Cannabinoid receptor type 1 antagonist, AM251, attenuates mechanical allodynia and thermal hyperalgesia after burn injury

Abstract

Background: Burn injury causes nociceptive behaviors, and inflammation-related pathologic pain can lead to glial cell activation. This study tested the hypothesis that burn injury activates glial cells, and cannabinoid receptor 1 (CB1R) antagonist, AM251, will decrease burn pain.

Methods: Anesthetized rats received 0.75-cm third-degree burn on dorsal hind paw. Vehicle or AM251 30 μg intrathecally (older rats, n=6 per group) or, either vehicle, 0.1 or 1.0 mg/kg intraperitoneally (younger rats, n=6 per group), started immediate postburn, was administered for 7 days. Mechanical allodynia and thermal hyperalgesia were tested on ventral paw for 14 days. Microglial and astroglial activity was assessed by immunocytochemistry.

Results: Allodynia, observed on burn side from day 1 to 14, was significantly (P<0.05) attenuated by intrathecal and intraperitoneal AM251 (1 mg/kg) starting from 3 to 14 days. Hyperalgesia, observed from day 3 to 12, was completely (P<0.05) reversed by intrathecal and intraperitoneal AM251 (1 mg/kg). AM251 0.1 mg/kg had no effect. Microglial activity (n=3 per time point) increased (P<0.05) 18.5±7.5 and 12.3±1.6 (mean±SD) fold at 7 and 14 days, respectively. Astroglial activity (n=4 per time point) increased 2.9±0.3 fold at day 7 only. Glial activities were unaltered by AM251.

Conclusions: AM251 inhibited nociceptive behaviors after burn even beyond 7-day period of administration. Although many studies have documented the utility of CB1R agonists, this study indicates that endogenous cannabinoids may have an unexpected pronociceptive effect during development of burn pain, explaining why CB1R antagonist, AM251, improves nociceptive behaviors. The decreased nociception with AM251 without altering glial activity indicates that AM251 acts further downstream of activated glial cells.

Figure 1. Intrathecal AM-251 attenuates mechanical allodynia of burn injury

Mechanical allodynia using Von Frey Filaments was tested preburn, and at 1–14 days postburn. Burn injury resulted in significant allodynia in the vehicle group throughout the observation period on the ipsilateral compared to contralateral side. Intrathecal AM-251 treatment, started immediately after burn and continued daily for 7 days attenuated the allodynia starting at 3 days. The beneficial effect of AM251 lasted up to 14 days beyond the infusion period of AM251.

Figure 2. Intrathecal AM-251 completely reverses thermal hyperalgesia of burn

Following hind paw burn, thermal hyperalgesia was measured preburn, and at 1–14 days postburn using beam of radiant heat. On vehicle treated ipsilateral (burn) side, hyperalgesia was induced at day 3, 5, 7 10, and 12, and spontaneously recovered by day 14. Intrathecal AM-251 treatment, started immediately after burn, and continued for 7 days after burn completely reversed the hyperalgesia even after termination of drug administration at day 7.

Figure 3. Intraperitoneal AM-251 to younger rats attenuates mechanical allodynia

Following hind paw burn, mechanical allodynia was observed as early as day 1 after burn and persisted even at day 14 on the untreated ipsilateral side of burn. The administration of AM-251 (1 mg/kg or 0.1 mg/kg daily) was started immediately postburn and continued up to day 7. The dose of 1.0 mg/kg of AM-251 attenuated the allodynia at 3, 5, 7, and 14 days. The administration of 0.1 mg/kg AM-251 showed no beneficial effects on mechanical alloydynia.

Figure 4. Interperiotneal AM-251 to younger rats reverses thermal hyperalgesia

Following hind paw burn injury, thermal hyperalgesia was measured using beam of radiant heat on the ipsilateral and contralateral sides. Hyperalgesic nociceptive behaviors were not observed at day 1 but observed at days 3, 5, and 7 postburn on the vehicle treated ipsilateral (burn) side. The hyperalgesia spontaneously reversed by day 14 after burn. Treatment with 1 mg/kg AM-251 completely reversed the development of hyperalgesia. The hyperalgesia persisted in the groups AM-251 0.1 mg/kg had no beneficial effect on hyperalgesia.

Figure 5. Burn injury increases ionized calcium-binding protein-1 (Ibal) expression in microglia on the ipsilateral side

Using Ibal antibody, microglial activity was determined at 3, 7, and 14 days after burn (n = 3 for each of burn and sham burn). Microglial activity did not differ between sham-burn and contralateral side of burn. At day 3, a diffuse 2–3 fold (nonsignificant) increase in microglial activity was observed; at day 7 and 14, microglial activity (indicated by arrows) was significantly (p < 0.05) increased 18.5 ± 7.5 and 12.5 ± 1.6 fold, respectively, on the dorsal horn of the ipsilateral side compared to contralateral side. Scale bars indicate 200μm

Figure 6. Increased glial fibrillary acidic protein (GFAP) expression at 7 days after burn is unaltered by treatment with AM251

Atroglial activity on the spinal cord dorsal horn was determined using fluorescent GFAP antibody at day 7. Groups studied (n = 4/group) included sham-burn treated with vehicle (Sham + Veh), ipsilateral side of burn treated with vehicle (Burn + Veh), contralateral side to burn treated with vehicle (Contra + Veh), ipsilateral to burn treated with AM-251 (Burn + AM-251), contralateral to burn treated with AM-251 (Contra + AM-251). Scale bar for 6A and B indicate 100 μm and for 6Ca, 6Cb, 6Da, 6Db indicate 25 μm. Burn injury significantly increases astroglia activity 2.9 ± 0.3 fold (p < 0.05)on the ipsilateral side compared to contralateral side at day 7. Treatment with AM-251 does not change the activity/expression of astroglia on the ipsilateral side compared to vehicle treatment of ipsilateral side (n = 4/group).

Figure 7. Increased ion calcium-binding adaptor molecule-1 (Ibal) expression in microglia of the spinal cord dorsal horn is unaltered by the administration of AM251

Activity of microglia with and without AM251 in spinal dorsal horn at level of lumbar 1–2 was studied at day 7 after burn using specific fluorescent tag, Iba1. Groups studied included sham burn animals treated with vehicle (Sham + Veh), ipsilateral side of burn treated with vehicle (Burn + Veh), contralateral side to burn treated with vehicle (Contra + Veh), burn injured side treated with AM-251 (Burn + AM-251), contralateral side to burn treated with AM-251 (Contra + AM-251). Scale bars for 7A and B are 100 μm and for 7ca, 7cb, 7Da, and 7DB are 25 μm. Burn injury, relative to contralateral side, activates microglia on the ipsilateral side. Treatment with AM-251 did not alter activation of microglia on the ipsilateral side of burned mice compared to ipsilateral side treated with vehicle (n = 3/group).