Neuropathology Of Multiple Sclerosis Research Articles

Investigating cortical hypoxia in multiple sclerosis via time-domain near-infrared spectroscopy.

Hypoperfusion and tissue hypoxia have been implicated as contributory mechanisms in the neuropathology of multiple sclerosis (MS). Our objective has been to study cortical oxygenation in vivo in patients with MS and age-matched controls. A custom, multiwavelength time-domain near-infrared spectroscopy system was developed for assessing tissue hypoxia from the prefrontal cortex. A cross-sectional case-control study was undertaken assessing patients with secondary progressive MS (SPMS) and age-matched controls. Co-registered magnetic resonance imaging was used to verify the location from which near-infrared spectroscopy data were obtained through Monte Carlo simulations of photon propagation. Additional clinical assessments of MS disease severity were carried out by trained neurologists. Linear mixed effect models were used to compare cortical oxygenation between cases and controls, and against measures of MS severity. Thirty-three patients with secondary progressive MS (median expanded disability status scale 6 [IQR: 5-6.5]; median age 53.0 [IQR: 49-58]) and 20 age-matched controls were recruited. Modeling of photon propagation confirmed spectroscopy data were obtained from the prefrontal cortex. Patients with SPMS had significantly lower cortical hemoglobin oxygenation compared with controls (-6.0% [95% CI: -10.0 to -1.9], P = 0.004). There were no significant associations between cortical oxygenation and MS severity. Using an advanced, multiwavelength time-domain near-infrared spectroscopy system, we demonstrate that patients with SPMS have lower cortical oxygenation compared with controls.

Cellular architecture of evolving neuroinflammatory lesions and multiple sclerosis pathology

Multiple sclerosis (MS) is a neurological disease characterized by multifocal lesions and smoldering pathology. Although single-cell analyses provided insights into cytopathology, evolving cellular processes underlying MS remain poorly understood. We investigated the cellular dynamics of MS by modeling temporal and regional rates of disease progression in mouse experimental autoimmune encephalomyelitis (EAE). By performing single-cell spatial expression profiling using in situ sequencing (ISS), we annotated disease neighborhoods and found centrifugal evolution of active lesions. We demonstrated that disease-associated (DA)-glia arise independently of lesions and are dynamically induced and resolved over the disease course. Single-cell spatial mapping of human archival MS spinal cords confirmed the differential distribution of homeostatic and DA-glia, enabled deconvolution of active and inactive lesions into sub-compartments, and identified new lesion areas. By establishing a spatial resource of mouse and human MS neuropathology at a single-cell resolution, our study unveils the intricate cellular dynamics underlying MS.

The potential therapeutic effect of statins in multiple sclerosis: beneficial or detrimental effects.

Multiple sclerosis (MS) is a chronic progressive disabling disease of the central nervous system (CNS) characterized by demyelination and neuronal injury. Dyslipidemia is observed as one of the imperative risk factors involved in MS neuropathology. Also, chronic inflammation in MS predisposes to the progress of dyslipidemia. Therefore, treatment of dyslipidemia in MS by statins may attenuate dyslipidemia-induced MS and avert MS-induced metabolic changes. Therefore, the present review aimed to elucidate the possible effects of statins on the pathogenesis and outcomes of MS. Statins adversely affect the cognitive function in MS by decreasing brain cholesterol CoQ10, which is necessary for the regulation of neuronal mitochondrial function. However, statins could be beneficial in MS by shifting the immune response from pro-inflammatory Th17 to an anti-inflammatory regulatory T cell (Treg). The protective effect of statins against MS is related to anti-inflammatory and immunomodulatory effects with modulation of fibrinogen and growth factors. In conclusion, the effects of statins on MS neuropathology seem to be conflicting, as statins seem to be protective in the acute phase of MS through anti-inflammatory and antioxidant effects. However, statins lead to detrimental effects in the chronic phase of MS by reducing brain cholesterol and inhibiting the remyelination process.

DNA methylation changes in glial cells of the normal-appearing white matter in Multiple Sclerosis patients

ABSTRACT Multiple Sclerosis (MS), the leading cause of non-traumatic neurological disability in young adults, is a chronic inflammatory and neurodegenerative disease of the central nervous system (CNS). Due to the poor accessibility to the target organ, CNS-confined processes underpinning the later progressive form of MS remain elusive thereby limiting treatment options. We aimed to examine DNA methylation, a stable epigenetic mark of genome activity, in glial cells to capture relevant molecular changes underlying MS neuropathology. We profiled DNA methylation in nuclei of non-neuronal cells, isolated from 38 post-mortem normal-appearing white matter (NAWM) specimens of MS patients (n = 8) in comparison to white matter of control individuals (n = 14), using Infinium MethylationEPIC BeadChip. We identified 1,226 significant (genome-wide adjusted P-value < 0.05) differentially methylated positions (DMPs) between MS patients and controls. Functional annotation of the altered DMP-genes uncovered alterations of processes related to cellular motility, cytoskeleton dynamics, metabolic processes, synaptic support, neuroinflammation and signaling, such as Wnt and TGF-β pathways. A fraction of the affected genes displayed transcriptional differences in the brain of MS patients, as reported by publically available transcriptomic data. Cell type-restricted annotation of DMP-genes attributed alterations of cytoskeleton rearrangement and extracellular matrix remodelling to all glial cell types, while some processes, including ion transport, Wnt/TGF-β signaling and immune processes were more specifically linked to oligodendrocytes, astrocytes and microglial cells, respectively. Our findings strongly suggest that NAWM glial cells are highly altered, even in the absence of lesional insult, collectively exhibiting a multicellular reaction in response to diffuse inflammation.

Cognition and disease characteristics in adult onset versus late onset multiple sclerosis

Background: Cognitive impairment is common sequelae of multiple sclerosis (MS); however, relatively little is known about cognitive impairment in late-onset multiple sclerosis (LOMS). Objective: To investigate differences in disease characteristics and cognition in LOMS and adult-onset multiple sclerosis (AOMS) patients. Methods: Archival medical records and neuropsychological evaluations from an MS specialty center were reviewed. Differences in disease characteristics between 53 LOMS and 124 AOMS were compared using chi-square or analysis of variance (ANOVA). To investigate differences in cognitive functioning, age-adjusted standardized scores were compared via analysis of covariance (ANCOVA), using cardiac risk factors and disease duration as covariates. Results: Compared to AOMS, LOMS patients had significantly more cardiac risk factors, shorter disease duration, and shorter time to diagnosis. LOMS patients had similar Expanded Disability Status Scale scores as AOMS patients. LOMS patients demonstrated significantly more impairment on tasks of visual learning and memory, and working memory than AOMS patients. Conclusion: Despite a shorter disease duration, LOMS and AOMS patients had similar levels of physical impairment. However, even after accounting for differences in disease duration and cardiac risk, LOMS patients showed a greater burden of cognitive impairment than AOMS patients, suggesting MS diagnosed later in life may progress faster due to the interaction between MS neuropathology and aging.

Mechanism of demyelination and remyelination in multiple sclerosis

AbstractRecent insights into its molecular neuropathology and immunology have provided a comprehensive overview of the pathology of multiple sclerosis (MS), including demyelination and axonal loss. To date, neuropathology in MS as the human autoimmune disease is considered to be mainly mediated by autoreactive leukocytes. By contrast, accumulating evidence has also suggested that the inflammation and tissue damage in MS and its animal model, experimental autoimmune encephalomyelitis, (EAE) is also critically regulated by glial cells in the central nervous system. However, the molecular mechanism of the glial cells‐mediated pathology of MS/EAE has not been fully understood. We examined the oligodendrocyte‐mediated demyelination and axonal injury. To address this issue, we established a new scanning electron microscopy analysis to observe ultrastructural myelin morphology. In addition, we focused on kallikrein 6, a serine protease, secreted by oligodendrocytes in the central nervous system and proposed a new molecular mechanism of kallikrein 6‐mediated demyelination. In this article, we discuss the pathological roles of oligodendrocytes in mouse models of EAE. We also highlight recent findings of abnormal myelin formation and axonal injury in EAE.

Diffusion basis spectrum imaging for identifying pathologies in MS subtypes.

Diffusion basis spectrum imaging (DBSI) combines discrete anisotropic diffusion tensors and the spectrum of isotropic diffusion tensors to model the underlying multiple sclerosis (MS) pathologies. We used clinical MS subtypes as a surrogate of underlying pathologies to assess DBSI as a biomarker of pathology in 55 individuals with MS. Restricted isotropic fraction (reflecting cellularity) and fiber fraction (representing apparent axonal density) were the most important DBSI metrics to classify MS using brain white matter lesions. These DBSI metrics outperformed lesion volume. When analyzing the normal‐appearing corpus callosum, the most significant DBSI metrics were fiber fraction, radial diffusivity (reflecting myelination), and nonrestricted isotropic fraction (representing edema). This study provides preliminary evidence supporting the ability of DBSI as a potential noninvasive biomarker of MS neuropathology.

IL-15 expression on B cells and myeloid cells is distinctly modulated by inflammatory factors in MS and healthy donors

Abstract Although the salience of the immune system in multiple sclerosis (MS) pathology has been established, the mechanisms involved are incompletely characterized. We have identified IL-15 as a relevant contributor to MS neuropathology; a greater proportion of antigen presenting cells (APC) (B cells and myeloid cells) from MS patients express IL-15 vs. controls. Our aim is to identify factors and mechanisms that contribute to elevated IL-15 levels in the context of MS. The addition of CpG, a TLR9 agonist, to CD40L-cultured human B cells increased the proportion of IL-15+ B cells. Whereas other TLR ligands (e.g. polyI:C) did not alter B cell IL-15 levels, they nevertheless increased CD40 expression. Monocytes from MS patients and age/sex-matched healthy controls (HC) were differentiated into M0 macrophages prior to M1 or M2 polarization. M2 polarization significantly enhanced the percentage of IL-15+macrophages; in contrast, M1 polarization did not. GM-CSF, which is produced by encephalitogenic T cells, induced a major increase in the proportion of IL-15+ M0 and M1 macrophages and decreased the proportion of IL-15+ monocytes from HC and MS donors. While IL-15+ M2 macrophages from HC are increased upon GM-CSF stimulation, IL-15+ M2 from MS donors are not. GM-CSF did not alter IL-15 expression on microglia. Pharmacological inhibitors of STAT5 (Pimozide) and JAK2 (AG490) phosphorylation decreased the GM-CSF-induced increase of IL-15 on M0 macrophages. Thus, GM-CSF is a potent trigger for IL-15 expression on human M0, M1 and M2 macrophages, but not monocytes or microglia. Our data suggest that specific stimuli mediate IL-15 expression on human APC and that cells from MS donors exhibit altered susceptibility to IL-15 modulators compared to HC.

Coping style as a protective factor for emotional consequences of structural neuropathology in multiple sclerosis

ABSTRACTIntroduction: In people with multiple sclerosis (MS), depression symptoms could be a direct consequence of neuropathological processes or a secondary consequence of coping with debilitating illness. We investigated the interaction of white matter structure and patient coping style in predicting positive and negative emotion symptoms of depression.Method: Participants completed a neuropsychological battery, including the Chicago Multiscale Depression Inventory (CMDI) and a measure of coping strategies that has Active Coping (more adaptive) and Avoidant Coping (less adaptive) scales. Participants also completed a diffusion tensor imaging (DTI) scan, from which fractional anisotropy (FA) was calculated to assess integrity in tracts of interest, and the interaction of FA and coping style was analyzed to predict depression symptoms.Results: Significant FA and Active Coping interaction effects for predicting CMDI Negative Emotion scores were found for the anterior thalamic radiation and uncinate fasciculus white matter tracts. For people with MS who showed relatively reduced integrity of these tracts, use of more Active Coping moderated the relationship of microstructure and negative emotion symptoms of depression. This moderating relationship was not seen with other tracts of interest or with positive emotion.Conclusion: There was a protective effect of adaptive coping style against the experience of negative emotion among people with MS who showed compromised regional white matter integrity of certain tracts that connect temporal and thalamic regions to frontal cortex.

Brain activity, regional gray matter loss, and decision-making in multiple sclerosis

Background: Decision-making (DM) abilities deteriorate with multiple sclerosis (MS) disease progression which impairs everyday life and is thus clinically important. Objective: To investigate the underlying neurocognitive processes and their relation to regional gray matter (GM) loss induced by MS. Methods: We used a functional magnetic resonance imaging (fMRI) Iowa Gambling Task to measure DM-related brain activity in 36 MS patients and 21 healthy controls (HC). From this activity, we determined neural parameters of two cognitive stages, a deliberation (“choice”) period preceding a choice and a post-choice (“feedback”) stage reporting decision outcomes. These measures were related to DM separately in intact and damaged GM areas as determined by a voxel-based morphometry analysis. Results: Severely affected patients (with high lesion burden) showed worse DM-learning than HC (t = −1.75, p = 0.045), moderately affected (low lesion burden) did not. Activity in the choice stage in intact insular (t = 4.60, pFamily-Wise Error [FWE] corrected = 0.034), anterior cingulate (t = 4.50, pFWE = 0.044), and dorsolateral prefrontal areas (t = 4.43, pFWE = 0.049) and in insular areas with GM loss (t = 3.78, pFWE = 0.011) was positively linked to DM performance across patients with severe tissue damage and HC. Furthermore, activity in intact orbitofrontal areas was positively linked to DM-learning during the feedback stage across these participants (t = 4.49, pFWE = 0.032). During none of the stages, moderately affected patients showed higher activity than HC, which might have indicated preserved DM due to compensatory activity. Conclusion: We identified dysregulated activity linked to impairment in specific cognitive stages of reward-related DM. The link of brain activity and impaired DM in areas with MS-induced GM loss suggests that this deficit might be tightly coupled to MS neuropathology.

GM-CSF upregulates IL-15 expression on specific human myeloid cells.

Abstract Although the salience of the immune system in multiple sclerosis (MS) pathology has been established, the mechanisms and mediators involved are incompletely characterized. We have identified IL-15, which aids in the development, activation, and survival of immune cells, as a relevant contributor to MS neuropathology. We previously showed that greater proportions of myeloid cells, including peripheral monocytes and brain macrophages/microglia, from MS patients express IL-15 versus controls. Thus, we aim to identify the factors that contribute to these elevated IL-15 levels in the context of MS. Primary human monocytes from MS patients and age/sex matched healthy donors are treated with M-CSF to trigger differentiation into macrophages (considered M0 macrophages) prior to being polarized into M1 or M2 macrophages. While a small proportion of M0 macrophages express IL-15, M2 polarization significantly enhances the percentage of IL-15+ macrophages. In contrast, M1 polarization does not affect IL-15 levels although it significantly increases MHCI and CD80 expression. GM-CSF is produced by pathogenic encephalitogenic T cells; monocytes and macrophages express the corresponding receptor (GM-CSF receptor). GM-CSF induces a major increase in the proportion of IL-15+ M0 and M2 macrophages from both donor groups. In contrast, GM-CSF does not modify IL-15 expression by monocytes although it induces STAT5 phosphorylation. In conclusion, GM-CSF is a potent trigger for IL-15 expression on M0/M2 macrophages but not monocytes. Moreover, M2 polarization of macrophages increases IL-15 expression. Identification of IL-15 triggers and further characterization of IL-15+ immune cells may provide novel targets for therapies for MS patients.

Altered Functional Connectivity of Striatal Subregions in Patients with Multiple Sclerosis.

Abnormal corticostriatal resting-state functional connectivity (rsFC) has been implicated in the neuropathology of multiple sclerosis. The striatum, a component of the basal ganglia, is involved in diverse functions including movement, cognition, emotion, and limbic information processing. However, the brain circuits of the striatal subregions contributing to the changes in rsFC in relapsing–remitting multiple sclerosis (RRMS) patients remain unknown. We used six subdivisions of the striatum in each hemisphere as seeds to investigate the rsFC of striatal subregions between RRMS patients and matched healthy controls (HCs). In addition, we also scanned a subcohort of RRMS patients after an average of 7 months to test the reliability of our findings. Compared to HCs, we found significantly increased dorsal caudal putamen (DCP) connectivity with the premotor area, dorsal lateral prefrontal cortex (DLPFC), insula, precuneus, and superior parietal lobule, and significantly increased connectivity between the superior ventral striatum and posterior cingulate cortex (PCC) in RRMS patients following both scans. Furthermore, we found significant associations between the Expanded Disability Status Scale and the rsFC of the left DCP with the DLPFC and parietal areas in RRMS patients. Our results suggest that the DCP may be a critical striatal subregion in the pathophysiology of RRMS.

Chemical exchange saturation transfer of the cervical spinal cord at 7 T.

High-magnetic-field (7 T) chemical exchange saturation transfer (CEST) MRI provides information on the tissue biochemical environment. Multiple sclerosis (MS) affects the entire central nervous system, including the spinal cord. Optimal CEST saturation parameters found via simulation were implemented for CEST MRI in 10 healthy controls and 10 patients with MS, and the results were examined using traditional asymmetry analysis and a Lorentzian fitting method. In addition, T1 - and T2 *-weighted images were acquired for lesion localization and the transmitted B1 (+) field was evaluated to guide imaging parameters. Distinct spectral features for all tissue types studied were found both up- and downfield from the water resonance. The z spectra in healthy subjects had the expected z spectral shape with CEST effects apparent from 2.0 to 4.5 ppm. The z spectra from patients with MS demonstrated deviations from this expected normal shape, indicating this method's sensitivity to known pathology as well as to tissues appearing normal on conventional MRI. Examination of the calculated CESTasym revealed increased asymmetry around the amide proton resonance (Δω = 3.5 ppm), but it was apparent that this measure is complicated by detail in the CEST spectrum upfield from water, which is expected to result from the nuclear Overhauser effect. The z spectra upfield (negative ppm range) were also distinct between healthy and diseased tissue, and could not be ignored, particularly when considering the conventional asymmetry analysis used to quantify the CEST effect. For all frequencies greater than +1 ppm, the Lorentzian differences (and z spectra) for lesions and normal-appearing white matter were distinct from those for healthy white matter. The increased frequency separation and signal-to-noise ratio, in concert with prolonged T1 at 7 T, resulted in signal enhancements necessary to detect subtle tissue changes not possible at lower field strengths. This study presents CEST imaging metrics that may be sensitive to the extensive and temporally varying biochemical neuropathology of MS in the spinal cord. Copyright © 2016 John Wiley & Sons, Ltd.

Preclinical Models of Multiple Sclerosis: Advantages and Limitations Towards Better Therapies.

Multiple sclerosis (MS) is a disease of the central nervous system (CNS) with an unknown etiology. MS complex pathophysiology-characterized by CNS inflammation, demyelination and axonal injury-has made its modeling in experimental systems particularly problematic. Moreover, the evidence that MS does not naturally occur in other species has further complicated MS preclinical studies. Through the years, several MS in vivo models have been developed. Experimental autoimmune encephalomyelitis (EAE) represents the most widely used MS experimental model and relies upon the autoimmune paradigm to explore MS neuropathology. Although EAE has been instrumental in understanding the molecular events which take place upon neuroinflammation, not all MS hallmarks can be efficiently shaped within this conceptual frameshift. Thus, alternative models of CNS demyelination have been characterized, either based on viral infection or neurotoxin administration. However imperfect, these models have greatly improved our knowledge of the immune system&#039;s function in health and disease. On the other side, their intrinsic distance from MS has often led to misinterpreting and overestimating the data gleaned from these experimental systems. In this review, each model will be discussed in the light of its potentiality to mimic MS and translate the most promising therapies to patients. In addition, we will address how new genomic technologies can help improve the existing models.

Interactions between microglia and T cells in multiple sclerosis pathobiology.

Brain-resident microglia and T lymphocytes recruited into the central nervous system both play important roles in the neuropathology of multiple sclerosis. The microglia and recruited T cells are in close proximity in lesions of multiple sclerosis and in animal models, suggesting their potential for interactions. In support, microglia and T cells express a number of molecules that permit their engagement. Here we describe the interactions between T cells and microglia and the myriad responses that can result. These interactions include antigen presentation by microglia to activate T cells, the T cell activation of microglia, their progressive stimulation of one another, and the production of injurious or neurotrophic outcomes in their vicinity. Important considerations for the future include the nature of the T helper cell subsets and the M1 and M2 polarized nature of microglia, as the interactions between different subsets likely result in particular functions and outcomes. That T cells and microglia are in proximity and that they interact in lesions in the central nervous system implicate them as modifiers of pathobiology in multiple sclerosis.

Participation of interleukin 17A in neuroimmune interactions

Inflammation involving the helper T cell 17 (Th17) subset of lymphocytes has been implicated in a number of diseases that affect the nervous system. As the canonical cytokine of Th17 cells, interleukin 17A (IL-17A) is thought to contribute to these neuroimmune interactions. The main receptor for IL-17A is expressed in many neural tissues. IL-17A has direct effects on neurons but can also impact neural function via signaling to satellite cells and immune cells. In the central nervous system, IL-17A has been associated with neuropathology in multiple sclerosis, epilepsy syndromes and ischemic brain injury. Effects of IL-17A at the level of dorsal root ganglia and the spinal cord may contribute to enhanced nociception during neuropathic and inflammatory pain. Finally, IL-17A plays a role in sympathetic axon growth and regeneration of damaged axons that innervate the cornea. Given the widespread effects of IL-17A on neural tissues, it will be important to determine whether selectively mitigating the damaging effects of this cytokine while augmenting its beneficial effects is a possible strategy to treat inflammatory damage to the nervous system.

Cognitive linguistic deficits in relapsing–remitting multiple sclerosis

Background: Recent years have witnessed increasing reports of language dysfunction associated with the neuropathology of multiple sclerosis (MS). Although linguistic compromise is not traditionally thought to be a significant clinical manifestation of MS, a number of published case and group reports have uncovered the presence of higher-level language and isolated general language deficits in samples of patients with both chronic progressive and relapsing–remitting (RR) subtypes of the disease. To the present day however, the precise nature and extent of a language compromise in MS remains largely controversial and unclear.Aims: The present study aims to profile the cognitive linguistic abilities of a cohort of fifteen RR-subtype MS patients against an age- and education-matched group of neurologically normal control participants.Methods & Procedures: MS participants were assessed using a comprehensive battery of cognitive linguistic assessments targeting general and higher-level language behaviours.Outcomes & Results: The results revealed reduced performance on higher-level language subtests including: listening comprehension (making inferences), oral expression (recreating sentences), semantic absurdities and definitions. For the general language behaviours, a reduced performance was found for spontaneously elicited speech, repetition and naming.Conclusions: The findings are suggestive of both expressive language and higher-level language dysfunction in RR subtype MS and highlight deficits in linguistic organisation, retrieval mechanisms and semantic manipulation and processing.

Xanthine Oxidase Mediates Axonal and Myelin Loss in a Murine Model of Multiple Sclerosis

ObjectivesOxidative stress plays an important role in the pathogenesis of multiple sclerosis (MS). Though reactive oxygen species (ROS) are produced by various mechanisms, xanthine oxidase (XO) is a major enzyme generating ROS in the context of inflammation. The objectives of this study were to investigate the involvement of XO in the pathogenesis of MS and to develop a potent new therapy for MS based on the inhibition of ROS.MethodsXO were assessed in a model of MS: experimental autoimmune encephalomyelitis (EAE). The contribution of XO-generated ROS to the pathogenesis of EAE was assessed by treating EAE mice with a novel XO inhibitor, febuxostat. The efficacy of febuxostat was also examined in in vitro studies.ResultsWe showed for the first time that the expression and the activity of XO were increased dramatically within the central nervous system of EAE mice as compared to naïve mice. Furthermore, prophylactic administration of febuxostat, a XO inhibitor, markedly reduced the clinical signs of EAE. Both in vivo and in vitro studies showed infiltrating macrophages and microglia as the major sources of excess XO production, and febuxostat significantly suppressed ROS generation from these cells. Inflammatory cellular infiltration and glial activation in the spinal cord of EAE mice were inhibited by the treatment with febuxostat. Importantly, therapeutic efficacy was observed not only in mice with relapsing-remitting EAE but also in mice with secondary progressive EAE by preventing axonal loss and demyelination.ConclusionThese results highlight the implication of XO in EAE pathogenesis and suggest XO as a target for MS treatment and febuxostat as a promising therapeutic option for MS neuropathology.

A SELDI mass spectrometry study of experimental autoimmune encephalomyelitis: sample preparation, reproducibility, and differential protein expression patterns

BackgroundExperimental autoimmune encephalomyelitis (EAE) is an autoimmune, inflammatory disease of the central nervous system that is widely used as a model of multiple sclerosis (MS). Mitochondrial dysfunction appears to play a role in the development of neuropathology in MS and may also play a role in disease pathology in EAE. Here, surface enhanced laser desorption ionization mass spectrometry (SELDI-MS) has been employed to obtain protein expression profiles from mitochondrially enriched fractions derived from EAE and control mouse brain. To gain insight into experimental variation, the reproducibility of sub-cellular fractionation, anion exchange fractionation as well as spot-to-spot and chip-to-chip variation using pooled samples from brain tissue was examined.ResultsVariability of SELDI mass spectral peak intensities indicates a coefficient of variation (CV) of 15.6% and 17.6% between spots on a given chip and between different chips, respectively. Thinly slicing tissue prior to homogenization with a rotor homogenizer showed better reproducibility (CV = 17.0%) than homogenization of blocks of brain tissue with a Teflon® pestle (CV = 27.0%). Fractionation of proteins with anion exchange beads prior to SELDI-MS analysis gave overall CV values from 16.1% to 18.6%. SELDI mass spectra of mitochondrial fractions obtained from brain tissue from EAE mice and controls displayed 39 differentially expressed proteins (p≤ 0.05) out of a total of 241 protein peaks observed in anion exchange fractions. Hierarchical clustering analysis showed that protein fractions from EAE animals with severe disability clearly segregated from controls. Several components of electron transport chain complexes (cytochrome c oxidase subunit 6b1, subunit 6C, and subunit 4; NADH dehydrogenase flavoprotein 3, alpha subcomplex subunit 2, Fe-S protein 4, and Fe-S protein 6; and ATP synthase subunit e) were identified as possible differentially expressed proteins. Myelin Basic Protein isoform 8 (MBP8) (14.2 kDa) levels were lower in EAE samples with advanced disease relative to controls, while an MBP fragment (12. 4kDa), likely due to calpain digestion, was increased in EAE relative to controls. The appearance of MBP in mitochondrially enriched fractions is due to tissue freezing and storage, as MBP was not found associated with mitochondria obtained from fresh tissue.ConclusionsSELDI mass spectrometry can be employed to explore the proteome of a complex tissue (brain) and obtain protein profiles of differentially expressed proteins from protein fractions. Appropriate homogenization protocols and protein fractionation using anion exchange beads can be employed to reduce sample complexity without introducing significant additional variation into the SELDI mass spectra beyond that inherent in the SELDI- MS method itself. SELDI-MS coupled with principal component analysis and hierarchical cluster analysis provides protein patterns that can clearly distinguish the disease state from controls. However, identification of individual differentially expressed proteins requires a separate purification of the proteins of interest by polyacrylamide electrophoresis prior to trypsin digestion and peptide mass fingerprint analysis, and unambiguous identification of differentially expressed proteins can be difficult if protein bands consist of several proteins with similar molecular weights.

Low antioxidant status of serum uric acid, bilirubin and albumin in patients with neuromyelitis optica

Oxidative stress plays a central role in neuropathology of multiple sclerosis (MS). The patients with MS have low antioxidant status. Antioxidant therapy may represent an attractive treatment of MS. However, the relationship between neuromyelitis optica (NMO), a distinct nosologic entity or a form of MS, and antioxidant status has not fully been investigated. To investigate the correlation between NMO and serum antioxidant status of uric acid (UA), bilirubin and albumin. The serum levels of antioxidant molecules UA, bilirubin (Tbil and Ibil) and albumin were measured in 236 individuals comprising healthy subjects and patients with NMO or MS. We found that serum levels of UA, Tbil, Ibil and albumin in patients with NMO were significantly lower than those in healthy control group. Moreover, these results were also observed when the male and female cohorts were investigated separately. Likewise, the serum levels of UA, Tbil, Ibil and albumin in patients with NMO were also lower than those of patients with MS. In all groups, women had lower serum UA, Tbil, Ibil and albumin levels than men. In UA groups, women had significantly lower serum UA levels than men. We concluded that there were reducing serum levels of UA, bilirubin and albumin in patients with NMO. The study showed patients with NMO had low antioxidant status. As a replacement therapy to patients, administration of albumin, bilirubin and UA or theirs precursors, such as inosine of UA precursor, may be beneficial to the patients with NMO.