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Neuropathology 2015; 35, 481–486

doi:10.1111/neup.12216

Workshop: New perspectives in MS, NMO, and PML

Significance of gray matter brain lesions in multiple sclerosis and neuromyelitis optica Izumi Kawachi and Masatoyo Nishizawa Department of Neurology, Brain Research Institute, Niigata University, Niigata, Japan Multiple sclerosis (MS) and neuromyelitis optica (NMO) are the two main autoimmune diseases of the CNS. In patients with NMO, the target antigen is aquaporin-4 (AQP4), the most abundant water channel protein in the CNS. AQP4 is mainly expressed on astrocytic endfoot processes at the blood-brain barrier and in subpial and subendymal regions. MS and NMO are distinct diseases, but they have some common clinical features: both have long been considered autoimmune diseases that primarily affect the white matter (WM). However, because WM demyelination by itself cannot explain the full extent of the clinical disabilities, including cognitive decline in patients with MS and NMO, renewed interest in gray matter (GM) pathology in MS and NMO is emerging. Important hallmarks of WM and GM lesions in MS and NMO may differentially influence neuronal degeneration and demyelination in the brain and spinal cord, given different detrimental effects, including cytokine diffusion, disruption of water homeostasis associated with or without AQP4 (the target antigen in NMO) dynamics, or other unidentified mechanisms. An increase in knowledge of the structure of GM and WM lesions in MS and NMO will result in more targeted therapeutic approaches to these two diseases.

relapsing, autoimmune disease that can be distinguished from MS based on epidemiological, clinical, radiological, immunological and pathological evidence.2,5,6 NMO is characterized by three major lesions:2,5 (i) myelopathy in NMO is associated with spinal cord lesions detected with MRI that extend more than three spinal segments (longitudinally extensive spinal cord lesions (LEM)) and that primarily involve the central part of the spinal cord on axial sections5,7; (ii) optic neuritis in NMO is bilateral and severe and is associated with swollen optic nerves, a chiasmatic lesion, or an altitudinal scotoma, as well as a thin retinal nerve fiber layer that is observed with optical coherence tomography5,8; and (iii) periaqueductal medullary lesions in NMO are associated with intractable hiccoughs or nausea/vomiting that are present for > 2 days.5,9 More recently, supratentorial lesions have been reported in NMO including extensive white matter (WM) lesions,10 cortical gray matter (GM) lesions,11 brainstem lesions, or cerebellar lesions. Thus, CNS lesions in NMO may be much more widespread than previously thought. To address the region-specific and disease-specific mechanisms of NMO and MS pathogenesis in the CNS, we summarized and reviewed the current concepts of WM and GM lesions in NMO and MS and further clarified some controversial issues (Fig. 1).6,11–13

Key words: gray matter lesions, neurodegeneration, neuromyelitis optica, multiple sclerosis, white matter lesions.

GM INVOLVEMENT IN MS INTRODUCTION Neuromyelitis optica (NMO) and multiple sclerosis (MS) are the two main autoimmune diseases of the CNS typically following a relapsing and remitting course.1,2 In patients with NMO, the specific autoantibody targets the water channel aquaporin-4 (AQP4).3,4 Details of the disease mechanisms, pathology and clinical characteristics of NMO have emerged. Currently, NMO is widely recognized as a distinct, Correspondence: Izumi Kawachi, MD, PhD, Department of Neurology, Brain Research Institute, Niigata University, 1-757 Asahimachi, Chuo-ku, Niigata 951-8585, Japan. Email: [email protected] Received 7 February 2015; revised and accepted 5 April 2015; published online 16 June 2015.

© 2015 Japanese Society of Neuropathology

MS has long been considered an autoimmune disease that primarily affects the WM. However, because WM demyelination by itself cannot explain the full extent of clinical disabilities, including cognitive decline in patients with MS, renewed interest in GM pathology in MS14,15 is emerging. From a historical perspective, GM lesions in MS were disregarded for a long time since their description by the neuropathologist James W. Dawson in 1916.16 In 1962, Brownell et al. reported that among a total of 1594 cerebral plaques in 22 postmortem MS brains, 80 (5%), 65 (4%), 265 (17%) and 1184 (74%) plaques were found in cortical GM, central GM, the junction of cortical GM and WM and WM, respectively, according to pathological assessment

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Fig. 1 Models of lesion formation in multiple sclerosis (MS) (A) and neuromyelitis optica (NMO) (B). Demyelination in cortical gray matter (GM) is evident in MS (A), but not in NMO (B). The pathological hallmarks of cortical GM lesions in MS are largely restricted to demyelination, subtle neuroaxonal degeneration, transection of neurites, and synaptic loss. On the other hand, cortical GM lesions are evident in NMO brains and are distinct from lesions in MS brains. Neuropathological assessment has demonstrated neuronal loss in cortical layers II, III and IV with nonlytic reaction of aquaporin-4 (AQP4)-negative astrocytes in layer I, massive activation of microglia in layer II and meningeal inflammation in NMO brains. No NMO cases showed evidence of cortical GM demyelination or ectopic B-cell follicle-like structures in the meninges. These data indicate that pathological processes consist not only of inflammatory demyelinating events characterized by pattern-specific loss of AQP4 immunoreactivity in the spinal cord and optic nerves, but also cortical neurodegeneration in NMO brains.

using conventional myelin staining such as Luxol Fast-Blue or Klüver-Barrera staining.17 Recent studies using more sensitive immunohistochemical staining with antibodies against myelin basic protein, myelin oligodendrocyte glycoprotein, or proteolipid protein demonstrated that GM demyelination was much more extensive in patients with MS than previously thought.18 Cortical GM demyelination in MS is classified into four lesion types:19,20 Type I lesions are mixed GM/WM lesions; Type II lesions are located within the cortical GM and do not extend to the surface of the brain; Type III lesions are subpial lesions, which reach from the pia downwards into the cortical GM but do not reach the WM-GM border; and Type IV lesions extend throughout the full width of the cortical GM, but do not reach into the subcortical WM.19,20 Pathological studies using postmortem samples have shown that the percentages of total demyelinated lesions are 14%, 1%, 67% and 17% in Types I, II, III and IV, respectively.19 A pathological study using biopsy samples in a cohort of patients with early-stage MS, including definite MS and clinically isolated syndrome, indicated that cortical demyelination is also evident in 38% of samples and that the percentages of total demyelinated lesions are 50%, 16%, 34% and 0%, in Types I, II, III and IV, respectively.14 Band-like subpial demyelination (Type III lesion) is invariably oriented toward the pial surface of the cortex and penetrates into the cortex at variable depths, is particularly

widespread in deep infoldings of the brain surface, affects the largest cortical areas and several adjacent gyri, is associated with inflammatory infiltrates in the meninges, and is mainly found in patients with secondary progressive (SPMS) or primary progressive MS (PPMS).21 On the other hand, Type I or II lesions are present in all stages and courses of MS, including acute MS, relapsing and remitting MS (RRMS), PPMS and SPMS.21 The pathology of cortical GM lesions is distinct from that of WM lesions in MS brains;15 the typical pathological hallmarks of WM lesions, including lymphocyte and macrophage infiltration, complement deposition, microglial activation, and blood-brain barrier disruption, are all unusual in cortical GM lesions.19,20,22,23 However, these differences may be dependent on lesion stage, because most of the cortical demyelination in patients with early-stage MS shows parenchymal inflammatory infiltrates including CD3+ T-cell infiltrates and macrophage-associated demyelination with meningeal inflammation,14 which was much more extensive than previously thought in cohorts of most patients with SPMS or PPMS.19,20,22,23 A focal experimental autoimmune encephalitis model of cortical demyelination, which is induced by stereotactically targeting the cerebral cortex by injecting proinflammatory mediators into myelin oligodendrocyte glycoprotein-sensitized rats, shows more rapid resolution of inflammation and more extensive remyelination of GM demyelination compared to WM demyelination.24 © 2015 Japanese Society of Neuropathology

Gray matter lesions in MS and NMO More recently, a clear gradient of neuronal loss was evident not only in GM demyelination but also in normalappearing GM when accompanied by meningeal B-cell follicle-like structures.25 These data indicate that some neurodegeneration in the cortex may be independent of demyelination and may more diffusely occur.25 Deep GM demyelination is also present in patients with MS, as shown by pathological assessment of postmortem brains.26,27 Deep GM demyelination is most often seen in the thalamus, hypothalamus and caudate nuclei, but is also present in the putamen, pallidum, claustrum, amygdala and substantia nigra. Deep GM inflammation is intermediate between low inflammatory cortical lesions and active WM lesions.27 The deep GM in MS shows not only focal demyelinated plaques, but also diffuse neurodegeneration27 analogous with cortical GM lesions. Moreover, nonneocortical lesions with prominent GM pathology are also present in the hippocampus.28 Demyelinated hippocampi show minimal neuronal loss, but significant decreases in synaptic density. Neuronal proteins essential for axonal transport, synaptic plasticity, glutamate neurotransmission, glutamate homeostasis and memory, are significantly decreased in demyelinated hippocampi, but not in demyelinated motor cortices in MS brains.28 Conventional MRI are usually not optimal for detecting cortical GM lesions in MS brains.29 Recently, a multi-slab three-dimensional (3D) double inversion recovery (DIR) technique has been developed and has enabled around a five-fold increase in the detection of cortical GM lesions in MS brains compared to conventional MRI.15,29–34 However, in a postmortem verification study, 3D-DIR detected only 9.1% of all intracortical GM lesions (Types II, III and IV lesions) that were identified with pathological methods, suggesting that 3D-DIR has poor sensitivity for detecting intracortical GM lesions, although it has excellent pathological specificity.31 Importantly, band-like subpial demyelination (Type III lesions), which is the most common type of cortical GM lesion in MS autopsied brains, is still undetectable with DIR sequences.30,31 Future radiological assessment methods are needed to improve the detection of cortical GM lesions.6

GM INVOLVEMENT IN NMO NMO is an autoimmune astrocytopathy characterized by severe attacks of optic neuritis and LEM.3,4 The target antigen in patients with NMO is AQP4,3,4 the most abundant water channel protein in the CNS that is mainly expressed on astrocytic endfoot processes at the blood-brain barrier and in subpial and subependymal regions.35 Historically, severe optic neuritis and LEM with a negative brain MRI are considered common features of NMO.7 LEM primarily involves central GM.5,35 However, increasing evidence indicates that © 2015 Japanese Society of Neuropathology

483 typical/atypical MS-type brain lesions are present in NMO.10,36–40 Sixty percent of NMO patients have brain abnormalities on MRI, most of which are nonspecific. However, 10% have MS-typical lesions, and 8% have MS-atypical lesions such as diencephalic, brainstem or cerebral lesions.10 Moreover, magnetization transfer and diffusion tensor MRI studies of patients with NMO have found abnormalities in normal-appearing GM as well as normal findings or minimal changes in normal-appearing WM, regardless of abnormalities seen with conventional MRI.36–40 These findings suggest that brain and spinal cord abnormalities, including not only WM, but also GM, are evident in NMO. The reason may be because the GM of the brain and spinal cord shows high AQP4 expression. The pathological hallmarks of NMO are pattern-specific loss of AQP4 immunoreactivity and intense vasculocentric immune complex depositions with activated complement.41–43 These findings in the spinal cord and optic nerves of the definite form of NMO are identical in the limited form of NMO.12 Recently, we revealed that cortical GM lesions are evident in NMO brains and are distinct from MS brains.11,44 Neuropathological assessments have demonstrated neuronal loss in cortical layers II, III and IV, with nonlytic reaction of AQP4-negative astrocytes in layer I, massive activation of microglia in layer II, and meningeal inflammation in NMO brains.11 Importantly, all NMO cases show no evidence of cortical GM demyelination or ectopic B-cell follicle-like structures in the meninges.11,44 These data indicate that pathological processes consist not only of inflammatory demyelinating events characterized by patternspecific loss of AQP4 immunoreactivity in the spinal cord and optic nerves, but also of cortical neurodegeneration in NMO brains.11 Irrespective of abnormalities seen with conventional MRI, examinations using magnetization transfer and diffusion tensor MRI have demonstrated that patients with NMO show a reduced magnetization transfer ratio and an increased mean diffusivity of the normal-appearing GM.36–38,40 Two voxel-based morphometry studies have shown regional atrophy in the sensorimotor, visual and frontotemporal cortices.39,40 These data provide evidence that GM pathology is present during the disease processes of NMO. Moreover, consistent with our pathological findings indicating that unique cortical GM lesions are evident, but that cortical GM demyelination is absent,11,44 a previous paper45 demonstrated that DIR sequences did not detect any cortical GM lesions in NMO brains. 3D-DIR has poor sensitivity for detecting cortical GM lesions, in particular, subpial lesions in MS.30,31 Because NMO brains have unique cortical GM lesions without any cortical demyelination and more severe damage in the subpial layers than deep layers,11 cortical GM lesions in NMO may not be detected with DIR sequences.

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CONCLUSIONS The involvement of not only WM, but also GM in the brain and spinal cord is seen in MS and NMO. However, studies of GM involvement in MS and NMO have only just begun, and no direct evidence of clinical relevance for GM involvement, including cognitive and physical impairment in MS and NMO, has been reported. As an example of the clinical relevance of GM involvement, common cognitive impairments, including impairment in attention, processing speed, executive functioning, and memory, are seen in MS46 and NMO.11 With radiological and clinical assessments, a correlation between cortical atrophy and cognitive decline in patients with MS47–49 has been observed, and cortical GM lesions detected with DIR sequences are considered to be a better predictor of cognitive impairment than WM lesions in patients with RRMS.49–52 However, 3D-DIR has poor sensitivity for detecting cortical GM demyelinating lesions in MS31 and neurodegeneration in cortical GM in NMO,45 because band-like subpial demyelination (Type III lesion) is the most common type of cortical GM lesions in MS brains, and NMO brains have unique cortical GM lesions without cortical demyelination and more severe damage in the subpial layers than in deep layers.11,44 Full understanding of the relationships among GM lesions, cognitive impairment and disease progression awaits future studies.

ACKNOWLEDGEMENTS This work was supported in part by JSPS KAKENHI Grant Number 26461289 (IK), and a Health and Labour Sciences Research Grant on Rare and Intractable Diseases (Evidence-based Early Diagnosis and Treatment Strategies for Neuroimmunological Diseases) from the Ministry of Health, Labour and Welfare of Japan (MN). We wish to thank S. Kawaguchi and M. Kaneko (Department of Neurology, Brain Research Institute, Niigata University, Japan) for technical assistances.

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