Utilizing Mutual Information Analysis to Explore the Relationship Between Gray and White Matter Structural Pathologies in Schizophrenia.

Fusion of white and gray matter geometry: a framework for investigating brain development.

Despite the many neuroimaging studies that suggest gray matter volume reductions in schizophrenia, there is no compelling postmortem evidence to suggest neuronal loss, nor is there a distinctive or specific signature of gray matter abnormalities in schizophrenia. Instead, there appears to be no observed focal lesion that characterizes gray matter pathology in schizophrenia. This state of affairs has led to a movement away from conceptualizing schizophrenia as resulting from a single lesion, to conceptualizing schizophrenia as arising from abnormal communication between brain regions. Abnormal connectivity between brain regions is not a new concept, as Bleuler (1911) postulated such abnormalities. What is new, however, is an appreciation that brain regions that are not spatially proximal may be connected functionally into neural networks and that to understand altered neural connectivity we need to have a better understanding of the connections between brain regions which are made possible by white matter, the main infrastructure in the brain that makes possible long distance communication among neurons. Accordingly, a focus on white matter connections in the brain has become increasingly of interest in schizophrenia, particularly in the areas of imaging studies, postmortem studies, and new animal models of schizophrenia. This special issue focuses on schizophrenia and oligodendrocytes, the latter a class of neuroglia that give rise to myelin. The primary aim of this special issue is to understand and to clarify further white matter pathology in schizophrenia and how it may contribute to disconnectivity among brain regions, which results in the observed cognitive, behavioral, and clinical symptoms in this disorder. The first paper begins with a description of the theory that schizophrenia reflects a disorder of connectivity. The proposition put forth is that schizophrenia is, at least in part, the result of abnormal communication between brain regions that may be spatially distant but nonetheless functionally connected. As white matter is the main infrastructure for brain connectivity, this paper focuses on the use of a magnetic resonance imaging (MRI) technique called diffusion tensor imaging (DTI) to gain insight into the role of white matter abnormalities in schizophrenia. This paper also focuses on the functional implications of white matter abnormalities, particularly with regard to the possible role of myelin in modulating the transmission velocity of neural discharges. The paper ends with a speculative hypothesis about the relationship between gray matter and white matter abnormalities in schizophrenia. The second paper also focuses on altered neuronal connectivity. Here, however, the focus is on altered connectivity and impaired myelination in a postmortem study of schizophrenia and normal controls, where electron microscopy is used to study myelinated fibers and oligodendrocytes. This morphometric study examines myelinated fibers in the prefrontal cortex in both gray and white matter. Six types of abnormal fibers and ultrastructural alterations are described in the schizophrenia sample. The study reveals increased pathological fibers in gray matter in both young and elderly patients that are associated with positive symptoms, while in elderly patients, the frequency of pathological fibers in white matter is increased and these changes are associated with more negative symptoms. The key implication here is that altered myelinated fibers in white matter in schizophrenia likely follow alterations of myelinated fibers in gray matter that occur earlier in the course of illness. The third paper is a postmortem study on age-related changes in the number of oligodendrocytes in prefrontal cortex in schizophrenia, bipolar disorder and major depression. The study reveals an age-related increase in numerical density of oligodendrocytes in layer VI and adjacent white matter of BA9 and 10 that is seen only in normal controls and not in schizophrenia or in mood disorders. The absence of the normal age-related increase in oligodendrocytes in patients suggests that this aspect of normal brain development is dysregulated in schizophrenia and in mood disorders, and confirms postmortem and imaging data, further highlighting the fact that the absence of a normal age-related increase in oligodendrocytes is a key shared feature in these psychiatric populations. The fourth paper reviews white matter abnormalities in schizophrenia, including imaging and postmortem findings on the disconnectivity theory of schizophrenia, a major theme that underlies the three previous papers in this special issue. This paper then reviews some of the white matter diseases that commonly lead to psychotic symptoms and then reviews transgenic and mutant mouse models of schizophrenia. More specifically, genetic mouse models such as Plp1 transgenic mice and mutant mice heterogeneous for either NRG1 or its receptor erbB4, as well as Nogo-A- deficient mice are reviewed. This is followed by a focus on the myelin toxicity model of mice fed cuprizone. In the early stage (the second and third week) of the cuprizone feeding, mice show higher dopamine levels and lower norepinephrine levels in their prefrontal cortex, along with behavioral changes indicative of increased CNS activity. In the late stage (weeks 4–6), when demyelination and oligodendrocyte loss are obvious, mice display cognitive deficits as well as deficits in social interactions that are reminiscent of social withdrawal seen in patients with schizophrenia. Importantly, this cuprizone-feeding mouse may be used as an in vivo platform for psychopharmacological studies testing the effects of antipsychotic drugs on white matter alterations and associated behavioral changes, in addition to providing a new animal model of schizophrenia. The fifth and final paper of this special issue focuses on abnormal behavior and microstructural changes in juvenile mice that are repeatedly exposed to nonneurotoxic levels of amphetamine. These mice are compared with nonamphetamine treated mice. Oligodendrocyte numbers and three proteins expressed in mature oligodendrocytes are investigated in these young male mice at sacrifice. Treated mice showed higher locomotion and impaired spatial working memory, along with lowered Noga-A and GST-pi proteins, and lower MBP proteins, in frontal cortex and hippocampus and fewer mature oligodendrocytes in frontal cortex and corpus callosum, as well as lower MBP staining in frontal cortex, corpus callosum, and hippocampus, than untreated mice. These differences suggest that in wild-type mice late developing white matter is vulnerable to amphetamine and may lead to compromised white matter with increased dopamine in specific brain regions. Not only do these findings explain well the increased locomotion and impaired spatial working memory observed in amphetamine-treated mice, but also may serve as a principle for the amphetamine-treated mouse being used as a novel animal model of schizophrenia. To summarize, compared to the focus on gray matter and neurons, research on white matter and myelin/glial has been sparse in schizophrenia. In this special issue, neuroimaging findings are presented that suggest that myelin abnormalities in schizophrenia may underlie the white matter abnormalities observed using diffusion tensor imaging techniques. Data from postmortem studies also confirm the presence of oligodendrocyte and myelin abnormalities in schizophrenia as well as mood disorders. Oligodendrocytes provide protection and improve communication between brain regions and the oligodendroglia-producing myelin is approaching maturity in the same time frame as the onset of symptoms. Further, normal age-related white matter increases and related gray matter changes that likely predate the white matter changes are dysregulated in schizophrenia and may contribute to the clinical symptomatology of the disease. Finally, the recently developed animal models are not only of help in understanding connectivity abnormalities in schizophrenia but also provide novel platforms to test novel therapeutic approaches for schizophrenic patients. Treatments specifically targeting white matter, for example, may improve on the efficacy of those treatments currently in use. Martha E. Shenton Haiyun Xu Vahram Haroutunian George Bartzokis

Structural MRI findings of abnormalities in the prefrontal cortex in schizophrenia and affective disorder have been inconsistent likely due to small, heterogeneous samples, the evaluation of prefrontal gray and white matter combined, and the fact that prefrontal cortex is typically not delineated into separate gyri (e.g., Shenton et al., 2001; Strakowski et al., 2002). We previously reported smaller prefrontal gray matter in first-episode schizophrenia relative to first-episode affective psychosis and controls (Hirayasu et al., 2001). One unresolved question in the literature is whether or not further volume reduction will be observed over time, the focus of this report. Prefrontal gray and white matter volumes were measured (see Fig. 1) in patients at the time of first hospitalization for schizophrenia (n=12, 3 females) or affective psychosis (n=10, 1 female, 9 bipolar, 1 unipolar), and psychiatrically well subjects (n=15, 1 female). Subjects were rescanned approximately 1.5 years later. Seven schizophrenia, six affective, and four comparison subjects were previously described solely at first scan (Hirayasu et al., 2001). Samples did not differ in age (28.1±8.4; 22.9±2.8; 25.4±4.5; F(2,34)=2.2, p=0.12) or WAIS Information scores (11.8±3.4; 12.2±3.1, 11.8±2.0; F(2.32)=0.6, p=0.9), nor in parental socioeconomic status (F(2.34)=2.32, p=0.11) or handedness (F(2.33)=1.6, p=0.22). BPRS scores were higher in schizophrenics (41.2±13.2) than affectives (33.8±8.0) (F(1,20)=5.71, p=0.027). Similar neuroleptics were prescribed for the patient groups (schizophrenics: 7 typical, 4 atypical, 1 none; affectives: 6 typical, 3 atypical, 1 none). Fig. 1 Prefrontal ROI definitions and volumes. (a) Coronal slice, (b) axial slice showing prefrontal gray and white matter, and (c) three. Three-dimensional reconstruction of the extent of gray and white matter boundaries. MR images were acquired with a 1.5-T ... Relative volumes [i.e., absolute volumes divided by intracranial contents (ICC)] were used in the analyses. Repeated-measures ANOVA was performed with diagnosis as the between-subjects factor and time and side as the within-subjects factors for gray and white matter separately. Prefrontal gray matter at time 1 was significantly different among groups (F2,30=5.56, p=0.009), with schizophrenics smaller than controls and affectives, who did not differ from each other. There was no interaction for group by time by side for prefrontal gray matter (F2,34=0.65, p=0.53). All groups showed larger gray matter on the left (F2,30=8.72, p=0.006). White matter volumes did not differ among groups (p>0.87), and all groups showed more white matter on the right (F1,30=39.20, p<0.001) and a reduction over time (F1,30=10.902, p=0.002), there was a trend for this effect to be larger on the right (F1,30=3.14, p=0.086) (%change over time for right white matter: schizophrenics 2.8%; affectives 6.5%; controls 2.1%). Neither removing the unipolar depression subject nor removing the females altered the results. There was no significant correlation between ROI volume change and medication dosage at time 1 for either patient group (Time 2 data came from out patients based on self-report and was for this reason not used). These data suggest that prefrontal cortical gray matter is selectively smaller at first hospitalization for schizophrenia relative to affective psychosis and controls, but this volume difference did not change over the relatively short post-first hospitalization time examined. Small gray matter volumes may be due to reduced dendritic arborization or increased neural density in prefrontal cortex in schizophrenia (Benes et al., 1992; Selemon et al., 1998). White matter, which was not different among groups, showed a decline with time in all groups, possibly consistent with normal aging (Good et al., 2001, although Bartzokis et al., 2001). Potential reasons for our not observing selective gray matter reductions point to several limitations of this study including the gender distribution, as females may have greater dorsolateral and orbitofrontal lobe involvement and males greater dorsomedial changes in schizophrenia (Gur et al., 2000); the use of large ROI, as sub-regions within the prefrontal cortex may change in volume at different rates (Gur et al., 1998; DeLisi et al., 1997); and lack of exact medication dosages and compliance histories during the intrascan interval cannot be ruled out as possible confounds. Although the sample size was small, the effect size was also small (Fig. 1) suggesting that even enlarging the sample would not result in a significant change in volume over time for either gray or white matter.

Differentiation of absolute metabolite concentrations between gray and white matter in the occipital region of normal human brain was performed by in vivo localized single-voxel 1H magnetic resonance spectroscopy at 1.5 Tesla with long echo time (136 ms). With the combination of image segmentation between white and gray matter and cerebrospinal fluid, signal compensation of T1 and T2 effects, tissue water signal as the internal concentration reference, as well as compensation by different water contents in gray and white matters, it was determined that the levels of N-acetylaspartate (NAA), creatine and/or phosphocreatine (Cr), and choline-containing compounds (Cho) in gray matter were significantly higher than in white matter. The averaged NAA, Cr, and Cho concentrations in gray matter were 11.0, 9.7, and 1.9 mM/liter, respectively, in comparison with 7.5, 5.2, and 1.6 mM/liter in white matter. These results suggest that precise composition of white and gray matter and cerebrospinal fluid is necessary to avoid partial voluming effect in a single voxel and to accurately quantify the metabolite concentrations.

Apolipoprotein E (apoE) and cholesterol play a critical role in synapse and myelin maintenance and integrity and are thus appealing candidates in the pathogenesis of schizophrenia and bipolar disorder. To explore the role of these 2 molecules, we quantified cholesterol and apoE levels in prefrontal grey and white matter in patients with schizophrenia, bipolar disorder and healthy controls. Furthermore, we investigated the relations between apoE and cholesterol levels and the APOE genotype. We obtained dorsolateral prefrontal grey and white matter from the Stanley Medical Research Institute Brain Collection (schizophrenia n = 35, bipolar disorder n = 35 and controls n = 35). Cholesterol levels were quantified using high-pressure liquid chromatography, whereas apoE was measured by enzyme-linked immunosorbent assay. We found no significant differences in cholesterol or apoE levels among the groups. ApoE levels were higher in grey matter than in white matter in all groups; conversely, levels of cholesterol were higher in white matter than in grey matter. We observed a significant inverse correlation between apoE and cholesterol levels in both grey and white matter. Furthermore, in grey matter, apoE levels were significantly higher in APOE ε2 carriers compared with APOE ε3 or APOE ε4 carriers, with cholesterol levels following the opposite trend. LIMITATIONS of our study include our inability to control for potential confounding variables and the small numbers of APOE ε2 and ε4 carriers in each group. Although large amounts of cholesterol are present in white matter, apoE expression is limited. The APOE genotype may play a role in the regulation of both cholesterol and apoE levels in grey matter. The impact of APOE polymorphisms on lipid homeostasis in people with psychiatric disorders warrants further investigation.

To measure the volumetric changes of gray and white matters in patients with temporal lobe epilepsy(TLE)using voxel-based morphometric study(VBM)and correlate the changes with clinical parameters. A total of 71 TLE patients were enrolled in the study,and 22 healthy subjects served as normal controls. Routine brain MRI and 3D fast spoiled gradient echo(FSPGR)T1-weighted images of all the subjects were acquired. The 3D structural images were co-registered,segmented and smoothed,and then the images were analyzed using the optimized VBM with preprocessed using Diffeomorphic Anatomical Registration using Exponentiated Lie algebra(DARTEL)algorithm. The global and local gray matter and white matter volume of each subject were calculated and compared between the TLE patients and normal controls. The potential correlations between the changes of the global and local gray and white matters in the TLE patients and the clinical parameters including the age at onset and the duration of epilepsy were explored. Compared to the normal controls,the TLE patients had diffuse volumetric reduction of gray and white matters in cerebrum both ipsilateral and contralateral to the seizure focus(P<0.05). Local gray matter reduction was found extensively in bilateral cerebral lobes,especially in the temporal and frontal lobes. Local white matter reduction was found in bilateral temporal,parietal and frontal lobes,in addition to the cingulate gyrus. The global gray matter volume(Global GMV)and the global white matter volume(Global WMV)were negatively correlated to the duration of epilepsy with the most significant change occurring in the first year of epilepsy. Global WMV dropped more quickly than Global GMV during the prolonged disease course. TLE patients have diffuse gray matter and white matter reduction,particularly in the early stage of epilepsy. The reduction of the white matter is more obvious than the gray matter.

Despite clinical and neuropsychological similarities between Parkinson's disease dementia (PDD) and dementia with Lewy bodies (DLB), recent studies have demonstrated that structural and pathological changes are more severe in DLB than in PDD. We used voxel-based morphometry using a 3-T MRI scanner to compare gray and white matter densities in 20 patients with probable PDD and 18 patients with probable DLB, who had similar overall severity of dementia and similar demographic characteristics. The gray matter density was significantly decreased in the left occipital, parietal, and striatal areas in patients with DLB compared with patients with PDD. The white matter density was significantly decreased in bilateral occipital and left occipito-parietal areas in patients with DLB compared with those with PDD. The degree of white and gray matter atrophy was similar in patients with DLB; in contrast, there was markedly less atrophy in the white matter than in the gray matter in patients with PDD. On analyzing the change of WM density relative to that of GM density in patients with DLB compared to those with PDD, the area of WM atrophy in the occipital areas was more extensive than that of GM atrophy. Our data demonstrate that atrophy of both gray and white matter was more severe in patients with DLB and that white matter atrophy relative to gray matter atrophy was less severe in patients with PDD. These data may reflect a difference in the underlying nature of PDD and DLB.

In vitro studies suggest that adenosine may attenuate anoxic white matter damage as an intrinsic protective substance. The authors investigated ischemic alterations of purines in relation to tissue depolarization and extracellular calcium and amino acid concentrations in vivo using microdialysis and ion-selective electrodes in cortical gray and subcortical white matter of 10 cats during 120 minutes of global brain ischemia. Immediately on induction of ischemia, regional cerebral blood flow ceased in all cats in both gray and white matter. The direct current potential rapidly decreased, the decline being slower and shallower in white matter. Extracellular calcium levels decreased in gray matter. In contrast, they first increased in white matter and started to decrease below control levels only after approximately 30 minutes. Adenosine levels transiently increased in both tissue compartments; the peak was delayed by 30 minutes in white matter. Thereafter, levels declined faster in gray than in white matter and remained elevated in the latter tissue compartment. Inosine and hypoxanthine elevations were progressive in both regions but smaller in white matter. Levels of gamma-aminobutyric acid, another putatively protective agent, steadily increased, starting immediately in gray matter and delayed by almost 1 hour in white matter. The delayed and prolonged accumulation of adenosine correlates with a slower adenosine triphosphate breakdown in white matter ischemia and may result in protection of white matter by suspending cellular calcium influx.

With quantitative electroencephalogram (EEG) and neuroimaging methods, we examined delta activity, atrophy, and neuronal-axonal dysfunction of the cerebral gray and white matter in patients with intractable temporal lobe epilepsy (TLE). Based on evidence that lesions of the white matter result in EEG delta activity, we postulated that background abnormalities in patients with TLE are related to changes of the temporal lobe white matter. We measured interictal delta activity in 34 TLE patients and 10 controls. Spike-free and artifact-free EEG samples were selected by visual inspection. A spectral analysis was used to compute the energy in the delta frequency band. We compared the results of the spectral analysis to magnetic resonance imaging- (MRI) based volumes of the temporal lobe white and gray matter, the hippocampus and the amygdala; and N-acetyl aspartate (NAA) in the lateral and posterior temporal lobe by using proton magnetic resonance spectroscopic imaging (1H-MRSI). The degree of correlation between delta activity and the neuroimaging measurements was assessed by using the Pearson correlation coefficient (r). An analysis of variance (ANOVA) was used to examine the influence of the seizure-focus lateralization on the delta activity and the neuroimaging parameters. There was no significant difference in the amount of delta activity in the temporal lobe between the controls and patients. We found no correlation between delta activity and the neuroimaging measures (p>0.05). The ANOVA showed significant differences between the patients and controls for the volume of the gray and white matter of the temporal lobe and for the NAA in the lateral and posterior temporal lobe (p<0.002). The interictal background delta activity was not explained by reduced volume of the temporal lobe white matter, gray matter, or by abnormalities seen in 1H-MRSI.

Brain extracellular space (ECS) provides an important microenvironment for neurons and glial cells. In the present study, we investigated differences in ischemic changes of ECS in gray and white matter during global ischemia in cats (n = 8). Diffusion capacity of tetramethylammonium ion (TMA+, 74.1Da) was evaluated by measuring extracellular TMA+ concentration applied via microdialysis. In both gray and white matter, TMA+ concentration significantly increased within 10 minutes after the induction of ischemia, and sustained during 120 min of ischemia. The increase, however, was slower and smaller in white matter than in gray matter. In other two animals, volume fraction and tortuosity of ECS were determined by iontophoresis during global ischemia. After 30 min of ischemia induction, volume fraction was decreased and tortuosity was increased in both gray and white matter. In white matter, decrease in volume fraction and increase in tortuosity were smaller than in gray matter. The present study demonstrates that cerebral ischemia induces not only significant shrinkage of ECS volume but also restricts molecular diffusion within. Smaller changes of diffusion capacity within white matter ECS during ischemia may be relevant for lower ischemic vulnerability of the region.

The remarkable success of magnetic resonance imaging of adult brain relates to the unusually large ratio of the longitudinal relaxation rates 1/T1 of white and gray matter, approximately 2:1 at physiological temperature and traditional imaging fields. Several investigators have conjectured that myelin is the source of the greater 1/T1 of white matter without, however, suggesting details of the molecular mechanisms responsible. From measurements of the magnetic field dependence of 1/T1 (NMRD profiles) of adult and neonatal gray and white matter at 5 and 35 degrees C, we find a thermally activated contribution to the NMRD profile of adult white matter that is not present in the profiles of either adult gray or neonatal gray and white matter. We attribute this contribution to myelin and develop a quantitative model that accounts for the unique relaxation behavior of myelinated white matter. We find that myelin water, 15% of the total, has a relatively short T1 that arises from an unexpectedly large interaction with myelin lipid; when cast in terms of an interaction over the entire myelin bilipid-water interface, it is sevenfold greater than the analogous protein-water interfacial interaction. Its magnitude remains to be accounted for, but cholesterol, known to alter the relaxation rates of lipid protons, may play an important role. The contribution of myelin to 1/T1 at physiological temperatures is attributed to thermally activated transmembrane diffusion of water and, hence, more rapid mixing of axonal and the rapidly relaxing myelin water molecules.

Objective To explore whether gray matter and white matter presented abnormalities in neural circuit in patients with OCD by using multimodal imaging strategies. Methods Magnetic resonance imaging (MRI) scans from 54 OCD patients and 54 healthy controls (HC) were acquired. Gray matter volume was compared using the voxel-based morphometry (VBM) approach based on the SPM software. Fractional anisotropy (FA) difference was also evaluated using tract based spatial statistics (TBSS) based on the FSL software. Results Compared with the HC, Patients with OCD showed decreased gray matter volume in the left middle frontal gyrus, left anterior cingulate and paracingulate gyri, left precental gyrus and right inferior temporal gyrus (P<0.05, Alphasim corrected); meanwhile, decreased FA values in the body and genu of corpus callosum were found. Conclusions Patients with OCD demonstrated widespread gray matter and white matter alterations especially in the incortico-striatal-thalamic-cortical (CSTC)circuit. Therefore, Patients with OCD might present altered gray and white matter structures simultaneously. Key words: Obsessive-compulsive disorder; Magnetic resonance imaging; Diffusion magnetic resonance imaging

Neurofibrillary pathology of abnormally hyperphosphorylated tau spreads along neuroanatomical connections, underlying the progression of Alzheimer's disease (AD). The propagation of tau pathology to axonally connected brain regions inevitably involves trafficking of seeding-competent tau within the axonal compartment of the neuron. To determine the seeding activity of tau in cerebral gray and white matters of AD. Levels of total tau, hyperphosphorylation of tau, and SDS- and β-mercaptoethanol-resistant high molecular weight tau (HMW-tau) in crude extracts from gray and white matters of AD frontal lobes were analyzed by immuno-blots. Tau seeding activity was quantitatively assessed by measuring RIPA buffer-insoluble tau in HEK-293FT/tau151-391 cells treated with brain extracts. We found a comparable level of soluble tau in gray matter versus white matter of control brains, but a higher level of soluble tau in gray matter than white matter of AD brains. In AD brains, tau is hyperphosphorylated in both gray and white matters, with a higher level in the former. The extracts of both gray and white matters of AD brains seeded tau aggregation in HEK-293FT/tau151-391 cells but the white matter showed less potency. Seeding activity of tau in brain extracts was positively correlated with the levels of tau hyperphosphorylation and HMW-tau. RIPA-insoluble tau, but not RIPA-soluble tau, was hyperphosphorylated tau at multiple sites. Both gray and white matters of AD brain contain seeding-competent tau that can template aggregation of hyperphosphorylated tau, but the seeding potency is markedly higher in gray matter than in white matter.

Catecholaminergic activation of G-protein coupling in rat spinal cord: Further evidence for the existence of dopamine and noradrenaline receptors in spinal grey and white matter

The regeneration of oligodendrocytes is a crucial step in recovery from demyelination, as surviving oligodendrocytes exhibit limited structural plasticity and rarely form additional myelin sheaths. New oligodendrocytes arise through the differentiation of platelet-derived growth factor receptor α (PDGFRα) expressing oligodendrocyte progenitor cells (OPCs) that are widely distributed throughout the CNS. Although there has been detailed investigation of the behavior of these progenitors in white matter, recent studies suggest that disease burden in multiple sclerosis (MS) is more strongly correlated with gray matter atrophy. The timing and efficiency of remyelination in gray matter is distinct from white matter, but the dynamics of OPCs that contribute to these differences have not been defined. Here, we used in vivo genetic fate tracing to determine the behavior of OPCs in gray and white matter regions in response to cuprizone-induced demyelination. Our studies indicate that the temporal dynamics of OPC differentiation varies significantly between white and gray matter. While OPCs rapidly repopulate the corpus callosum and mature into CC1 expressing mature oligodendrocytes, OPC differentiation in the cingulate cortex and hippocampus occurs much more slowly, resulting in a delay in remyelination relative to the corpus callosum. The protracted maturation of OPCs in gray matter may contribute to greater axonal pathology and disease burden in MS.