Neurotoxicity Comparison Of Local Anesthetics:Amide-bupivacaine Versus Ester-procaine

Local anaesthetics(LAs),which are divided into amide and ester types based on their chemical structure,are frequently used in clinical anaesthesia.However,extensively studies indicated that LAs has displayed cytotoxicity when high concentrations or prolonged use of LAs in many types of cells experiments.There are reports on the toxic effects of LAs in clinical applications.Although toxicity caused by clinically relevant concentrations is rare,it may produce severe complications when it occurs.Specifically,transient neurological symptoms,persistent lumbosacral neuropathy and,the most severe complication,cauda equine syndrome after spinal anaesthesia have attracted attention for many years.For these complications,LA neurotoxicity is thought to be the principle elements.However,the exact mechanisms of LA neurotoxicity are still not fully understood.Many neurotoxicity studies have examined different LAs,but none have comprehensively explored the distinct mechanisms of neurotoxicity caused by amide-(bupivacaine)and ester-(procaine)type LAs.Based on it,we designed this issue.Objects:1.Explore neurotoxicity between bupivacaine and procaine on SH-SY5Y cells.2.Comprehensively compare the mechanism distinction of neurotoxicity caused by bupivacaine and procaine on SH-SY5Y cells and DRG neurons.Methods:1.SH-SY5Y cells were treated with bupivacaine and procaine at different concentrations respectively.Cell viability and cytotoxicity were measured by CCK-8 and LDH assay,and then LD50 of two drugs was calculated by curve fitting which was chosen as the drug concentration for the subsequent cell model.2.SH-SY5Y cells were treated with LD50 values of bupivacaine and procaine,respectively.Subsequently,the mitochondrial calcium content was detected by Rhod-2-AM probe and the mitochondrial membrane potential was detected by JC-1 kit,which reflected the mitochondrial function;the intracellular peroxide content was measured using a DCFH-DA probe,and the intracellular superoxide content was measured by DHE probe,which reflected the ROS production;DNA damage was detected by comet assay,and the content of DNA damage marker protein p-?-H2AX was detected by Western Blot,which reflected the DNA damage;apoptosis was detected by TUNEL assay,the expression of apoptosis related proterin cleaved caspase 3 and cleaved caspase 9 were detected by Western Blot,which reflected the apoptosis.Some of which were also affirmed in DRG neurons.Results:1.Cell viability of SH-SY5Y declined in a concentration-dependent manner caused by bupivacaine and procaine.2.Both bupivacaine and procaine significantly induced mitochondrial calcium overload and mitochondrial membrane potential declined,overproduction of ROS,DNA damage,and apoptosis(P<0.05).There was no statistical difference on mitochondrial injury and apoptosis between bupivacaine and procaine subgroup(P>0.05).But the superoxide anionic level after treated with bupivacaine,which led to more severe early DNA damage,was higher than that with procaine,while procaine produced more peroxidase than bupivacaine.Conclusions:Bupivacaine and procaine exerted neurotoxicity mechanism may refer to mitochondrial dysfunction,ROS overproduction,DNA damage,and apoptosis.The variance of superoxidation and peroxidation remind us that different types of LAs perform neurotoxicity via different pathways.We can make more accurate treatment measures according to their different mechanisms of neurotoxicity.