbleeds in cerebral amyloid angiopathy correspond to hemorrhages and vasculopathies
Three cortical microbleeds in the postmortem brain tissue of an 84-year-old woman with severe cerebral amyloid angiopathy. The atypical microbleed 1 in (A) corresponds to the lesion in (B), which shows a ruptured vessel with extravasation of partly lysed erythrocytes. Microbleeds 2 and 3 in (A) correspond to the lesions in (C) and (D), respectively, which show 2 severely dilated thrombotic vessels with fibrin deposition and erythrocyte accumulation in the vessel wall, suggestive of fibrinoid necrosis (confirmed by a phosphotungstic acid/hematoxylin stain on an adjacent section). Neurology 86
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3
Figure 3
Presence of chronic ischemic tissue injury underlying MRI-observed microbleeds in cerebral amyloid angiopathy
Two cortical microbleeds in the postmortem brain tissue of an 81-year-old man with severe cerebral amyloid angiopathy. The atypical microbleed in (A) corresponds to the lesion in (B), which shows an area with pronounced tissue loss, accompanied by gemistocytic astrocytes (arrows), and a few dispersed siderophages (brown dots; broken arrows), suggestive of a hemorrhagic microinfarct. The adjacent section was iron-positive (inset in B; scale bar indicates 100 mm). Interestingly, this microbleed appeared more typical on lower resolution T2* (i.e., the resolution achieved on in vivo images). The microbleed in (C) corresponds to the lesion in (D), which shows a focal accumulation of siderophages (brown dots), gemistocytic astrocytes (arrows), and mild tissue loss, more suggestive of an old hemorrhage with a general gliotic response. The adjacent section was iron-positive (inset in D; scale bar indicates 100 mm).
accompanied by iron-positive siderophages. The basal ganglia MBs corresponded to accumulations of ironpositive siderophages, without erythrocytes and none to a mild degree of gemistocytic astrocytes and tissue loss (figure e-1). DISCUSSION The main finding of this study is that the underlying histopathology of MRI-observed cortical MBs in patients with severe CAA and Alzheimer pathology is heterogeneous, both in terms of lesion stage and the nature of the lesion. MBs represented vasculopathies, (sub) acute hemorrhages, old hemorrhages with mild to moderate degrees of ischemic tissue injury, or hemorrhagic microinfarcts. The vast majority (.98%) of observed MBs were located within the cortex. This is consistent with the fact that CAA predominantly affects cortical vessels, and has recently also been demonstrated in an in vivo 7T MRI study in CAA patients.6 Despite the fact that MBs have characteristic and well-defined imaging features,1 their underlying histopathology is 4
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heterogenous.3 In our study, only a small number of MBs corresponded to recent hemorrhagic events. The majority corresponded to focal accumulations of siderophages. Although evidence of a ruptured vessel —which would be proof of bleeding as underlying event—is often lacking, these lesions are generally interpreted as old hemorrhages. Siderophages were generally accompanied by reactive astrocytes, as has been reported before.7 This could be interpreted as a gliotic response to primary hemorrhagic injury. However, when reactive astrocytes are accompanied by pronounced tissue loss, an alternative hypothesis could be that this reflects primary ischemic tissue injury with hemorrhagic transformation. Remaining siderophages would then show up as MBs on MRI. Indeed, in our study we identified lesions resembling hemorrhagic microinfarcts, which have also been reported in previous studies.8–10 It should be noted that the cross-sectional nature of a histopathology study, such as ours, does not allow assessing the order of events, where the nature of the initial injury based
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on static histopathologic observations can be subjective. This should be further investigated in experimental studies, in which primary and secondary consequences of vessel rupture or occlusion on tissue can be assessed. When translating our findings to in vivo MRI, a few issues inherently related to ex vivo MRI need to be taken into consideration. First, ex vivo MRI may contain artefacts related to postmortem conditions (e.g., postmortem thrombi in perforating vessels, mistaken for MBs on ex vivo T2- and T2*-weighted MRI). Although we did not identify any falsepositives based on our histopathologic observations, it cannot be excluded that MBs not sampled for histology in our study may have been the result of such an artefact. Second, perimortem events may cause hemorrhages, which potentially could have resulted in an overestimation of acute hemorrhages in our sample. However, this is unlikely as we only observed one hemorrhage recent enough to be explained by such events (figure 1E). Finally, sensitivity issues play a role. With ex vivo MRI, a higher resolution can be obtained compared to in vivo, which may also explain the high number of observed MBs in this study, compared to reported MB number on clinical in vivo MRI.2 Therefore, it also remains to be seen to what extent heterogeneity of MBs may be distinguished on in vivo MRI. In this context, it would be interesting to pursue acute microinfarcts, as detected with diffusion-weighted MRI in patients, and examine whether a subset may evolve into a lesion manifesting itself as an MB on follow-up imaging. A limitation of this study is the small sample size. Nevertheless, within the 5 cases, we observed many MBs. Although there was heterogeneity in clinical histories, all cases included in this study were specifically selected for presence of severe CAA. The fact that all patients also proved to have concomitant Alzheimer pathology may be due to the fact that many patients undergoing autopsy were derived from the memory clinic at the VUMC. The histopathology of CAA-related MBs in other patient populations, for example patients with intracerebral hemorrhage, might be different, and should be studied further. Another limitation is that we did not obtain whole brain coverage by scanning approximately 3 slabs per case. This may explain why we did not observe any MBs on MRI in 1 case. Our study highlights that uniform-appearing MBs in the context of severe CAA have heterogeneous pathologic substrates likely reflecting different etiologies. Further studies into the role of ischemia in MB formation may improve our understanding of the etiology of CAA-related tissue injury.
AUTHOR CONTRIBUTIONS Susanne J. van Veluw was involved in the concept and design of the study, acquisition, analysis, and interpretation of the data, drafting and revising of the manuscript. Geert Jan Biessels was involved in study supervision, obtaining funding, concept and design of the study, interpretation of the data, drafting and revising of the manuscript. Catharina J.M. Klijn was involved in concept and design of the study, interpretation of the data, revising the manuscript. Annemieke J.M. Rozemuller was involved in acquisition of the data, interpretation of the data, and revising of the manuscript.
STUDY FUNDING Supported by grants from ZonMw (Vidi), The Netherlands Organization for Health Research and Development (91711384), and the Netherlands Heart Foundation (2010 T073) to G.J.B. C.J.M.K. is supported by a clinical established investigator grant of the Netherlands Heart Foundation (2012 T077) and an ASPASIA grant from ZonMw (015008048).
DISCLOSURE The authors report no disclosures relevant to the manuscript. Go to Neurology.org for full disclosures.
Received June 29, 2015. Accepted in final form November 5, 2015. REFERENCES 1. Gregoire SM, Chaudhary UJ, Brown MM, et al. The Microbleed Anatomical Rating Scale (MARS): reliability of a tool to map brain microbleeds. Neurology 2009;73: 1759–1766. 2. Vernooij MW, van der Lugt A, Ikram MA, et al. Prevalence and risk factors of cerebral microbleeds: the Rotterdam Scan Study. Neurology 2008;70:1208–1214. 3. Shoamanesh A, Kwok CS, Benavente O. Cerebral microbleeds: histopathological correlation of neuroimaging. Cerebrovasc Dis 2011;32:528–534. 4. Fisher M. Cerebral microbleeds: where are we now? Neurology 2014;83:1304–1305. 5. Reijmer YD, van Veluw SJ, Greenberg SM. Ischemic brain injury in cerebral amyloid angiopathy. J Cereb Blood Flow Metab Epub 2015 May 6. 6. Ni J, Auriel E, Martinez-Ramirez S, et al. Cortical localization of microbleeds in cerebral amyloid angiopathy: an ultra high-field 7T MRI study. J Alzheimers Dis 2015;43: 1325–1330. 7. Schrag M, McAuley G, Pomakian J, et al. Correlation of hypointensities in susceptibility-weighted images to tissue histology in dementia patients with cerebral amyloid angiopathy: a postmortem MRI study. AJNR Am J Neuroradiol 1999;20:637–642. 8. Van Veluw SJ, Zwanenburg JJ, Rozemuller AJ, Luijten PR, Spliet WG, Biessels GJ. The spectrum of MR detectable cortical microinfarcts: a classification study with 7-tesla postmortem MRI and histopathology. J Cereb Blood Flow Metab 2015;35:676–683. 9. Transkanen M, Mäkelä M, Myllykangas L, Rastas S, Sulkava R, Paetau A. Intracerebral hemorrhage in the oldest old: a population-based study (Vantaa 851). Front Neurol 2012;3:103. 10. Janaway BM, Simpson JE, Hoggard N, et al. Brain haemosiderin in older people: pathological evidence for an ischaemic origin of magnetic resonance imaging (MRI) microbleeds. Neuropathol Appl Neurobiol 2014;40: 258–269.
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Heterogeneous histopathology of cortical microbleeds in cerebral amyloid angiopathy Susanne J. van Veluw, Geert Jan Biessels, Catharina J.M. Klijn, et al. Neurology published online February 3, 2016 DOI 10.1212/WNL.0000000000002419 This information is current as of February 3, 2016 Updated Information & Services
including high resolution figures, can be found at: http://www.neurology.org/content/early/2016/02/03/WNL.0000000000 002419.full.html
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Supplementary material can be found at: http://www.neurology.org/content/suppl/2016/02/03/WNL.000000000 0002419.DC1.html
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This article, along with others on similar topics, appears in the following collection(s): Alzheimer's disease http://www.neurology.org//cgi/collection/alzheimers_disease Infarction http://www.neurology.org//cgi/collection/infarction Intracerebral hemorrhage http://www.neurology.org//cgi/collection/intracerebral_hemorrhage MRI http://www.neurology.org//cgi/collection/mri
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