基于基因组学探讨鞘内注射罗哌卡因对大鼠脊髓的影响

Effects of intrathecal injection of ropivacaine on spinal cord in rats based on genomics

目的:临床上有罗哌卡因致神经损伤的病例报道,但罗哌卡因致神经损伤的机制仍未完全阐明。本研究探讨0.5%和1.0%的罗哌卡因鞘内注射后6和24 h,大鼠机械痛阈的改变以及脊髓L_(3)基因组学的变化,以探究罗哌卡因致神经损伤的机制。方法:选用30只成年雄性SD大鼠,在鞘内置管成功后随机分为对照组和罗哌卡因1~4组(n=6)。对照组鞘内置管后以8.3μL/h速度连续鞘内注射生理盐水24 h,注射完成后停药24 h。罗哌卡因1组、罗哌卡因2组在鞘内置管后以8.3μL/h的速度连续于鞘内注射1%罗哌卡因24 h,罗哌卡因3组、罗哌卡因4组于鞘内置管后以8.3μL/h的速度连续于鞘内注射0.5%罗哌卡因24 h;罗哌卡因1组、罗哌卡因3组注射完成后停药6 h,罗哌卡因2组、罗哌卡因4组注射完成后停药24 h。各组大鼠在给完药物即刻以及相应停药时间后进行行为学观察,并使用压力探针法观察大鼠机械痛阈的改变,完成行为学观察后取脊髓L_(3)样本使用Illumina双端测序构建基因文库,运用统计学方法寻找差异表达基因,并对差异表达基因进行基因本体富集分析和京都基因与基因组百科全书信号通路分析,再用real-time RT-PCR法验证部分差异表达基因。结果:与对照组比较,罗哌卡因1~4组大鼠在注射完成时痛阈均增加;停药对应时间后的痛阈恢复程度与给药浓度呈负相关、与停药时间呈正相关。与对照组比较,罗哌卡因1组差异表达的基因有488个,其中456个上调,32个下调;罗哌卡因2组差异表达的基因1194个,其中1092个上调,102个下调;罗哌卡因3组差异表达的基因518个,其中384个上调,134个下调;罗哌卡因4组差异表达的基因68个,其中46个上调,22个下调。基因本体富集分析和京都基因与基因组百科全书信号通路分析显示差异表达的基因主要与炎症反应相关信号通路有关。结论:0.5%和1.0%罗哌卡因鞘内注射24 h后,大鼠脊髓L_(3)的差异表达基因主要与炎症反应相关信号通路有关。

Objective:There are clinical reports of nerve injury caused by ropivacaine.The mechanism for nerve injury induced by ropivacaine has not been fully clarified.This study aims to investigate the changes of pain threshold and L_(3)spinal cord genomics at 6 h and24 h after intrathecal injection of 0.5%and 1.0%ropivacaine,and to explore the underlying mechanisms for nerve injury caused by ropivacaine.Methods:A total of 30 male Sprague Dawley rats weighing 220-260 g were successfully implanted with microspinal catheter.The rats were randomly divided into 5 groups(each n=6):a control group(given saline),a ropivacaine group 1 and a ropivacaine group 2(both given 1%ropivacaine),a ropivacaine group 3 and a ropivacaine group 4(both given 0.5%ropivacaine).The rats received continuous intrathecal injection of corresponding drugs at8.3μL/h for 24 h via an implanted intrathecal catheter followed by 24 h-pause of injection for the ropivacaine group 2,the ropivacaine group 4 and the control group,6 h-pause of injection for the ropivacaine group 1 and the ropivacaine group 3.For each group,the observation of behavioral change and the paw withdrawal mechanical threshold(PWMT)was conducted immediately after the injection and again after the pause of injection.After the PWMT observation,the rats were dissected to acquire L_(3)spinal cords.Illumina sequencing was applied to construct gene libraries.Then the statistical methods were used to find out differentially expressed genes between the groups.Gene Ontology(GO)enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)signaling pathway analysis were conducted for those genes.Real-time RT-PCR was used to determine different expressions of some of those genes.Results:Compared with control group,the PWMT got higher in the ropivacaine group 1-4and was positively correlated with concentration,negatively correlated with discontinuation duration.Compared with control group,the ropivacaine group 1 had 488 differentially expressed genes,of which 456 were up-regulated and 32 were down-regulated;the ropivacaine group 2 had 1194 differentially expressed genes,of which 1092 were upregulated and 102 were down-regulated;the ropivacaine group 3 had 518 differentially expressed genes,of which 384 were up-regulated and 134 were down-regulated;and the ropivacaine group 4 had 68 differentially expressed genes,of which 46 were up-regulated and 22 were down-regulated.GO enrichment analysis and KEGG signaling pathway analysis showed that most of these differentially expressed genes were related to signaling pathways of inflammatory response.Conclusion:After intrathecal injection of 0.5%ropivacaine and 1.0%ropivacaine for 24 h,the differentially expressed genes in L_(3)spinal cord of rats are mainly related to signaling pathways of inflammatory response.