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3R-Project 101-06
Organotypic CNS slice cultures as an in-vitro model for immune mediated tissue damage and repair in multiple sclerosis
Norbert Goebels
University Hospital, Dept. of Neurology, Clinical Neuroimmunology
8091 Zürich, Switzerland
norbert.goebels@usz.ch
Keywords: mice; brain; slices; reduction; replacement
Duration: 3 years End of the Project: 2009
Background and Aim
Multiple sclerosis (MS) is a chronic demyelinating disease of the central nervous system (CNS) affecting about one out of 700 young adults in Switzerland. Histological data and results from animal experiments indicate that T lymphocytes, B cells, macrophages and antibodies contribute to the formation of immune-mediated lesions in the brain and spinal cord of MS patients. The presence of clonally expanded immunoglobulins in the cerebrospinal fluid (CSF) of MS patients is one of the most prominent hallmarks of the disease. However, neither the specificity of these immunoglobulins nor the triggers causing demyelination and axonal damage are known (Sospedra and Martin, 2004)
A large proportion of current MS research is currently performed in a model system called experimental autoimmune encephalomyelitis (EAE). For the induction of EAE, rodents or nonhuman primates are actively immunized with myelin antigens, thus inducing an inflammation of the CNS. This inflammation leads to a progressive paralysis of the animals before they eventually are sacrificed. While CNS inflammation can also be caused by adoptive transfer of MOG-reactive T-cells alone, antibodies are additionally required for the induction of demyelination in rats and nonhuman primates. Likewise, deposits of immunoglobulins and complement can be detected in MS lesions, supporting the role of antibodies in MS pathogenesis. Although EAE has provided many valuable insights into the pathology of MS, it only partially reflects MS and it is difficult to discern the role of different arms of the immune system with respect to the induction of CNS damage. Using organotypic CNS slice cultures, we have therefore established an experimental model system in which the complex architecture of the CNS is maintained and which allows the detailed investigation of interactions between the CNS and components of the immune system, as well as mechanisms of CNS damage and repair.
Method and Results
in progress (present status)
Organotypic slice cultures provide an excellent tool to characterize the role of humoral and cellular effector mechanisms in MS. In order to establish antibody-mediated tissue damage, we used an antibody specific for myelin (against MOG, myelin oligodendrocyte glycoprotein) in the presence of complement (Figure 1, B). Addition of the antibody alone or of an isotype antibody with and without complement did not induce morphological changes (Figure 1).
In addition, we established confocal live imaging of transgenic mice expressing green fluorescent protein (GFP) in myelin (PLP-GFP). Time-lapse imaging showed that demyelination starts already after 4 hours of antibody/complement addition (Figure 2, lower panel). After removal of the detrimental factors antibody/complement, marked remyelination was observable (Figure 3, upper panel). In ongoing experiments, we are currently evaluating the role of cellular immune components (macrophages, T cells) and cytokines in axonal damage. Additionally, we have started to evaluate different strategies to improve remyelination.
Conclusions and Relevance for 3R
Reduce:
Using slice cultures of hippocampus and cerebellum, several experimental conditions can be tested with tissue obtained from a single mouse. In contrast, for the induction of EAE, several mice have to be included per experimental group. Using transgenic mice expressing GFP in myelin, experiments can be investigated by (confocal) live imaging.
Refine:
Induction of EAE leads to severe disability and suffering of mice. Organotypic slice cultures are performed after euthanasia, which substantially refines the experimental procedures. Bystander reactions are less likely to happen in an in-vitro setup than
in-vivo. Additionally, organotypic slice cultures reflect the three-dimensional in-vivo structure more precisely than conventional cell cultures.
Replace:
Organotypic slice cultures present an alternative tool to study the interactions between the CNS and specific components of the immune system. Therefore, many questions, which are now investigated in the animal model of MS, can alternatively be studied using this in-vitro system, thus replacing many in-vivo experiments.
References
1. Sobottka, B; Harrer, M D; Ziegler, U; Fischer, K; Wiendl, H; Hünig, T; Becher, B; Goebels, N (2009). Collateral bystander damage by myelin-directed CD8+ T cells causes axonal loss. American Journal of Pathology, 175(3):1160-1166.
2. Harrer M.D. (2008) Organotypic CNS Slice Cultures as an in vitro Model for Damage and Repair in Multiple Sclerosis, Dissertation at the University of Zürich, Switzerland, 1-88.
Figures
Figure 1: Antibody/complement-mediated demyelination. Demyelination was induced by the addition of a myelin-specific antibody and complement (B). The antibody alone (A) or an isotype antibody without (C) or with (D) complement did not induce morphological changes. Myelin is visualized by staining against myelin basic protein (MBP).
Figure 2: Live imaging of cerebellar slices from transgenic mice expressing GFP in myelin. Pictures show the extent of demyelination in 4-hour intervals. Upper panel: Untreated slice cultures. Lower panel: Slices treated with the anti-myelin antibody and complement.
Figure 3: Live imaging of remyelination after antibody/complement-mediated demyelination in PLP-GFP transgenic mice. Addition of the anti-MOG antibody and complement results in a strong demyelination (A versus B). Pronounced remyelination can be observed after removal of the detrimental factors anti-myelin antibody and complement (C). No marked changes were observed by the addition of an irrelevant antibody with complement (D-F). Minor changes are due to the long cultivation period of the organotypic CNS slices. Original magnification 4x.
| Dernières modifications: 29.08.2010 |  |