Role Of Three Opioid Receptors In The Mechanisms Of Morphine Tolerance

Morphine, nowadays, as the most effective opioids, remains a overwhelming drug in the management of moderate or severe pain. But its clinical utility is severely hindered by the development of drug tolerance and physical dependence when administered chronically. Recently, with every effort in the study of opioid tolerance, it has made a great progress in the mechanisms of morphine tolerance. Many kinds of morphine tolerant models were established in vivo or in vitro, and the possible cellular signal trasduction pathways in morphine tolerance were postulated. However, because of the complexity mechanism in morphine tolerance, results from vitro experiments still cannot explain some phenomena in vivo, conclusions from many studies seem inconsistence and need further verification.μ,δ,κare the most common opioid receptors in nervous system which mediate various effects of morphine and were cloned successively. But the role of these three opioid receptors in morphine tolerance and whether the interactions of them are important to morphine tolerance has not been elucidated. This experiment was designed based on the initial component, opioid receptors, to evaluate the changes of three opioid receptors in various regions in nervous system of a classical morphine tolerant rat model by the method of immunohistochemistry and real time PCR, and to provide an experimental evidence to further explore the mechanisms of morphine tolerance.PartⅠEstablishment of Morphine Tolerant Rat Model32 healthy and similar pain threshold adult male Wistar rats were randomly allocated equally to 2 groups. Group NS (n=16), the control group, received a subcutaneous (s.c.) injection of 1ml/kg normal saline (NS) in the nape; group MS (n=16) received a s.c. injection of morphine 10mg/kg. All rats were housed in a same enviroment and received s.c. injection twice a day (8:00 and 16:00) for 7 days. In the morning of the 8th day, 8 control rats and 8 morphine rats were killed. The remaining rats, which named as NSN subgroup and MSN subgroup respectively, received no further morphine injections, bur received 2 injections of naloxone (0.4mg/kg) on the 8th day twice (8:00 and 16:00), and were killed on the 9th day. The tail-flick latency (TFL) as an index of pain threshold was obtained with the rat tail immersed in hot water (52±0.5℃) and was measured in each rat 30 min before and after every morning injection, with the average TFL before the first injection as the baseline value. The cutoff time was 12s to avoid burn of the tail skin. Morphine tolerance was developed, as TFL returned to the baseline level.The rats' weight which were measured every morning in the observation period increased gradually, and there was no significant changes between two groups. The pain behaviors indicated that TFLs before injection every morning were not significantly different within or between groups. The TFL of Group MS increased significantly than baseline value and Group NS after first morphine injection. But as injection increasing, the average TFL after injection of Group MS shortened gradually and returned to baseline value after the 7th day injection. There were no significant difference in TFL after naloxone injection between groups and baseline value.Part II Protein expression of three opioid receptors in central nervous system of morphine tolerant rats16 healthy and similar pain threshold adult male Wistar rats were randomly allocated equally to 2 groups, named Group NS (n=8) and Group MS (n=8). The drug administration, pain threshold measurement and standard of morphine tolerance are same as Part I . The subcutaneous injection conducted consecutively for 7 days and 4 rats in each group were killed on the 8th morning. The remaining rats, which recorded as NSN subgroup and MSN subgroup, s.c. naloxone 0.4mg/kg twice in the 8th day, and were killed on the 9th morning. All rats were killed with 4℃4% polyformaldehyde perfusion and fixation under deep anesthesia. The brain and spinal cord were anatomised and prepared of paraffin sections for immunohistochemistry (IHC). Analyse protein expression of three opioid receptors in thalamus, hypothalamus, hippocampus, locus ceruleus, periaqueductal gray (PAG) and layer I -V of lumber-sacral spinal cord.The analysis of IHC stain suggested thatμopioid receptor decreased significantly in PAG of morphine tolerant rats.δopioid receptor also had a lower expression in PAG and LC than control group. Butκopioid receptor increased highly in LC and layer I - V of lumber-sacral spinal cord.Part III mRNA expression of three opioid receptors in nervous system of morphine tolerant rats16 healthy and similar pain threshold adult male Wistar rats were randomly allocated equally to 2 groups, named Group NS (n=8) and Group MS (n=8). The drug administration, pain threshold measurement and standard of morphine tolerance are same as Part I . The subcutaneous injection conducted consecutively for 7 days and 4 rats in each group were killed on the 8th morning. The remaining rats, which recorded as NSN subgroup and MSN subgroup, s.c. naloxone 0.4mg/kg twice in the 8th day, and were killed on the 9th morning. All rats were killed decapitation under deep anesthesia. The brain, spinal cord and dorsal root ganglion were anatomised and refrigerated in liquid nitrogen. Extract total RNA of thalamus, hypothalamus, hippocampus, LC, PAG, lumber-sacral spinal cord and dorsal root ganglion. Analyse mRNA expression of three opioid receptors in these regions using real time PCR method.The result of real time PCR indicated that mRNA expression ofμopioid receptor decreased significantly in PAG of morphine tolerant rats.δopioid receptor also had a lower expression in PAG and LC than control group. Thoughκopioid receptor mRNA expression increased highly in LC and lumber-sacral spinal cord, it had a down-regulation in dorsal root ganglion.Based on the results of this study, we may draw the following conclusions:1 .Morphine tolerance may be a result of several opioid receptors coregulation;2.μopioid receptor decreased significantly in PAG of morphine tolerant rats;3.δopioid receptor decreased in PAG and LC of morphine tolerant rats;4.κopioid receptor increased statisticly in LC and lumber-sacral spinal cord, but it had a down-regulation in dorsal root ganglion;5.The involved regions of morphine tolerance should be PAG, LC, spinal cord and dorsal root ganglion.