| Object:To investigate the expression and functions of matrix metalloproteinase-9 (MMP-9)in the Wistar rat of experimental autoimmune encephalomyelitis(EAE), which is the model of human multiple sclerosis(MS).EAE is a myelin antigen-induced, T cell-mediated autoimmune disease, which is characterized by perivascular inflammatory cell infiltration, BBB breakdown and white matter demyelination. Lewis rat is utilized in the studies of EAE at abroad, which now is recognised as the ideal animal model of MS. Matrix metalloproteinases(MMPs) belong to a large subgroup of proteinases which includes the collagenases, gelatinases and stromelysins,etc. Some studies have discovered that MMPs have several functions such as destroy blood-brain barrier(BBB), degradate extracellular matrix and myelin basic protein(MBP).Researchers have found that microglia, astrocytes, endothelial cells and immune cells such as T cells and macrophages can secrete various MMPs. There are increased expression of MMPs in and around EAE/MS plaques, especially of MMP-9. The present study uses Wistar rat, which is the nonsensitive species for EAE, and detects the expression of MMP-9 in the brain and spinal cord of EAE rats to identify the role of MMP -9 in the cause of EAE. Materials and methods:1. Animals, reagents and apparatus: Wistar rat, female, 180-220g; guinea pig,female, 250-3 50g. Wistar rats were divided into the following 3 groups by random: EAE group,CFA (complete Freund's adjuvant) group and NS group. The reagents such as Goat-anti rat MMP-9 antibody, Streptavidin/Peroxidase Plus Kits were provided by Beijing Zhongshan Biotechnology Company, the apparatus such as UltrasonicCrusher Machine, microtome were provided by the Second Hospital affilated of Zhejing University.2. EAE model was made by subcutaneous injection at the nape with 0.4ml guinea pig brain-spinal cord homogenate in CFA and intrademal injection with l x l010 pertussis simultaneously. CFA and NS groups was injected subcutaneously with 0.4ml CFA or NS respectively. At the nineth day post inoculation(p.i), 10% guinea pig spinal-cord homogenate(GPSCH) or NS was injected subcutaneously, the result was objected at the 12th day.3. Detect the expression of MMP -9 by immunohistochemistry in brain and spinal cord of EAE rat (scale 2-3 ) at 3d, 6-8d and 2weeks after clinical syndrome onset, the result was compared with that of CFA and NS groups.Results:1. The EAE model was induced successfully by injection of guinea pig brain-spinal cord homogenate in CFA and pertussis with morbidity 29.5%, The onset of clinical syndrome begins at 8-10d post-inoculation. The average scale at 14d p.i which reflects the severity of clinical syndrome is 1.94 0.87,whereas no rat falling ill in CFA/NS groups. The result of skin test showed that the average diameter of rash of EAE group is larger than that of CFA/NS groups significantly. HE and Weil staining display pathological changes such as immune mononuclear cells infiltration surround blood vessel and demyelination.2. Immunohistochemistry showed specific MMP-9 expression in EAE brain and spinal cord. The positive signal can be found in infiltrated mononuclear cells and extracellular matrix, long menings and around little blood vessels 3d. But positive infiltrated mononuclear cells reduced at 6-8d post onset. There is few positve MMP -9 signal in CFA/NS groups. The statistics showed significant differences between different groups or different clinical stages. The MMP-9 positive mononuclear cells (3d post onset) are more than that of 6-8d(P<0.05) or 2weeks post onset(P<0.01). Conclusions:1. The EAE model was induced successfully with Wistar rat, which is the nonsensitive species for EAE, whereas the morbidity lower than Lewis rat.Simultaneously, the similarity between EAE and MS wae declared in pathology withthe immune mononuclear cells infiltration and demyelination.2. Specific MMP -9 expression is found by immunohistochemistry in infiltratedmononuclear cells and menings in EAE... |
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