Descending Facilitation From the Rostral Ventromedial Medulla Maintains Visceral Pain in Rats With Experimental Pancreatitis
2006; Elsevier BV; Volume: 130; Issue: 7 Linguagem: Inglês
10.1053/j.gastro.2006.03.025
ISSN1528-0012
AutoresLouis P. Vera–Portocarrero, Jennifer X. Yie, Justin Kowal, Michael H. Ossipov, Tamara King, Frank Porreca,
Tópico(s)Apelin-related biomedical research
ResumoBackground & Aims: Pain is a main complaint of patients with pancreatitis. We hypothesized that such pain is mediated through ascending pathways via the nucleus gracilis (NG) and is dependent on descending facilitatory influences from the rostral ventromedial medulla (RVM). Methods: A rat model of persistent experimental pancreatitis was used. After establishment of pancreatitis, rats received microinjection of lidocaine in the NG or in the RVM to determine the importance of neural activity at these supraspinal sites in the expression of abdominal hypersensitivity evoked by von Frey filaments (ie, pancreatic pain). Rats also were pretreated for 28 days before induction of pancreatitis with a single RVM microinjection of dermorphin–saporin to eliminate cells that drive descending facilitation. Dynorphin content was measured in the spinal cord of pancreatitic rats and the effects of spinal antidynorphin antiserum in pancreatic pain were assessed. Results: Microinjection of lidocaine into either the NG or the RVM produced a time-related reversal of pancreatitis-induced pain. Pancreatitis significantly increased thoracic spinal dynorphin content and spinal antidynorphin antiserum elicited a time-related reversal of abdominal hypersensitivity. RVM dermorphin–saporin injection prevented the maintenance, but not the expression, of pancreatitis abdominal hypersensitivity and also prevented the increase of spinal dynorphin content in animals with pancreatitis. Conclusions: Our findings suggest that descending facilitation from the RVM plays a critical role in the maintenance, but not the expression, of pancreatic pain. These results provide a novel insight into the role of descending pathways and spinal plasticity in the maintenance of visceral pain from pancreatitis. Background & Aims: Pain is a main complaint of patients with pancreatitis. We hypothesized that such pain is mediated through ascending pathways via the nucleus gracilis (NG) and is dependent on descending facilitatory influences from the rostral ventromedial medulla (RVM). Methods: A rat model of persistent experimental pancreatitis was used. After establishment of pancreatitis, rats received microinjection of lidocaine in the NG or in the RVM to determine the importance of neural activity at these supraspinal sites in the expression of abdominal hypersensitivity evoked by von Frey filaments (ie, pancreatic pain). Rats also were pretreated for 28 days before induction of pancreatitis with a single RVM microinjection of dermorphin–saporin to eliminate cells that drive descending facilitation. Dynorphin content was measured in the spinal cord of pancreatitic rats and the effects of spinal antidynorphin antiserum in pancreatic pain were assessed. Results: Microinjection of lidocaine into either the NG or the RVM produced a time-related reversal of pancreatitis-induced pain. Pancreatitis significantly increased thoracic spinal dynorphin content and spinal antidynorphin antiserum elicited a time-related reversal of abdominal hypersensitivity. RVM dermorphin–saporin injection prevented the maintenance, but not the expression, of pancreatitis abdominal hypersensitivity and also prevented the increase of spinal dynorphin content in animals with pancreatitis. Conclusions: Our findings suggest that descending facilitation from the RVM plays a critical role in the maintenance, but not the expression, of pancreatic pain. These results provide a novel insight into the role of descending pathways and spinal plasticity in the maintenance of visceral pain from pancreatitis. Pain is a prominent and difficult-to-manage complaint of patients with pancreatitis. Recent models of pancreatic pain appear to mimic closely some aspects of the human condition.1Houghton A.K. Kadura S. Westlund K.N. Dorsal column lesions reverse the reduction of homecage activity in rats with pancreatitis.Neuroreport. 1997; 8: 3795-3800Crossref PubMed Scopus (63) Google Scholar, 2Vera-Portocarrero L.P. Yu Y. Westlund K.N. Nociception in persistent pancreatitis in rats effects of morphine and neuropeptides alterations.Anesthesiology. 2003; 98: 474-484Crossref PubMed Scopus (70) Google Scholar, 3Winston J.H. He Z.-J. Shenoy M. Xiao S.-Y. Pasricha P.J. Molecular and behavioral changes in nociception in a novel rat model of chronic pancreatitis for the study of pain.Pain. 2005; 117: 214-222Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar In these models, hyperalgesia is experienced in the abdominal area and persists for an extended period of time, allowing for investigation of mechanisms of pancreatic pain. Descending pain modulatory systems arising in the brainstem have been shown to be important in the expression and maintenance of some persistent pain conditions.4Ossipov M.H. Lai J. Malan Jr, T.P. Porreca F. Spinal and supraspinal mechanisms of neuropathic pain.Ann N Y Acad Sci. 2000; 909: 12-24Crossref PubMed Scopus (228) Google Scholar, 5Millan M.J. Descending control of pain.Prog Neurobiol. 2002; 66: 355-474Crossref PubMed Scopus (2439) Google Scholar The rostral ventromedial medulla (RVM) has been implicated as an important source of descending modulation of pain transmission.6Vanegas H. Schaible H.-G. Descending control of persistent pain inhibitory or facilitatory?.Brain Res Rev. 2004; 46: 295-309Crossref PubMed Scopus (409) Google Scholar Microinjection of lidocaine into the RVM reversed experimental neuropathic pain,7Pertovaara A. Wei H. Hamalainen M.M. Lidocaine in the rostroventromedial medulla and the periaqueductal gray attenuates allodynia in neuropathic rats.Neurosci Lett. 1996; 218: 127-130Crossref PubMed Scopus (152) Google Scholar, 8Kovelowski C.J. Ossipov M.H. Sun H. Lai J. Malan Jr, T.P. Porreca F. Supraspinal cholecystokinin may drive tonic descending facilitation mechanisms to maintain neuropathic pain in the rat.Pain. 2000; 87: 265-273Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar revealing the critical role for descending facilitation in this type of pain. Selective ablation of μ-opioid receptor–expressing cells in the RVM, presumed to be important for descending facilitation, prevented the maintenance, but not initiation, of experimental neuropathic pain.4Ossipov M.H. Lai J. Malan Jr, T.P. Porreca F. Spinal and supraspinal mechanisms of neuropathic pain.Ann N Y Acad Sci. 2000; 909: 12-24Crossref PubMed Scopus (228) Google Scholar, 9Porreca F. Burgess S.E. Gardell L.R. Vanderah T.W. Malan Jr, T.P. Ossipov M.H. Lappi D.A. Lai J. Inhibition of neuropathic pain by selective ablation of brainstem medullary cells expressing the mu-opioid receptor.J Neurosci. 2001; 21: 5281-5288PubMed Google Scholar, 10Burgess S.E. Gardell L.R. Ossipov M.H. Malan Jr, T.P. Vanderah T.W. Lai J. Porreca F. Time-dependent descending facilitation from the rostral ventromedial medulla maintains, but does not initiate, neuropathic pain.J Neurosci. 2002; 22: 5129-5136PubMed Google Scholar At the spinal cord level, dynorphin also appears to be critical for maintaining, but not initiating, experimental neuropathic pain.11Wang Z. Gardell L.R. Ossipov M.H. Vanderah T.W. Brennan M.B. Hochgeschwender U. Hruby V.J. Malan Jr, T.P. Lai J. Porreca F. Pronociceptive actions of dynorphin maintain chronic neuropathic pain.J Neurosci. 2001; 21: 1779-1786Crossref PubMed Google Scholar, 12Vanderah T.W. Gardell L.R. Burgess S.E. Ibrahim M. Zhong C.M. Zhang E.T. Malan Jr, T.P. Ossipov M.H. Lai J. Porreca F. Dynorphin promotes abnormal pain and spinal opioid antinociceptive tolerance.J Neurosci. 2000; 18: 7074-7079Google Scholar, 13Malan Jr, T.P. Ossipov M.H. Gardell L.R. Ibrahim M. Bian D. Lai J. Porreca F. Extraterritorial neuropathic pain correlates with multisegmental elevation of spinal dynorphin in nerve-injured rats.Pain. 2000; 86: 185-194Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 14Gardell L.R. Ibrahim M. Wang R. Wang Z. Ossipov M.H. Malan Jr, T.P. Porreca F. Lai J. Mouse strains that lack spinal dynorphin upregulation after peripheral nerve injury do not develop neuropathic pain.Neuroscience. 2004; 123: 43-52Crossref PubMed Scopus (50) Google Scholar Spinal dynorphin is up-regulated reliably after peripheral injuries and up-regulation is secondary to activation of descending facilitation mechanisms.15Gardell L.R. Wang R. Burgess S.E. Ossipov M.H. Vanderah T.W. Malan Jr, T.P. Lai J. Porreca F. Sustained morphine exposure induces a spinal dynorphin-dependent enhancement of excitatory transmitter release from primary afferent fibers.J Neurosci. 2002; 22: 6747-6755PubMed Google Scholar Descending modulation is known to play an important role in acute visceral pain. Electrical stimulation of the RVM produces biphasic modulation of spinal cord neuronal responses to colorectal distention.16Zhou M. Sengupta J.N. Gebhart G.F. Biphasic modulation of spinal visceral nociceptive transmission from the rostroventral medial medulla in the rat.J Neurophysiol. 2002; 87: 2225-2236PubMed Google Scholar, 17Zhou M. Gebhart G.F. Facilitation and attenuation of a visceral nociceptive reflex from the rostroventral medulla in the rat.Gastroenterology. 2002; 122: 1007-1019Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar Low doses of RVM neurotensin facilitate whereas higher doses inhibit acute visceral reflexes.18Urban M.O. Coutinho S.V. Gebhart G.F. Biphasic modulation of visceral nociception by neurotensin in rat rostral ventromedial medulla.J Pharmacol Exp Ther. 1999; 290: 207-213PubMed Google Scholar RVM N-methyl-d-aspartate (NMDA) antagonists block facilitation of visceral reflexes whereas non-NMDA antagonists injected in the RVM block descending inhibition.19Coutinho S.V. Urban M.O. Gebhart G.F. Role of glutamate receptors and nitric oxide in the rostral ventromedial medulla in visceral hyperalgesia.Pain. 1998; 78: 59-69Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar However, the role of descending modulation in more persistent states of visceral pain is not well understood. Nociceptive signals from the pancreas are known to reach supraspinal centers in part via the postsynaptic dorsal column pathway,20Houghton A.K. Wang C.C. Westlund K.N. Do nociceptive signals from the pancreas travel in the dorsal column?.Pain. 2001; 89: 207-220Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 21Westlund K.N. Zhang L. Wei J. Quast M.J. Vera-Portocarrero L.P. Cleeland C.S. fMRI of supraspinal areas after one week of pancreatic inflammation and morphine in rats.E J Neurosci. 2006; (submitted).Google Scholar which relays in the nucleus gracilis (NG).22Wang C.C. Westlund K.N. Responses of rat dorsal column neurons to pancreatic nociceptive stimulation.Neuroreport. 2001; 12: 2527-2530Crossref PubMed Scopus (26) Google Scholar Such information may be relayed to sites involved in descending modulation of pain21Westlund K.N. Zhang L. Wei J. Quast M.J. Vera-Portocarrero L.P. Cleeland C.S. fMRI of supraspinal areas after one week of pancreatic inflammation and morphine in rats.E J Neurosci. 2006; (submitted).Google Scholar, 23Wang C.C. Willis W.D. Westlund K.N. Ascending projections from the area around the spinal cord central canal a Phaseolus vulgaris leucoagglutinin study in rats.J Comp Neurol. 1999; 415: 341-367Crossref PubMed Scopus (109) Google Scholar including the RVM and the periaqueductal gray (PAG). Here, a model of persistent pancreatitic pain has been used to explore the possible role of descending pain facilitation mechanisms arising in the RVM and the contribution of spinal dynorphin to such visceral pain. Adult male Sprague–Dawley rats (Harlan, Indianapolis, IN) weighing 150–200 g were maintained in a climate-controlled room with food and water ad libitum on a 12-hour light/dark cycle (light on at 7:00 am). All procedures followed the policies of the International Association for the Study of Pain and the National Institutes of Health guidelines for the handling and use of laboratory animals. Studies were approved by the University of Arizona Institutional Animal Care and Use Committee. Pancreatitis was produced by a tail-vein injection of dibutyltin dichloride (DBTC; Aldrich, Milwaukee, WI) dissolved in 100% ethanol at a dose of 8 mg/kg under isofluorane anesthesia (2–3 L/min, 4.0%/vol until anesthetized, then 2.5%/vol throughout the procedure).2Vera-Portocarrero L.P. Yu Y. Westlund K.N. Nociception in persistent pancreatitis in rats effects of morphine and neuropeptides alterations.Anesthesiology. 2003; 98: 474-484Crossref PubMed Scopus (70) Google Scholar Controls were injected with the same volume (0.25 mL) of ethanol. Abdominal mechanical thresholds were quantified by measuring the number of withdrawal events evoked by application of a calibrated von Frey filament (to elicit either abdominal withdrawal, licking of the abdominal area, or whole-body withdrawal). Rats were placed inside Plexiglas boxes (Plastic Plus, Tucson, AZ) on an elevated fine fiberglass screen mesh and acclimated for 60 minutes before testing. A 4-g von Frey filament was applied from underneath, through the mesh floor, to the abdominal area at different points on the surface. A single trial consisted of 10 applications of this filament applied once every 10 seconds to allow the animals to cease any response and to return to a relatively inactive position. The mean occurrence of withdrawal events in each trial is expressed as the number of responses to 10 applications.2Vera-Portocarrero L.P. Yu Y. Westlund K.N. Nociception in persistent pancreatitis in rats effects of morphine and neuropeptides alterations.Anesthesiology. 2003; 98: 474-484Crossref PubMed Scopus (70) Google Scholar After the conclusion of the experiments, animals were killed using a CO2 chamber and blood and pancreatic tissue were collected for confirmation of pancreatitis. Amylase and lipase levels were measured from blood serum using amylase and lipase-PS kits (Sigma, St. Louis, MO). Pancreatic tissue was placed in 4% paraformaldehyde overnight and then in 30% phosphate-buffered saline/sucrose. Paraffin-embedded sections were cut at 8-μm thickness and stained with H&E to visualize the pancreatic inflammation. In addition, heart, liver, lungs, and kidneys were harvested and processed at the same time to confirm the presence or absence of inflammation in these organs. Rats were anesthetized with ketamine/xylazine (100 mg/kg) and placed in a stereotaxic headholder. For the RVM cannula, the skull was exposed and two 26-gauge guide cannula separated by 1.2 mm (Plastics One Inc., Roanoke, VA) were directed at the lateral portions of the RVM (anteroposterior, −11.0 mm from bregma; lateral ± 0.6 mm from midline; dorsoventral, −8.5 mm from the cranium)24Paxinos G. Watson C. The rat brain in stereotaxic coordinates. Academic Press, San Diego1986Google Scholar and secured to the skull with dental cement. For the NG, a 26-gauge guide cannula was implanted with the following coordinates: anteroposterior, −15.0 mm from bregma; ± 0.6 mm mediolateral; dorsoventral −6.5 mm from the cranium.24Paxinos G. Watson C. The rat brain in stereotaxic coordinates. Academic Press, San Diego1986Google Scholar The guide cannula was secured to the skull with dental cement. After recovery (5 days), animals were injected with intravenous (IV) DBTC for induction of pancreatitis. On day 6 after DBTC injection, animals received lidocaine microinjection either into the RVM or the NG. Lidocaine administration was performed slowly, expelling 0.5 μl of 4% lidocaine through a 33-gauge injection cannula inserted through the guide cannula and protruding an additional 1 mm into fresh brain tissue to prevent backflow. Animals were tested for abdominal mechanical sensitivity every 20 minutes after injection for a period of 60 minutes. Animals then were euthanized and brain, blood, and pancreas were harvested for confirmation of cannula placement and pancreatitis, respectively. Five days after cannula placement, dermorphin–saporin conjugate (Advanced Targeting Systems, San Diego, CA) was injected into the RVM (1.5 pmol in 0.5 μl on each side). Vehicle (distilled water), dermorphin alone, or saporin alone were injected as controls in the same volume. After 28 days, baseline behavioral measures were taken and rats then received IV DBTC to induce pancreatitis. Behavioral testing then took place daily to determine mechanical sensitivity of the abdominal area until day 7 post-DBTC when rats were euthanized and brain, blood, and pancreas were harvested for confirmation of cannula placement and pancreatitis, respectively. On day 6 after IV DBTC or saline, and after behavioral evaluation, the spinal cord was taken and the dorsal half of the lumbar, thoracic, and cervical cord was dissected rapidly. Tissue samples were frozen immediately on dry ice and stored at −70°C. Thawed tissue was placed in 1 N acetic acid, disrupted with a Polytron homogenizer (Brinkman Instruments, Inc, Westbury, NY), and incubated for 20 minutes at 95°C. After centrifugation at 10,000 × g for 20 minutes (4°C), the supernatant was lyophilized and stored at −70°C. Protein concentrations were determined by the use of the bicinchoninic acid method with bovine serum albumin as a standard. Immunoassay was performed by the use of a commercial enzyme immunoassay kit with an antibody specific for dynorphin A(1-17) (Peninsula Laboratories, Belmont, CA). Standard curves were constructed and dynorphin content was determined with Graph Pad Prism software (San Diego, CA). A separate group of rats underwent surgeries to implant RVM cannulas and received microinjection of the dermorphin–saporin conjugate or vehicle, dermorphin alone, or saporin alone. After 28 days, rats were injected with DBTC to induce pancreatitis and were monitored for mechanical sensitivity of the abdominal area. On day 6 after DBTC injection, animals were euthanized and the spinal cord was harvested as described earlier to measure dynorphin content. Rats were anesthetized with ketamine/xylazine (100 mg/kg) and the nape of the neck and the back of the head were shaved. Rats were placed in a stereotaxic headholder and an incision at the back of the skull was made to expose the atlanto-occipital membrane. A PE-10 catheter (Beckton Dickinson, Sparks, MD), 4.5–5.0 cm in length, was inserted into the subarachnoid space and advanced to the midthoracic level of the spinal cord. After a 3-day recovery period, rats were injected with IV DBTC to induce pancreatitis. On day 6 after DBTC injection, either antidynorphin antiserum (200 μg followed by a 9-μl saline flush) or control antiserum was given intrathecally as previously described.15Gardell L.R. Wang R. Burgess S.E. Ossipov M.H. Vanderah T.W. Malan Jr, T.P. Lai J. Porreca F. Sustained morphine exposure induces a spinal dynorphin-dependent enhancement of excitatory transmitter release from primary afferent fibers.J Neurosci. 2002; 22: 6747-6755PubMed Google Scholar Rats were tested for mechanical sensitivity of the abdominal area every 20 minutes for 2 hours and then were euthanized for confirmation of intrathecal catheter placement and the presence of pancreatitis. Significant differences within each experimental group for the behavioral tests over time were detected by 1-factor analysis of variance (ANOVA) followed by the Fisher least significant difference post hoc test. Two-factor ANOVA was used to detect significant differences in behavioral outcomes among treatment groups and across time. Pair-wise comparisons for dynorphin content between treatments were determined by Student t test. Significance was established at a P value of less than .05. Before receiving any treatments, or after IV vehicle, probing the abdomen with the von Frey filament elicited very few withdrawal responses (Figure 1, pre-DBTC). After DBTC, rats showed significantly increased withdrawal frequency to mechanical stimulation of the abdomen compared with rats injected with vehicle, indicating the development of pancreatitis and associated referred abdominal hypersensitivity (P < .05; Figure 1, DBTC). On day 6 after IV injection, NG lidocaine did not alter responses to abdominal stimulation in vehicle controls but produced a time-related reversal of abdominal hypersensitivity in DBTC rats, showing a similar number of withdrawals to groups injected with IV vehicle and receiving NG saline or lidocaine. Rats showed significantly increased withdrawals to mechanical stimulation of the abdomen only after DBTC (P < .05; Figure 2, DBTC). RVM lidocaine did not alter responses in control rats but produced a time-related reversal of DBTC-induced referred abdominal hypersensitivity on day 6 after intravenous injection. Rats receiving DBTC and RVM lidocaine showed similar responses to groups receiving vehicle and RVM saline or lidocaine (Figure 2). Thoracic dynorphin content was increased significantly when measured 6 days after DBTC compared with levels seen in control rats (Figure 3, P < .05). Dynorphin content tended to be higher, but did not reach significance in lumbar tissues of DBTC-treated rats (Figure 3). Dynorphin content in the cervical spinal cord did not change between vehicle and DBTC-injected rats. On day 6 after injection of DBTC, when pancreatitis-induced abdominal mechanical hypersensitivity was prominent, suggesting that pancreatitis was well established, rats were given a single intrathecal injection of antidynorphin antiserum (antiDYN, 200 μg, Bachem, Torrance, CA) or control serum (200 μg) as previously described15Gardell L.R. Wang R. Burgess S.E. Ossipov M.H. Vanderah T.W. Malan Jr, T.P. Lai J. Porreca F. Sustained morphine exposure induces a spinal dynorphin-dependent enhancement of excitatory transmitter release from primary afferent fibers.J Neurosci. 2002; 22: 6747-6755PubMed Google Scholar and were tested for mechanical hypersensitivity every 20 minutes for 2 hours. At 20 minutes after intrathecal injection, rats receiving antidynorphin antiserum showed a decrease in withdrawals from mechanical stimulation of the abdominal area to basal levels, indicating a reversal of DBTC-induced abdominal hypersensitivity (Figure 4). Injection of control antiserum did not have any effects on the presence of mechanical hypersensitivity. None of the intrathecal injections had any effects on vehicle-treated rats. The effect of dynorphin antiserum still was evident at 40 minutes after injection but had dissipated at 60 minutes after injection. A single RVM injection of dermorphin–saporin was shown previously to elicit a loss of RVM μ-opioid receptor–expressing cells by postinjection day 28.9Porreca F. Burgess S.E. Gardell L.R. Vanderah T.W. Malan Jr, T.P. Ossipov M.H. Lappi D.A. Lai J. Inhibition of neuropathic pain by selective ablation of brainstem medullary cells expressing the mu-opioid receptor.J Neurosci. 2001; 21: 5281-5288PubMed Google Scholar, 10Burgess S.E. Gardell L.R. Ossipov M.H. Malan Jr, T.P. Vanderah T.W. Lai J. Porreca F. Time-dependent descending facilitation from the rostral ventromedial medulla maintains, but does not initiate, neuropathic pain.J Neurosci. 2002; 22: 5129-5136PubMed Google Scholar None of the RVM pretreatments alone altered responses to probing the abdomen with von Frey filaments at day 28 post-RVM injection. After intravenous DBTC injection but not vehicle injection, all rats showed the development of referred abdominal hypersensitivity that was established fully by post-DBTC injection day 4 and sustained through postinjection day 5. Rats previously microinjected with RVM vehicle, dermorphin, or saporin, and treated with DBTC showed sustained referred abdominal hypersensitivity throughout the experiment (ie, through day 7, P < .05). In contrast, rats previously injected with dermorphin–saporin and receiving DBTC showed a similar time course of increased abdominal withdrawals through post-DBTC day 5, but this increase diminished on day 6 after pancreatitis induction and returned to basal withdrawal levels by day 7 (Figure 5). Twenty-eight days after RVM pretreatments, rats received intravenous DBTC (or vehicle) to induce pancreatitis and referred abdominal hypersensitivity was monitored. On day 6 after DBTC or vehicle, rats were euthanized and thoracic spinal dynorphin content was measured. Rats pretreated with RVM vehicle, dermorphin alone, or saporin alone, and with DBTC-induced pancreatitis showed significantly increased dynorphin content compared with rats with the same RVM pretreatments and with intravenous vehicle injection (Figure 6, P < .05). In contrast, rats receiving RVM pretreatment with dermorphin–saporin and DBTC had similar levels of dynorphin content to rats with vehicle injection (Figure 6). Rats with DBTC injections showed pancreatic inflammation and increased blood serum concentrations of amylase and lipase at post-DBTC day 6. Pancreatic enzyme levels still were increased in comparison with the vehicle-injected group after lidocaine microinjection either into the NG or RVM (Table 1, Table 2). Treatment with dynorphin antiserum did not modify the concentrations of amylase and lipase (Table 1, Table 2). Treatment in the RVM of dermorphin–saporin or the respective controls did not modify the increase of pancreatic enzyme levels (Table 1, Table 2).Table 1Concentration of Amylase in the Blood Serum of Rats From All Experimental GroupsNo treatmentNG lidocaineRVM lidocaineDermorphin-SaporinSaporinDermorphinWaterDynorphin antiserumDBTC3766 ± 3863699 ± 3153953 ± 3783599 ± 3653457 ± 3583357 ± 3873490 ± 3353896 ± 312Vehicle1410 ± 2451357 ± 3181507 ± 2101538 ± 2351690 ± 2151479 ± 2181590 ± 2321914 ± 208NOTE. Amylase concentrations are increased in rats with DBTC compared with vehicle-injected rats. None of the experimental manipulations affected these DBTC-induced changes. Data are expressed as mean SEM U/L. The data are taken from 6 animals per group. Open table in a new tab Table 2Concentrations of Lipase in the Blood Serum of Rats From All Experimental GroupsNo treatmentNG lidocaineRVM lidocaineDermorphin-SaporinSaporinDermorphinWaterDynorphin antiserumDBTC190 ± 20190 ± 41216 ± 80207 ± 29185 ± 26189 ± 36197 ± 35195 ± 43Vehicle27 ± 632 ± 722 ± 516 ± 718 ± 917 ± 416 ± 315 ± 5NOTE. Lipase concentrations are increased in rats with DBTC compared with vehicle-injected rats. None of the experimental manipulations affected these DBTC-induced changes. Data are expressed as mean SEM U/L. The data are taken from 6 animals per group. Open table in a new tab NOTE. Amylase concentrations are increased in rats with DBTC compared with vehicle-injected rats. None of the experimental manipulations affected these DBTC-induced changes. Data are expressed as mean SEM U/L. The data are taken from 6 animals per group. NOTE. Lipase concentrations are increased in rats with DBTC compared with vehicle-injected rats. None of the experimental manipulations affected these DBTC-induced changes. Data are expressed as mean SEM U/L. The data are taken from 6 animals per group. On day 6 after injection of DBTC, the pancreas, liver, heart, lungs, and kidney were harvested and processed for histology using H&E staining. The pancreas showed signs of inflammation including edema formation, inflammatory cell infiltration, and acinar cell atrophy (Figure 7 A–B). Inflammatory signs were not present in the liver, lungs, heart, and kidneys of rats injected with DBTC or vehicle (Figure 7C–J). None of the experimental treatments modified the histologic appearance of the different organs examined either in the DBTC or vehicle-injected group (data not shown). Chronic pancreatitis can produce abdominal pain for extended periods of time even after the initial inflammation has subsided and pain is the main complaint of these patients.25Ammann R.W. Muellhaupt B. The natural history of pain in alcoholic chronic pancreatitis.Gastroenterology. 1999; 116: 1132-1140Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar Mechanical hypersensitivity of the abdominal area is a validated method to measure referred pain from visceral organs.2Vera-Portocarrero L.P. Yu Y. Westlund K.N. Nociception in persistent pancreatitis in rats effects of morphine and neuropeptides alterations.Anesthesiology. 2003; 98: 474-484Crossref PubMed Scopus (70) Google Scholar, 3Winston J.H. He Z.-J. Shenoy M. Xiao S.-Y. Pasricha P.J. Molecular and behavioral changes in nociception in a novel rat model of chronic pancreatitis for the study of pain.Pain. 2005; 117: 214-222Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 26Al-Chaer E.D. Kawasaki M. Pasricha P.J. A new model of chronic visceral hypersensitivity in adult rats induced by colon irritations during postnatal developments.Gastroenterology. 2000; 119: 1276-1285Abstract Full Text Full Text PDF PubMed Scopus (677) Google Scholar, 27Winston J.H. Toma H. Shenoy M. He Z.-J. Zou L. Xiao S.-Y. Micci M.-A. Pasricha P.J. Acute pancreatitis results in referred mechanical hypersensitivity and neuropeptide up-regulation that can be suppressed by the protein kinase inhibitor k252a.J Pain. 2003; 4: 329-337Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 28Vera-Portocarrero L.P. Westlund K.N. Attenuation of nociception in a model of acute pancreatitis by an NK-1 antagonist.Pharmacol Biochem Behav. 2004; 77: 631-640Crossref PubMed Scopus (27) Google Scholar Tail-vein injection of DBTC has been characterized as a model of persistent pancreatitic pain.2Vera-Portocarrero L.P. Yu Y. Westlund K.N. Nociception in persistent pancreatitis in rats effects of morphine and neuropeptides alterations.Anesthesiology. 2003; 98: 474-484Crossref PubMed Scopus (70) Google Scholar, 28Vera-Portocarrero L.P. Westlund K.N. Attenuation of nociception in a model of acute pancreatitis by an NK-1 antagonist.Pharmacol Biochem Beh
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