Study Of MRP1 Expression And Drug Effect To Hippocampus Of Intractable Epilepsy

Objective:To investigate the effects of topiramate,topira-mate and probenecid combine used of them on GFAP protein and MRP1 protein expression in hippocampal CA3 region of the rats with coriaria la-ctone-induced intractable epilepsy and to explore the best method among antiepilepsy treatment. Methods:32 adult male Sprague-Dawley rats,4 group. To model group, union group, topiramate group intramusular injected CL lml/(kg-84h) to preparation rat model intractable epilepsy, Epileptic seizures(ES)were observed within after injection and all rats reached over classⅢseizures according to Racine standard,classⅠ: chewing frequently,classⅡ:muscle of neck convulsion,classⅢ: monolateral foreleg tic,class IV:standing and bilateral foreleg tic, class V: hysterical convulsion. To group model after kinding,then continue to inject CL 1ml/(kg·168h) for four times.To group union after kinding,do gastric lavage with solution topiramate 200mg/(kg·d) and solution probenecid 100mg/(kg·d) for 30 days, continue to inject CL lml/(kg·168h) for four times. To group topiramate after kinding, do gastric lavage with solution topiramate 200mg/(kg·d),continue to inject CL 1ml/(kg·168h) for four times.To group control, do gastric lavage with saline 2ml for 30 days,inject saline 1 mg/kg for four times.All of rats were conventional perfusion fixing by 4% paraformaldehyde,obtaining the specimen and sectioning(4um), hematoxylineasin staining. Apply immunohistochemical staining to sliced specimens with GFAP(1:200), MRP 1(1:100). Around the vascular lesions in biopsy specimens of hippocampus CA3, Ax07TRF-A imaging system is applied,automatic white balance function is turned off while maintaining the same light source and optical conditions,randomly took 5 pictures in this area, analyze with ImagePro-Plus 6.0 image analysis software, to measure the number of MRP1 positive cells and integrated optical density Similarly, around the hippocampus CA3,take picture to measure the number of GFAP positive cells and integrated optical density. Take the average value of each index as representative value of one sample. Statistical analysis is done to these data.Result:1.H-E staining:Neurons in CA3 region in hippocampus of control group rats were observed many in number,and arranged regularly in zonal shape. These cells were complete in conformation with normal extracellular spaces. The pyramidal cells of CA3 region were relatively large, each had a large,round centrally located nucleus surrounded by homogeneous cytoplasm.The nucleus with distinct nucleolus.Nissl's body in these cells appeared as very thin granules.Some cells could be seen with their processes.Arrangement of neurons in CA3 region in hippocampus of model group rats was disordered and had changed in conformation. Neurons of CA3 region were sparse and had obvious neuron loss.Surviving neurons became smaller and shrinked into polygonal or very irregular shape,whose cytoplasm and nuclei were dark-blue stained, obscure in structure. Some could be seen with shortened and dark stained processes. The pericellular and perivascular spaces became large. The number of Neurons in union group and topiramate group increased than that model group,Neurons conformation of Neurons in union group and topiramate group better than that model group.2.Apply GFAP immunohistochemical staining:observe central anterior cortex of the model group in the light microscope positive GFAP immunoreactive astrocytes were seen.The cytoplasm is brown, the shape of cell was spider-like, with radial protrusions around some protrusions were chunky with no branch; some protrusions were slender with a number of very tiny branches.Cellullar swelling can also be seen, appeared as enlarged cell body, light staining of cytoplasmic deep staining of nucleus and some vacuoles inside. The number of immunoreactive astrocytes in model group GFAP is obviously increased than that in control group, increased protrusions and branches and enhanced GFAP immunoreactivity. The GFAP immunoreactivity and integrated optical density in topiramate group and combined treatment group were decreased than that in model group,However,there is no statistical difference between the numbers of positive cells and integrated optical density in the two treatment groups. 3.Apply MRP1 immunohistochemical staining:for control group, only part of the epithelial cell expression of vascular endothelial could be seen; for model group, enhanced epithelial cell staining of blood vessels in the hippocampus could be seen, and brown cytoplasm ofgliocytes could be seen around part of the blood vessels. Enhanced epithelial cell staining of blood vessels in the hippocampus could be seen in topiramate group and combined treatment group, and brown cytoplasm of gliocytes could be seen around part of the blood vessels, there was no statistical difference between the numbers of positive cells and IOD in two treatment groups and in model group.Conclusion:1.Intractable epilepsy caused by intramuscular injections of CL may damage nerve cells, enhanced GFAP expression, and expression of MRP1 is also found.2. In the rat model of intractable epilepsy, topiramate and topiramate plus probenecid treatment can both protect hippocampal neurons, weakening GFAP expression.However,there is no statistical difference between the numbers of positive cells and integrated optical density in the two treatment groups.3. Compared with model group, there is no decrease of gliocytes and IOD for MRP1 expression in topiramate group and combined treatment group.