Massive astrocyte destruction in neuromyelitis optica despite natalizumab therapy.

Auto-antibody mediated astrocyte injury is implicated as a primary event in neuromyelitis optica (NMO) by biomarker, post-mortem and experimental studies that differentiate the condition from multiple sclerosis. We describe the clinical, radiological and neuropathological features of a severe cerebral attack in a natalizumab-treated patient with relapsing myelitis and serum aquaporin-4 antibodies. Our findings support autopsy evidence that abrupt astrocyte destruction precedes demyelination in NMO, and emphasize the importance of serological testing in patients with limited disease. Adherence to current NMO diagnostic criteria may delay treatment, or lead to inappropriate therapy with beta-interferon or natalizumab.

MS: is it one disease?

Neuropathological studies of early multiple sclerosis (MS) tissue have shaped prevailing views of the pathogenesis of the disease. The hallmark of the acute MS lesion, inflammatory demyelination, has been largely accepted as evidence of a macrophage-mediated attack on normal myelin, driven by perivascular and parenchymal autoreactive CD4+ Th1 cells primed in the periphery by an unknown self or foreign antigen(s). Predicated largely upon comparisons with experimental allergic encephalomyelitis, this paradigm has, in recent years, been recognized as a simplification of the events that constitute and perhaps presage lesion formation in the human disease; and the importance of the innate immune cells of the central nervous system, humoral factors, cytotoxic CD8+ T-cells and regulatory T-cells has been emphasized. An influential series of publications by one group, based on histopathological examination of actively demyelinating lesions in selected autopsy and biopsy MS tissue, defined four early lesion subtypes. In a given individual, these subtypes were reported to be mutually exclusive, suggesting that disparate pathogenetic pathways separate patients with clinically indistinguishable syndromes. This schema, which has considerable therapeutic implications, has not been independently verified and has recently been questioned by the finding of a uniform pre-phagocytic pathology and overlap of lesion subtypes in individual patients with typical relapsing and remitting disease. The latter findings would suggest that the heterogeneous features observed in active MS lesions sampled at different time-points are a reflection of the evolution of a single pathophysiological process, perhaps modified in part by genetic factors in individual cases.

The macrophage in MS: just a scavenger after all? Pathology and pathogenesis of the acute MS lesion.

Advances in the neuropathology of multiple sclerosis (MS) have contributed greatly to our understanding of the mechanisms of tissue injury in the condition. Particular interest has focussed on the active MS lesion, defined by macrophage activity in the presence of partially demyelinated axons. This has led to the prevailing consensus that a T-cell dependent, macrophage-mediated, autoimmune attack on constituents in the normal myelin sheath underlies the disease. This hypothesis, which has been largely supported by comparisons with the animal model, experimental allergic encephalomyelitis, has recently been questioned by an analysis of the pathological events preceding myelin phagocytosis in nascent MS lesions. The prephagocytic changes in evolving lesions examined shortly after the onset of an MS relapse raise the possibility that oligodendrocyte cell death and associated changes within the myelin sheath initiate local macrophage scavenger activity, with subsequent amplification of the inflammatory response. The presence of such lesions in patients with a spectrum of pathological changes in nearby or distant active phagocytic plaques suggests that pathological heterogeneity in MS is largely due to evolution of lesional pathology, rather than pathogenic heterogeneity.

Immunopathology of secondary-progressive multiple sclerosis.

Twenty-three plaques obtained at early autopsy from 2 patients with secondary-progressive multiple sclerosis were examined immunohistochemically for microglia/macrophages, and for immunoglobulins and components of activated complement. Most of the lesions examined in both cases exhibited evidence of low-grade active demyelination of an unusual type (frustrated phagocytosis) in periplaque white matter. This included linear groups of microglia engaging short segments of disrupted myelin that were associated with deposits of C3d, an opsonin formed during complement activation. Similar microglia/C3d/myelin profiles were not observed in newly forming lesions in cases of acute multiple sclerosis or other central white matter diseases. As C3d coupling is known to increase the immunogenicity of potential antigens enormously, present findings point to disrupted myelin close to plaques as a possible source of the putative multiple sclerosis antigen. Ongoing myelin destruction found in a high proportion of old, established plaques was surprising. It suggests that slowly expanding lesions (progressive plaques), in which ongoing myelin breakdown occurs in the absence of florid perivascular cell cuffing or other histological signs of acute inflammation, contribute to disease progression in cases of secondary-progressive multiple sclerosis.

Blood-brain barrier abnormalities in longstanding multiple sclerosis lesions. An immunohistochemical study.

Thirty-five randomly selected plaques from five patients with longstanding multiple sclerosis were examined immunohistochemically for evidence of extravascular serum proteins. One lesion showed histological evidence of active demyelination and 34 were inactive. In the one active lesion and in 26 of the 34 inactive lesions, serum proteins were detected outside blood vessels in a distribution consistent with leakage during life. The findings suggest that the blood-brain barrier (BBB) is permanently damaged in many old plaques, although to a degree not often detectable by current gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA)-enhanced magnetic resonance imaging (MRI). The findings also suggest that in patients with multiple sclerosis, a breached BBB is not by itself sufficient to induce active demyelination. Continuous exposure of demyelinated axons and glia to cytokines, antibody or other factors present in the circulation might be important, however, in preventing oligodendrocyte regeneration and new myelin formation in longstanding lesions.

Phosphatidylserine suppresses myelin-induced experimental allergic neuritis (EAN) in Lewis rats.

Phosphatidylserine administered as an aqueous dispersion to myelin-induced experimental allergic neuritis rats had a significant effect on disease course. Intraperitoneal injections of 30 mg/kg were given daily beginning at the onset of disease and continued for 14 days. Clinical severity and mortality were markedly reduced by this treatment as compared to saline controls. Improved clinical outcome was associated with a reduction in peripheral nerve pathology. A possible mechanism involving tumor necrosis factor is discussed.

Pathology of inflammatory demyelinating neuropathies.

GBS and CIDP are multifocal demyelinating diseases with lesions widely disseminated throughout the peripheral nervous system including cranial nerves, terminal intramuscular nerves and autonomic nerves. In both conditions myelin destruction is mediated by macrophages that attack myelin without disturbing the integrity of Schwann cells. The same pattern of myelin breakdown occurs in EAN, providing additional evidence that these disorders are immune-mediated. Recent autopsy and biopsy studies have challenged the view that lymphocytes are always present in newly forming lesions, an observation supporting a role for humoral immunity in the pathogenesis of some cases of GBS and CIDP. In both conditions neurological dysfunction is related to both myelin destruction and axonal loss. In some cases of CIDP there is the additional factor of impaired remyelination. Other chronic demyelinating neuropathies that may be immune-mediated differ from CIDP in presenting as solitary or multiple mononeuropathies. These can be classified into cases with and without evidence of widespread demyelination, cases with and without focal nerve enlargements, and a pure motor variant, multiple motor neuropathy associated with elevated anti-GM1 ganglioside antibody titres.

Electrolyte-induced demyelination in rats. 1. Role of the blood-brain barrier and edema.

The blood-brain barrier (BBB) was studied in rats with electrolyte-induced demyelination (EID), an experimental model for central pontine myelinolysis. Intravenously injected peroxidase was extravasated at 3 h post hypertonic saline injection (PHS) into regions frequently involved in EID. Increased pinocytotic activity and focal interendothelial gaps were seen at 3 h PHS and less frequently at 48 h PHS. Measurement of total cerebral water content revealed an increase during the hyponatremic phase. This was followed by a marked increase at 3 h PHS with continued increment at 48 h PHS. Intracellular edema with accumulation of fluid within neurites and astrocytic processes was noted during the hyponatremic phase, whereas extracellular edema developed after hypertonic saline injection. The implications of disrupted BBB and its role in the pathogenesis of EID are discussed.

Electrolyte-induced demyelination in rats. 2. Ultrastructural evolution.

This study presents the electron microscopic evolution of lesions in electrolyte-induced demyelination (EID) in rats, a lesion which bears striking histological and clinical similarity to central pontine myelinolysis. The earliest change was observed during the hyponatremic phase and consisted of minimal intracellular edema present throughout the brain. Following the injection of hypertonic saline, additional changes were observed which were restricted to sites previously reported to be frequently involved in EID. Early dilatation of the inner tongue of oligodendrocyte cytoplasm in myelinated nerve fibers was observed at 3 h post hypertonic saline injection (PHS). This was followed, at 48 h PHS, by the appearance of degenerative changes consistent with dying oligodendrocytes. Well-delineated, vacuolar and spongy lesions, seen by light microscopy, were present by 48 h PHS at the same sites as above. Electron microscopically, this appearance was found to be due to striking intramyelinic edema. By 96 h PHS, macrophages containing myelin and other cellular debris were frequently present at these sites. Concomitantly, myelin sheaths underwent vesicular disruption and disintegration. This sequence of events suggests a lesion of the oligodendrocyte-myelin complex, secondary to initial blood-brain barrier damage and edema.

Multiple sclerosis. Pathology of recurrent lesions.

Recent autopsy studies suggest that remyelinated shadow plaques located in otherwise intact white matter are the outcome of a previous single episode of acute demyelination. In the present study, of 98 remyelinated plaques examined in 15 patients with multiple sclerosis who died between 27 days and 5 years after clinical onset, 15 showed evidence of a superimposed new demyelinating lesion. Inspection of old shadow plaques in a separate series of patients with subacute and long-standing multiple sclerosis revealed that such lesions sometimes exhibit punched-out areas of demyelination and gliosis similar in size and shape to fresh lesions located within or overlapping remyelinated shadow plaques. The findings support magnetic resonance imaging evidence that local recurrence may be as important or more important than progressive edge activity in determining plaque growth and the conversion of nascent lesions into classical demyelinated plaques. The findings also support experimental evidence that recurrent demyelination of the same area of white matter may be one of the factors underlying failed remyelination in multiple sclerosis.

Multiple sclerosis: remyelination of nascent lesions.

The relationship between plaque pathology and disease duration was examined in 15 patients with multiple sclerosis who died early in the course of their illness. Myelin-stained sections revealed that most plaques examined in patients who died during the first month of their illness showed evidence of ongoing myelin destruction accompanied by a loss of oligodendrocytes. Plaques containing large numbers of oligodendrocytes were not observed in these patients, but were relatively common in patients who died more than 1 month after clinical onset. Remyelination affecting more than 10% of the plaque area was observed in 3 of 82 plaques in 5 patients who died within 10 weeks of clinical onset, in 38 of 105 plaques in 5 patients who died 3 to 10 months after clinical onset, and in 19 of 92 plaques in 5 patients who died 18 months or longer after clinical onset. The study provides new evidence that both oligodendrocytes and myelin are destroyed in new lesions, that this activity ceases completely in many lesions within a few weeks, and that remyelination frequently ensues following repopulation of the plaque by oligodendrocytes. The findings suggest that new lesions normally remyelinate unless interrupted by recurrent activity and that remyelinated shadow plaques are the outcome of a single previous episode of focal demyelination.

The longstanding MS lesion. A quantitative MRI and electron microscopic study.

Important questions remain unanswered about the sequence of events leading to progressive and ultimately irreversible tissue damage in MS. This study was designed to investigate the pathological characteristics of, and function of, the blood-brain barrier within longstanding MS lesions using quantitative and Gd-DTPA enhanced MRI techniques. The ultrastructural appearances of postmortem lesions from a single, separate case of MS have been correlated with the MRI findings. Both MRI and ultrastructural analysis revealed considerable heterogeneity in the chronic lesions: some are 'closed' with no detectable extracellular water, but most are 'open' and show expansion of the extracellular space to as much as 87% of tissue area. This variable expansion probably results from differing degrees of axonal loss. Evidence of blood-brain barrier damage was found in only 17% of lesions, was less severe than that seen in acute lesions, and may result from repeated previous inflammatory insults. The findings imply progressive axonal loss in lesions as they age. It is possible that this loss is related to clinical progression of the disease.

Interaction of astrocytes and newly formed oligodendrocytes in resolving multiple sclerosis lesions.

Cells resembling oligodendrocytes are sometimes seen within reactive astrocytes in fresh lesions in multiple sclerosis. Using immunostained paraffin and epoxy sections of fresh plaques obtained at autopsy from a series of cases of short clinical duration, it was found that small cells with round nuclei are commonly observed within reactive astrocytes in some hypercellular plaques and that these cells are phenotypically undifferentiated oligodendrocytes, i.e., nonmyelinating cells expressing intensely the oligodendrocyte determinants 2',3'-cyclic nucleotide 3'-phosphohydrolase and the carbohydrate epitope present on the family of cell adhesion molecules recognized by monoclonal antibody HNK-1. They also stain positively for IgG. This unusual astrocyte-oligodendrocyte interaction, which appears to be restricted to nonmyelinating oligodendrocytes in lesions of several weeks' to several months' duration, has not been described during normal oligodendrocyte differentiation or in experimental central remyelinating lesions. It bears some resemblance, however, to a pattern of slow oligodendrocyte destruction seen previously in organotypic perinatal central nervous tissue cultures exposed to multiple sclerosis serum. It is concluded that the evolution of some multiple sclerosis lesions early in the course of the disease is associated with abnormal binding and/or destruction of newly generated oligodendrocytes by reactive astrocytes. These observations raise new questions concerning mechanisms underlying failed remyelination in multiple sclerosis, including the novel possibility of an immune response directed against a developmentally restricted oligodendrocyte antigen.

Sera from MS patients and normal controls opsonize myelin.

Fc receptor-dependent myelin phagocytosis has been proposed as a mechanism of demyelination in multiple sclerosis (MS). The object of this study was to determine whether MS patients' sera are more opsonic for myelin than normal controls' sera. Opsonization was tested by culturing thioglycollate elicited murine peritoneal macrophages with 125I-labelled, serum-sensitized bovine central myelin. The two groups of sera were found to opsonize myelin equally well after 30 and 120 min incubations. We conclude that MS patients' sera are not more opsonic for myelin than sera from normal controls.

Multiple sclerosis. Oligodendrocyte proliferation and differentiation in fresh lesions.

Fresh lesions in the brain and spinal cord of patients with multiple sclerosis who died shortly after the onset of symptoms were examined immunocytochemically for myelin and oligodendrocyte antigens that are known to be sequentially expressed during normal development. Cells with oligodendrocyte-like morphology that appear in large numbers throughout fresh lesions after acute myelin breakdown and before new myelin formation were found to express galactocerebroside, carbonic anhydrase, and 2',3'-cyclic nucleotide 3'-phosphohydrolase but not myelin-associated glycoprotein or myelin basic protein. They also exhibit intense surface reactivity for a carbohydrate epitope associated with the family of cell adhesion molecules recognized by the monoclonal antibody HNK-1. With the onset of remyelination and the appearance of myelin-associated glycoprotein, myelin basic proteins, CNP, and the HNK-1 epitope is newly formed myelin sheaths, perikaryon CNP and HNK-1 reactivity diminished. A possible oligodendrocyte precursor cell in the form of a large HNK-1 positive glial fibrillary acidic protein negative glial cell was observed among interfascicular oligodendrocytes in white matter bordering these hypercellular plaques. Because a similar progression in the expression of CNP and the HNK-1 epitope occurs during normal oligodendrocyte differentiation, these observations are additional evidence that extensive oligodendrocyte regeneration occurs in some plaques early in the course of the disease. The finding of large numbers of immature oligodendrocytes, presumably expressing many developmentally restricted antigens not normally present in the mature nervous system, in plaques at a particular stage in their evolution may be important in understanding why remyelination eventually fails in multiple sclerosis.

Opsonization of normal myelin by anti-myelin antibodies and normal serum.

Fc receptor-dependent myelin phagocytosis has been proposed as a possible important effector mechanism in several immune-mediated demyelinating diseases. The present study was designed to determine whether myelin is opsonizable by anti-myelin antibodies. Thioglycolate-elicited mouse peritoneal macrophages were cultured with 125I-labelled bovine central myelin pretreated with normal or immune serum. Serum opsonic activity was determined by a kinetic study comparing macrophage uptake of opsonized and untreated 125I-myelin. Heat-stable and heat-labile myelin opsonins were detected in normal rabbit serum. Myelin was also opsonized by normal rabbit gamma globulin and by heat-inactivated normal mouse, human, and guinea pig serum. Increased opsonic activity was detected in rabbit anti-myelin antiserum and the gamma globulin fraction prepared from this serum, in anti-myelin basic protein and anti-galactocerebroside antiserum but not in anti-myelin-associated glycoprotein antiserum or in serum from rabbits injected with Freund's adjuvant alone. One out of three anti-sheep red blood cell antisera tested also showed increased myelin opsonic activity. It is concluded that anti-myelin antibodies can promote opsonic phagocytosis, and that normal serum and normal serum gamma globulin also opsonize myelin.

Protective effect of steroids in electrolyte-induced demyelination.

Electrolyte-induced demyelination (EID), an experimental model for central pontine myelinolysis was produced in rats by inducing hyponatremia followed by hypernatremia. There was a marked reduction in the number and size of lesions developing in animals that were bled repeatedly by tail transection during induction of the disease. Subsequently a similar protective effect was produced in animals by injecting a single dose of dexamethasone, one hour before the induction of hypernatremia. These findings suggest that steroids may be useful in preventing central pontine myelinolysis from developing in high risk patients requiring urgent correction of hyponatremia.

Measles virus nucleic acid sequences in human brain.

We constructed a measles virus genomic recombinant DNA library, and used clones coding for portions of the viral P, M and H proteins to probe for measles virus nucleic acid sequences in post-mortem multiple sclerosis, SSPE and control brains. By dot blot hybridization, the probes detected measles virus nucleic acid sequences in as little as 3 nanograms of total RNA extracted from measles virus-infected cells and also in highly diluted RNA extracted from SSPE brain, but did not detect measles virus sequences in RNA extracted from 11 multiple sclerosis or 8 control brains, even at a 1 000-fold higher concentration of RNA. By in situ hybridization, these probes detected measles virus nucleic acid sequences in virtually every cell and the surrounding neuropile of SSPE brain, but again did not detect such sequences in multiple sclerosis or control brains. Our findings using these highly specific probes confirm that measles virus is found in SSPE brains and indicate that measles virus genome is unlikely to be present in multiple sclerosis or normal brains.

The distribution of myelin-associated glycoprotein and myelin basic protein in actively demyelinating multiple sclerosis lesions.

Active plaques from 4 patients with multiple sclerosis were examined for myelin-associated glycoprotein (MAG) and myelin basic protein (MBP) using the peroxidase-antiperoxidase (PAP) immunocytochemical procedure applied to paraffin sections. MBP loss was intimately related to the presence of infiltrating macrophages which appeared to remove MBP-positive fragments directly off intact myelin sheaths. Phagocytosis of MAG-positive myelin sheaths was also observed. These findings support previous morphological studies that suggest that phagocytosis by macrophages of myelin attached to axons is an important mechanism of demyelination in multiple sclerosis.