The Effects Of Chloroquine On Cell Cycle Expression Of Gfap In The Brain Of Rat With Seizures Induced By Pentylenetrazole

Objective:Observation of different intervention dose of chloroquine on cell cycle and expression of GFAP,c-Fos and CyclinD1 in hippocampus and cerebral cortex of rats with seizures induced by pentylenetrazole. State of chronic epilepsy and chloroquine treatment in brain astrocyte proliferation and activation status,the role of antiepileptic mechanism of chloroquine and the best dose.Methods:Experiment1, An experimental animal model experiments, group,behavioral observation and cell cycle.sixty healthy male SD rats randomly divided into control group(10 rats injected with saline according to 40mg/kg for 4 weeks);50 for the production of animal models of chronic epilepsy(at 40mg/kg injection of PTZ).According to Racine marking where a row of at least 5 times made by two or more are considered modeling success.After the success of modeling pentylenetetrazol injection every five days timeto be consolidated.The longest continuous injection of pentylenetetrazol two weeks,fifty rate were all modeling success.The fifty rats were randomly divided into PTZ-induced epilepsy group(Sham intervention group,10 to 40mg/kg normal saline for 2 weeks),chloroquine,an intervention group(20mg/kg),2chloroquine intervention group(30mg/kg injection,3 intervention in chloroquine intervention group(40mg/kg),4chloroquine intervention group(50mg/kg).After PTZ injection2 hours,, respectively,2 hours after chloroquine treatment to observe and record the behavior of 6 groups and the EEG changes in classification by Racine score standards;3 weeks after intervention treatment with 3% chloral hydrate(1ml/100g)anesthesia,brains were removed quickly,seperating the cerebral cortex and hippocampus.Kit using DNA ploidy chromosome staining on glial cells,flow cytometry cell cycle,cell cycle changes observed in each group.Experiment2, Immunohistochemical GFAP,C-Fos, CyclinD1expression.Preparation of animal model by grouping,methods,ibid.Extraction of bilateral cerebral cortex and hippocampus,4% paraformaldehyde,conventional tissue processing production.Detected by immunohistochemistry GFAP protein expression in different tissues,and observed in each group GFA-positive astrocytes in shape,number and changes in expression levels of GFAP,simultaneous detection of C-Fos,CyclinD1protein in astrocytes.By HPLAS-1000 high-definition imaging analysis system for image analysis using GFAP antibody for glial cytoplasm stained GFAP-positive glial cells in each group shape,number and expression level of GFAP.Manual count:total 7 rats in each group of 20 slices,ecch slice in the 10×20 times in 10 randomly selected perspective view,count the number of positive cells,whichever is average,simultaneous determination of integrated optical density of positive cells,contained The mean value.Follow the steps to block animal brain tissue extracts, detected by Western blot CyclinD1 protein.Results:1, behavioral observations, the majority of animals injected with nine days (d) above GradeⅡafter onset, all animals were injected with 14d, andⅡand above attack.Control group had no epileptic seizures,chloroquine treatment group 1 compared with the epilepsy group was no significant difference(P<0.05).The other three in the intervention group compared with the epilepsy group were significantly different(P<0.05)2.EEG records, the control group without epileptiform discharges,epilepsy group showed frequent high-amplitude sharp waves,spikes and slow wave or spike-slow wave complex,the intervention group showed slow wave or spike-slow wave complx,the intervention group showed slow waves and small spikes,with the intervention dose increase,epilepsy waves weakened.3.Flow cytometry tests showed,PTZ-induced epileptic group of cortex and hippocampus the strongest proliferative activity of astrocytes,S phase percentage of an average of 47%,control group,the proportion of S phase average 0.5%.With the intervention dose of indicators of normalcy.4.Immunohistochenistry revealed that the epilepsy group than the normal control group,the number of astrocytes significantly increased(P < 0.01),and unequally distributed, The intervention group compared with the epilepsy group started to decrease the the number of glial cells,the cell morphology has beenrestored.With the intervention dose increased ,the number of glial cells,a progressive decline,stabilization on relatively fixed five value. The difference between the group and the epilepsy group was significantly(P<0.05 ,P<0.01).GFAP expression was higher in the epileptic group strong,and to the hippocampus as a,compared with the control group there was significant difference in(P<0.05).5.Western blot display the content of CyclinD1 up the epileptic group,followed by four in the intervention group decreased in all groups compared with the control group, there were significant differences(P<0.05).Conclusion:1, using 0.5% can be successfully rat pentylenetetrazol model of chronic epilepsy.2. Effect of chloroquine on chronic epileptic seizures in rats with the intervention in the intervention increased dose at frist and then became a stable increase.3.Chloroquine inhibited the activation of Ast and epilepsy state,by restraining the proliferation of astrocyte and the expression of GFAP CyclinD1 and C-Fos in the Ast..4.Chloroquine treatment dose was increased to certain values,adverse drug reactions increased,while the intervention did not significantly increase,the optimum dose range is 30mg/kg-40mg/kg.In this study,the frist 2,3,4conclusions have not been reported at homa and abroad.