Cellular Mechanisms Of Bupivacaine Induced Neurotoxcity Injury In Type 2 Diabetic Mice

BackgroundBupivacaine is widely used in clinical for peripheral nerve block,intrathecal anesthesia and post-operative analgesia.However,it can induce transient neurologic syndrome and hyperalagesia,and even cause cellular swelling,mitochondrial dysfunction,or even apoptosis in some severe cases,which is called "local anesthetics neurotoxicity".The incidence of this type of neurotoxicity is even higher in diabetic patients,yet the overall mechanisms are unknown.It is suggested that bupicacaine induced calcium overload is the major source of neurotoxicity,which can induce cellular hypersensitivity and open the downstream pathways of cellular injury and apoptosis.However,the cellular mechanism for the calcium overload is obscure.Transient receptor potential vanilloid 1(TRPV1)can be activated by bupivacaine and hence induce calcium influx.However,whether TRPV1 is involved in bupivacaine induced neurotoxicity and the enhancement under diabetic,is unknown.We aimed to investisgate the role of TRPV1 in bupivacaine-induced neurotoxicity.ObjectiveWT and Trpvl-/-dorsal root ganglion neurons(DRG)were cultured to test the sensitivity of bupivacaine induced calcium overload.To test whether bupivacaine induce more calcium influx under hyper glucose environment via TRPV1,WT andTrpvl-/-DRG neurons were tested after incubation in high concentration glucose culture medium for 6 hours.We also test the TRPV1 sensitivity after high glucouse incubation.Furthermore,in order to miminic the diabetic environment,we induced a mouse model of type 2 diabetes by high fat diet and streptozotocin(STZ)intrapeitoneal injection.Diabetic WT and Trpv-/-mice were tested by using behavior assay,immunofluoresence staining,quantitative real-time PCR,Western-blot assay and calcium imging.We aimed to test whether the bupivacaine neurotoxicity is more severe due to the increased sensitivity and expression of TRPV1 in diabetic mice.Material and methods1.AnimalsAll animal experiments were conducted under the Protocol of Sourthern Medical University.C57BL/6J mice were used as WT.Trpvl-/-mice were gifted from Dr.Qin Liu of Department of Anesthesiology,Washington Univesity in St.Louis.PCR assay was used for genotyping.Littermates from heterozygous breeding cages were used for behavior assay and experimenters were blind to all mouse genotypes.PCR results and foot tattoo markers were used to identify the groups and genotypes after data analysis.2.Bupivacaine induced calcium overload in cultured DRG neurons via TRPV1DRGs were harvested from 3 to 4 weeks old WT and Trpvl-/-mice.After 24 hours' incubation,neurons were applied to Fura-2AM loading for 3 Omins before calcium imging assay.Bupivacaine and capsicin(a specific agonist of TRPV1)were applied in the calium imaging buffer to test the response in WT neurons.Bupivacaine buffer was applied for 2 min to test the calcium overload in WT and Trpvl-/-neurons.The peak of 340/380 ratio was analysed and the area under curve was evaluated for the calcium response in the software.3.Bupivacaine induced calcium overload in high glucose via TRPV1DRGs were harvested from 3 to 4 weeks old WT and Trpvl-/-mice.After 18 hours' incubation,neurons were applied to high glucose medium or normal glucose medium for 6 hours's incubation.Fura-2AM loading for 30 mins before calcium imging assay.Bupivacaine buffer was applied for 2 min to test the calcium overload in WT and Trpvl-/-neurons.Different concentrations of capsicin were applied in the calium imaging buffer to test the TRPV1 sensitivity in WT neurons.The peak of 340/380 ratio was analysed and the area under curve was evaluated for the calcium response in the software.4.Bupivacaine induced neurotoxicity in diabetic mouse via TRPV1Type 2 diabetic mouse was induced by STZ intraperitoneal injection after 3 weeks' high fat diet.The control group received normal fat diet and the vehicle injection at the same period.Blood glucose over lO.Ommol/L was considered as the successful diabetic model 4 weeks after streptozotocin injection.WT and Trpvl-/-mice were included in the diabetic group and the control group.Von-frey hair test was administrated 5 hours after bupivacaine(15mM,10?L)intrathecal to test the hyperalagesia different between WT and Trpvl-/-mice.Calcium imaging and TUNEL assay were applied on the cultured neurons from diabetic and control WT and Trpvl-/-mice.Before TUNEL assay,cultured neurons were treated with 1.5mM bupivacaine for 3 hours,and incubated for 21 hours.5.TRPV1 sensitivity and expreesion examination in diabetic miceWT mice were induced into type 2 diabetic mouse model by STZ intraperitoneal injection after 3 weeks' high fat diet.Control group received normal fat diet and the vehicle injection at the same period.Blood glucose over 10.0mmol/L was considered as the successful diabetic model 4 weeks after streptozotocin injection.Behavioral assasy were tested in WT control and diabetic mice.Tail immersion test was applied to the tails of the mice with 48? heat bath to test the heat sensitivity.Capsaicin intraplantar injection was applied to the hindpaw to test the TRPV1 acitivation behavior.DRGs from WT control group and diabetic group were harvested to incubate for 24 hours before Fura-2AM calcium imaging assay.Different concentrations of capsacin were applied to test the sensitivity of TRPV1.Immunofluoresence staining was employed to test the expression level of TRPV1 in purfused DRG sections from control and diabetic mice.The ratio of TRPV1 positive neurons was counted compared to the total neurons in the same section of the L4 and L5 DRG sentions.To compare the mRNA expression of TRPV1 in control and diabetic mice,quantitative real-time PCR was employed.All DRGs from two groups were collected on ice and keep in-80? until RNA extraction.cDNA was compiled using the Transcript First Strand cDNA Synthesis Kit(Roche,Basel,Switzerland).Real-time PCR was performed using Light Cycler 480 SYBR Green I Master Mix(Roche,Basel,Switzerland)and a Light Cycler 480 II Real-Time PCR instrument(Roche,Basel,Switzerland),according to the manufacturer's recommended protocol.Primers were TRPV1-F:5'-CCA CTG GTGTTGAGA CGC C-3',TRPV1-R:5'-TCT GGG TCT TTG AAC TCG CTG-3'.The cDNA is 167bp.Western blot was used to analysis the TRPV1 protein expression in control and diabetic mice.6.Bupivacaine induced calcium overload is relayed on extracellular calciumCulured DRG neurons from WT and Trpvl-/-control and diabetic mice were tested after 24 hours' incubation.Calcium free and calcium buffer were applied to the neurons together with bupivacaine.Of note,calcium was replaced with EGTA in the calcium free imaging buffer.Different concentrations of bupivacaine were tested and the dose-curve response was analysed.7.Statistics analysisData was arranged by mean±sem.All data was calculated with GraphPad Prism 5.0 software.An un-paired t-ttest was used to compare two groups,and one-way ANOVA analysis was administred between three groups.Statistic difference was met when P<0.05.Results1.TRPV1 is involved in bupivacaine induced calcium overload in DRG neurons1.1 Bupivacaine induced calcium influx in capsaicin positive DRG neuronsWT cultured DRG neurons were tested by calcium imaging 24 hours after incubation.Bupivacaine can directly activate capsaicin(agonist of TRPV1)positive DRG neurons.1.2 Bupivacaine induced calcium overload was reduced in Trpvl-/-miceBupivacaine was applied for 2min after a baseline observation.And the response curve of neurons kept elevating while the Trpvl-/-neurons showed a decline trend,P<0.001.2.TRPV1 is involved in bupivacaine induced calcium overload in high glucose environment2.1 Bupivacaine induced calcium overload in high glucose environment is TRPV1 dependent.Cultured neurons of WT and Trpv1-/-mice were treated with high glucose(50mM)for 6 hours before Fura-2AM calcium imaging assay.Bupivacaine induced calcium response was larger in high glucose incubated WT neurons,P<0.05.Bupivacaine induced calcium response was the same in high glucose incubated Trpv1-/-neurons.WT neurons showed larger response then Trpv1-/-neurons in both normal and high glucose condictions,P<0.05.2.2 Capsaicin sensitivity was increased in high glucose environmentWT neurons was dissociated and incubated for 18 hours,then 6 hours of high glucose medium before Fura-2AM calcium imaging assay.The result indicated that more neurons were capsaicin sensitive in high glucose incubated then normal glucose,P<0.05.3.Bupivacaine induced neurotoxicity in diabetic mice is related to TRPV13.1 The fat-fed STZ mouse modelHigh fat diet followed by strptozotocin intraperitoneal injection induced type 2 diabetes model in WT and Trpvl-/-mice.There was no difference in the blood glucose level between two genotypes before the high fat diet feeding.Blood glucose level increased significantly in diabetic group at week 3 and week 7,when compared to the baseline,as well as to the control group.There was no difference in blood glucose level between week 3 and week 7 in diabetic WT and Trpvl-/-mice,P<0.001.3.2 Motor block after bupivacaine intrathecalBupivacaine(15mM,10 ? L)was intrathecally injection in control and diabetic WT and Trpvl-/-mice.Motor block time prolonged in diabetic group when compared to the control group(P<0.05),there is no difference between genotypes(P>0.05)·3.3 Mechanical stumilus test after bupivacaine intrathecalBupivacaine(15mM,10 ?L)was intrathecally injection in control and diabetic WT and Trpvl-/-mice.Five hours after bupivacaine injection,von-Frey hair test was applied and hyprealgesia(P<0.05)was induced in both control and diabetic mice in two genotypes.Diabetic Trpvl-/-mice showed no difference when compared to the control group(P>0.05).3.4 Calcium imagingCalcium imaging assay was conducted after 24 hours' cultured of WT and Trpvl-/-control and diabtes DRG neurons.The response of the diabetic group was significantly higher than the control group in both genotypes(P<0.001).But the increasement was less in the Trpv1-/-diabetic neurons than WT(P<0.01).3.5 TUNEL assayBupivacaine treatment(1.5mM,3 hours)was applied in cultured neurons from WT and Trpvl-/-control and diabtes DRG neurons which were cultured for 24 hours.TUNEL assay indicated more apoptosis neurons were found in diabeties group,when compared to the control group(P<0.05).Less apoptosis neurons were found in Trpvl-/-in both diabtes and control group(P>0.05),when compared to the WT group,respectively(P<0.05).4.TRPV1 sensitivity but not expression was increased in diabetic mice4.1 Tail immersion test is hypersensitive in diabetic miceWT mice showed reduced tail flicking(48?)threshold in diabetic group when compared to the control group,P<0.001.4.2 WT diabetic mice showed increased response to capsaicin intraplantarFliching response was increased in diabetic WT after capsaicin intraplantar in the hindpaw,when compared to the control group(P<0.001).Licking time was longer in diabetic WT when compared to the control group(P<0.01).4.3 Diabetic WT neurons exhibit hypersensitivity to capsaicinControl and diabetic WT DRGs were dissociated and cultured for 24 hours before calcium imaging assay.The proportion of capsaicin positive neurons was significant higher in the diabetic group when compared to the control group(P<0.05).4.4 TRPV1 immunofluoresence positive neurons is unchanged in diabetic miceThe proportion of TRPV1 immunofluoresence positive neurons was the same in L4 DRG in diabetic WT mice and the control mice(P>0.05).4.6 TRPV1 mRNA and protein expression in diabetic WT DRGsThe expression of TRPV1 mRNA and protein of the fat-fed streptozotocin WT mice showed no significant difference when compared to the control group(P>0.05).5 Bupivacaine induced calcium influx is extracellular calcium and dose dependent5.1 Bupivacaine induced calcium overload is extracellular calcium dependentCultured DRG neurons from control and diabetic mice were test on calcium imaging buffer and calcium free imaging buffer.Bupivacaine can only induced calcium influx in calcium imaging buffer,but not calcium free imaging brffer.5.2 Bupivacaine dose-dependent induced calcium overload in DRG neuronsBupivacaine dose-dependent induced calcium overload in cultured DRG neurons.There were significant difference between the different proportion of the responsed neurons,respetively:0.1mM(35.81±4.10)%,1mM(66.54±3.49)%,10mM(65.85±4.23)%,.The proportion is significant higher in 1mM and 10mM when compared to 0.1mM(P<0.01).However,there is no significant difference bwtween 1mM and 10mM(P>0.05).Conclusion1.Bupivacaine induced calcium overload is related to TRPV1,and dependent on extracellular calcium concentration.2.Bupivacaine induced calcium overload is increased in high glucose incubation,which related to the increased sensitivity of TRPV1.3.TRPV1 is involved in bupivacaine induced calcium overload,apoptosis and mechanical hypersensitivity in mice.In type 2 diabetic mice,the sensitivity of TRPV1 enhances,which result is higher calcium overload,apoptosis and mechanical hypersensitivity.