Neuropathology and Applied Neurobiology

The fine anatomy of the perivascular compartment in the brain. Relevance to dilated perivascular spaces in cerebral amyloid angiopathy

Journal:For Neuropathology Peer and ReviewApplied Neurobiology Manuscript ID NAN-2018-0001.R1

Manuscript Type: Scientific Correspondence

Date Submitted by the Author: 13-Feb-2018

Complete List of Authors: MacGregor Sharp, Matthew; University of Southampton, Faculty of Medicine Bulters, Diederik; University of Southampton, Faculty of Medicine Brandner, Sebastian; UCL, Research Center, Department of Brain Repair and Rehabilitation, UCL Institute of and the National Hospital for Neurology and Neurosurgery Holton, Janice; UCL, Stroke Research Center, Department of Brain Repair and Rehabilitation, UCL Institute of Neurology and the National Hospital for Neurology and Neurosurgery Verma, Ajay; Biogen Carare, Roxana; University of Southampton, Faculty of Medicine Werring, David; UCL, Stroke Research Center, Department of Brain Repair and Rehabilitation, UCL Institute of Neurology and the National Hospital for Neurology and Neurosurgery

Dilated perivascular spaces, cerebral amyloid angiopathy, Keywords: , small vessel , intramural periarterial drainage

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1 2 3 The fine anatomy of the perivascular compartment in the : 4 relevance to dilated perivascular spaces in cerebral amyloid angiopathy 5 6 1 1 2 2 7 Matthew MacGregor Sharp , Diederik Bulters , Sebastian Brandner , Janice Holton , 8 3 2* 1* 9 Ajay Verma , David J Werring , Roxana O Carare 10 11 12 13 1 Faculty of Medicine, University of Southampton, Southampton, UK; 14

15 2 16 Stroke Research Centre, Department of Brain Repair and Rehabilitation, UCL 17 Institute of Neurology and the National Hospital for Neurology and Neurosurgery, 18 19 London, UK. For Peer Review 20 21 3 Biogen, USA. 22 23 24 *Contributed equally to the study 25 26 27 28 29 Corresponding author: 30 31 Prof Roxana O Carare, University of Southampton, Faculty of Medicine, 32 33 Southampton General Hospital, South Academic Block, MP806, Tremona Road, 34 Southampton, Hampshire, SO16 6YD, UK. Email: [email protected] 35 36 37 Keywords: Dilated perivascular spaces, cerebral amyloid angiopathy, white matter 38 hyperintensities, small vessel disease, intramural periarterial drainage 39 40 41 Running Title: The fine anatomy of the perivascular compartment in the brain 42 43 44 Word Count: 1309 Figures: 1 45 46 47 48 List of Abbreviations: 49 50 Aβ, , CAA, cerebral amyloid angiopathy, CT, computed tomographic 51 52 scanning, IPAD, intramural periarterial drainage, ISF, interstitial fluid, MRI, magnetic 53 54 55 resonance imaging, PVS, perivascular spaces, WMH, white matter hyperintensities. 56 57 58 59 60 Neuropathology and Applied Neurobiology Page 2 of 10

1 2 3 Cerebral white matter hyperintensities (WMH) observed on magnetic resonance 4 5 imaging (MRI), or low attenuation on computed tomographic scanning (CT), are the 6 7 most frequent brain imaging finding in patients with small vessel disease or 8 9 . It has been assumed that WMH are due to or blood-brain 10 11 barrier breakdown, though recently it was demonstrated that WMH have distinct 12 13 14 molecular signatures in Alzheimer’s disease (AD) where markers of Wallerian 15 16 degeneration are present, compared to normal ageing [1]. Dilated perivascular 17 18 spaces (PVS) are of particular interest for the study of interstitial fluid (ISF) dynamics 19 For Peer Review 20 because they are related to the intramural periarterial drainage (IPAD) of ISF and 21 22 solutes along arterial basement membranes and can potentially be detected by MRI. 23 24 MRI-visible dilated PVS have been described in the white matter of patients with AD 25 26 27 or cerebral amyloid angiopathy (CAA) [2, 3]. There are no detectable PVS around 28 29 in the , even in pathological conditions, whereas PVS can 30 31 dilate in the white matter and may indicate failure of drainage of ISF and solutes [4]. 32 33 Recently we demonstrated in an elderly dog that arterioles are enveloped by one 34 35 complete layer of leptomeninges in both grey and white matter, often with a second 36 37 incomplete layer in the white matter, whereas venules have incomplete layers of 38 39 40 leptomeningeal cells [5]. This suggests that arterioles in the white matter in young 41 42 brains may have a distinct perivascular compartment formed between a) the 43 44 leptomeningeal layer adjacent to the side of tunica media facing side the 45 46 parenchyma and b) the leptomeningeal layer adjacent to the glia limitans ( 47 48 end feet) that potentially could be expanded to allow the formation of a dilated PVS. 49 50 One limitation of establishing the underlying anatomy of the dilated PVS, both in 51 52 53 CAA and more generally, has been the lack of fixed post-mortem material from large 54 55 mammals, suitable for ultrastructural studies. Here using biopsy tissue from the white 56 57 58 59 60 Page 3 of 10 Neuropathology and Applied Neurobiology

1 2 3 matter of a patient with CAA and a young control patient with no signs of CAA or 4 5 dementia, we identify by transmission electron microscopy (TEM) the exact 6 7 anatomical location of the dilated PVS to further clarify IPAD mechanisms. TEM was 8 9 performed following our standard protocols [6]. Briefly, fresh tissue was submersion 10 11 fixed overnight in 3.4% formaldehyde plus 3% glutaraldehyde in 0.1M PIPES buffer, 12 13 14 cut into 1mm cube sections and processed for TEM [6]. Tissue blocks were trimmed 15 16 and ultrathin 80-nm sections cut. The ultrathin sections were collected onto copper 17 18 grids and viewed using a FEI Technai T12 electron microscope operating a EMSIS 19 For Peer Review 20 MegaView III digital camera and EMSIS image capture software (formerly iTEM 21 22 software, Universal TEM Imaging platform, Soft Imaging System, Münster, 23 24 Germany). A series of high power images of arterial walls (from n=3 arteries/case) 25 26 27 was acquired and then reconstructed as a montage using Adobe Photoshop CS6 28 29 with an automated photomerge script set to auto layout with image blend, vignette 30 31 removal and geometric Distortion Correction. 32 33 34 Control tissue was obtained from a 26-year-old female patient undergoing 35 36 frontal lobectomy for seizures due to an underlying cavernoma. Tissue was obtained 37 38 after patient consent under ethical approval REC 12/NW/0794. Clinical 39 40 neuropathology in Southampton General Hospital reported that the cortical samples 41 42 43 from the white subcortical region showed evidence of reactive gliosis, with no other 44 45 abnormality observed. Analysis of subcortical white matter at the posterior resection 46 47 margin most distant from the cavernoma by TEM revealed arteries in the white 48 49 matter with closely apposed layers of cells and their basement membranes (Figure 1 50 51 A). A complete layer of endothelial cells with tight junctions and an intact basement 52 53 membrane was observed. Layers of smooth muscle cells were separated by 54 55 56 basement membranes from the endothelium and from the pial glial basement 57 58 59 60 Neuropathology and Applied Neurobiology Page 4 of 10

1 2 3 membrane. There were no spaces observed between the basement membranes of 4 5 the smooth muscle and the pial glial basement membranes. Two layers of intact pia 6 7 mater were observed either side of the pial glial basement membranes (Figure 1 B). 8 9 10 11 12 CAA tissue was obtained from a 68-year-old man who initially presented with rapidly 13 14 progressive dementia, over several months. A brain MRI including T2*-weighted 15 16 17 gradient echo (Figure 1 C) and susceptibility-weighted imaging sequences (Figure 1 18 19 D) showed multipleFor strictly Peer lobar cerebral Review microbleeds and widespread cortical 20 21 superficial siderosis. Diffusion-weighted imaging showed a small cortical 22 23 hyperintense lesion in the left occipital region, suggesting an active occlusive 24 25 vasculopathy (Figure 1 E). As part of his investigation for treatable causes of 26 27 dementia, he underwent a non-dominant frontal lobe brain biopsy. The right frontal 28 29 30 lobe specimen was divided for diagnostic histology and TEM. Histological 31 32 assessment showed severe CAA and frequent diffuse and mature amyloid beta (Aβ) 33 34 plaques in the neural parenchyma. There was no evidence of vasculitis. TEM 35 36 revealed arteries in the white matter with thick and distorted walls, with an enlarged 37 38 PVS. The PVS had an intact pial-glial basement membrane separating it from the 39 40 surrounding parenchyma (Figure 1 F). One layer of was observed adjacent 41 42 43 to the pial-glial basement membrane. This layer appeared highly disrupted with only 44 45 fragments visible (Figure 1 G). 46 47 48 Insert Figure 1 here 49 50 51 52 53 54 Dilatation of PVS occurs in the white matter of the cerebral hemispheres, in 55 56 the and in the grey matter of the [7]. The potential space 57 58 59 60 Page 5 of 10 Neuropathology and Applied Neurobiology

1 2 3 between the outer aspect of an wall and the brain parenchyma, can be 4 5 expanded experimentally by the intracerebral injection of particles [8]. As cortical 6 7 arteries enter the surface of the cerebral cortex, the pia mater is reflected from the 8 9 surface of the brain on to the vessels in the subarachnoid space, thus separating the 10 11 subarachnoid space from the cerebral cortex [8]. In the present study we confirmed 12 13 14 that there is no PVS around cortical arteries in the normal brain as layers of smooth 15 16 muscle, basement membrane and one layer of leptomeninges are all compressed 17 18 together. The CAA patient is not age-matched to the control, so therefore both 19 For Peer Review 20 ageing and CAA may have destroyed the pia mater adjacent to the pial-glial 21 22 basement membrane resulting in a dilated PVS. This arrangement may provide the 23 24 anatomical substrate for fluid to accumulate as MRI-visible PVS, possibly 25 26 27 contributing to WMH. Furthermore, the control tissue was from a patient with a 28 29 vascular lesion, although care was taken to use tissue that had normal appearance, 30 31 from the wide resection. Based on our observations we propose a mechanism by 32 33 which the IPAD pathways become compromised in the cortical arteries, such that 34 35 fluid from the white matter is unable to drain efficiently with increasing age, or after 36 37 38 immunization against Aβ, resulting in axonal destruction and dilated PVS [1, 9, 10]. 39 40 41 Conflict of Interest 42 43 The authors confirm that this article content has no conflicts of interest. 44 45 46 Acknowledgments: 47 48 49 The study was conducted with tissue obtained from the UK Brain Archive Information 50 51 52 Network (BRAIN UK) ethical approval number 14/SC/0098. This work was supported 53 54 and funded by Biogen. 55 56 57 58 59 60 Neuropathology and Applied Neurobiology Page 6 of 10

1 2 3 Ethical Approval: 4 5 6 The study was conducted with tissue obtained from the UK Brain Archive Information 7 8 Network (BRAIN UK) ethical approval number 14/SC/0098 (CAA patient). Control 9 10 tissue was obtained after patient consent under ethical approval REC 12/NW/0794. 11 12 13 14 15 16 Author Contributions: 17 18 19 ROC, AV and DJW Fordesigned Peer the study. MM-SReview performed the electron microscopy. 20 21 DB provided the control tissue. SB and JH performed the histopathology. All authors 22 23 contributed to the interpretation of data and writing the manuscript. 24 25 26 27 28 29 References 30 31 32 1. McAleese KE, Walker L, Graham S, Moya ELJ, Johnson M, Erskine D, et al. 33 Parietal white matter lesions in Alzheimer's disease are associated with cortical 34 35 neurodegenerative pathology, but not with small vessel disease. Acta 36 neuropathologica. 2017;134(3):459-73. 37 38 2. Charidimou A, Meegahage R, Fox Z, Peeters A, Vandermeeren Y, Laloux P, 39 40 et al. Enlarged perivascular spaces as a marker of underlying arteriopathy in 41 intracerebral haemorrhage: a multicentre MRI cohort study. Journal of neurology, 42 43 neurosurgery, and psychiatry. 2013;84(6):624-9. 44 45 3. Banerjee G, Kim HJ, Fox Z, Jager HR, Wilson D, Charidimou A, et al. MRI- 46 visible perivascular space location is associated with Alzheimer's disease 47 48 independently of amyloid burden. Brain. 2017;140(4):1107-16. 49 4. Ramirez J, McNeely AA, Scott CJ, Masellis M, Black SE, Alzheimer's Disease 50 51 Neuroimaging I. White matter burden in elderly cohort studies: The 52 53 Sunnybrook Dementia Study, Alzheimer's Disease Neuroimaging Initiative, and 54 Three-City Study. Alzheimer's & dementia : the journal of the Alzheimer's 55 56 Association. 2016;12(2):203-10. 57 58 59 60 Page 7 of 10 Neuropathology and Applied Neurobiology

1 2 3 5. Criswell TP, Sharp MM, Dobson H, Finucane C, Weller RO, Verma A, et al. 4 The structure of the perivascular compartment in the old canine brain: a case study. 5 6 Clin Sci (Lond). 2017;131(22):2737-44. 7 8 6. Sharp MM, Page A, Morris A, Weller RO, Carare RO. Quantitative 9 Assessment of Cerebral Basement Membranes Using Electron Microscopy. In: 10 11 Clausen BE, Laman JD, editors. Inflammation: Methods and Protocols. New York, 12 13 NY: Springer New York; 2017. p. 367-75. 14 7. Salzman KL, Osborn AG, House P, Jinkins JR, Ditchfield A, Cooper JA, et al. 15 16 Giant tumefactive perivascular spaces. AJNR Am J Neuroradiol. 2005;26(2):298- 17 305. 18 19 8. Pollock H, HutchingsFor M,Peer Weller RO, Review Zhang ET. Perivascular spaces in the 20 21 basal ganglia of the human brain: their relationship to lacunes. J Anat. 1997;191 ( Pt 22 3):337-46. 23 24 9. Hawkes CA, Hartig W, Kacza J, Schliebs R, Weller RO, Nicoll JA, et al. 25 26 Perivascular drainage of solutes is impaired in the ageing mouse brain and in the 27 presence of cerebral amyloid angiopathy. Acta neuropathologica. 2011;121(4):431- 28 29 43. 30 10. Carare RO, Teeling JL, Hawkes CA, Puntener U, Weller RO, Nicoll JA, et al. 31 32 Immune complex formation impairs the elimination of solutes from the brain: 33 34 implications for immunotherapy in Alzheimer's disease. Acta neuropathologica 35 communications. 2013;1(1):48. 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Neuropathology and Applied Neurobiology Page 8 of 10

1 2 3 Figure Legends 4 5 6 Figure 1: A) Reconstructed montage of an artery from the posterior frontal 7 subcortical white matter of a control patient. The endothelium (en), smooth muscle 8 9 cells (sm) and pia mater (pm) appear normal. The pial-glial basement membrane 10 11 (pgbm) is only expanded in the presence of (m). B) High power of the 12 vessel wall highlighted by the box seen in (A). Note the two layers of pia mater (pm) 13 14 with no dilated perivascular space. C – E) MRI of a 68-year-old with rapidly 15 16 progressing dementia. The ventricles are dilated (C). Susceptibility-weighted imaging 17 revealed multiple strictly lobar cerebral microbleeds (arrows) and widespread cortical 18 19 superficial siderosisFor (D). Diffusion-weightedPeer Review imaging showed a small cortical 20 hyperintense lesion in the left occipital region (E). F) Reconstructed montage of an 21 22 artery from the right frontal subcortical white matter of a CAA patient. The 23 24 endothelium (en) appears enlarged. There is a dilated perivascular space (dpvs) 25 (white arrows) and expansion of the pial glial basement membrane (pgbm). The pia 26 27 mater (pm) appears disrupted with only fragments visible. E) High power of the 28 29 vessel wall highlighted by the box seen in (F). Note only one layer of pia mater (PM) 30 surrounded by a dilated perivascular space (dpvs) is visible. Other abbreviations; 31 32 smooth muscle (sm); parenchyma (p); lumen (lu). 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 9 of 10 Neuropathology and Applied Neurobiology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 For Peer Review 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Neuropathology and Applied Neurobiology Page 10 of 10

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 For Peer Review 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 Figure 1: A) Reconstructed montage of an artery from the posterior frontal subcortical white matter of a 40 control patient. The endothelium (en), smooth muscle cells (sm) and pia mater (pm) appear normal. The 41 pial-glial basement membrane (pgbm) is only expanded in the presence of macrophages (m). B) High power 42 of the vessel wall highlighted by the box seen in (A). Note the two layers of pia mater (pm) with no dilated 43 perivascular space. C – E) MRI of a 68-year-old with rapidly progressing dementia. The ventricles are dilated 44 (C). Susceptibility-weighted imaging revealed multiple strictly lobar cerebral microbleeds (arrows) and 45 widespread cortical superficial siderosis (D). Diffusion-weighted imaging showed a small cortical hyperintense lesion in the left occipital region (E). F) Reconstructed montage of an artery from the right 46 frontal subcortical white matter of a CAA patient. The endothelium (en) appears enlarged. There is a dilated 47 perivascular space (dpvs) (white arrows) and expansion of the pial glial basement membrane (pgbm). The 48 pia mater (pm) appears disrupted with only fragments visible. E) High power of the vessel wall highlighted 49 by the box seen in (F). Note only one layer of pia mater (PM) surrounded by a dilated perivascular space 50 (dpvs) is visible. Other abbreviations; smooth muscle (sm); parenchyma (p); lumen (lu). 51 173x175mm (300 x 300 DPI) 52 53 54 55 56 57 58 59 60