Volume 134, Issue 4, Pages 1094-1103 (April 2008) Activation of Extracellular Signal-Regulated Protein Kinase in Sensory Neurons After Noxious Gastric Distention and Its Involvement in Acute Visceral Pain in Rats  Jun Sakurai, Koichi Obata, Noriyuki Ozaki, Atsushi Tokunaga, Kimiko Kobayashi, Hiroki Yamanaka, Yi Dai, Takashi Kondo, Kan Miyoshi, Yasuo Sugiura, Takayuki Matsumoto, Hiroto Miwa, Koichi Noguchi  Gastroenterology  Volume 134, Issue 4, Pages 1094-1103 (April 2008) DOI: 10.1053/j.gastro.2008.01.031 Copyright © 2008 AGA Institute Terms and Conditions

Figure 1 Visceromotor response (vmr) to GD. (A) Representative tracings show EMG activity recorded from the acromiotrapezius muscle 7 days after balloon implantation. (B) A summary of intensity-dependent changes in visceromotor response after noxious mechanical stimulation of the stomach. Although 20 and 40 mm Hg of GD did not differ significantly from control, higher distention pressures caused a significant aversive response. Data represent mean ± SEM; n = 10 for each pressure. *P < .05 compared with control rats (0 mm Hg). **P < .05 compared with 60 mm Hg of GD. Gastroenterology 2008 134, 1094-1103DOI: (10.1053/j.gastro.2008.01.031) Copyright © 2008 AGA Institute Terms and Conditions

Figure 2 Mechanical stimulus intensity–dependent p-ERK1/2 expression in DRG and NG neurons. (A) p-ERK1/2 labeling in the T9/10 DRG neurons 2 minutes after GD of 0, 40, 60, and 100 mm Hg. (B) Quantification of the percentage of p-ERK1/2–IR neurons in the T9/10 DRG neurons after mechanical stimulation of the stomach. (C) p-ERK1/2 labeling in the NG neurons 2 minutes after GD of 0, 40, 60, and 100 mm Hg. (D) Quantification of the percentage of p-ERK1/2–IR neurons in the NG after mechanical stimulation of the stomach. Data represent mean ± SD; n = 4 for each pressure. *P < .05 compared with control rats (0 mm Hg). **P < .05 compared with 60 mm Hg of GD. Scale bars, 50 μm (A and C, arrows indicate single-labeled p-ERK1/2–IR neurons). Gastroenterology 2008 134, 1094-1103DOI: (10.1053/j.gastro.2008.01.031) Copyright © 2008 AGA Institute Terms and Conditions

Figure 3 Time course of 100 mm Hg of GD–evoked p-ERK1/2 expression in the (A) T9/10 DRG neurons and (B) NG neurons. Data represent mean ± SD; n = 4 for each time point. *P < .05 compared with naive control. Gastroenterology 2008 134, 1094-1103DOI: (10.1053/j.gastro.2008.01.031) Copyright © 2008 AGA Institute Terms and Conditions

Figure 4 (A and B) Double labeling for p-ERK1/2–IR and FG in the (A) T9/10 DRG neurons and in the (B) NG neurons 2 minutes after 100 mm Hg GD. FG-labeled neurons were labeled by p-ERK1/2–IR (open arrows). (C) Quantification of the percentage of p-ERK1/2–IR neurons of FG-labeled DRG neurons or the percentage of FG-labeled neurons of p-ERK1/2–IR DRG neurons after mechanical stimulation of the stomach. (D) Quantification of the percentage of p-ERK1/2–IR neurons of FG-labeled NG neurons or the percentage of FG-labeled neurons of p-ERK1/2–IR NG neurons after mechanical stimulation of the stomach. Data represent mean ± SD; n = 4 for each pressure. *P < .05 compared with control rats (0 mm Hg). **P < .05 compared with 60 mm Hg of GD. Scale bars, 50 μm. Gastroenterology 2008 134, 1094-1103DOI: (10.1053/j.gastro.2008.01.031) Copyright © 2008 AGA Institute Terms and Conditions

Figure 5 Colocalization of p-ERK1/2 (green) and NF200, TRPV1, or ASIC3 (red) in the (A) T9/10 DRG and (B) NG 2 minutes after 100 mm Hg of GD. Double staining of p-ERK1/2 with NF200, a marker for myelinated A fibers, showed no colocalization in DRG and NG neurons, indicating that ERK1/2 is activated predominantly in unmyelinated C-fiber nociceptors. Open arrows indicate double-labeled neurons with p-ERK1/2– and TRPV1- or ASIC3-IR. Scale bars, 50 μm. Gastroenterology 2008 134, 1094-1103DOI: (10.1053/j.gastro.2008.01.031) Copyright © 2008 AGA Institute Terms and Conditions

Figure 6 (A) Effect of nerve dissection on the visceromotor response to noxious mechanical stimulation. Seven days after surgery, rats underwent noxious GD. Compared with sham-operated rats, subdiaphragmatic vagotomy did not alter the response to 100 mm Hg of GD significantly, whereas it was blunted after splanchnic nerve dissection. Data represent mean ± SEM. *P < .05 compared with control rats (0 mm Hg). #P < .05 compared with sham-operated rats. (B–D) Effect of nerve dissection on ERK1/2 activation in the DRG and NG after noxious mechanical stimulation. (B) p-ERK1/2 labeling in the T9/10 DRG and NG neurons 2 minutes after GD of 100 mm Hg (arrows indicate single-labeled p-ERK1/2–IR neurons). (C) Quantification of the percentage of p-ERK1/2–IR neurons of T9/10 DRG neurons after 100 mm Hg of GD. (D) Quantification of the percentage of p-ERK1/2–IR neurons of NG neurons after 100 mm Hg of GD. Data represent mean ± SD; n = 4 each group. #P < .05 compared with sham-operated rats. Scale bars, 50 μm. Gastroenterology 2008 134, 1094-1103DOI: (10.1053/j.gastro.2008.01.031) Copyright © 2008 AGA Institute Terms and Conditions

Figure 7 (A) Effect of the MEK1/2 inhibitor U0126 on the visceromotor response to noxious mechanical stimulation. Intrathecal U0126 injections (10 μg) 30 minutes before stimulation significantly changed the response to 100 mm Hg of GD. Data represent mean ± SEM. #P < .05 compared with the vehicle control group. (B) Quantification of the percentage of p-ERK1/2–IR neurons in the T9/10 DRG neurons after 100 mm Hg of GD. (C) Quantification of the percentage of p-ERK1/2–IR neurons in the NG neurons after 100 mm Hg of GD. Data represent mean ± SD; n = 4 each group. #P < .05 compared with the vehicle control group. (D) p-ERK1/2 labeling in the T9/10 spinal dorsal horn 2 minutes after GD of 100 mm Hg. U0126 decreased the increase in the number of p-ERK1/2–IR cells in the superficial dorsal horn after noxious GD, compared with vehicle-treated rats. Scale bars, 50 μm. Gastroenterology 2008 134, 1094-1103DOI: (10.1053/j.gastro.2008.01.031) Copyright © 2008 AGA Institute Terms and Conditions