Neuropathology 2011; ••, ••–•• C a se Repor t neup_1283 doi:10.1111/j.1440-1789.2011.01283.x 1..6 Multiple cerebral infarcts with a few vasculitic lesions in the chronic stage of cerebral amyloid angiopathy-related inflammation Kenji Sakai,1,2 Shintaro Hayashi,1 Kazuhiro Sanpei,3 Masahito Yamada2 and Hitoshi Takahashi1 1 Department of Pathology, Brain Research Institute, University of Niigata, Niigata, 2Department of Neurology and Neurobiology of Aging, Kanazawa University Graduate School of Medical Science, Kanazawa and 3Department of Neurology, Sado General Hospital, Sado, Japan We report a 75-year-old man with a 3.5-year history of cerebral amyloid angiopathy (CAA)-related inflammation. His initial symptom was headache and sensory aphasia appeared 1 month later. Brain MRI revealed features compatible with meningoencephalitis involving the right frontal, parietal and temporooccipital lobes. A brain biopsy sample from the right parietal lobe showed thickening of the leptomeninges, and granulomatous vasculitis with multinucleated giant cells and vascular Ab deposits. No vascular lesions were evident by cerebral angiography. Serological examination revealed an elevated level of proteinase 3 anti-neutrophil cytoplasmic autoantibodies (PR3-ANCA). The patient was treated with corticosteroids, but this was only partially and temporarily effective. Autopsy revealed marked leptomeningeal thickening with inflammatory cell infiltrates and hemosiderin deposits, many superficial predominantly small infarcts at various stages in the cerebral cortex and only a few cerebral active vasculitic lesions. Immunohistochemically, CAA showing widespread Ab-positive blood vessels with double-barrel formations was demonstrated. In conclusion, we consider that, although the association of PR3-ANCA with the pathogenesis of Ab-associated vasculitis remained unclear, the present case represents a rare example of CAA-related inflammation at the chronic stage. Key words: Ab, CAA-related inflammation, cerebral amyloid angiopathy, cerebral infarct, PR3-ANCA. Correspondence: Kenji Sakai, MD, PhD, Department of Neurology and Neurobiology of Aging, Kanazawa University Graduate School of Medical Science, 13-1 Takara-machi, Kanazawa 920-8640, Japan. Email: ksakai@med.kanazawa-u.ac.jp Received 23 September 2011; revised and accepted 8 November 2011. © 2011 Japanese Society of Neuropathology INTRODUCTION Primary angiitis of the central nervous system (PACNS) is a form of vasculitis confined to the brain and spinal cord without apparent evidence of systemic vasculitis. Recently, it has became clear that a proportion of patients with PACNS show cerebral amyloid angiopathy (CAA) associated with Ab-induced vasculitic lesions (Ab-related angiitis).1–3 This pathological condition is now designated as CAA-related inflammation, and affected patients develop subacute- or acute-onset headache, seizures, focal cortical deficits and rarely, recurrent cerebral lobar hemorrhage.4 It is also important to note that immunosuppressive therapy can be partly effective.4,5 The neuropathology of CAA-related inflammation is characterized by vascular Ab amyloid deposition accompanied by granulomatous vasculitis in the subarachnoid space and cortices, infiltration of lymphocytes and macrophages, and occurrence of multinucleated giant cells. The lesions may be associated with acute thrombosis and/or evidence of old thrombosis with recanalization.2–4,6–15 In addition to the inflammatory lesions, necrotic foci, brain edema with herniation, hemorrhages and multiple infarcts may be evident in the affected brain.2–4,6–15 In most patients, the histological diagnosis is obtained only by brain biopsy in the acute stage,4 and the majority of post mortem brain examinations have been performed in patients after a clinical course of several months after onset.2,3,7–9,11–15 Up to now, post mortem studies of patients with a clinical course of several years have been rare.6,10 Therefore, the neuropathological features of this disorder in the chronic stage still remain unclear. Here we describe the clinicopathological findings of a biopsy-confirmed case of CAA-related inflammation after a disease duration of 3.5 years in a 75-year-old man who 2 K Sakai et al. exhibited a partial, temporary clinical response to corticosteroid therapy during the illness. The evident neuropathology was considered to represent the advanced, chronic stage of the disease. CLINICAL SUMMARY A 71-year-old Japanese man developed headache and 1 month later became unable to speak, read or write. On examination, sensory aphasia was evident. Brain MRI revealed hyperintense and hypointense mosaic lesions with swelling of the right temporo-occipital lobe (Fig. 1a). Two weeks later, he developed left-sided hemiparesis and became unable to walk. The lesions extended to the right frontal lobe with contrast enhancement along the affected gyri (Fig. 1b). Serological examination revealed an elevated level of proteinase 3 anti-neutrophil cytoplasmic autoantibodies (PR3-ANCA).The serum levels of myeloperoxidase ANCA (MPO-ANCA) and angiotensin converting enzyme were unremarkable. Examination of the CSF revealed mononuclear pleocytosis (31/mL) and an elevated protein level (115 mg/dL). Cerebral angiography showed no abnormality. Biopsy samples of the brain and leptomeninges taken from the right parietal lobe region demonstrated inflammatory granulomatous vasculitis with perivascular infiltration of lymphocytes and occurrence of multinucle- ated giant cells, leptomeningeal thickening and reactive astrocytosis in the adjacent brain parenchyma (Fig. 2a). Immunostaining revealed Ab-amyloid deposits in the affected blood vessel walls (Fig. 2b). No inflammatory cells containing Ab in their cytoplasm were detected.The patient was treated with corticosteroids, which elicited a partial, temporary improvement of the symptoms. Thereafter, he became bedridden with advanced hemiparesis and subsequently developed flexion contracture in the extremities. By the age of 73 years, the patient was maintained by tube feeding, and showed flexion contracture with complete quadriparesis and mutism. Although the elevated serum PR3-ANCA level was maintained, the immunosuppressive therapy was abandoned 33 months prior to death because of repeated infection (particularly pneumonia). CT scans with contrast enhancement revealed progression of the cerebral atrophy with extension of the low-density, non-enhanced lesions (Fig. 1c). At the age of 75 years, the patient died due to pneumonia, about 3 years and 6 months after disease onset. There was no familial history of neurological disorders and the patient’s history was non-contributory. A general autopsy was performed 5 h after death. Severe pneumonia in both lungs was confirmed. There was no histological evidence of systemic vasculitis in the visceral organs. The brain weighed 1170 g before fixation. Fig. 1 Brain MRI and CT scan. Axial T2-weighted image, showing hypointense and hyperintense mosaic lesions in the right temporo-occipital lobe (a). T1-weighted image, showing enhanced lesions along the affected gyri in the right frontal lobe (b). CT scan with contrast enhancement at the age of 73 years (about 26 months after disease onset), showing low-density, non-enhanced areas that are more severe in the right hemisphere (c). Fig. 2 Biopsied tissue from the right parietal lobe, showing inflammatory granulomatous vasculitis and Ab-amyloid deposits in the walls. Note a multinucleated giant cell (arrow) (a, b). (a) HE (b) Ab-amyloid immunostain. Bar = 50 mm (a, b). © 2011 Japanese Society of Neuropathology Infarcts with CAA-related inflammation PATHOLOGICAL FINDINGS The brain and spinal cord were fixed with 20% buffered formalin. Thickening of the leptomeninges and generalized cerebral atrophy with an uneven surface appearance were evident (Fig. 3a). In sections, multiple small necrotic foci were evident in the frontal and temporal cortices. The cerebral white matter also appeared to be atrophic and discolored. The basal ganglia, thalamus, hippocampal formation, brainstem and cerebellum were unremarkable. Fig. 3 Post mortem neuropathological findings. (a) Lateral view of the right cerebral hemisphere. A tissue defect due to the brain biopsy is indicated by an arrow. (b) Coronal section of the right parietal lobe, showing meningeal thickening, multiple lacunae and myelin pallor. (c) The motor cortex, showing a lesion consisting of marked leptomeningeal thickening, cortical rarefaction with hemosiderin deposits, mild inflammatory cell infiltration, and blood vessels with double-barrel formation (arrows). (d) Superficial cortical infarct, old and cystic, in the temporal lobe. (e) A blood vessel in the frontal lobe, showing evidence of vasculitis and associated surrounding ring hemorrhage. No multinucleated giant cells were evident here. (f, g) Many blood vessels, both subarachnoidal and intracerebral show Ab-amyloid deposits in their walls. A blood vessel with vasculitis shows no immunoreactivity for Ab-amyloid (arrow). (b) KB (c–e) HE (f, g) Ab-amyloid immunostaining. Bars = 1 cm (a, b), 100 mm (c–g). © 2011 Japanese Society of Neuropathology 3 For histological examination, multiple tissue blocks were embedded in paraffin, and sections 4 mm thick were cut and stained with HE, KB, Congo Red and Elastica-Goldner. Selected sections were also immunostained using the ABC method with a Vectastain ABC kit (Vector, Burlingame, CA, USA) with diaminobenzidine as the chromogen. The immunostained sections were counterstained with hematoxylin. The primary antibodies used were rabbit polyclonal antibodies against GFAP (Dako; Glöstrup, Denmark; 1:2000) and ubiquitin (Dako; 1:800), and mouse monoclonal antibodies against 4 K Sakai et al. phosphorylation-dependent tau (AT8; Innogenetics, Ghent, Belgium; 1:200) and Ab-amyloid (Dako; 1:100). In the cerebrum, leptomeningeal thickening was found in association with hemosiderin deposits and infiltration of inflammatory cells, including plasma cells, small lymphocytes and macrophages, being more apparent in the posterior frontal and parietal cortices (Fig. 3b,c). Multiple widespread infarcts, both recent and old, and located more frequently in the cerebral superficial cortical layers, were a notable feature (Fig. 3b,d). Active vasculitis was evident in only a few blood vessels of the frontal cortex, exhibiting mural destruction with infiltration of small lymphocytes (Fig. 3e). A few other blood vessels also exhibited evidence of old thrombus formation with recanalization, presumably due to vasculitis. A large number of blood vessels were found to show hyalinous thickening and splitting of the walls (double barrel) (Fig. 3c,d).These affected vessel walls were stained with Congo Red and displayed apple-green birefringence in polarized light, being also immunoreative for Ab-amyloid (Fig. 3f,g). In the blood vessels showing active vasculitis, no Ab immunoreactivity was evident (Fig. 3g). Many Ab-amyloid deposits (diffuse-type senile plaques) were observed throughout the entire cerebral cortex. Mild neuronal loss was noted in the entorhinal cortex, amygdaloid nucleus and Meynert nucleus. Scattered tau-positive pretangles/tangles were encountered in the above areas and part of the cerebral neocortex (Braak & Braak stage III).16 DISCUSSION In the present patient, headache developed as the initial manifestation, followed shortly by higher cerebral dysfunction (sensory aphasia) with corresponding cerebral organic lesions (ischemia). An elevated serum PR3-ANCA level, which continued during the illness, was a major feature reflecting the patient’s disease condition. The neuropathology was characterized by severe CAA showing blood vessels often with double-barrel formation, marked fibrous meningeal thickening with inflammatory cell infiltration and hemosiderin deposits, and multiple cerebral infarcts at various stages predominantly in the superficial cortical layers. The apparent lack of vasculitic lesions in the visceral organs was noteworthy. The findings of brain biopsy performed in the acute stage strongly suggested that the vasculitis was granulomatous in nature. Although there were no macrophages or multinucleated giant cells containing Ab in their cytoplasm, co-occurrence of CAA and granulomatous vascullitis leads to a neuropathological diagnosis of CAA-related inflammation. The presence of these cells is one of the hallmarks of this disorder;2,7–9,11–13 however, these findings would be related to the stage of disease, the degree of inflammation and the antibody against Ab. In Alzheimer’s disease tissue, the granular structures in glial cells (microglia and astrocyte) are recognized by antibodies specific for C-terminal sequences of Ab fragments.17 In our study, we used a clone 6F/3D which recognizes N-terminal side fragment of Ab protein. The second unusual point of CAA-related inflammation is lack of Ab immunostaining at the blood vessels showing active inflammation (Fig. 3g). Several autopsy cases show negative findings of Ab immunostaining at part of the blood vessels with active vasculitis.2,9,11,15 The absence of Ab immunoreactivity at the vessels would be a result of the elimination of Ab through the active inflammation. In addition, the absence of amyloid P component at the affected blood vessels was noted in a case of CAA-related vasculitis.1 Although many previous reports have documented the neuropathology of CAA-related inflammation at the acute stage,2–4,7–9,11–15 no autopsy cases that can be considered examples of the chronic stage, after a clinical course of several years, have so far been reported. In 1974, Reid and Maloney reported an autopsied patient with Down syndrome, in whom a 4-year antemortem history of psychiatric change was a feature; autopsy revealed amyloid deposits in many subarachnoidal arteries and arterioles accompanied by multinucleated giant cells, as well as associated multiple small infarcts in the cerebral cortex, subcortical white matter and corpus striatum.6 However, unfortunately Ab immunohistochemistry was not available at that time. Another noteworthy report has documented the autopsy findings of PACNS with CAA in a 56-year-old woman who had received long-term immunosuppressive therapy.10 In that case, although a brain biopsy specimen showed no evidence of amyloid deposits, autopsy revealed small infarcts and hemorrhages in the basal ganglia without signs of active vascular inflammation or post-inflammatory changes in the leptomeningeal and intraparenchymal arteries, but with evidence of CAA.10 In the present case, only a few vascular lesions showed active inflammation with absence of multinucleated giant cells, although immunosuppressive therapy was abandoned 33 months prior to death. It is possible to consider that CAA-related inflammatory activity might have decreased during the patient’s long clinical course. Thickening of the leptomeninges with hemosiderin deposits in the present case can be interpreted as traces of preceding leptomeningeal inflammation and hemorrhage. The fact that infarcts involving the superficial cerebral cortex were much more numerous than those associated with vasculitic lesions suggested that the development of most of these infarcts might have been independent of vascular inflammation. It is known that the severe vascular alterations induced by CAA can themselves lead to chronic brain hypoperfusion, and may cause multiple cere© 2011 Japanese Society of Neuropathology Infarcts with CAA-related inflammation bral (especially small) infarcts.18,19 Some patients with CAA-related inflammation have been reported to show superficial cortical infarcts.6,13,15 The co-presence of vasculitic lesions and leptomeningeal inflammation in advanced CAA must accelerate the development of superficial multiple infarcts. In addition, all reported cases of this disorder examined by autopsy have shown only a small number of vessels with vasculitic changes in comparison to the widespread presence of vessels with Ab deposits, suggesting unknown factors in the development of inflammation and an immune response to vascular Ab-amyloid. It is well known that an elevated level of serum PR3ANCA has high specificity and sensitivity for diagnosis of patients with Wegener’s granulomatosis.20,21 However, in Wegener’s granulomatosis, the association between an increase of PR3-ANCA and disease relapse has not been confirmed.22 In vivo evidence of a pathogenic role of PR3ANCA is uncertain.23 In the present patient, the pathological diagnosis of Wegener’s granulomatosis was very difficult.21 Values of inflammatory markers, including erythrocyte sedimentation rate, C-reactive protein level and plasma viscosity are known to be elevated in 33% of patients with CAA-related inflammation.4 However, to our knowledge, no serum ANCA-positive patients with CAArelated inflammation have so far been reported.2 At present, we can only speculate that PR3-ANCA might be involved in the disease process of Ab-associated, nonWegener’s granulomatous vasculitis. In conclusion, we have described an autopsied patient with CAA-related inflammation, in whom widespread superficial-predominant cortical multiple infarcts and only a few foci of active vasculitic lesions were characteristic features. We consider that the present case represents the first autopsy-proven example of the chronic stage of the disease. ACKNOWLEDGMENTS The authors thank C. Tanda, J. Takasaki, S. Egawa, Y. Kakuda, and Y. Yamaguchi for technical assistance, M. Machida for secretarial assistance and Professor James Nicoll of the University of Southampton for helpful comments. REFERENCES 1. Yamada M, Itoh Y, Shintaku M et al. Immune reactions associated with cerebral amyloid angiopathy. Stroke 1996; 27: 1155–1162. 2. Scolding NJ, Joseph F, Kirby PA et al. Ab-related angiitis: primary angiitis of the central nervous system associated with cerebral amyloid angiopathy. Brain 2005; 128: 500–515. © 2011 Japanese Society of Neuropathology 5 3. 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