Melatonin prevents morphine-induced hyperalgesia and tolerance in rats: role of protein kinase C and N-methyl-D-aspartate receptors

Abstract

Background: Morphine-induced hyperalgesia and tolerance significantly limits its clinical use in relieving acute and chronic pain. Melatonin, a pineal gland neurohormone, has been shown to participate in certain neuropsychopharmacological actions. The present study investigated the effect of melatonin on morphine-induced hyperalgesia and tolerance and possible involvement of protein kinase C (PKC)/N-methyl-D-aspartate (NMDA) pathway in melatonin-mediated.

Methods: Experiments were performed on adult, male Sprague-Dawley rats. Melatonin (10 mg/kg, intraperitoneal, i.p.) or saline was administrated 10 min after morphine injection (10 mg/kg, subcutaneous, s.c.) each day for consecutive 14 days. Withdrawal threshold of the hindpaw to mechanical and thermal stimulation was measured before any drug administration and one hour after melatonin or saline on each designated test day. On the 15(th) day, thermal withdrawal was measured after s.c. morphine (20 mg/kg), but not melatonin, and morphine tolerance was measured and expressed by MPAE% (percent of maximal possible anti-nociceptive effect) of morphine. Levels of expression of protein kinase C gamma (PKCγ) and NMDA receptor subtype NR1 in spinal cord were detected by Western blotting.

Results: The mechanical withdrawal threshold and thermal withdrawal latency decreased and shortened significantly (i.e., threshold decreased) in rats that received morphine treatment for two weeks compared with that in rats receiving saline. This morphine-induced mechanical and thermal hyperalgesia were greatly attenuated by co-administration of morphine with melatonin. The MPAE% representing morphine analgesic effect was reduced approximately 60% in rats that received morphine treatment. However, following the treatment of morphine with melatonin, the MPAE% was reduced only about 30%, comparing with those that received saline treatment as control. Administration of morphine alone resulted in significantly increased expression of PKCγ and NR1 proteins in the spinal cord. These increased levels of expression of PKCγ and NR1 were significantly inhibited by co-administration of morphine with melatonin.

Conclusions: Our findings demonstrate that melatonin have potential to attenuate repetitive morphine-induced hyperalgesia and tolerance, possibly by inhibiting PKCγ and NR1 activities in the spinal cord.

Keywords: Melatonin; Morphine tolerance; Morphine-induced hyperalgesia; NR1; PKCγ.

Figure 1

Melatonin attenuated morphine-induced mechanical and thermal hyperalgesia. A: Hindpaw withdrawal threshold to mechanical stimulation. B: Hindpaw withdrawal latency to thermal stimulation. Mechanical withdrawal threshold and thermal withdrawal latency were both gradually decreased and shortened, respectively, in rats that received morphine (10 mg/kg, s.c.) alone from day 3 to 14. Co-administration of morphine with melatonin(10 mg/kg, i.p.) significantly prevented the decreased withdrawal threshold and latency from day 3 to day 14. Six rats were included in each group. *P < 0.05 and **P < 0.01, compared with SAL-SAL; # P <0.05 and ## P < 0.01, compared with MOR-SAL. Data are presented as mean ± SEM.

Figure 2

Effect of melatonin on the morphine tolerance. The development of the tolerance to morphine’s analgesic effect was assessed by the hindpaw thermal withdrawal latency at 60 min after co-administration (10 mg/kg melatonin and/or 10 mg/kg morphine) from day 1 to 14 (A), as well as MPAE% of morphine at 60 min after administration of morphine (20 mg/kg) alone on day 15 (B). A: Injection of morphine (10 mg/kg) significantly increased the thermal withdrawal latency in rats receiving administration of morphine on day 1. However, such an analgesic effect gradually decreased and then disappeared (tolerance) from day 3 to 14 after repeated treatment of morphine. Co-administration of 10 mg/kg melatonin reversed the analgesic effect of morphine. B: The MPAE% of morphine (20 mg/kg) significantly decreased in rats receiving repeated administration of morphine on day 15. However, the decreased MPAE% owing to morphine tolerance was reversed by consecutive 14 days co-administration of morphine with melatonin. MPAE% = [(test latency - basal latency)/(20–basal latency) × 100% (20 s as the cut-off time). *P < 0.05 and **P < 0.01, as compared with the SAL-SAL group at the same time point; # P <0.05 and ## P < 0.01, as compared with the MOR-SAL group at the same time point. Data are presented as mean ± SD for 6 rats per group.

Figure 3

Effects of melatonin on morphine-induced increased expression of PKCγ and NR1 in the spinal cord. Western blot shows expression of PKCγ (A) and NR1 (B) in the spinal cord dorsal horn (n = 3) in each sample following treatment of morphine with or without melatonin for consecutive 14 days. Data are presented as mean ± SEM. *P < 0.05, compared with SAL-SAL group # P < 0.05, compared with MOR-SAL group. C: Co-localization of spinal PKCγ and NR1. There was co-localization of PKCγ and NR1 immunoreactivity in the superficial layers (I and II) of the spinal cord dorsal horns at the lumbar (L4) level. Spinal cord samples were taken from rats receiving a combination of morphine and melatonin for consecutive 14 days (n = 3). Blue: DAPI for nucleus. Scale bar: 100 μm. DL: the dorsolateral part of the spinal cord dorsal horn.