Descending Facilitation From the Rostral Ventromedial Medulla Maintains Visceral Pain in Rats With Experimental Pancreatitis

GASTROENTEROLOGY 2006;130:2155–2164

Descending Facilitation From the Rostral Ventromedial Medulla Maintains Visceral Pain in Rats With Experimental Pancreatitis LOUIS P. VERA–PORTOCARRERO, JENNIFER X. YIE, JUSTIN KOWAL, MICHAEL H. OSSIPOV, TAMARA KING, and FRANK PORRECA Department of Pharmacology, College of Medicine, University of Arizona, Tucson, Arizona

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.

ain 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.1–3 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

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some persistent pain conditions.4,5 The rostral ventromedial medulla (RVM) has been implicated as an important source of descending modulation of pain transmission.6 Microinjection of lidocaine into the RVM reversed experimental neuropathic pain,7,8 revealing the critical role for descending facilitation in this type of pain. Selective ablation of ␮-opioid receptor– expressing cells in the RVM, presumed to be important for descending facilitation, prevented the maintenance, but not initiation, of experimental neuropathic pain.4,9,10 At the spinal cord level, dynorphin also appears to be critical for maintaining, but not initiating, experimental neuropathic pain.11–14 Spinal dynorphin is up-regulated reliably after peripheral injuries and up-regulation is secondary to activation of descending facilitation mechanisms.15 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.16,17 Low doses of RVM neurotensin facilitate whereas higher doses inhibit acute visceral reflexes.18 RVM N-methyl-d-aspartate (NMDA) antagonists block facilitation of visceral reflexes whereas non-NMDA antagonists injected in the RVM block descending inhibition.19 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,20,21 which relays in the nucleus gracilis (NG).22 Such information may be relayed to sites involved in descending modulation of pain21,23 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 Abbreviations used in this paper: RVM, rostral ventromedial medulla; NG, nucleus gracilis; DBTC, dibutyltin dichloride. © 2006 by the American Gastroenterological Association Institute 0016-5085/06/$32.00 doi:10.1053/j.gastro.2006.03.025

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facilitation mechanisms arising in the RVM and the contribution of spinal dynorphin to such visceral pain.

Materials and Methods Animals Adult male Sprague–Dawley rats (Harlan, Indianapolis, IN) weighing 150 –200 g were maintained in a climatecontrolled 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 Model 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).2 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.2 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-␮m 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.

Supraspinal Lidocaine Microinjection Procedures 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 can-

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nula 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)24 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.24 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 ␮l 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.

RVM Pretreatment With Dermorphin–Saporin 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 ␮l 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.

Dynorphin Content in the Spinal Cord 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.

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Results NG Lidocaine Reverses Pancreatitic Pain

Figure 1. Time-course of the effects of lidocaine microinjection into the NG on referred abdominal hypersensitivity as measured by frequency of withdrawal from mechanical stimulation of the abdominal area. Before any manipulation, rats presented negligible withdrawals to von Frey probing. On day 6 after DBTC injection, rats presented increased frequency of withdrawals compared with vehicle-injected rats (baseline, before NG microinjections). NG lidocaine reduced the frequency of withdrawals at 20 minutes after injection to baseline levels. This effect still was present at 40 minutes after lidocaine injection. ⫹P ⬍ .05 vs DBTC–NG–saline group; n ⫽ 8 per group.

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.

Intrathecal Antidynorphin Antiserum Injection 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 ␮g followed by a 9-␮l saline flush) or control antiserum was given intrathecally as previously described.15 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.

Before receiving any treatments, or after IV vehicle, probing the abdomen with the von Frey filament elicited very few withdrawal responses (Figure 1, preDBTC). 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. RVM Lidocaine Reverses Pancreatitic Pain 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 timerelated 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). Spinal Dynorphin Content Thoracic dynorphin content was increased significantly when measured 6 days after DBTC compared

Statistical Procedures 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.

Figure 2. Time-course of the effects of lidocaine microinjection into the RVM on referred abdominal hypersensitivity as measured by frequency of withdrawal from mechanical stimulation of the abdominal area. Before any manipulation, rats presented negligible withdrawals to von Frey probing. On day 6 after DBTC injection, rats presented increased frequency of withdrawals (baseline, before RVM microinjections). RVM lidocaine reduced the frequency of withdrawals at 20 minutes after injection. This effect was not seen by 40 minutes after lidocaine injection. ⫹P ⬍ .05 vs DBTC–RVM–saline group; n ⫽ 8 per group.

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Figure 3. Dynorphin levels in the spinal cord dorsal horn of rats 6 days after DBTC-induced pancreatitis and control vehicle rats. Dynorphin levels were increased significantly in rats with DBTC-induced pancreatitis only in the thoracic spinal cord dorsal horn. *P ⬍ .05 vs vehicle group; n ⫽ 10 per group.

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. Spinal Antidynorphin Antiserum Reverses Pancreatitic Pain 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 ␮g, Bachem, Torrance, CA) or control serum (200 ␮g) as previously described15 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

Figure 4. Intrathecal injection of antidynorphin antiserum blocks the increased referred abdominal hypersensitivity seen 6 days after DBTC. Rats injected with DBTC presented increased numbers of withdrawals compared with vehicle control rats. At 20 minutes after spinal antidynorphin antiserum injection, the frequency of withdrawal was similar to the control group. The effect still was evident at 40 minutes after injection. Injection of control antiserum did not have any effects on withdrawal responses seen in either the DBTC or vehicle rats. ⫹P ⬍ .05 vs DBTC group; n ⫽ 8 per group.

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Figure 5. Time-course of the DBTC-induced referred abdominal hypersensitivity in rats receiving a single RVM pretreatment with vehicle, dermorphin, saporin, or conjugated dermorphin–saporin. All groups developed referred abdominal hypersensitivity starting on day 3 after DBTC injection. On day 6 after DBTC injection, rats pretreated with dermorphin–saporin showed reduced referred abdominal hypersensitivity and this effect continued through day 7. *P ⬍ .05 vs control groups; n ⫽ 8 per group.

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. RVM Dermorphin–Saporin Prevents Maintenance of Pancreatitic Pain A single RVM injection of dermorphin–saporin was shown previously to elicit a loss of RVM ␮-opioid receptor– expressing cells by postinjection day 28.9,10 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).

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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). Figure 6. Pretreatment with RVM dermorphin–saporin prevents the increase in dynorphin content seen in the thoracic cord dorsal horn of rats 6 days after DBTC-induced pancreatitis. Dynorphin content was increased in rats with RVM pretreatment with water, dermorphin alone, or saporin alone. ⫹P ⬍ .05 vs RVM control groups (dermorphin, saporin, and water). There were 10 rats per group.

RVM Dermorphin–Saporin Prevents Pancreatitis-Induced Up-Regulation of Spinal Dynorphin 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). Presence of Pancreatic Inflammation and Pancreatitis Markers 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 (Tables 1 and 2). Treatment with dynorphin antiserum did not modify the concentrations of amylase and lipase (Tables 1 and 2). Treatment in the RVM of dermorphin–saporin or the respective controls did not modify the increase of pancreatic enzyme levels (Tables 1 and 2). Inflammatory Signs Are Confined to the Pancreas 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

Figure 7. Representative photographs of histologic sections of different internal organs of rats 6 days after IV injection of either vehicle or DBTC. Pancreas tissue section from (A) vehicle and (B) DBTC animals. The pancreas from DBTC-injected animals showed signs of inflammation including edema, inflammatory cell infiltration, and acinar cell atrophy. There were no signs of inflammation in the (C, D) liver, (E, F) heart, (G, H) lungs, and (I, J) kidneys.

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Table 1. Concentration of Amylase in the Blood Serum of Rats From All Experimental Groups

DBTC Vehicle

No treatment

NG lidocaine

RVM lidocaine

DermorphinSaporin

Saporin

Dermorphin

Water

Dynorphin antiserum

3766 ⫾ 386 1410 ⫾ 245

3699 ⫾ 315 1357 ⫾ 318

3953 ⫾ 378 1507 ⫾ 210

3599 ⫾ 365 1538 ⫾ 235

3457 ⫾ 358 1690 ⫾ 215

3357 ⫾ 387 1479 ⫾ 218

3490 ⫾ 335 1590 ⫾ 232

3896 ⫾ 312 1914 ⫾ 208

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.

Discussion 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.25 Mechanical hypersensitivity of the abdominal area is a validated method to measure referred pain from visceral organs.2,3,26 –28 Tail-vein injection of DBTC has been characterized as a model of persistent pancreatitic pain.2,28 In this model, the inflammation is limited to the pancreatic tissue and does not extend to other organs. Injection of DBTC has been reported to have effects on the liver but the methodology used and the mode of administration are different from our study.29,30 In the time course of our experiments, the inflammation was limited to the pancreas, suggesting that the referred abdominal hypersensitivity is specific for pancreatitis as previously shown.2 Mechanisms of acute visceral pain have been studied more thoroughly than those underlying chronic visceral pain. An important aspect is understanding mechanisms that mediate the transition from acute to chronic stages of visceral pain. One important mechanism proposed for chronic somatic pains is the development of central sensitization.31,32 Similar processes of sensitization have been suggested to influence the advent of chronic visceral pain.33,34 Unlike previous models of pancreatic pain that have a short time course,35–37 DBTC-induced abdominal hypersensitivity persists for 7–10 days after induction, allowing for study of plasticity of the nervous system that might contribute to the persistence of this type of visceral pain. Previous work has shown that descending modulation of visceral pain processing occurs. During cervical cord block, viscerosomatic neurons showed reduced responses to splanchnic nerve stimulation,38 colorectal,39 and esophageal distention.40 Electrical stimula-

tion of the RVM produced biphasic modulation of spinal cord neuronal responses to colorectal distention16 and of colorectal distention–induced nociceptive reflexes,17 with facilitation and inhibition seen at low and high electrical intensities, respectively. Lidocaine microinjection, or ibotenic acid injection to produce lesion of the RVM, decreased spontaneous activity and responses of spinal neurons to colorectal distention.16 Although most of these studies investigated acute visceral responses, the present investigation explored the role of descending pain facilitatory influences, and subsequent spinal plasticity, in the expression of persistent visceral pain of an inflammatory origin. Our data show that the persistence of pancreatitis pain depends on activation of descending pain facilitatory mechanisms that arise in the RVM and subsequent upregulation of spinal dynorphin. DBTC produced referred abdominal mechanical hypersensitivity that lasted through 7 days postinjection and such hyperalgesia corresponded with an increase in markers of pancreatitis and a significant up-regulation of dynorphin in the thoracic spinal cord. Referred abdominal hypersensitivity was abolished by microinjection of lidocaine in the RVM, suggesting a requirement for descending pain facilitation for the expression of pancreatitis pain. Pancreatitis pain also was abolished by spinal administration of antidynorphin antiserum, suggesting a prominent pronociceptive role for this peptide in visceral pain. The role of descending facilitation was supported by data indicating a requirement for RVM ␮-opioid receptor– expressing cells, hypothesized to be important in generating descending pain facilitation.41– 43 Dermorphin–saporin ablation of RVM ␮-opioid receptor– expressing neurons prevented the up-regulation of spinal dynorphin and, although not

Table 2. Concentrations of Lipase in the Blood Serum of Rats From All Experimental Groups

DBTC Vehicle

No treatment

NG lidocaine

RVM lidocaine

DermorphinSaporin

Saporin

Dermorphin

Water

Dynorphin antiserum

190 ⫾ 20 27 ⫾ 6

190 ⫾ 41 32 ⫾ 7

216 ⫾ 80 22 ⫾ 5

207 ⫾ 29 16 ⫾ 7

185 ⫾ 26 18 ⫾ 9

189 ⫾ 36 17 ⫾ 4

197 ⫾ 35 16 ⫾ 3

195 ⫾ 43 15 ⫾ 5

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.

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affecting the initial expression of pancreatitis pain, prevented the maintenance of this condition. Descending influences from the RVM can exert facilitatory influences in nociceptive transmission at the spinal cord level.42 In somatic pain models, inactivation of the RVM with lidocaine reveals a facilitatory drive that enables increased nociceptive sensitivity.7,8,10 The descending facilitation pathway travels in the dorsolateral funiculus, and lesions of this tract block neuropathic and opioid-induced hyperalgesia.10,15 Activation of descending facilitation in some injury states can be time dependent because RVM lidocaine blocked nerve injury–induced behavioral hypersensitivity at 6, but not 3, days after injury. In addition, specific ablation of RVM ␮-opioid receptor– expressing cells similarly blocks neuropathic pain in a time-dependent manner.9,10 In these studies, it also was observed that ablation of this population of cells did not affect acute pain thresholds. This evidence suggests that this population of facilitatory cells has a relatively minor role in the modulation of physiologic pain but a more prominent role in persistent pathologic pain. These studies indicate that the descending facilitation from the RVM plays a critical role in the maintenance, but not the induction, of nerve injury– induced pain. Parallel findings were observed in the present study with pancreatitis-induced pain. At the peak time of pancreatitis-induced abdominal hypersensitivity, microinjection of lidocaine into the RVM produced a time-related reversal of such hyperalgesia, supporting a requirement for descending facilitation for the expression of pancreatitis pain. A possible role for descending inhibition in the modulation of pancreatic nociception cannot be excluded because DBTC-induced inflammation of the pancreas is observed within 24 hours,2 but referred abdominal hypersensitivity is not present until day 3. It is possible that descending inhibition is activated early after inflammation of the pancreas, delaying behavioral manifestation of mechanical hypersensitivity. As the inflammation persists, the descending inhibitory influence may be overwhelmed and the balance shifts to descending pain facilitation as has been suggested in other models of somatic inflammation.44,45 Indeed, descending inhibition has an important role in the modulation of acute visceral pain.46 –51 Dynorphin is an endogenous opioid that may act via ␬ receptors.52 More recently, dynorphin also has been shown to be pronociceptive in chronic pain states.13,14,53 Dynorphin is up-regulated after nerve injury, inflammation, and prolonged opioid treatment.10,11,14,54 –56 Dynorphin also is up-regulated after abdominal inflammation produced by carrageenan.57 Antiserum against dynor-

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phin reduces neurologic impairment after nerve injury,58,59 and blocks neuropathic60 and opioid-induced pain,12 suggesting a prominent pronociceptive role after up-regulation. Mice lacking the precursor gene for prodynorphin develop nerve injury–induced pain normally, but do not sustain such pain.11 The up-regulation of spinal dynorphin in chronic pain states is secondary to descending facilitation from the RVM.61 Lesions of the dorsolateral funiculus or ablation of RVM cells expressing the ␮-opioid receptor prevent the up-regulation of dynorphin in the lumbar spinal cord after nerve injury10,62 or sustained morphine.15 In the present study, dynorphin was up-regulated only in the thoracic cord of rats with pancreatitis and this up-regulation was important functionally because antiserum against dynorphin produced a time-related reversal of pancreatitis-induced abdominal hypersensitivity. The significant increase was not seen at cervical or lumbar levels, suggesting that the dynorphin up-regulation is not a systemic effect of DBTC. Consistent with this observation, ablation of ␮-opioid receptor– expressing cells in the RVM by microinjection of dermorphin–saporin prevented the upregulation of dynorphin in the thoracic cord of pancreatitic rats and pancreatitis-induced abdominal hypersensitivity. Visceral nociception can reach higher brain centers by multiple pathways including the spinothalamic, spinoreticular, and spinohypothalamic pathways.63– 67 Clinical evidence has indicated that the postsynaptic dorsal column pathway also is important in transmission of visceral pain.68 –72 In animal studies, it has been shown that the postsynaptic dorsal column cell pathway (PSDC) mediates nociceptive signals from the colon73–75 and duodenum.76 For the pancreas, it also has been established that nociceptive signals travel in the PSDC pathway because lesions of the dorsal columns block nociceptive behaviors induced by pancreatic inflammation.1 Nociceptive signals travel to the thalamus by the PSDC pathway,20 with a relay in the NG.22 These findings are consistent with data from the present study in which inactivation of the NG with lidocaine elicited a timerelated reversal of pancreatic nociception. These observations suggest that interrupting ascending pathways also may interrupt descending pain facilitation mechanisms, a concept already proposed in inflammatory pain77 and visceral pain,78 which may be driven by either sustained afferent input from the inflamed organ and/or evoked inputs from the von Frey filaments. It is important to note that other pathways also may have a role in the activation of descending facilitation. The spinoparabrachial pathway has been shown to promote descending facilitation75 and vagal pathways also can activate brainstem influences.79,80

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In conclusion, the present study has shown that descending facilitation plays an important role in the maintenance, but not initiation, of pancreatic nociception. Engagement of descending facilitation in the RVM may be the result of afferent input through the dorsal columns, likely involving postsynaptic dorsal column cells with relays in the NG. Such descending pain facilitation appears critical for the up-regulation of spinal dynorphin and for the expression of pancreatitis pain. Critically, descending pain facilitation mechanisms maintain, but do not initiate, pancreatitis pain. These findings reveal a novel mechanism by which pancreatitis pain is sustained and provide a path for the investigation of mechanisms of descending pain facilitation and eventual therapeutic interventions in chronic visceral pain.

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Received October 19, 2005. Accepted February 22, 2006. Address requests for reprints to: Frank Porreca, PhD, Department of Pharmacology, College of Medicine, University of Arizona Health Sciences Center, Tucson, Arizona 85724. e-mail: [email protected]; fax: (520) 626-4182. Supported by DA11823 and NS051011.