Multiple sclerosis and cerebral endothelial dysfunction: Mechanisms

Pathophysiology 18 (2011) 3–12

Review

Multiple sclerosis and cerebral endothelial dysfunction: Mechanisms J. Steven Alexander a , Robert Zivadinov b , Amir-Hadi Maghzi c,d , Vijay C. Ganta a , Meghan K. Harris e , Alireza Minagar e,∗ b

a Department of Cellular and Molecular Physiology, Louisiana State University Health Sciences Center, Shreveport, LA 71130, USA Buffalo Neuroimaging Analysis Center, The Jacobs Neurological Institute, Department of Neurology, School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, NY, USA c Isfahan Research Committee of Multiple Sclerosis (IRCOMS), Isfahan University of Medical Sciences, Isfahan, Iran d Neuroimmunology Unit, Neuroscience Center, Institute of Cell and Molecular Science, Barts and the London School of Medicine and Dentistry, London, UK e Department of Neurology, Louisiana State University Health Sciences Center, 1501 Kings Highway, Shreveport, LA 71130, USA

Received 16 March 2010; received in revised form 30 March 2010; accepted 8 April 2010

Abstract Multiple sclerosis (MS) is believed to be an immune-mediated neurodegenerative disorder of the human central nervous system which usually affects younger adults with certain genetic backgrounds. The causes and cure for MS remain elusive. Based on the recent advances in our understanding of the pathogenic mechanisms of MS, it appears to represents a heterogeneous group of disorders with dissimilar pathophysiology and neuropathology. Currently, there is no unifying hypothesis to explain the pathogenesis of this complex disease. The three prevailing concepts on the pathogenesis of MS include viral, immunological, and vascular hypotheses. This review presents MS as a neuroinflammatory disease with a significant vascular component and examines the existing evidence for the role of cerebral endothelial cell dysfunction in the pathogenesis of this progressive central nervous system (CNS) inflammatory disorder. © 2010 Elsevier Ireland Ltd. All rights reserved. Keywords: Multiple sclerosis; Endothelial cell; MRI; Blood–brain barrier

Contents 1. 2. 3. 4. 5. 6. 7. 8. 9.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Cerebral endothelial cells: unique features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Vascular pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Role of endothelial microparticles in pathogenesis of MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Endothelial cell adhesion molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Chemokines regulation of immune cell extravasation in MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Blood-borne factors in MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Inflammation and the blood–brain barrier disruption in MS: MR imaging perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

1. Introduction

∗

Corresponding author. Tel.: +1 318 675 4679; fax: +1 318 675 7805. E-mail address: [email protected] (A. Minagar).

0928-4680/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.pathophys.2010.04.002

Multiple sclerosis (MS) is a chronic inflammatory demyelinating and neurodegenerative disease of the human central nervous system (CNS), which affects mainly young

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adults during the most productive periods of their life [1]. Despite major advances in our understanding of the pathophysiology of this progressive neurologic disease and introduction of new disease-modifying agents for its treatments, the etiology and cure for MS remain unidentified. Existing data suggest that pathogenesis of MS has two, often parallel, components: the inflammatory arm versus the neurodegenerative arm [2]. The inflammatory response in MS is associated with an excess activation of cellular and humoral components of the immune system often against possible central nervous system antigens, which results in “the inflammatory demyelination” and loss of oligodendrocytes. The neurodegenerative process, which is responsible for increasing disability in MS, evokes neuronal and axonal loss and leads to brain atrophy [3]. A myriad of hypotheses have been projected to elucidate the origin and pathophysiology of MS as well as the underlying mechanisms for MS relapses. While there is ample evidence to support each hypothesis, none can individually fully explain the specific circumstances and the sequence of events which typify MS and its clinical manifestations. The three prevailing hypotheses for pathophysiology of MS are: viral, immunological, and vascular. Based on the “vascular hypothesis of MS”, disruption of the blood–brain barrier (BBB) which in turn stems from profound abnormalities of the cerebral endothelial cells (CECs) a crucial step in pathogenesis of MS. The authors examine the most recent findings about the abnormalities of cerebral endothelium as well as their contribution towards transendothelial migration of the activated leukocytes into the CNS environment.

2. Cerebral endothelial cells: unique features The BBB, which restricts the immune traffic of cells, drugs, and many blood-borne substances from the peripheral circulation into the CNS milieu, is formed by two major cellular components: highly specialized CECs and astrocytes. CECs exhibit exclusive structural features which differentiate them from other endothelial beds. Unlike nonCNS endothelial cells, CNS endothelial cells possess highly organized tight and adherens junctions. The major components of these endothelial junctions are occludin, claudins, VE-cadherin, zonula-occludens-1, -2, -3, and junctional associated molecules (JAMs), which restrict the exchange of materials across cerebral vessels and creates a complex which normally prevents entry of peripheral immune cell into the CNS [4]. While tight junctions, as the main anatomic barrier within the BBB, remarkably restrict the paracellular traffic of hydrophilic molecules, the lipophilic compounds, such as CO2 and O2 , freely move across along their concentration gradients and enter the CNS. Ultrastructural studies of these tight junctions present them as a constellation of continuous and anastomosing fibrils with tight apposition of the outer leaflets of plasma membranes of neighboring CECs. Of the tight junction proteins, occludin has received intense atten-

tion by endothelial biologists. Human occludin, a 65-kDa (504-amino acid polypeptide), is encoded by OCLN gene and as an integral component of plasma membrane is situated uniquely at tight junctions [5,6]. Claudins are another critical group of transmembrane proteins which, together with occludin, establish tight junctions. This multigene family of small transmembrane proteins (20–27 kDa), with more than 24 members, constitutes tight junction strands through homophilic and heterophilic claudin–claudin interactions mediated by the extracellular loop 2 of claudins [7]. Claudin-5, claudin-3, and claudin12 have been identified in the tight junctions of the BBB at the protein level [8–11]. The role of claudin-5 has been assessed in claudin-deficient mice. In these animals the BBB lacked barrier function against passage of small molecules (<800 Da) which is lethal [10]. Vascular cadherin (VE-cadherin; also known as cadherin5/CD144) is a member of the cadherin superfamily which is encoded by the human gene CDH5. This specific endothelial molecule is situated at complexus adherens junctions between endothelial cells and plays a critical role in the maintenance of endothelial cell contacts and to regulate endothelial permeability and leukocyte extravasation [12]. During activation of the neuroinflammatory cascade of MS dysregulation of these molecular complexes occurs, which in turn permits progressive immune cell entry into the CNS milieu. Inflammatory and immune cell signaling and molecular mechanisms originating from either innate (within CNS) or systemic (peripheral immune system) result in modifications in the integrity and organization of these junctional molecules and BBB permeability and are thought to contribute to the etiogenesis and worsening of multiple sclerosis. Previous immunohistochemical examinations have demonstrated the alterations in occludin and zonula occludens-1 protein in chronic active and inactive lesions as well as normal-appearing white matter [13–15].

3. Vascular pathogenesis Is MS an endothelial disease? And to what degree do abnormally activated CECs play a role in pathogenesis of MS? While the accurate answers for these questions as well as the etiology of MS remain elusive, a number of neuropathological and in vitro observations indicate that pathophysiology of MS, at least in the early stages, includes strong interactions among the cerebral endothelial cells, activated CD4+ T lymphocytes, CD8+ T lymphocytes, macrophages, cytokines and chemokines [16–18]. Neuropathologically, MS lesions are characterized by the presence of perivenular self reactive leukocytes, activated cerebral endothelial cells, areas of demyelination with loss of oligodendrocytes, and neurodegeneration [19]. The presence of these inflammatory perivenular lesions indicates disruption of the normal structure of the BBB in MS patients [20]. Based on the endothelial hypothesis for MS, activation of

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the CECs by inflammatory cytokines such as IFN-␥ and TNF-␣ may alter their anatomic structure and function via different mechanisms. Two major structural proteins of the tight junction that maintain solute barrier between adjacent CECs are occludin and VE-cadherin. A number of observations have demonstrated that the tight junctions’ proteins as well as their adaptor molecules, which anchor them to the cytoskeleton, are affected adversely during neuroinflammatory disorders such as MS [15,21]. Experiments based on the incubation of serum specimens from MS patients who were in acute exacerbation with cultured endothelial cells exhibited decreased expression of occludin and to a lesser degree of VE-cadherin by the endothelial cells [15]. It is possible that during initiation and relapses of MS, elevated serum levels of inflammatory cytokines, particularly IFN-␥, promotes downregulation of synthesis of components of the tight junctions, which in turn translates into a less effective barrier against invading leukocytes [22].

4. Role of endothelial microparticles in pathogenesis of MS Under normal circumstances the endothelial luminal surface provides an anti-thrombogenic surface for blood circulation. However, during active MS, exposure of the endothelial cells to inflammatory cytokines (particularly IFN␥) or changes in the levels of growth and nutritional factors, causes these cells to become activated. An early sign of endothelial activation consists of blebbing and shedding of fragments of their plasma membrane into circulation. These tiny cell-fragments which may also carry certain adhesion molecules or other characteristic molecules of their parent cells are known as endothelial microparticles (EMP). Apart from endothelial cells, microparticles may also be shed from platelets, leukocytes, and erythrocytes. The released EMP are sub-microscopic fragments with a size <1.5 ␮m [23] and based on their size and the type of adhesion molecules they carry, they can be detected and measured by flow cytometry. Elevated plasma levels of EMP carrying CD31 (EMPCD31+ ) and CD51 [vitronectin receptor] (EMPCD51+ ) have been reported in MS patients [23]. The plasma levels of EMPCD31+ were significantly increased in MS patients with relapses and their elevated values revealed a significant association with the presence of contrast-enhancing lesions on brain MRI [23]. On the other hand, the presence of plasma levels of EMPCD51+ may reflect chronic and ongoing damage to the underlying endothelium. Interestingly, the plasma levels of the EMPCD31+ were significantly decreased after treatment of MS patients with interferon-␤1a [24]. Jy et al. further investigated the role of EMP in pathogenesis of MS and reported that plasma EMP, mainly EMPCD54+ make conjugates with monocytes which can activate them [25]. In this way, binding of plasma EMP with monocytes may lead to their activation and greatly enhances the migration of these leukocytes into the CNS.

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Jimenez et al. [26] has studied the role of EMP in migration of activated monocytes through monolayers of CECs. Using an in vitro model, the investigators studied transendothelial migration of U937 monocytes in the presence of plasma obtained from MS patients in relapse versus plasma obtained from normal individuals. They found that MS patient derived plasma increased migration of monocytes through the CECs monolayer and that pre-treatment of monocytes with EMP enhanced their migratory capability through enhanced binding and motility of the EMP–monocyte complexes. Based on the results of this study we may conclude that formation of EMP from stress or activated CECs promotes the inflammatory reaction of MS and leads to greater access of the activated leukocytes to the CNS milieu.

5. Endothelial cell adhesion molecules During normal conditions, resting leukocytes do not cross the BBB in significant numbers because non-activated leukocytes do not avidly bind to or penetrate healthy BBB. However, the inflammatory pathogenesis of MS is characterized by the development of an activated, adhesive and inflamed endothelial phenotype which supports the binding and transendothelial migration of activated T lymphocytes and monocytes [27]. The endothelial pathophysiology of MS is also characterized by the localized disintegration of the normal BBB which in turn facilitates leukocyte extravasation but also permits blood-borne products including neuroactive hormones to enter the parenchyma with destructive and often disastrous consequences. The binding interactions between inflamed endothelium and activated leukocytes are promoted by the expression of adhesion molecules, which include selectins, integrins, immunoglobulin family cell adhesion molecules, chemokines, cytokines, matrix metalloproteinases (MMPs) and members of JAMs family. Leukocyte extravasation into the CNS or into any organ involves a cascade of tethering, rolling, adhesion and eventually transendothelial migration (diapedesis). This chain of events is initiated by transient contact between the circulating activated leukocyte and the underlying cerebral endothelium, which is mediated by adhesion molecules of the selectin family which leads to subsequent interaction between leukocyte integrins and endothelial Ig-CAM integrin ligands (in MS often ␣4␤1 binding to VCAM-1 expressed by activated endothelium). Following the initial tethering, the leukocyte rolls on the endothelium with reduced speed which in turn enhances the possibility of the binding to the underlying endothelium. Next, the rolling leukocyte binds to the chemokines which are expressed at the surface of the activated cerebral endothelial cells. The expressed endothelial chemokines bind to the serpentine receptors located on the surface of activated leukocytes and deliver a G-protein-mediated inside-out signal to the integrins. This in turn enhances the integrins’ avidity for potent adhesion to the leukocytes [28].

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The integrins are a large group of cell surface glycoproteins which also play significant roles in endothelial cell signaling, leukocyte migration, and also cerebral capillary tube formation during angiogenesis [29]. While immunohistochemical analysis has demonstrated a higher expression of vascular cell adhesion molecule (VCAM-1), expression of mucosal addressin cell adhesion molecule-1 (MAdCAM1) was very low or absent, unlike that in non-MS controls, indicating the potential participation of VCAM-1 in the etiology of multiple sclerotic pathology [33]. While a4integrin blockade using the drug Tysabri is now being used to prevent a4b1 and a4b7 dependent lymphocyte adhesion to inflamed brain vessels expressing VCAM-1 and MAdCAM1 respectively, Allavena et al. [30] failed to find significant elevation of MAdCAM-1 in the MS-inflamed cerebrum, suggesting that VCAM-1 may be the most important therapeutic target of Tysabri. The participation of intercellular adhesion molecule-1 (ICAM-1) and VCAM-1 in recruitment of CD4+ T-cell infiltrates in multiple sclerosis is further demonstrated in a mouse model of virus infection (Theiler’s virus infection). Selectins play an important role in leukocyte infiltration into the tissue. Even though increased expression of P-selectin on inflamed endothelium was observed in a mouse model of autoimmune encephalitis, E-selectin and P-selectin deficient mice developed similar disease pathology to that of controls indicating that these adhesion molecules might not be active participants in autoimmune encephalitis [31]. However, due to the potential involvement of these selectins in immune cell trafficking and constitutive expression of P-selectin on choroid plexus epithelium (one of the major routes of immune cell entry into CNS tissue) the roles of these selectins in human disease pathology might be different from that of mouse models. P-selectin glycoprotein ligand-1 (PSGL-1/CD162), a ligand for L-, E- and P-selectin, appears to play an important role in lymphocyte extravasation in MS. Previous reports have indicated that PSGL-1 is not involved in the pathology of autoimmune encephalitis in mouse model [31,32]. Neither PSGL-1 inhibition with antibody or PSGL-1 deficient mice decreased CD8+ T-cell migration across the BBB in EAE [33]. However, a recent report by Bahbouhi et al. [34], showed that CD4+ cells in relapsing remitting MS (RRMS) express higher levels of PSGL-1 than control CD4+ cells and have a greater capacity to penetrate brain endothelial monolayers. These migrating cells apparently use PSGL-1, LFA-1 and VLA-4 (␣4␤1) to cross TNF-␣ activated brain endothelial monolayers [33,34]. PSGL-1 can also bind L-selectin on platelets and E-selectin on activated endothelial cells. PSGL-1 interactions with P-selectin are known to mediate rolling in T-cell rolling in EAE [35,36]. This upregulation of PSGL-1 can be induced by IL-12 [37], which has been shown to be elevated in MS. Because the choroid plexus has high persistent expression of P-selectin, this area may be particularly susceptible to invasion by PSGL1 expressing cells. However, ∼10% of PSGL-1 negative CD4+ T-cells migrated across in vitro HCMEC/D3 barrier

indicating the existence of PSGL-1 independent mechanisms of leukocyte transmigration across inflamed MS cerebral endothelium [34,35]. As P-selectin, E-selectin and L-selectin were shown to be not involved in leukocyte transmigration across the brain endothelium, PSGL-1 specific molecular and biochemical signaling still remains to be elucidated in MS etiology.

6. Chemokines regulation of immune cell extravasation in MS Chemokines and adhesion molecules usually function cooperatively in regulating leukocyte adhesion and migration across endothelial monolayers [28,38]. MS is associated with the increased expression of several chemokines and chemokine receptors on reactive cerebral endothelium. Several lymphoid chemokines e.g. CCL19 and CCL21 as well as their common receptor CCR7 were observed in the inflamed brain vasculature in a mouse model of MS, autoimmune encephalitis [39]. While CCL21 was mainly expressed by inflamed blood vessels, CCL19 was expressed within extravasated leukocytes, astrocytes and microglia. CCR7 expression was observed at the sites of inflammatory lesions and was correlated with the infiltration of mature dendritic cells, B-cells and naïve T-cells. Encephalitogenic T-cells also expressed CCR7 and CXCR3, an alternative receptor for CXCL21. In EAE CCL19 and CCL21 expression were observed in mouse brain endothelium and inhibition of CCL19, CCL21 or CCR7 receptor decreased the adhesion of encephalitogenic T-cells to the inflamed cerebral vasculature [32]. Increased levels of these chemokines and their receptors help leukocytes home into and penetrate the inflamed CNS in MS. Levels of CXCL13, which mediates B-cell and T-cell recruitment in MS, were elevated in the CSF of patients with clinically isolated syndrome (CIS), RRMS, primary progressive MS and secondary progressive MS (SPMS) compared with CSF from patients with non-inflammatory neurologic disease (CXCL12 was not altered) [40]. CSF levels of CXCL13 were proportionate with CSF B-cell concentrations, immune activation, and disease activity (in CIS and RRMS) [41]. CXCL13 was inversely related to IL-10 and TGFb levels and was reduced by high-dose steroids + natalizumab (Tysabri). Expression of CXCL12 and CXCL13 was also implicated differentially in immune cell extravasation into the CNS in MS. CXCL12 is constitutively expressed on CNS parenchyma and microvasculature and is highly upregulated on astrocytes and microvasculature in active/chronic MS lesions. TNF-␣ and IL-1␤, but not IFN-␥ induce CXCL13 [38]. Apart from contributing to leukocyte extravasation CXCL12 may also mediate axonal loss by promoting neurotoxic MMP proteolysis. CXCL13 was upregulated in acute, but apparently not in chronic MS and increased CXCL13 levels were correlated with increased B-cell, CD4+ T-cells and

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plasma blasts [40]. Increased CXCL13 in MS patients’ CSF has a strong correlation with infiltrated leukocyte subsets in MS and suggests that CXCL13 as well as its receptor CXCR5 are important potential therapeutic targets. Chemokines promote MS pathology, and paradoxically IFN-␤ treatment for MS has been shown to upregulate gene expression and serum levels of CCL1, CCL2, CCL7, CXCL10, CXCL11 and CCR1 [42]. The functional importance of this effect requires further study and may provide important new insights applicable in for MS. For example, apart from their detrimental effects, chemokines like CXCL1 also protect the CNS against demyelination (in chronic EAE, equivalent benefit in human MS is not yet obvious). Transgenic mice which overexpress CXCL1 exhibited decreased inflammation and demyelination, decreased Wallerian degeneration with a prominent improvement in remyelination and oligodendrocyte numbers [43].

7. Blood-borne factors in MS Sera from MS patients contain a mixture of proinflammatory cytokines which disturb vascular functions. We previously reported that MS serum leads to a decreased expression of occludin and VE-cadherin [15]. This might reflect effects of cytokines e.g. IFN-␥ effects on the endothelial barrier, which are blocked by IFN-␤1a and IFN-␤1b [15]. Proia et al. [44] reported that sera from SPMS contain factors which are toxic to neurons, suggesting that neurodegeneration in MS could be a primary event, not secondary to demyelination. In another study, Cunnea et al. [45] examined genes expressed within the vascular compartment during

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active MS episodes. Fifty-two of 113 different endothelial specific transcripts were altered in MS. ICAM-2 in particular, was increased in MS possibly reflecting increased vascular remodeling in this condition. Using proteomics, we studied alterations of the protein expression in cerebral endothelial cells in response to exposure to serum obtained from patients with relapsing MS (while they were in relapse) and found elevated levels of protein 14-3-3, metavinculin, myosin-9, plasminogen, reticulocalbin-2 and-3, ribonuclease/angiogenin inhibitor 1, annexin A1, tropomyosin and Ras-related protein Rap-1A [46]. Another significant serum factor which may play a role in pathogenesis is vascular endothelial growth factor (VEGF). VEGF is a potent angiogenic and vasoactive compound which increases endothelial permeability and serves as a mitogen and survival factor for endothelial cells [47–49]. During pathogenesis of MS, VEGF may promote angiogenesis. The issue of neo-angiogenesis in MS lesions has been connected to the presence of enhancement of the ring of the contrast-enhancing lesions on post-gadolinium T1-weighted images, while such phenomenon is not observed at the center of these lesions [50]. In addition, animal studies have shown that chronic overexpression of VEGF in the rat’s brain promoted disruption of the BBB, enhanced expression of major histocompatibility complex class I and II molecules and ICAM-1 [51]. Furthermore, it has been shown that expression of VEGF in both acute and chronic MS plaques is consistently elevated [52]. While the role of other members of the VEGF family in pathogenesis of MS is still under investigation, microvessel proliferation could potentially lead to formation of new and abnormally highly permeable vessels which promotes inflammation, scarring, lesion persistence and leukocyte extravasation.

Fig. 1. Representative axial FLAIR image shows widespread white matter lesions in the periventricular region (a). Supratentorial periventricular lesions are especially well visualized by FLAIR imaging compared to T2-WI where cerebrospinal fluid may mask the visualization of these lesions (a). White matter lesions may also be seen at the cortical level (b). Three different types of cortical lesions are found in patients with MS: (1) juxtacortical lesions (Type 1) (b), located on the border between GM and WM; (2) intracortical lesions (Type 2) (b); and (3) subpial cortical lesions (Type 3), a class of GM lesion recently identified using new myelin basic protein-based pathologic staining methods.

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Fig. 2. Enhancing lesions typical of MS on axial T1-WI post-contrast scans after gadolinium injection (0.1 mmol/kg). Two types of enhancing lesions are present in a 35-year-old female with RRMS with significant residual deficit and disability: homogenous lesions (hyperintense on T1-WI) and open-ring lesions (with central hypointense and external hyperintense rim).

8. Inflammation and the blood–brain barrier disruption in MS: MR imaging perspectives The presence and accumulation of T2-weighted (Fig. 1) and gadolinium (Gd) enhancing T1-weighted lesions (Fig. 2) have represented the MRI gold standard for making a diagnosis and evaluating long-term prognosis in multiple sclerosis (MS). These measures have also been used for a long time as principal MRI outcomes in clinical trials. One of the principal limitations of these techniques is that they incompletely reveal the pathophysiologic process in this disease. At least twenty clinical trials have demonstrated very pronounced inhibition of these inflammatory MRI measures without concurrent clinical benefit over the long term. A meta-analysis study [53] analyzed data from five natural history studies plus the placebo groups from four clinical trials including a total of 307 patients. A moderate correlation was found between the mean cumulative number of Gd-enhancing lesions (Fig. 2) observed during the first six months and relapse rate during the first year. More recent meta-analysis approaches showed that 80% of the variance in the effect on relapses between trials is explained by the variance in MRI effects [54]. Smaller and shorter phase II studies based on MRI lesion end points may give indications also on the effect of the treatment on relapse end points. Nevertheless, little correlation was observed between the number of these lesions at baseline or over the first six months and progression of disability, as measured by change in the Expanded Disability Summary Scale (EDSS) score at one or two years. Therefore, Gd-enhancing lesions are very good short term (6–12 months) predictors of clinical activity but not of long-term development of disability [55]. A number of studies have followed patients over time with serial MRI scans, and these have allowed the evolution of individual lesion appearance over time to be mapped [56–59]. Approximately 98% of the lesions first appear on

T1-WI as bright Gd-enhancing spots, which represent acute inflammatory lesions where the blood–brain barrier (BBB) has broken down locally [60]. On T2-WI, these lesions may or may not appear concomitantly hyperintense. A small part of these lesions (∼5%) will disappear completely and no longer are visible on either T1-WI or T2-WI. They correspond to those lesions that achieve complete remyelination with no residual inflammation or edema [61]. Enhancement duration is short (around three to six weeks) [56], and after four to six weeks, most lesions do not enhance. These lesions thus disappear from T1-WI but remain visible in most cases as hyperintense areas on T2-WI. From a histologic point of view, these lesions are characterized by limited inflammatory activity but are heterogeneous in terms of remyelination status, ranging from extensively demyelinated to fully remyelinated lesions [62,63]. Approximately two-thirds of lesions remain visible on T1-WI as hypointense areas after Gd-enhancement is lost one or two months after their first appearance. These lesions, called “acute black holes”, may evolve over the following months into demyelinated or partially/completely remyelinated lesions invisible on T1-WI (Fig. 3). This process presumably corresponds to acute phase remyelination. Approximately one-third of these acute black holes persist on T1-WI as hypointense areas. These T1 chronic black holes are characterized histopathologically by extensive axonal loss and gliosis and their density is associated with the degree of chronic neurologic impairment [64]. Prevention of evolution of initially Gd-enhancing lesions into T1 chronic black holes may thus be considered as a key treatment objective in MS therapy [65–67]. Different pathologic lesion states may give rise to a similar appearance on conventional MRI imaging and, conversely, lesions with similar myelination status may take different forms on MRI images. This suggests that lesion appearance on conventional MRI images may not be specific in terms of underlying pathology, and probably accounts in part for the mismatch between these MRI measures and clinical outcome.

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Fig. 3. Axial T1-WI in a 43-year-old female with RRMS and EDSS score of 5.5. On T1-WI, multiple lesions are hypointense or appear as “black holes”.

Several strategies have been proposed to overcome the limitations of conventional MRI scanning for better visualization of the global active inflammatory pathology in MS. One is related to development of more advanced conventional sequences and application of newer contrast agents that are able to detect, in real time on the scanner, pathology that is currently invisible with standard conventional sequences. Newer imaging hardware strategies (such as high field strength scanners) (Fig. 4) may increase the sensitivity of conventional scanning paradigms for the detection of invisible pathology. Inflammatory lesions observed in patients with MS are characterized by activation of microglia and infiltration of lymphocytes and macrophages across the BBB. Preclinical studies of the experimental autoimmune encephalomyelitis rat model of MS detected intracellular phagocytosis occurring in inflammatory lesions by imaging with ultra-small particle iron oxide (USPIO), a new contrast agent that accumulates in phagocytic cells. One prospective, open-label,

phase 2 imaging study investigated macrophage infiltration into the CNS in patients with clinically diagnosed RRMS [68]. Ten patients experiencing an acute relapse of MS were injected with intravenous USPIO 2.6 mg Fe/kg. Gadolinium enhancement, a marker of increased BBB permeability, was also evaluated in the same patients. Twenty-four hours following injection, 33 USPIO-enhanced lesions were observed on T1-WI images in 9 of 10 patients, and 55 Gd-enhanced lesions were visualized in 7 of 10 patients. Additionally, findings were similar in 4 patients receiving long-term treatment with IFN beta or immunosuppressive therapy (13 USPIO lesions; 25 Gd-lesions) compared with 6 untreated patients (20 USPIO lesions; 30 Gd-lesions). In a phase 2 clinical study of 19 patients with RRMS, lesion enhancement with the novel USPIO nanoparticle SHU555C occurred more frequently and remained visible for a longer period of time compared with Gd-lesion enhancement [69]. Overall, application of MRI using USPIO permitted visualization of different inflammatory aspects of CNS lesions and provided evidence that

Fig. 4. Evaluation of T2 hyperintense lesions in patients with RRMS on FLAIR images. 1.5 T scanner (a) shows less T2 lesions than 3 T (b) (arrows).

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infiltration of macrophages and other inflammatory cells can be evaluated either with or without increased permeability of the BBB. Another recently published study explored use of USPIO as a marker for diffuse inflammation in MS normalappearing white matter (NAWM), using quantitative MRI [70]. The study suggested that USPIO-enhanced MRI may be a new potential marker for subtle inflammatory activity in MS NAWM. High-resolution micro-autoradiography using USPIO is emerging as an important new imaging tool that may accurately predict progression of MS. However, longitudinal studies are needed to further investigate the pathologic processes occurring within USPIO-enhanced lesions in patients with relapsing and progressive forms of MS and their relation to disease-modifying treatments. Another way to increase the sensitivity of conventional MRI sequences for detection of inflammatory pathology in patients with MS is to use high field strength (Fig. 4). In this respect, 3T scanners are being used increasingly for visualizing inflammatory pathology. Use of a 3T scanner allows the detection of around 20–50% more Gd-enhancing and T2 lesions compared to a 1.5 T scanner [71–73].

[6]

[7]

[8]

[9]

[10]

[11]

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9. Conclusion While the cause(s) and pathogenic mechanisms underlying MS are still unclear and intensely debated, the vascular inflammation associated with the cerebral endothelium now clearly represents a central and early abnormality which undoubtedly underlies several features of MS including BBB disturbances and enhanced leukocyte emigration which drive inflammatory neurodegeneration typical of this condition. Proof for this concept is found in current MS therapies targeting cerebral endothelial cells which are now the most effective treatments for MS that can diminish the inflammatory cascade of MS. Further research into defining the changes in cerebral endothelial cells, particularly adhesive determinants, chemokines and tight and adherens junction dysregulation, could lead to future identification of even more highly selective therapeutic markers and targets in MS.

References [1] J.H. Noseworthy, C. Lucchinetti, M. Rodriguez, B.G. Weinshenker, Multiple sclerosis, N. Engl. J. Med. 343 (2000) 938–952. [2] B.D. Trapp, J. Peterson, R.M. Ransohoff, R. Rudick, S. Mörk, L. Bö, Axonal transection in the lesions of multiple sclerosis, N. Engl. J. Med. 338 (1998) 278–285. [3] J.J. Geurts, P.K. Stys, A. Minagar, S. Amor, R. Zivadinov, Gray matter pathology in (chronic) MS: modern views on an early observation, J. Neurol. Sci. 282 (2009) 12–20. [4] H. Wolburg, A. Lippoldt, Tight junctions of the blood–brain barrier: development, composition and regulation, Vascul. Pharmacol. 38 (2002) 323–337. [5] Y. Ando-Akatsuka, M. Saitou, T. Hirase, M. Kishi, A. Sakakibara, M. Itoh, S. Yonemura, M. Furuse, S. Tsukita, Interspecies diversity

[13]

[14]

[15]

[16] [17] [18] [19] [20] [21]

[22]

[23]

[24]

[25]

of the occludin sequence: cDNA cloning of human, mouse, dog, and rat-kangaroo homologues, J. Cell. Biol. 133 (1996) 43–47. M. Furuse, T. Hirase, M. Itoh, A. Nagafuchi, S. Yonemura, S. Tsukita, S. Tsukita, Occludin: a novel integral membrane protein localizing at tight junctions, J. Cell. Biol. 123 (1993) 1777–1788. J. Piontek, L. Winkler, H. Wolburg, S.L. Müller, N. Zuleger, C. Piehl, B. Wiesner, G. Krause, I.E. Blasig, Formation of tight junction: determinants of homophilic interaction between classic claudins, FASEB J. 22 (2008) 146–158. H. Wolburg, K. Wolburg-Buchholz, J. Kraus, G. Rascher-Eggstein, S. Liebner, S. Hamm, F. Duffner, E.H. Grote, W. Risau, B. Engelhardt, Localization of claudin-3 in tight junctions of the blood–brain barrier is selectively lost during experimental autoimmune encephalomyelitis and human glioblastoma multiforme, Acta Neuropathol. 105 (2003) 586–592. T. Nitta, M. Hata, S. Gotoh, Y. Seo, H. Sasaki, N. Hashimoto, M. Furuse, S. Tsukita, Size-selective loosening of the blood–brain barrier in claudin-5-deficient mice, J. Cell. Biol. 161 (2003) 653–660. S. Liebner, U. Kniesel, H. Kalbacher, H. Wolburg, Correlation of tight junction morphology with the expression of tight junction proteins in blood–brain barrier endothelial cells, Eur. J. Cell. Biol. 79 (2000) 707–717. K. Morita, H. Sasaki, M. Furuse, S. Tsukita, Endothelial claudin: claudin-5/TMVCF constitutes tight junction strands in endothelial cells, J. Cell. Biol. 147 (1999) 185–194. D. Vestweber, VE-cadherin: the major endothelial adhesion molecule controlling cellular junctions and blood vessel formation, Arterioscler. Thromb. Vasc. Biol. 28 (2008) 223–232. J. Kirk, J. Plumb, M. Mirakhur, S. McQuaid, Tight junctional abnormality in multiple sclerosis white matter affects all calibres of vessel and is associated with blood–brain barrier leakage and active demyelination, J. Pathol. 201 (2003) 319–327. J. Plumb, S. McQuaid, M. Mirakhur, J. Kirk, Abnormal endothelial tight junctions in active lesions and normal-appearing white matter in multiple sclerosis, Brain. Pathol. 12 (2002) 154–169. A. Minagar, D. Ostanin, A.C. Long, M. Jennings, R.E. Kelley, M. Sasaki, J.S. Alexander, Serum from patients with multiple sclerosis downregulates occludin and VE-cadherin expression in cultured endothelial cells, Mult. Scler. 9 (2003) 235–238. M.A. Friese, L. Fugger, Pathogenic CD8(+) T cells in multiple sclerosis, Ann. Neurol. 66 (2009) 132–141. E.M. Frohman, M.K. Racke, C.S. Raine, Multiple sclerosis—the plaque and its pathogenesis, N. Engl. J. Med. 354 (2006) 942–955. M. Sospedra, R. Martin, Immunology of multiple sclerosis, Annu. Rev. Immunol. 23 (2005) 683–747. S. Love, Demyelinating diseases, J. Clin. Pathol. 59 (2006) 1151–1159. A. Minagar, J.S. Alexander, Blood–brain barrier disruption in multiple sclerosis, Mult. Scler. 9 (2003) 540–549. S.D. Bamforth, U. Kniesel, H. Wolburg, B. Engelhardt, W. Risau, A dominant mutant of occludin disrupts tight junction structure and function, J. Cell. Sci. 112 (1999) 1879–1888. A. Minagar, A. Long, T. Ma, T.H. Jackson, R.E. Kelley, D.V. Ostanin, M. Sasaki, A.C. Warren, A. Jawahar, B. Cappell, J.S. Alexander, Interferon (IFN)-beta 1a and IFN-beta 1b block IFN-gamma-induced disintegration of endothelial junction integrity and barrier, Endothelium 10 (2003) 299–307. A. Minagar, W. Jy, J.J. Jimenez, W.A. Sheremata, L.M. Mauro, W.W. Mao, L.L. Horstman, Y.S. Ahn, Elevated plasma endothelial microparticles in multiple sclerosis, Neurology 56 (2001) 1319–1324. W.A. Sheremata, W. Jy, S. Delgado, A. Minagar, J. McLarty, Y. Ahn, Interferon-beta1a reduces plasma CD31+ endothelial microparticles (CD31+ EMP) in multiple sclerosis, J. Neuroinflammation 3 (2006) 23. W. Jy, A. Minagar, J.J. Jimenez, W.A. Sheremata, L.M. Mauro, L.L. Horstman, C. Bidot, Y.S. Ahn, Endothelial microparticles (EMP) bind and activate monocytes: elevated EMP–monocyte conjugates in multiple sclerosis, Front. Biosci. 9 (2004) 3137–3144.

J.S. Alexander et al. / Pathophysiology 18 (2011) 3–12 [26] J. Jimenez, W. Jy, L.M. Mauro, L.L. Horstman, E.R. Ahn, Y.S. Ahn, A. Minagar, Elevated endothelial microparticle–monocyte complexes induced by multiple sclerosis plasma and the inhibitory effects of interferon-beta 1b on release of endothelial microparticles, formation and transendothelial migration of monocyte–endothelial microparticle complexes, Mult. Scler. 11 (2005) 310–315. [27] W.F. Hickey, B.L. Hsu, H. Kimura, T-lymphocyte entry into the central nervous system, J. Neurosci. Res. 28 (1991) 254–260. [28] T.S. Olson, K. Ley, Chemokines and chemokine receptors in leukocyte trafficking, Am. J. Physiol. Int. Comp. Physiol. 283 (2002) R7–R28. [29] G.J. del Zoppo, R. Milner, Integrin–matrix interactions in the cerebral microvasculature, Arterioscler. Thromb. Vasc. Biol. 26 (2006) 1966–1975. [30] R. Allavena, S. Noy, M. Andrews, N. Pullen, CNS elevation of vascular and not mucosal addressin cell adhesion molecules in patients with multiple sclerosis, Am. J. Pathol. 176 (2010) 556–562. [31] A. Doring, M. Wild, D. Vestweber, U. Deutsch, B. Engelhardt, E- and P-selectin are not required for the development of experimental autoimmune encephalomyelitis in C57BL/6 and SJL mice, J. Immunol. 179 (2007) 8470–8479. [32] B. Engelhardt, Molecular mechanisms involved in T cell migration across the blood–brain barrier, J. Neural. Transm. 113 (2006) 477–485. [33] B. Engelhardt, Immune cell entry into the central nervous system: involvement of adhesion molecules and chemokines, J. Neurol. Sci. 274 (2008) 23–26. [34] B. Bahbouhi, B.L. Berthelot, S. Pettré, L. Michel, S. Wiertlewski, B. Weksler, I.A. Romero, F. Miller, P.O. Couraud, S. Brouard, D.A. Laplaud, J.P. Soulillou, Peripheral blood CD4+ T lymphocytes from multiple sclerosis patients are characterized by higher PSGL-1 expression and transmigration capacity across a human blood–brain barrier-derived endothelial cell line, J. Leukoc. Biol. 86 (2009) 1049–1063. [35] L. Battistini, L. Piccio, B. Rossi, S. Bach, S. Galgani, C. Gasperini, L. Ottoboni, D. Ciabini, M.D. Caramia, G. Bernardi, C. Laudanna, E. Scarpini, R.P. McEver, E.C. Butcher, G. Borsellino, G. Constantin, CD8+ T cells from patients with acute multiple sclerosis display selective increase of adhesiveness in brain venules: a critical role for P-selectin glycoprotein ligand-1, Blood 101 (2003) 4775–4782. [36] S. Kerfoot, P. Kubes, Overlapping roles of P-selectin and alpha 4 integrin to recruit leukocytes to the central nervous system in experimental autoimmune encephalomyelitis, J. Immunol. 169 (2002) 1000–1006. [37] P. Deshpande, I.L. King, B.M. Segal, IL-12 driven upregulation of Pselectin ligand on myelin-specific T cells is a critical step in an animal model of autoimmune demyelination, J. Neuroimmunol. 173 (2006) 35–44. [38] N.M. Rebenko-Moll, L. Liu, A. Cardona, R.M. Ransohoff, Chemokines, mononuclear cells and the nervous system: heaven (or hell) is in the details, Curr. Opin. Immunol. 18 (2006) 683–689. [39] C. Alt, M. Laschinger, B. Engelhardt, Functional expression of the lymphoid chemokines CCL19 (ELC) and CCL 21 (SLC) at the blood–brain barrier suggests their involvement in G-protein-dependent lymphocyte recruitment into the central nervous system during experimental autoimmune encephalomyelitis, Eur. J. Immunol. 32 (2002) 2133–2144. [40] M. Krumbholz, D. Theil, S. Cepok, B. Hemmer, P. Kivisäkk, R.M. Ransohoff, M. Hofbauer, C. Farina, T. Derfuss, C. Hartle, J. Newcombe, R. Hohlfeld, E. Meinl, Chemokines in multiple sclerosis: CXCL12 and CXCL13 up-regulation is differentially linked to CNS immune cell recruitment, Brain 129 (2006) 200–211. [41] F. Sellebjerg, L. Börnsen, M. Khademi, M. Krakauer, T. Olsson, J.L. Frederiksen, P.S. Sørensen, Increased cerebrospinal fluid concentrations of the chemokine CXCL13 in active MS, Neurology 73 (2009) 2003–2010. [42] S. Cepok, H. Schreiber, S. Hoffmann, D. Zhou, O. Neuhaus, G. von Geldern, S. Hochgesand, S. Nessler, V. Rothhammer, M. Lang, H.P.

[43]

[44]

[45]

[46]

[47] [48]

[49] [50]

[51]

[52]

[53]

[54]

[55] [56]

[57]

[58]

[59]

[60]

11

Hartung, B. Hemmer, Enhancement of chemokine expression by interferon beta therapy in patients with multiple sclerosis, Arch. Neurol. 66 (2009) 1216–1223. K.M. Omari, S.E. Lutz, L. Santambrogio, S.A. Lira, C.S. Raine, Neuroprotection and remyelination after autoimmune demyelination in mice that inducibly overexpress CXCL1, Am. J. Pathol. 174 (2009) 164–176. P. Proia, G. Schiera, G. Salemi, P. Ragonese, G. Savettieri, I. Di Liegro, Neuronal and BBB damage induced by sera from patients with secondary progressive multiple sclerosis, Int. J. Mol. Med. 24 (2009) 743–747. P. Cunnea, J. McMahon, E. O’Connell, K. Mashayekhi, U. Fitzgerald, S. McQuaid, Gene expression analysis of the microvascular compartment in multiple sclerosis using laser microdissected blood vessels, Acta Neuropathol. (2009). J.S. Alexander, A. Minagar, M. Harper, S. Robinson-Jackson, M. Jennings, S.J. Smith, Proteomic analysis of human cerebral endothelial cells activated by multiple sclerosis serum and IFNbeta-1b, J. Mol. Neurosci. 32 (2007) 169–178. D.O. Bates, S.J. Harper, Regulation of vascular permeability by vascular endothelial growth factors, Vascul. Pharmacol. 39 (2002) 225–237. J.M. Isner, et al., Clinical evidence of angiogenesis after arterial gene transfer of phVEGF165 in patient with ischaemic limb, Lancet 348 (1996) 370–374. R.K. Jain, Molecular regulation of vesselmaturation, Nat. Med. 9 (2003) 685–693. J.F. Hiehle Jr., et al., Correlation of spectroscopy and magnetization transfer imaging in the evaluation of demyelinating lesions and normal appearing white matter in multiple sclerosis, Magn. Reson. Med. 32 (1994) 285–293. M.A. Proescholdt, J.D. Heiss, S. Walbridge, J. Mühlhauser, M.C. Capogrossi, E.H. Oldfield, M.J. Merrill, Vascular endothelial growth factor (VEGF) modulates vascular permeability and inflammation in rat brain, J. Neuropathol. Exp. Neurol. 58 (1999) 613–627. M.A. Proescholdt, S. Jacobson, N. Tresser, E.H. Oldfield, M.J. Merrill, Vascular endothelial growth factor is expressed in multiple sclerosis plaques and can induce inflammatory lesions in experimental allergic encephalomyelitis rats, J. Neuropathol. Exp. Neurol. 61 (2002) 914–925. L. Kappos, D. Moeri, E.W. Radue, A. Schoetzau, K. Schweikert, F. Barkhof, et al., Predictive value of gadolinium-enhanced magnetic resonance imaging for relapse rate and changes in disability or impairment in multiple sclerosis: a meta-analysis. Gadolinium MRI Meta-analysis Group, Lancet 353 (1999) 964–969. M.P. Sormani, L. Bonzano, L. Roccatagliata, G.R. Cutter, G.L. Mancardi, P. Bruzzi, Magnetic resonance imaging as a potential surrogate for relapses in multiple sclerosis: a meta-analytic approach, Ann. Neurol. 65 (2009) 268–275. R. Zivadinov, T.P. Leist, Clinical-magnetic resonance imaging correlations in multiple sclerosis, J. Neuroimaging 15 (2005) 10S–21S. F. Cotton, H.L. Weiner, F.A. Jolesz, C.R. Guttmann, MRI contrast uptake in new lesions in relapsing–remitting MS followed at weekly intervals, Neurology 60 (2003) 640–646. C.R. Guttmann, S.S. Ahn, L. Hsu, R. Kikinis, F.A. Jolesz, The evolution of multiple sclerosis lesions on serial MR, Am. J. Neuroradiol. 16 (1995) 1481–1491. A. Rovira, J. Alonso, G. Cucurella, C. Nos, M. Tintore, S. Pedraza, et al., Evolution of multiple sclerosis lesions on serial contrast-enhanced T1weighted and magnetization-transfer MR images, Am. J. Neuroradiol. 20 (1999) 1939–1945. J.H. van Waesberghe, M.A. van Walderveen, J.A. Castelijns, P. Scheltens, A. Lycklama, G.J. Nijeholt, C.H. Polman, et al., Patterns of lesion development in multiple sclerosis: longitudinal observations with T1-weighted spin-echo and magnetization transfer MR, Am. J. Neuroradiol. 19 (1998) 675–683. R. Zivadinov, Evidence for neuroprotection in multiple sclerosis: can imaging techniques measure neuroprotection and remyelination? Neurology 68 (2007) S72–S82.

12

J.S. Alexander et al. / Pathophysiology 18 (2011) 3–12

[61] D. Cadavid, L.J. Wolansky, J. Skurnick, J. Lincoln, J. Cheriyan, K. Szczepanowski, et al., Efficacy of treatment of MS with IFNbeta-1b or glatiramer acetate by monthly brain MRI in the BECOME study, Neurology 72 (2009) 1976–1983. [62] F. Barkhof, W. Bruck, C.J. De Groot, E. Bergers, S. Hulshof, J. Geurts, et al., Remyelinated lesions in multiple sclerosis: magnetic resonance image appearance, Arch. Neurol. 60 (2003) 1073–1081. [63] P. Patrikios, C. Stadelmann, A. Kutzelnigg, H. Rauschka, M. Schmidbauer, H. Laursen, et al., Remyelination is extensive in a subset of multiple sclerosis patients, Brain 129 (2006) 3165–3172. [64] F. Bagnato, N. Jeffries, N.D. Richert, R.D. Stone, J.M. Ohayon, H.F. McFarland, et al., Evolution of T1 black holes in patients with multiple sclerosis imaged monthly for 4 years, Brain 126 (2003) 1782–1789. [65] F. Bagnato, S. Gupta, N.D. Richert, R.D. Stone, J.M. Ohayon, J.A. Frank, et al., Effects of interferon beta-1b on black holes in multiple sclerosis over a 6-year period with monthly evaluations, Arch. Neurol. 62 (2005) 1684–1688. [66] C.M. Dalton, K.A. Miszkiel, G.J. Barker, D.G. MacManus, T.I. Pepple, M. Panzara, et al., Effect of natalizumab on conversion of gadolinium enhancing lesions to T1 hypointense lesions in relapsing multiple sclerosis, J. Neurol. 251 (2004) 407–413. [67] M. Filippi, M. Rovaris, M.A. Rocca, M.P. Sormani, J.S. Wolinsky, G. Comi, Glatiramer acetate reduces the proportion of new MS lesions evolving into “black holes”, Neurology 57 (2001) 731–733.

[68] V. Dousset, B. Brochet, M.S. Deloire, L. Lagoarde, B. Barroso, J.M. Caille, et al., MR imaging of relapsing multiple sclerosis patients using ultra-small-particle iron oxide and compared with gadolinium, Am. J. Neuroradiol. 27 (2006) 1000–1005. [69] M.M. Vellinga, R.D. Oude Engberink, A. Seewann, P.J. Pouwels, M.P. Wattjes, S.M. van der Pol, et al., Pluriformity of inflammation in multiple sclerosis shown by ultra-small iron oxide particle enhancement, Brain 131 (2008) 800–807. [70] M.M. Vellinga, H. Vrenken, H.E. Hulst, C.H. Polman, B.M. Uitdehaag, P.J. Pouwels, et al., Use of ultrasmall superparamagnetic particles of iron oxide (USPIO)-enhanced MRI to demonstrate diffuse inflammation in the normal-appearing white matter (NAWM) of multiple sclerosis (MS) patients: an exploratory study, J. Magn. Reson. Imaging 29 (2009) 774–779. [71] C. DiPerri, M. Dwyer, D. Wack, J.L. Cox, K. Hashmi, E. Saluste, et al., Lesion characteristics at 1.5 and 3 Tesla in multiple sclerosis patients and healthy controls: a morphological and topological quantitative comparison study, Mult. Scler. 14 (2008) S102. [72] M.K. Erskine, L.L. Cook, K.E. Riddle, J.R. Mitchell, S.J. Karlik, Resolution-dependent estimates of multiple sclerosis lesion loads, Can. J. Neurol. Sci. 32 (2005) 205–212. [73] N.L. Sicotte, R.R. Voskuhl, S. Bouvier, R. Klutch, M.S. Cohen, J.C. Mazziotta, Comparison of multiple sclerosis lesions at 1.5 and 3.0 Tesla, Invest. Radiol. 38 (2003) 423–427.