Local anaesthetics (LAs) may lead to neurological complications, but the underlying mechanism is still unclear. Many neurotoxicity research studies have examined different LAs, but none have comprehensively explored the distinct mechanisms of neurotoxicity caused by amide- (bupivacaine) and ester- (procaine) type LAs. Here, based on a CCK8 assay, LDH assay, Rhod-2-AM and JC-1 staining, 2′,7′-dichlorohy-drofluorescein diacetate and dihydroethidium probes, an alkaline comet assay, and apoptosis assay, we show that both bupivacaine and procaine significantly induce mitochondrial calcium overload and a decline in the mitochondrial membrane potential as well as overproduction of ROS, DNA damage and apoptosis (P < 0.05). There were no significant differences in mitochondrial injury and apoptosis between the bupivacaine and procaine subgroups (P > 0.05). However, to our surprise, the superoxide anionic level after treatment with bupivacaine, which leads to more severe DNA damage, was higher than the level after treatment with procaine, while procaine produced more peroxidation than bupivacaine. Some of these results were also affirmed in dorsal root ganglia neurons of C57 mice. The differences in the superoxidation and peroxidation induced by these agents suggest that different types of LAs may cause neurotoxicity via different pathways. We can target more accurate treatment based on their different mechanisms of neurotoxicity.

Bupivacaine, a commonly used local anesthetic, has potential neurotoxicity through diverse signaling pathways. However, the key mechanism of bupivacaine-induced neurotoxicity remains unclear. Cultured human SH-SY5Y neuroblastoma cells were treated (bupivacaine) or untreated (control) with bupivacaine for 24 h. Compared to the control group, bupivacaine significantly increased cyto-inhibition, cellular reactive oxygen species, DNA damage, mitochondrial injury, apoptosis (increased TUNEL-positive cells, cleaved caspase 3, and Bcl-2/Bax), and activated autophagy (enhanced LC3II/LC3I ratio). To explore changes in protein expression and intercommunication among the pathways involved in bupivacaine-induced neurotoxicity, an 8-plex iTRAQ proteomic technique and bioinformatics analysis were performed. Compared to the control group, 241 differentially expressed proteins were identified, of which, 145 were up-regulated and 96 were down-regulated. Bioinformatics analysis of the cross-talk between the significant proteins with altered expression in bupivacaine-induced neurotoxicity indicated that phosphatidyl-3-kinase (PI3K) was the most frequently targeted protein in each of the interactions. We further confirmed these results by determining the downstream targets of the identified signaling pathways (PI3K, Akt, FoxO1, Erk, and JNK). In conclusion, our study demonstrated that PI3K may play a central role in contacting and regulating the signaling pathways that contribute to bupivacaine-induced neurotoxicity.

Bupivacaine has been shown to induce neurotoxicity through inducing excessive reactive oxygen species (ROS), but the underlying mechanism remains unclear. NOX2 is one of the most important sources of ROS in the nervous system, and its activation requires the membrane translocation of subunit p47phox. However, the role of p47phox in bupivacaine-induced neurotoxicity has not been explored. In our in vitro study, cultured human SH-SY5Y neuroblastoma cells were treated with 1.5 mM bupivacaine to induce neurotoxicity. Membrane translocation of p47phox was assessed by measuring the cytosol/membrane ratio of p47phox. The effects of the NOX inhibitor VAS2870 and p47phox-siRNA on bupivacaine-induced neurotoxicity were investigated. Furthermore, the effect of VAS2870 on bupivacaine-induced neurotoxicity was assessed in vivo in rats. All these changes were reversed by pretreatment with VAS2870 or transfection with p47phox-siRNA in SH-SY5Y cells. Similarly, pretreatment with VAS2870 attenuated bupivacaine-induced neuronal toxicity in rats. It is concluded that enhancing p47phox membrane translocation is a major mechanism whereby bupivacaine induced neurotoxicity and that pretreatment with VAS2870 or local p47phox gene knockdown attenuated bupivacaine-induced neuronal cell injury.