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Cortical demyelinated lesions are frequent and widespread in chronic multiple sclerosis (MS) patients, and may contribute to disease progression. Inflammation and related oxidative stress have been proposed as central mediators of cortical damage, yet meningeal and cortical inflammation is not specific to MS, but also occurs in other diseases. The first aim of this study was to test whether cortical demyelination was specific for demyelinating CNS diseases compared to other CNS disorders with prominent meningeal and cortical inflammation. The second aim was to assess whether oxidative tissue damage was associated with the extent of neuroaxonal damage. We studied a large cohort of patients diagnosed with demyelinating CNS diseases and non-demyelinating diseases of autoimmune, infectious, neoplastic or metabolic origin affecting the meninges and the cortex. Included were patients with MS, acute disseminated encephalomyelitis (ADEM), neuromyelitis optica (NMO), viral and bacterial meningoencephalitis, progressive multifocal leukoencephalopathy (PML), subacute sclerosing panencephalitis (SSPE), carcinomatous and lymphomatous meningitis and metabolic disorders such as extrapontine myelinolysis, thus encompassing a wide range of adaptive and innate cytokine signatures. Using myelin protein immunohistochemistry, we found cortical demyelination in MS, ADEM, PML and extrapontine myelinolysis, whereby each condition showed a disease-specific histopathological pattern. Remarkably, extensive ribbon-like subpial demyelination was only observed in MS, thus providing an important pathogenetic and diagnostic cue. Cortical oxidative injury was detected in both demyelinating and non-demyelinating CNS disorders. Our data demonstrate that meningeal and cortical inflammation alone accompanied by oxidative stress are not sufficient to generate the extensive subpial cortical demyelination found in MS, but require other MS-specific factors., (© 2020 The Authors. Brain Pathology published by John Wiley & Sons Ltd on behalf of International Society of Neuropathology.)

X-linked adrenoleukodystrophy (X-ALD) and metachromatic leukodystrophy (MLD) are two relatively common examples of hereditary demyelinating diseases caused by a dysfunction of peroxisomal or lysosomal lipid degradation. In both conditions, accumulation of nondegraded lipids leads to the destruction of cerebral white matter. Because of their high lipid content, oligodendrocytes are considered key to the pathophysiology of these leukodystrophies. However, the response to allogeneic stem cell transplantation points to the relevance of cells related to the hematopoietic lineage. In the present study, we aimed to better characterize the pathogenetic role of microglia in the above-mentioned diseases. Applying recently established microglia markers to human autopsy cases of X-ALD and MLD we were able to delineate distinct lesion stages in evolving demyelinating lesions. The immune-phenotype of microglia was altered already early in lesion evolution, and microglia loss preceded full-blown myelin degeneration both in X-ALD and MLD. DNA fragmentation indicating phagocyte death was observed in areas showing microglia loss. The morphology and dynamics of phagocyte decay differed between the diseases and between lesion stages, hinting at distinct pathways of programmed cell death. In summary, the present study shows an early and severe damage to microglia in the pathogenesis of X-ALD and MLD. This hints at a central pathophysiologic role of these cells in the diseases and provides evidence for an ongoing transfer of toxic substrates primarily enriched in myelinating cells to microglia., (© 2019 The Authors. Glia published by Wiley Periodicals, Inc.)

In patients with Charcot-Marie-Tooth disease 1A (CMT1A), peripheral nerves display aberrant myelination during postnatal development, followed by slowly progressive demyelination and axonal loss during adult life. Here, we show that myelinating Schwann cells in a rat model of CMT1A exhibit a developmental defect that includes reduced transcription of genes required for myelin lipid biosynthesis. Consequently, lipid incorporation into myelin is reduced, leading to an overall distorted stoichiometry of myelin proteins and lipids with ultrastructural changes of the myelin sheath. Substitution of phosphatidylcholine and phosphatidylethanolamine in the diet is sufficient to overcome the myelination deficit of affected Schwann cells in vivo. This treatment rescues the number of myelinated axons in the peripheral nerves of the CMT rats and leads to a marked amelioration of neuropathic symptoms. We propose that lipid supplementation is an easily translatable potential therapeutic approach in CMT1A and possibly other dysmyelinating neuropathies.

Remyelination is in the center of new therapies for the treatment of multiple sclerosis to resolve and improve disease symptoms and protect axons from further damage. Although remyelination is considered beneficial in the long term, it is not known, whether this is also the case early in lesion formation. Additionally, the precise timing of acute axonal damage and remyelination has not been assessed so far. To shed light onto the interrelation between axons and the myelin sheath during de- and remyelination, we employed cuprizone- and focal lysolecithin-induced demyelination and performed time course experiments assessing the evolution of early and late stage remyelination and axonal damage. We observed damaged axons with signs of remyelination after cuprizone diet cessation and lysolecithin injection. Similar observations were made in early multiple sclerosis lesions. To assess the correlation of remyelination and axonal damage in multiple sclerosis lesions, we took advantage of a cohort of patients with early and late stage remyelinated lesions and assessed the number of APP- and SMI32- positive damaged axons and the density of SMI31-positive and silver impregnated preserved axons. Early de- and remyelinating lesions did not differ with respect to axonal density and axonal damage, but we observed a lower axonal density in late stage demyelinated multiple sclerosis lesions than in remyelinated multiple sclerosis lesions. Our findings suggest that remyelination may not only be protective over a long period of time, but may play an important role in the immediate axonal recuperation after a demyelinating insult., (© 2017 The Authors GLIA Published by Wiley Periodicals, Inc.)

Oligodendrocyte damage is a central event in the pathogenesis of the common neuroinflammatory condition, multiple sclerosis (MS). Where and how oligodendrocyte damage is initiated in MS is not completely understood. Here, we use a combination of light and electron microscopy techniques to provide a dynamic and highly resolved view of oligodendrocyte damage in neuroinflammatory lesions. We show that both in MS and in its animal model structural damage is initiated at the myelin sheaths and only later spreads to the oligodendrocyte cell body. Early myelin damage itself is characterized by the formation of local myelin out-foldings-'myelinosomes'-, which are surrounded by phagocyte processes and promoted in their formation by anti-myelin antibodies and complement. The presence of myelinosomes in actively demyelinating MS lesions suggests that oligodendrocyte damage follows a similar pattern in the human disease, where targeting demyelination by therapeutic interventions remains a major open challenge.

Objective: To assess, in a surgical biopsy cohort of active demyelinating lesions, the diagnostic utility of aquaporin-4 (AQP4) immunohistochemistry in identifying neuromyelitis optica (NMO) or NMO spectrum disorder (NMOSD) and describe pathologic features that should prompt AQP4 immunohistochemical analysis and AQP4-immunoglobulin G (IgG) serologic testing., Methods: This was a neuropathologic cohort study of 20 surgical biopsies (19 patients; 11 cord/9 brain), performed because of diagnostic uncertainty, interpreted as active demyelinating disease and containing 2 or more of the following additional features: tissue vacuolation, granulocytic infiltrates, or astrocyte injury., Results: AQP4 immunoreactivity was lost in 18 biopsies and increased in 2. Immunopathologic features of the AQP4 loss cohort were myelin vacuolation (18), dystrophic astrocytes and granulocytes (17), vascular hyalinization (16), macrophages containing glial fibrillary acid protein (GFAP)-positive debris (14), and Creutzfeldt-Peters cells (0). All 14 cases with available serum tested positive for AQP4-IgG after biopsy. Diagnosis at last follow-up was NMO/NMOSD (15) and longitudinally extensive transverse myelitis (1 each relapsing and single). Immunopathologic features of the AQP4 increased cohort were macrophages containing GFAP-positive debris and granulocytes (2), myelin vacuolation (1), dystrophic astrocytes (1), Creutzfeldt-Peters cells (1), and vascular hyalinization (1). Diagnosis at last follow-up was multiple sclerosis (MS) and both tested AQP4-IgG seronegative after biopsy., Conclusions: AQP4 immunohistochemistry with subsequent AQP4-IgG testing has diagnostic utility in identifying cases of NMO/NMOSD. This study highlights the importance of considering NMOSD in the differential diagnosis of tumefactive brain or spinal cord lesions. AQP4-IgG testing may avert biopsy and avoid ineffective therapies if these patients are erroneously treated for MS., (© 2014 American Academy of Neurology.)

Multiple sclerosis (MS) is the most frequent demyelinating disease of the central nervous system (CNS) that affects worldwide about 2.5 million people. The morphological correlates of the disease are multiple lesions in brain and spinal cord which are characterized by demyelination, inflammation, gliosis and axonal damage. The underlying cause for the permanent neurological deficits in MS patients is axonal loss. Demyelinated axons are prone to damage due to the lack of trophic support by myelin sheaths and oligodendrocytes, as well as the increased vulnerability to immune mediated attacks. Remyelination occurs, but especially in chronic lesions is frequently limited to a small rim at the lesion border. Current treatment strategies are based on anti-inflammatory or immunomodulatory drugs and have the potential to reduce the numbers of newly evolving lesions, although as yet no treatment strategy exists to influence or prevent the progressive disease phase. Therefore, the development of neuroprotective treatment options, such as the promotion of endogenous remyelination is an attractive strategy. A prerequisite for the development of such new treatments is the understanding of the mechanisms leading to remyelination and the reasons for insufficient endogenous repair in chronic MS. This review will therefore provide an overview of the current concepts regarding remyelination in the rodent and human CNS. We will also summarize a selected number of inhibitory pathways and non-disease related factors which may contribute to remyelination failure in chronic MS.

Objective: To investigate the impact of single or repeated episodes of reversible demyelination on long-term locomotor performance and neuroaxonal integrity, and to analyze the myelin proteome after remyelination and during aging., Methods: Long-term locomotor performance of previously cuprizone-treated animals was monitored using the motor skill sequence (MOSS). Quantitative analysis of myelin proteome and histopathological analysis of neuronal/axonal integrity was performed after successful remyelination. Histopathological findings observed in experimental chronic remyelinated lesions were verified in chronic remyelinated lesions from multiple sclerosis (MS) patients., Results: Following cessation of cuprizone treatment, animals showed an initial recovery of locomotor performance. However, long after remyelination was completed (approximately 6 months after the last demyelinating episode), locomotor performance again declined in remyelinated animals as compared to age-matched controls. This functional decline was accompanied by brain atrophy and callosal axonal loss. Furthermore, the number of acutely damaged amyloid precursor protein-positive (APP+) axons was still significantly elevated in long-term remyelinated animals as compared to age-matched controls. Confocal analysis revealed that a substantial proportion of these APP+ spheroids were ensheathed by myelin, a finding that was confirmed in the chronic remyelinated lesions of MS patients. Moreover, quantitative analysis of myelin proteome revealed that remyelinated myelin displays alterations in composition that are in some aspects similar to the myelin of older animals., Interpretation: We propose that even after completed remyelination, axonal degeneration continues to progress at a low level, accumulating over time, and that once a threshold is passed axonal degeneration can become functionally apparent in the long-term. The presented model thus mimics some of the aspects of axonal degeneration in chronic progressive MS., (Copyright © 2011 American Neurological Association.)

The IκB kinase complex induces nuclear factor kappa B activation and has recently been recognized as a key player of autoimmunity in the central nervous system. Notably, IκB kinase/nuclear factor kappa B signalling regulates peripheral myelin formation by Schwann cells, however, its role in myelin formation in the central nervous system during health and disease is largely unknown. Surprisingly, we found that brain-specific IκB kinase 2 expression is dispensable for proper myelin assembly and repair in the central nervous system, but instead plays a fundamental role for the loss of myelin in the cuprizone model. During toxic demyelination, inhibition of nuclear factor kappa B activation by conditional ablation of IκB kinase 2 resulted in strong preservation of central nervous system myelin, reduced expression of proinflammatory mediators and a significantly attenuated glial response. Importantly, IκB kinase 2 depletion in astrocytes, but not in oligodendrocytes, was sufficient to protect mice from myelin loss. Our results reveal a crucial role of glial cell-specific IκB kinase 2/nuclear factor kappa B signalling for oligodendrocyte damage during toxic demyelination. Thus, therapies targeting IκB kinase 2 function in non-neuronal cells may represent a promising strategy for the treatment of distinct demyelinating central nervous system diseases.

Oligodendrocytes are the myelin-forming cells of the CNS and guarantee proper nerve conduction. Sphingosine, one major component of myelin, has recently been identified to activate TRPM3, a member of the melastatin-related subfamily of transient receptor potential (TRP) channels. TRPM3 has been demonstrated to be expressed in brain with unknown cellular distribution. Here, we show for the first time that TRPM3 is expressed in oligodendrocytes in vitro and in vivo. TRPM3 is present during oligodendrocyte differentiation. Immunohistochemistry of adult rat brain slices revealed staining of white matter areas, which co-localized with oligodendrocyte markers. Analysis of the developmental distribution revealed that, prior to myelination, TRPM3 channels are localized on neurons. On oligodendrocytes they are found after the onset of myelination. RT-PCR studies showed that the transcription of TRPM3 splice variants is also developmentally regulated in vitro. Ca(2+) imaging approaches revealed the presence of a sphingosine-induced Ca(2+) entry mechanism in oligodendrocytes - with a pharmacological profile similar to the profile published for heterologously expressed TRPM3. These findings indicate that TRPM3 participates as a Ca(2+)-permeable and sphingosine-activated channel in oligodendrocyte differentiation and CNS myelination.

Objective: Cortical myelin can be severely affected in patients with demyelinating disorders of the central nervous system. However, the functional implication of cortical demyelination remains elusive. In this study, we investigated whether cortical myelin influences cortical spreading depression (CSD)., Methods: CSD measurements were performed in rodent models of toxic and autoimmune induced cortical demyelination, in neuregulin-1 type I transgenic mice displaying cortical hypermyelination, and in glial fibrillary acidic protein-transgenic mice exhibiting pronounced astrogliosis., Results: Cortical demyelination, but not astrogliosis or inflammation per se, was associated with accelerated CSD. In contrast, hypermyelinated neuregulin-1 type I transgenic mice displayed a decelerated CSD propagation., Interpretation: Cortical myelin may be crucially involved in the stabilization and buffering of extracellular ion content that is decisive for CSD propagation velocity and cortical excitability, respectively. Our data thus indicate that cortical involvement in human demyelinating diseases may lead to relevant alterations of cortical function.

The type I interferons, interferon-beta and alpha (IFN-beta, IFN-alpha), are widely used for the treatment of autoimmune demyelination in the central nervous system (CNS). Their effects on de- and remyelination through the broadly expressed type I IFN receptor (IFNAR), however, are highly speculative. In order to elucidate the role of endogenous type I interferons for myelin damage and recovery we induced toxic demyelination in the absence of IFNAR1. We demonstrate that IFNAR signalling was induced during acute demyelination since the cytokine IFN-beta as well as the IFN-dependent genes IRF7, ISG15 and UBP43 were strongly upregulated. Myelin damage, astrocytic and microglia response, however, were not significantly reduced in the absence of IFNAR1. Furthermore, motor skills of IFNAR1-deficient animals during non-immune demyelination were unaltered. Finally, myelin recovery was found to be independent from endogenous IFNAR signalling, indicating a redundant role of this receptor for non-inflammatory myelin damage and repair.

Statins are widely prescribed drugs in cardiovascular diseases. Recent studies also demonstrated anti-inflammatory and immunomodulatory properties of statins by modulating the activity of small GTPases. Statins are thus considered as potential therapeutic drug for the inflammatory demyelinating disease multiple sclerosis (MS). However, little is known about the effects of statins on myelin-forming oligodendrocytes. Here, we show that statins hamper process and myelin formation in vitro by interfering with Ras and Rho signaling in mature oligodendrocytes and provide evidence that statins impair ongoing remyelination in vivo. Our findings may have significant implications for the application of statins in MS patients and in other demyelinating diseases of the CNS.

Impaired function/differentiation of progenitor cells might provide an explanation for the limited remyelination observed in the majority of chronic multiple sclerosis lesions. Here, we establish that in the normal adult human CNS, the transcription factors Nkx2.2 and Olig2 are strongly expressed in progenitor cells while mature oligodendrocytes are characterized by low levels of Olig2 or Nkx2.2. In vitro studies confirmed the expression of Olig2 in oligodendroglial progenitor cells and mature oligodendrocytes while astrocytes, microglial cells and neurons were negative for Olig2. In early multiple sclerosis lesions, we found Olig2-positive progenitor cells throughout all lesion stages and in periplaque white matter (PPWM). The number of progenitors in PPWM was significantly increased compared with the white matter from controls. In chronic multiple sclerosis lesions progenitor cells were still present, however, in significantly lower numbers than in early multiple sclerosis lesions. A subpopulation of progenitor cells in early multiple sclerosis lesions and PPWM but not in control cases co-expressed NogoA, a marker of mature oligodendrocytes. The co-expression of these two markers suggested that these cells were maturing oligodendrocytes recently recruited from the progenitor pool. In contrast, in chronic multiple sclerosis lesions maturing progenitors were only rarely present. In summary, we provide evidence that a differentiation block of oligodendroglial progenitors is a major determinant of remyelination failure in chronic multiple sclerosis lesions.

Macrophages are key effectors in demyelinating diseases of the central and peripheral nervous system by phagocytosing myelin and releasing immunoregulatory mediators. Here, we report on a distinct, a priori anti-inflammatory reaction of macrophages phagocytosing myelin upon contact with damaged nerve tissue. Macrophages rapidly invaded peripheral (sciatic) and central (optic) nerve tissues in vitro, readily incorporated myelin and expressed high levels of phagocytosis-associated molecules (e.g., Fc and scavenger receptors). In contrast, factors involved in antigen presentation (MHC class-II, CD80, CD86) revealed only a restricted expression. In parallel, a highly ordered appearance of cytokines and chemokines was detected. IL-10, IL-6, CCL22, and CXCL1 were immediately but transiently induced, whereas CCL2, CCL11, and TGFbeta revealed more persisting levels. Such a profile would attract neutrophils, monocytes/macrophages, and Th2 cells as well as bias for a Th2-supporting environment. Importantly, proinflammatory/Th1-supporting factors, such as TNFalpha, IL-12p70, CCL3, and CCL5, were not induced. Still the simultaneous presence of TGFbeta and IL-6 could assist Th17 development, further depending on yet not present IL-23. The release pattern was clearly distinct from reactive phenotypes induced in isolated macrophages and microglia upon treatment with IL-4, IL-13, bacterial lipopolysaccharide, IFNgamma, or purified myelin. Nerve-exposed macrophages thus commit to a unique functional orientation.

Nitric oxide (NO) and peroxynitrite (ONOO(-)) are potential mediators of the injury and cytotoxicity occurring over time to oligodendrocytes in multiple sclerosis (MS) lesions. Our in vitro results indicate that human adult CNS-derived oligodendrocytes are relatively resistant to NO-mediated damage. In contrast, human oligodendrocytes are highly susceptible to peroxynitrite-mediated injury. In situ, we found that inducible nitric oxide synthase (iNOS) was expressed in astrocytes and macrophages in all active demyelinating and remyelinating MS lesions examined, yet no correlation was found between numbers of glial cells expressing iNOS and the extent of oligodendrocyte cell death. Nitrotyrosine groups, indicative of the presence of peroxynitrite in vivo, could be detected on astrocytes, macrophages, and oligodendrocytes in MS lesions. High numbers of nitrotyrosine-positive oligodendrocytes were found in one MS case that featured extensive oligodendrocyte cell death. Our results indicate that NO alone is unlikely to induce oligodendrocyte injury, whereas its more potent byproduct peroxynitrite is a potential mediator of injury to oligodendrocytes in MS., ((c) 2007 Wiley-Liss, Inc.)

Recent studies revealed prominent cortical demyelination in patients with chronic multiple sclerosis (MS). Demyelination in white matter lesions is frequently accompanied by remyelination. This repair process, however, often remains incomplete and restricted to the lesion border. In the present study, we examined the frequency and extent of remyelination in cortical and white matter lesions in autopsy brain tissue of 33 patients with chronic MS. The majority of patients (29 of 33) harbored cortical demyelination. Remyelination of cortical lesions was identified light microscopically by the presence of thin and irregularly arranged myelin sheaths, and confirmed by electron microscopy. Extensive remyelination was found in 18%, remyelination restricted to the lesion border in 54%, and no remyelination in 28% of cortical lesions. A direct comparison of the extent of remyelination in white matter and cortical lesions of the same patients revealed that remyelination of cortical lesions was consistently more extensive. In addition, g-ratios of fibers in areas of "normal appearing cortex" yielded values consistent with remyelination. Our data confirm the high prevalence of cortical demyelination in chronic MS and imply that the propensity to remyelinate is high in cortical MS lesions.

Neocortical demyelination in the forebrain has recently been identified as an important pathological feature of multiple sclerosis (MS). Here we describe that the cerebellar cortex is a major predilection site for demyelination, in particular in patients with primary and secondary progressive MS. In these patients, on average, 38.7% of cerebellar cortical area is affected, reaching in extreme examples up to 92%. Cerebellar cortical demyelination occurs mainly in a band-like manner, affecting multiple folia. The lesions are characterized by primary demyelination with relative axonal and neuronal preservation, although some axonal spheroids and a moderate reduction of Purkinje cells are present. Although cortical demyelination sometimes occurs together with demyelination in the adjacent white matter (leukocortical lesions), in most instances, the cortex was affected independently from white matter lesions. We found no correlation between demyelination in the cortex and the white matter, and in some cases, extensive cortical demyelination was present in the near absence of white matter lesions. Our data identify cortical demyelination as a potential substrate of cerebellar dysfunction in MS.

Although spontaneous remyelination does occur in multiple sclerosis lesions, its extent within the global population with this disease is presently unknown. We have systematically analysed the incidence and distribution of completely remyelinated lesions (so-called shadow plaques) or partially remyelinated lesions (shadow plaque areas) in 51 autopsies of patients with different clinical courses and disease durations. The extent of remyelination was variable between cases. In 20% of the patients, the extent of remyelination was extensive with 60-96% of the global lesion area remyelinated. Extensive remyelination was found not only in patients with relapsing multiple sclerosis, but also in a subset of patients with progressive disease. Older age at death and longer disease duration were associated with significantly more remyelinated lesions or lesion areas. No correlation was found between the extent of remyelination and either gender or age at disease onset. These results suggest that the variable and patient-dependent extent of remyelination must be considered in the design of future clinical trials aimed at promoting CNS repair.

Recent studies have revealed widespread demyelination in the cortex of patients with chronic multiple sclerosis. In contrast to white matter lesions, cortical multiple sclerosis lesions are accompanied by only minor inflammation. Research into the pathogenesis of cortical lesion formation has been hampered by the fact that the conventional rodent model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), does not regularly affect the cortex. To overcome this limitation we developed a new rat model of cortical multiple sclerosis. Lesions were stereotactically targeted to the cerebral cortex by injection of pro-inflammatory mediators in animals that were immunized subclinically with myelin oligodendrocyte glycoprotein (MOG). We thus generated highly reproducible demyelinated lesions in the neocortex with remarkable histological similarities to cortical multiple sclerosis lesions. The focal cortical EAE model led to the typical pattern of intracortical and subpial demyelination, infiltration with inflammatory cells, complement deposition, acute axonal damage and neuronal cell death. Surprisingly, extensive cortical inflammation largely resolved within 2 weeks. Furthermore, cortical demyelination was readily compensated by rapid remyelination. Our data thus suggest that cortical inflammation is a transient phenomenon, and that remyelination of cortical inflammatory-demyelinating lesions may occur rapidly.

Macrophages/microglia are the key effector cells in myelin removal. Differences exist in the amount and time course of myelin uptake in the central (CNS) and peripheral nervous system (PNS), the basis of this difference, however, is not yet clarified. In the present experiments we studied the phagocytosis rate of CNS or PNS myelin by macrophages and microglia in vitro. Additionally, the effects of intravenous immunoglobulins (IVIg) on this process were investigated. In the PNS experiments, sciatic nerves were cocultured with peritoneal macrophages. Optic nerve fragments were used to characterize the myelin-removing properties of microglia. Cocultures with peritoneal macrophages aimed at investigating the differences in phagocytosis between resident microglia and added macrophages. The myelin phagocytosis in sciatic nerve fragments was higher than in optic nerves, indicating differences in the myelin uptake rate between peripheral macrophages and microglia. IVIg increased the phagocytosis of PNS myelin by macrophages, but not by microglia in optic nerves. The addition of peritoneal macrophages to optic nerve fragments did not lead to an increase in the phagocytosis of CNS myelin either. The IVIg induced phagocytosis of PNS myelin by peripheral macrophages was associated with an increased expression of macrophage Fc receptors measured by FACS. Blocking of Fc receptors by anti-Fc receptor antibody reduced the IVIg induced PNS myelin phagocytosis to basic levels, indicating that the induced but not the basic myelin uptake by macrophages is Fc receptor dependent. In contrast to peripheral macrophages, IVIg did not increase Fc receptor density on microglia. These data indicate that phagocytosis of PNS and CNS myelin by macrophages or microglia is differentially regulated. Local factors within the CNS or PNS may affect this process by modulating the surface receptor profile and activation state of the phagocytic cell or the structure of the myelin sheath., (Copyright 2002 Wiley-Liss, Inc.)

We report on a case of dementia and extensive cerebral white matter abnormalities seen on magnetic resonance-images which meet the criteria for leukoencephalopathy with vanishing white matter. This is an inherited condition that was first thought to occur only in children. Our patient shows that vanishing white matter should be considered in adult patients with early-onset dementia and extensive white matter changes seen on magnetic resonance images.

Wallerian degeneration of a peripheral nerve is mainly characterized by axon and myelin degradation and is paralleled by a massive invasion of peripheral macrophages into the nerve. These cells enter the nerve attracted by a cascade of chemokines and cytokines but require proteolytic and enzymatic factors which enables them to cross the blood-nerve barrier. Here we investigated whether alpha-naphthyl (alpha-NA) esterases -- which have been shown to be exclusively expressed in human monocytes -- play a role during Wallerian degeneration. These enzymes were blocked by the specific inhibitor bis(4-nitrophenyl)-phosphate (BNPP) in an established in vitro model of Wallerian degeneration. Sciatic nerve segments of mice were co-cultured with peritoneal macrophages and BNPP was added to the cultures in various concentrations and at different timepoints. The macrophage numbers and myelin density in the nerve segments and the myelin load of macrophages were evaluated. After BNPP treatment the macrophage number within the nerve was significantly diminished and the myelin load within the macrophages was decreased, resulting in elevated levels of preserved myelin within the nerves. These experiments clearly showed a double effect of the alphaNA esterase inhibitor BNPP on macrophages. First, it suggests a role for alphaNA esterases on the migratory potential of macrophages since their invasion into the nerves was diminished. Second, the reduced myelin uptake is due to the inhibition of phagocytic capacity of these cells by BNPP. The therapeutical use of this inhibitor for treatment of autoimmune diseases such as multiple sclerosis or Guillain-Barré syndrome remains to be investigated.

Wallerian degeneration after peripheral nerve transection leads to the phagocytosis of degenerated myelin and axon components by macrophages. These phagocytes are recruited from the systemic circulation and Wallerian degeneration may therefore be used as a model for myelin removal by hematogenous macrophages, a feature that is also a hallmark of demyelinating diseases of the central and peripheral nervous system. The immunomodulator linomide has been shown to be effective in the treatment of experimental demyelinating diseases although the exact mode of its action is not yet defined. The present study investigated the effect of linomide on monocyte invasion and myelin phagocytosis after sciatic nerve transection. Linomide had a dual effect in Wallerian degeneration. Monocyte migration from the circulation to the damaged nervous system was significantly reduced. Additionally, the myelin phagocytic capacity of macrophages was impaired, finally resulting in a significant delay in the removal of myelin. The present experiments may provide an explanation for the effects of linomide during the course of demyelinating diseases of the nervous system.

In multiple sclerosis (MS), remyelination of demyelinated lesions diminishes with disease progression for unknown reasons. Oligodendrocyte progenitor cells contribute to remyelination; however, antibodies specific for oligodendrocyte progenitor antigens could block remyelination by eliminating or impeding these cells. In myelinating cultures, cell lysis with antibody recognizing a progenitor cell-specific surface glycoprotein (AN2) suppressed the synthesis of myelin proteins. Cerebrospinal fluid from patients with relapsing-remitting active MS contains antibodies against AN2, whereas cerebrospinal fluid from patients with nonactive disease does not. This is the first report describing antibodies in MS against a progenitor cell-specific antigen that may contribute to the development and progression of chronically demyelinated lesions.

Multiple sclerosis (MS) is a disease with profound heterogeneity in clinical course, neuroradiological appearance of the lesions, involvement of susceptibility gene loci, and response to therapy. These features are supported by experimental evidence, which demonstrates that fundamentally different processes, such as autoimmunity or virus infection, may induce MS-like inflammatory demyelinating plaques and suggest that MS may be a disease with heterogeneous pathogenetic mechanisms. From a large pathology sample of MS, collected in three international centers, we selected 51 biopsies and 32 autopsies that contained actively demyelinating lesions defined by stringent criteria. The pathology of the lesions was analyzed using a broad spectrum of immunological and neurobiological markers. Four fundamentally different patterns of demyelination were found, defined on the basis of myelin protein loss, the geography and extension of plaques, the patterns of oligodendrocyte destruction, and the immunopathological evidence of complement activation. Two patterns (I and II) showed close similarities to T-cell-mediated or T-cell plus antibody-mediated autoimmune encephalomyelitis, respectively. The other patterns (III and IV) were highly suggestive of a primary oligodendrocyte dystrophy, reminiscent of virus- or toxin-induced demyelination rather than autoimmunity. At a given time point of the disease--as reflected in autopsy cases--the patterns of demyelination were heterogeneous between patients, but were homogenous within multiple active lesions from the same patient. This pathogenetic heterogeneity of plaques from different MS patients may have fundamental implications for the diagnosis and therapy of this disease.

Macrophages play a crucial role in myelin removal during nerve degeneration and demyelination. The exact mechanisms of myelin recognition and uptake are not yet defined. The present experiments aimed at defining the role of intercellular adhesion molecule-1 (ICAM-1) in this process. Myelin phagocytosis was studied in an established in vitro model of cultured macrophages and sciatic nerves. Cocultures of wild-type C57BL macrophages with sciatic nerves resulted in a massive invasion of the nerves by macrophages with subsequent removal of myelin. In contrast, when macrophages of ICAM-1-deficient animals were cocultured with wild-type nerves, myelin phagocytosis was significantly retarded, whereas cell invasion was completely undisturbed. These data indicate that the ICAM-1 molecule acts as a costimulatory signal in myelin recognition and uptake by macrophages.

The present study investigated the effect of human immunoglobulins on migration and myelin phagocytosis by macrophages. Mouse sciatic nerves and macrophages were cocultured and treated with 1, 10 and 20 mg/ml immunoglobulins for 10 days in vitro. Numbers of invading macrophages, myelin density within the nerves and macrophage myelin load were determined in semithin sections. Human immunoglobulins lead to an increased myelin removal by macrophages as proven by a statistically significant higher myelin load of the macrophage cytoplasm when compared with untreated control macrophages. The results suggest that one possible action of immunoglobulins in demyelinating diseases is an improved clearance of lesional debris with the removal of myelin-associated inhibitory molecules.

The effects of pentoxifylline (POX) on macrophage migration and myelin uptake were studied in an in vitro model of myelin phagocytosis. The POX is a phosphodiesterase inhibitor which inhibits TNF-alpha (tumor necrosis factor alpha) production and reduces ICAM-1 (intercellular adhesion molecule-1) expression by macrophages. Both of these molecules have earlier been shown to be involved in the process of myelin recognition and degradation. In the present series of experiments, cocultured peripheral nerves and macrophages were treated with different concentrations of POX. Untreated controls were massively invaded by macrophages which ingested the degenerating myelin sheaths. High concentrations of POX (100 microg ml(-1)) inhibited macrophage invasion of the nerves. Lower POX concentrations (50 microg ml(-1)), in contrast, lead to an increased myelin uptake by phagocytic cells without affecting macrophage migration. These data indicate that POX may regulate different effector functions of macrophages such as migration and myelin phagocytosis during Wallerian degeneration. This is important for inflammatory demyelinating conditions in the central or peripheral nervous system (PNS) in which macrophages are also important effector cells. Since POX is used as an immunomodulatory drug in demyelinating diseases, its effects on the described macrophage functions may be of high relevance. An increased myelin uptake during Wallerian degeneration may also support a more efficient axonal regeneration by removing axonal outgrowth inhibitors.

Myelin phagocytosis during Wallerian degeneration and immune-mediated demyelination depends on the action of mononuclear cells of the monocyte/macrophage system. The present study investigated the role of the macrophage scavenger receptor, a trimeric membrane glycoprotein, in myelin uptake by macrophages. Two in vitro models of myelin phagocytosis were used: an organ culture model of mouse peripheral nerves exposed to cocultured macrophages and a quantitative flow cytometric assay. Different concentrations of the monoclonal rat anti-mouse scavenger receptor antibody (2F8) were applied to these systems to selectively block the macrophage scavenger receptor. Concentration-dependent effects on macrophage migration and myelin uptake were seen when the macrophage scavenger receptor was blocked by the antibody 2F8. Low concentrations reduced myelin phagocytosis by the invading macrophages; higher concentrations completely abolished macrophage invasion of the nerves. Using a quantitative flow cytometric assay it was also shown that the 2F8 antibody inhibits phagocytosis of myelin in a dose-dependent manner. These data indicate that the macrophage scavenger receptor is involved in myelin phagocytosis by macrophages.

Stimulation of remyelination in multiple sclerosis lesions may be one possible therapeutic approach. However, since the pathology and pathogenesis of multiple sclerosis may be heterogeneous, it is important to analyze in what patients and at what stages of the disease such a therapy can be reasonable and feasible. Spontaneous remyelination is pronounced in some MS patients. The extent of remyelination correlates well with the presence of oligodendrocytes in the lesions. Yet, the source and origin of the remyelinating cells is still unclear. These may be derived from either undifferentiated progenitor cells or, in part from mature oligodendrocytes, that have escaped destruction during lesional activity. Complete remyelination of plaques is possible, leading to the formation of 'shadow plaques'. No reliable data are available at present on the frequency of remyelination in different forms of MS. However, most studies agree that remyelination is especially prominent at the early stages of the disease, whereas it is sparse after several years of disease duration. In addition, very little remyelination is found in cases of primary progressive MS.

Newly recruited hematogenous mononuclear cells of the monocyte/macrophage system are suggested to be important effector cells in myelin removal during Wallerian degeneration. Their role has extensively been studied in various in vitro and in vivo models. However, there has been much controversy concerning the role of hematogenous vs. resident cells of the peripheral nervous system in Wallerian degeneration. The present study used a recently established technique to deplete the hematogenous monocyte population by application of dichloromethylene diphosphonate-containing liposomes. Intravenously injected liposomes containing dichloromethylene diphosphonate (Cl2MDP) are ingested by macrophages and monocytes and cause temporary and selective depletion of these cells. The number of LFA-1- and Mac-1- positive macrophages within the nerves was significantly reduced when liposomes were injected shortly after nerve transsection. In these nerves, myelin degradation was significantly less, indicating an essential role of newly recruited phagocytes in this process. Macrophage invasion of degenerating nerves occurred within the first 2 days after transsection. Resident cells of the peripheral nerve participate in myelin removal since macrophage depletion did not completely abolish myelin degradation. These results confirm the important role of hematogenous phagocytes in myelin removal during Wallerian degeneration.

The present study used cocultures of rat dorsal root ganglia (DRG) and peritoneal macrophages to define the role of activated complement components during demyelination. The complement cascade was activated in vitro by treatment of the cultures with natural rat serum and lipopolysaccharides. Complement activation was examined by detection of the membrane attack complex of complement (MAC) with an antibody directed against rat C5-9. Detection of MAC in vitro by immunoelectron microscopy was associated with morphological changes of the myelin sheath. The sheath's regular structure was disrupted. Myelin lamellae were split and showed signs of decompaction. These changes were followed by a selective macrophage attack on myelin sheaths resulting in demyelination. Schwann cell viability was not affected by complement activation. Axons and sensory ganglion cells also survived this attack. The specificity of the complement effect was tested in experiments using treatment regimens with natural rat serum or lipopolysaccharides alone. In these experiments, no morphological changes of the myelin sheath were observed as well as no macrophage attack on myelin.

Mononuclear cells of the monocyte/macrophage system play an important role in myelin ingestion during Wallerian degeneration. The present in vitro study clarifies the role in this process of two macrophage-secreted cytokines, TNF-alpha and interleukin-1. Treatment with TNF-alpha massively reduced the amount of myelin ingested by macrophages via their complement receptor type 3 (CR3). Anti-TNF-alpha antibodies reversed the effect. Immunofluorescence of macrophages indicated that TNF-alpha caused a reduced expression of the CR3 by phagocytic cells. Further experiments revealed an interaction of TNF-alpha with its receptor on the macrophage cell membrane. Interleukin-1 had no effect on myelin ingestion in the in vitro system used in these experiments.

Myelin removal in nerves undergoing wallerian degeneration mainly depends on invading, non-resident macrophages. The present study clarifies the role of serum complement components in this process in vitro and in vivo. Macrophages cocultured with degenerating nerves in vitro were unable to invade these nerves in the presence of C3-deficient serum. Application of C3-deficient serum subsequent to cellular invasion abolished the myelin phagocytic capacity of the invaded macrophages. This indicates that opsonization of myelin by complement components is necessary in myelin ingestion via macrophage receptors. In vivo, a monoclonal antibody to the macrophage complement receptor type 3 (CR3) significantly reduced myelin phagocytosis. Immunohistochemistry with anti-C3 antibodies showed a marked reaction in degenerating nerves. Immunoelectron microscopy localized C3 particles at the degenerating myelin sheaths. Haematogenous cells, invading the degenerating nerves, also showed a strong reaction for C3 in their cytoplasm. These results indicate that complement components play a critical role both in macrophage invasion of degenerating nerves and in the ingestion of myelin by these cells.

Myelin phagocytosis in nerves undergoing Wallerian degeneration has been shown to depend on their invasion by non-resident, hematogenous macrophages. This process can be studied in vitro using organ cultures of peripheral nerves exposed to cultured peritoneal macrophages. The present report concerns the role of cell surface carbohydrates in the invasion of degenerating nerves and in the recognition and ingestion of myelin by the phagocytic cells. Additional experiments explored the effect of pH, calcium and cytochalasin D on myelin phagocytosis. Organ cultures with peritoneal macrophages were treated with 14 simple or complex sugars or with eight sugar-splitting enzymes. Macrophage invasion was diminished by many simple or complex sugars, but exposure to sugars had no effect on the recognition or ingestion of myelin by the invading macrophages. Macrophage invasion was abolished upon treatment with beta-mannosidase. Exposure to L-fucosidase abolished the myelin phagocytic capacity of invading macrophages completely without affecting their capacity to ingest carbon or latex particles. The results indicate that the phagocytosis of myelin by macrophages is an L-fucosidase-sensitive process, probably by interaction with their complement receptor type C3.

Myelin phagocytosis in Wallerian degeneration of peripheral nerves depends on invasion of nerves by non-resident macrophages. The present study was done to clarify the role of the macrophage complement receptor type 3 (CR3) in myelin removal. Myelin phagocytic capacity of invading macrophages was abolished by treatment of cultured nerves and macrophages with anti-CR3 antibody or by serum complement depletion with cobra venom factor. This indicates that myelin phagocytosis is mediated by the macrophage CR3.

Myelin phagocytosis in nerves undergoing Wallerian degeneration was shown to depend on their invasion by non-resident, hematogenous macrophages. This process can be studied in vitro using organ cultures of peripheral nerves exposed to cultured peritoneal macrophages. The present report concerns the effect of recombinant interferon-gamma (rIFN-gamma) on luminol-dependent chemiluminescence, macrophage migration and myelin phagocytosis in organ cultures. Chemiluminescence was activated by rIFN-gamma compared to untreated cells. The macrophage population was capable of activation at any phase of exposure to organ cultures. The engagement of macrophages in myelin phagocytosis, however, varied with the timing of the application of rIFN-gamma. Application from the start of the experiment led to activation of chemiluminescence and also to a complete inhibition of macrophage invasion of the organ culture, thus preventing myelin removal. Application of rIFN-gamma at a later phase of the experiment had no effect on cell invasion and also no detectable effect on the efficiency of myelin phagocytosis. There was no indication that myelin phagocytosis by itself activated chemiluminescence in untreated cultures. Phagocytosis of myelin appears to be a function of macrophages independent of activation causing production of oxygen radicals.