Acta Neuropathol (2011) 122:155170 DOI 10.

1007/s00401-011-0840-0

REVIEW

The neuropathological basis of clinical progression in multiple sclerosis

Richard Reynolds Federico Roncaroli Richard Nicholas Bishan Radotra Djordje Gveric Owain Howell

Received: 30 April 2011 / Accepted: 18 May 2011 / Published online: 28 May 2011 Springer-Verlag 2011

Abstract Multiple sclerosis is the major inammatory condition affecting the central nervous system (CNS) and is characterised by disseminated focal immune-mediated demyelination. Demyelination is accompanied by variable axonal damage and loss and reactive gliosis. It is this pathology that is thought to be responsible for the clinical relapses that often respond well to immunomodulatory therapy. However, the later secondary progressive stage of MS remains largely refractory to treatment and it is widely suggested that accumulating axon loss is responsible for clinical progression. Although initially thought to be a white matter (WM) disease, it is increasingly apparent that extensive pathology is also seen in the grey matter (GM) throughout the CNS. GM pathology is characterised by demyelination in the relative absence of an immune cell inltrate. Neuronal loss is also seen both in the GM lesions and in unaffected areas of the GM. The slow progressive nature of this later stage combined with the presence of extensive grey matter pathology has led to the suggestion that neurodegeneration might play an increasing role with increasing disease duration. However, there is a paucity of studies that have correlated the pathological features with clinical milestones during secondary progressive MS. Here, we review the contributions that the various types of

pathology are likely to make to the increasing neurological decit in MS. Keywords Demyelination Remyelination Axon loss Neurodegeneration Inammation Clinical progression

Introduction Multiple sclerosis (MS) is the commonest neurological disease that affects young adults and is characterised in the majority of patients by episodes, or relapses, of neurological dysfunction, which are related to the site of the pathology [22]. Full recovery from these relapses usually occurs during the early stages of the disease course, but after a variable period of relapsing-remitting phase (RRMS) a progressive build-up of residual neurological dysfunction develops. This phase of the disease is termed secondary progression (SPMS). Although the nature and length of the initial relapsing-remitting course can be extremely variable, the accumulation of disability during the secondary progressive phase appears to occur at a similar rate in the majority of patients, independently of the initial course [23]. A proportion of individuals, accounting for 1020% of patients with MS, exhibit a progressive disease from onset without episodes of relapse and remission, termed primary progressive MS (PPMS). There is a wide variation in the age of almost all clinical milestones in MS, but many studies now show that the age at which these landmarks are reached is not substantially inuenced by the initial course of the disease, including the frequency of relapses [24, 73]. Clinical progression in both SP-MS and PP-MS is widely suggested to start at around 40 years of age and then proceed at approximately the same rate [51], suggesting the same or similar pathogenetic mechanisms underlying the progression.

R. Reynolds and F. Roncaroli contributed equally to the review. R. Reynolds (&) F. Roncaroli R. Nicholas B. Radotra D. Gveric O. Howell Wolfson Neuroscience Laboratories, Division of Experimental Medicine, UK Multiple Sclerosis Tissue Bank, Centre for Neuroscience, Imperial Faculty of Medicine College London, Hammersmith Hospital Campus, London W12 0NN, UK e-mail: r.reynolds@imperial.ac.uk

123

156

Acta Neuropathol (2011) 122:155170

Pathological hallmarks of MS MS is characterised by multiple foci of inammation in the white matter (WM), consisting of perivascular lymphocyte inltrates and accompanying loss of oligodendrocytes and demyelination. Astrogliosis is a prominent feature of the demyelinated lesions and eventually results in a gliotic scar. Axon damage occurs during active inammationinduced demyelination [33, 80], although there is a relative sparing of axons in the majority of lesions. Demyelination is also present to a varying degree in the cerebral cortex and deep grey matter (GM) and is associated with neuronal loss in the absence of signicant inammatory inltrates [5, 43, 60, 69]. However, the presence of immune cell inltrates in the leptomeninges overlying the cerebrum is a common feature in postmortem MS brain [38, 46, 53, 59]. Away from the macroscopically visible lesions, diffuse changes to the normal appearing white matter (NAWM) and grey matter (NAGM), in the form of axon damage/loss [53], microglial activation [48, 49], changes at the node of Ranvier [49] and neuronal loss [30, 60, 83] have been shown to occur. Repair of demyelinated lesions by remyelination has been shown to occur but is highly variable in extent [67, 68]. Although it is suggested that remyelination fails as the disease progresses, very little is known about the natural history of the repair process. Pathogenetic mechanisms during the early stages of MS It is widely accepted that inammation is closely associated with and responsible for acute relapses in RRMS and is associated with changes in blood brain barrier (BBB) permeability [54], as determined by the presence of gadolinium (Gd-DTPA) enhancing lesions on magnetic resonance imaging (MRI) scans. Histologically, these are seen as focal inammatory lesions of the white matter characterised by perivascular inltrates of predominantly T-lymphocytes and monocytes/macrophages [54]. Demyelination results from active myelin phagocytosis by macrophages, with or without the deposition of immunoglobulins and complement components. The presence of myelin debris at various stages of degradation in macrophages can be used to dene the relative age of the individual lesion [14]. Although studies of experimental allergic encephalomyelitis (EAE) have suggested that the early events in the genesis of lesions involve a predominantly CD4? T-cell-mediated process, it is not clear whether this is the case in MS. Studies of postmortem MS tissues have shown that CD8? MHC class I restricted T cells outnumber CD4? cells [10], which are predominantly found in the perivascular inltrates [2], but one must bear in mind that the earliest events in the genesis of MS lesions are not readily

accessible to detailed study. There is considerable debate concerning whether T-lymphocyte inltration is a primary event or is secondary to oligodendrocyte apoptosis and microglial activation [3]. B-lymphocytes are present at all disease stages [38], predominantly in perivascular inltrates and leptomeninges but rarely in the central nervous system (CNS) parenchyma [59]. In contrast, plasma cells are suggested to be few in number in the CNS during the early stages of disease, but become more prominent in lesions, NAWM and the leptomeninges as the disease progresses [38]. Studies of biopsy samples of early lesions and autopsy material from acute cases [58] have proposed that different mechanisms may be involved in the destruction of myelin in inammatory lesions during the early stages of disease: I. demyelination by cytotoxic lymphocytes and activated macrophages in the absence of antibody and complement deposition, II. immunoglobulin and complement-mediated demyelination, III. hypoxia-like injury to distal processes causing oligodendrocyte apoptosis, and IV. primary oligodendrocyte degeneration. However, whether this represents true disease heterogeneity or stage-dependent complexity of immune effector mechanisms is still open to debate and other studies suggest that antibody and complement-mediated myelin phagocytosis become the dominant mechanism in established MS [12]. It is now accepted that acute axonal damage accompanies the active focal inammatory demyelination that is prominent during the early stages of MS [33, 80] and recent studies have indicated that it decreases in extent with disease progression [52]. However, the extent of axon damage and loss is extremely variable and appears linked to the severity of the inammatory process causing demyelination. Pathological substrates of clinical progression Although MRI ndings suggest that inammatory lesions become less prominent as MS enters the progressive phase, neuropathology studies show the persistence of focal inltrates of peripheral immune cells and actively demyelinating lesions [38]. It is thought that inammation becomes increasingly compartmentalised in the perivascular and subarachnoid spaces, behind a relatively intact BBB [47, 59, 62]. It is widely suggested that an increasing burden of axonal loss in white matter tracts of the brain and spinal cord is the major pathological substrate of progressive clinical disability. However, diffuse changes and tissue damage to the NAWM and NAGM and an increasing degree of cortical GM pathology, unrelated to axonal loss in focal WM lesions, are also likely to play an important role in the accumulation of motor, sensory and cognitive disability that characterises SPMS.

123

Acta Neuropathol (2011) 122:155170

157

Despite our detailed knowledge of the pathological features of MS that has accumulated from many decades of the study of postmortem samples, we know relatively little about the relationship between these features and clinical milestones during the progressive phase. A major restriction of all postmortem tissue studies is the limited availability of well-characterised tissues from patients with ongoing disease activity and many studies have attempted to correlate neuropathological features with clinical milestones using small numbers of cases. Because of the inherent variability of the clinical course of MS and the extent and location of pathology in individual cases (Fig. 1), clinico-pathological correlations are extremely difcult and require the study of large numbers of cases. Here, we review what is known about the pathological substrates that underlie the progressive phase of the disease course in MS and show how detailed lifetime clinical information is essential in order to correlate pathological features with clinical milestones.

Neuroimaging and clinical progression A substantial number of neuroimaging studies have been carried out on the temporal changes that can be identied in the MS brain and how they correlate with clinical progression. MRI has proven powerful for investigating the temporal and spatial development of WM lesions and changes in BBB permeability and their association with neurological dysfunction [13, 34, 63]. However, until recently GM pathology has been difcult to identify with any accuracy. MRI is not often performed during the later stages of MS and so few correlations have been drawn between changes seen on imaging and the pathological substrates of clinical progression. Despite the sensitivity of T2-weighted MRI for detecting focal inammation and demyelination, there is only a weak correlation between lesion load and clinical disability measured by the expanded disability status scale (EDSS) in both the brain [34] and spinal cord [56]. Although the number of Gd-DTPA enhancing lesions may prove useful in predicting subsequent relapse rate [64], longitudinal studies have shown that a moderate correlation between T2 lesion volume and disability is only present over the rst 5 years [36] and clinically detectable relapses have only a weak effect on clinical progression [24]. A number of studies have now demonstrated that measures of GM atrophy, alongside spinal cord atrophy, provide the best correlation with accumulating disability [9, 35, 36, 56]. GM atrophy increases at a much greater rate during the secondary progressive phase of MS [35, 36] and associates with clinical disability independently of WM lesion load, suggesting that GM pathology becomes the major component of disease burden as the disease progresses. In contrast, the rate of increase in WM atrophy remains constant through the disease stages and does not correlate with increasing disability [35]. The introduction of double inversion recovery (DIR) MRI has allowed the in vivo study of the contribution of cortical lesions to GM atrophy and clinical progression [40] and has demonstrated that they are present from the early relapsing remitting stage of the disease [17]. Recent longitudinal studies show that the number of cortical lesions increases at a signicantly greater rate in patients with actively progressive disease and correlates with increasing EDSS score [15, 17]. Although a large proportion of cortical lesions are still undetected by DIR-MRI, in particular subpial demyelination, the baseline cortical lesion volume was found to predict the progression of disability [17]. Thus, there is increasing evidence that accumulating cortical GM pathology plays a major role in the severity of both physical and cognitive disability [18]. In order to validate the in vivo ndings and rene the MRI diagnostic criteria of MS, MRI has been applied to

Fig. 1 Bi-hemispheric sections of an MS brain stained with Luxol Fast Blue (a) and anti-MOG antibody (b) to illustrate the gross distribution of demyelinated lesions

123

158

Acta Neuropathol (2011) 122:155170

examine postmortem brains and to compare the imaging features to histopathology. These studies have considerably improved our understanding of the pathological substrate of lesion formation and the dynamic changes of demyelination and remyelination and have unveiled the extent of neocortical, hippocampal and deep GM pathology [6, 74 76]. MRI of postmortem brains can be used to improve the yield of pathological tissue and particularly to increase the accuracy of sampling of lesions that are difcult to identify or that are not present on the cut surfaces of the slices. De Groot et al. [26] studied 29 MS brains of patients with a mean disease duration of 25 years and showed an increase of 44% in sensitivity using MRI-guided sampling of lesions that were not macroscopically visible. In a study of spinal cord, Nijeholt et al. [66] documented an increased yield in sensitivity for diffuse WM pathology using high-eld MRI compared to histopathology and observed that the lesion area detected on MRI was larger than indicated by the neuropathology [66]. Thus, increasing renements to the specicity of MRI imaging and increasing eld strengths [76] should in the future allow an increased understanding of the pathological substrates of clinical progression. However, MRI of postmortem brains is not free of limitations. Postmortem delay, improper handling of the tissue, tissue preservation and dehydration and changes in pH are the major limiting factors that can hamper the evaluation of imaging ndings [6, 74].

Clinico-pathological correlations in progressive MS Inammation and clinical progression Perivascular and diffuse lymphocytic inltration is a common nding in active lesions and largely consists of clonally expanded CD8? T cells [2]. CD4? lymphocytes, B-lymphocytes and plasma cells are also present, but they usually represent a minor component of the inammatory inltrate [37]. The extent of lymphocytic inltration varies between patients, between different plaques in the same patient and according to the degree of activity, being usually orid at the earliest stages of demyelination. Perivascular cufng of T and B lymphocytes can also be seen in the NAWM [1], whereas lymphocytic inammation is much less common in cortical lesions and NAGM [5]. The occurrence of leptomeningeal inammation and its significance in clinical progression is discussed in a later section. Focal uptake of Gd-DTPA on T1-weighted sequences is regarded as an indication of disruption of the BBB and is commonly considered evidence for acute inammation, but MRI and spectroscopy studies documented only a modest correlation between inammation identied by contrastenhanced lesions and disease progression [34]. Such

discrepancy may depend on the fact that perivascular inammation does not always occur after a disruption of the BBB [47, 82]. Virchow-Robin spaces (VRS) have also been investigated in MS patients as a possible marker of inammation with the assumption that perivascular spaces should be wider when lymphocytes and macrophages transmigrate from the vascular lumen. One study has shown that VRS volumes differ between MS patients and controls and correlate with the number of gadoliniumenhancing lesions. But no correlation with other MRI parameters, including the number and volume of T2 lesions or the number and volume of T1-hypointensities, was observed [86]. In addition, no correlation of VRS number and volume was found with disease duration, EDSS score or the time since last relapse. Although lymphocytic inltration is a constant nding in MS brains, very few studies have attempted to correlate its presence with any measures of disease progression. In their early study, Guseo and Jellinger [46] observed a striking correlation between increasing severity of active demyelination and severe inammation present at the time of death and a shorter disease course. More recently, Frischer et al. [38] investigated autopsy material from 67 MS cases and 28 controls, including 17 cases with large hemispheric or double hemispheric sections, with the aim of correlating the extent of inammatory inltration and neurodegeneration. They found a signicant correlation between acute axonal injury and the presence of lymphocytic inltrates among patients with progressive disease and showed that inammation and ongoing axonal injury decline in the late stage of the disease to levels that are similar to age-matched controls, thus strongly supporting the view that inammation has a role in the progression of MS and clinical disability. An example of differing degrees of perivascular inammation is given in Fig. 2. Axonal loss and diffuse white matter pathology Axonal injury and loss are important features of MS and one of the most important correlates of clinical progression. Numerous studies of postmortem tissues have demonstrated that extensive axonal injury occurs in both the brain and spinal cord [32, 33, 50, 57, 80]. Axonal loss is particularly evident during the early stages of MS but then proceeds throughout the lifetime of the patient so that the cumulative axonal loss becomes one of the major pathological substrates for permanent disability [4, 38, 52]. Acute damage can be detected by the presence of swellings and end bulbs accumulating the amyloid precursor protein (APP) and indicating impaired axonal transport [33]. APPpositive spheroids can be found in active inammatory demyelinating lesions irrespective of the disease duration but appear to be more extensive during the rst year from

123

Acta Neuropathol (2011) 122:155170

159

Fig. 2 Extensive demyelination of the MS cerebral cortical grey matter identied in an anti-MOG stained section (a). A bi-hemispheric section labelled with anti-MOG antibodies (b) illustrates the

predilection of grey matter lesions for the cortical GM in contact with the longitudinal cerebral ssure (cingulate gyrus) and the lateral sulcus

onset, with the number of acutely injured axons decreasing with disease durations longer than 10 years [52]. Several studies have now demonstrated a positive correlation between axonal damage and the degree of inammation in active cerebral MS lesions [38, 50, 52]. Others have shown that the density of activated macrophages within MS lesions is predictive of the extent of axonal injury [33, 80], and that the extent of axonal loss correlates well with numbers of both CD8? T cells and activated microglia/ macrophages in close proximity [52]. The production of inammatory mediators and reactive oxygen and nitrogen species by these cells is likely to contribute to the acute suppression of axonal function and cause mitochondrial impairment [30], which is suggested to play a key role in the consequent axonal degeneration [65, 78]. In addition to the axonal pathology occurring within active plaques, there is also a highly signicant correlation between inammation and the extent of axonal damage in the NAWM [38] that appears in part to occur independently from focal plaques [53]. Multiple episodes of demyelination over the course of the disease and subclinical attacks may cause permanent injury that leads to progressive axonal destruction and eventually to secondary neuronal loss due to Wallerian degeneration, but the extent of these global changes does not show any correlation with the number, size and location of focal brain and spinal cord WM lesions [32, 53, 54]. Axonal loss can be studied in vivo by measuring the levels of the glutamate metabolite, N-acetyl-aspartate (NAA) by MR-spectroscopy (MRS) [27, 39]. Reduced levels of NAA in postmortem tissues correlate well with the decrease in axon numbers seen in spinal cord lesions, although no correlation was found with disease duration [4]. In a longitudinal patient study, decreases in brain NAA correlated with an increase in lesion load at all stages of

MS, but a signicant correlation between the decrease in NAA and disability measured using the EDSS score was seen only in RRMS patients [27]. Thus, although the extensive loss of axons in SPMS and PPMS patients with a long disease duration, together with the demonstration of reduced NAA levels that correlate with functional impairment, argues for a major role for axonal loss in clinical progression, it does not fully explain the slow build-up of neurological decit that cannot be assigned to focal WM lesions. Cortical grey matter pathology Over the last two decades, several neuroimaging and neuropathological studies have documented cortical demyelination in patients with MS and have shown that GM lesion burden is often much greater than that found in the WM [18]. Damage and atrophy of the GM has been shown to occur from the earliest stages of the disease in both RRMS and PPMS, becoming more prominent in the secondary progressive phase [35, 36, 53, 83]. It can be predominant and unrelated to the load and extension of WM plaques and can evolve faster than WM pathology [18, 42]. Neocortical GM pathology (Fig. 2) can be broadly classied into three types: type I lesions involve the WM and the lower layers of GM (leucocortical); type II lesions are entirely intracortical; and type III lesions affect the subpial GM and extend to a variable depth through the cortical layers [7, 69]. It is now widely accepted that accumulating cortical GM damage is likely to play an important role in clinical progression [18] and patients with a younger age at death have greater GM demyelination [43], but few correlative studies have yet been carried out. Unlike WM demyelination, cortical pathology is likely to have a signicant impact on both cognitive and motor

123

160

Acta Neuropathol (2011) 122:155170

disability [28, 29, 44] and its accumulation is suggested to represent a key event in the transition from RRMS into SPMS [79]. Cortical involvement shows marked regional variations and is usually more extensive in the cingulate gyrus, the temporal and frontal cortices and the hippocampus (Fig. 3) and less severe in the occipital lobe and primary motor cortex [41, 43, 53]. Such predilection for the frontal lobe and hippocampus accounts for the predominance of cognitive over motor disability related to cortical pathology and in one study cognitive dysfunction was noted in all cases with extensive subpial demyelination [8]. The occurrence of cortical pathology was also found to correlate with epilepsy in patients with RRMS and one study found that up to 90% of RRMS patients with seizures had GM demyelination compared with 48% of patients without epilepsy [16]. In addition to neocortical pathology, demyelination can also involve the subcortical grey matter such as the thalamus, basal ganglia, hypothalamus, cerebellum, and spinal cord and, similar to neocortical plaques, be the neuropathological substrate of clinical disability [20, 43, 44]. Although the relationship between GM pathology seen by MRI and permanent disability is well established [36], the correlation between clinical progression and the occurrence of cortical demyelination and neuronal loss has received little attention [60]. Studies of postmortem brains can only reect the situation in vivo at a single time point after a variable duration of progressive disease and therefore cannot report on the temporal appearance of cortical pathology [42]. On the other hand, correlative neuroimaging studies are hampered by technical limitations in detecting cortical pathology and the absence of lifelong clinical histories with which to correlate nal outcomes.

Conventional MRI techniques do not reliably identify intracortical or subpial lesions and, although more sophisticated techniques have recently been introduced, they still do not detect the full extent of cortical pathology and only identify about 20% of the actual lesions [18]. In contrast to WM lesions, established cortical plaques are characteristically accompanied by mild peripheral immune cell inltration [5, 69]. In established plaques, perivascular inltrates are rare and the density of lymphocytic inltrate as well as the lymphocyte subsets identied with CD4, CD8, CD20 is often similar to the density of inltrating lymphocytes in the normal appearing GM. In contrast, early, newly formed GM lesions identied in biopsy specimens may contain a orid lymphocytic inltrate [70]. Other differences between cortical and WM demyelination include the lack of signicant leakage of plasma proteins, suggesting a preserved BBB, the absence of signicant signs of complement activation and the presence of an increased number of activated microglial cells, suggesting a role for these cells in modulating the local inammatory response [7, 25, 59, 60]. Neuronal pathology can be prominent in cortical plaques and in the NAGM. Cortical lesions contain apoptotic neurons, mostly pyramidal cells in cortical layers 3 and 5 [60] and subpopulations of neurons appear more vulnerable than others. Interneurons expressing parvalbumin within layer 2 of the precentral gyrus can be signicantly reduced without a corresponding loss of calretinin-positive cells [21]. The synaptic density can also be substantially reduced within demyelinating lesions [31, 85]. Neuronal pathology is not restricted to demyelinated lesions and considerable neuronal loss is also observed in the NAGM [60], suggesting that the two may not be directly linked. The

Fig. 3 Perivascular lymphocytic inltrates consist of a few to several layers of mature lymphocytes disrupting the vessel wall and lling the perivascular space. A mild (a LFB/PAS, 109) and a more

conspicuous (b LFB/PAS, 109) inltrate are seen in two different late active plaques of the same patient. A orid inltrate is observed in an early active plaque (c LFB/PAS, 109) (bar 50 lm)

123

Acta Neuropathol (2011) 122:155170

161

expression of several nuclear encoded mitochondrial genes was substantially reduced in NAGM and the reduction occurred predominantly in neurons [30] and mitochondrial DNA deletions were identied away from cortical plaques [19]. To further support the relevance of damage to the NAGM, pathological and imaging studies have shown that a general volume loss of the neocortical ribbon, indicating widespread cortical atrophy, is an independent predictor of cognitive decline [18]. The evidence of substantial cortical pathology in patients with MS associated with steady functional decline, with little if any response to treatment in the absence of notable inammatory episodes on MRI, has led to the suggestion that neurodegeneration plays a key role in clinical progression and permanent disability in MS (see later section) and therefore might be a primary event [77, 79]. It is widely accepted that neurodegeneration in MS is not an autonomous process, as it is thought to be in Alzheimers and Parkinsons diseases, but is secondary to inammation that becomes compartmentalised behind an intact BBB [60, 79]. However, the evidence of a similar rate of progression from a certain threshold of disability also suggests a common mechanism underlying progressive dysfunction that is likely to be determined by a steady accumulation of neural and axonal damage, irrespective of the initial inammatory insult [23, 55]. Remyelination and disability Many studies have demonstrated that demyelination in the MS brain and spinal cord can be followed by remyelination, although the extent of this repair is highly variable [11, 67, 68, 71]. In their study of 51 postmortem brains, Patrikios et al. [68] observed remarkable differences in the incidence of shadow plaques and extent of remyelination between patients. In 10 patients 6096% of total plaque area was remyelinated while in 34 patients remyelination accounted for only 025% of total plaque area and was restricted to small zones at the edge of inactive plaques. Areas of remyelination appear as sharply demarcated, homogenous discoloured plaque-like lesions usually centred by a small or medium sized vein with disrupted wall. Myelin sheaths are thinner than those in the adjacent NAWM and there is some degree of axonal loss. In some lesions the replacement of myelin is nearly complete and shadow plaques are barely distinguishable from the normal white matter. In many instances, shadow plaques exhibit reactivation of the inammatory response at their edge with subsequent enlargement of the lesion and some shadow plaques may contain inactive areas as well as areas of early activity with orid myelin breakdown. Notably, Bramow et al. [11] observed that remyelinated areas are more prone to undergo a second episode of demyelination and that this

was more common in SPMS than PPMS. It had previously been thought that remyelination is sparse in patients with long-standing progressive disease and usually restricted to the borders of inactive plaques. However, a number of recent studies have used extensive tissue sampling protocols to demonstrate extensive remyelination in some long standing cases [67, 68]. On the basis that remyelination should both restore action potential conduction and protect axons against further inammatory attack, it might be expected that increasing remyelination would correlate with a longer disease duration and greater age at death. Neuroimaging studies of the correlation between clinical progression and remyelination have been very difcult due to the lack of adequate MRI criteria to dene myelin repair. For this reason, almost all studies on remyelination are based on tissue samples of postmortem brains. Patrikios et al. [68] reported that shadow plaques were rare in early active cases, accounting for only 013% of total plaque area and that shadow plaques were slightly more common in RRMS and PPMS compared to SPMS. No correlation was observed between the extent of shadow plaque formation and the age at disease onset or gender, but they found a signicant positive correlation between the extent of remyelinated plaque area and the age of death. Patients who died at an older age showed signicantly more shadow plaques than those who died at a younger age. A signicant correlation was also found between remyelination and disease duration as might have been expected. Bramow et al. [11] compared the extent of demyelination and remyelination in a large number of SPMS and PPMS cases and demonstrated that the PPMS brains had a lower number of inammatory active lesions and more complete remyelination than SPMS. Although there was no difference in the number of active lesions and the degree of remyelination in the spinal cord, a reduction in the degree of remyelination did correlate with increased disability, thereby providing a possible link between the balance of demyelinationremyelination and the severity of clinical disease.

The UK MS Tissue Bank experience A population-based cohort The majority of studies of the neuropathology of MS have been carried out on postmortem brains and spinal cords that have been collected as a result of routine medico-legal autopsies and many of these collections are archival in nature. This has led to a number of limitations when wishing to correlate neuropathological ndings with clinical events as detailed lifetime clinical histories are not

123

162

Acta Neuropathol (2011) 122:155170 Table 1 Population and clinical milestones data for the UK MS Tissue Bank cases Total cases Sex Male Female Age onset Mean Median Minimum Maximum Age progressive Mean Median Minimum Maximum Age wheelchair Mean Median Minimum Maximum Age death Mean Median Minimum Maximum MS type Unknown RRMS SPMS PPMS MS on death certif. Yes No 258 173 13 22 345 46 63.4 62 30 98 50.3 50 18 86 44.8 44 16 79 33.0 31.5 5 66 125 306 431

often available. Brain banks that operate prospective tissue donor schemes, such as the UK MS Tissue Bank (UKMSTB) and the Netherlands Brain Bank, have been able to overcome some of these limitations and can supply detailed clinical information to accompany well-characterised tissue samples from population-based cohorts. The UKMSTB operates a nationwide donor scheme with more than 5,000 potential donors, with and without MS, currently registered to donate brain and spinal cord after death. The donor scheme is community-based and thus closely reects the whole UK MS population. The tissue collection now contains more than 500 brains, spinal cords and CSF samples, 450 of which are from MS cases that closely reect the range and ratio of PPMS and SPMS cases in the UK (Table 1). The majority of the tissues are preserved within 24 h postmortem for frozen samples and within 48 h for formalin-xed samples. The quality of collected tissue is ensured by rapid preservation, thorough neuropathological examination and subsequent screening of every single brain and spinal cord sample. A full clinical and neuropathology report accompanies every tissue sample supplied to a research project. The UKMSTB is thus a valuable resource of human postmortem brain and spinal cord tissue and one of the few brain banks specically supporting worldwide scientic efforts to elucidate the pathogenesis of MS. The extensive tissue collection accompanied by excellent clinical information has allowed us to carry out clinico-pathological correlations using a large unbiased cohort of MS cases. Clinical milestones All cases of MS entering the UKMSTB are accompanied by an extensive set of general practitioners (GP) notes that include detailed consultant neurologist reports. These histories are summarised to provide information on the age at disease onset, the number and nature of clinical relapses, an estimate of the time to conversion to secondary progression, the time at which the patient required the use of a wheelchair, the age at death and the total disease duration (Table 1). A record of individual medical problems, symptoms and medication is also produced. The extent of the data varies in each case but each history is graded in quality from 0 to 5 (0, no information; 1, minimal notes birth/death; 2, milestones; 3, milestones/some relapses gap in history; 4, full history available of all relapses/ milestones from either GP or hospital notes; 5, full history available of all relapses/milestones from both GP and hospital letters). Ninety-two percent of the UKMSTB notes are graded 4 or 5. These data then allow an analysis of the individual lifetime history of MS and can be correlated with the individual and global neuropathological features. In addition, we have been able to compare the population

data from the UKMSTB with published clinical cohort studies [23, 73]. Analysis of the clinical milestones and MS disease sub-types (Table 1) indicates that they are in good agreement with the previously published natural history studies. Neuropathological assessment The brains collected by the UKMSTB are examined by the neuropathologist following the guideline for brain cut-up proposed by BrainNet Europe (http://www.brainet-europe. org). Although these guidelines are intended for neurodegenerative diseases, each or most of the samples will also include demyelinating plaques. Sections are stained with haematoxylineosin, LFB/PAS and LFB/ MHC class II antigen. Additional histochemical and

123

Acta Neuropathol (2011) 122:155170

163

immunohistochemical stains are performed when required. Although the terms that describe the different stages of activity of plaques vary between authors and studies, there is a general agreement on the sequence of events and the pathological changes that occurs from an early lesion to an inactive plaque. The terminology used at the UKMSTB is therefore in line with the current concept that plaques start with an active, orid phase of lymphocytic and microglial/ macrophagic inltrate that declines over time to result eventually in an inactive area of demyelination. Although there are several criteria proposed to classify disease activity, it is the evidence of myelin degradation products within macrophages that reliably indicates active demyelination. A more rigorous assessment of demyelination would be based on the assessment of the composition of myelin degradation products in macrophages or on the identication of markers of macrophage activation [14], but such an evaluation is not routinely performed at the

UKMSTB. The classication of plaques used at the UKMSTB has been proposed by Professor I. Allen (http:// www.ICDNS.org) and is reported in Table 2. Cortical pathology, axonal damage and lymphocytic inltrate are also thoroughly assessed and graded when possible in order to allow a correlation with clinical parameters. The baseline pathological assessment of brains donated to the UKMSTB is summarised in Table 3. In contrast to other classications, we avoid the use of acute plaque and describe the rst stage of inammation and myelin breakdown as early active. However, the question of how MS lesions start remains open and it is unclear if a distinct pathologically dened lesion that precedes active demyelination, the breakdown of the BBB and the migration of leucocytes in the brain tissue exists. Van der Valk and Amor [81] have recently described lesions in otherwise NAWM that are composed of clusters of activated but still ramied microglial cells without accompanying

Table 2 Pathological changes of plaques at the different stages of demyelination: criteria used at the UKMSTB

Type of plaque Preactive Early active

Key pathological features Aggregates of microglia in absence of or minimal myelin breakdown Hypercellularity with microglial activation Alteration in myelin staining Myelin phagocytosis and degradation Luxol-fast blue positive fragments of myelin or positivity for MOG in macrophages Relative axonal preservation Perivascular inammation

Late active

Variable cellularity Demyelination with myelin and variable oligodendrocyte loss Fat-lled macrophages Astrocytosis Perivascular inammation (slight) Relative axonal preservation Microglial activation at plaque edge

Chronic active Hypocellular in lesion centre with hypercellular edge Active myelin breakdown at lesion edge HLA-DR? macrophages containing early stage myelin breakdown products at lesion edge (LFB or MOG?) Chronic inactive Hypocellularity Myelin and oligodendrocyte loss No phagocytosis Paucity of microglia Shadow Relative axonal preservation An extensive area of partial reduction in myelin density, often at the edge of a demyelinated lesion Palely staining myelin, caused by reduction in the number and thickness of individual myelin sheaths (Courtesy of Prof. Dame I Allen, http://www.ICDNS.org)

123

164 Table 3 Baseline assessment of postmortem brains at the UKMSTB Quality assessment Tissue preservation Quality of sampling Quality of histochemical and immunohistochemical stains Type of lesion Preactive Early active Late active Chronic active Chronic inactive Shadow Site of pathology Supratentorial Hypothalamus Midbrain Cerebellum Ponsmedulla Spinal cord Specify where predominant Cortical involvement Subcortical Intracortical Subpial Mildmoderatesevere Axonal damage Present or absent Site of axonal damage Degree of perivascular inammation Grade 05 If within or outside demyelinating plaques Leptomeningeal Coexistent pathology Possible anterograde or retrograde axonal damage Vascular changes Neurodegeneration Primary or metastatic tumours

Acta Neuropathol (2011) 122:155170

Active disease at death is associated with shorter disease duration and earlier onset The availability of data on clinical milestones and semiquantitative data on neuropathological features for the UKMSTB MS cases has allowed us to produce some clinico-pathological associations (Table 1; Fig. 5). An association was found between disease activity at postmortem and clinical progression; increased early active lesions present at death are associated with a shorter time from wheelchair use to death (Fig. 5a), indicating active progressive disease. Increasing inammation at postmortem is clearly associated with an earlier age at wheelchair use and death, but not, as expected, age at onset (Fig. 5b). Greater involvement of the cortical GM is also associated with the earlier age at onset, use of a wheelchair and age at death (Fig. 5c). The relationship between cortical pathology, meningeal inammation, neuronal loss and clinical progression In contrast to the many cellular and molecular pathogenetic mechanisms that are suggested to be responsible for the evolution of focal WM lesions, relatively little is known concerning the mechanisms of lesion formation in the cortical GM and how the accumulation of cortical pathology might contribute to clinical progression. Hypotheses need to take into account the lack of peripheral cell inltrates and the predominant sub-pial localisation of the lesions. Many studies have reported that inammatory inltrates are commonly found in the cerebral leptomeninges in SPMS cases at autopsy [38, 46, 53, 59], particularly in cases with ongoing active cortical demyelination [46] and extensive sub-pial cortical demyelination [53]. In addition to diffuse inammatory inltrates in the cerebral meninges, large B cell aggregates with some of the characteristics of ectopic B cell follicles have been identied in the subarachnoid space in a proportion of SPMS cases [59]. Similar immune cell aggregates have been noted in a number of other studies [38, 46]. Subsequent studies in London and Rome identied the presence of these ectopic lymphoid-like structures in the meninges of approximately 40% of a large sample of the SPMS cases in the UKMSTB [59, 60]. The lymphoid-like structures are characterised by CD20? B cell aggregates interspersed with CD35?/CD21?/CXCL13? follicular dendritic cells (FDCs) and CD4? and CD8? T cells. Dividing CD20? B cells and Ig?/CD138? plasma cells are present in variable numbers. These ectopic B cell follicle-like structures can be found distributed throughout the cerebral meninges and are variable in number and size. Their predominant presence in the depths of cerebral sulci [59, 60] suggests that the reduced CSF ow and protected microenvironment in

inammation and without or only minimal demyelination and they have proposed the term preactive lesion to suggest that they might represent the earliest stage of plaque formation. Interestingly, these lesions are often unrelated to a vein. So dened, preactive lesions are rarely seen in postmortem brains and their identication on tissue sections can be challenging. Preactive lesions can be considerably more numerous than active lesions suggesting that many of them are likely to resolve without progressing into an active inammatory stage. The key histopathological features of MS plaques are represented in Fig. 4.

123

Acta Neuropathol (2011) 122:155170 Fig. 4 The key pathological features of demyelination. A preactive lesion with only minimal demyelination and perivascular microglial accumulation but no lymphocytic inltrate is represented in ac. dg An early active plaque showing orid macrophagic inltrate with active myelin breakdown and preservation of axons. hj An inactive plaque (a HE, 109; b LFB/PAS, 109; c LFB/antiMHCII, 109; d LFB/PAS, 209; e LFB/anti-MHCII, 209; f antiMOG, 209; g anti-NF, 209; h LFB/PAS, 109; i LFB/ MHCII, 109; j anti-MOG, 109) (bar 50 lm)

165

123

166

Acta Neuropathol (2011) 122:155170

Fig. 5 Clinico-pathological correlations for the UKMSTB cases. Data from the 431 MS cases in the UKMSTB show that the presence of active inammatory disease at postmortem is associated with an earlier age at wheelchair use and death (a). An increasing degree of perivascular inltration (assessed as described in the text) is also associated with an earlier age at wheelchair use and death (b). An increasing extent of grey matter pathology is associated with earlier age at onset, wheelchair use and death (c)

these locations favour the homing and retention of inammatory cells that will then give rise to a highly inammatory milieu in the CSF. MS cases with this more extensive and organised meningeal inammation are characterised by increased cortical GM atrophy and a greater degree of GM pathology, in particular a substantial increase in subpial demyelination with little difference in the WM lesion load [60]. B cell follicle-like cellular aggregates were always found in close association with subpial lesions, although not all subpial lesions showed a direct relationship with meningeal inammatory inltrates. The increased pathology was associated with increased numbers and activation state of microglia, damage to the glia limitans, increased axonal and neuritic injury and a gradient of neuronal loss, greatest closer to the pial surface, that is not only present in areas of demyelination but also present more diffusely in the NAGM [59, 60]. Loss of layer III and V pyramidal neurons in these cases exceeded 40 and 50%, respectively, and was accompanied by loss of interneurons in the other cortical layers [60]. These ndings suggest that the meningeal inltrates give rise to increased concentrations of pro-inammatory and cytotoxic cytokines in the CSF that might then be responsible for cortical tissue damage, either directly or indirectly via chronically activating microglia. Our further studies have demonstrated that the B cell follicle-like structures represent one extreme of a continuum of meningeal inammation and that the level of this inammation correlates with the degree of cortical GM demyelination and microglial activation (Howell et al., submitted). These studies show that disease milestones are reached at a younger age in cases with more extensive meningeal inammation and GM demyelination. MS cases with meningeal lymphoid-like structures are associated with a younger age at disease onset, a shorter time to progression and wheelchair use and shorter disease duration [59, 60]. Correlation of our data on neuronal loss in cortical GM of SPMS cases [60] with disease milestones clearly shows that an increasing loss of cortical neurons is signicantly correlated with the presence of meningeal inammation, earlier age at wheelchair use and death and a shorter disease duration (Fig. 6). The presence of meningeal inammation, and in particular B cell aggregates, not only provides a rational explanation for the presence of oligoclonal immunoglobulin bands in the CSF of MS patients but may also explain the increase in cortical GM pathology and consequent atrophy [18] that accompanies the slow and general accumulation of neurological and cognitive dysfunction that characterises the progressive stage of MS.

123

Acta Neuropathol (2011) 122:155170 Fig. 6 Quantitative estimation of total neuronal and pyramidal cell loss and correlations with clinical milestones. Increasing total neuronal loss (n = 20) in the precentral gyrus GM of MS cases with (red dots) and without (black dots) meningeal B cell follicle-like structures [60] shows a correlation with age at death (a) and age at which a wheelchair is rst required (b). Increasing loss of pyramidal neurons in layer V of the precentral gyrus correlates with a shorter disease duration (c) and a shorter time between age at wheelchair use and death (d)

167

Benign MS A minority of patients with MS show a remarkably slowly progressive disease progression and remain stable for long periods of time. Broadly dened, benign MS (BMS) describes a form of MS with indolent course and a longer relapse free time than RRMS [61]. The reported incidence of BMS varies broadly between studies, with gures ranging between 5 and 64%. Such variability is obviously articial and depends on the different denitions given to the condition, the differences between the patient populations, the different EDSS cut-off values, the length of follow-up, the difference in disease severity between population-based cohorts and hospital-based studies, the end-point considered, and the changing consensus diagnostic criteria of MS [45]. The use of EDSS to categorise BMS may not be helpful because patients with BMS may develop non-motor symptoms, which can be as debilitating as motor disability. A diagnosis of BMS can only be made retrospectively because there are no clinical, demographic, genetic, laboratory or imaging features that are predictive of a benign course. In addition, only a few dated studies have investigated the neuropathological substrate of the disease. Nevertheless, despite the controversies reported above, there is a little doubt that a benign, slowly progressive form

of MS exists. The pathological substrate of BMS has almost exclusively been investigated using neuroimaging techniques with only one study examining the neuropathological features of the condition [84]. A number of clinicopathological features appear to dene BMS. For example, there is a positive correlation of BMS with female gender, an initial exacerbating-remitting course, onset before the age of 40, onset with optic neuritis, absence of pyramidal signs at presentation, predominant sensory symptoms, duration of the rst remission of more than a year and only one relapse in the rst 5 years after clinical onset. Cortical involvement and brain atrophy are reportedly less common in BMS than RRMS and the changes in the white matter away from demyelinating areas seem less prominent [72]. Given the lack of pathological studies on BMS, at the UKMSTB we have analysed the neuropathological features of ten cases of BMS compared to RRMS and SPMS for the purpose of this review. Interestingly, visual symptoms were more common in BMS than SPMS patients. Similar to neuroimaging studies, we observed a signicantly lower number of active plaques in BMS, considerably less cortical pathology and less prominent perivascular and leptomeningeal inltrates (Fig. 7). In contrast, there was no signicant difference in the number of shadow plaques between the two groups. Interestingly, 9 out of 10 BMS

123

168

Acta Neuropathol (2011) 122:155170 and the Medical Research Council (Grant No. G0700356 to RR and OH). Conict of interest of interest. The authors declare that they have no conict

References
1. Allen IV, McKeown SR (1979) A histological, histochemical and biochemical study of the macroscopically normal white matter in multiple sclerosis. J Neurol Sci 41:8191 2. Babbe H, Roers A, Waisman A et al (2000) Clonal expansions of CD8(?) T cells dominate the T cell inltrate in active multiple sclerosis lesions as shown by micromanipulation and single cell polymerase chain reaction. J Exp Med 192:393404 3. Barnett MH, Prineas JW (2004) Relapsing and remitting multiple sclerosis: pathology of a newly forming lesion. Ann Neurol 55:458468 4. Bjartmar C, Kidd G, Mork S et al (2000) Neurological disability correlates with spinal cord axonal loss and reduced N-acetyl aspartate in chronic multiple sclerosis patients. Ann Neurol 48:893901 5. Bo L, Vedeler CA, Nyland H, Trapp BD, Mork SJ (2003) Intracortical multiple sclerosis lesions are not associated with increased lymphocyte inltration. Multiple Scler 4:323331 6. Bo L, Geurts JJ, Ravid R, Barkhof F (2004) Magnetic resonance imaging as a tool to examine the neuropathology of multiple sclerosis. Neuropathol Appl Neurobiol 30:106117 7. Bo L, Geurts JJ, Mork SJ, van der Valk P (2006) Grey matter pathology in multiple sclerosis. Acta Neurol Scand Suppl 183:4850 8. Bo L, Geurts J, Van der Valk P, Polman C, Barkhof F (2007) Lack of correlation between cortical demyelination and white matter pathologic changes in multiple sclerosis. Arch Neurol 64:7480 9. Bonati U, Fisniku LK, Altmann DR et al (2011) Cervical cord and brain grey matter atrophy independently associate with long term MS disability. J Neurol Neurosurg Psychiatry 82:471472 10. Booss J, Esiri MM, Tourtellotte WW, Mason DY (1983) Immunohistological analysis of T lymphocyte subsets in the central nervous system in chronic progressive multiple sclerosis. J Neurol Sci 62:219232 11. Bramow S, Frischer JM, Lassmann H et al (2010) Demyelination versus remyelination in progressive multiple sclerosis. Brain 133:29832998 12. Breij ECW, Brink BP, Veerhuis R, van den Berg C et al (2008) Homogeneity of active demyelinating lesions in established multiple sclerosis. Ann Neurol 63:1625 13. Brex PA, Ciccarelli O, ORiordan JI et al (2002) A longitudinal study of abnormalities on MRI and disability from multiple sclerosis. N Engl J Med 346:158164 14. Bruck W, Porada P, Poser S et al (1995) Monocyte/macrophage differentiation in early multiple sclerosis lesions. Ann Neurol 38:788796 15. Calabrese M, de Stefano N, Atzori M et al (2007) Detection of cortical inammatory lesions by double inversion recovery magnetic resonance imaging in patients with multiple sclerosis. Arch Neurol 64:14161422 16. Calabrese M, de Stefano N, Atzori M et al (2008) Extensive cortical inammation is associated with epilepsy in multiple sclerosis. J Neurol 255:581586 17. Calabrese M, Rocca MA, Atzori M et al (2010) A 3-year magnetic resonance imaging study of cortical lesions in relapse-onset multiple sclerosis. Ann Neurol 67:376383

Fig. 7 Distribution of lesion load and type in BMS versus SPMS

patients died of causes unrelated to MS. Further proof of the existence of BMS is the observation that clinically silent demyelinating plaques can be incidentally found in postmortem brain, accounting for a prevalence of 0.10.2% in large autopsy series. Incidental plaques can also be discovered at MRI performed for other reasons than a neurological decit and not all these subjects subsequently develop a RRMS. These cases can be considered examples of BMS at its benign end of the spectrum.

Conclusions Whereas acute focal perivascular inltration of peripheral immune cells into the CNS white matter, followed by resolution of the inammation, provides a good explanation for the rapid onset of symptoms during a relapse and the subsequent remission, the progressive stage of MS is characterised by a slow accumulation of neurological deficit and disability that cannot easily be associated with individual demyelinated lesions. Axonal loss and chronic expanding demyelination clearly play a role in the accumulating clinical decit from the early stages of MS but cannot alone explain the slow progressive increase in symptoms that often do not correlate with the location and number of brain and spinal cord focal WM lesions. Cortical GM pathology, including neuronal degeneration, and diffuse WM and GM changes that are not the result of inammatory WM lesions must now also be considered to play a major role in the accumulation of motor, sensory and cognitive disability that characterises secondary progression. The relative contribution of these events to the overall neurological progression will almost certainly be highly variable from patient to patient.
Acknowledgments Work described in this review was funded by the Multiple Sclerosis Society (Grant No. 747/02 to RR, FR and RN)

123

Acta Neuropathol (2011) 122:155170 18. Calabrese M, Filippi M, Gallo P (2010) Cortical lesions in multiple sclerosis. Nat Rev Neurol 6:438444 19. Campbell GR, Ziabreva I, Reeve AK et al (2011) Mitochondrial DNA deletions and neurodegeneration in multiple sclerosis. Ann Neurol 69:481492 20. Cifelli A, Arridge M, Jezzard P, Esiri MM, Palace J, Matthews PM (2002) Thalamic neurodegeneration in multiple sclerosis. Ann Neurol 52:650653 21. Clements RJ, McDonough J, Freeman EJ (2008) Distribution of parvalbumin and calretinin immunoreactive interneurons in motor cortex from multiple sclerosis post-mortem tissue. Exp Brain Res 187:459465 22. Compston A, Coles A (2008) Multiple sclerosis. Lancet 372:1502 1517 23. Confavreux C, Vukusic S (2006) Age at disability milestones in multiple sclerosis. Brain 129:595605 24. Confavreux C, Vukusic S, Moreau T, Adeleine P (2000) Relapses and progression of disability in multiple sclerosis. N Engl J Med 343:14301438 25. Dal Bianco A, Bradl M, Frischer J, Kutzelnigg A, Jellinger K, Lassmann H (2008) Multiple sclerosis and Alzheimers disease. Ann Neurol 63:174183 26. de Groot CJ, Bergers E, Kamphorst W et al (2001) Post-mortem MRIguided sampling of multiple sclerosis brain lesions: increased yield of active demyelinating and (p)reactive lesions. Brain 124:16351645 27. De Stefano N, Matthews PM, Fu L et al (1998) Axonal damage correlates with disability in patients with relapsingremitting multiple sclerosis. Results of a longitudinal magnetic resonance spectroscopy study. Brain 121:14691477 28. de Stefano N, Matthews PM, Filippi M et al (2003) Evidence of early cortical atrophy in MS: relevance to white matter changes and disability. Neurology 60:11571162 29. Deloire MS, Ruet A, Hamel D, Bonnet M, Dousset V, Brochet B (2011) MRI predictors of cognitive outcome in early multiple sclerosis. Neurology 76:11611167 30. Dutta R, McDonough J, Yin X et al (2006) Mitochondrial dysfunction as a cause of axonal degeneration in multiple sclerosis patients. Ann Neurol 59:478489 31. Dutta R, Chang A, Doud MK et al (2011) Demyelination causes synaptic alterations in hippocampi from multiple sclerosis patients. Ann Neurol 69:445454 32. Evangelou N, Esiri MM, Smith S et al (2000) Quantitative pathological evidence for axonal loss in normal appearing white matter in multiple sclerosis. Ann Neurol 47:391395 33. Ferguson B, Matyszak MK, Esiri MM, Perry VH (1997) Axonal damage in acute multiple sclerosis lesions. Brain 120:393399 34. Filippi M, Paty DW, Kappos L et al (1995) Correlations between changes in disability and T2-weighted brain MRI activity in multiple sclerosis: a follow-up study. Neurology 45:255260 35. Fisher E, Lee JC, Nakamura K, Rudick RA (2008) Gray matter atrophy in multiple sclerosis: a longitudinal study. Ann Neurol 64:255265 36. Fisniku LK, Chard DT, Jackson JS et al (2008) Gray matter atrophy is related to long-term disability in multiple sclerosis. Ann Neurol 64:247254 37. Friese MA, Fugger L (2009) Pathogenic CD8? T cells in multiple sclerosis. Ann Neurol 66:132141 38. Frischer JM, Bramow S, Dal-Bianco A et al (2009) The relation between inammation and neurodegeneration in multiple sclerosis brains. Brain 132:11751189 39. Fu L, Matthews PM, De Stefano N (1998) Imaging axonal damage of normal-appearing white matter in multiple sclerosis. Brain 121:103113 40. Geurts JJ, Pouwels PJ, Uitdehaag BM et al (2005) Intracortical lesions in multiple sclerosis: improved detection with 3D double inversion-recovery MR imaging. Radiology 236:254260

169 41. Geurts JJ, Bo L, Roosendaal SD et al (2007) Extensive hippocampal demyelination in multiple sclerosis. J Neuropathol Exp Neurol 66:819827 42. Geurts JJ, Barkhof F (2008) Grey matter pathology in multiple sclerosis. Lancet Neurol 7:841851 43. Gilmore CP, Donaldson I, Bo L, Owens T, Lowe J, Evangelou N (2009) Regional variations in the extent and pattern of grey matter demyelination in multiple sclerosis: a comparison between the cerebral cortex, cerebellar cortex, deep grey matter nuclei and the spinal cord. J Neurol Neurosurg Psychiatry 80:182187 44. Giorgio A, de Stefano N (2010) Cognition in multiple sclerosis: relevance of lesions, brain atrophy and proton MR spectroscopy. Neurol Sci 31(Suppl 2):S245S248 45. Glad SB, Aarseth JH, Nyland H, Riise T, Myhr KM (2010) Benign multiple sclerosis: a need for a consensus. Acta Neurol Scand Suppl 190:4450 46. Guseo A, Jellinger K (1975) The signicance of perivascular inltrations in multiple sclerosis. J Neurol 211:5160 47. Hochmeister S, Grundtner R, Bauer J et al (2006) Dysferlin is a new marker for leaky brain blood vessels in multiple sclerosis. J Neuropathol Exp Neurol 65:855865 48. Howell OW, Palser A, Polito A et al (2006) Disruption of neurofascin localisation reveals early changes preceding demyelination and remyelination in multiple sclerosis. Brain 129:31733185 49. Howell OW, Rundle JL, Garg A, Komada M, Brophy PJ, Reynolds R (2010) Activated microglia mediate axoglial disruption that contributes to axonal injury in multiple sclerosis. J Neuropathol Exp Neurol 69:10171033 50. Kornek B, Lassmann H (1999) Axonal pathology in multiple sclerosis: a historical note. Brain Pathol 9:651656 51. Kremenchutzky M, Rice GP, Baskerville J, Wingerchuk DM, Ebers GC (2006) The natural history of multiple sclerosis: a geographically based study 9: observations on the progressive phase of the disease. Brain 129:584594 52. Kuhlmann T, Lingfeld G, Bitsch A, Schuchardt J, Bruck W (2002) Acute axonal damage in multiple sclerosis is most extensive in early disease stages and decreases over time. Brain 125:22022212 53. Kutzelnigg A, Lucchinetti CF, Stadelmann C et al (2005) Cortical demyelination and diffuse white matter injury in multiple sclerosis. Brain 128:27052712 54. Lassmann H, Wekerle H (2005) The pathology of multiple sclerosis. McAlpines multiple sclerosis, vol 4. Churchill Livingstone, London, pp 557599 55. Leray E, Yaouanq J, Le Page E et al (2010) Evidence for a twostage disability progression in multiple sclerosis. Brain 133:19001913 56. Losseff NA, Webb SL, ORiordan JI et al (1996) Spinal cord atrophy and disability in multiple sclerosis. A new reproducible and sensitive MRI method with potential to monitor disease progression. Brain 119:20092019 57. Lovas G, Szilagyi N, Majtenyi K et al (2000) Axonal changes in chronic demyelinated cervical spinal cord plaques. Brain 123:308317 58. Lucchinetti C, Bruck W, Parisi J, Scheithauer B, Rodriguez M, Lassmann H (2000) Heterogeneity of multiple sclerosis lesions: implications for the pathogenesis of demyelination. Ann Neurol 47:707717 59. Magliozzi R, Howell O, Vora A et al (2007) Meningeal B-cell follicles in secondary progressive multiple sclerosis associate with early onset of disease and severe cortical pathology. Brain 130:10891104 60. Magliozzi R, Howell OW, Reeves C et al (2010) A Gradient of neuronal loss and meningeal inammation in multiple sclerosis. Ann Neurol 68:477493

123

170 61. McAlpine D (1961) The benign form of multiple sclerosis. A study based on 241 cases seen within three years of onset and followed up until the tenth year or more of the disease. Brain 84:186203 62. Meinl E, Krumholtz M, Derfuss T, Junker A, Hohlfeld R (2008) Compartmentalization of inammation in the CNS: a major mechanism driving progressive multiple sclerosis. J Neurol Sci 274:4244 63. Miller DH (1995) Magnetic resonance imaging and spectroscopy in multiple sclerosis. Curr Opin Neurol 8:210215 64. Molyneux PD, Filippi M, Barkhof F et al (1998) Correlations between monthly enhanced MRI lesion rate and changes in T2 lesion volume in multiple sclerosis. Ann Neurol 43:332339 65. Neumann H, Medana I, Bauer J, Lassmann H (2002) Cytotoxic T lymphocytes in autoimmune and degenerative CNS diseases. Trend Neurosci 25:313319 66. Nijeholt GJ, Bergers E, Kamphorst W et al (2001) Post-mortem high-resolution MRI of the spinal cord in multiple sclerosis: a correlative study with conventional MRI, histopathology and clinical phenotype. Brain 124:154166 67. Patani R, Balaratnam M, Vora A, Reynolds R (2007) Remyelination can be extensive in multiple sclerosis despite a long disease course. Neuropath App Neurobiol 33:277287 68. Patrikios P, Stadelmann C, Kutzelnigg A et al (2006) Remyelination is extensive in a subset of multiple sclerosis patients. Brain 129:31653172 69. Peterson JW, Bo L, Mork S, Chang A, Trapp BD (2001) Transected neuritis, apoptotic neurons and reduced inammation in cortical multiple sclerosis lesions. Ann Neurol 50:389400 70. Pirko I, Lucchinetti CF, Sriram S, Bakshi R (2007) Gray matter involvement in multiple sclerosis. Neurology. 68:634642 71. Prineas JW, Barnard RO, Revesz T, Kwon EE, Sharer L, Cho ES (1993) Multiple sclerosis. Pathology of recurrent lesions. Brain 116:681693 72. Rovaris M, Barkhof F, Calabrese M et al (2009) MRI features of benign multiple sclerosis: toward a new denition of this disease phenotype. Neurology 72:16931701 73. Scalfari A, Neuhaus A, Degenhardt A et al (2010) The natural history of multiple sclerosis: a geographically based study 10: relapses and long-term disability. Brain 133:19141929

Acta Neuropathol (2011) 122:155170 74. Seewann A, Kooi EJ, Roosendaal SD, Barkhof F, van der Valk P, Geurts JJ (2009) Translating pathology in multiple sclerosis: the combination of post-mortem imaging, histopathology and clinical ndings. Acta Neurol Scand 119:349355 75. Schmierer K, Scaravilli F, Altmann DR, Barker GJ, Miller DH (2004) Magnetization transfer ratio and myelin in post-mortem multiple sclerosis brain. Ann Neurol 56:407415 76. Schmierer K, Parkes HG, So P-W et al (2010) High eld (9.4 Tesla) magnetic resonance imaging of cortical grey matter lesions in multiple sclerosis. Brain 133:858867 77. Siffrin V, Vogt J, Radbruch H, Nitsch R, Zipp F (2010) Multiple sclerosiscandidate mechanisms underlying CNS atrophy. Trends Neurosci 33:202210 78. Smith KJ, Kapoor R, Hall SM, Davies M (2001) Electrically active axons degenerate when exposed to nitric oxide. Ann Neurol 49:470479 79. Stadelmann C (2011) Multiple sclerosis as a neurodegenerative disease: pathology, mechanisms and therapeutic implications. Curr Opin Neurol Adv. Access Mar 31 80. Trapp BD, Peterson J, Ransohoff RM, Rudick R, Mork S, Bo L (1998) Axonal transection in the lesions of multiple sclerosis. N Engl J Med 338:278285 81. Van der Valk P, Amor S (2009) Preactive lesions in multiple sclerosis. Curr Opin Neurol 22:207213 82. Vellinga MM, Oude Engberink RD, Seewann A et al (2008) Pluriformity of inammation in multiple sclerosis shown by ultrasmall iron oxide particle enhancement. Brain 131:800807 83. Vercellino M, Plano F, Votta B, Mutani R, Giordana MT, Cavalla P (2005) Grey matter pathology in multiple sclerosis. J Neuropathol Exp Neurol 64:11011107 84. Vuia O (1977) The benign form of multiple sclerosis. Anatomoclinical aspects. Acta Neurol Scand 55:289298 85. Wegner C, Esiri MM, Chance SA, Palace J, Matthews PM (2006) Neocortical neuronal, synaptic, and glial loss in multiple sclerosis. Neurology 67:960967 86. Wuerfel J, Haertle M, Waiczies H et al (2008) Perivascular spacesMRI marker of inammatory activity in the brain? Brain 131:23322340

123