Gene Expression Profiles Of Bupivacaine-induced Neurotoxicity In The Rat Hippocampus

Objective To observe the gene expression profiles of bupivacaine-induced neurotoxicity in the rat hippocampus and to research the mechanism of bupivacaine-induced neurotoxicity at gene level.Methods 6 Sprague-Dawley(SD)rats were allocated randomly to 2 groups: control group(n = 3)and bupivacaine group(n = 3).Rats in each group were randomly numbered.Rats received water and food ad libitum in one week of acclimation.After that rats were exposed to sevoflurane in self-made anesthesia box.Bupivacaine group was received intravenous infusion of 0.75% bupivacaine via tail vein;control group was injected with saline via tail vein.Hippocampal tissue was immediately removed when rats in bupivacaine group appeared irritability,ataxia,convulsions,other toxic reactions and convulsive waveform appeared in EEG.Rats were broken the neck to death.The hippocampal tissue was taken under the anatomical microscope and taken into physiological saline with RNase-Free.And then it was cleaned and stored in liquid nitrogen.The total RNA of hippocampal tissue was isolated by Trizol reagent.The purity and content of RNA were measured by UV spectrophotometer.According to Oligotex m RNA Midi Kit,m RNA was purified.The single-stranded c DNA was amplified through reverse transcription reaction by using m RNA as template and four kinds of deoxyribonucleic acid as raw material;another single-stranded c DNA was amplified by using the single-stranded c DNA as template and four kinds of deoxyribonucleic acid as raw material.Two DNA chains synthesized double stranded c DNA.The labeled c RNA in bupivacaine group was synthesized from Cy3-DTP by using c DNA as template and labeled c RNA in control group was synthesized from Cy5-DTP by using c DNA as template.After the c RNA was purified and fragmentated by RNeasy kit,it was tested by Test3.And then the c RNA was hybridized with affymetrix rat genome 230 2.0 array.When rats in the bupivacaine group appeared convulsions,the dosage of bupivacaine was recorded.Using SPSS 17.0 statistical software,the measurement data were expressed by mean±standard deviation((?)±s).Chips were scanned by using Gene Chip Scanner 3000 software.Using Microarray Suite Version 5.0 software we analyzed those differentially expressed genes to find the altered gene expression profile of hippocampus between two groups.Results(1)The dosage of bupivacaine: when rats in the bupivacaine group appeared convulsions,the dosage of bupivacaine was(5.50±0.66)mg/kg.(2)The results of differentially expressed genes: a total of 107 genes in the bupivacaine group changed significantly compared with the control group,of which 80 genes(e.g.Arid5 a,Atf3,Bax,Caspase-3,Egr1,Fcgr3,Fgl2,Fos,Hspb1,Jun,Klf10,Lp1,Litaf,Mt,Nans,Nr4a1,Pde4 b,Plaa,Ptgs2 and Timpl)were up-regulated and 27 genes(e.g.Bcl-2,Eef2 k,Fmo5,Grm3,Lrrfip2,Npy5 r,Orc4,PI3 K,Rrag B,Sdpr,Tpmt,Strbp,Wasl and Znf386)were down-regulated.(3)Analysis of biological information: the differentially expressed genes were main classified into apoptosis related genes,signal transduction related genes,transcription related genes,metabolism related genes and enzymes related genes.Conclusion(1)Bupivacaine could induce the significant alteration of gene expression profile in rats hippocampus.It suggested that bupivacaine-induced neurotoxicity was the result of multiple genes.(2)The apoptosis related genes and signal transduction related genes were dominant in those differentially expressed genes by analyzing function of protein encoded by genes.It came to a conclusion that neurotoxicity was caused by neuronal apoptosis through multiple signaling pathways and multiple targets.Our current study laid the foundation for a more thorough study of those genes that caused neurotoxicity.