Demyelination and remyelination in the rat central nervous system following ethidium bromide injection

Intracisternal injection of ethidium bromide induced status spongiosus with prominent degenerative changes in oligodendroglia in the subpial regions of the central nervous system of the rat. Chronologic investigation of the lesions has revealed that status spongiosus resulted in myelin degeneration, and by the 6th day postinjection many axons were demyelinated. At this time, numerous debris-filled phagocytic cells were observed among the totally naked axons. Vesicular transformation of myelin was the common degenerative change. Features suggestive of separation of myelin lamellae by phagocytic cells were also observed. In the demyelinated areas, oligodendroglial cells disappeared completely. By the 12th day postinjection, remyelination was apparent and numerous active oligodendroglia appeared in association with thinly myelinated axons. Some central nervous system axons were myelinated by Schwann cells. These patterns of demyelination and remyelination observed in ethidium bromide-treated rats were compared with those observed in other demyelinating conditions of varied etiology such as experimental allergic encephalomyelitis, diphtheria toxin, or lysolecithin injection and cuprizone intoxication.     

Schwann cell remyelination and recurrent demyelination in the central nervous system of mice infected with attenuated Theiler's virus.

Theiler's murine encephalomyelitis virus (TMEV) infection produces a chronic demyelinating disease in mice, and myelin breakdown appears to be immune-mediated. By using an attenuated TMEC strain, WW virus, to infect mice, the course of the disease was slowed and the severity of the inflammatory and glial responses were reduced. In this circumstance, most of the demyelinating lesions showed extensive remyelination, predominantly by Schwann cells. In addition, it was demonstrated that there was recurrent demyelinating activity in the central nervous system (CNS) of infected animals. It is suggested that the rapidity and intensity of demyelinating lesions may influence the potential for remyelination and that Schwann cell participation may be a more important mechanism of myelin repair than it is now thought to be. The fact that there is a recurrent demyelination in TMEV infection increases its relevance as an experimental animal model for multiple sclerosis.     

Multicontrast MRI of remyelination in the central nervous system

Although magnetic resonance imaging (MRI) represents the most sensitive tool for the detection of white matter abnormalities in patients with multiple sclerosis (MS), the heterogeneity of MS placques severely hampers the elucidation of specific pathophysiological processes. In order to identify putative MRI markers for de- and remyelination, we employed the cuprizone mouse model which leads to a selective and reversible demyelination of the corpus callosum with little or no axonal damage. Apart from histopathology, animals were studied with high-resolution three-dimensional MRI in vivo using multiple contrasts. While individual MRI findings significantly correlated with electron microscopy, the differentiation of regions with normal, demyelinated or remyelinated white matter by one contrast alone was less specific than by histology or electron microscopy. However, an accurate MRI prediction of the in vivo myelin status was achieved by a discriminant function analysis using a combination of T1, T2 and magnetization transfer contrast. With a correct assignment of 95% of all animals examined, the procedure will allow for the survey of new therapeutic approaches aiming at improved remyelination.     

Presence of Schwann cells in neurodegenerative lesions of the central nervous system

Ultrastructural analysis of neurodegenerative CNS lesions produced by an excitotoxic substance revealed that the majority of cells ensheathing axons were not oligodendrocytes. By their morphology and the presence of both a basal lamina and collagen fibers they were identified as Schwann cells. The presence of Schwann cells, whose growth-promoting role in the peripheral nervous system has been largely documented, may account for the development of regenerating growth cones which have been observed in the excitotoxically lesioned central nervous system. Further support for this hypothesis came from the analysis of fetal neural transplants implanted into the lesioned area. Schwann cells ensheathing axons were indeed numerous in the neuron-depleted area surrounding the transplants, where neurite outgrowth of graft origin occurred.     

On the occurrence of Schwann cells within the normal central nervous system

Summary The presence of Schwann cells and P.N.S. myelin are reported in subpial areas of apparently normal spinal cord from one control rabbit, two experimental rabbits and one experimental guinea pig. These P.N.S. elements exerted no adverse effects upon local C.N.S. components. The occurrence of ectopic Schwann cells in the normal C.N.S. has also been reported elsewhere in studies on normal human spinal cord tissue. The propensity for Schwann cells to reside in the normal C.N.S. of several species makes it necessary for experiments and hypotheses on the aetiology of Schwann cell invasion into the abnormal C.N.S. to take the present phenomenon into consideration.     

An autoradiographic study of cellular proliferation in remyelination of the central nervous system.

The proliferation and origin remyelinating oligodendrocytes was studied by light and electron miscrosopic autoradiography in the superior cerebellar peduncles of mice demyelinated by Cuprizone. In the early phases of demyelination, the cells undergoing mitotic activity were macrophages and astrocytes. In the later phases of demyelination, immature proliferating oligodendrocytes appeared; these differentiated into mature (dark) oligodendrocytes which were responsible for the remyelination of axons seen when animals were again placed on normal diets. The pattern of differentiation recapitulated that seen in developing oligodendrocytes in normal animals. Dark oligodendrocytes did not show mitotic activity. There was no mitotic activity in the subependymal cells around the fourth ventricle adjacent to the superior cerebellar peduncles. This study demonstrates the regenerative capacity of oligodendrocytes and their ability to carry out remeylination in the central nervous system.     

Immunoglobulins stimulate central nervous system remyelination: electron microscopic and morphometric analysis of proliferating cells

Infection with the Daniel strain of Theiler's murine encephalomyelitis virus results in immunemediated primary demyelination in the spinal cords of susceptible SJL/J mice. Treatment of chronically infected mice (3 to 7 months) with purified immunoglobulins directed against spinal cord homogenate resulted in an increase in the number and average size of lesions that were undergoing remyelination by oligodendrocytes. In vivo autoradiography with [3H]thymidine demonstrated labeling of many lymphocytes in areas of demyelination and remyelination. A direct correlation was found between number of labeled lymphocytes infiltrating the lesion and size of demyelinating lesions. Remyelinated areas contained proliferating cells that resembled immature oligodendrocytes or progenitor glial cells morphologically. The number of labeled presumptive glial cells correlated with the area of remyelination. However, central nervous system remyelination occurred even in the presence of proliferating lymphocytes and astrocytic hypertrophy. In addition, treatment of normal uninfected SJL/J mice with antiserum to spinal cord homogenate resulted in increased numbers of proliferating cells in the spinal cord. These experiments suggest that immunoglobulins to a spinal cord antigen may induce proliferation of cells in the central nervous system to promote remyelination.     

Hyaluronectin is produced by oligodendrocytes and Schwann cells in vitro

A hyaluronectin (HN)-like antigen was found in rat O-2A progenitors and oligodendrocytes, as well as in Schwann cells and in their culture medium. The HN-like antigen secreted in culture supernatants had a higher molecular mass than HN extracted from rat brain at acidic pH. In vitro the secreted HN-like antigen was spontaneously and slowly degraded into species whose Mr was close to that of HN found in acidic brain extract. In brain or nerve neutral pH extracts, both HN-like antigen and HN were present. The high Mr of the secreted antigen, the homology in amino acid sequences between HN and N-terminal domain of PG-M/versican, in addition to a positive hybridization between Schwann cell RNAs and a probe obtained with primers derived from HN sequences also found in versican suggested that HN is closely related to the large proteoglycan PG-M/versican. The presence in Schwann cell extract of a HN mRNA whose Mr was compatible with the size expected for HN showed that HN may be directly secreted by cells and not only the consequence of a proteolytic cleavage. The similarity of HN with PG-M (V3) suggested that HN found in vivo could be the result of an alternative splicing of a single gene. We conclude that HN as other members of the PG-M/versican family is a marker of oligodendrocytes and Schwann cells in culture.     

Generating neuronal diversity in the Drosophila central nervous system

Generating diverse neurons in the central nervous system involves three major steps. First, heterogeneous neural progenitors are specified by positional cues at early embryonic stages. Second, neural progenitors sequentially produce neurons or intermediate precursors that acquire different temporal identities based on their birth-order. Third, sister neurons produced during asymmetrical terminal mitoses are given distinct fates. Determining the molecular mechanisms underlying each of these three steps of cellular diversification will unravel brain development and evolution. Drosophila has a relatively simple and tractable CNS, and previous studies on Drosophila CNS development have greatly advanced our understanding of neuron fate specification. Here we review those studies and discuss how the lessons we have learned from fly teach us the process of neuronal diversification in general.     

The neurotoxicant, cuprizone, as a model to study demyelination and remyelination in the central nervous system

Myelin of the adult CNS is vulnerable to a variety of metabolic, toxic, and autoimmune insults. That remyelination can ensue, following demyelinating insult, has been well demonstrated. Details of the process of remyelination are, however difficult to ascertain since in most experimental models of demyelination/remyelination the severity, localization of lesion site, or time course of the pathophysiology is variable from animal to animal. In contrast, an experimental model in which massive demyelination can be reproducibly induced in large areas of mouse brain is exposure to the copper chelator, cuprizone, in the diet. We review work from several laboratories over the past 3 decades, with emphasis on our own recent studies, which suggest an overall picture of cellular events involved in demyelination/remyelination. When 8 week old C57BL/6 mice are fed 0.2% cuprizone in the diet, mature olidgodendroglia are specifically insulted (cannot fulfill the metabolic demand of support of vast amounts of myelin) and go through apoptosis. This is closely followed by recruitment of microglia and phagoctytosis of myelin. Studies of myelin gene expression, coordinated with morphological studies, indicate that even in the face of continued metabolic challenge, oligodendroglial progenitor cells proliferate and invade demyelinated areas. If the cuprizone challenge is terminated, an almost complete remyelination takes place in a matter of weeks. Communication between different cell types by soluble factors may be inferred. This material is presented in the context of a model compatible with present data -- and which can be tested more rigorously with the cuprizone model. The reproducibility of the model indicates that it may allow for testing of manipulations (e.g. available knockouts or transgenics on the common genetic background, or pharmacological treatments) which may accelerate or repress the process of demyelination and or remyelination.     

Chronic toxic demyelination in the central nervous system leads to axonal damage despite remyelination

The majority of multiple sclerosis lesions fail to remyelinate after chronic demyelinating episodes resulting in neurologic disability. In the current study, chronic demyelination was investigated by using the cuprizone model, a toxic demyelination model. C57BL/6 mice were administered a 0.2% cuprizone diet up to 16 weeks to induce chronic demyelination. For comparison, another group was maintained only for 6 weeks on cuprizone to model acute demyelination. Both groups were analysed regarding the remyelination process after withdrawal of the toxin. Reexpression of myelin proteins after chronic demyelination was reduced by a factor of two as judged by LFB and myelin protein stainings compared to acute demyelination after 2 weeks on remyelination. During chronic demyelination mature oligodendrocytes (Nogo-A positive cells) were severely depleted by 90% compared to age matched controls. Nevertheless, extensive remyelination occurred after withdrawal of cuprizone and was nearly complete after 12 weeks. There was only minimal acute axonal damage as judged by APP staining, with the course of APP positive axons correlating with macrophage/microglia accumulation. Chronic axonal damage detected by SMI-32 positive staining was only seen after chronic demyelination and was still observable during the whole remyelination period. These data suggest that two pattern of axonal injury occur in the cuprizone model.     

Generating neuronal diversity in the Drosophila central nervous system: a view from the ganglion mother cells.

The generation of cellular diversity in the developing embryonic central nervous system of Drosophila melanogaster requires the precise orchestration of several convergent molecular and cellular mechanisms. Most reviews have focused on the formation and specification of neuroblasts (NBs), the putative neural stem cell in the Drosophila central nervous system. NBs divide asymmetrically to regenerate themselves and produce a secondary precursor cell called a ganglion mother cell (GMC), which divides to produce neurons and glia. Historically, our understanding of GMC specification has arisen from work involving asymmetric localization of intrinsic factors in the NB and GMC. However, recent information on NB lineages has revealed additional intrinsic factors that specify general and specific GMC fates. This review addresses what has been revealed about these intrinsic cues with regard to GMC specification. For example, Prospero, an asymmetrically localized determinant, plays a general role to enable GMC development and to distinguish GMCs from NBs. In contrast, the temporal gene cascade functions within NB lineages to ensure that each GMC in a lineage acquires a different fate. Two different mechanisms used to make the progeny of GMCs different will also be discussed. One is a generic mechanism, regulated by Notch and Numb, that allows sibling cells to adopt different fates. The other mechanism involves genes, such as even-skipped and klumpfuss that specify the fate of individual GMCs. All of these mechanisms converge within a GMC to bestow upon it a unique fate.     

Schwann cell invasion of the central nervous system of the myelin mutants

Schwann cells are excluded from the CNS during development by the glial limiting membrane, an area of astrocytic specialisation present at the nerve root transitional zone, and at blood vessels in the neuropil. This barrier, however, can be disrupted and, with the highly migratory nature of Schwann cells, can result in their invasion and myelination of the CNS in many pathological situations. In this paper we demonstrate that this occurs in a number of myelin mutants, including the myelin deficient ( md ) and taiep rats and the canine shaking ( sh ) pup. While it is still relatively uncommon in the rodent mutants, the sh pup shows extensive Schwann cell invasion along the neuraxis. This invasion involves the spinal cord, brain stem, and cerebellum and increases in amount and distribution with age. In situ hybridisation studies using a P₀ riboprobe suggest that the likely origin of these cells in the sh pup is the nerve roots, primarily the dorsal roots. Paradoxically, Schwann cell myelination of the CNS increases with time in the sh pup despite a marked, progressive gliosis involving the glia limitans and neuropil. Thus the mechanism by which these cells migrate into the CNS through the gliosed nerve root transitional zone or from vasa nervorum remains unknown. Extensive Schwann cell CNS myelination may have therapeutic significance in human myelin disease.     

Focal neuronal gigantism and cerebral cortical thickening after therapeutic irradiation of the central nervous system

An exceptional type of cortical dysplasia is described in the brain of a 32-year-old woman who had received radiation therapy for a large pituitary adenoma 6 years before death. Markedly thickened gyri of the left inferior frontal, insular, and temporal cortex were found grossly. Microscopically, these gyri showed laminar disorganization and many unusually large and abnormally shaped ganglion cells. These neurons were heavily impregnated with silver preparations; were strongly reactive for neuron-specific enolase, synaptophysin, and neuronal cytoskeletal proteins (68- and 200-kd subunits of neurofilament protein, microtubule-associated protein 2, and tau); and ultrastructurally contained numerous perikaryal neurofilaments. Collectively, these findings suggest that the abnormally large, misshapen neurons contained excessive accumulations of cytoskeletal intermediate filaments. The present case and a similar one described in 1964 are the only two documented instances of neuronal gigantism apparently related to therapeutic irradiation of the brain.     

Changes in the distribution of a calcium-dependent ATPase during demyelination and remyelination in the central nervous system

Summary A calcium-adenosine triphosphatase (Ca²⁺-ATPase) activity expressed by CNS nerve fibres has been examined during demyelination and remyelination in rats, 21–26 days after an intraspinal injection of ethidium bromide. The Ca²⁺-ATPase distribution was determined cytochemically, using a technique believed primarily to reflect the presence of ecto-ATPases. We confirm that in normal nerve fibres Ca²⁺-ATPase activity was present on the external surface of the myelin sheath, and on the axolemma at the nodes of Ranvier. Labelling of the internodal axolemma was restricted to small, scattered, punctate regions. However, following demyelination the Ca²⁺-ATPase activity was expressed continuously along both the exposed, previously internodal axolemma of entirely naked axons, and it was particularly prominent at sites of contact between axons and glial-cell processes. During remyelination (which in this lesion is accomplished predominantly by Schwann cells) the proportion of the axonal surface exhibiting Ca²⁺-ATPase activity decreased in concert with the progressive thickening of the new myelin sheath. The non-myelin forming plasmalemma of Schwann cells was positive for the Ca²⁺-ATPase activity, but activity was abruptly lost at the site of compaction between the inner and outer leaflets of the forming myelin sheath. Ecto-ATPase activity is a property of some cell adhesion molecules, and it follows that the changes observed in the distribution of ATPase activity in this study may reflect changes in the axolemma which are important for the successful repair of the lesion by remyelination. The ATPase activity may, for example, reflect the changing distribution of molecules important in aiding axo-glial recognition and the establishment of axo-glial contacts.     

Nestin in central nervous system cells

This literature review reflects current knowledge on the intermediate filament protein nestin, which most authors regard as a marker of “neural stem/progenitor cells.” The structural-functional characteristics of nestin and its presence in various central nervous system cells at different stages of ontogenesis in normal and pathological conditions are discussed. __________ Translated from Morfologiya, Vol. 131, No. 1, pp. 85–90, January–February, 2007. Director: Corresponding Member of the Russian Academy of Medical Sciences Professor V. A. Otellin     

Peripheral nerve grafts lacking viable Schwann cells fail to support central nervous system axonal regeneration

Summary Peripheral nerve grafts were implanted bilaterally into the diencephalon of adult hamsters. One graft segment contained both viable Schwann cells and their basal lamina tubes. The Schwann cell population in the second graft segment was killed by freezing prior to implantation. Seven weeks after graft implantations, the extracranial end of each graft segment was exposed, transected and labelled with a fluorescent tracer substance. One week after the labelling procedure each animal was perfused and the diencephalon and midbrain were examined. Ultrastructural analyses of both types of graft demonstrated the persistence of the Schwann cell-derived basal lamina tubes. Retrogradely labelled neurons were found in all cases in which an intact graft remained in place for two months, but were seen in only one case with a frozen graft. Large numbers of myelinated and unmyelinated axons were seen within the intact grafts, but no axons were found in the previously frozen grafts. These results indicate that lesioned CNS axons are able to regenerate vigorously when provided with an environment which includes viable Schwann cells. But, CNS axons regenerate less well, if at all, when Schwann cells are absent. Further, it appears that Schwann cell-derived basal lamina tubes, when isolated from their parent cells, are insufficient to initiate or sustain CNS axonal regeneration.This material is based upon work supported by the National Science Foundation under grant BNS-8416911