Nociception in persistent pancreatitis in rats: effects of morphine and neuropeptide alterations

Abstract

Background: Most animal models of pancreatitis are short-lived or very invasive. A noninvasive animal model of pancreatitis developed in highly inbred rats by Merkord with symptoms persisting for 3 weeks was adopted in the current study to test its validity as a model of visceral pain in commercially available rats.

Methods: The persistent pancreatitis model was established by tail vein injection of dibutyltin dichloride. Animals were given 10% alcohol in their drinking water to enhance the pancreatitis attack. Blood serum pancreatic enzymes and nociceptive state were monitored for 3 weeks after dibutyltin dichloride or vehicle. Behavioral testing included reflexive withdrawal to mechanical and thermal stimulation of the abdominal area. The effect of morphine on nociceptive behaviors was tested. Histologic analysis of the pancreas and immunohistochemical analysis of substance P and calcitonin gene-related peptide in the spinal cord are included in the study.

Results: Compared with naïve and vehicle-only injected control groups, rats receiving dibutyltin dichloride demonstrated an increase in withdrawal events after von Frey stimulation and decreased withdrawal latency after thermal stimulation, signaling a sensitized nociceptive state through 7 days. These pain-related measures were abrogated by morphine. Blood serum concentrations of amylase and lipase as well as tissue inflammatory changes and substance P were also significantly elevated during this same time period.

Conclusions: These results indicate that animals with the dibutyltin dichloride-induced experimental pancreatitis expressed serum, histologic, and behavioral characteristics similar in duration to those present during acute attacks experienced by patients with chronic pancreatitis. These findings and responsivity to morphine suggest the utility of this model developed in a commercially available strain of rats for study of persistent visceral pain.

Fig. 1

Measurement of serum concentrations of amylase and lipase from naive, vehicle-injected, and dibutyltin dichloride (DBTC)-injected rats. (A) Time course of amylase serum concentrations through 3 weeks after tail vein injection. *P < 0.05 compared with vehicle-injected group. (B) Time course of lipase serum concentrations through 3 weeks after tail vein injection. *P < 0.05 compared with vehicle-injected group. Concentrations are expressed in units per liter. Error bars denote SEM.

Fig. 2

Paraffin sections of pancreas stained with hematoxylin and eosin from rats injected with dibutyltin dichloride (DBTC; 4-μm sections). (A) Normal pancreas (from naive and vehicle-injected animals) were similar in appearance. (B) Pancreas 3 days after DBTC injection in the tail vein (8 mg/kg body weight). The pancreas shows acinar atrophy (arrowhead), stromal proliferation (thick arrow), and edema in the interlobular and intralobular ducts (thin arrow). (C) Liver tissue section from a control Lewis rat stained with hematoxylin and eosin. (D) Liver section from a Lewis rat injected with DBTC taken 7 days after injection. No signs of inflammation are apparent in the liver. Scale bar = 100 mm.

Fig. 3

Time course of nociceptive behaviors. The y-axis indicates the average number of reflexive withdrawals from von Frey stimuli applied to the abdominal surface of rats (AWE). (A) Rats with dibutyltin dichloride (DBTC) injection demonstrate a greater sensitivity to von Frey stimuli (4.86 mN) compared with naive and vehicle-injected animals at 3 and 7 days after DBTC injection. (B) Similar results were obtained with von Frey stimuli with an applied force of 9.96 mN. (C) Suprathreshold stimuli (von Frey 204.1 mN) also evoked greater reactivity from rats with DBTC injection compared with rats from the other groups. +P < 0.05 when comparing the respective animal group behavior to its baseline measure at time point 0. *P < 0.05 when compared with corresponding controls (naive and vehicle-injected groups). Error bars denote SEM.

Fig. 4

Time course for the withdrawal latencies of rats injected with dibutyltin dichloride (DBTC), vehicle, or neither (naïve animals). Rats with DBTC injection demonstrated more rapid reflexive withdrawal to abdominal stimulation with a radiant heat source 3 and 7 days after injection compared with the vehicle-injected and naive groups. *P < 0.05 compared with vehicle control. +P < 0.05 compared with baseline (time point 0). Error bars denote SEM.

Fig. 5

Dose–response curves for nociceptive responses to von Frey stimuli (cumulative scores) and heat stimuli at days 3 and day 7 after either saline or morphine systemic injection in animals treated previously with either vehicle (v) injection or dibutyltin dichloride (DBTC) injection. *P < 0.05 versus that group’s own baseline (Student paired t test). +P < 0.05 comparing saline and morphine groups (Student unpaired t test). **,++P < 0.01. (A) Day 3 responses to von Frey stimuli. (B) Day 3 responses to heat stimuli. (C) Day 7 responses to von Frey stimuli. (D) Day 7 responses to heat stimuli. AWE = average withdrawal event (the summation of responses to three von Frey filaments; see Methods). Error bars denote SEM.

Fig. 6

Representative photomicrographs of spinal cord sections showing staining for substance P (SP; A and B) and calcitonin gene-related peptide (CGRP; C and D) in the dorsal horn. (A and C) Sections taken from control animals; (B and D) sections taken from animals with pancreatic inflammation induced by tail vein injection of dibutyltin dichloride.

Fig. 7

Time course for immunostaining density of substance P (SP) and calcitonin gene-related peptide (CGRP) in the superficial dorsal horn. (A) Quantitative immunohistochemistry demonstrates expression increases for SP at days 3 and 7 from animals injected with dibutyltin dichloride for comparison to the other groups (*P < 0.05). (B) The CGRP immunostaining showed a trend of increased density at all time points, but the increase did not reach statistical significance. Error bars denote SEM.