Imaging in mice and men: Pathophysiological insights into multiple sclerosis from conventional and advanced MRI techniques

Accepted Manuscript Title: Imaging in mice and men: pathophysiological insights into multiple sclerosis from conventional and advanced MRI techniques Authors: Julia Kr¨amer, Wolfgang Bruck, ¨ Frauke Zipp, Manuela Cerina, Sergiu Groppa, Sven G. Meuth PII: DOI: Article Number:

S0301-0082(19)30058-9 https://doi.org/10.1016/j.pneurobio.2019.101663 101663

Reference:

PRONEU 101663

To appear in:

Progress in Neurobiology

Received date: Revised date: Accepted date:

28 February 2019 17 June 2019 17 July 2019

Please cite this article as: Kr¨amer J, Bruck ¨ W, Zipp F, Cerina M, Groppa S, Meuth SG, Imaging in mice and men: pathophysiological insights into multiple sclerosis from conventional and advanced MRI techniques, Progress in Neurobiology (2019), https://doi.org/10.1016/j.pneurobio.2019.101663 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Imaging in mice and men: pathophysiological insights into multiple sclerosis from conventional and advanced MRI techniques a*

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Julia Krämer , [email protected] Wolfgang Brück [email protected], c

a

Frauke Zipp [email protected], Manuela Cerina [email protected], c

a

Sergiu Groppa [email protected], Sven G. Meuth [email protected] a

Department of Neurology with Institute of Translational Neurology, University Hospital Münster,

Albert-Schweitzer-Campus 1, Building A1, 48149 Münster, Germany. b

Department of Neuropathology, University Medical Center Göttingen, Robert-Koch-Str. 40, 37075

Göttigen, Germany. c

Department of Neurology, Focus Program Translational Neuroscience, University Medical Center of

the Johannes Gutenberg University Mainz, Langenbeckstraße 1, 55131 Mainz, Germany.

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For submission to Progress in Neurobiology as Review Article *Correspondence to: Julia Krämer, Department of Neurology with Institute of Translational Neurology, University Hospital Münster, Albert-Schweitzer-Campus 1, Building A1, 48149 Münster, Germany, Phone: 0049-251-8346811, Fax: 0049-251-8344414. Highlights

This review summarizes MRI and histopathological studies in mice and humans with MS.

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Hereby it partially elucidates the underlying pathophysiology behind imaging findings.

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It recommends imaging protocols for the future examination of different aspects of MS.

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Further in vivo imaging and histopathological studies in MS mouse models are necessary.

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A combination of imaging techniques is necessary to examine the pathophysiology of MS.

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Abstract Magnetic resonance imaging (MRI) is the most important tool for diagnosing multiple sclerosis (MS). However, MRI is still unable to precisely quantify the specific pathophysiological processes that underlie imaging findings in MS. Because autopsy and biopsy samples of MS patients are rare and biased towards a chronic burnt-out end or fulminant acute early stage, the only available methods to identify human disease pathology are to apply MRI techniques in combination with subsequent histopathological examination to small animal models of MS and to transfer these insights to MS patients. This review summarizes the existing combined imaging and histopathological studies performed in MS mouse models and humans with MS (in vivo and ex vivo), to promote a better understanding of the pathophysiology that underlies conventional MRI, diffusion tensor and magnetization transfer imaging findings in MS patients. Moreover, it provides a critical view on imaging capabilities and results in MS patients and mouse models and for future studies recommends how to

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combine those particular MR sequences and parameters whose underlying pathophysiological basis could be partly clarified. Further combined longitudinal in vivo imaging and histopathological studies on rationally selected, appropriate mouse models are required.

Keywords MRI; multiple sclerosis; mouse model; pathophysiology; histopathology; translational research.

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Abbreviations

ABH = acute black hole; AD = axial diffusivity; ADC = apparent diffusion coefficient; APP= anti-amyloid

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precursor protein; BBB = blood–brain barrier; BH = black hole; BSCB = blood–spinal cord barrier; CC = corpus callosum; CFA = complete Freund’s adjuvant; CIS = clinically isolated syndrome; CL = cortical lesion; CNPase = cyclic nucleotide phosphodiesterase; CNS = central nervous system; Cre =

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creatine/phosphocreatine; CSF = cerebrospinal fluid; Cup = cuprizone; “Cup-EAE” = cuprizone-fed mice with additional immunization with MOG35-55; “Cup” mice = cuprizone-fed mice; DAPI = 4,6diamidino-2-phenylindole; DD = disease duration; DIR = double inversion recovery; DTI = diffusion

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tensor imaging; EAE = experimental autoimmune encephalomyelitis; EDSS = Expanded Disability Status Scale; EPI = echo-planar imaging; FA = fractional anisotropy; f = female; FFE = fast field echo; FLAIR = fluid-attenuated inversion recovery; FLASH = Fast Low-Angle Shot; FSE = fast spin-echo; GAP = growth associated protein; Gd = Gadolinium; DTPA = diethylenetriamine penta-acetic acid; FOV = field of view; GEL = Gadolinium-enhancing lesion; GFAP = Glial fibrillary acidic protein; GLUT = glucose transporter; GM = grey matter; HC = healthy control; H & E = Hematoxylin and eosin; Ig =

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Immunoglobulin; LFB = Luxol fast blue; LFB/PAS = Luxol Fast Blue and Periodic Acid Schiff; LMI = leptomeningeal inflammation; L-N = Luxol-Nissl; MAP = microtubule associated protein; MBP = Myelin basic protein; MD = mean diffusivity; MOG = myelin-oligodendrocyte-glycoprotein; MRI = magnetic resonance imaging; MRP = myeloid-related protein; MRS = magnetic resonance spectroscopy; MS = multiple sclerosis; MT = magnetization transfer; MTI = magnetization transfer imaging; MTR = magnetization transfer ratio; MWF = myelin water fraction; N = number; NAA = N-acetyl aspartate; NABT = normal-appearing brain tissue; NAGM = normal-appearing grey matter; NAWM = normalappearing white matter; NeuN = neuronal nuclei; NF = neurofilament protein; N/P = not performed; OPC = oligodendrocyte precursor cell; PBH = permanent black hole; PCR = polymerase chain reaction; PD = proton density; PDGF = platelet-derived growth factor; PDw = proton density-weighted;

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PLP = proteolipid protein; pNF = non-phosphorylated neurofilament; PPMS = primary progressive multiple sclerosis; PRESS = Point-RESolved Spectroscopy; PSIR = phase-sensitive inversion recovery; PTX = pertussis toxin; qMT = quantitative magnetization transfer; RA = relative anisotropy; RAG = recombination activation gene; RARE = rapid acquisition with relaxation enhancement; RD = radial diffusivity; RF = radiofrequency; RIS = radiologically isolated syndrome; RNS = reactive nitrogen species; ROS = reactive oxygen species; ROI = region of interest; RRMS = relapsing-remitting multiple sclerosis; SDGM = subcortical deep grey matter; SE = spin-echo; SNR = signal-to-noise-ratio; SPMS = secondary progressive multiple sclerosis; STEAM = single-shot stimulated echo acquisition mode; SYN = Synaptophysin; TBH = transient black hole; TE = echo time; TR = relaxation time; TMEV = Theiler’s murine encephalitis virus; TSE = turbo spin-echo; TUNEL = terminal deoxynucleotidyl transferase dUTP nick end labelling; T1w = T1-weighted; T2w = T2-weighted; USPIOs = ultrasmall superparamagnetic iron oxides; VSOP = very small superparamagnetic iron oxide particle; WM =

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white matter; WML = WM lesion. 1 Introduction

Multiple sclerosis (MS) is a chronic, immune-mediated, inflammatory, demyelinating, and neurodegenerative disease of the human central nervous system (CNS). It leads to demyelination and progressive axonal and neuronal loss in white and grey matter (WM, GM) (Figure 1) and,

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subsequently, results in irreversible physical disability and cognitive impairment. Even though magnetic resonance imaging (MRI) has become the tool of choice for MS diagnosis and disease

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monitoring over time, it is still limited in its ability to uncover and precisely quantify the specific neuropathophysiological alterations. A deeper understanding of the highly complex pathophysiology of MS is however essential for developing highly effective anti-inflammatory, neuroprotective, and

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reparative therapies (Filippi et al., 2019). As has been stated, “Ideally, the best way to study MS is by studying the human disease itself” (Pirko and Johnson, 2008). But patients are only rarely biopsied for differential diagnosis in unclear and fulminant cases and autopsy samples give a “snapshot in time” of

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an often chronic and burnt-out end stage of MS (Filippi et al., 2012; Klawiter et al., 2011; Wuerfel et al., 2007). Therefore, small animal models of the disease are the most common path to investigate relevant pathomechanisms (Pirko and Johnson, 2008; Wuerfel et al., 2007). In the last few decades, small animal imaging, both in vivo and ex vivo, has become a promising and widely available technique in MS research. In combination with histopathological examinations, this approach offers the possibility to correlate imaging findings with histopathological data in a manner that is highly

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translational to human studies (Denic et al., 2011a; Driehuys et al., 2008; Garcia-Alloza and Bacskai, 2004; MacKenzie-Graham et al., 2012; Merkler et al., 2005; Oguz et al., 2012; Pirko and Johnson, 2008).

When developing advanced MRI techniques, neuroimmunologists constantly strive to increase sensitivity and specificity of these measures in order to be able to look into the human brain and spinal cord while MS is ongoing. Currently, there is a great disparity in knowledge between the clinic and the bench. On one hand, physicians are able to accurately detect MS-induced alterations by applying conventional and advanced MRI techniques. However, it is often challenging for them to also have a firm grasp of the underlying pathophysiological basis of these findings. On the other hand, benchbased researchers working with mouse models of MS may have a deeper understanding of the

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disease at a cellular level. However, these researchers may be largely unaware of the different imaging techniques used in MS and examples of typical features seen with MS patients in the real world. The purpose of our review is to bridge the knowledge gap. Step by step, the review summarizes those combined imaging and histopathological studies in MS mouse models and humans with MS (in vivo and ex vivo) which use conventional MRI (T1- & T2-weighted (T1w & T2w) sequences), magnetization transfer imaging (MTI), and diffusion tensor imaging (DTI). We directly compare and discuss the imaging and histopathophysiological findings of the most commonly used MS mouse models with findings in MS patients to better understand the underlying pathophysiological processes behind MRI findings that have not yet been clarified (Denic et al., 2011b). For scientists who may be unfamiliar with mouse imaging techniques per se, we first describe relevant

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technical, practical, and challenging aspects. Box 1 describes the technical aspects of the considered conventional and advanced MRI techniques.

2 Technical aspects, special requirements, and challenges of mouse MRI

Mouse MRI studies are most commonly conducted in dedicated narrow-bore magnets operating at a high field strength, namely in the range of 4.7–17.6 T, of which the 7 T magnet is the most commonly

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used (Denic et al., 2011b; Pirko and Johnson, 2008). The two leading manufacturers of these systems are Bruker BioSpin (Ettlingen, Germany) and Varian Medical Systems (Palo Alto, CA). Resolutions of 3

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Johnson, 2008). 2.1 Advantages of in vivo mouse imaging

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100 μm are feasible in living animals and 10 μm in fixed animals (Driehuys et al., 2008; Pirko and

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In vivo mouse imaging has the advantage of being interactive in a single animal over a prolonged disease course without the need for sacrifice. It offers the possibility to control for many variables (environmental conditions, duration and dose of treatments/intoxication, etc.), specify the timing of pathological processes, and develop new effective therapies (Aung et al., 2013; Denic et al., 2011b;

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Driehuys et al., 2008; MacKenzie-Graham et al., 2012; Merkler et al., 2005; Pirko and Johnson, 2008; Zhang et al., 2012). The generation of new, genetically modified mice from well-characterized “standard” strains allows a focus on specific aspects of pathology, e.g. inflammation, demyelination or remyelination. Compared to human imaging, MS mouse models permit much longer scan times, resulting in improved resolution and increased signal-to-noise-ratio (SNR). Despite the generally lower spatial resolution of in vivo compared to ex vivo MRI (as a result of shorter acquisition time), images

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are derived from a more natural context due to the maintenance of cerebral and spinal in vivo morphology and absence of tissue deformation associated with histological embedding, sectioning, and staining procedures (MacKenzie-Graham et al., 2012; Zhang et al., 2012).

2.2 Challenges of in vivo mouse imaging To conduct MRI on mice in vivo, several important factors need to be considered (for reviews see (Denic et al., 2011b; Driehuys et al., 2008; Pirko and Johnson, 2008)). Some of these factors are (i) usage of ultra-high field small animal MR scanners that are difficult to access and associated with high costs of purchase, service, and maintenance, (ii) the need for specific radiofrequency (RF) coils, (iii) expenses for special equipment, (iv) the need for specialized MR physicists, and (v) longer acquisition

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time that exceeds that of most other imaging systems. To achieve a resolution comparable to the 3

1mm voxel size in human neuroimaging, images from mice must be acquired with a voxel size of 3

100μm or less (Tagge et al., 2016). Figure S1 depicts the striking difference in voxel size between a human brain and that of a mouse. However, the decreased 3D voxel volume leads to a decreased SNR. Approaches to win back SNR loss include optimizing RF coils and using a higher gradient field strength (increasing B0), the use of many available contrast enhancers not approved for humans, and longer image acquisition times (Pirko and Johnson, 2008). Moreover, during the imaging session it is crucial to monitor animals’ vital parameters, stabilize them in a repeatable position without causing any trauma, and use exogenous heating systems. Living organisms comprising multiple compartments with different physico-chemical properties can generate artifacts due to susceptibility and relaxation properties (for review see (Pirko and Johnson, 2008)). Subtle biological movements including e.g. pulsation of blood vessels, cerebrospinal fluid (CSF) motion, heart, respiration, and involuntary muscle

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activity, represent a major obstacle in obtaining good quality in vivo images. Such movements can occur even with optimized anesthesia and in animals that are completely immobilized. Combined with the longer image acquisition times needed to attain a high-resolution, these movements not only produce image blurring, but can also result in artefacts due to spatial encoding and registration errors of the same anatomic structures occupying different positions during the scans. A further challenge

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are the necessary modification and adaptation of software tools used for image pre-processing, processing, and analysis in humans for use on mouse images. Pipelines for processing and analyzing

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mouse MR images are far less standardized than those used for human scans (Pallast, 2019). 3 Mouse models of MS

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The most commonly used mouse models of MS to monitor WM lesion (WML) pathogenesis include: (1) experimental autoimmune or allergic encephalomyelitis (EAE),

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(2) viral-induced models like Theiler’s Murine Encephalomyelitis Virus (TMEV) infection, (3) general or focal toxin-induced models achieved with cuprizone (Cup, biscyclohexanone oxaldihydrazone) diet or injection of lysolecithin (lyso-phosphatidyl choline)/ethidium bromide typically in the spinal cord or the corpus callosum (CC). Both compounds target mature oligodendrocytes and are typically used to induce demyelination

(for reviews on mouse models of MS see (Constantinescu et al., 2011; Denic et al., 2011a; Garcia-

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Alloza and Bacskai, 2004; Lassmann and Bradl, 2017; Matsushima and Morell, 2001; McCarthy et al., 2012; Procaccini et al., 2015; Ransohoff, 2012)). EAE, the most commonly studied and oldest MS model applied in rodents and monkeys, can be either actively induced (“active EAE”) by immunization with an emulsion of complete Freund’s adjuvant (CFA) and myelin components such as myelin basic protein (MBP), myelin-oligodendrocyteglycoprotein (MOG, peptide or full length protein), and proteolipid protein (PLP), or passively induced (“passive” or “adoptive EAE”) by intravenous transfer of in vitro pre-stimulated autoreactive CD4+ Tlymphocytes to the host animals (Aharoni et al., 2013; Baxter, 2007; Boretius et al., 2012; Constantinescu et al., 2011; M.J., 2005; Schellenberg et al., 2007; t Hart et al., 2011; Terry et al.,

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2016). Diseases with different severity and disease courses may be evoked depending on the peptide used for immunization and the mouse strain chosen. In this respect, EAE induction in SJL/J mice with the PLP139–155 peptide induces a relapsing-remitting-like disease, while immunization of C57BL/6 mice with the MOG35–55 peptide produces a monophasic or chronic disease course (Terry et al., 2016). Although EAE pathogenesis shares some pathophysiological hallmarks to human MS pathogenesis, this model has a number of weaknesses and limitations (for further information see (Denic et al., 2011a; Lassmann and Bradl, 2017; Ransohoff, 2012)). First, already the sole induction method renders the model very heterogeneous. The same holds true when clinical and pathological features and amenability to treatment are taken into account (Constantinescu et al., 2011). In more detail, the classical MOG35-55-induced EAE is mainly mediated by CD4+ reactive T cells, whereas in MS patients CD8+ T cells play a predominant role (t Hart et al., 2011). To overcome this limitation, researchers have developed EAE models for which the role of T and B cells can be calibrated depending on the

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specific myelin antigen and its concentration (Lyons et al., 2002; Palumbo and Pellegrini, 2017; Steinman, 2001). Moreover, EAE is usually characterized by the appearance of small inflammatory demyelinating lesions which are abundant and extended in the spinal cord (Lassmann and Bradl, 2017) and smaller and more randomly distributed in the brain (Levy et al., 2010; Steinman and Zamvil, 2005). In mice, lesions in the spinal cord are mainly associated with physical disabilities, while lesions

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in the brain are associated with cognitive disorders. However, it is difficult to further analyze the observed impairments or underlying pathophysiological events in vivo as lesions appear randomly in

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WM and/or GM regions. It is therefore essential to know the precise localization of the lesion within a specific area of the CNS to associate symptoms with brain regions (Lassmann and Bradl, 2017). The pathology and distribution of lesions varies for different animal strains (Lassmann and Bradl, 2017).

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Immunization of C57BL/6 mice with MOG35–55 largely generates spinal cord lesions with low affection of the brain stem and the cerebellum and very little inflammation or tissue damage in the forebrain (Levy et al., 2010; Steinman and Zamvil, 2005). In contrast, immunization of SJL/J mice with PLP139– typically causes lesions in the optic nerve, brainstem, spinal cord, cerebellum and cerebral cortex

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(Constantinescu et al., 2011; Lassmann and Bradl, 2017). In most chronic EAE models, cortical lesions are small, randomly distributed, and difficult to identify histologically (Palumbo and Pellegrini, 2017). The major limitation of lesion localization can be overcome by targeting specific brain regions and performing local stereotaxic injection of cytokines (typically INF-gamma and TNF-alpha) in preimmunized mice (Merkler et al., 2006b). In this way, the activated immune cells that already infiltrated

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the CNS upon immunization can be attracted to a specific region by the cytokines. Another possibility it the use of the common marmoset (Callithrix jacchus) EAE model (Merkler et al., 2006a; Pomeroy et al., 2005). However, the highly demanding requirements for infrastructure and animal husbandry, the ethical constraints, and the lack of specific transgenic animals make the availability of rodent models more attractive (Kap et al., 2010; Merkler et al., 2006a). With regards to the TMEV model, intracerebral injection of TMEV can produce a mono or biphasic disease pattern depending on the mouse strain used. TMEV infection induces an acute monophasic meningo-encephalomyelitis in all mouse strains with a peak on day 7 after infection, and results in full recovery. However, the disease is biphasic in some susceptible strains, producing a second stage of

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late chronic-progressive demyelination and disability occurring over 9 to 12 months after infection and reflecting neurodegenerative aspects of MS (progressive demyelination and axonal degeneration). Similar to EAE, the TMEV model is characterized by a predominance of spinal cord lesions over brain lesions; although cerebral atrophy has also been observed in this model (Lassmann and Bradl, 2017; Paz Soldan et al., 2015; Pirko et al., 2012; Pirko et al., 2011). While viral models such as the TMEV model reflect key features of human MS lesions, they are difficult to induce and dissect afterwards due to their very complex pathogenesis. A shortcoming of both the focal EAE and TMEV model are the strong imaging artifacts due to intracerebral injection that interfere with mouse MRI data analysis. The toxin-induced models are the most suitable tools to study experimental demyelination and remyelination. The cuprizone model is the most commonly used one. Acute Cup treatment (Cup administered for a maximum of 8 weeks) results in a reproducible, extensive, and confluent

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demyelination in all basic structures of the cerebral WM, especially the CC, and in cortical and subcortical deep GM (SDGM), with concomitant activation of astrocytes and microglia/macrophages and significant acute axonal injury (Boretius et al., 2012; Fjaer et al., 2015; Matsushima and Morell, 2001; Song et al., 2005; Tagge et al., 2016; Xie et al., 2010; Zaaraoui et al., 2008). An advantage of using this approach is that, once the compound is not administered, spontaneous remyelination

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occurs. Hence, the process is reversible and the same animal can be investigated before and after myelin loss. However, there are also limitations of this model. The gender of the animals, their age,

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and the duration of exposure to the compound are important determinants for reproducibility and occurrence of de- and remyelination. Moreover, this model does not reflect other important aspects of MS pathology and pathogenesis such as blood-brain barrier (BBB) damage, edema, and T-cell

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mediated inflammation (Abakumova et al., 2015; Boretius et al., 2012; Fjaer et al., 2013; Lassmann and Bradl, 2017; Mason et al., 2001; Matsushima and Morell, 2001; Merkler et al., 2005; Song et al., 2005; Tagge et al., 2016; Turati et al., 2015; Xie et al., 2010; Zaaraoui et al., 2008).

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Each of the described MS mouse models can only portray certain aspects of the human disease. None of them covers the entire spectrum of clinical, pathological, or immunological features of human MS (Lassmann and Bradl, 2017; Denic et al., 2011b). To generate a model that mimics MS in humans more closely, combinations of different mouse models have been attempted. One example is the established “Cup-EAE” animal model which generates MS-like damage through immunization of Cup-

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fed mice (“Cup” mice) with MOG35-55 (Boretius et al., 2012). 4 In vivo and ex vivo histopathological and imaging studies investigating MS in humans and mouse models

For our extensive literature research on histopathological and imaging studies conducted in mice and humans, we searched PubMed, Scopus, Ovid MEDLINE, and Google Scholar regularly over the st

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course of 2.5 years (September, 1 2016 to January, 1 2019) for relevant articles written in German and English. The keywords used as search terms were: “multiple sclerosis” and “human” or “mice” or “mouse” or “mouse models of MS” and “histopathology”, in conjunction with several other keywords, e.g. “conventional MRI”, “magnetic resonance imaging”, “magnetization transfer imaging”, “diffusion

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tensor imaging”, “imaging”, “gadolinium enhancement”, “white matter hyperintensities”, “cortical lesion”. Reference lists of already published articles were screened for additional studies to be included. Articles were screened for eligibility by one of the authors (JK) and only studies which met all of the following criteria were included: a. gave detailed descriptions of the analyzed human MS sample, its characteristics and demographic variables (this means e.g. the applied MS diagnostic criteria, the type of multiple sclerosis, age, sex, Expanded Disability Status Scale (EDSS), and disease duration) b. gave detailed descriptions of the analyzed mouse sample (this means e.g. the strain used, the induced mouse model, age, and sex) c.

gave detailed descriptions of the MR scanner and sequences used, the kind of MR data analysis and/or immunohistochemical/histopathological examinations chosen (this means e.g. the staining

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methods, the mean postmortem interval in case of postmortem MRI examination)

d. reported histopathological and/or MRI data as unadjusted means and standard deviations, or in an equivalent format

The reported histopathological and/or MRI data were evaluated qualitatively. A quantitative

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assessment was not possible due to inter-study differences. A flowchart of the study selection process is depicted in Figure S2.

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Tables S1 and S2 offer a summary of results for each cited study on MS mouse models and humans with MS, including information on subjects, applied mouse model, type of study, methods, MRI

in histopathological examinations. 4.1 Conventional MRI

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sequences used, and histopathological and imaging results. Table S3 explains staining methods used

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4.1.1 Conventional MRI studies in humans

MRI is the gold standard method for MS diagnosis and disease monitoring (see Box 1 for an overview of classical MRI sequences used in everyday clinical practice and MS research). Despite its high sensitivity,

conventional

MRI

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characterize

the

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pathophysiological processes behind individual MS lesions (Bittner et al., 2009; Boretius et al., 2012; Filippi and Rocca, 2011; Mallik et al., 2014; Merkler et al., 2005; Ontaneda and Fox, 2017; Song et al.,

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2005; Zaaraoui et al., 2008). T1w pre- and post-contrast and T2w imaging used in combination can, however, differentiate between lesions occurring as a consequence of acute inflammation, and those that are more representative of permanent damage. Newly appearing or enlarging T2 hyperintensities together with contrast enhancement on T1w images are mainly associated with “active” MS lesions (Figure 2A). However, T2 hyperintensity is pathologically non-specific and may theoretically be caused by edema, demyelination, remyelination, inflammation, gliosis, or axonal loss (Table 1, Figure 2B) (Bakshi, 2005a; Filippi and Rocca, 2011; Mallik et al., 2014). This is because signal intensity on T2w imaging is altered by subtle changes in the macromolecular environment of water protons (Traboulsee et al., 2005). Nevertheless, its strong association with myelin content (LFB and Klüver staining), which is superior to T1w imaging and magnetization transfer ratio (MTR) after fixation, was

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demonstrated in several cerebral and spinal postmortem studies of MS patients (Barkhof et al., 2003; Bot et al., 2004; Schmierer et al., 2008) (Table 1, Table S2). Gadolinium (Gd) enhancement occurs as a result of increased permeability of the BBB, which enables the contrast agent to enter the CNS and highlight areas with an acute infiltration of inflammatory cells (e.g. macrophages and lymphocytes) and soluble factors (Figure 1B, Figure 2A, Table 1) (Bruck et al., 1997; Nesbit et al., 1991; own unpublished results). Enhancing lesions are primarily nodular with homogenous enhancement (68%), although a large percentage can be ring-like (23%). The remaining 9% comprise a variety of different shapes (Sahraian and Radue, 2008a). Gd enhancement was demonstrated to be not only selective for “early active” lesions but also for those that are “late active”, “demyelinated”, and “remyelinating” (Bruck et al., 1997) (Table S2). While most individual new MS lesions enhance for 2–6 weeks, a small number of lesions demonstrate enhancement for up to 2–3 months (Cotton et al., 2003; Guttmann et al., 1995; Harris et al., 1991; Miller et al., 1988; Sahraian and Radue, 2008b; Silver et al., 1999) and,

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in rare cases, for longer than 6 months (He et al., 2001). Recently, MRI contrast agents composed of iron particles known as ultrasmall superparamagnetic iron oxides (USPIOs), which are taken up by cells of the monocyte-macrophage lineage, were shown to provide significant advantages compared to conventional Gd-based contrast agents, as they have a prolonged blood circulation time, depict different aspects of the MS inflammatory process (i.e. cellular infiltration associated with inflammation

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vs. abnormalities of BBB permeability), and can be used safely even for patients with chronic kidney disease (Gkagkanasiou et al., 2016; Schindler et al., 2017; Vellinga et al., 2008). Black holes (BHs),

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hypointense T1w lesions concordant with T2w hyperintense lesions (Sahraian and Radue, 2008b; Sahraian et al., 2010), are considered to be acute black holes (ABHs) when they coincide with a gadolinium enhancing lesion (GEL) and to be chronic or persistent (“permanent/persistent black

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holes”, PBHs) when no corresponding GEL exists (Figure 2C) (Sahraian and Radue, 2008b; Sahraian et al., 2010). While most ABHs resolve without “visible” tissue damage and again become isointense on T1w images (“transient black holes”, TBHs), a few of them progress to become PBHs after 12

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months of follow-up (Filippi and Rocca, 2011; Naismith et al., 2010; Sahraian and Radue, 2008b; Sahraian et al., 2010). The formation and duration of PBHs was demonstrated to correlate with the duration of enhancement. Thus, GELs persisting for more than one month have a greater chance of having an associated ABH that evolves into a PBH (Bagnato et al., 2003). While ABHs have a multifactorial origin, occurring as a result of inflammation, active demyelination, edema, cellular infiltration, early remyelination, axonal transection, glial activation, and astrogliosis, PBHs seem to

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reflect an irreversible state of tissue destruction including axonal loss and persistent demyelination (Figure 1E, Figure 2C, Table S2, Table 1) (Bitsch et al., 2001; Sahraian and Radue, 2008b; Sahraian et al., 2010; own unpublished results). In postmortem studies on patients with progressive MS, histopathological and imaging analysis of unfixed brain tissue revealed an association between the level of T1 hypointensity of lesions and axonal density (Bodian-staining); moderately for the degree of tissue destruction (hematoxylin and eosin (H & E) staining) and inversely for the number of reactive astrocytes in the surrounding tissue (van Waesberghe et al., 1999; van Walderveen et al., 1998). T1 hypointensity did not correlate with the degree of demyelination (Klüver-Barrera staining) or the number of reactive astrocytes (glial fibrillary acidic protein (GFAP) immunostaining) within the plaque (Table S2). While in these studies T1 hypointensity was found to primarily reflect axonal damage and

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tissue destruction, later studies found links between T1 hypointensity and levels of myelination. A longitudinal MRI analysis of T1 hypointensity performed on biopsy-defined demyelinating lesions from MS patients, demonstrated that the future evolution of MS lesions to PBHs over time depends on the initial demyelinating activity (luxol fast blue (LFB) staining, anti-MBP, anti-MOG, anti-PLP immunostaining) and the extent of acute axonal damage (anti-amyloid precursor protein (APP)) or axonal loss (Bielschowsky’s silver impregnation) (Table S2) (Bitsch et al., 2001). After the initial demyelination of a lesion, it seems that a decision is rapidly made whether the lesion is to undergo remyelination (Bitsch et al., 2001). Another postmortem histopathological and imaging study examining T1 hypointensity in lesions from patients with progressive MS demonstrated that strong T1 hypointensity was significantly associated with demyelinated and partially remyelinated lesions compared with fully remyelinated lesions (Barkhof et al., 2003) (Table S2). This suggested that BHs present an irreversible state of tissue destruction with both demyelination and axonal loss (Table 1,

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Figure 2C).

Cortical lesions (CLs), which can be detected at the earliest clinical stages of MS, were shown to be independent predictors of subsequent disease evolution and disability progression and to correlate more strongly with physical and cognitive disability than WMLs (Calabrese et al., 2010; Harrison et al.,

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2015; Strijbis et al., 2017). Against this background, reliable in vivo detection of cortical lesions is highly relevant in everyday clinical practice (Kilsdonk et al., 2015; Seewann et al., 2012). However, imaging cortical lesions is difficult, especially with conventional MRI sequences (including dual-echo

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T2w and fluid-attenuated inversion recovery (FLAIR)), due to their small size, poor contrast with the surrounding normal-appearing GM (NAGM), and partial volume effects with WM and CSF (Calabrese

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et al., 2010; Filippi et al., 2019; Filippi et al., 2016; Kilsdonk et al., 2015; Reynolds et al., 2011; Seewann et al., 2012). MRI techniques such as double inversion recovery (DIR) (Figure 3A) and phase-sensitive inversion recovery (PSIR) (see section 4.3.1.2 for more details), or magnetizationprepared rapid acquisition with gradient echo sequences have been developed to improve detection of

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CLs (Filippi et al., 2019). Recently, the ratio of T1w to T2w image intensities has been shown to be sensitive to cortical demyelination in a postmortem MRI and histopathology study on six MS patients (Nakamura et al., 2017). However, the specificity of this technique for cortical demyelination needs further validation since another postmortem imaging and histopathology study performed on autopsy material from 9 patients with progressive MS revealed a strong correlation between decreased cortical T1/T2 ratio and dendrite density, but not myelin content (Righart et al., 2017). The study of cortical GM

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pathology remains challenging even when using more advanced imaging techniques or when imaging at ultra-high field strengths, and a high proportion of intracortical lesions still remains undetected as demonstrated in postmortem MRI and histopathology verification studies (Geurts et al., 2008; Jonkman et al., 2016; Kilsdonk et al., 2015; Reynolds et al., 2011; Seewann et al., 2012). On the basis of their topographical localization, CLs identified using immunohistochemistry can be classified as leukocortical (type I), intracortical (type II) or subpial (type III) (Peterson et al., 2001). The most common CL type spreads subpially, involving many adjacent gyri (Bo et al., 2003). Several pathological studies demonstrated that CLs (Figure 3A) generally lack the classic pathological hallmarks observed in WMLs, showing little inflammation, only mild peripheral immune cell infiltration,

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no BBB-disruption or complement activation, and an increased number of activated microglial cells (Table 1, Table S2, Figure 3A) (Peterson et al., 2001; Reynolds et al., 2011; Strijbis et al., 2017; Wegner et al., 2006; own unpublished results). The latter was also confirmed by in vivo positronemission tomography imaging with the radioligand 11C-PK11195 (Politis et al., 2012). In postmortem studies on patients with progressive MS, relapsing-remitting MS (RRMS), and healthy controls (HCs), light and electron microscopy of immunohistochemical stained paraffin-embedded brain tissue showed more extensive remyelination in CLs than in WMLs (Albert et al., 2007; Chang et al., 2012; Strijbis et al., 2017). Thus, Albert et al. estimated that approximately 75% of CLs show signs of remyelination (Albert et al., 2007). An abundance of oligodendrocyte precursor cells (OPCs) were found in CLs, refuting an OPC recruitment failure in cortical GM (Chang et al., 2012; Strijbis et al., 2017). Moreover, neuronal pathology was prominent in CLs and in the cortical NAGM (Reynolds et al., 2011; Magliozzi et al., 2010). An analysis of 112 CLs from 50 MS patients revealed axonal and dendritic transection,

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apoptotic loss of neurons (TUNEL and anti-SMI-32 immunostaining), and activation of microglia (Table 1, Table S2, Figure 3A) (Peterson et al., 2001). Klaver et al. demonstrated significant signs of neurodegeneration in both subpial (type III) lesions and cortical NAGM. Wegner et al. found substantial glial, neuronal, and synaptic loss (anti-MBP, Nissl, anti-synaptophysin, and anti-neural GAP-43 staining) in leukocortical (type I) lesions, whereas in NAGM the only described pathologic

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alteration was the appearance of rounded neurons (Table 1, Table S2) (Klaver et al., 2015; Wegner et al., 2006). By combining a modified Golgi-Cox impregnation technique with high-resolution confocal

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microscopy, widespread and extensive loss of dendritic spines was recently demonstrated in demyelinated cortex and cortical NAGM, occurring independently of cortical demyelination and axonal loss (Figure 3A) (Jurgens et al., 2016). Neurodegeneration appears to follow a gradient, from the pial

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surface to the deep grey matter layers (Magliozzi et al., 2010).

Recently, cortical pathology (cortical demyelination and neuroaxonal damage, global and focal cortical thinning) was linked to leptomeningeal inflammation (LMI) (Absinta et al., 2015; Bergsland et al., 2019;

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Howell et al., 2011; Magliozzi et al., 2007). Post-contrast 3D-FLAIR imaging was identified as sensitive method for detecting focal leptomeningeal contrast enhancement which may correspond to leptomeningeal inflammation (LMI) as seen histopathologically (Absinta et al., 2015; Wicken et al., 2018; Zivadinov et al., 2018). LMI was associated with more severe disease course in MS (Howell et al., 2011; Magliozzi et al., 2007; Magliozzi et al., 2018).

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Brain atrophy is another feature of MS that can be measured by MRI as acquired loss in brain volume due to tissue destruction (Figure 2D). By using a wide variety of automated, freely available MRbased segmentation techniques, e.g. the statistical parametric mapping (Ashburner and Friston, 2000), FreeSurfer (Fischl et al., 2002), FIRST (Patenaude et al., 2011), SIENA and SIENAX (Smith et al., 2002) as parts of FSL, global and regional (WM, cortical GM and SDGM) brain atrophy was demonstrated to occur early in the disease course across all MS subtypes. It is already present in patients with radiologically and clinically isolated syndrome (RIS and CIS) and early RRMS (Chaudhuri, 2013; Deppe et al., 2016a; Deppe et al., 2014; Deppe et al., 2016b; Kramer et al., 2015; Rocca et al., 2017; Rocca et al., 2016; Varosanec et al., 2015). Putamen atrophy starts directly after manifestation of the first symptoms, or even years earlier in patients with RRMS, progressing in a

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degressive manner (Kramer et al., 2015). Recently, early thalamic atrophy in patients with CIS and RRMS was shown to be caused mainly by silent (non-lesional) microstructural destructive processes within the thalamus and not by retrograde neuroaxonal degeneration or anterograde transsynaptic changes occurring secondary to WMLs (Wallerian degeneration) as thought previously (Deppe et al., 2016a). Moreover, selective WM atrophy in patients with CIS and early RRMS (disease duration (DD) < 24 months) with a nearly intact cerebral cortex was shown to result in an increased cortical extrinsic curvature compared to HCs (Deppe et al., 2014). GM atrophy was demonstrated to be frequent and widespread not only in neocortical areas (Kroth et al., 2017), but also in the hippocampus, cerebellum, and in SDGM structures, starting in the SDGM and then progressively extending to the cortical GM (Rocca et al., 2017). Postmortem studies of patients with progressive MS revealed that cortical atrophy is predominantly

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explained by neurodegeneration (neuroaxonal loss and neuronal shrinkage as assessed by anti-NeuN and anti-SMI32 staining), and is at least to a large extent independent of demyelination (myelin or oligodendrocyte density as assessed by anti-PLP and anti-olig2 staining) (Klaver et al., 2015; Popescu et al., 2015) (Table 1, Table S2).

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A postmortem study performed on paraffin-embedded autopsy material from 75 MS cases and 12 HCs showed that SDGM was affected by two different processes: by formation of focal demyelinating lesions and by diffuse neurodegeneration (Haider et al., 2014). SDGM lesions developed on the of

inflammation

with

perivascular

and

parenchymal

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background

lymphocytic

infiltration.

Neurodegeneration was reflected by neuronal loss, acutely injured axons, and the accumulation of

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oxidized phospholipids and DNA in neurons, oligodendrocytes, and axons (Haider et al., 2014). Using in vivo MRI and MRS and postmortem histopathology, substantial thalamic atrophy was demonstrated to be accompanied by neuronal loss (reduced N-acetylaspartate concentration; cresyl violet and Weil’s

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myelin staining) in MS patients (Table S2) (Cifelli et al., 2002). 4.1.2 Conventional MRI studies in mice

There are conflicting reports in the literature concerning the relationship between BBB disruption and inflammation in mice. While some studies have indicated that BBB disruption occurs prior to the onset of inflammation and cellular infiltration, other studies have found that BBB disruption is present only during inflammation (Schellenberg et al., 2007). Longitudinal and cross-sectional in vivo MRI studies

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of mice with MOG35-55 -induced EAE and of “Cup” mice with additional immunization with MOG35-55 (“Cup-EAE” mice) demonstrated that blood-spinal cord barrier (BSCB) and BBB disruption histopathologically corresponded to areas with marked infiltration of immune cells and tissue deposition of IgG and fibrinogen (anti-CD3, anti-IgG, and anti-fibrinogen immunostaining) (Table 1, Table S1, Figure 1B, Figure 2A) (Boretius et al., 2012; Schellenberg et al., 2007; Nessler et al., 2007; own unpublished results). BBB breakdown generally occurred in areas where activated microglia cells and reactive astrocytes (anti-Mac-3 and anti-GFAP immunostaining) were located (Table 1, Table S1, Figure 1B) (Nessler et al., 2007). In two in vivo 7T MRI studies on SJL⁄J mice with passive transfer EAE, very small superparamagnetic iron oxide particles (VSOPs) (visualized by

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Prussian Blue staining) were capable of showing both BBB breakdown through extravasation and the diffuse infiltration of extracellular macrophages/microglia (anti-Iba1 immunostaining) into active inflammatory plaques. Regarding the former, VSOPs are much smaller than conventional magnetic nanoparticles, meaning they are able to cross the disrupted BBB. The latter is due to the fact that VSOPs are incorporated into local phagocytic cells by endocytosis labelling them during infiltration (Tysiak et al., 2009; Wuerfel et al., 2007). BHs, namely non-active EAE lesions, in CNS slices obtained from MOG35-55-immunized mice were characterized by damaged tissue (H&E staining) (Figure 2C) (own unpublished results). The presence of specific immune cells (epitope-specific cytotoxic CD8+ T cells), effector molecules (perforin), and an adaptive immune system (recombinase activating gene 1) were required for BH formation in TMEVinfected mice, suggesting that CD8+ T cells and perforin are critical mediators of axonal and neuronal damage (Table 1, Table S1, Figure 1E) (Pirko et al., 2008). This is in line with previous studies

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demonstrating that CD8+ T cells, which are the second most abundant cell type besides macrophages and destroy only cells that display major histocompatibility complex I class surface antigens (e.g. somatic cells), are the most prevalent immune cell type observed in newly forming MS lesions and in NAGM and normal-appearing WM (NAWM) (Babbe et al., 2000; Lassmann and Ransohoff, 2004; Pirko et al., 2008; Skulina et al., 2004). In contrast, CD4+ T cells were shown to play a preventive role

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in BH formation (Table 1, Table S1) (Pirko et al., 2012; Pirko et al., 2008). Resolution of BHs by day 45, which was paralleled by normalization of magnetic resonance spectroscopy (MRS) findings, was

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demonstrated in a longitudinal in vivo cerebral MRI and MRS study on TMEV-infected mice, suggesting active repair of the compromised axonal/neuronal integrity (Table S1) (Pirko et al., 2004). Examination of “Cup” and “Cup-EAE” mice with cerebral MRI 34 days after starting with Cup diet

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revealed that T2w imaging is a sensitive but rather non-specific indicator of tissue injury (Table 1). T2w intensities of the CC differed from control and diseased mice, but not from “Cup” and “Cup-EAE” mice, while T1w intensities of the CC were more strongly reduced in the “Cup-EAE” compared to the

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“Cup” mice. The latter was ascribed to the additional presence of T cell infiltrates and acutely damaged axons (as assessed by anti-CD3 and APP immunostaining) (Table S1) (Boretius et al., 2012). In a novel mouse model of oligodendrogliopathy, which induces myelin and axonal damage through tamoxifen-inducible expression of diphtheria toxin fragment A in PLP-positive myelinating glia, pronounced T2w hyperintensities in cerebellum and brain stem matched areas of pronounced vacuolation and demyelination as visualized on Luxol-Nissl (L-N) stained histological sections (Table

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1, Table S1) (Mueggler et al., 2012). In a longitudinal combined histopathological/imaging study on mice that were fed 0.2% Cup for 6 weeks, T2-weighted images provided qualitative and semiquantitative estimates of myelin content (Tagge et al., 2016). By combining multiple MRI contrasts (T1w, T2w and MTI), prediction of in vivo myelin status of a lesion could be dramatically improved (Merkler et al., 2005). Longitudinal (over 12 months) in vivo 7T MRI studies on TMEV-infected mice documented the development of thalamic T2 hypointensity and significant brain and spinal cord atrophy (measured by lateral ventricular volume and C4-5 spinal cord cross-sectional area) (Figure 1E, Figure 2D, Table S1) (Paz Soldan et al., 2015; Pirko et al., 2011; Pirko et al., 2009). Additionally, TMEV-infected mice experienced severe disability that was correlated with the degree of thalamic T2 hypointensity (Pirko

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et al., 2009). Progressive loss of whole brain, whole cerebellum, and cerebral cortex was found in MOG35-55-induced EAE mice compared to controls over an observation period of 80 days post disease induction. GM atrophy in the cerebral cortex correlated with myelin density (anti-MBP staining), neurofilament density (NF200 staining), synaptic density (anti-synapsin-1 staining), and neuronal number (anti-NeuN staining), whereby neuronal numbers correlated most strongly (Table S1) (MacKenzie-Graham et al., 2012). This is consistent with the finding that Purkinje cell loss (Calbindin-2 immunostaining) was associated with atrophy of the molecular layer of the cerebellar cortex (MacKenzie-Graham et al., 2009). Astrocytosis (anti-GFAP immunostaining), microglia activation and infiltration (anti-CD11b immunostaining), and neuronal apoptosis (TUNEL and anti-NeuN staining) were found in the lesioned cerebral cortex of MOG35-55 -immunized mice with focal EAE (Figure 3B) (own unpublished results). In addition, several pathological studies confirmed the presence of LMI in the brains and spinal cords of mice from various EAE models (Columba-Cabezas et al., 2006; Dang et

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al., 2015; Pikor et al., 2015; Magliozzi et al., 2004). Using serial contrast-enhanced MRI, leptomeningeal contrast enhancement was shown to be associated with clinical symptoms and high meningeal inflammatory cell density (macrophage/microglia, B and T cells) in EAE mice (Pol et al., 2019; Bhargava et al., 2016).

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4.1.3 Comparing and discussing results of mice and human studies using conventional MRI

Mice with adoptive transfer EAE showed focal BBB disruption mostly in infratentorial areas (Nessler et

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al., 2007), “Cup-EAE” mice only in the CC (Boretius et al., 2012), and MOG35-55-induced EAE mice throughout the lumbar spinal cord (Schellenberg et al., 2007). In contrast, mice with adoptive transfer

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EAE had GELs periventricular, in the brainstem, midbrain, cerebellum, and cranial nerves, with a distribution resembling human MS lesions. Similar as in human MS, these GELs preceded or coincided with clinical symptoms (Tysiak et al., 2009; Wuerfel et al., 2007). From the histopathological point of view, the BBB disruption corresponded to areas with IgG and fibrinogen deposition and

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infiltration of inflammatory cells (Figure 2A) (Boretius et al., 2012; Bruck et al., 1997; Nessler et al., 2007; Schellenberg et al., 2007; Tysiak et al., 2009; Wuerfel et al., 2007; own unpublished results). This finding was confirmed by imaging and histopathological studies on cerebral autopsies and biopsies from MS patients (Bruck et al., 1997; Nesbit et al., 1991; own unpublished results), for which, in contrast to MS mouse models, several types of GELs at different stages of demyelination could be analyzed histologically (Kuhlmann et al., 2017). Tissue destruction was suspected behind BHs in

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MOG35-55-immunized and TMEV-infected mice and confirmed by several postmortem studies on patients with progressive MS (Figure 2C) (own unpublished results), (Barkhof et al., 2003; Bitsch et al., 2001; Pirko et al., 2004; van Waesberghe et al., 1999; van Walderveen et al., 1998). While cortical GM atrophy correlated with myelin, neurofilament, synaptic, and neuronal density in MOG35-55-induced EAE mice (MacKenzie-Graham et al., 2012), (MacKenzie-Graham et al., 2009), it was only associated with neuronal and axonal density in patients with progressive MS. This was explained by the fact that GM lesions were excluded from analyses (Popescu, 2015). Significant signs of neurodegeneration such as activation of microglia, and axonal and neuronal loss were shown in the cerebral lesioned cortex of MOG35-55-immunized mice (Figure 3B) (own unpublished results) and CLs of humans with MS (Figure 3A) (Jurgens et al., 2016; Klaver et al., 2015; Peterson et al., 2001; Politis et al., 2012;

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Reynolds et al., 2011; Strijbis et al., 2017; Wegner et al., 2006). Histopathological differences between CLs and cortical NAGM which were investigated in humans with MS, were not examined further in MS mouse models. While “Cup” mice showed confluent, non-focal T2 hyperintense signals in the whole CC (Boretius et al., 2012; Merkler et al., 2005; Tagge et al., 2016), novel mouse models demonstrated more focal T2w hyperintensities in brain stem and cerebellar structures (Mueggler et al., 2012). Despite the different lesion distribution of T2w hyperintensities in brain, both studies on autopsies and biopsies of MS patients and on MS mouse models identified T2w imaging as sensitive but rather nonspecific indicator of tissue injury (Barkhof et al., 2003; Bruck et al., 1997; van Waesberghe et al., 1999; van Walderveen et al., 1998) with, however, a particularly strong association with myelin content (Boretius et al., 2012; Merkler et al., 2005; Schmierer et al., 2008; Tagge et al., 2016).

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4.2 Magnetization transfer imaging (MTI) 4.2.1 MTI studies in humans

MTI has emerged over the last few decades as a method to improve tissue contrast and thereby quantify tissue damage in an effort to overcome problems with conventional MRI. MTR is known to decrease in intensity in a manner that is proportional to macromolecule density (for further technical

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details on MTI and MTR, see Box 1). Therefore, it is a quantitative measure of the magnetization transfer effect on tissues. Various studies have examined MTR alterations in T1w and T2w lesions,

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normal-appearing brain tissue (NABT), NAWM, and NAGM (Filippi and Agosta, 2007). Variable degrees of MTR reduction have been reported in acute and chronic MS lesions, with the most

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prominent changes being found in BHs (Filippi and Rocca, 2011) (Figure 4). T1 hypointense lesions were demonstrated to have lower MTR values and lower myelin content than T1 isointense lesions or NAWM (Barkhof et al., 2003; Horsfield, 2005). The MTR of T1 hypointense lesions was inversely correlated with the degree of T1 hypointensity (Filippi and Agosta, 2007). Reduced MTR values have

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been found in NAWM days to weeks before lesion formation and were followed by a rapid MTR drop at the moment of Gd-enhancement; this can recover partially or completely during the subsequent 1–6 months to reflect demyelination and remyelination (Filippi and Agosta, 2007; Filippi and Rocca, 2011; Horsfield, 2005; Moll et al., 2011; Ontaneda and Fox, 2017). In a longitudinal study of GELs with monthly MTI, the degree of MTR depression at the time of initial Gd-enhancement was predictive of

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BH persistence and continued MTR depression after 6 months (Table S2) (van Waesberghe et al., 1998).

Decreased MTR values have been found in NABT, NAWM, and GM of patients with CIS and different MS phenotypes compared to HCs (Filippi and Rocca, 2011; Horsfield, 2005). Such MTI abnormalities increased progressively with DD and were more pronounced in patients with progressive MS than in patients with other phenotypes (Table S2) (Filippi et al., 2000; Harrison et al., 2011). Lower MTR values were found in cortical NAGM of MS patients when compared to HCs and in cortical MS lesions when compared with cortical NAGM (Table S2) (Yaldizli et al., 2016). These findings should, however, be considered with caution since a high proportion of intracortical lesions can still remain undetected despite using advanced MRI techniques such as DIR and PSIR (see 4.1.1). NAWM MTR changes

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vary with distance from lesions, such that NAWM around T2w lesions has a significantly lower MTR than NAWM that is more distant (Table S2) (Moll et al., 2011). MTR was significantly lower in demyelinated than in remyelinated lesions, although the MTR of remyelinated lesions was still lower than that of NAWM (Table S2) (Schmierer et al., 2004). A number of studies have analyzed the histopathology behind MTR changes in WMLs and NAWM in MS. A postmortem study examining the pathologic basis of MTR abnormalities in WMLs and different NAWM types with immunohistochemistry in SPMS patients showed the following: MTR abnormalities in NAWM close to WMLs can be attributed to axonal swelling and microglial activation (increased numbers of enlarged microglia/macrophages); abnormalities in NAWM far from WMLs are connected to marked microglial activation associated with proximity to cortical lesions, but not with axonal pathology (Table S2) (Moll et al., 2011). When considering WMLs and different types of NAWM

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together, MTR correlated moderately with myelin density (anti-MBP immunostaining), axonal area, and axonal counts (anti-Neurofilament-H immunostaining), although when excluding lesions on FLAIR, +

T1, and MTR images from analysis, MTR was only correlated with activated microglia (anti-MHCII

immunostaining), but not with axonal or myelin integrity (Table S2) (Moll et al., 2011). In combined MTI and histopathological studies on the postmortem brains and spinal cords of patients with

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progressive MS, MTI was more strongly correlated with axonal density (Bielschowsky’s silver and Bodian staining) than with myelin density (LFB and Klüver staining), not just in lesions but also in NAWM (Table S2) (Mottershead et al., 2003; van Waesberghe et al., 1999). Using a multivariate

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regression analysis, Schmierer et al. revealed that MTR was the best predictor of myelin (LFB staining) in unfixed brains of 15 patients with progressive MS, which was followed by T2w and T1w

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imaging, and fractional anisotropy (FA) (Schmierer et al., 2008), and was not primarily associated with axonal count (Bielschowsky’s silver staining) (Table S2). These results confirmed earlier findings that suggested the association between axonal count (Bielschowsky’s silver staining) and MTR to be caused by the correlations of both measures with myelin content (LFB staining) (Table S2) (Barkhof et

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al., 2003; Schmierer et al., 2004; Schmierer et al., 2008). No association was detected between the extent of gliosis (anti-GFAP immunostaining) and MTI measures (Table S2) (Schmierer et al., 2004). 4.2.2 MTI studies in mice

Numerous studies on “Cup” mice showed a correlation between MTR and myelin content as assessed

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by Black Gold II, LFB, Luxol Fast Blue and Periodic Acid Schiff (LFB/PAS), and anti-MBP/anti-PLP immunohistochemical staining (Table S1) (Fjaer et al., 2013; Merkler et al., 2005; Tagge et al., 2016; Zaaraoui et al., 2008). Longitudinal in vivo 2.35–11.75 T MTI studies on mice that were fed a diet of 0.2% Cup over varying time periods (between 4 and 12 weeks) followed by recovery phases of varying length (between 2 and 7 weeks) revealed reduced MTR within the CC and SDGM, associated with demyelination and partial remyelination (Table S1, Figure 4) (Boretius et al., 2012; Fjaer et al., 2013; Merkler et al., 2005; Tagge et al., 2016; Zaaraoui et al., 2008). In a study examining MTR in the “Cup-EAE” model, significantly reduced MTR was only observed in the CC of “Cup-EAE” mice compared to control but not when compared to “Cup” mice, despite both

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Progress in Neurobiology

groups showing a comparable extent of demyelination. Lesions in “Cup-EAE” mice are more destructive and harbor demyelinating and inflammatory components and acute axonal damage, thus comprising all basic pathologic aspects of an acute MS lesion, whereas “Cup” mice exhibit pure demyelination. Hence, the stronger MTR decrease in “Cup-EAE” mice was proposed to be due to axonal swelling (anti-APP immunostaining), infiltration with T-lymphocytes (anti-CD3 staining), and edema (Table S1) (Boretius et al., 2012). In a novel mouse model of oligodendrogliopathy, where temporally and spatially controlled ablation of oligodendrocytes was achieved by selective expression of diphtheria toxin fragment A in PLP-positive myelinating glia, a minor MTR decrease was found at the end stage of disease in the brainstem, but not in cerebellar WM, frontal cortex, or olfactory bulb. This minor MTR reduction was thought to be due to inefficient removal of myelin debris (Table S1) (Mueggler et al., 2012). This result was further

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investigated by Fjaer et al., who demonstrated that MTR is not sensitive enough to detect myelin changes in areas with low myelin content, such as the cerebellum, olfactory bulb, and cerebral cortex (Table S1) (Fjaer et al., 2013). These results were confirmed and extended by another longitudinal in vivo 7.0 T quantitative magnetization transfer (qMT) imaging study on two mouse strains (C57BL/6 and SJL/J with different sensitivities to Cup) which were fed 0.2% Cup for 5 (C57BL/6) or 7 weeks

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(SJL/J), and then either allowed to recover without Cup diet for 10 weeks or continuously fed with Cup diet. Macromolecular pool size ratio F, which was shown to correlate significantly with myelin content as captured by Black-Gold II staining and MBP immunofluorescence (in contrast to Black-Gold II

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staining, MBP immunostaining also confirmed remyelination in SJL/J mice), was reduced during Cup diet, deteriorated when diet was continued at 10 weeks, and partially recovered after suspension of

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diet in C57BL/6, but not SJL/J mice. In contrast to F, MTR only detected demyelination following initial Cup diet in C57BL/6 and SJL/J mice but no other effects, suggesting that F is more sensitive to Cupinduced demyelination and remyelination than MTR (Table S1) (Turati et al., 2015).

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A longitudinal in vivo MTI study on the dorsal column of the C5 spinal cord segment of lysolecithintreated mice revealed that myelin water fraction (MWF) reflected the time course of de- and partial remyelination (evaluated by eriochrome cyanine staining) more closely than MTR, although MWF detection of early demyelination was also delayed in comparison to histological examination (Table S1) (McCreary et al., 2009). It was postulated that MTR is sensitive to demyelination, although it is not specific, and that it is also influenced by axonal swelling, axonal density, cellular inflammation,

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infiltration of reactive astrocytes, activated microglia and macrophages, CD3 positive T cells, and edema (Table 1) (Boretius et al., 2012; Merkler et al., 2005; Tagge et al., 2016). 4.2.3 Comparing and discussing results of mice and human studies using MTI Because of BBB damage, substantial edema, and lymphocyte mediated inflammation are missing in the most commonly used “Cup” mouse model (Goldberg et al., 2015; Kipp et al., 2009; Praet et al., 2014; Torkildsen et al., 2008), changes observed upon MTR in this mouse model were considered to be more reflective of de- and remyelination than of any other pathology (Fjaer et al., 2013; Merkler et al., 2005; Tagge et al., 2016; Zaaraoui et al., 2008). In the “Cup-EAE” mouse model, with more

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marked axonal damage and inflammation compared to the “Cup” mouse model, MTR was influenced by axonal swelling, infiltration with T cells, and edema (Boretius et al., 2012). This finding was supported by postmortem studies on MS patients. In contrast to mouse studies, these studies allowed examination of human plaques at different stages of demyelination (Kuhlmann et al., 2017; Reynolds et al., 2011) and NAWM. Depending on which kind of lesions were analyzed, e.g. active or chronic inactive, whether lesions and NAWM were analyzed separately or together, and whether imaging/histopathological analyses were conducted on the brain tissue in situ or after cutting slices, or in fixed or unfixed conditions, MTR was associated stronger with either myelin or axonal density, or equally with both parameters, and was additionally influenced by activated microglia and macrophages, cellular inflammation, or infiltration of reactive astrocytes (Table 1). To summarize, numerous previous MTI and histopathological studies have identified MTR as a sensitive and quantitative measure of the degree of myelination in MS (Table 1) – which is plausible because myelin

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contributes to the motion-restricted proton pool (see Box 1) (Vavasour et al., 2011). However, each pathophysiological process that leads to changes in water content, e.g. edema, inflammation, changes in pH and temperature, can influence MTR (Schmierer et al., 2008; Vavasour et al., 2011). Therefore, “MTR determination of MS pathology is somewhat non-specific” and “caution should be used when relating changes in MTR exclusively with changes in myelin content.” (Bakshi et al., 2005b; Vavasour

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et al., 2011).

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4.3 Diffusion tensor imaging (DTI) 4.3.1 DTI studies in humans

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DTI has proven to be an effective means for quantifying demyelination and axonal loss (Sbardella et al., 2013). In DTI, a diffusion tensor that characterizes three-dimensional water movement within tissues is calculated for each voxel and provides in vivo data on processes that influence diffusion as

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a result of microstructural damage to the brain (see Box 1 for more detail). DTI metrics include mean diffusivity (MD), FA, axial diffusivity (AD), and radial diffusivity (RD) (Sbardella et al., 2013). Using a number of different strategies, such as region-of-interest analysis (Calabrese et al., 2011; Deppe et al., 2014, 2016a,b; Droby et al., 2015) , optimized voxel-based analysis (Ceccarelli et al., 2009), tract-based spatial statistics (Raz et al., 2010; Rocca et al., 2016; Roosendaal et al., 2009), histograms (Rovaris et al., 2002; Rovaris et al., 2008), and brain atlas-based analysis (Hasan et al.,

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2012), numerous, mostly cross-sectional DTI studies could demonstrate widespread damage in global (whole brain, whole WM and GM) and regional brain structures (SDGM, cortical GM, specific WM tracts and regions, corticospinal tract) in patients with CIS and different MS phenotypes. Global and regional brain damage was present from the earliest stages of MS and became more pronounced with increasing DD (Cappellani et al., 2014a, b; Filippi and Rocca, 2011; Sbardella et al., 2013). 4.3.1.1 DTI alterations in WM lesions Numerous studies examining DTI changes within WMLs showed higher MD and lower FA values in T2 hyperintense lesions than in NAWM, with the most abnormal values seen in BHs (Figure 5A) (Rovaris et al., 2005; Sbardella et al., 2013). These differences in diffusion parameters described in vivo

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between WMLs and NAWM were further confirmed in two combined imaging and histopathological cerebral postmortem studies of patients with progressive MS. MD, AD, and RD were higher and FA lower in WMLs than in NAWM, regardless of whether data were acquired in unfixed brain tissue or after fixation (Table S2). In addition, FA, MD, RD, but not AD were all demonstrated to be independent predictors of myelin content, with FA as the single best predictor. None of the indexes were associated with axonal count or gliosis (Table 1, Table S2) (Schmierer et al., 2007; Schmierer et al., 2008). Pronounced NAWM RD, FA, and MD changes precede lesion formation and were found in GELs (Filippi and Rocca, 2011; Rovaris et al., 2005; Sbardella et al., 2013). Quantification of DTI parameters within GELs of 22 MS patients longitudinally over 15 months showed a nonsignificant progressive alteration of RD, FA, and MD within the future lesion one to two months prior to enhancement, and a significant RD and MD increase and FA decrease during enhancement. Two months after

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enhancement, these three parameters began to normalize towards pre-enhancement baseline values but remained significantly altered at 12 months in PBHs. A 40% RD elevation within an individual GEL was associated with a 5-fold increased risk for PBHs at 12 months, leading to the speculation that elevated RD within active MS lesions may be indicative of more severe tissue injury (Table 1, Table S2) (Naismith et al., 2010).

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4.3.1.2 Cortical DTI alterations

Calabrese et al. longitudinally investigated alterations of diffusion parameters within cortical lesions detected by DIR and cortical NAGM in MS patients (Figure 3A shows an example of cortical lesions

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detected by DIR). DTI results examining cortical NAGM showed higher FA and unchanged MD values in RRMS patients compared to HCs. Following 166 MS patients longitudinally, revealed significantly

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increased cortical NAGM FA and MD values three years after baseline measurements, whereas cortical lesion FA and MD values did not show a significant difference (Table S2) (Calabrese et al., 2011). However, contrasting PSIR imaging results from Yaldizli et al. showed lower FA and higher MD values in cortical NAGM of the MS group (RRMS and SPMS) compared to HCs; a finding which was

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consistent with other studies (Table S2) (Yaldizli et al., 2016). In three separate studies, higher FA and higher (Yaldizli et al., 2016), lower (Jonkman et al., 2016), and unchanged (Calabrese et al., 2011) MD was shown in cortical lesions relative to cortical NAGM (Table S2). A 7T post-mortem MRI and histopathology study of 14 MS patients demonstrated that the FA increase in cortical lesions is not due to lesional and non-lesional differences in microglia activation and/or proliferation. Rather it is due to increased cellular density without a notable difference in cellular size, that is, tissue compaction

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resulting from ongoing neurodegeneration (Table S2) (Jonkman et al., 2016). Data on DTI alterations in cortical NAGM and cortical lesions have to be interpreted with caution because a high proportion of intracortical lesions still remain undetected, even when using advanced MRI techniques such as DIR and PSIR (see section 4.1.1). 4.3.1.3 Spinal DTI alterations Increased AD, MD, and RD and decreased FA were found in spinal NAWM and T2 lesions in MS patients (Table S2) (Freund et al., 2010; Klawiter et al., 2011; Sbardella et al., 2013). Freund et al. recently showed higher corticospinal tract RD and MD values and lower FA values in RRMS and SPMS patients compared to HCs. While RD and FA normalized in the left lateral corticospinal tract over 6 months following cervical cord relapse, they remained abnormal in the anterior and posterior

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columns. Lower corticospinal tract RD at baseline was found to be predictive of clinical recovery after a spinal cord relapse in MS (Table S2) (Freund et al., 2010). A 4.7 T ex vivo MRI and histopathology study showed increased AD, MD, RD and unchanged relative anisotropy (RA) in cervical spinal WM tracts containing chronic lesions, and in the NAWM of 9 SPMS patients compared to 5 HCs. Coregistration between histological and MRI images was performed to map regions of interest identified in histological sections to respective areas on the DTI map (Figure 5C). RD increased with a rise in demyelination and was the only parameter that could differentiate between normal, mild, and moderate/severe states of demyelination (Figure 5C). Increased AD and MD also reflected demyelination, although they were not as sensitive as RD. Increased RD, increased MD, and decreased RA indicated axonal loss; axonal and myelin pathology were shown to contribute independently to changes in RD, RA, and MD. AD did not correlate with axonal density. Because RD was the only parameter that predicted the level of demyelination, even when controlling for axonal loss

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in the statistical model, and was also altered with axon injury, RD was considered as a marker of overall tissue integrity within chronic MS lesions (Table 1, Table S2) (Klawiter et al., 2011). 4.3.1.4 DTI alterations in NAWM and NAGM

Previous DTI studies revealed widespread abnormalities of microstructural integrity. This was shown by MD, RD, AD increase and FA decrease in whole and regional NAWM and specific WM tracts, and

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MD increase and FA increase/decrease in cortical GM and SDGM in patients with CIS and all MS phenotypes compared to HCs (Table S2) (Ceccarelli et al., 2009; Ciccarelli et al., 2001; Deppe et al.,

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2014, 2016a,b; Harrison et al., 2011; Kolasa et al., 2015; Raz et al., 2010; Rovaris et al., 2005; Sbardella et al., 2013). Both decreased FA and increased MD were demonstrated to be mainly caused by increased RD, implying the predominant role of RD in reflecting the pathological changes (Table

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S2) (Liu et al., 2012; Roosendaal et al., 2009). Using a brain atlas-based analysis and considering the age-dependence of DTI metrics, MD, AD, and RD were increased in normal-appearing subcortical and cortical GM and lobar WM in RRMS patients compared to HCs. FA was reduced in most but not all

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examined regions, leading to the conclusion that FA is a less sensitive measure than MD, AD, and RD (Table S2) (Hasan et al., 2012).

Longitudinal DTI studies revealed worsening of microstructural WM and GM damage over time in patients with different MS types, with more pronounced damage in patients with SPMS than in other MS phenotypes (Table S2) (Cappellani et al., 2014a; Filippi and Rocca, 2011; Harrison et al., 2011; Rovaris et al., 2002; Rovaris et al., 2005; Sbardella et al., 2013). Using a new smoothing technique for

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diffusion-weighted images, reduced cerebellar FA could be demonstrated even in patients with mild and early RRMS without cerebellar lesions (median EDSS 1.5, median DD 28 months), and decreased further with increasing DD, EDSS, and WML load (Table S2) (Deppe et al., 2016b). Significantly reduced thalamic FA was found in patients with CIS and RRMS, even in the absence of thalamic and extensive WMLs, and correlated with the relative thalamic volume (Figure 5B) (Deppe et al., 2016a). Moreover, a unilateral temporary FA increase was found in the normal-appearing left thalamus of one RRMS patient and could explain an episode of central pain and sensory deficits of the contralateral side of the body (Figure 5B) (Deppe et al., 2013). Cross-sectional (Cappellani et al., 2014a; Raz et al., 2010) and longitudinal DTI studies over 2–4 years (Kolasa et al., 2015; Rocca et al., 2016; Rovaris et al., 2008) demonstrated widespread, diffuse WM and GM damage throughout the

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whole brain in patients with CIS. Damage was already present at 2–3 months after symptom onset and worsened with increasing DD (Table S2). No significant associations were found between baseline DTI values and conversion to MS, although DTI alterations in NAWM and NAGM tended to progress more rapidly in patients who converted to definite MS than in those who remained stable with CIS (Table S2) (Kolasa et al., 2015). 4.3.2 DTI studies in mice Using the “Cup” or MOG35-55-induced EAE mouse model, numerous in vivo and ex vivo DTI studies have further explored the pathological correlates behind alterations of cerebral and spinal DTI metrics. Although FA, RA, and MD, widely used as summary parameters, are sensitive markers of pathology, they do not permit specific assessment of underlying injury mechanisms and are not capable of differentiating axonal versus myelin damage (Table 1, Table S1) (Budde et al., 2008; Kim et al., 2006;

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Song et al., 2003; Sun et al., 2006). In contrast, RD increased in response to demyelination and dysmyelination (LFB or MBP immunostaining) in the CC, optic nerve, and optic tract, presumably due to the loss of myelin membrane integrity allowing increased water diffusion perpendicular to the fiber orientations (Table 1, Table S1, Figure 5D) (Aung et al., 2013; Song et al., 2005; Sun et al., 2007; Sun et al., 2006). A decrease in AD was indicative of axonal damage, swelling, and

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integrity/neurofilament dysfunction detected by Bielschowsky’s silver impregnation, or anti-APP or non-phosphorylated neurofilament protein (pNF, SMI-32) immunostaining (Table 1, Table S1, Figure 5D) (Boretius et al., 2012; Budde et al., 2008; Kim et al., 2006; Sun et al., 2007; Sun et al., 2006; Wu

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et al., 2007). AD reduction was attributed to axon fragmentation, which creates barriers to the

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longitudinal movement of water (Aung et al., 2013).

Recent cross-sectional and longitudinal in vivo DTI studies examining the optic nerve of MOG35-55induced EAE mice revealed that a decreased AD was associated with axonal damage (pNF immunostaining), and that AD continued to decrease further with increasing DD from day 8 to day 19

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after immunization (Table 1, Table S1) (Sun et al., 2007; Wu et al., 2007). While Wu et al. detected no RD changes and no demyelination in the optic nerve 19 days after immunization (identified by methylene blue staining and electron microscopy) (Wu et al., 2007), Sun et al. observed increased RD correlating with demyelination (MBP immunostaining) three months after immunization (Table 1, Table S1) (Sun et al., 2007).

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Another longitudinal 4.7 T DTI study examined alterations of the diffusion parameters AD and RD with biweekly in vivo DTI in mice that were fed a diet of 0.2% Cup for 12 weeks followed by 12 weeks of recovery. This DTI and histopathological study revealed reduced AD in the CC in weeks 2–6 of Cup diet, associated with early axonal damage as confirmed by intense SMI-32 immunostaining (Figure 5D). Increased CC RD was seen between weeks 6 and 12 of the ingestion phase and was followed by gradual, partial normalization during the recovery phase. Changes in RD over time were paralleled by a diminished LFB staining at week 6, which progresses in loss till week 12 reflecting demyelination and increases at the end of the recovery phase (week 12+12) reflecting remyelination (Table S1, Figure 5D) (Sun et al., 2006). At 4 weeks of diet, RD appeared within the normal range even though mild demyelination of the CC was suggested by the slightly diminished LFB staining. These results

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were extended by Xie et al., who demonstrated reduced AD in CC only during initial stages of demyelination (after 4 weeks of Cup diet) characterized by extensive microglia/macrophage activation, non-uniform axonal swellings, varicosities, neurofilament dephosphorylation, and reduced axon diameters. AD was not reduced during chronic demyelination during which axonal atrophy was most notable (Table 1, Table S1). The acute axon damage did not progress to discontinuity or loss of axons even after a period of chronic demyelination, but was instead shown to recover. This was paralleled by AD normalization after 10 weeks of Cup diet. RD increased in chronically demyelinated CC regions after 12 weeks (most extensive demyelination) but not after 4 weeks of Cup diet at which time point differences in the proportions of myelinated fibers were found already (Table S1) (Xie et al., 2010). The latter finding was confirmed by a recent DTI and histopathological study on fixed brains of “Cup” mice. Axonal damage detected by beta-APP immunoreactivity was, however, not reflected by AD alterations, suggesting that the tissue fixation process may reduce the sensitivity of AD (Table 1,

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Table S1) (Song et al., 2005).

In two in vivo DTI studies examining the spinal cord WM of chronic EAE mice (MOG 35-55-induced EAE or adoptively transferred and MOG35-55-induced EAE, respectively), AD correlated with axonal damage (anti-beta-APP or anti-pNF immunostaining) and with neurological impairment (Table S1) (Budde et

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al., 2008; Kim et al., 2006). While Kim et al. found no WM RD alterations, suggesting that axonal damage is more widespread than myelin damage in the spinal cord WM of EAE mice, Budde et al. revealed no correlation between demyelinated areas detected by anti-MBP immunostaining and RD

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(Table S1) (Budde et al., 2008; Kim et al., 2006). Boretius et al. suggested that the sensitivity of RD for myelin damage is reduced in the presence of significant demyelination, inflammation (including

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microglia/macrophage activation and astrogliosis), and acute axonal damage (Table 1) (Boretius et al., 2012; Budde et al., 2008). This is because the aforementioned mechanisms that decrease AD also decrease RD during axonal injury, so that opposing actions of axonal damage reducing RD and of

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demyelination increasing RD offset each other (Song et al., 2005; Sun et al., 2006). 4.3.3 Comparing and discussing results of mice and human studies using DTI Overall, mouse models of MS are more suitable to investigate pathophysiological processes behind DTI alterations than postmortem imaging/histopathological studies on humans with MS. Thus, very specific pathophysiological aspects can be analyzed individually in mouse models, while in humans

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with MS concurrent processes are more complex. For example, in mice fed with Cup for 12 weeks a non-focal progredient CC demyelination was paralleled by an increased RD in CC, and early axonal damage with microglia/macrophage activation was paralleled by a reduced CC AD (Figure 5D) (Song et al., 2005; Sun et al., 2006; Xie et al., 2010). When analyzing cerebral WMLs and non-lesional WM in combination in patients with long-standing progressive MS, FA, MD, AD, and RD correlated with myelin and axonal density in some studies (Moll, 2011), (Schmierer, 2007), but were associated with only myelin and not axonal density in another study (Schmierer, 2008). While DTI alterations of different WML stages and NAWM could be investigated in postmortem studies on humans (Schmierer, 2007), (Schmierer, 2008), this was not possible in the mouse models. Furthermore, the pathophysiology behind FA and MD alterations was not analyzed (Sun, 2006), (Xie, 2010), (Song,

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2005). Moreover, the “Cup” mouse model lacks neuroaxonal loss and low-grade inflammation, two typical lesion features in patients with long-standing progressive MS. DTI studies on mice only focused on WM structures while those on MS patients also examined GM structures. Despite these differences between mouse and human studies, and the difficulty to resolve the underlying mechanisms of DTI parameter changes in response to a complex CNS disease such as MS (Budde et al., 2009; Xie et al., 2010), FA and MD were identified as sensitive but nonspecific markers of tissue integrity and injury, and as predictors of myelin content (Table 1). AD and RD were shown to be useful surrogate markers of acute axonal and myelin damage in the CNS, respectively (Table 1). 5. Discussion By visualizing what happens beneath the surface of MS pathology, advanced MRI techniques offer insights into MS pathophysiology beyond WMLs (Louapre, 2018). However, even though the field of

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neuroimaging has made great advances in the last few decades, we are still unable to determine the exact underlying pathophysiology behind imaging alterations detected in MS. By systematically analyzing the existing in vivo and ex vivo imaging and histopathological studies in MS mouse models and humans with MS, we could partially uncover the underlying pathophysiology behind conventional MRI, MTI, and DTI findings (Table 1). The systematic analysis of current literature revealed that Gd

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enhancement on T1w images, resulting from increased permeability of the BBB, corresponds to infiltration of inflammatory cells, soluble factors, and IgG and fibrinogen deposition. While ABHs can have various causes, PBHs seem to reflect severe tissue destruction, including axonal loss and

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persistent demyelination. Despite its strong association with myelin content, T2w imaging seems to be a sensitive but rather non-specific marker of tissue injury. Concerning DTI, the literature review

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identified AD and RD as useful surrogate markers of axonal and myelin damage, respectively; although RD sensitivity is reduced in the presence of acute axonal damage, demyelination, inflammation, and astrogliosis, and AD sensitivity is reduced in the presence of axonal atrophy. Even if FA and MD as derived from DTI are strong predictors of myelin content, they were demonstrated to be

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sensitive but non-specific markers of tissue pathology. With regard to MTI, MTR emerged as a sensitive and quantitative measure of the degree of myelin density, where extent and time course reflect de- and remyelination. However, MTR can also be influenced by other processes that lead to changes in water content.

It is important to note, that our arguments are based only on a relatively small number of combined

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imaging and histopathological studies in MS mouse models and humans with MS (Table 1). Therefore, our conclusions should be considered with caution. They provide a good orientation but are unable to uncover the exact pathophysiological processes behind human imaging findings to the last detail. As previously stated by Rovaris et al. regarding changes in diffusion MRI “at present we can only speculate on their possible pathologic substrates in the MS brain” (Rovaris et al., 2005). Moreover, none of the available imaging techniques are able to provide a complete picture of the MS disease process in all its complexity if used in isolation (Filippi and Rocca, 2011). Therefore, it is beneficial to combine pre- and post-contrast T1w and T2w MRI with MTR, and FA, MD, and RD as derived from DTI for the examination of inflammatory, demyelinating processes and indirect characterization of the axonal compartment. T1w MRI together with DIR, or better PSIR, can be

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applied to quantify cortical processes such as cortical atrophy and lesions, and together with AD, FA, and MD to examine neurodegenerative processes. Further longitudinal in vivo combined imaging and histopathological studies on rationally selected, appropriate mouse models that combine different suitable pathomechanism-specific sequences (Filippi et al., 2019; Inglese and Petracca, 2018), as in our provided MRI protocols, are necessary to further elucidate the pathological correlate behind human imaging alterations. Moreover, better communication and collaboration between pathologists and MRI researchers is needed, including a direct association between pathology and imaging close to autopsy, with the acquisition of MRI sequences of brain and spinal cord in situ and within a short timespan post mortem (Filippi et al., 2019). Additional efforts should be directed towards making these techniques more widely available and feasible for everyday clinical practice, in order to achieve personalized patient care adapted to the

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various pathological mechanisms in MS (Louapre, 2018).

There are several limitations that we would like to point out. We focused our literature review only on those studies in MS mouse models and humans with MS (in vivo and ex vivo) that used conventional MRI, MTI and DTI. However, there are many other advanced imaging modalities adopted in humans and mice that also allow quantification of several pathological processes in MS in vivo. The reason for

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analyzing only conventional MRI, MTI and DTI studies is that these imaging techniques are the most commonly used modalities in the clinical setting. In addition, these imaging modalities have yielded most of the insights into MS pathophysiology and capture the majority of imaging pathologies. There is

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no single mouse model that completely captures the complex pathogenesis and entire spectrum of heterogeneity of human MS and its variety in clinical and radiological presentation. Concerning MS

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mouse models, it was noted that “their comparability to the human disease is limited” and that “some features … are inconsistent with basic observations in human MS” (Boretius et al., 2012; Pirko et al., 2012). However, over the last several decades, new, useful and relevant mouse models of MS have been developed that exhibit specific aspects of the human disease. Each of these models can be used

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to study some pathogenetic mechanisms of MS in the context of a particular research task and to develop new approaches towards the diagnosis and therapy of MS (Abakumova et al., 2015; Denic et al., 2011b). “Depending on the specific research question, the rational selection of appropriate animal models is likely to yield outcomes that will result in translatable findings applicable to MS” (Denic et al., 2011b). Concerning studies of the pathological correlates of neuroimaging in humans, the results are compromised by several limitations such as small group size and inclusion of heterogeneous patient

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groups that differ in type of MS, age, and DD (Table S2). Usually, elderly patients with progressive MS and long DD were examined. For some cases, the type of MS was unknown or the diagnosis of MS not confirmed. Imaging results were not adjusted for age or DD and the influence of treatment on results was not examined. Therefore, results have to be interpreted with caution. Moreover, autopsy and biopsy samples can only display a single time point of disease (Filippi et al., 2019). Frequently, human and mouse studies included no control group. Additionally, fixation in both animal and human postmortem studies presents a potential confounder for imaging and histopathology correlation studies. Thus, T1, T2, MTR, and diffusivity measures were shown to drop following fixation (Schmierer et al., 2007; Schmierer et al., 2008). This is due to dehydration of postmortem tissue, tissue decay due to autolysis, lower temperature during postmortem imaging, and breakdown of energy-dependent ion

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transport mechanisms resulting in a net influx of water into cells and thereby reducing the extracellular space (Horsfield, 2005; Sun et al., 2007; van Walderveen et al., 1998; Zhang et al., 2012). However, the unfixed postmortem brain is very soft; hence, it may be difficult to achieve accurate matching between regions of interest detected on MRI and in the tissue, and the specimen is prone to be damaged due to manual handling (Schmierer et al., 2007). Another limitation is the long postmortem delay until imaging which leads to structural damage due to autolysis (Schmierer et al., 2007). Moreover, study differences in imaging data acquisition and analysis strategies, the manner of tissue sampling for histopathological analyses, and the staining methods used must also be considered. Hence, multiple confounding aspects challenge the translation of results onto human imaging findings. Other difficulties are the various, and often concomitant, pathologic changes occurring in the MS brain that might affect the diffusivity and anisotropy characteristics of tissues in

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opposite ways, thereby reducing the sensitivity and specificity of DTI findings. 6. Conclusion

Even if the exact pathological basis of abnormal human imaging findings in MS is still far from being understood, our literature review aids a better understanding of the different pathophysiological

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processes that underlie MRI findings in MS. Further longitudinal combined in vivo imaging and histopathological studies on rationally selected, appropriate mouse models are necessary. These

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behind human imaging alterations.

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studies should combine several different MRI sequences to further explore the pathological correlates

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Severe irreversible tissue destruction, including permanent demyelination and axonal loss.

Barkhof et al., 2003 It is not possible to differentiate between axonal and van Waesberghe et al., 1999 myelin loss in permanent BHs. van Walderveen et al., 1998 Bitsch et al., 2001

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Pre- and post- Permanent BH contrast T1w

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Conventional MRI Suspected pathophysiology Positive correlation based on Positive correlation based Caveats / Other comments findings study data on mice models on study data on humans with MS with MS Post-contrast T1w GadoliniumIncreased BBB permeability Boretius et al., 2012 Bruck et al., 1997 enhancement with diffuse infiltration of Schellenberg et al., 2007 Nesbit et al., 1991 inflammatory cells Nessler et al., 2007 (macrophages and T- Tysiak et al., 2009 lymphocytes), soluble factors, Wuerfel et al., 2007 IgG and fibrinogen deposition. T1w VSOP enhancement Tysiak et al., 2009 T2w Wuerfel et al., 2007 T2w*

Cortical GM lesions Axonal and dendritic transection, glial, neuronal, synaptic, and axonal loss, and activation of microglia.

Jurgens et al., 2016 Klaver et al., 2015 Peterson et al., 2001 Wegner et al., 2006

DIR, PSIR

Cortical NAGM

Wegner et al., 2006 Jurgens et al., 2016 Klaver et al., 2015

3D T1w

Postcontrast FLAIR

T2w

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DIR, PSIR

Rounded and shrunken neurons and axonal loss.

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MRI

Sequences/ Indices

Cortical atrophy

3D- Leptomeningeal contrast enhancement

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Imaging technique

T2 hyperintensity

Neuroaxonal loss and neuronal MacKenzie-Graham shrinkage, i.e., reduced myelin, 2012 neurofilament, synaptic density MacKenzie-Graham and neuronal number. 2009 Leptomeningeal inflammation Pol et al., 2019 Bhargava et al., 2016

Oedema, demyelination, Boretius et al., 2012 remyelination, inflammation, Mueggler et al., 2012 gliosis, and axonal loss. Tagge et al., 2016 Merkler et al., 2005

et

al., Popescu et al., 2015

et

al.,

Absinta et al., 2015

These findings should be considered with caution because a high proportion of intracortical lesions can still remain undetected despite using advanced MRI techniques such as DIR and PSIR. Therefore, it is difficult to differentiate between cortical lesions and NAGM. These findings should be considered with caution because a high proportion of intracortical lesions can still remain undetected despite using advanced MRI techniques such as DIR and PSIR. Therefore, it is difficult to differentiate between cortical lesions and NAGM.

These findings should be considered with caution because only a few MRI studies with subsequent histopathological examination looked at leptomeningeal inflammation Bruck et al., 1997 High sensitivity and low specificity with regard to Barkhof et al., 2003 tissue pathology. Strong association with myelin van Waesberghe et al., 1999 content, which was superior to T1 and MTR. T2 Mottershead et al., 2003 hyperintensity correlated with axonal content, which Schmierer et al., 2008 was explained by the strong association of both Bot et al., 2004 parameters (T2 hyperintensity and axonal content) with myelin content.

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-

Myelin density

Zaaraoui et al.,2008 Fjaer et al., 2013 Tagge et al., 2016 Merkler et al.,2005 Boretius et al., 2012 Turati et al.,2015 Mueggler et al.,2012 McCreary et al., 2009

DTI

FA

-

Myelin content

MD

-

Myelin content

AD

-

RD

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Acute axonal damage (non- Boretius et al., 2012 uniform swelling, varicosities, Wu et al., 2007 neurofilament Budde et al., 2008 dephosphorylation/dysfunction) Budde et al., 2009 and microglia/macrophage Kim et al., 2006 activation. Sun et al., 2006 Sun et al., 2007 Xie et al., 2010 Myelin damage Song et al., 2005 Sun et al., 2006 Sun et al., 2007 Xie et al., 2010 Thiessen et al., 2013

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Moll et al., 2011 Schmierer et al., 2004 Schmierer et al., 2008 Barkhof et al., 2003 Mottershead et al., 2003 van Waesberghe et al., 1999

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MTR

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MTI

Schmierer et al., 2007 Schmierer et al., 2008

Schmierer et al., 2007 Schmierer et al., 2008 Klawiter et al., 2011

Schmierer et al., 2008 Klawiter et al., 2011

Sensitive to alterations of myelin content, but not specific. MTR can also be influenced by axonal density, axonal swelling, cellular inflammation, infiltration of reactive astrocytes, activated microglia/macrophages, CD3 positive T cells, and edema. MTR correlated with axonal density, which was explained by the strong association of both parameters (MTR and axonal density) with myelin density. Sensitive, but non-specific marker of pathology. FA correlated with axonal content, which was explained by the strong association of both parameters (FA and axonal content) with myelin content. Sensitive, but non-specific marker of pathology. MD correlated with axonal content, which was explained by the strong association of both parameters (MD and axonal content) with myelin content. Reduced sensitivity of AD in the presence of axonal atrophy and by tissue fixation.

Marker of overall tissue integrity and severe tissue injury. Reduced sensitivity of RD in the presence of significant demyelination, inflammation including microglia/macrophage activation, astrogliosis, and acute axonal damage.

Table 1 The suspected pathophysiology behind conventional magnetic resonance imaging findings, magnetization transfer and diffusion tensor indices are presented. All combined imaging and histopathological studies on MS mouse models and humans with MS that support a positive correlation between the suspected pathophysiology and the demonstrated MRI, MTI, and DTI findings are listed. Reviews or case reports that also underline these

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positive correlations were not cited. The abbreviations used in the table are explained in the glossary at the beginning of the manuscript.

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Acknowledgements/Funding statement This work was supported by the medical Faculty of the University of Münster (18-002 fellowship to JK). This research was funded by SFB-TR 128, B05 to SG, FZ, and SGM.

Conflicts of interest Dr. J. Krämer has received honoraria for lecturing from Biogen, Novartis, Mylan, Merck Serono, Roche, Sanofi Genzyme, and Teva and financial research support from Sanofi Genzyme.

Prof. W. Brück has received honoraria for lectures from Bayer Vital, Biogen, Merck Serono, Teva, Genzyme, Roche and Novartis. He is a member of scientific advisory boards for Teva, Biogen, Novartis, MedDay, Celgene, and Genzyme and receives research support from Teva, Genzyme,

Prof. S. Groppa and M. Cerina have no competing interests.

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MedDay, and Novartis.

Prof. F. Zipp has received research grants and/or consultation funds from DFG, BMBF, PMSA, MPG,

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Genzyme, Merck Serono, Roche, Novartis, Sanofi-Aventis, Celgene, ONO, and Octapharma.

Prof. S. G. Meuth has received honoraria for lecturing, travel expenses for attending meetings, and

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financial research support from Almirall, Amicus Therapeutics GmbH Deutschland, Bayer Health Care, Biogen, Celgene, Diamed, Genzyme, MedDay Pharmaceuticals, Merck Serono, Novartis, Novo

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1. Conventional MRI

The conventional MRI techniques typically used for MS diagnosis and disease monitoring include T2weighted (T2w), pre- and postcontrast T1-weighted (T1w), and fluid attenuated inversion recovery (FLAIR) imaging. T1w imaging demonstrates differences in the T1 relaxation times of tissues and

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relies upon the longitudinal relaxation (also called spin-lattice relaxation) of a tissue’s net magnetization vector. A T1w sequence uses a short repetition time (TR) to produce the T1 contrast (TR < ~ 600 ms) and a short echo time (TE < 30 ms) to minimize T2 relaxation effects. Contrast agents shorten the longitudinal relaxation time of tissue and thus increase the T1 signal. For MRI, contrast agents based on the paramagnetic Gd ion are usually selected. Based on the chemical structure of the ligand, Gd-containing contrast agents can be classified as linear versus macrocyclic.

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Linear agents have an elongated organic molecular ligand wrapped around the Gd ion. Macrocyclic agents form a cage-like ligand structure with the Gd ion trapped in a preformed central cavity. The dissociation rates of Gd from macrocyclic ligands are slower than dissociation from linear ligands and are thus considered to be more “stable”. If a Gd ion dissociates from the chelating molecule, the released Gd ion is picked up by a variety of competing anions and cation-binding proteins in the circulating blood. The in vivo dissociation rate of the complex is an important factor that determines, at least in part, the likelihood for associating specific Gd-based contrast agents with the serious adverse event nephrogenic systemic fibrosis (NSF). Clinical and experimental studies suggest that macrocyclic and linear Gd-based contrast agents have different characteristics with regard to Gd deposition in the brain. Thus, it was suggested that only the less tightly bound Gd of linear Gd-based contrast agents is

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deposited in the brain. The T2w sequence highlights differences in the T2 relaxation time of tissues and relies upon the transverse relaxation (also known as “spin-spin” relaxation) of the net magnetization vector. T2 weighting requires long TE times to produce the T2 contrast (TE > 80 ms) and long TR times (TR > 2000 ms) to minimize T1 relaxation effects. For some time T2w images were acquired as 2

nd

echo in a dual echo sequence. The first short echo (TE < 30 ms) was then proton

density (PD) weighted. At present, with fast (Turbo-) spin-echo (SE) sequences the PD image is omitted, and FLAIR sequences are used. FLAIR images are T2w images with a preceding inversion pulse with a long inversion time (TI) of about 70% of the T1 of cerebrospinal fluid (CSF), for which longitudinal magnetization thus crosses the zero line at the time when the first excitation pulse for SEimaging is transmitted. Therefore, the CSF signal is suppressed while signals from all tissues with shorter T1 contribute to the image. Without CSF suppression the T2w image would be dominated by the CSF with its long T2 time, possibly impeding the differentiation between CSF and lesions with

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increased T2. 2. Magnetization transfer imaging

Magnetization transfer imaging (MTI) is a technique that is based on a two-pool model for the protons

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in tissue. The “mobile” pool consists of protons in free water, and the “bound” pool consists of protons in water molecules bound to macromolecules including myelin. Magnetization is exchanged between both pools by cross-relaxation and to a certain extent by (chemical) exchange of water molecules

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between the pools. By studying the transfer of magnetization between the pools it is possible to deduce properties of the macromolecular pool, for which the signal cannot be directly observed in

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conventional MRI. This is achieved practically by applying off-resonance radiofrequency (RF) pulses which exclusively excite the bound pool, such that the protons in this pool are “magnetically saturated”. Depending on the degree of coupling between these pools, the free water pool also becomes partially saturated. If the (conventionally visible) free water pool is subsequently imaged

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using standard imaging pulses, its signal will be reduced. The signal loss depends on the extent of magnetization exchange between the free and the bound pool; more effective exchange will result in more signal loss, e.g. in the presence of a larger pool of macromolecules with bound water. The magnitude of this MT effect can be determined from the relative difference between the signal intensity of two sets of images, acquired with and without application of an off-resonance saturation pulse that decreases signal intensity proportional to the density of macromolecules. The magnetization transfer

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ratio (MTR) for a given voxel can be computed as: MTR = (M 0 − MS)/M0 x 100 where M0 is the magnitude of tissue signal before the MT pulse and MS is the signal after the MT pulse has been applied.

3. Diffusion tensor imaging

Diffusion tensor imaging (DTI) provides information on the magnitude and directionality of free water diffusion in biological tissues in vivo. In an environment without obstacles, e.g. in a glass of water, molecules that jostle around due to thermal motion will disperse equally in all directions (isotropic

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diffusion). However, in tissues with a complex (internal) structure, molecular diffusion is not free and water will diffuse more rapidly along the longitudinal direction of axonal fibers and more slowly perpendicularly to them. Thus, water diffusion is usually more anisotropic in white matter regions, and basically isotropic in CSF. The properties of each voxel of a single DTI image are usually given by a diffusion tensor which describes the relative diffusion in all spatial directions. In order to describe the full diffusion tensor, diffusion gradients need to be applied in at least 6 directions. The diffusion tensor, which can geometrically be visualized using a 3D ellipsoid, can be mathematically decomposed into eigenvectors (Σ1, Σ2 and Σ3) representing the direction of diffusion anisotropy, and eigenvalues (λ1, λ2 and λ3) representing the magnitude of diffusion anisotropy. The axial diffusivity (AD, λ||, longitudinal, or parallel diffusivity) represents the diffusion along the fiber tract and the radial diffusivity (RD, λ⊥, transverse or perpendicular diffusivity) represents the diffusion perpendicular to the axis of

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the fiber tract. The mean diffusivity (MD, apparent diffusion coefficient (ADC)) is a measure of the overall diffusivity in a particular voxel regardless of direction. The degree of anisotropy within a given voxel can be represented by the fractional anisotropy (FA), a scalar dimensionless measure, ranging

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between 0 (completely isotropic) and 1 (maximally anisotropic, i.e. diffusion in only one direction).

Figure 1 Pathophysiological features of multiple sclerosis. (A, left side) Healthy brain with numerous cortical neurons (yellow), intact myelinated axons and intact BBB. Lymphocytes (green) and IgG are

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present within the blood. Processes from parenchymal astrocytes (orange) extend to neuronal synapses, nodes of Ranvier, and BBB. Processes from parenchymal oligodendrocytes (grey) that form myelin sheaths wrapped around axons are depicted. (B, middle above) BBB disruption is one of the earliest abnormalities seen in MS patients. Breakdown of the BBB allows for influx of blood (red), IgG, and soluble factors into the brain, and for transmigration of inflammatory cells (lymphocytes (green), macrophages (blue)) through the inflamed endothelial cell layer of the BBB into the CNS. Proinflammatory soluble factors in the CNS upregulate expression of adhesion molecules on endothelial cells. The process of transendothelial migration is regulated by the complex interaction between

adhesion

molecules,

cytokines,

chemokines,

chemokine

receptors,

and

matrix

metalloproteinases. There are traces of IgG surrounding vessels, and tissue deposition of IgG and

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fibrinogen. (C, center-left) Demyelination is thought to be mediated by resident microglial cells, or infiltrating monocytes/macrophages secreting neurotoxic products including ROS and RNS (pink squares), glutamate, cytokines, and chemokines (yellow dots). These immune cells destroy myelin and, to a variable degree, oligodendrocytes. (D, center-right) If the inflammatory process is arrested in an early phase, demyelinated axons can be remyelinated. OPCs are the main cells responsible for the remyelination. Their migration, proliferation, survival, maturation, and differentiation into myelinating cells is supported by astrocytes. Remyelinated axons are characterized by uniformly thin and shortened internodes. (E, middle below) Chronic CNS inflammation along with oxidative stress, mitochondrial injury, and ion channel redistribution and dysfunction with subsequent ionic imbalances leads to axonal and neuronal damage. The consequences are axonal and dendritic transection, glial, neuronal, synaptic, and axonal loss. (F, right side) All pathophysiological features of MS in Figure 1B-

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E are summarized in the diseased brain (microglia are colored in pink; red squares=ROS/RNS).

Figure 2 Conventional MRI images with classical features of multiple sclerosis in humans and mice,

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and corresponding histopathological images of brain slices showing the suspected underlying pathophysiological processes behind MRI findings. (A) T1w/Gadolinium enhancement labelling of an active lesion and correlating histological sections. (A, left side) Axial T1-weighted post-contrast image of a patient with MS demonstrates a Gd-enhancing lesion. (A, center-left above) Histological examination of the biopsied, Gd-enhancing lesion showed dense inflammatory infiltrates (left image, H & E staining) and T cell infiltrates (right image, anti-CD3 immunostaining) (own unpublished results). (A,

center-left

below)

Dense infiltration by macrophages/microglia infiltrates (left image,

immunohistochemistry for Ki-M1P) and diffuse IgG deposition due to BBB leakage (right image, antiIgG immunostaining) were found in the biopsied, Gd-enhancing lesion (own unpublished results). (A, center-right) Murine Gd enhancement (white arrowheads) in MOG35-55-immunized mice treated with

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Nle4-D-Phe7-α-melanocyte-stimulating hormone (NDP-MSH) or PBS (control) (Mykicki et al., 2016). (A, right above) Exemplary pictures of coronal brain slices depicting active EAE lesions in MOG35-55 immunized mice in the CNS upon H&E staining. Broad perivascular infiltrating immune cells in white and grey matter regions as indicated by the arrows and the star, respectively, in the magnification of the right picture (own unpublished results). (A, right below) Tissue deposition of IgG (IgG) and fibrinogen (Fibrinogen) within a Gadolinium-enhancing lesion in the corpus callosum of a “Cup-EAE” mouse (Boretius et al., 2012). (B) T2w hyperintense regions (non-specific MS lesions) in a 34-year-old male patient with RRMS (EDSS 1.5) and a single murine lesion displaying T2 hyperintensity (Nessler et al., 2007). (C) T1w hypointense black holes and correlating histological sections. (C, left side) Numerous black holes in a 53-year-old female patient with longstanding MS (EDSS 3). (C, center-left above) Bielschowsky’s silver impregnation showing significant axonal loss in a chronic white matter lesion (own unpublished results). (C, center-left below) The same chronic white matter lesion complete

demyelination

(immunohistochemistry

for

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blue)

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macrophages/microglial cells (immunohistochemistry for Ki-M1P in brown) (own unpublished results). (C, center-right) T1w hypointensities located around the ventricles in wild-type C57BL/6 mice infected with TMEV and imaged 7 days after infection (Pirko et al., 2008). (C, right side) Black holes/non active EAE lesions in CNS slices obtained from MOG35-55 -immunized mice upon H&E staining. Arrows

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and dashed round areas indicate areas containing black holes, namely non active lesions characterized by damaged tissue. Horizontal dashed area in the first picture from the right shows a

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demyelinated area (CC = corpus callosum) (own unpublished results). (D) Atrophy. 53-year-old female patient with longstanding MS (EDSS 5.0) showing extensive white matter atrophy (lateral ventricular enlargement). Murine images show the effect of anandamide treatment on progressive brain damage

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(Bittner et al., 2009).

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as assessed by MRI. Preventative Ana treatment prevents lateral ventricle enlargement in EAE mice

Figure 3 (A, left side) 3T DIR images (black and white and colored) showing cortical lesions (white arrows) in a 31-year-old male patient with RRMS (EDSS 1.0). (A, right side, (a)-(e)) Histopathological images of human brain slices showing the suspected underlying pathophysiology behind cortical lesions. (A, (a)) Subpial cortical lesion showing subpial demyelination (immunohistochemistry for MBP in blue) and microglial activation (immunohistochemistry for Ki-M1P in brown) (own unpublished results). (A, (b)) Confocal microscopy demonstrated that many ferritin-positive microglia (red) with

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elongated shapes were closely apposed to neurites (green) (arrowheads) (Peterson et al., 2001). (A, (c)) Confocal images of proximal segments of Golgi-Cox impregnated dendrites of frontotemporal cortical layer IV–VI neurons demonstrate widespread spine loss in multiple sclerosis cortex compared to control cortex. Arrowheads indicate branches of the main dendrite (Jurgens et al., 2016). (A, (d)-(e)) The

subpial

lesion

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axonal

loss

and

minor

loss

of

neuronal

cell

bodies

((d):

immunohistochemistry for neurofilament, (e): Bielschowsky’s silver impregnation) (own unpublished results). (B) Exemplary immunofluorescence staining showing astrocytosis (upper row), microglia activation and infiltration (middle row), and apoptosis (lower row) in lesioned cortex (Cx) of brain slices obtained from MOG35-55 -immunized mice (own unpublished results). (B, upper row) Exemplary pictures showing staining in Cx using DAPI (blue, marker for cell nuclei), GFAP (red, marker for astrocytes), and merged pictures depicting high numbers of astrocytes in the lesioned Cx (high magnification; GFAP positive cells are indicated by white stars). (B, middle row, (a)-(d)) Left image

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(a) shows an exemplary overview picture of one brain hemisphere from MOG 35-55 immunized mice with cortical focal EAE containing glial cells (Cd11b, in red) in the lower part of the hippocampus (hippo, rectangular dashed area) and in the cortex (Cx, circular dashed area). (b-d) Higher magnification exemplary picture showing cell nuclei in blue (DAPI), CD11b-positive cells in red, and merged pictures indicating accumulation of these cells in lesioned grey matter areas. (B, lower row) Exemplary

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overview picture of cortical lesions obtained from MOG35-55 immunized mice with cortical focal EAE. Cell nuclei are depicted in blue (DAPI) and neurons in green (specific neuronal marker NeuN). The

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apoptotic cells in the inflamed region.

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apoptotic marker TUNEL is depicted in red. Merged picture (first picture from the right) shows

Figure 4 (Left side) Human and murine MTR maps highlighting demyelinated regions. Human MTR

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images (A) without MT saturation pulse, (B) with off-resonance saturation pulse applied, and (C) the MTR image (Horsfield, 2005). A low MTR value is seen in the MS lesion posterior to the ventricular horn (white arrow). (Right side, upper row) Population averaged MTR maps from control (left side) and cuprizone-fed mice (right side). Concurrent caudal-to-rostral and medial-to-lateral gradients of MTR decrease within the corpus callosum after 6 weeks of cuprizone treatment (yellow arrows). (Right side, lower row) Black Gold II Stain for myelin in genu of CC. Age-matched control with normal myelinated lateral CC/external capsule (left side) and substantial demyelination in rostral lateral CC/external capsule (arrows) after 6 weeks of cuprizone diet (right side). Blue arrows indicate demyelination in external capsule beyond the extent of MRI-visible lateral CC/external capsule (Tagge

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et al., 2016).

Figure 5 A-C Human DTI images showing MS lesions and alterations in NAGM/NAWM. (A) Fractional

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anisotropy (FA), axial diffusivity (AD), radial diffusivity (RD), and mean diffusivity (MD) alterations within a permanent black hole (white arrows) from a 25-year-old male patient with CIS (EDSS 1.0). (B) FA maps from a healthy control, a 38-year-old patient with early RRMS, and a 39-year-old patient with

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advanced RRMS. The middle FA map shows an area of FA increase that developed in the left thalamus (circle) and was observed only during an episode of contralateral pain (Deppe et al., 2013). The right FA map shows an FA decrease in the thalamus which otherwise appeared normal with conventional MRI (Deppe et al., 2016a). Colored FA scale (values between zero and one). (C) Results of spinal cord MRI image coregistered and warped to a histological section (LFB staining) showing three regions of interest. Shown are axial and radial diffusivity (AD, RD) maps. RD increased with a

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rise in demyelination and was the only parameter that could differentiate between normal, mild, and moderate/severe states of demyelination. Increased AD also reflected demyelination, although it was not as sensitive as RD (Klawiter et al., 2011). (D) Temporal evolution of the diffusion parameters axial diffusivity (AD) and radial diffusivity (RD) and of the corresponding axonal and myelin damage of the corpus callosum (CC) of mice that were fed a diet of 0.2% Cup for 12 weeks followed by 12 weeks recovery. (D, upper two rows) The CC signal in the AD map, which is hyperintense prior to cuprizone feeding (week 0), becomes hypointense after 4 weeks of cuprizone diet (week 4), and subsequently becomes hyperintense even with continuation of cuprizone diet up to 12 weeks. The intensity of the CC signal in the AD map seen after 12 weeks recovery (week 12+12) is similar to that observed at 12 weeks of cuprizone diet. This is associated with early axonal damage in the CC as confirmed by

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intense SMI-32 immunostaining which is evident after 4 weeks of cuprizone diet. Specific SMI-32 immunoreactivity is not marked for other time points. (D, lower both rows) The hypointense CC signal in the RD map prior to cuprizone diet (weeks 0 and 4) becomes hyperintense after 12 weeks of continuous cuprizone diet, and partially normalizes during the recovery period (week 12+12). Changes in RD over time were paralleled by a gradual loss of LFB staining at 4–12 weeks indicating demyelination, and an increase at the end of the recovery phase (week 12+12) reflecting

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remyelination. Arrowheads point to the CC (Sun et al., 2006).