Volume 130, Issue 6, Pages 1729-1742 (May 2006) The Role of Neurokinin 1 Receptors in the Maintenance of Visceral Hyperalgesia Induced by Repeated Stress in Rats  Sylvie Bradesi, Efi Kokkotou, Simos Simeonidis, Simona Patierno, Helena S. Ennes, Yash Mittal, James A. McRoberts, Gordon Ohning, Peter McLean, Juan Carlos Marvizon, Catia Sternini, Charalabos Pothoulakis, Emeran A. Mayer  Gastroenterology  Volume 130, Issue 6, Pages 1729-1742 (May 2006) DOI: 10.1053/j.gastro.2006.01.037 Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 1 Effect of peripheral administration of the NK1R antagonist SR140333 (1 mg/kg IP) on the VMR to CRD (day 11). (A) EMG amplitude expressed as mean change from baseline after treatment with vehicle or SR140333 in rats previously exposed to repeated WA stress. IP injection of SR140333 abolished the WA stress-induced increase in the VMR to CRD at pressures of 40 and 60 mm Hg. (B) EMG amplitude expressed as mean change from baseline after treatment with vehicle or SR140333 in rats previously exposed to repeated sham WA stress. SR140333 did not affect the EMG response after chronic sham WA stress compared with vehicle. Data are expressed as mean ± SE, n = 6 in each group, *P < .05 significantly different compared with vehicle, Student t test. Gastroenterology 2006 130, 1729-1742DOI: (10.1053/j.gastro.2006.01.037) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 2 Effect of central administration of the NK1R antagonist SR140333 (20 μg/kg intrathecally) on the VMR to CRD (day 11). (A) EMG amplitude expressed as mean change from baseline after treatment with vehicle or SR140333 in rats previously exposed to repeated WA stress. Intrathecal injection of SR140333 abolished the chronic stress-enhanced VMR to CRD compared with vehicle at the pressures of distention of 40 and 60 mm Hg. (B) EMG amplitude expressed as mean change from baseline after treatment with vehicle or SR140333 in rats previously exposed to chronic sham WA stress. SR140333 did not affect the EMG response after chronic sham WA stress compared with vehicle. Data are expressed as mean ± SE, n = 6 in each group, *P < .05 significantly different compared with vehicle, Student t test. Gastroenterology 2006 130, 1729-1742DOI: (10.1053/j.gastro.2006.01.037) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 3 Expression of NK1Rs in L1–L2 spinal cord segments following repeated WA stress. (A) Levels of mRNA for NK1R measured by real-time quantitative RT-PCR. We observed a significantly higher level of NK1R mRNA in samples from chronic WA stress rats compared with control. Data are expressed as the relative NK1R PCR product to that of the housekeeping gene GAPDH, mean ± SEM, n = 8 in each group, *P < .05, significantly different from sham WA, Student t test. (B) Levels of NK1R protein measured by Western blotting. A significant increase in the level of NK1R proteins in samples from chronic WA stress rats compared with control was observed. Data are expressed as normalized optical density, mean ± SEM, n = 8 in each group, *P < .05, significantly different from sham WA, Student t test. (C) Representative Western blotting for NK1R in spinal cord extracts showing a prominent band at ∼65 kilodaltons. Gastroenterology 2006 130, 1729-1742DOI: (10.1053/j.gastro.2006.01.037) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 4 Representative illustration of Western blots showing the migration of spinal cord extracts incubated either with the primary anti-NK1R serum alone (A1) or the primary anti-NK1R serum preabsorbed with the receptor peptide fragment (B1); preincubation with the antigenic receptor fragment reduces the intensity of staining compared with A1. Similar results were observed with a positive control load migrating at the same level (∼65 kilodaltons); preabsorption with the receptor peptide fragment attenuated the intensity of staining (B2) compared with incubation with primary antibody alone (A2). Gastroenterology 2006 130, 1729-1742DOI: (10.1053/j.gastro.2006.01.037) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 5 Representative confocal images of NK1R IR neurons in the dorsal horn of the L1–L2 spinal segment of rats subjected to (A) chronic sham WA stress or (B) chronic WA stress. Images in the main panels were taken with a 20× objective (scale bars = 50 μm) and consist of 2 optical sections 2.53 μm thick separated 2.48 μm. Images in the insets were taken with a 100× objective (scale bars = 10 μm) and consist of 2 or 3 optical sections 0.62 μm thick separated 0.57 μm. The densest NK1R immunostaining is found in laminae I of the dorsal horn. In both sham WA and WA stress conditions, immunoreactivity was predominant on the plasma membrane. Gastroenterology 2006 130, 1729-1742DOI: (10.1053/j.gastro.2006.01.037) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 6 Quantification of NK1R IR neurons in the laminae I of the dorsal spinal cord from rats previously subjected to chronic WA stress or control sham WA stress. There are an increased number of NK1R-expressing neurons in the spinal segment from stressed rats compared with control. Results are expressed as the mean number of NK1R IR-positive cells in the laminae I of one side of the dorsal horn from 5 sections/rat; mean ± SEM, n = 8 rats in each group, *P < .05, significantly different from sham WA, Student t test. Gastroenterology 2006 130, 1729-1742DOI: (10.1053/j.gastro.2006.01.037) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 7 Expression of NK1Rs in the distal colon. (A) Levels of mRNA for NK1Rs measured by semiquantitative RT-PCR. A significant reduction in the level of NK1R mRNA was observed in samples from chronic WA stress rats compared with controls. Data are expressed as the amount of NK1R PCR product relative to that of 18S PCR product (arbitrary units, mean ± SEM, n = 8 in each group, *P < .05, significantly different from sham WA, Student t test). (B) Levels of NK1R protein measured by Western blotting. A significant reduction in the level of NK1R proteins in samples from chronic WA stress rats compared with controls was observed. Data are expressed as normalized optical density, mean ± SEM, n = 8 in each group, *P < .05, significantly different from sham WA, Student t test. (C) Representative Western blotting for NK1R in colon extracts shows a prominent band at ∼65 kilodaltons. Gastroenterology 2006 130, 1729-1742DOI: (10.1053/j.gastro.2006.01.037) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions