Neurotoxicity induced by bupivacaine via T-type calcium channels in SH-SY5Y cells.

There is concern regarding neurotoxicity induced by the use of local anesthetics. A previous study showed that an overload of intracellular calcium is involved in the neurotoxic effect of some anesthetics. T-type calcium channels, which lower the threshold of action potentials, can regulate the influx of calcium ions. We hypothesized that T-type calcium channels are involved in bupivacaine-induced neurotoxicity. In this study, we first investigated the effects of different concentrations of bupivacaine on SH-SY5Y cell viability, and established a cell injury model with 1 mM bupivacaine. The cell viability of SH-SY5Y cells was measured following treatment with 1 mM bupivacaine and/or different dosages (10, 50, or 100 µM) of NNC 55-0396 dihydrochloride, an antagonist of T-type calcium channels for 24 h. In addition, we monitored the release of lactate dehydrogenase, cytosolic Ca(2+) ([Ca2+]i), cell apoptosis and caspase-3 expression. SH-SY5Y cells pretreated with different dosages (10, 50, or 100 µM) of NNC 55-0396 dihydrochloride improved cell viability, reduced lactate dehydrogenase release, inhibited apoptosis, and reduced caspase-3 expression following bupivacaine exposure. However, the protective effect of NNC 55-0396 dihydrochloride plateaued. Overall, our results suggest that T-type calcium channels may be involved in bupivacaine neurotoxicity. However, identification of the specific subtype of T calcium channels involved requires further investigation

Inhibition of T-type calcium channels prevents bupivacaine-induced cleavage of caspase-3.

<p>SH-SY5Y cells were either pretreated with the indicated concentrations of NNC 55-0396 dihydrochloride or left untreated prior to 1 mM bupivaine exposure for 24 h. Procaspase-3 (inactive form) and cleaved caspase-3 (active form) expression was measured by western blot analysis (mean+S.D, n = 6). Lane 1 = S group; Lane 2 = S+NNC 100 group; Lane 3 = S+B group; Lane 4 = S+B+ NNC 10 group; Lane 5 = S+B+NNC 50 group; Lane 6 = S+B+NNC 100 group. <i>^aP</i><0.05 vs. S group; <i>^bP</i><0.05 vs. S+NNC 100 group; <i>^cP</i><0.05 vs. S+B group; <i>^dP</i><0.05 vs. S+B+NNC 10 group.</p

Apoptosis measured by Hoechst 33258 staining (%, mean±S.D, n = 6).

a<p><i>P</i><0.05 vs. S group;</p>b<p><i>P</i><0.05 vs. S+NNC 100 group;</p>c<p><i>P</i><0.05 vs. S+B group;</p>d<p><i>P</i><0.05 vs. S+B+NNC 10 group.</p

Bupivacaine treatment leads to an increase in cytosolic Ca²⁺ ([Ca2+]_i).

<p>SH-SY5Y cells were either pretreated with the indicated concentrations of NNC 55-0396 dihydrochloride or left untreated prior to 1 mM bupivaine treatment for 24 h. [Ca2+]_i levels were measured by Quest Fluo-8 AM ester (mean±SD, n = 6)). A: Representative image of Quest Fluo-8 AM ester flow cytometry analysis. B: [Ca2+]_i levels in the different treatment groups. <i>^aP</i><0.05 vs. S group; <i>^bP</i><0.05 vs. S+NNC 100 group; <i>^cP</i><0.05 vs. S+B group; <i>^dP</i><0.05 vs. S+B+NNC 10 group.</p

NNC 55-0396 dihydrochloride protects SH-SY5Y cells from bupivacaine-induced apoptosis.

<p>Cells were either treated with the indicated concentrations of NNC 55-0396 dihydrochloride or left untreated prior to 1 mM bupivaine treatment for 24 h. Apoptosis was measured by Annexin-V staining with flow cytometry (%, mean±SD, n = 6). A: Representative image from the flow cytometric analysis. B: Rates of apoptosis in the different treatment groups. <i>^aP</i><0.05 vs. S group; <i>^bP</i><0.05 vs. S+NNC 100 group; <i>^cP</i><0.05 vs. S+B group; <i>^dP</i><0.05 vs. S+B+NNC 10 group.</p

The effect of increasing concentrations of bupivacaine on SH-SY5Y cell viability.

<p>SH-SY5Y cells were exposed to different concentrations of bupivacaine (0.1, 0.5, 0.75, 1, 2, 5, and 10 mM). The viability of the cells declined with increasing bupivacaine concentration.</p

Bupivacaine treatment leads to the release of LDH.

<p>SH-SY5Y cells were either pretreated with the indicated concentrations of NNC 55-0396 dihydrochloride or left untreated prior to 1 mM bupivaine treatment for 24 h. LDH release was determined by the level of LDH activity present in the culture media. (%, mean±S.D, n = 6). <i>^aP</i><0.05 vs. S group; <i>^bP</i><0.05 vs. S+NNC100 group; <i>^cP</i><0.05 vs. S+B group; <i>^dP</i><0.05 vs. S+B+NNC 10 group; <i>^eP</i><0.05 vs. time point of 6 h; <i>^fP</i><0.05 vs. time point of 12 h.</p

SH-SY5Y cell viability following treatment with 1 mM bupivacaine (%, mean±S.D, n = 6).

<p><i>^aP</i><0.05 vs. S group; <i>^bP</i><0.05 vs. S+NNC 100 group; <i>^cP</i><0.05 vs. S+B group; <i>^dP</i><0.05 vs. S+B+NNC 10 group; <i>^eP</i><0.05 vs. time point of 6 hours; <i>^fP</i><0.05 vs. time point of 12 h.</p