Role of Muscarinic Receptors in Hypoalgesia Induced by Crocin in Neuropathic Pain Rats

Abstract

Objective: Crocin as an important constituent of saffron has antineuropathic pain properties; however, the exact mechanism of this effect is not known. The aim of this study was whether the hypoalgesic effect of crocin can be exerted through muscarinic receptors.

Materials and methods: In the present project, 36 male Wistar rats (200 ± 20 g) were used. Animals randomly divided into six groups (sham, neuropathy, neuropathy + crocin, neuropathy + atropine 0.5 mg/kg, neuropathy + atropine 1 mg/kg, and neuropathy + atropine 1 mg/kg + crocin). Neuropathy was induced by the chronic constriction injury (CCI) method on the sciatic nerve. Crocin and atropine was administered intraperitoneally during 14 days following the 14^th day after surgery. Pain response was detected every three days, two hours after each injection and 3 days following last injection. Mechanical allodynia and thermal hyperalgesia were detected using the Von Frey filaments and plantar test device, respectively.

Results: CCI significantly reduced the paw withdrawal response to mechanical and thermal stimulus (P < 0.01 and P < 0.05, respectively). Crocin therapy significantly reduced mechanical allodynia and thermal hyperalgesia induced by CCI (P < 0.05). Atropine pretreatment significantly blocked the hypoalgesic effect of crocin (P < 0.05 in mechanical allodynia and P < 0.01 in thermal hyperalgesia). Fourteen days administration of atropine alone at a dose of 0.5 mg/kg but not 1 mg/kg significantly reduced CCI-induced mechanical allodynia at day 30 after surgery.

Conclusion: Crocin significantly decreased CCI-induced neuropathic pain. The hypoalgesic effect of crocin was blocked by atropine pretreatment, which indicates an important role for muscarinic receptors in the effect of crocin.

Figure 1

Timeline of experiments. Days 0, 1, 2, 3,…, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30.

Figure 2

Effect of crocin on CCI-induced neuropathic pain. CCI significantly reduced mechanical (a) and thermal (b) paw withdrawal threshold compared to the sham group. Fourteen days crocin therapy significantly reduced CCI-induced mechanical allodynia and thermal hyperalgesia. Hypoalgesic effect of crocin was observed at the end of therapy (day 27) and continued on the day 30 after surgery. All data are expressed as mean ± SEM. n = 6. And sign and num sign compares crocin with the CCI group. Asterisks compare CCI with the sham group. #P < 0.05, ^∗∗P < 0.01, ^∗∗∗∗P < 0.0001, ^&&&P < 0.01, and ^&&&&P < 0.001.

Figure 3

Effect of atropine on CCI-induced neuropathic pain. Effect of atropine with two doses, 0.5 and 1 mg/kg, were examined on the mechanical allodynia (a) and thermal hyperalgesia (b) during 2 weeks of treatment from 14^th day to 27^th day after surgery. Atropine with a dose of 0.5 mg/kg significantly (P < 0.001) reduced mechanical allodynia at day 30 after surgery (3 days after stopping treatments); however, atropine 1 mg/kg did not change CCI-induced neuropathic pain during days in which pain behaviors were examined. Asterisks compare atropine 1 mg/kg with the CCI group at day 30 after surgery. All data are expressed as mean ± SEM. n = 6.^∗∗∗P < 0.001.

Figure 4

Effect of atropine pretreatment on the crocin-induced hypoalgesia. Atropine at 1 mg/kg significantly blocked the hypoalgesic effect of crocin. Fourteen days pretreatment with atropine significantly inhibited the effect of crocin on the mechanical allodynia (a) and thermal hyperalgesia (b), and these effects continued on to the 30^th day after surgery, while all treatments were stopped three days before. Asterisks compare atropine + crocin against that crocin group at days 27 and 30 after surgery. All data are expressed as mean ± SEM. n = 6. ^∗∗P < 0.01; ^∗∗∗P < 0.001.