Figure 1.
The effect of increasing concentrations of bupivacaine on SH-SY5Y cell viability.
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.
Figure 2.
SH-SY5Y cell viability following treatment with 1 mM bupivacaine (%, mean±S.D, n = 6).
aP<0.05 vs. S group; bP<0.05 vs. S+NNC 100 group; cP<0.05 vs. S+B group; dP<0.05 vs. S+B+NNC 10 group; eP<0.05 vs. time point of 6 hours; fP<0.05 vs. time point of 12 h.
Figure 3.
Bupivacaine treatment leads to the release of LDH.
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). aP<0.05 vs. S group; bP<0.05 vs. S+NNC100 group; cP<0.05 vs. S+B group; dP<0.05 vs. S+B+NNC 10 group; eP<0.05 vs. time point of 6 h; fP<0.05 vs. time point of 12 h.
Figure 4.
Bupivacaine treatment leads to an increase in cytosolic Ca2+ ([Ca2+]i).
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. aP<0.05 vs. S group; bP<0.05 vs. S+NNC 100 group; cP<0.05 vs. S+B group; dP<0.05 vs. S+B+NNC 10 group.
Figure 5.
NNC 55-0396 dihydrochloride protects SH-SY5Y cells from bupivacaine-induced apoptosis.
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. aP<0.05 vs. S group; bP<0.05 vs. S+NNC 100 group; cP<0.05 vs. S+B group; dP<0.05 vs. S+B+NNC 10 group.
Figure 6.
NNC 55-0396 dihydrochloride protects SH-SY5Y cells from bupivacaine-induced nuclear alterations during apoptosis.
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. Nuclear morphology was evaluated by Hoechst 33258 staining (×200). Apoptotic cells were observed to have condensed or segmented nuclei accompanied by bright blue fluorescence.
Table 1.
Apoptosis measured by Hoechst 33258 staining (%, mean±S.D, n = 6).
Figure 7.
Inhibition of T-type calcium channels prevents bupivacaine-induced cleavage of caspase-3.
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. aP<0.05 vs. S group; bP<0.05 vs. S+NNC 100 group; cP<0.05 vs. S+B group; dP<0.05 vs. S+B+NNC 10 group.