1. Volume 131, Issue 3, Pages 900-910 (September 2006)
Endogenous Opioids Inhibit Early-Stage Pancreatic Pain in a Mouse Model of Pancreatic Cancer 
Molly A. Sevcik, Beth M. Jonas, Theodore H. Lindsay, Kyle G. Halvorson, Joseph R. Ghilardi, Michael A. Kuskowski, Pinku Mukherjee, John E. Maggio, Patrick W. Mantyh 
Gastroenterology 
Volume 131, Issue 3, Pages (September 2006)
DOI: /j.gastro
Copyright © 2006 American Gastroenterological Association (AGA) Institute Terms and Conditions

2. Figure 1
The gross pathology and histopathologic changes associated with the development of pancreatic cancer. (A–C) Low-power photographs of the pancreas and (D–F) confocal photomicrographs of CGRP-ir small-diameter peptidergic sensory nerve fibers (white) and CD31-ir blood vessels (blue) in sections of WT and ET pancreas. (C) On dissection, major pathologic changes were not observed until 14–24 weeks, (B) although palpation of the pancreas at 9–12 weeks revealed small nodules corresponding to small well-circumscribed tumors or adenomas. (D) In the pancreas of WT mice at 9–12 weeks there is a smooth and organized appearance of capillaries and CGRP-ir fibers. (E) At early stage in ET mice (9–12 wk), there is a significant increase in capillary and CGRP fiber density within the parenchyma of the pancreas as compared with the age-matched WT controls. (F) At late time points (14–24 wk), capillary and CGRP fiber density in central necrotic areas of the tumor decreased; however, areas of higher capillary and CGRP fiber density still were observed near the margins of the tumor (shown) where active growth was still occurring. Scale bars: A–C, 5 mm; D–F, 100 μm.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association (AGA) Institute Terms and Conditions

3. Figure 2
Quantification of the gross and histopathologic changes associated with the development of pancreatic cancer. (A) Pancreatic volume remains constant until 14–24 weeks, at which time a significant increase is observed. ■, WT; ▨, ET 5–7 wk; ▩, ET 9–12 wk; □, ET 14–24 wk. (B) The number of CD68-immunoreactive macrophages in the pancreas increases significantly at 5–7, 9–12, and 14–24 weeks of disease (P < .05 vs WT). (C) In the pancreas of ET mice at 5–7, 9–12, and 14–24 weeks there is an increased density of CD31-ir vascular endothelial cells. These increases in microvascular density were significant as compared with WT at all time points. (D) The density of CGRP-immunoreactive sensory nerve fibers increases with disease progression and first becomes significantly different from WT at 5–7 weeks and continues to be significantly different until 14–24 weeks or euthanasia criteria are met. Note, however, that although changes in all other (B–D) pathologic end points are increasing rapidly as early as 5–7 weeks, (A) changes in pancreatic volume do not become significant until 14–24 weeks. *P < .05 vs WT.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association (AGA) Institute Terms and Conditions

4. Figure 3
Representative images and silhouette outlines of the hunching behavioral characteristics of mice with varying stages of pancreatic cancer. The scores for the observed behaviors were as follows: 0 = lack of a rounded-back posture, displays exploratory behavior and normal coat luster (A and B display hunching silhouette for a score of 0). 1 = mild rounded-back posture, displays exploratory behavior, normal coat luster (C and D display hunching silhouette for a score of 1). 2 = severe rounded-back posture, displays slightly reduced exploratory behavior, slight piloerection, and intermittent abdominal contractions (E and F display hunching silhouette for a score of 2). 3 = severe rounded-back posture, displays considerably reduced exploratory behavior, moderate piloerection, and intermittent abdominal contractions (G and H display hunching silhouette for a score of 3). 4 = severe rounded-back posture, displays little or no exploratory behavior, whole body piloerection with head immobile, and intermittent abdominal contractions (I and J display hunching silhouette for a score of 4). Note the silhouette outlines (B, D, F, H, J; grey line) comparing the progressive rounded-back posture that occurs with disease progression in this mouse model of pancreatic cancer pain.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association (AGA) Institute Terms and Conditions

5. Figure 4
The effect of the CNS-penetrant opioid antagonist naloxone on pancreatic cancer pain-related behaviors. (A) Spontaneous hunching behavior first became evident in ET mice at approximately 14 weeks, increased in severity until euthanasia, and rarely was observed in WT mice at any time point examined (n ≥ 8 WT, n ≥ 8 ET). (B) When ET mice were administered the opioid antagonist, naloxone (3 mg/kg, sc), the hunching behavior became evident at 9–12 weeks, increased dramatically at 14–24 weeks, and was significantly different from WT controls that also received naloxone (n ≥ 8 WT, n ≥ 8 ET). (C) Spontaneous vocalization behavior first became slightly evident in ET mice at approximately 12 weeks, increased at 14–24 weeks, and was observed minimally in WT mice (n ≥ 5 WT, n ≥ 5 ET). (D) The number of vocalization events over a 1-minute observation period increased at 9–12 and 14–24 weeks in ET mice after injection of naloxone when compared with age-matched WT mice that also received naloxone (n ≥ 5 WT, n ≥ 5 ET). (A–D) Error bars represent SEM. *P < .05 vs WT. ■, WT; □, ET.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association (AGA) Institute Terms and Conditions

6. Figure 5
The effects of CNS-penetrant and CNS-nonpenetrant opioid-receptor agonists and antagonists on visceral pain-related behaviors in mice with early- and late-stage pancreatic cancer. (A) In WT mice, spontaneous, saline (Sal), naloxone (Nal), or naltrexone (Ntx)-induced hunching behavior rarely was observed at any time point, and there was no significant difference between WT + Sal vs WT + Nal or WT + Ntx (n ≥ 5). Spontaneous hunching of ET mice at 9–12 weeks was not increased significantly by sc injection of Sal alone; however, after injection of Nal or Ntx (3 mg/kg, sc), ET mice had a higher degree of hunching. After injection of the peripherally acting naloxone methiodide in early-stage disease, hunching behavior was not increased significantly from WT + Sal or from ET + Sal. ■, WT; □, ET. (B) After administration of Nal (3 mg/kg, sc) or Ntx (3 mg/kg, sc), ET mice at 9–12 weeks showed hunching behavior that continued for 105 or 195 minutes, respectively, and the area under each response-time curve was significantly larger than for Sal controls (n ≥ 5). In preliminary experiments, the naloxone- and naltrexone-induced visceral pain behaviors in mice with early-stage pancreatic cancer were robust at 10 minutes post-injection. ○, Sal; ■, Nal 3 mg/kg; ▴, Ntx 3 mg/kg. (C) In late-stage disease (14–24 wk), baseline hunching in ET mice was increased drastically from WT. Loperamide, a peripherally acting opioid agonist, did not reduce hunching behaviors in late-stage ET mice. Note that morphine sulfate (MS) administration (10 mg/kg, sc) attenuates hunching compared with Sal (P = .03); however, after Nal (3 mg/kg, sc) reversal of MS, this hunching behavior is increased significantly when compared with Sal. Naltrexone administration did not significantly increase hunching behaviors at late-stage disease compared with saline. ■, WT; □, ET. (A–C) Error bars represent SEM. *P < .05 vs Sal.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association (AGA) Institute Terms and Conditions