The Research Of The Calpain Expression In The Cortex Of Laboratory Rats With Experimental Autoimmune Encephalomyelitis

ObjectiveThe expressions of Calpain I and Calpain II in the brains of laboratory rats with experimental autoimmune encephalomyelitis (EAE) were investigated. EAE is mediated by the T-lymphocyte, and it is an autoimmune disease exhibited in laboratory animals, presenting with the following characteristics: demyelization of CNS, destruction of blood-brain barrier (BBB), and infiltration of mononuclear lymphocytes in cerebrospinal substance and peri-vascular regions. Calpain was a calcium-dependent, neutral protease, and it was already proved to participate in the development of EAE. In this study, Wistar rat, non-sensitive to EAE, was used to establish EAE model, and the changes in the expressions of Calpain I and Calpain II in the cerebral cortex during the progression of EAE were dynamically observed. It proved that the established EAE model using Wistar rats was plausible. The pathophysiological process of EAE was also investigated, to provide theoretical basis for the clinical treatment of acute diffuse encephalomyelitis (ADEM), as well as being evidence for the duration and the best applied time of calpastatin during the therapy.Method1. Fetal Wistar rats of 6 to 8 weeks, provided by our hospital research center, were selected as research subjects. The mixture of cerebrospinal mixture of guinea pig and complete freund's adjuvant was used as antigen, which was subcutaneously injected in the four footpads of Wistar rat, while the solution of Pertusis was injected on the back of foot, to establish EAE model. 2. The pathological changes of the brain of EAE lab rats were HE stained and observed under light microscope.3. For the normal group, the post-immunization 7d group, and the post-incidence 3d, 7d, 14d, 21d groups, they were all treated with BrdU and GFAP immunohistochemical staining, before dynamically observing the distribution and the pattern of expressions of Calpain I and Calpain II in the brains of EAE rats of different groups at different time periods.Results1. Under light microscope, the cerebral and spinal tissues of EAE group showed large scale of infiltration by inflammatory cells, and they were primarily lymphocytes. The vascular endothelial cells were proliferated, swollen, and destroyed. Glial cells also proliferated. Inflammatory cells aggregated around blood vessels to form a typical "cuff-like" change. In the meningeal and spinal membranes, there were infiltrations of inflammatory cells. The brains and spinal cords of rats of the control group showed no abnormalities.2. For the Calpainl-positive cells, their cytoplasm was stained to brownish yellow color. Few Calpainl-positive cells were seen in the brain tissues of lab rats of the normal control group. After the immunological administration of the mixture of spinal cord of guinea pig, the number of Calpainl-positive cells gradually increased, and reached maximum level at 7th day after the incidence, as well as an increase in the staining reaction was seen. It slowly decreased afterward, but even remained higher than normal level after 21 days, which had statistical significance when compared to the normal control group (p < 0.05). When comparing the number of Calpainl-positive cells in the normal group, the post-immunization 7d group, and the post-incidence 3d, 7d, 14d, 21d groups, they all showed statistical significance (p < 0.05). The comparison of the post-incidence 7d group and the rest also showed statistical significance as well (p<0.05).3. For the Calpainll-positive cells, their cytoplasm was stained to brownish yellow color. Few CalpainII-positive cells were seen in the brain tissues of lab rats of the normal control group. After the immunological administration of the mixture of spinal cord of guinea pig, the number of CalpainII-positive cells gradually increased and reached maximum level at 3rd days after the incidence. It slowly decreased afterward, but even remained higher than normal level after 21 days. When comparing the number of CalpainII-positive cells in the normal group, the post-immunization 7d group, and the post-incidence 3d, 7d, 14d, 21d groups, they all showed statistical significance (p < 0.05). The comparison of the post-incidence 3d group and the rest also showed statistical significance as well (p < 0.05). The comparison of the post-incidence 7d group and the post-incidence 14d group had no statistical significance (p > 0.05).ConclusionThe method for establishing EAE model was stable, reliable, and with high success rate. Calpain I and Calpain II were widely available in the CNS of normal laboratory rats. In the progression of EAE, the expressions of Calpain I and Calpain II were increased in the cerebral cortical regions of lab rats.