Microglia subtypes in acute, subacute, and chronic multiple sclerosis.

The study was designed to examine microglia morphology in early and late forms of multiple sclerosis (MS). Archival paraffin embedded tissue samples from 25 cases were examined immunohistochemically. Pío del Río Hortega reported that phagocytes in acute focal destructive CNS lesions develop from microglia with no early contribution from infiltrating monocytes. In this study, we were unable to identify the changes cited by del Río Hortega in support of his theory. Instead, myelin phagocytes in MS appear to originate chiefly from infiltrating monocytes. In 4 cases, walls composed of MHC class II antigen-positive "wall microglia" were observed at plaque margins separating demyelinated and bordering myelinated tissue. Wall microglia in 2 plaques were accompanied by AQP4-positive fiber-forming astrocytes. In chronic but not early disease MS cases, microglia were seen to interact with infiltrating monocytes to form microglial nodules of several types. Also, MHC II-positive "activated" microglia in bordering intact tissue were exceptionally prominent where there was little evidence of ongoing myelin loss. It is concluded that myelin phagocytes in MS derive entirely from infiltrating MRP14-positive monocytes and not from resident microglia and that Río Hortega's microglia play an anti-inflammatory role in MS and not the destructive role favored by the current literature.

Multiple Sclerosis: Microglia, Monocytes, and Macrophage-Mediated Demyelination.

This study examined the roles of microglia and monocytes in myelin destruction in patients with early multiple sclerosis (MS). Twenty-two cases were studied; the clinical duration was <9 weeks in 10 cases. Twenty myeloid cell subtypes or categories were identified including 2 cell types not known previously to occur in demyelinating diseases. Commencing myelin breakdown in plaques and in perivascular and subpial tissues occurred in the immediate presence of infiltrating monocytes and was effected by a homogeneous population of IgG-positive Fc receptor-bearing early phagocytes interacting with abnormal myelin. Oligodendrocyte apoptosis was observed in intact myelinated tissue bordering areas of active demyelination. Capillaries in the cerebral cortex plugged by large numbers of monocytes were common in acute cases of MS and in a patient with a neuromyelitis optica variant and extreme systemic recruitment of monocytes. In an MS patient with progressive disease, microglial nodules centered on MHC-II-positive capillaries plugged by monocytes were present in the cerebral cortex. This constitutes a new gray matter lesion in MS.

Multiple Sclerosis: Destruction and Regeneration of Astrocytes in Acute Lesions.

There are reports that astrocyte perivascular endfeet are damaged in some cases of multiple sclerosis (MS). This study was designed to determine the origin and outcome of astrocyte damage in acute, resolving, and inactive plaques. Ten acute plaques from 10 early MS cases and 14 plaques of differing histological age from 9 subacute and chronic cases were examined immunohistochemically. Also examined were nonnecrotic early lesions in 3 patients with neuromyelitis optica (NMO). Plaques from 3 MS cases were examined electron microscopically. The edge zones in each of the 10 acute MS lesions revealed a complete loss of astrocyte cell bodies and their pericapillary, perineuronal, and perivascular foot processes. Dendrophagocytosis of degenerate astrocytes was observed. Astrocyte precursors, similar to those that replace destroyed astrocytes in nonnecrotic NMO lesions, were present in areas depleted of astrocytes. Resolving plaques were repopulated initially by stellate astrocytes that stained negatively for the water channel molecule aquaporin4 (AQP4). In older lesions, astrocytes were predominantly AQP4-positive. Loss and recovery of astrocytes in new MS lesions may be as important as myelin loss as a cause of conduction block responsible for symptoms in patients with relapsing and remitting and secondary progressive MS.

Multiple sclerosis: Serum anti-CNS autoantibodies.

BACKGROUND: It is uncertain whether there are autoantibodies detectable by indirect immunofluorescence in the serum of patients with multiple sclerosis (MS). OBJECTIVE: To determine whether there are anti-central nervous system (CNS) autoantibodies detectable by indirect immunofluorescence in the serum of MS patients. METHODS: Sera and in some cases cerebrospinal fluid from 106 patients with multiple sclerosis, 156 patients with other neurological diseases, and 70 healthy control subjects were examined by indirect immunofluorescence using cryostat sections of rat cerebrum fixed by perfusion with paraformaldehyde. RESULTS: Autoantibodies were detected that recognized more than 30 neuronal, glial, and mesodermal structures in 28 of 106 MS cases. Most were also detected in patients with other related and unrelated neurological diseases and several were also found in healthy controls. Novel anti-CNS autoantibodies recognizing particular sets of interneurons were detected in both normal controls and in subjects with CNS diseases. INTERPRETATION: Serum anti-CNS autoantibodies of diverse specificities are common in MS patients. The same anti-CNS autoantibodies are not uncommon in patients with other neurological diseases. The findings provide no support for the proposition that myelin breakdown in MS is caused by exposure of intact myelin sheaths or oligodendrocytes to a pathogenic serum anti-myelin or anti-oligodendrocyte autoantibody.

Synaptic pathology in the cerebellar dentate nucleus in chronic multiple sclerosis.

In multiple sclerosis, cerebellar symptoms are associated with clinical impairment and an increased likelihood of progressive course. Cortical atrophy and synaptic dysfunction play a prominent role in cerebellar pathology and although the dentate nucleus is a predilection site for lesion development, structural synaptic changes in this region remain largely unexplored. Moreover, the mechanisms leading to synaptic dysfunction have not yet been investigated at an ultrastructural level in multiple sclerosis. Here, we report on synaptic changes of dentate nuclei in post-mortem cerebella of 16 multiple sclerosis patients and eight controls at the histological level as well as an electron microscopy evaluation of afferent synapses of the cerebellar dentate and pontine nuclei of one multiple sclerosis patient and one control. We found a significant reduction of afferent dentate synapses in multiple sclerosis, irrespective of the presence of demyelination, and a close relationship between glial processes and dentate synapses. Ultrastructurally, we show autophagosomes containing degradation products of synaptic vesicles within dendrites, residual bodies within intact-appearing axons and free postsynaptic densities opposed to astrocytic appendages. Our study demonstrates loss of dentate afferent synapses and provides, for the first time, ultrastructural evidence pointing towards neuron-autonomous and neuroglia-mediated mechanisms of synaptic degradation in chronic multiple sclerosis.

Fibrosis of the Choroid Plexus Filtration Membrane.

We report a previously undescribed inflammatory lesion consisting of deposition of activated complement (C3d and C9neo) in association with major histocompatibility complex type II (MHC2)-positive activated microglia in choroid plexus villi exhibiting classical fibrous thickening of the pericapillary filtration membrane. The proportion of villi affected ranged from 5% to 90% in 56 adult subjects with diseases of the CNS and 11 subjects with no preexisting disease of the CNS. In 3 of the 4 children studied, 2% or less of examined villi showed stromal thickening, complement deposition, and the presence of MHC2-positive microglia; in adults, the proportion of villi affected increased with age. Other features of the lesion included loss of capillaries and failure by macrophages to clear extracellular particulate electron-dense material by clathrin-mediated phagocytosis. This choroid plexus lesion may relate pathogenetically to age-related macular degeneration and to Alzheimer disease, 2 other conditions with no known risk factors other than increasing age. All 3 conditions are characterized by the presence of damaged capillaries, inflammatory extracellular aggregates of mixed molecular composition and defective clearance of the deposits by macrophages.

Oligodendrocytes and the early multiple sclerosis lesion.

There is little agreement among neuropathologists regarding the timing and nature of oligodendrocyte loss in multiple sclerosis (MS). This review describes changes that accompany acute oligodendrocyte loss in new lesions. Included is a description of the immunopathology of new lesions in 23 severe early cases selected from a bank of 300 MS autopsies. Oligodendrocytes in prephagocytic lesions exhibit cytopathic changes that include apoptosis of oligodendrocytes immunoreactive for caspase 3, phagocytosis of apoptotic oligodendrocytes, swelling of cells with abnormal nuclei, complement deposition, and lysis. These are nonspecific changes that provide no clue as to the cause of oligodendrocyte injury. Associated changes include the presence of enlarged immunoglobulin (IgG)(+) microglia and early macrophages, the presence nearby of a focus of inflammatory demyelination, an open blood-brain barrier, and the presence of rare CD8 T cells. Myelin contacted by IgG(+) macrophages is immunoreactive for complement but not for IgG. It is likely that macrophage activity in evolving white and gray matter plaques is scavenging activity directed at nonvital myelin secondary to oligodendrocytes loss. One feature of MS that is not understood is the extraordinarily close resemblance the disease shows pathologically to neuromyelitis optica (NMO), including that demyelination in both is secondary to a loss of caspase 3-positive apoptotic oligodendrocytes. These similarities raise the possibility that like NMO, MS is an autoimmune disease in which oligodendrocyte apoptosis is determined by injury to some other glial or mesenchymal component.

Comprehensive tissue processing strategy for quantitative proteomics of formalin-fixed multiple sclerosis lesions.

Formalin-fixed (FF) autopsy tissue comprises the bulk of existing Multiple Sclerosis (MSc) pathology archives, providing a rich pool of material for biomarker discovery and disease characterization. Here, we present the development of a heat-induced extraction protocol for the proteomic analysis of FF brain tissue, its application to the study of lesion remyelination and its failure in MSc. A 4-round extraction strategy was optimized using FF tissue leading to a 35% increase in the number of proteins identified compared to a single extraction; and a 65% increase in proteins identified with ≥4 peptides. Histological staining of sections with oil red O and luxol fast blue-periodic acid Schiff, required to characterize MSc lesions was found to have minimal effect on LC-MS/MS. The application of the optimized protocol to chronic demyelinated and remyelinated FF MSc lesions and the adjacent periplaque white matter, isolated through laser guided manual dissection from 3 patients, identified 428 unique proteins (0.2% FDR) using LC-MS/MS. Comparison of the lesion types using iTRAQ and 2-D LC-MS/MS revealed 82 differentially expressed proteins. Protein quantitation by iTRAQ and spectral counting was well-correlated (r(s)= 0.7653; p < 10(-30)). The data generated from this work illustrates the scope of the methodology and provides insights into the pathogenesis of MSc and remyelination.

Neuromyelitis optica: a demyelinating disease characterized by acute destruction and regeneration of perivascular astrocytes.

BACKGROUND: A serum antibody directed against astrocytes is present in a high proportion of patients with neuromyelitis optica (NMO). The pathogenicity of the antibody is uncertain because no consistent astrocyte lesion is known to occur in NMO. OBJECTIVE: To determine whether there is an astrocyte lesion in NMO and if this differs from astrocyte changes in multiple sclerosis (MS). METHODS: Astrocyte pathology in early (still-myelinated) lesions and subacute NMO and MS lesions was examined immunohistochemically and in sections stained for astrocytes using routine histological techniques. RESULTS: Demyelination in early NMO lesions is accompanied by oligodendrocyte apoptosis in a pattern identical to that seen in MS and this is preceded by an abrupt destruction of perivascular astrocytes. Reparative astrogliosis is effected by a population of unipolar, new astrocytes. Evidence of a different type of astrocyte lesion was found in MS. DISCUSSION: The findings add to experimental evidence that the antibody is pathogenic. They also raise the possibility that demyelination in MS may be a bystander effect of an astrocyte lesion, i.e. that MS is not a disease primarily of myelin and oligodendrocytes.

Multiple sclerosis: distribution of inflammatory cells in newly forming lesions.

OBJECTIVE: CD4 T-cell-dependent macrophage activation directed against a myelin or oligodendrocyte antigen is generally thought to be the mechanism causing myelin destruction in multiple sclerosis (MS). However, areas within expanding MS lesions may exhibit prominent oligodendrocyte loss and apoptosis in the absence of infiltrating lymphocytes. The present study was designed to further investigate the inflammatory profile of different regions within rapidly expanding MS lesions. METHODS: Twenty-six active lesions from 11 patients with early MS were serially sectioned and immunostained for T and B cells, plasma cells, ramified microglia, macrophages, monocytes, and CD209-positive dendritic cells. Cell counts were compared in prephagocytic, phagocytic, and immediately postphagocytic areas. RESULTS: Parenchymal T and B cells were largely absent in areas of initial oligodendrocyte loss and in areas of degenerate and dead myelin infiltrated by myelin phagocytes. In contrast, trailing areas of complete demyelination packed with lipid macrophages, and, in some lesions, regenerating oligodendrocytes, showed large numbers of T cells, B cells, and immunoglobulin G (IgG)-positive plasma cells. Lesions in 2 exceptionally early cases contained relatively few T and B cells, and no IgG-positive plasma cells. INTERPRETATION: Early loss of oligodendrocytes is a prominent feature in tissue bordering rapidly expanding MS lesions. Macrophage activity is largely an innate scavenging response to the presence of degenerate and dead myelin. Adaptive immune activity involving T and B cells is conspicuous chiefly in recently demyelinated tissue, which may show signs of oligodendrocyte regeneration. The findings suggest that plaque formation has some basis other than destructive cell-mediated immunity directed against a myelin or oligodendrocyte antigen.

Immunoglobulins and complement in postmortem multiple sclerosis tissue.

OBJECTIVE: To identify evidence of a discrete, specific immune response in multiple sclerosis (MS) by analyzing the distribution of immunoglobulins and complement in tissue derived from cases of MS, and from control inflammatory white matter diseases known to express viral and autoantigens in the brain and spinal cord. METHODS: Autopsy tissue from 25 MS patients and 24 patients with other neurological diseases was examined immunohistochemically for immunoglobulins and activated complement (C3d and C9neo). RESULTS: In tissue remote from focal lesions in MS and other neurological diseases, IgG was detected in many normal structures but not in myelin or ramified microglia. Disrupted myelin in areas of active myelin breakdown and in phagocytes stained positively for C3d and C9neo, and equivocally for IgG in MS and all other neurological diseases examined, including ischemic infarcts. Disease-specific deposits of IgG or complement were detected in virus-infected cells in progressive multifocal leukoencephalopathy, subacute sclerosing panencephalitis, and cytomegalovirus encephalitis; in glial-limiting membranes in neuromyelitis optica; and in senile plaques in Alzheimer's dementia. Specific to MS were unusual microglial nodules containing short, linear deposits of activated complement (C3d) on partly demyelinated axons located in normal-appearing periplaque white matter. INTERPRETATION: IgG and complement immunostaining of disrupted myelin in MS lesions, frequently cited as an indication of pathogenic anti-myelin antibodies, is a nonspecific feature that cannot be interpreted as evidence of a distinct pathogenesis or serve to define particular variants of the disease. The unusual microglial nodules described in this study may constitute a specific biomarker with pathogenetic significance in MS.

Relapsing and remitting multiple sclerosis: pathology of the newly forming lesion.

The study describes the clinical and pathological findings in 12 patients with relapsing and remitting multiple sclerosis, who died during or shortly after the onset of a relapse. Pathological changes not previously associated with the formation of new symptomatic lesions were observed in seven cases, namely, extensive oligodendrocyte apoptosis and microglial activation in myelinated tissue containing few or no lymphocytes or myelin phagocytes. No current laboratory model of multiple sclerosis, in particular, experimental allergic encephalomyelitis, is known with these features, which raises the possibility of some novel process underlying new lesion formation in multiple sclerosis.

Generation and characterization of antibodies to sulfated glucuronyl glycolipids in Lewis rats.

Antibodies to sulfated glucuronyl glycolipids (SGGLs) have been reported in sera of patients with peripheral neuropathies including patients with IgM gammopathy. However, the role of anti-SGGL antibodies in the pathogenesis of neuropathy remains unclear. In order to study the role of antibodies to SGGLs in the pathogenesis of neuropathy, Lewis female rats were injected with purified SGPG mixed with keyhole limpet hemocyanin (KLH) and emulsified with equal amount of complete Freund's adjuvant. High titer anti-SGPG antibodies were detected by ELISA in sera of all rats inoculated with SGPG. All anti-SGPG antibodies cross-reacted with human myelin-associated glycoprotein (MAG). None of the sensitized rats exhibited clinical signs of neuropathy. Histological examination showed that there was no demyelination or axonal damage in peripheral nerves. Our data demonstrate that SGPG is a highly immunogenic glycolipid but high titer antibodies against it do not produce an experimental autoimmune neuropathy in Lewis rats.