Thermal sensitivity as a measure of spontaneous morphine withdrawal in mice

Abstract

Introduction: Opioid withdrawal syndrome is a critical component of opioid abuse and consists of a wide array of symptoms including increases in pain sensitivity (hyperalgesia). A reliable preclinical model of hyperalgesia during opioid withdrawal is needed to evaluate possible interventions to alleviate withdrawal. The following study describes a method for assessing increases in thermal sensitivity on the hotplate in a mouse model of spontaneous morphine withdrawal.

Methods: C57BL/6J mice received 5.5days of 30, 56, or 100mg/kg morphine or saline (s.c., twice daily). In Experiment I, thermal sensitivity data were collected at baseline and at 8, 24, 32, 48h and 1week following the final injection. Thermal sensitivity was assessed by examining latency to respond on a hotplate across a range of temperatures (50, 52, 54, and 56°C). In Experiment II, 0.01mg/kg buprenorphine was administered 30min prior to each testing session during the withdrawal period. In Experiment III, jumping during a 30min period was assessed at baseline and at 0, 8, 24, 32, and 48h following the final morphine injection.

Results: During the withdrawal period, thermal sensitivity increased significantly in all morphine-treated mice as compared to saline-treated mice. Thermal sensitivity was greater in mice treated with 56mg/kg morphine compared to 30mg/kg and peaked earlier than in mice treated with 100mg/kg (32h v 1wk). The increase in thermal sensitivity following 56mg/kg morphine was attenuated by a dose of buprenorphine that did not produce antinociception alone (i.e., 0.01mg/kg). In general, the results of the jumping experiment paralleled those obtained in Experiment I.

Discussion: Response latency on the hotplate is a reliable and sensitive measure of spontaneous morphine withdrawal in mice, making it an ideal behavior for assessing the potential of medications and environmental interventions to alleviate opioid withdrawal.

Fig 1. Effects of 30, 56 or 100 mg/kg morphine or saline treatment on latency (mean ±SEM) to respond on the hotplate at 50, 52, 54, and 56° C

Morphine or saline treatment consisted of 5.5 days of twice daily injections (s.c.). Latency on the hotplate was determined at baseline and at 8, 24, 32, 48 hrs, and 1 wk after the final injection. Abscissa: hotplate temperature in ° C. Ordinate: latency to respond in seconds. N=7–8. Statistically significant differences (p<0.001) are indicated as follows: A= 30 mg/kg v. sal, B= 56 mg/kg v sal, C= 100 mg/kg v sal, X= 56 mg/kg v 30 and 100 mg/kg, Y= 100 mg/kg v 30 mg/kg.

Fig. 2. ET10 values (mean ±SEM) for mice following 5.5 days of 30, 56 or 100 mg/kg morphine or saline treatment

ET10 values represent the temperature that would produce a 10 sec response on the hotplate. Response latency on the hotplate was determined at baseline and at 8, 24, 32, 48 hrs and 1 wk after the final injection. N=8. Statistically significant differences are indicated as follows: *= a difference from the group’s baseline, # = a difference between morphine and saline treated mice at a particular time point. p<0.001

Fig. 3. The effect of 0.01 mg/kg buprenorphine on withdrawal from 5.5 days of 56 mg/kg morphine

A. Dose effect curves for buprenorphine (0.01– 0.32 mg/kg) at 50, 52, 54, and 56°C. Mean latencies (±SEM) are presented as % maximum possible effect (%MPE). B. ET10 values (mean ±SEM) for mice treated with 0.01 mg/kg buprenorphine or saline following 5.5 days of 56 mg/kg morphine. ET10 values represent the temperature that would produce a 10 sec response on the hotplate. Response latency on the hotplate was determined at baseline and at 8, 24, 32, and 48 hrs after the final morphine injection. Mice received 0.01 mg/kg buprenorphine (s.c.) 30 min prior to each hotplate test session. N=8. Statistically significant differences are indicated as follows: *= a difference from the group’s baseline, # = a difference between buprenorphine and saline treated mice. p<0.001

Fig. 4. Jumps (mean ±SEM) adjusted for baseline following 30, 56 or 100 mg/kg morphine or saline

Morphine or saline treatment consisted of 5.5 days of twice daily injections (s.c.). Jumping was determined at baseline and at 0, 8, 24, 32, and 48 hrs after the final injection. Baseline jumps indicate total jumping in 30 min at 10am and 6pm. Jumps at 0, 8, 24, 32, and 48 hrs indicate jumps observed during the 30-min withdrawal period minus the average number of jumps that occurred during the corresponding baseline period. Data obtained for the 0, 24, and 48 hrs withdrawal period fell in the AM; therefore, total jumps were adjusted using baseline measures from the AM period. Data obtained for the 8 and 36 hrs withdrawal period fell in the PM; therefore, total jumps were adjusting using baseline measures from the PM period. N=8. * = a statistically significant difference compared to saline treated mice. p<0.001.