Activation of p47phox as a Mechanism of Bupivacaine-Induced Burst Production of Reactive Oxygen Species and Neural Toxicity

Figure 2

NOX inhibition protected cells and rats from bupivacaine-induced toxicity via reducing excessive production of ROS. SH-SY5Y cells were pretreated with VAS2870 (10 μM) 30 min prior to bupivacaine treatment. (a) SH-SY5Y cells were treated with bupivacaine (1.5 mM) for 1 h, 2 h, and 3 h; the production of cytoplasmic peroxide (DCFH-DA) and O₂^.− (DHE) was measured by a microplate reader. (b, c) SH-SY5Y cells were treated with bupivacaine for 3 h. The level of cytoplasmic peroxide (DCFH-DA, green) and O₂^.− (DHE, red) was measured by microscopy. (d) The production of cytoplasmic peroxide (DCFH-DA) and O₂^.− (DHE) was measured by a microplate reader. (e, f) The protein level of cleaved caspase-3 and phospho-γ-H2A.x after bupivacaine treatment. (g, h) The ratio of apoptotic cells was detected by TUNEL staining. Cells tagged by white arrows were TUNEL positive. (i) The basic lines of paw mechanical thresholds were tested before treatment. Paw mechanical threshold was tested by the electronic von Frey system after different treatments. (j, k) Schematic diagram indicates the area of tested sections in the spinal dorsal horn. The level of cytoplasmic O₂^.− (DHE, red) was measured by microscopy. (l, m) The ratio of apoptotic cells was detected by TUNEL staining. Cells tagged by white arrows were TUNEL positive. (n, o) The protein level of cleaved caspase-3 was measured by Western blot. Data represent mean ± SD of at least 3 independent experiments or for 6 rats each group. Scale bar: 50 μm. versus control group. ^# versus Bup group. ^& versus VAS group.

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

(j)

(k)

(l)

(m)

(n)

(o)