Volume 133, Issue 4, Pages 1282-1292 (October 2007) Transient Receptor Potential Vanilloid 1 Mediates Hyperalgesia and Is Up-Regulated in Rats With Chronic Pancreatitis  Guang–Yin Xu, John H. Winston, Mohan Shenoy, Huaizhi Yin, Swaroop Pendyala, Pankaj Jay Pasricha  Gastroenterology  Volume 133, Issue 4, Pages 1282-1292 (October 2007) DOI: 10.1053/j.gastro.2007.06.015 Copyright © 2007 AGA Institute Terms and Conditions

Figure 1 Morphologic changes, expression of NGF-like immunoreactivity in the pancreas and time course of prolonged pancreatic hyperalgesia in rats after TNBS infusion. (A–D) H&E-stained pancreatic sections at (A and B) 3 and (C and D) 12 weeks after infusion of the pancreatic duct with (A and C) saline or (B and D) TNBS. (A and C) No significant morphologic changes were observed in control rats. In TNBS-treated rats, inflammatory infiltrates with large regions of acinar loss and periductular and intralobular fibrosis were seen. (B) Acinar tissue was replaced with tubular structures, but islets of Langerhans appeared to be intact. (D) Similar pathology was observed in the pancreata of TNBS-treated rats up to 12 weeks postinfusion. (E–H) NGF immunohistochemistry in pancreatic sections from control and TNBS-treated rats 3 and 12 weeks postinduction. In the pancreatic sections from (E and G) saline-treated rats, NGF-like immunoreactivity was detected primarily in the islets and in the endothelium of blood vessels and ducts. In contrast, in pancreatic sections at (F) 3 and (H) 12 weeks after TNBS infusion, NGF-like immunoreactivity was observed in tubular structures in addition to duct and blood vessel endothelium and islets. (I–K) The number of nocifensive behaviors in response to a 5-minute application of (I) 2-mA, (J) 5-mA, and (K) 10-mA current to the pancreas were measured at 3, 8, and 12 weeks after pancreatic duct infusion with TNBS (•) or with saline (○). The number of nocifensive behaviors started to increase significantly at 3 weeks post-TNBS infusion compared with controls. This pancreatic hyperalgesia persisted for at least 12 weeks. *P < .05, **P < .01, ***P < .001. n = 6–10 per group. Gastroenterology 2007 133, 1282-1292DOI: (10.1053/j.gastro.2007.06.015) Copyright © 2007 AGA Institute Terms and Conditions

Figure 2 TNBS-induced CP potentiates CAP-activated currents. (A) An example of a sustained current evoked by CAP application (1 μmol/L) to a pancreas-specific DRG neuron from control rats (con). The membrane potential was held at −60 mV. The solid line above each trace indicates the period of CAP application. This inward current was blocked by pretreatment with capsazepine (czp, 10 μmol/L). (B) An example of a sustained current evoked by CAP (1 μmol/L) application to a pancreas-specific DRG neuron from rats 3 weeks after TNBS injection. This inward current was blocked by pretreatment with capsazepine (10 μmol/L). (C) Bar graph shows the mean peak current densities in pancreas-specific DRG neurons from control and TNBS-treated rats. The mean peak current density measured in TNBS-treated rats was 4.4 times larger than that measured in control rats (control, 6.8 ± 2.6 pA/pF, n = 8; TNBS, 29.6 ± 5.5 pA/pF, n = 13; *P < .02). (D) Activation time of CAP-evoked current was reduced significantly from 10.5 ± 0.5 seconds (n = 6) in control to 3.4 ± 0.5 seconds (n = 7) in TNBS-treated rats (*P < .001). (E) The percentage of CAP-responding DRG neurons was increased significantly in TNBS-treated rats when compared with controls (*P < .05). (F) In CAP-responding pancreas-specific DRG neurons, the percentage of small (diameter, <25 μm), medium (diameter, >25 μm but <35 μm), and large (diameter, >35 μm) size neurons were the same in both groups. (C–E) □, CON; ■, TNBS. (F) ■, Small; □, medium; , large. Gastroenterology 2007 133, 1282-1292DOI: (10.1053/j.gastro.2007.06.015) Copyright © 2007 AGA Institute Terms and Conditions

Figure 3 Enhanced expression of TRPV1 protein in DRGs after 3 weeks post-TNBS treatment. (A) Western blots showing TRPV1 expression from T9–13 DRGs of rats from vehicle (con) and TNBS treatment (tnbs). Glyceraldehyde-3-phosphate dehydrogenase was used as loading control. (B) Mean densitometry levels for TRPV1 expression relative to control animals. The expression level of TRPV1 was enhanced significantly in DRGs from TNBS-treated rats (n = 6) as compared with the controls (n = 5, *P < .05). (C–H) Immunocytochemical analysis of TRPV1-positive neurons. An example of TRPV1-positive neurons (green) in T11 DRG section from (C) control and (F) TNBS-treated rats. DiI-labeled neurons (red) in the same section of DRGs from (D) control and (G) TNBS-treated rats as in C and F, respectively. (E and H) Merge of TRPV1-positive staining and DiI labeling (yellow). (I) Bar graph shows the percentage of TRPV1-positive, DiI-labeled neurons in total DiI-labeled DRG neurons. The percentage of pancreas-specific neurons expressing TRPV1 was significantly higher in the TNBS-treated group than in controls (*P < .05). Gastroenterology 2007 133, 1282-1292DOI: (10.1053/j.gastro.2007.06.015) Copyright © 2007 AGA Institute Terms and Conditions

Figure 4 Up-regulation of TRPV1 mRNA expression in pancreas-specific DRG neurons. (A–C) Illustration of the LCM process: (A) before LCM, 2 neurons labeled with CTB in blue; (B) after LCM, CTB-labeled neurons were lifted from the tissue section (note the empty space). (C) CTB-labeled cells in DRG section were captured and transferred to the film. (D) Bar graph shows TRPV1 mRNA levels in CTB-labeled neurons from control and TNBS-treated rats. TNBS treatment significantly enhanced mRNA level of TRPV1 compared with controls (*P < .05). Gastroenterology 2007 133, 1282-1292DOI: (10.1053/j.gastro.2007.06.015) Copyright © 2007 AGA Institute Terms and Conditions

Figure 5 TRPV1 antagonism diminishes TNBS-induced pancreatic hyperalgesia. (A) Three and (D) 12 weeks after TNBS injection, 200 nmol (in 500 μL) of SB-366791, selective TRPV1 antagonist was injected intraperitoneally into TNBS-treated rats (n = 8 and 6, respectively). The numbers of nocifensive behaviors induced by 2-, 5-, and 10-mA current stimulation of the pancreas were reduced significantly 30 minutes after SB-366791 injection (*P < .05). Vehicle treatment had no significant effect on the numbers of nocifensive behaviors both (B) 3 and (E) 12 weeks after TNBS injection. In saline-injected control rats (C, 3 wk; F, 12 wk), SB-366791 treatment did not have any significant effect. □, Pretreatment; ■, posttreatment. Gastroenterology 2007 133, 1282-1292DOI: (10.1053/j.gastro.2007.06.015) Copyright © 2007 AGA Institute Terms and Conditions

Figure 6 TRPV1 antagonism attenuates referred somatic hyperalgesia associated with CP. Response frequencies to mechanical stimulation of the abdomen with VFF of various strengths before (PRE) and after intraperitoneal injection of (A) SB-366791 or (B) vehicle in TNBS-treated rats (n = 4). SB-366791 significantly reduced the response frequency to mechanical stimulation of the abdomen in TNBS-treated rats. *P < .05, **P < .01 compared with PRE. □, PRE; ■, SB-366791. Gastroenterology 2007 133, 1282-1292DOI: (10.1053/j.gastro.2007.06.015) Copyright © 2007 AGA Institute Terms and Conditions