Centrally mediated antinociceptive effects of cannabinoid receptor ligands in rat models of nociception

Abstract

The endogenous nonapeptide hemopressin (HE) demonstrates potent block of the cannabinoid subtype-1 (CB1) receptor in vitro and robust antinociception in vivo. The current study evaluated the effects of centrally administered HE in mechanistically distinct pre-clinical rat models of pain-the hot plate test and the hind paw formalin test. The non-subtype selective CB receptor agonist WIN 55,212-2 was tested concurrently as a positive control. In the hot plate test, neither intrathecal (i.t.) HE nor WIN 55,212-2 significantly altered the latency to respond to noxious heat. By contrast, i.t. HE and WIN 55,212-2 significantly reduced pain-related behaviors in the formalin test. Possible HE functionality as a CB1 receptor antagonist at the spinal level was evaluated in the formalin test. Intrathecal pretreatment with HE did not attenuate the antinociceptive effect of i.t. WIN 55,212-2. However, pretreatment with the CB1 receptor antagonist rimonabant did; i.t. rimonabant pretreatment was not antinociceptive. Potential supraspinal antinociceptive activity of HE was also evaluated. Whereas intracerebroventricular (i.c.v.) injection of WIN 55,212-2 reduced pain-related behaviors in the formalin test, interestingly, i.c.v. HE increased behaviors. In the current study, an antinociceptive effect with the CB receptor ligand HE was obtained under the specific condition of tissue injury and not in the uninjured state. Thus, HE could be a useful analgesic peptide with a novel spinal mechanism of action.

Figure 1

Maximum possible effect of intrathecal and intracerebroventricular injection of WIN 55,212-2, hemopressin and rimonabant in the hot plate test in rats. Rats were either intrathecally (IT) or intracerebroventricularly (ICV) injected with either 30 μg WIN 55,212-2 (WIN), 10 μg hemopressin (HE), 30 μg rimonabant (Rim) or an equal volume of vehicle. The effect of the compound 30 min following injection is shown. A significant antinociceptive effect of WIN was observed 30 min following ICV injection. Vehicle injection did not significantly withdrawal latencies (data not shown). Data are expressed as mean ± S.E.M. n = 3-8/group. *p < 0.05 vs. Vehicle. NT, not tested.

Figure 2

Effect of intrathecal WIN 55,212-2 on pain-related behavior in the formalin test in rats. A. The horizontal axis is time post-formalin injection (min) and the vertical axis is total number of formalin-induced behaviors counted per minute. Rats were intrathecally (i.t.) injected with 5 μl of a dose (in μg) of WIN 55,212-2 (WIN) or vehicle 10 min prior to hind paw formalin injection. B. Total number of formalin-induced behaviors during phase 1 (0-1 min) and phase 2 (15-61 min). Data are expressed as mean ± S.E.M. n = 7/group. *p < 0.05 vs. Vehicle.

Figure 3

Effect of intrathecal hemopressin on pain-related behavior in the formalin test in rats. A. The horizontal axis is time post-formalin injection (min) and the vertical axis is total number of formalin-induced behaviors counted per minute. Rats were i.t. injected with 5 μl of a dose (in μg) of hemopressin (HE) or vehicle 10 min prior to hind paw formalin injection. B. Total number of formalin-induced behaviors during phase 1 (0-1 min) and phase 2 (15-61 min). Data are expressed as mean ± S.E.M. N = 6/group. *p < 0.05 vs. Vehicle.

Figure 4

Best-fit dose-response curves of intrathecal hemopressin and WIN 55,212-2 in the formalin test. The horizontal axis is log-dose (μg) and the vertical axis is percent inhibition (i.t. vehicle treatment = 0% inhibition). The solid lines in (A) and (B) are best-fit curves for WIN 55,212-2 (WIN) A. Increasing i.t. doses of WIN and Hemopressin (HE) reduced formalin-evoked pain-related behaviors in Phase 1. However, 10 μg HE did not significantly affect formalin-induced behaviors. The A₅₀ (95% C.L.) of WIN is 2.2 (1.2-4.2 ) μg. The estimated A₅₀ for HE is 0.6 μg. B. The doses of 30 μg WIN 55,212-2 and 3 μg HE significantly reduced pain-related behaviors in Phase 2. The dose of 10 μg HE tended to increase formalin-induced behaviors in phase 2. Data are expressed as mean ± S.E.M.

Figure 5

Effect of intracerebroventricular (i.c.v.) injection of WIN 55,212-2 and hemopressin on pain-related behaviors in the formalin test in rats. Total number of formalin-evoked behaviors during phase 1 (0-1 min) and phase 2 (15-61 min) are shown. Five μl of either (A) WIN 55,212-2, (B) HE, or vehicle were i.c.v. injected 10 min prior to hind paw formalin injection. Data are expressed as mean ± S.E.M. N = 3-7/group. *p < 0.05 vs. Vehicle.

Figure 6

Effect of intrathecal hemopressin and rimonabant pretreatment on the antinociceptive effect of intrathecal WIN 55,212-2 in the formalin test. A. Rats were i.t. injected with either 1 μg HE or vehicle (Veh) 10 min prior to i.t. injection of 30 μg WIN (or Veh). Ten min following the second i.t. injection, the left hind paw was injected with formalin. B. Rats were i.t. injected with either 30 μg rimonabant or Veh 10 min prior to i.t. injection of 30 μg WIN (or Veh). Ten min following the second i.t. injection, the left hind paw was injected with formalin. Data expressed as total number of formalin-induced behaviors in phase 1 (0-1 min) and phase 2 (15-61 min); mean ± S.E.M. N = 7/group. *p < 0.05 vs. Veh/Veh.

Figure 6

Effect of intrathecal hemopressin and rimonabant pretreatment on the antinociceptive effect of intrathecal WIN 55,212-2 in the formalin test. A. Rats were i.t. injected with either 1 μg HE or vehicle (Veh) 10 min prior to i.t. injection of 30 μg WIN (or Veh). Ten min following the second i.t. injection, the left hind paw was injected with formalin. B. Rats were i.t. injected with either 30 μg rimonabant or Veh 10 min prior to i.t. injection of 30 μg WIN (or Veh). Ten min following the second i.t. injection, the left hind paw was injected with formalin. Data expressed as total number of formalin-induced behaviors in phase 1 (0-1 min) and phase 2 (15-61 min); mean ± S.E.M. N = 7/group. *p < 0.05 vs. Veh/Veh.