Butorphanol Decreases Edema Following Carrageenan-Induced Paw Inflammation in Rats

PASCAL VACHON, DMV, PHD,1* AND JEAN-PIERRE MOREAU, RLATR2

The objectives of this study were to evaluate the anti-inflammatory effects and possible drug interactions of butorphanol by using carrageenan-induced paw inflammation in rats. We injected the right hind footpads of 64 female Sprague-Dawley rats (approximate weight, 100 g) with 50 µL of a 0.5% solution of carrageenan. Indomethacin (0, 1, 2.5, or 5 mg/kg) was administered by gavage to each of four groups 1 h prior to carrageenan injections. Half of the animals in each group received butorphanol (2 mg/kg) subcutaneously 1 h prior to carrageenan injections. Butorphanol decreased paw inflammation following carrageenan injections with or without concurrent administration of indomethacin. No drug interaction with indomethacin was observed. In conclusion, when using butorphanol for its analgesic properties, investigators should be mindful of the drug’s anti-inflammatory effects, which may interfere with the experimentation.

Carrageenan injected into the hindpaws of rats induces an Ancare, Bellmore, N.Y.) and acclimated for 1 week prior to the inflammatory response (1) that has been used widely to test anti- study. Hardwood bedding (Beta Chips, Northeastern Products inflammatory drugs. Opioid analgesics are known to have Co., Warrensburg, N.Y.) was changed twice weekly. Rats received anti-inflammatory properties (2). Mu (µ) receptor-activating reverse osmosis-purified, ultraviolet-treated water and a certified opioids (e.g., morphine) decrease edema, hyperalgesia, and pain laboratory diet (Harlan Teklad Mouse/Rat Diet, Bartonville, Ill.) in various animal models of inflammation. However, very little ad libitum. The MDS Pharma animal facility is accredited by the attention has been given to kappa (κ) receptor-activating opio- Canadian Council of Animal Care, and the experimental proto- ids. Experimental drugs such as U 50488 and tifluadom, which col was approved by the Institutional Animal Care and Use are specific κ receptor agonists, decrease carrageenan-induced Committee prior to animal use. paw swelling (3). Analgesic drugs used in small rodents are mainly Carrageenan-induced paw inflammation and indomethacin opioids and non-steroidal anti-inflammatory drugs (4), and the administration. The protocol we used for carrageenan-induced opioid analgesics typically used are mainly µ agonists and the mixed paw inflammation has been published previously (5). In our κ and µ agonist, butorphanol. Although the anti-inflammatory and study, we injected 50 µl of a 0.5% solution of carrageenan (Lamda analgesic properties of non-steroidal anti-inflammatory drugs and Type IV, Sigma, Inc., St. Louis, Mo.) in the right hind footpad of µ agonists have been evaluated (2), the anti-inflammatory proper- all animals, and they were returned to their cage thereafter. In- ties of butorphanol have not. domethacin (Sigma, Inc.) in 0.5% carboxymethylcellulose The main objective of this study was to evaluate the effect of (Sigma, Inc.) was prepared at doses of 1.0 mg/kg for groups 2 concurrent butorphanol administration on the indomethacin- and 6, 2.5 mg/kg for groups 3 and 7, and 5 mg/kg for groups 4 associated reduction of carrageenan-induced paw inflammation. and 8; groups 1 and 5 received the vehicle only. The carboxym- Indomethacin is a non-steroidal anti-inflammatory drug that has ethylcellulose solution and indomethacin solutions were been used as a standard in the evaluation of the anti-inflamma- administered by gavage, at a dose volume of 3 ml/kg, 1 h prior tory properties of new drugs. If butorphanol minimally affects to the carrageenan injections. In addition, groups 1 through 4 carrageenan-induced inflammation and the effects of indometha- received saline subcutaneously (s.c.), and groups 5 through 8 cin on that process, butorphanol could be used to increase the received 2 mg/kg butorphanol tartrate (Torbugesic, Ayerst Labo- well-being of animals involved when screening for new anti-in- ratories, Montreal, Quebec, Canada) at a dose volume of 2 ml/kg flammatory drugs by using the carrageenan-induced paw s.c. 1 h prior to carrageenan injections. inflammation model. The rats were euthanized 3 h after the carrageenan injections by using an overdose of isoflurane. Both hind paws were severed Materials and Methods at the tarsotibial joint, removed, and placed in preweighed weigh- Animals. We used 64 (groups 1 through 8; eight animals each) boats. The difference in weight between the right (injected) and female Sprague-Dawley rats (approximate weight, 100 g; age, 4 left (uninjected) paws for each animal (right hind paw—left hind weeks) from Charles River (St-Constant, Quebec, Canada). The paw) was calculated. sex and weight were chosen according to a previously published Histologic preparations. Paws were placed in a 10% formalin protocol (5). These rats were specific pathogen-free (SPF) and solution. After 2 days, the tissues were embedded in paraffin. originated from a barrier facility. After their arrival at MDS Thin (5-µm) sections were stained with hematoxylin and eosin Pharma Services, the animals were kept in a SPF animal facility (H&E) and mounted for microscopic observation. (15 fresh, filtered air changes/h; 21° ± 3°C; 40% to 60% humidity; Statistical analysis. Unpaired t tests were performed to assess 12:12-h light:dark cycle). They were housed in groups of five in paw edema differences between group pairs 1 and 5, 2 and 6, 3 polycarbonate cages (height, 21 cm; width, 40 cm; length, 51 cm; and 7, and 4 and 8, which were matched for the carrageenan concentrations (0, 1, 2.5, and 5 mg/kg, respectively) injected into the right hind paw. The effects of subcutaneous butorphanol 1 Faculty of Veterinary Medicine, Department of Biomedicine, University of Montreal, 3200 (groups 5 through 8) on carrageenan-induced paw edema were Sicotte, St-Hyacinthe, Quebec J2S 7C6, Canada, and 2MDS Pharma Services, 2350 Cohen, St-Laurent (Montréal), Quebec H4R 2N6, Canada therefore compared to those of subcutaneous saline (groups 1 *Corresponding author through 4). Statistical significance was set at P < 0.05. All statisti-

Volume 41, No. 6 / November 2002 CONTEMPORARY TOPICS © 2002 by the American Association for Laboratory Animal Science 15 Figure 1. Photomicrographs showing transverse sections of the (A) control left hindpaw and (B) carrageenan-induced inflamed right hindpaw from an animal that received saline subcutaneously (group 1). Note the edematous accumulation present in subcutaneous tissue of the right hindpaw (arrows). cal analyses were performed by using Statistica software (version 4.3, Statsoft, Tulsa, Okla.).

Results Figure 1 shows a typical example of edema following carrageenan injection into the right hindpaw; Fig. 2 highlights the increases in right hindpaw edema. Animals that received a subcutaneous injection of butorphanol (group 5) had a decrease in paw edema (P < 0.02) when compared to those receiving saline (group 1). For animals receiving indomethacin by gavage and butorphanol subcutaneously (groups 6, 7, and 8), only group 7 (which received 2.5 mg/kg) showed a significant difference (P < 0.01) in paw weights when compared with those of control groups (2, 3, and 4). However, decreases in paw weights occurred in all groups and varied between 17% and 38%. No clear drug interaction was observed because paw weights with increasing indomethacin concentrations with and without butorphanol Figure 2. Increase in right hindpaw weight following the intraplantar behaved in a parallel fashion (slope, 0.23 versus 0.22). Variation injection of 50 µL of a 0.5% solution of carrageenan and the effects of (% coefficient of variability) in the dissection procedure is re- different oral doses of indomethacin (0, 1, 2.5, and 5 mg/kg) without flected by the differences in left hindpaw weights, which ranged and with subcutaneous administration of butorphanol (2 mg/kg). The from 3% to 7% for all groups. The dissection procedure was data are shown are the mean ± 1 standard deviation from eight animals. therefore relatively similar between groups. tion of the peripheral sympathoadrenal system is activated by Discussion intracerebroventricular and intracisternal injections of µ opio- Butorphanol with or without concurrent administration of ids (10, 11). In contrast, intracisternal injections of κ agonists indomethacin decreases paw edema following carrageenan in- produce a decrease in sympathoadrenal tone (12). This last jections. However, no clear drug interaction occurred because finding is supported by the peripheral inhibition of the release indomethacin decreased edema in a similar fashion with or with- of epinephrine by κ but not µ agonists (13). Because catechola- out butorphanol. This decrease was relatively consistent (17% mines can inhibit the inflammatory response (14, 15), the to 38%) independent of indomethacin concentrations. This anti-inflammatory potential of µ opioids might therefore be ex- observation should be taken with caution and not generalized plained by increases in epinephrine and perhaps corticosterone. to all anti-inflammatory drugs because butorphanol and in- For κ agonists, the anti-inflammatory potential may be explained domethacin act via different mechanisms. by an increase in circulating corticosterone only. When injected Morphine decreases paw edema by 24% to 36% (6), a range peripherally, κ agonists such as U50488, trifluadom, and similar to that we observed with butorphanol. This result can butorphanol, increase plasma levels of corticosterone significantly be explained in part by a contribution of the central nervous (16). These findings strongly support the anti-inflammatory prop- system. Opioid agonists (µ and κ) stimulate the hypothalamo- erties of butorphanol. pituitary-adrenal axis (HPA) in rats, causing a release of In conclusion, when using opioids for their analgesic proper- corticosterone from the adrenal glands (7-9) which may account ties, one should remember their anti-inflammatory properties, the anti-inflammatory properties of opioid drugs. However, a which may help in the healing process but also interfere with decrease in plasma corticosterone is observed when the activa- the experimental results when studying inflammatory processes.

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