Entwicklung eines Mausmodells für die intrazerebrale Induktion einer zerebralen Immunreaktion

Multiple sclerosis (MS) is an autoimmune inflammatory disorder of the central nervous system (CNS) showing focal demyelinating lesions of the white brain substance which contain T-cells against the endogenous myelin oligodendrocyte protein (MOG). There are different clinical disease’s symptoms such as impaired balance, paralysis or in general cognitive deficits. The exact aetiology of the MS remains unknown. The experimental autoimmune encephalomyelitis (EAE) is an animal model for the MS which allows to examine the disease’s pathogenesis. The laboratory animals get injected subcutaneously above the flanks with an emulsion consisting of complete Freund Adjuvans (CFA) and MOG. In order to intensify the following immune reaction, it’s possible to administer additionally Pertussis toxin (PT) which is known to enhance the permeability of the blood-brain-barrier (BBB). The MOG-containing emulsion leads to an immune response activating naïve CD4+T-cells which differentiate towards Th1- and Th17-cells. These MOG-specific T-cells are now able to migrate into the CNS and destroy the neuron’s myelin sheaths. The inflammation is located in the cerebellum and the spinal cord of the animals who consequently develop impaired balance and paralysis. In this work the induction of the EAE was modified so far as the immune reaction was limited to only the CNS level. To this, the MOG-containing emulsion was injected with help of a stereotactic device directly into the cerebellum of the laboratory mice. The emulsion’s volume was limited to 3 µl/mouse and the MOG concentration to 37 mg/ml. Thus it was guaranteed that despite of the low emulsion’s volume there was still enough MOG-antigen to induce an immune reaction. In order to limit the immune response to the CNS, it was necessary to “turn off” the peripheral immune system. For this, the transgenic mice of the CA-line were used in the experiment. They express specifically the subunits of the IL-12, p35 and p40, under transcriptional control of the GFAP-promotor only in the astrocytes. The p40 subunit is also a part of IL-23 which is why both interleukins 12 and 23 may be synthesised only in the CNS. Thus, the activation of the naïve CD4+T-cells in the peripheral immune system is no longer possible. Stereotactic cerebellum surgery was performed on 88 mice of the CA-line. 14 mice of the C57Bl/6-line, 17 mice of the IL-12p40-/--line and 3 mice of the MyD88-/--line received surgery as well and served as negative control. The animal’s clinical symptoms were evaluated by an EAE score. Within the time of observation of maximal 42 days nearly all the mice of the CA-line developed an ataxia and often circling. Still it’s difficult to evaluate these symptoms as a sign of a specific immune reaction, as the mice of the CA-line tend to develop a spontaneous ataxia beyond the age of 12 weeks. Some of the mice have been observed over 30 days so it was very likely for them to develop a spontaneous inflammation. Furthermore, the cerebellum surgery might have led to symptom’s increase or an earlier onset of the ataxia. Lasting paralysis were not recorded. The histological results and immunochemistry showed clear signs of infection such as dispersed brain tissue and a diffuse local infiltration around the injection spot of CD4+, CD8+, CD45+-lymphocytes and macrophages. Only a few specific activated T-cells could be detected in the FACS-analysis. Altogether 0,2-3% of Th1- and Th17-cells were producing IFN-γ or IL-17. These findings correspond well with the peripheral EAE induction, where similar few specific MOG-activated CD4+-T-cells are found in fresh lesions. In summary, the induction of cerebral immune reaction is possible but there is a clearly weaker immune response compared to a peripheral inducted EAE. In the future, one possibility to prove the MOG-specific activated CD4+-T-cells could be an adoptive transfer. To perform this, immunized T-lymphocytes (Th1- and/ or Th17-cells) should be extracted from the mice after cerebral induction and injected to a wildtype mouse. If there would be any signs of EAE symptoms, a specific T-cell activation would have been proved. In the big picture, the understanding of the cerebral autoimmune reaction may lead towards a therapy in autoimmune diseases such as MS.