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On-line version ISSN 1678-4227
Arq. Neuro-Psiquiatr. vol.59 no.2B São Paulo June 2001
ABSTRACT - Oligodendrocytes and Schwann cells are engaged in myelin production, maintenance and repairing respectively in the central nervous system (CNS) and the peripheral nervous system (PNS). Whereas oligodendrocytes act only within the CNS, Schwann cells are able to invade the CNS in order to make new myelin sheaths around demyelinated axons. Both cells have some limitations in their activities, i.e. oligodendrocytes are post-mitotic cells and Schwann cells only get into the CNS in the absence of astrocytes. Ethidium bromide (EB) is a gliotoxic chemical that when injected locally within the CNS, induce demyelination. In the EB model of demyelination, glial cells are destroyed early after intoxication and Schwann cells are free to approach the naked central axons. In normal Wistar rats, regeneration of lost myelin sheaths can be achieved as early as thirteen days after intoxication; in Wistar rats immunosuppressed with cyclophosphamide the process is delayed and in rats administered cyclosporine it may be accelerated. Aiming the enlightening of those complex processes, all events concerning the myelinating cells in an experimental model are herein presented and discussed.
KEY WORDS: demyelination, remyelination, oligodendrocytes, Schwann cells, ethidium bromide, immunosuppression, cyclophosphamide, cyclosporine.
Comportamento de oligodendrócitos e células de Schwann em modelo experimental de desmielinização tóxica do sistema nervoso central
RESUMO - Oligodendrócitos e células de Schwann realizam a produção e manutenção das bainhas de mielina, respectivamente no sistema nervoso central (SNC) e periférico (SNP). As células de Schwann, à diferença dos oligodendrócitos, são capazes de invadir o SNC para remielinizar axônios desmielinizados, sempre que os astrócitos tenham sido destruídos. O brometo de etídio é uma droga gliotóxica usada para induzir desmielinização com o desaparecimento precoce de astrócitos, de modo que as células de Schwann têm liberdade para invadir o SNC. Em ratos Wistar normais, a remielinização é detectada treze dias após desmielinização; em ratos Wistar imunossuprimidos com ciclofosfamida a reparação do tecido é tardia, enquanto que em animais tratados com ciclosporina ela é acelerada. O objetivo do artigo é discutir todas as etapas do processo de destruição e reparação da mielina em um modelo experimental de desmielinização em ratos.
PALAVRAS-CHAVE: desmielinização, remielinização, oligodendrócito, células de Schwamm, brometo de etídio, imunossupressão, ciclosporina, ciclofosfamida.
Oligodendrocytes and Schwann cells perform the unique task of producing and maintaining myelin sheaths around selected axons respectively in the central nervous system (CNS) and in the peripheral nervous system (PNS)1,2. Although both cells have been exhaustively investigated either in normality or in disease1,3 , many questions remain unanswered concerning the proliferating capacity of oligodendrocytes within demyelinating lesions3-5 and the origin of those Schwann cells that invade the CNS to repair the lost myelin sheaths6-8. To get a deeper insight into those issues, the behaviour of both cells in variants of the Etmidium Bromide (EB) model of demyelination was studied6,9-13. EB is an intercalating gliotoxic dye extensively used to induce demyelination in the CNS6,7,10,13. The area of demyelination is larger than in other models (i.e. lysolecithin14) and glial cells, namely astrocytes, are destroyed early in the process. Central axons are chiefly remyelinated by Schwann cells, reflecting the degree of astrocytic damage 14 and disruption of the glial limiting membrane15,16.
Animals and ethidium bromide injection - Immunossupressed9,10 and normal7 Wistar rats of different ages12,17 were injected with either single or multiple local doses of 0.5 to 10 µl of 0,1 % ethidium bromide in saline within the brainstem9,10,12,13 and the spinal cord6,7. (Table 1).
Immunosuppressive treatment - The administratiom of the immunosuppressive drugs began at the same time of the EB injection. Animals treated with cyclophosphamide (CY) had an intraperitoneal injection of two weekly doses (respectively 50 and 30 mg/kg every three days), along the length of the experiment. Animals immunosuppressed with cyclosporine (CsA) had a daily injection of 10 mg/kg in the first week and thereafter the injections were given three times a week, every 48 hours, along the length of the experiment.
Perfusions and sampling of the tissues - The rats were killed by intraaortic perfusion of 4 % glutaraldehyde from 24 h to up to 150 days after injection in the spinal cord and from 7 to 30 days in the brainstem. Transverse slices 1 mm thick of the brain and the spinal cord from the area of the lesions were sampled for light and electron microscopy studies.
Processing of the samples - The samples were washed in phosphate buffer and post-fixed in Millonig's Osmium tetroxide for 2 hours. Routine dehydration and resin embedding procedures were performed 7. Semi-thin sections were stained with Toluidine blue (Methilene blue) and thin sections selected from the former were stained with Uranyl acetate and Lead citrate and examined under an electron microscope.
Demyelinated lesions were induced because of the early disappearance of glial cells7,10,13,17. The loss of astrocytes determined a breach in the glial limiting membrane (GLM) that allowed the free entry of Schwann cells within the CNS territory2,7,8, feature reported elsewhere15,16 .The bulk of the invading Schwann cells is larger than in most toxic demyelinating models where the target of the chemical is the myelin sheath itself14. In the spinal cord the number of invading Schwann cells was much more marked than in the brainstem8,18. The origin of the invading Schwann cells was not definitely sorted out. The proposed origins include the pial and cranial nerves, the dorsal roots and the sympathetic nerves that surround the blood vessels7,18.
In the spinal cord the GLM was restored after the recovery of astrocytes by day 13 after injection in the spinal cord7 and by day 15 in the brainstem10. The new myelin sheaths were easily detected because of their reduced thickness related to axonal diameter7,10,19. The sheaths produced by Schwann cells also showed a basement membrane and collagen fibres around the cell boundaries7,9,18. Excluding minor differences in the morphology and timing of remyelination between the brainstem and the spinal cord, the whole process was very much alike. The burden of myelin repair was carried out by Schwann cells in areas away from the normal tissue7-10 chiefly in subpial and perivascular areas.
Oligodendrocytes produced the new sheaths close to the normal tissue where astrocytic processes were conspicuous (Fig 1A). Oligodendrocytes showed different degrees of activation: in weanling rats they produced huge intracytoplasmic scrolls12 (Fig 1C); in normal adult rats it was detected the degree of slight activation reported in most experimental models when they repair lost myelin sheaths10; in cyclosporine immunosuppressed adult rats oligodendrocytes were more conspicuous and although exhibited a round appearance, they showed a marked increase in the number of rough endoplasmic cisternae9 (Fig 1D). When cyclophosphamide was administered to the rats the whole process of removal of dead cells and disrupted myelin sheaths was delayed, suggesting an interference with macrophagic scavenging activities10.
Glial transplantation studies have shown that minor cell migration is detected when cell cultures enriched for oligodendrocytes precursors are placed within areas of normal and X-irradiated adult CNS20, a situation that mimics what is observed in the EB-induced lesions. Thus, intended transplants must be placed within the lesion area in spontaneous demyelinating diseases. In the EB-induced lesions a thin rim of oligodendrocyte remyelinated axons lined the normal tissue. Although it is known for more than a decade that totipotent neural stem cells occur in the CNS of adult mammals21 full detection of those cells has been elusive. Therefore the origin of the oligodendrocytes that remyelinated the naked axons in our model remains to be defined.
Schwann cells kept a similar behaviour despite the anatomical location and age of the rats, producing myelin for CNS axons in a fast and efficient manner, frequently forming redundant myelin loops (Fig 1B). Other than promoting axonal regeneration in the CNS Schwann cells are extremely effective in repairing areas of central demyelination and restoring conduction. They do so by their remarkably ability to produce trophic factors and cell adhesion molecules22.
In most lesions, either in the brainstem or in the spinal cord lymphocytes have been depicted. They were interpreted as part of the general inflammatory influx induced by the chemical11.Those experiments using multiple injections in the spinal cord23 confirmed the suspicion that there is no immune interference in the EB lesions. The presence of lymphocytes in the early stages of demyelination addresses these cells as a component of the whole inflammatory response that have a brief interaction with macrophages who might be the actual effector cells within the CNS24.
The EB model of experimental demyelination has been useful to demonstrate that Schwann cells are able to repair CNS axonal sheaths of myelin in any situation when astrocytes and the GLM are destroyed even when they are induced to produce their own collagen fibres7,8. Likewise it was possible to assess oligodendrocytes activation according to the age of the rats and under selective immunosuppression and also their dependence on astrocytes to produce the new myelin sheaths.
Acknowledgments - The authors are indebted to the Electron Microscopy Laboratories of the Veterinary Pathology Unit, UFSM, Santa Maria, RS, Histology and Embriology Department, UNICAMP, Campinas, SP and Department of Veterinary Pathology, FMVZ/USP, São Paulo, SP, Brazil
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1Departamento de Patologia, Universidade Federal de Santa Maria (UFSM), Santa Maria RS, 2Universidade Bandeirante (UNIBAN), São Paulo, SP; 3Departamento de Histologia e Embriologia, Universidade Estadual de Campinas (UNICAMP), Campinas, SP; 4Departamento de Patologia Animal, Universidade Federal de Pelotas (UFPel), Pelotas, RS; 5Departamento de Patologia Veterinária, Universidade São Paulo (USP), São Paulo SP, Brasil.
Received 6 October 2000, received in final form 24 January 2001. Accepted 1 February 2001.
Dra. Dominguita L. Graça - Departamento de Patologia UFSM - 97105-900 Santa Maria RS - Brasil. FAX 55 220 8284. E-mail dlgraça@lince.hcv.ufsm.br