Endogenous Opioids Inhibit Early-Stage Pancreatic Pain in a Mouse Model of Pancreatic Cancer
2006; Elsevier BV; Volume: 131; Issue: 3 Linguagem: Inglês
10.1053/j.gastro.2006.06.021
ISSN1528-0012
AutoresMolly 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,
Tópico(s)Pediatric Pain Management Techniques
ResumoBackground & Aims: The endogenous opioid system is involved in modulating the experience of pain, the response to stress, and the action of analgesic therapies. Recent human imaging studies have shown a significant tonic modulation of visceral pain, raising the question of whether endogenous opioids tonically modulate the pain of visceral cancer. Methods: Transgenic mice expressing the first 127 amino acids of simian virus 40 large T antigen, under the control of the rat elastase-1 promoter, that spontaneously develop pancreatic cancer were used to investigate the role of endogenous opioids in the modulation of pancreatic cancer pain. Visceral pain behaviors were assessed as degree of hunching and vocalization. Results Although mice with late-stage pancreatic cancer displayed spontaneous, morphine-reversible, visceral pain-related behaviors such as hunching and vocalization, these behaviors were absent in mice with early-stage pancreatic cancer. After systemic administration of the central nervous system (CNS)-penetrant opioid receptor antagonists naloxone or naltrexone, mice with early-stage pancreatic cancer displayed significant visceral pain-related behaviors, whereas systemic administration of the CNS-nonpenetrant opioid antagonist naloxone-methiodide did not induce an increase in visceral pain behaviors. Conclusions Our findings suggest that a CNS opioid-dependent mechanism tonically modulates early and late-stage pancreatic cancer pain. Understanding the mechanisms that mask this pain in early stage disease and drive this pain in late-stage disease may allow improved diagnosis, treatment, and care of patients with pancreatic cancer. Background & Aims: The endogenous opioid system is involved in modulating the experience of pain, the response to stress, and the action of analgesic therapies. Recent human imaging studies have shown a significant tonic modulation of visceral pain, raising the question of whether endogenous opioids tonically modulate the pain of visceral cancer. Methods: Transgenic mice expressing the first 127 amino acids of simian virus 40 large T antigen, under the control of the rat elastase-1 promoter, that spontaneously develop pancreatic cancer were used to investigate the role of endogenous opioids in the modulation of pancreatic cancer pain. Visceral pain behaviors were assessed as degree of hunching and vocalization. Results Although mice with late-stage pancreatic cancer displayed spontaneous, morphine-reversible, visceral pain-related behaviors such as hunching and vocalization, these behaviors were absent in mice with early-stage pancreatic cancer. After systemic administration of the central nervous system (CNS)-penetrant opioid receptor antagonists naloxone or naltrexone, mice with early-stage pancreatic cancer displayed significant visceral pain-related behaviors, whereas systemic administration of the CNS-nonpenetrant opioid antagonist naloxone-methiodide did not induce an increase in visceral pain behaviors. Conclusions Our findings suggest that a CNS opioid-dependent mechanism tonically modulates early and late-stage pancreatic cancer pain. Understanding the mechanisms that mask this pain in early stage disease and drive this pain in late-stage disease may allow improved diagnosis, treatment, and care of patients with pancreatic cancer. Spinal cord excitability is influenced directly by descending inputs originating in higher brain centers and this descending modulation can be inhibitory or facilitatory in nature.1Vanegas H. Schaible H.G. 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A major reason why pancreatic cancer remains such a lethal disease is that visceral pain is not perceived by the patient until the disease is highly advanced and the tumor has metastasized to other organs, at which point therapeutic treatment no longer has a significant effect on survival.13Pour P.M. The silent killer.Int J Pancreatol. 1991; 10: 103-104PubMed Google Scholar, 14Kelsen D.P. Portenoy R. Thaler H. Tao Y. Brennan M. Pain as a predictor of outcome in patients with operable pancreatic carcinoma.Surgery. 1997; 122: 53-59Abstract Full Text PDF PubMed Scopus (134) Google Scholar, 15White R.R. Hurwitz H.I. Morse M.A. Lee C. Anscher M.S. Paulson E.K. Gottfried M.R. Baillie J. Branch M.S. Jowell P.S. McGrath K.M. Clary B.M. Pappas T.N. Tyler D.S. 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Responses of rat dorsal column neurons to pancreatic nociceptive stimulation.Neuroreport. 2001; 12: 2527-2530Crossref PubMed Scopus (26) Google Scholar or pathology of the pancreas can produce significant pain,21Tenenbein M.S. Tenenbein M. Acute pancreatitis due to erythromycin overdose.Pediatr Emerg Care. 2005; 21: 675-676Crossref PubMed Scopus (18) Google Scholar it has remained unclear why pain resulting from pancreatic cancer is perceived only in late-stage disease. The present study investigates the possibility that the endogenous opioid system modulates the initial presentation and long-term experience of pancreatic cancer pain. Transgenic mice expressing the N-terminal 127 amino acids of simian virus 40 large T antigen, under the control of the rat elastase-1 promoter (ET), were generated as previously described.22Tevethia M.J. Bonneau R.H. Griffith J.W. Mylin L. A simian virus 40 large T-antigen segment containing amino acids 1 to 127 and expressed under the control of the rat elastase-1 promoter produces pancreatic acinar carcinomas in transgenic mice.J Virol. 1997; 71: 8157-8166PubMed Google Scholar, 23Ornitz D.M. Hammer R.E. Messing A. Palmiter R.D. Brinster R.L. Pancreatic neoplasia induced by SV40 T-antigen expression in acinar cells of transgenic mice.Science. 1987; 238: 188-193Crossref PubMed Scopus (204) Google Scholar Homozygosity for the mutant allele is embryonic lethal; consequently, heterozygous mice (ET) and their wild-type (WT) littermates were used for all experiments and breeding. A total of 221 mice were used in the present experiments. ET mice with a C57BL/6J background (Mayo Clinic, Scottsdale, AZ) were bred with female C57BL/6J mice (The Jackson Laboratory, Bar Harbor, ME). Pups were weaned at 3–4 weeks, and the DNA extracted from tail sections was screened for the presence of the transgene using reverse-transcription polymerase chain reaction analysis. Mice included in this study were genotyped twice to ensure accuracy of the experimental condition. All mice were maintained in a vivarium at 22°C with a 12-hour alternating light-dark cycle and given food and water ad libitum. The mice were housed in accordance with The National Institutes of Health guidelines and all procedures were approved by the Institutional Animal Care and Use Committee at the University of Minnesota. Mice with no embryologic or developmental abnormalities and male mice not used in colony development were included. Testing and data analysis exclusion criteria included observation of physical abnormalities such as blindness or severe alopecia, or data resulting in greater than 2 SDs from the mean test variable. Mice were not assessed behaviorally before 5 weeks of age owing to low body weight and small body structure that resulted in difficulty in assessing visceral pain-related behaviors. Euthanasia criteria included weight loss of 20%–25%, weakness, inability to obtain food or water, loss of coat luster, and/or excessive bite or scratch wounds. These criteria have been set by and are followed in accordance with Research Animal Resources of the University of Minnesota. Male ET mice and age-matched WT littermates were observed behaviorally from 5 weeks of age for visceral pain-related symptoms. Visceral pain-related behaviors were assessed as degree of hunching, time spent hunching (>300 s), and the number of palpation-induced vocalization events (>60 s). The hunching behavior is similar to what has been described previously as hunching or hump-backed behavior in rats with visceral pain.24Wesselmann U. Czakanski P.P. Affaitati G. Giamberardino M.A. Uterine inflammation as a noxious visceral stimulus: behavioral characterization in the rat.Neurosci Lett. 1998; 246: 73-76Crossref PubMed Scopus (97) Google Scholar Displays of vocalization also have been described as measures of pain in other rodent pain models.25Han J.S. Bird G.C. Li W. Jones J. Neugebauer V. Computerized analysis of audible and ultrasonic vocalizations of rats as a standardized measure of pain-related behavior.J Neurosci Methods. 2005; 141: 261-269Crossref PubMed Scopus (101) Google Scholar, 26Swedberg M.D. The mouse grid-shock analgesia test: pharmacological characterization of latency to vocalization threshold as an index of antinociception.J Pharmacol Exp Ther. 1994; 269: 1021-1028PubMed Google Scholar, 27Giamberardino M.A. Valente R. de Bigontina P. Vecchiet L. Artificial ureteral calculosis in rats: behavioural characterization of visceral pain episodes and their relationship with referred lumbar muscle hyperalgesia.Pain. 1995; 61: 459-469Abstract Full Text PDF PubMed Scopus (135) Google Scholar To monitor the general health of the mice, body weights were recorded weekly before behavioral tests. Hunching behavior was assessed at 5–7, 9–12, and 14–24 weeks or until euthanasia criteria were met. In this study, the same mice were tested at 5–7, 9–12, and 14–24 weeks. In addition, we also used a separate group of mice that were tested only at each time period. We observed the same results in both groups of animals. Mice were placed individually in the center of an open field arena and observed over a 300 s period. The hunching score is based on the amount of accumulated time, in seconds, engaged in the hunching behavior (up to 300 s observation period) multiplied by the scoring factor associated with that behavior's observed degree of impairment. The possible range of scores is from 0 (300 s × 0 = 0 for normal animals that do not hunch) to 1200 (300 s × 4 for animals that continuously hunch throughout the entire 300 s test period). The scoring factors for the observed hunching behaviors were as follows: 0 = normal coat luster, displays exploratory behavior, lack of a rounded-back posture; 1 = mild rounded-back posture, displays exploratory behavior, normal coat luster; 2 = severe rounded-back posture, displays slightly reduced exploratory behavior, slight piloerection, intermittent abdominal contractions; 3 = severe rounded-back posture, displays considerably reduced exploratory behavior, moderate piloerection, intermittent abdominal contractions; and 4 = severe rounded-back posture, displays little or no exploratory behavior, whole-body piloerection with head immobile, intermittent abdominal contractions. For example, mice that spent the entire 5-minute period in a severe rounded-back posture, displayed considerably reduced exploratory behavior, and had moderate piloerection with intermittent abdominal contractions would have a score of 900. These methods were adapted from Lanteri-Minet et al.28Lanteri-Minet M. Bon K. de Pommery J. Michiels J.F. Menetrey D. Cyclophosphamide cystitis as a model of visceral pain in rats: model elaboration and spinal structures involved as revealed by the expression of c-Fos and Krox-24 proteins.Exp Brain Res. 1995; 105: 220-232Crossref PubMed Scopus (135) Google Scholar The arena was washed between each trial with a 10% bleach solution to eliminate possible bias caused by odors left by the previous mouse. In all cases, observations were performed by 2 independent observers blinded as to the experimental status of the mouse. Quantification of ultrasonic and audible vocalization was performed before and 15 minutes after drug or saline administration. Mice were restrained gently and, using a 5-mm diameter cotton-tipped applicator (Solon MFG, Solon, ME), palpated approximately 1 cm below the xiphoid process to the left of the midline with a palpation occurring approximately every 3 seconds over a 1-minute observation period. Data recording was performed in a manner similar to methods described previously.25Han J.S. Bird G.C. Li W. Jones J. Neugebauer V. Computerized analysis of audible and ultrasonic vocalizations of rats as a standardized measure of pain-related behavior.J Neurosci Methods. 2005; 141: 261-269Crossref PubMed Scopus (101) Google Scholar Briefly, recordings were made with a D 980 bat detector (Pettersson Elektronik AB, Uppsala, Sweden) (detection range, 20 Hz to 180 kHz ± 5 kHz). This range was chosen because others29Gourbal B.E. Barthelemy M. Petit G. Gabrion C. Spectrographic analysis of the ultrasonic vocalisations of adult male and female BALB/c mice.Naturwissenschaften. 2004; 91: 381-385Crossref PubMed Scopus (85) Google Scholar, 30Ko S.W. Chatila T. Zhuo M. Contribution of CaMKIV to injury and fear-induced ultrasonic vocalizations in adult mice.Mol Pain. 2005; 1: 10Crossref PubMed Scopus (31) Google Scholar have reported mice to vocalize in both the audible and high ultrasonic ranges. The recorded signals were filtered and amplified (UltraVox 4-channel filter system; Noldus Information Technology, Wageningen, The Netherlands). Analyses of the number of palpation-evoked vocalizations over the observation period were made using UltraVox 2.0 software (Noldus Information Technology). The detector shifts a user-defined, high-frequency band to the audible range while suppressing nonrelevant sounds and ignoring ultrasounds outside the defined frequency range. For as long as the animal is emitting sound, the detector and audio filter records the signal. Only sounds within the frequency range of 10–180 kHz and exceeding a −6 dBm amplitude threshold value are recorded by the UltraVox software as an event. Experiments were performed in a quiet area and appropriate filtering levels were used to avoid the recording of background noise. For recordings, only 1 mouse was in the testing room at a time. Mice were administered opioid antagonists naloxone hydrochloride (3 mg/kg, subcutaneously [sc]; Sigma, St. Louis, MO), naloxone methiodide (3 mg/kg, sc; Sigma), naltrexone hydrochloride (10 mg/kg, sc; Sigma), opioid agonist morphine sulfate (10 mg/kg, sc; Elkins-Sinn, Cherry Hill, NJ), opioid agonist loperamide (10 mg/kg, sc; Sigma), or vehicle (0.9% saline) after baseline hunching and vocalization quantification at each time point. Dose-response experiments using 0.1, 1.0, and 3.0 mg/kg naltrexone; 1.0, 1.5, and 3.0 mg/kg naloxone; and 1.0 and 3.0 mg/kg naloxone methiodide were performed to assess side-effect profiles. These experiments, in addition to doses of naloxone methiodide up to 30.0 mg/kg, did not produce any observable side effects. After drug administration, mice again were placed in the open field arena and the degree of hunching and the time spent hunching over a 300 s period were quantified. 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A simian virus 40 large T-antigen segment containing amino acids 1 to 127 and expressed under the control of the rat elastase-1 promoter produces pancreatic acinar carcinomas in transgenic mice.J Virol. 1997; 71: 8157-8166PubMed Google Scholar At 18–24 weeks, which was the age at which ET mice were euthanized as a result of weight loss or deterioration of their general health, 12% of the ET mice showed visual evidence of metastases to the liver, duodenum, or peritoneum. Mice found to have visual evidence of lesions or metastases at the time of euthanasia were analyzed separately. The pancreas was dissected from the peritoneal viscera, leaving the spleen and duodenum associated for anatomic reference. The pancreas was embedded in TissueTek (Sakura, Torrance, CA) and cut on a cryostat at a thickness of 40 μm in the frontal plane. Serial tissue sections were collected, thaw mounted, and processed on gelatin-coated slides. These sections were incubated for 30 minutes at room temperature in a blocking solution of 3.0% normal donkey serum in PBS with 0.3% Triton X-100, and then incubated overnight at room temperature in the primary antiserum. Small- to medium-diameter peptidergic primary afferent sensory nerve fibers were immunostained for the neurotransmitter calcitonin gene-related peptide (CGRP) (polyclonal rabbit anti-rat CGRP, 1:15,000; Sigma). Blood vessels were immunostained for the platelet endothelial cell adhesion molecule CD31 (monoclonal rat anti-mouse CD31, 1:500; BD Pharmingen, San Diego, CA). Macrophages were immunostained for the myeloid-specific lysosomal antigen CD68 (monoclonal rat anti-mouse CD68, 1:5000; Serotec, Oxford, UK). After incubation, tissue sections were washed 3 times for 10 minutes each in PBS and incubated in the secondary antibody solution for 3 hours at room temperature. Secondary antibodies conjugated to fluorophores Cy3 and fluorescein isothiocyanate (Jackson ImmunoResearch, West Grove, PA) were used at 1:600 and 1:150, respectively. To confirm specificity of primary antibodies, controls included preabsorption with the corresponding antigen or omission of the primary antibody. To control for interactions between antibodies in double-immunostaining experiments, all results were confirmed on singly immunostained sections. After secondary incubation, sections were washed 3 times for 10 minutes each in PBS and coverslipped with p-phenylenediamine in glycerol. After immunohistochemical quantification, sections were counterstained with H&E for histologic identification of pancreatic structures. Quantification was performed on a total of 21 sections per marker; 3 sections were sampled at 400-μm intervals from the pancreas of 12 male mice (WT 9–12 wk, n = 3; ET mice: 5–7 wk, n = 3; 9–12 wk, n = 3; 14–24 wk, n = 3). To determine the association of nerve fibers with pancreatic structures, sections were viewed using an MRC-1024 confocal imaging system (Bio-Rad, Richmond, CA). Blood vessel, macrophage, and nerve fiber density and distribution was determined using an Olympus BH2 microscope (Olympus, Tokyo, Japan) equipped for epifluorescence and a SPOT2 digital camera (Diagnostic Instruments, Inc., Sterling Heights, MI). Quantification of CD31-immunoreactive (CD31-ir) blood vessels was performed using methodology adapted from previous studies.36Esposito I. Menicagli M. Funel N. Bergmann F. Boggi U. Mosca F. Bevilacqua G. Campani D. Inflammatory cells contribute to the generation of an angiogenic phenotype in pancreatic ductal adenocarcinoma.J Clin Pathol. 2004; 57: 630-636Crossref PubMed Scopus (217) Google Scholar, 37Weidner N. Semple J.P. Welch W.R. Folkman J. 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