Volume 139, Issue 2, Pages 553-563.e5 (August 2010) Evidence of Central and Peripheral Sensitization in a Rat Model of Narcotic Bowel-Like Syndrome  Simona Agostini, Helene Eutamene, Christel Cartier, Maria Broccardo, Giovanna Improta, Eric Houdeau, Carla Petrella, Laurent Ferrier, Vassillia Theodorou, Lionel Bueno  Gastroenterology  Volume 139, Issue 2, Pages 553-563.e5 (August 2010) DOI: 10.1053/j.gastro.2010.03.046 Copyright © 2010 AGA Institute Terms and Conditions

Figure 1 (A–E) Effect of morphine (10 mg/kg SC) or vehicle daily treatment (10 days) on abdominal response to CRD recorded at days 1, 2, 6, 8, and 10 of treatment. (F) Summary of abdominal response to CRD of morphine-treated rats vs vehicle at 30 mm Hg. We observed a morphine-induced visceral analgesic effect (days 1–2), tolerance period (day 6), and visceral hyperalgesia (days 8–10). *P < .05; **P < .01; ***P < .001 vs basal response. +P < .05; ++P < .01; +++P < .001 vs vehicle. Each group represents the mean ± standard error of the mean of 8 rats. Gastroenterology 2010 139, 553-563.e5DOI: (10.1053/j.gastro.2010.03.046) Copyright © 2010 AGA Institute Terms and Conditions

Figure 2 Effect of morphine (10 mg/kg SC) or vehicle daily treatment (8 days) on (A and B) abdominal response to CRD and (C and D) distension volume, recorded 8 and 14 days after the end of treatment. Morphine induced a delayed visceral hyperalgesia without affecting colonic muscular tone. Vehicle treatment did not show any effect. *P < .05; **P < .01; ***P < .001 vs basal response. Each group represents the mean ± standard error of the mean of 8 rats. Gastroenterology 2010 139, 553-563.e5DOI: (10.1053/j.gastro.2010.03.046) Copyright © 2010 AGA Institute Terms and Conditions

Figure 3 Effect of an acute morphine injection (10 mg/kg SC) on delayed morphine-induced colonic hypersensitivity response to CRD. Morphine showed a reduced analgesic effect compared with that observed the first day of treatment. *P < .05; **P < .01; ***P < .001 vs basal response. xP < .05 vs morphine. Each group represents the mean ± standard error of the mean of 8 rats. Gastroenterology 2010 139, 553-563.e5DOI: (10.1053/j.gastro.2010.03.046) Copyright © 2010 AGA Institute Terms and Conditions

Figure 4 Effect of co-treatment of minocycline (20 mg/kg IP), a microglia inhibitor, with morphine (10 mg/kg SC) on abdominal response to CRD. (A) At day 8 of treatment minocycline abolished morphine-induced visceral hyperalgesia. (B) Minocycline did not affect delayed (8 days) morphine-induced visceral hyperalgesia. *P < .05; **P < .01; ***P < .001 vs vehicle. xxxP < .001 vs morphine. P < .05; P < .001 vs vehicle + minocycline. Each group represents the mean ± standard error of the mean of 8 rats. Gastroenterology 2010 139, 553-563.e5DOI: (10.1053/j.gastro.2010.03.046) Copyright © 2010 AGA Institute Terms and Conditions

Figure 5 Representative micrograph (upper panels) and relative quantification (lower panels) of immunostaining for P-p38 and OX42 alone and co-stained on dorsal L6-S1 spinal cord sections. (A1, B1, C1, and right graph) Spinal P-p38 staining is increased in morphine-treated rats on day 8 of treatment, but not 8 days after the end of the treatment vs vehicle. (A2, B2, C2, and left graph) Spinal OX42 immunolabeling showed no difference between vehicle and morphine-treated rats. (A3, B3, and C3) P-p38 is found to colocalize with OX42-positive cells showing the microglia activation. % of labeling, calculated as the ratio of positive P-p38 or OX42 area/laminas I, II, III, IV total area. *P < .05 vs vehicle. Data are presented as mean ± standard error of the mean of 5 rats. Gastroenterology 2010 139, 553-563.e5DOI: (10.1053/j.gastro.2010.03.046) Copyright © 2010 AGA Institute Terms and Conditions

Figure 6 Effect of an acute treatment with nor-BNI (1 mg/kg SC), a κ-opioid antagonist, on abdominal response to CRD performed 8 days after morphine treatment. (A) Nor-BNI reduced delayed morphine-induced hyperalgesia without affecting basal sensitivity. *P < .05; **P < .01 vs vehicle. xP < .05 vs morphine. (B) Prodynorphin expression in dorsal spinal cord horn (L6–S1) 8 days after morphine treatment. Note a prodynorphin significant increase (P < .05) in morphine-treated rats vs vehicle. Each group represents the mean ± standard error of the mean of 8 rats. Gastroenterology 2010 139, 553-563.e5DOI: (10.1053/j.gastro.2010.03.046) Copyright © 2010 AGA Institute Terms and Conditions

Figure 7 Effect of an acute injection of doxantrazole (5 mg/kg IP) on abdominal response to CRD in rats receiving morphine treatment. Doxantrazole did not reduce morphine-induced colonic hyperalgesia to CRD (A) on day 8 under treatment but did reduce it (B) 8 days after treatment. **P < .01; ***P < .001 vs basal response. +P < .05; +++P < .001 vs doxantrazole alone. xP < .05 vs morphine. Each group represents the mean ± standard error of the mean of 8 rats. (C) Co-immunostaining for RMCPII (red) and PGP 9.5-immunoreactive fibers (green) in colon sections of rats treated with vehicle (top); morphine at day 8 of treatment (middle), and morphine 8 days after the end of treatment (bottom). The graph on the right shows the number of mast cells in close apposition to PGP 9.5 fibers. The number of mast cells tightly associated to nerve (yellow) is higher only at 8 days after the end of morphine treatment vs vehicle. *P < .05 vs vehicle. Data are presented as mean ± standard error of the mean of 5 rats. Gastroenterology 2010 139, 553-563.e5DOI: (10.1053/j.gastro.2010.03.046) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 1 Experimental protocols. Gastroenterology 2010 139, 553-563.e5DOI: (10.1053/j.gastro.2010.03.046) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 2 (A) Representative Western blots for P-p38 (upper panel) and β-actin (lower panel) for rat dorsal spinal samples 8 days under morphine treatment (m8d) and 8 days after morphine treatment (m+8d) in comparison with vehicle (ve). (B) Relative quantification for Western blot analysis. Each group represents the mean ± standard error of the mean of 5 rats. Gastroenterology 2010 139, 553-563.e5DOI: (10.1053/j.gastro.2010.03.046) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 3 Effect of chronic morphine or vehicle administration on colonic muscular tone. Each group represents the mean ± standard error of the mean of 8 rats. Gastroenterology 2010 139, 553-563.e5DOI: (10.1053/j.gastro.2010.03.046) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 4 Effect of chronic morphine treatment on colonic transit. Each group represents the mean ± standard error of the mean of 8 rats. **P < .01; ***P < .001 vs vehicle. Gastroenterology 2010 139, 553-563.e5DOI: (10.1053/j.gastro.2010.03.046) Copyright © 2010 AGA Institute Terms and Conditions