The Fuction Of MicroRNA On The Memory Disorders In PTZ-induced Epileptic Rats

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Grouping pentylenetetrazol-induced epileptic rats according to memory impairment AIMTLE-related cognitive disorders have been characterized by experimental models and memory and learning deficits were compared after preconditioning.However,standardized neuropsychological tests to analyze memory impairment in epileptic rat models remain elusive.Material and Method1.TLE preconditioning was performed by a single intraperitoneal injection of PTZ(60 mg/kg)on the first day,followed by repeated injections of PTZ(35 mg/kg)on alternating days between 8:00 and 10:00AM starting on the 3rd day,with a total of 14 injections.The control group rats received 0.9% physiological saline(10 ml/kg)i.p.every other48 hours as sham preconditioning,with 15 injections total.The rats were weighed daily,and the dosage was adjusted according to body weight.The animals were monitored for 30 min after each PTZ treatment.Seizures were rated according to Racine's scale.Kindling was defined as?4 consecutive stage 2 seizures,or two seizures that were stage 4 or higher.2.The MWM test consisted of a place navigation test and a spatial probe test.The memory performance of all successfully established TLE rats and normal rats was evaluated 24 h after the last PTZ administration.The memory-impairment evaluation system for epileptic rats was established as follows.Two scores were obtained as a reflection of normal memory function according to data collected from the 10 rats in the normal group during two MWM sessions(<15 s escape latency for the place navigation test on day 5 and >20 crossings in 400 s for the spatial probe test).Then,6 rats were selected from the 17 TLE animals because their data overlapped the scores of the normal rats;these 6 rats formed the TLE control group(TLE-C).3.The memory impairment in TLE(TLE-MI)group included the 10 rats with the worst performance in the MWM experiment(> 50 s escape latency for the place navigation test on day 5 and <10 crossings in 400 s for the spatial probe test).Then,6 rats each were randomly selected from the TLE-C group and from the TLE with memory impairment group(TLE-MI),and micro RNA expression patterns in these rats were investigated using micro RNA array and differential analyses.Results1.After the first kindling in response to PTZ(60 mg/kg)(i.p.),followed by 14 repeated kindling stimulations with 35 mg of PTZ/kg(i.p.)in every 48 hours,TLE models were successfully established in 52 of the60 rats in the PTZ group;2.In total,17 TLE rats obtained scores of <15 s for escape latency on day 5 and >20 crossings in 400 s;these scores overlapped with the scores calculated for the 10 rats that underwent normal sham preconditioning and had normal memory function.3.Additionally,10 rats with the worst performance in the MWM experiments obtained scores of >50 s for the escape latency on day and<10 crossings in 400 s.These rats were classified as exhibiting memory impairment according to the grouping method applied to the 52 TLE rats,representing an incidence of 19.23%(10/52)memory impairment.Conclusion1.In this study,a model of temporal lobe epilepsy(TLE)was induced via pentylenetetrazol(PTZ)kindling in Sprague-Dawley rats.2.We screened PTZ kindling-induced TLE rats using the Morris water maze test to identify the TLE rats suffering from memory impairment among the rats with normal cognitive function.Part ? micro RNA expression profiles and function analysis in the hippocampusAIM Previous studies have demonstrated a close relationship between abnormal regulation of micro RNA(miRNA)and various types of diseases,including epilepsy and other neurological disorders of memory.,the effects of miRNAs on memory deficits in epilepsy alone have not been reported.Therefore,we profiled miRNA expression in the hippocampus of a PTZ-kindled rat with TLE-associated memory deficits and characterized the effects of specific miRNAs.Material and Method1.The rats were then sacrificed,and the hippocampus were rapidly harvested and removed to cryogenic vials with RNAlater.Mi RNA microarray analysis was performed on total hippocampal RNA from TLE rats with memory disorders and from TLE rats with normal memory performance(n = 6 for each group).Both groups were analyzed to detect dysregulated miRNAs in the hippocampus;2.Quantitative real-time PCR validation of the initial results: To validate the initial results detected by miRNA microarray in this study,4miRNAs were selected for an additional q RT-PCR analysis from hippocampus and peripheral blood samples.3.miRNA Target Gene Prediction and Functional Analysis: Potential target genes of the differentially expressed miRNAs were predicted from data in the Micro Cosm,mi Randa,and mi RDB databases using our proprietary database.Using this identified target gene set,the significant Gene Ontology(GO)classifications and Kyoto Encyclopedia Genes and Genomes(KEGG)pathways over-represented among these target genes from two of three databases were provided to obtain useful information regarding the functions of the targets(a p-value < 0.05 is recommended).Results1.Four up-regulated miRNAs(mi R-34 c,mi R-374,mi R-181 a,and mi R-let-7c-1)and seven down-regulated miRNAs(mi R-1188,mi R-770-5p,mi R-127-5p,mi R-375,mi R-331,mi R-873-5p,and mi R-328a)were found.2.Some of the dysregulated miRNAs(mi R-34 c,mi R-1188 a,mi R-328 a,and mi R-331)were confirmed using q RT-PCR,and their blood expression patterns were identical to those of their counterparts in the rat hippocampus.3.Based on the data from the mi Randa,Micro Cosm,and mi RDB databases within our proprietary database,1971,3699,and 750 predicted target genes of the 11 dysregulated miRNAs were identified in the mi Randa,Micro Cosm and mi RDB databases,respectively,with 345 miRNA target genes integrated from two of these three databases and 23 target genes integrated from all three databases.The targets of these dysregulated miRNAs and other potentially enriched biological signaling pathways were analyzed using bioinformatics.Following these results,the MAPK,apoptosis and hippocampal signaling pathways might be involved in the molecular mechanisms underlying the memory disorders of TLE.Conclusion1.A distinct patterns of miRNA expression were observed in the TLE rats with memory disorders compared with those rats whose memory was not affected by TLE treatment.2.The MAPK,apoptosis and hippocampal signaling pathways might be involved in the molecular mechanisms underlying the memory disorders of TLE.Part ? p38 MAPK inhibitor restore memory impairment in epilepsyAIM We already used microarrays to compare the differences of miRNA expression profiles in the epilepsy rats with different memory function,and found that 11 miRNAs was deregulated and the MAPK signaling pathway was the most enriched pathway according to the KEGG pathway analysis regarding over-represented target genes.So we hypothesized that the deregulation of MAPK pathway may play an important role in memory impairment of epilepsy basing on miRNA regulation.Material and methodswe administrated a p38 MAPK inhibition(SB203580)treatment for rats with epilepsy induced by PTZ.1.SD rats were randomly separated to 3 groups: Normal control group(0.9 % Na Cl 5ml/Kg ip.for 30 days),PTZ + 0.9 % Na Cl group(PTZ 60mg/Kg ip.for the first day,PTZ 35mg/kg and 0.9 % Na Cl5ml/Kg ip.on alternative days for 14 times/28 days)and PTZ +SB203580 group(PTZ 60mg/Kg ip.for the first day,PTZ 35mg/kg and SB203580 2mg/kg ip.on alternative days for 14 times/28 days).2.Morris water maze test were performed 24 hours after the last injection;3.The expression of p-p38 MAPK and caspase-3 in the brain tissue from each group were detected using western blotting.Results1.We found that the inhibition caused a down-regulation of p-p38 MAPK expression,and a better studying and memory ability according to the performance in the Morris Water Maze(MWM)test.2.We also found a decreased expression of the caspase-3 after SB203580 treatment.So we propose that a intervention of p38 MAPK pathway may be a potential way to restore memory impairment and neurodegeneration in chronic epilepsy.There was nothing changed from PTZ + SB203580 compared to Normal control group.Conclusion1.MAPK pathway,especially the p38 MAPK pathway take part in the development of memory impairment in epilepsy.2.p38 MAPK inhibitor SB203580 could adverse the memory disorders by deregulating the expression of p-p38 MAPK and neuronal apoptosis.