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. 2018 Feb 19;37(8):e97349. doi: 10.15252/embj.201797349

Figure EV3. The inflammatory cocktail causes pain hypersensitivity through production of increased reactive oxygen species.

Figure EV3

  1. Imaging of DRG neurons loaded with the ROS‐sensitive probe H2DCFDA before (t = 0) and 30 min after inflammatory cocktail application.
  2. Left panel: Fluorescence intensity of cultured DRG neurons loaded with ROS‐sensitive probe H2DCFDA in control neurons (black bars, n = 114), neurons treated with the inflammatory cocktail (red bars, n = 119), and neurons co‐treated with inflammatory cocktail and 4 mM of the ROS scavenger alpha‐phenyl‐N‐tert‐butyl nitrone (PBN, green bars, n = 95). Measure of fluorescence intensity was made 12 and 30 min after drug application. Right panel: positive control for ROS detection. DRG neurons (n = 15) were treated with 0.3% H2O2 and imaged at 12 (light gray) and 30 min (dark gray) after drug application. Fluorescence intensity (B) was analyzed with a non‐parametric Mann‐Whitney U‐test. ***P < 0.001.
  3. Activation curves of Nav1.9 current fitted with single Boltzmann equations giving V 0.5 values of −35.02 ± 0.79 mV (n = 3, black line) and −26.34 ± 1.5 mV (n = 8, green line) for cells treated with the cocktail or with cocktail + NAC, respectively. Current were recorded 10 min after adding drugs.
  4. Comparison of mechanical hypersensitivity induced by intraplantar injection of NAC (n = 5, 20 mM), cocktail (20×, n = 4), and NAC + cocktail (n = 9). Note that the effects of cocktail were strongly reduced by NAC when injected simultaneously.
Data information: All values are shown as mean ± standard error of the mean (SEM).