Local uncoupling of the cerebrovascular and metabolic responses to somatosensory stimulation after neuronal nitric oxide synthase inhibition

In order to investigate the remyelinating potential of mature oligodendrocytes in vivo, we have developed a model of demyelination in the adult rat spinal cord in which some oligodendrocytes survive demyelination. A single intraspinal injection of complement proteins plus antibodies to galactocerebroside (the major myelin sphingolipid) resulted in demyelination followed by oligodendrocyte remyelination. Remyelination was absent when the spinal cord was exposed to 40 Grays of x-irradiation prior to demyelination, a procedure that kills dividing cells. Quantitative Rip immunohistochemical analysis revealed a similar density of surviving oligodendrocytes in x-irradiated and nonirradiated lesions 3 days after demyelination. Rip and bromodeoxyuridine double immunohistochemical analysis of demyelinated lesions indicated that Rip+ oligodendrocytes did not divide as an acute response to demyelination. Oligodendrocytes were also identified by Rip immunostaining and electron microscopy at late time points (3 weeks) within x-irradiated areas of demyelination. These oligodendrocytes extended processes that engaged axons, and on occasion formed myelin membranes, but did not lay down new myelin sheaths. These studies demonstrate that (a) oligodendrocytes that survive within a region of demyelination are not induced to divide in the presence of demyelinated axons, and (b) fully-differentiated oligodendrocytes are therefore postmitotic and do not contribute to remyelination in the adult CNS.     

An autoradiographic study of the renewal of mouse bronchiolar epithelium following bromobenzene exposure

We report an experimental model of germanium dioxide (GeO2)-induced neuropathy in rats. More than 6 months administration of GeO2 to young rats produced neuropathy characterized by segmental demyelination/remyelination and nerve edema. Electron microscopic studies demonstrated that changes in Schwann cells, such as an increased cytoplasmic volume or disintegration of the cytoplasm, were the earliest pathological findings. Schwann cell mitochondria contained high electron-dense materials. Subsequent removal of necrotic Schwann cell debris and myelin by invading macrophages was evident. These findings suggested that the Schwann cells themselves are the primary target of the toxin. The deposition of electron-dense granules in the intra-axonal vesicles, which was suggestive of glycogen granules in mitochondria, was observed in the advanced stage of the neuropathy. The findings of endoneurial edema with splitting of myelin lamellae were noted at the early stage of demyelination. Nerve edema may be the result of GeO2-induced endothelial cell injury.     

A new model of multiple sclerosis. Experimental vaccinia infection in the monkey

Experimental vaccinia infection in immunosuppressed and immunocompetent Rhesus monkeys has been studied for two years. The results show that vaccinia virus induces two forms of infection in the central nervous system, i.e. choriomeningitis and demyelination disease. The first form occurs in immunologically competent animals; the latter can develop in animals with defective immune response associated with an incomplete clearance of virus. These animals exhibit a noncytocidal persistent infection of glial cells, inducing a complex of potentially pathogenic immune mechanisms. Their direct, or by mediators mediated action is capable of damaging myelin sheaths.     

Trapidil-mediated inhibition of CNS remyelination results from reduced numbers and impaired differentiation of oligodendrocytes

In a previous study, we described the inhibitory effects of the growth factor-antagonist, trapidil, on spontaneously occurring oligodendrocyte remyelination in the rat spinal cord following lysolecithin-induced demyelination [30]. The objective of the present study was to further investigate the mechanisms of trapidil-mediated impairment of remyelination and thus obtain greater insight into the steps at which growth factors may be involved in remyelination. To this end, an ultrastructural analysis of the cellular composition of lesions from control and trapidil-treated animals was undertaken. Demyelination was created in the dorsal funiculus of 6-week-old female rats by the injection of 1.0 microliter of 1% lysolecithin. The animals received daily intraperitoneal injections of trapidil (80 mg/kg) or saline for 21 days, beginning on the day of lesion induction. Quantitative electron microscopic examination of lesions from both groups of animals showed that trapidil-treated lesions had reduced numbers of oligodendrocytes (P = 0.02) with a higher relative proportion of immature phenotypes, but increased numbers of microglia (P = 0.0009) and dystrophic axons (P0.02). In addition, the numbers of myelin lamellae around remyelinated axons were fewer in trapidil-treated animals. These results suggest that trapidil-mediated impairment of CNS remyelination is due to a blockage of growth factor-mediated proliferation and/or recruitment of remyelinating cells. Furthermore, the presence of oligodendrocytes with a more immature phenotype and the decreased thickness of the myelin sheaths of remyelination in the trapidil-treated animals indicate an impairment of growth factor-mediated differentiation.     

Perspectives in multiple sclerosis research]

There is little doubt that multiple sclerosis (MS) is an immune mediated disease, yet the exact immunological mechanisms, that are responsible for inflammation and demyelination in this disease are controversial. Recent evidence is summarized here, which suggests that heterogeneous pathogenetic mechanisms may lead to the inflammatory demyelinating plaques in different MS patients. This heterogeneity apparently involves the antigen specificity of the immune response as well as the mechanisms, responsible for the destruction of myelin sheaths. Since such a pathogenetic heterogeneity may have consequences for the design of therapeutic studies, strategies are discussed, which should allow a more accurate categorization of patient sub-groups in the future.     

Ultrastructural studies of an immune-mediated inflammatory response in the CNS parenchyma directed against a non-CNS antigen

We have shown previously that heat-killed bacillus Calmette-Guerin injected into the brain parenchyma becomes sequestered behind the blood brain barrier for months undetected by the immune system. However, independent peripheral sensitization of the immune system to bacillus Calmette-Guérin results in recognition of bacillus Calmette-Guérin in the brain and the induction of focal chronic lesions [Matyszak M. K. and Perry V. H. (1995) Neuroscience 64, 967 977]. We carried out ultrastructural studies of these lesions. Prior to subcutaneous challenge we used immunohistochemistry to detect bacillus Calmette-Guérin which was found in cells with the morphology of macrophages/microglia and in perivascular macrophages. Eight to 14 days after subcutaneous challenge there was a conspicuous leucocyte infiltration at the site of bacillus Calmette-Guérin deposits within the brain parenchyma. The majority of these cells were macrophages and lymphocytes, with some lymphocytes showing characteristic blast morphology. Dendritic cells in close contact with lymphocytes were prominent. Inflammatory cells were found in perivascular cuffs and within the brain parenchyma. The tissue was oedematous and some axons were undergoing Wallerian degeneration with associated myelin degeneration. Throughout the lesions, but more commonly at the edges, we detected macrophages containing myelin in their cytoplasm close to intact axons and axons with evidence of remyelinating sheaths, suggestive of primary demyelination. In older lesions, two to three months after the peripheral challenge, the oedema was less pronounced and there was little evidence of Wallerian degeneration. There were still many macrophages. lymphocytes and dendritic cells, although the number of these cells was lower than in earlier lesions. Late lesions also contained many plasma cells which were not present in early lesions. In these late lesions there were bundles of axons with no myelin or a few axons with thin myelin sheaths, suggestive of persistent or ongoing demyelination or remyelination. These observations show that, during a delayed-type hypersensitivity lesion in the CNS, the leucocyte populations change with time, and suggest that the mechanisms and type of tissue damage are different in the early and late stages of the lesion.     

A previously undescribed side effect of icodextrin: overestimation of glycemia by glucose analyzer

In a study designed to identify the neuropathological features typical of chronic inflammatory demyelinating polyneuropathy (CIDP), we reviewed the sural nerve biopsy findings in 105 patients with this disorder. The patients' mean age at biopsy was 49 years. In 65% of patients the disease had a progressive and in 35% a relapsing-remitting course. In 47% of cases the disorder was idiopathic; the remainder had various concurrent conditions. All sural nerve biopsy specimens showed varying amounts of active demyelination associated with onion bulbs (48% of cases), endoneurial edema (55%) and inflammatory infiltrates (25%). The immunopathological hallmarks were T cell infiltration with macrophagic activation and up-regulation of major histocompatibility complex (MHC) class II expression, without B cell infiltration or immunoglobulin deposition on myelin sheaths. In 30% of cases some myelin sheaths showed C3d deposition. Analysis of proinflammatory cytokine expression invariably showed interleukin-1 in perivascular and endoneurial ramified cells and tumor necrosis factor-alpha prevalently in epineurial macrophages, whereas it detected interferon-gamma only in samples with perivascular inflammatory cells. This immunological pattern suggests that the cellular components of immunity play the major role in CIDP. In 19% of cases the neuropathological changes had a focal distribution. This distinctive feature corresponded to more active demyelination, more frequent detection of inflammatory infiltrates and more prominent immunological activation, suggesting that focal involvement is a possible step in the course of the disease.     

Electron microscopical findings in the sural nerve of twins with metachromatic leukodystrophy. An attempt to classify the findings according to distinctive phases of the disease (author's transl)

Sural nerve biopsies taken from a pair of twins with metachromatic leukodystrophy were investigated by electron microscopy. The morphological findings showed distinct alterations. In the first twin the particularity was a large number of granules containing sulfatides. In the second twin, however, the main finding was a damage of myelin sheaths evidently caused by an excessive and morphologically as well as chemically defective myelin formation. According to the grading given by Ulrich the first case corresponds to the stadium one, in which a storage of sulfatides dominates, whereas case two showing a severe damage of myelin sheaths is characteristic of the second stadium of metachromatic leukodystrophy.     

Trochanteric hip fractures. Fixation failure commoner with femoral medialization, a comparison of 101 cases

Dejerine-Sottas disease is an uncommon hereditary neuropathy which has not been reported in Taiwan. We describe a 57-year-old woman who had slowly progressive weakness in her four limbs since adolescence. None of her close relatives had the disease and no consanguinity was noted. Neurologic examination showed severe weakness and vibratory sensation loss in the four limbs. The tendon reflexes were generally absent. Electrophysiologic studies suggested a systemic myelinopathic process. Light and electron microscopy of the sural nerve biopsy specimens revealed many onion-bulb shapes formed by the Schwann cell processes or basement membranes without any evidence of myelin sheaths around the axons, which are characteristic features of Dejerine-Sottas disease.     

Normal and aberrant functions of integrins in the adult central nervous system

Eighteen normal human eye-bank eyes (age: 18-81 years), five fetal eyes (16-24 weeks), 11 primary open-angle glaucoma (POAG) eyes (age: 76-89 years), and two Schnabel's cavernous optic atrophy eyes were examined using a biotinylated-hyaluronan binding protein to study the changes in the distribution of hyaluronic acid (HA) in the fetal, adult and glaucomatous optic nerve head. The vitreous body served as a positive control. Sections treated with Streptomyces hyaluronidase were used to confirm specificity. Monoclonal antibodies to myelin basic protein (MBP) and glial fibrillary acidic protein (GFAP) were used as additional controls. In fetal optic nerve, HA was localized in blood vessels, peripapillary sclera and the pial septae in the retrolaminar nerve. No staining was associated with axons. Staining for MBP was negative. In adults, HA was found surrounding the myelin sheaths in the retrolaminar nerve; staining decreased with age. In contrast, HA staining in myelinated peripheral nerves (e.g. ciliaries) remained unchanged with age. HA also was localized to the adventitia of arteries and veins throughout the posterior segment. Compared to age-matched normal eyes, HA staining was virtually absent around myelin sheaths of the retrolaminar nerve in POAG eyes. Similar changes were not found in other HA positive structures. In Schnabel's cavernous optic atrophy. HA was present in increased amount in the atrophic area, but virtually absent in the remaining retrolaminar nerve. HA staining was invariably positive in vitreous, and Streptomyces hyaluronidase treated sections were negative. In adults, staining of MBP was associated with the myelin sheath in the retrolaminar nerve. In contrast to HA, staining of MBP was unchanged with age and in POAG. In Schnabel's atrophy, MBP staining disappeared only in the atrophic area. HA in the retrolaminar optic nerve appears to be associate with the space-filling matrix between myelin sheaths. HA is not present in the axon bundles prior to myelination of the optic nerve. HA in the retrolaminar optic nerve appears to decrease with age and is further reduced in POAG; however, corresponding changes are not found in MBP or in peripheral nerves. Perhaps, decreased amounts of HA is related to a higher susceptibility to elevated intraocular pressure or to optic nerve atrophy. In Schnabel's cavernous optic atrophy, HA is present in increased amount only in the atrophic area while MBP is markedly decreased, suggesting in situ production of HA in areas of optic nerve atrophy.     

Overexpression of 2',3'-cyclic nucleotide 3'-phosphodiesterase in transgenic mice alters oligodendrocyte development and produces aberrant myelination

The function of the intracellular protein 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) of oligodendrocytes (ODC) is unknown. We have now generated several homozygous transgenic mouse lines in which the human CNP gene is overexpressed up to sixfold, revealing new insights into early stages of myelinogenesis. Although no behavioral phenotype is immediately apparent, abnormalities of ODC and their myelin sheaths are striking. These are manifested as redundant myelin membrane and intramyelinic vacuoles, as well as lack of myelin compaction concordant with failure of the cytoplasmic leaflets of compact myelin to fuse. Further, ODC that overexpress CNP appear to mature earlier in development, resulting in earlier maximum gene expression for myelin basic proteins and proteolipid protein. These results indicate that CNP is an early expressed regulator of cellular events that culminate in CNS myelination.     

Acceleration in the rate of CNS remyelination in lysolecithin-induced demyelination

One important therapeutic goal during CNS injury from trauma or demyelinating diseases such as multiple sclerosis is to develop methods to promote remyelination. We tested the hypothesis that spontaneous remyelination in the toxic nonimmune model of lysolecithin-induced demyelination can be enhanced by manipulating the inflammatory response. In PBS-treated SJL/J mice, the number of remyelinating axons per square millimeter of lesion area increased significantly 3 and 5 weeks after lysolecithin injection in the spinal cord. However, methylprednisolone or a monoclonal antibody (mAb), SCH94.03, developed for its ability to promote remyelination in the Theiler's virus murine model of demyelination, further increased the number of remyelinating axons per lesion area at 3 weeks by a factor of 2.6 and 1.9, respectively, but did not increase the ratio of myelin sheath thickness to axon diameter or the number of cells incorporating tritiated thymidine in the lesion. After 3 weeks, the number of remyelinating axons in the methylprednisolone or mAb SCH94.03 treatment groups was similar to the spontaneous remyelination in the 5 week PBS control-treated group, indicating that these treatments promoted remyelination by increasing its rate rather than its extent. To address a mechanism for promoting remyelination, through an effect on scavenger function, we assessed morphometrically the number of macrophages in lesions after methylprednisolone and mAb SCH94.03 treatment. Methylprednisolone reduced the number of macrophages, but SCH94.03 did not, although both enhanced remyelination. This study supports the hypothesis that even in toxic nonprimary immune demyelination, manipulating the inflammatory response is a benefit in myelin repair.     

Inflammation and the brain

Inflammation in the brain selectively damages the myelin sheath resulting in a variety of clinical syndromes of which the most common is multiple sclerosis. In these disorders, the areas of inflammation and demyelination can be identified in life by magnetic resonance imaging. Events occurring at the blood-brain barrier depend on T-cell activation, which increases immune surveillance within the central nervous system. T-cells activated against brain antigens persist to establish the conditions needed for inflammatory demyelination and this depends on local release of cytokines, culminating in removal of oligodendrocytes and their myelin lamellae by macrophages or microglia. These interactions involve binding between receptors present on microglia for the Fc portion of antibody and complement components to corresponding ligands on target cells. Taken together, the evidence from clinical and experimental studies provides a rationale for the issue of immunological treatments in patients with multiple sclerosis.     

Axonal regeneration and physiological activity following transection and immunological disruption of myelin within the hatchling chick spinal cord

Transections of the chicken spinal cord after the developmental onset of myelination at embryonic day (E) 13 results in little or no functional regeneration. However, intraspinal injection of serum complement proteins with complement-binding GalC or 04 antibodies between E9-E12 results in a delay of the onset of myelination until E17. A subsequent transection of the spinal cord as late as E15 (i.e., during the normal restrictive period for repair) results in neuroanatomical regeneration and functional recovery. Utilizing a similar immunological protocol, we evoked a transient alteration of myelin structure in the posthatching (P) chicken spinal cord, characterized by widespread "unravelling" of myelin sheaths and a loss of MBP immunoreactivity (myelin disruption). Myelin repair began within 7 d of cessation of the myelin disruption protocol. Long term disruption of thoracic spinal cord myelin was initiated after a P2-P10 thoracic transection and maintained for > 14 d by intra-spinal infusion of serum complement proteins plus complement-binding GalC or 04 antibodies. Fourteen to 28 d later, retrograde tract tracing experiments, including double-labeling protocols, indicated that approximately 6-19% of the brainstem-spinal projections had regenerated across the transection site to lumbar levels. Even though voluntary locomotion was not observed after recovery, focal electrical stimulation of identified brainstem locomotor regions evoked peripheral nerve activity in paralyzed preparations, as well as leg muscle activity patterns typical of stepping in unparalyzed animals. This indicated that a transient alteration of myelin structure in the injured adult avian spinal cord facilitated brainstem-spinal axonal regrowth resulting in functional synaptogenesis with target neurons.     

Inflammatory response of the spinal cord to multiple episodes of blood-brain barrier disruption and toxic demyelination in Wistar rats

Multiple episodes of blood-brain barrier disruption were induced by sequential intraspinal injections of ethidium bromide. In addition to the barrier disruption, there was toxic demyelination and exposure of myelin components to the immune system. Twenty-seven 3-month-old Wistar rats received 2, 3 or 4 injections of 1 microliter of either 0.1% ethidium bromide in normal saline (19 rats) or 0.9% saline (8 rats) at different levels of the spinal cord. The time intervals between the injections ranged from 28 to 42 days. Ten days after the last injection, all rats were perfused with 2.5% glutaraldehyde. The spinal sections were evaluated macroscopically and by light and transmission electron microscopy. All the lesions demonstrated a mononuclear phagocytic infiltrate apparently removing myelin. Lymphocytes were not conspicuous and were found in only 34% of the lesions. No perivascular cuffings were detected. In older lesions (38 days and older) they were found only within Virchow-Robin spaces. This result suggests that multiple blood-brain barrier disruptions with demyelination and exposure of myelin components to the immune system were not sufficient to induce an immune-mediated reaction in the central nervous system.     

Molecular cloning and characterization of RGC-32, a novel gene induced by complement activation in oligodendrocytes

Sublytic complement activation on oligodendrocytes (OLG) down-regulates expression of myelin genes and induces cell cycle in culture. Differential display (DD) was used to search for new genes whose expression is altered in response to complement and that may be involved in cell cycle activation. DD bands showing either increased or decreased mRNA expression in response to complement were identified and designated Response Genes to Complement (RGC) 1-32. RGC-1 is identical with heat shock protein 105, RGC-2 with poly(ADP-ribose) polymerase, and RGC-10 with IP-10. A new gene, RGC-32, that encodes a protein of 137 amino acids was cloned. RGC-32 has no homology with other known proteins, and contains no motif that would indicate its function. In OLG, the mRNA expression was increased by complement activation and by terminal complement complex assembly. RGC-32 protein was localized in the cytoplasm and co-immunoprecipitated with cdc2 kinase. Overexpression of RGC-32 increased DNA synthesis in OLGxC6 glioma cell hybrids. These results suggest that RGC-32 may play a role in cell cycle activation.     

Ultrastructural localization of relaxin in the corpus luteum of the pregnant and early lactating tammar wallaby, Macropus eugenii

Electron-microscope immunocytochemistry was used to determine the subcellular distribution and presence of immunoreactive relaxin throughout pregnancy and early lactation in the corpus luteum of a marsupial, the tammar wallaby. Membrane-bound, electron-dense granules were a prominent feature of the luteal cell cytoplasm. The highest numbers of granules were observed between days 20 and 24 of the 26-day gestation, with a rapid clearance immediately after birth. Relaxin immunogold particles were present only in small, electron-dense granules (200-350 nm in diameter), with no particles observed in larger granules (>400 nm diameter), nuclei or mitochondria. Relaxin immunoreactivity was low throughout early and mid pregnancy but increased markedly between days 21 and 22 and remained high over the last 4 days of pregnancy. The number of granules containing relaxin immunogold particles and the density of immunostaining were both reduced on the day of expected births (day 26). Our data demonstrate that electron-dense granules in the luteal cell cytoplasm of a pregnant marsupial contain relaxin. The peptide is produced in greatest amounts at the end of pregnancy, consistent with a role in parturition.     

An in-frame deletion in peripheral myelin protein-22 gene causes hypomyelination and cell death of the Schwann cells in the new Trembler mutant mice

Cloning and sequencing of the peripheral myelin protein-22 cDNA and genomic DNA from newly found Trembler mice revealed an in-frame deletion including exon IV which codes for the second (TM2) and a part of third (TM3) transmembrane domain of peripheral myelin protein-22. This mutation was distinct from those in both other allelic Trembler and Trembler-J mice, which carry point mutations within the putative transmembrane spanning regions of peripheral myelin protein-22. Inheritance was autosomal dominant. The affected mice revealed an abnormal gait, which appeared at 15-20 days of age, followed by motor and sensory ataxia, which remained throughout life. Most of the affected mice could survive more than one year. One of the most notable pathological phenotypes was a giant vacuolar formation in the sciatic nerve of homozygotes. They vary in size within the cytoplasm of Schwann cells, which failed to assemble myelin at any ages studied. Heterozygotes showed normal myelination during the early postnatal stages, followed by a segmental demyelination at an advanced stage. Vacuolar formation was not so frequent as in the homozygotes. These results suggest that the missing of transmembrane spanning region (TM2 and TM3) of peripheral myelin protein-22 may disturb a dual biological function of peripheral myelin protein-22, leading to a dysmyelination of axons and to a vacuolar formation within the cytoplasm of the Schwann cells. The latter phenotype is discussed in conjunction with the disruption of an intracellular transport system and subsequent cell death.     

The pathogenesis of multiple sclerosis. Cytotoxic antibodies against myelin sheath tissue in multiple sclerosis

Cytotoxic antibodies to myelin can be demonstrated by the method of 51Cr-release from chick erythrocytes coated with myelin basic protein. The cytotoxic antibody is inactivated by heating to 56 degrees C and needs complement in order to exert its action. The antibody was determined as IgM and IgG. It has relative specifity and shows cross-reaction with other basic proteins. The cytotoxic antibody was found in only 8% of healthy persons. Patients with multiple sclerosis were positive in 87% of the cases and in acute cases in 94%. In other neurological diseases cytotoxic antibody was present in 64%. The occurrence of cytotoxic antibody to myelin protein is not specific for a particular neurological disorder, especially not for multiple sclerosis. Cytotoxic antibodies arise as a secondary phenomenon, they are not the cause of the disease involved. They appear to be suitable, however, to determine, in association with cellular immunological reactions against myelin which may be regarded as the "primary" immunological processes, the demyelination process in the multiple sclerosis focus.     

Gene expression in brain during cuprizone-induced demyelination and remyelination

When C57BL/6J mice, 8 weeks of age, received 0.2% Cuprizone in their diet, extensive demyelination in corpus callosum was detectable after 3 weeks, and there was massive demyelination by 4 weeks. As expected, the accumulation of phagocytically active microglia/macrophages correlated closely with demyelination. When Cuprizone was removed from the diet, remyelination was soon initiated; after 6 weeks of recovery, myelin levels were near-normal and phagocytic cells were no longer prominent. Steady-state levels of mRNA for myelin-associated glycoprotein, myelin basic protein, and ceramide galactosyltransferase were already profoundly depressed after 1 week of Cuprizone exposure and were only 10-20% of control values after 2 weeks. Unexpectedly, upregulation of mRNA for these myelin genes did not correlate with initiation of remyelination but rather with accumulation of microglia/macrophages. After 6 weeks of exposure to Cuprizone, mRNA levels were at control levels or higher-in the face of massive demyelination. This suggests that in addition to effecting myelin removal, microglia/macrophages may simultaneously push surviving oligodendroglia or their progenitors toward myelination.