Long Term Interactions between Tea Polyphenols, Memantine and Morphine on Orofacial Pain in Mice

Morphine is one of the analgesics used in dental clinics, but morphine tolerance is a severe side effect. We attempted to delay the development of morphine tolerance by a week off two days model and enhanced the analgesic effects of morphine by Green Tea Polyphenols (GTP), memantine (m) either alone or in combination (GTPm). The results obtained showed that morphine was very effective in attenuating the orofacial pain induced by capsaicin in mice even after prolonged treatment in 92 days. GTP, memantine and GTPm all were effective in attenuating at the late stage of prolonged treatment in 92 days on the orofacial pain of mice. The behavioral changes in mice after chronic treatment of morphine included the increased locomotor activities and decreased the exploratory activities in the open field of elevated plus maze which could be apparently minimized by GTPm. In conclusion, we have demonstrated that week off two days model and the combination use with GTPm could be successfully in enhancement of morphine analgesic effect on orofacial pain as long as for 3 months in the mice. DOI: 10.29011/APCM-106. 000006


Introduction
The prevalence of orofacial pain in the dental clinic is estimated as high as about 25 % of the population in the world. Morphine is one of the most commonly used analgesics in dentistry [1]. However, the severe side effects of morphine analgesic tolerance limited of its use. It is urgent for us to investigate the better effective analgesic for treatment of orofacial pain.
Morphine is one of the opioid alkaloids from opium poppy [2]. It induced analgesic effects by acting on μ-receptors located at gabaergic nerve endings which subsequently activated the descending inhibitory serotonergic neurons at the brain stern and then produced analgesia. Moreover, morphine inhibited pain sensation by reducing the release of substance P, glutamate and nitric oxide from nociceptive afferent neurons at the spinal cord. Chronic morphine administrations may alter the μ-receptor-Gi protein-adenylate cyclase-cAMP signaling pathway which accounted for morphine analgesic tolerance.
Transient Receptor Potential Vanilloid 1 (TRPV1) is nonselective cation channels which could be activated by capsaicin isolated from hot chili peppers [3]. TRPV1 were highly expressed in trigeminal nerves, dorsal root ganglion cells and central nervous system (hippocampus, striatum, hypothalamus and cerebellum), where it was involved in thermal nociception and burning sensation [4]. It has been demonstrated that TRPV1 colocalized with glutamatergic NMDA receptors and evoked pain sensation by facilitating activation of NMDA receptors. Memantine, an uncompetitive NMDA receptor antagonist, was thus able to attenuate pain sensation [5][6][7].
In this study, we attempted to delay the development of morphine tolerance by intermittent administration by week off two days model, which was based on our proposal that the processes of analgesic effects of morphine on μ-opioid receptors [8,9] may restore to the normal state during morphine holiday. The results obtained in this study were in accordance with our proposal that morphine analgesic effect on capsaicin-induced orofacial pain sustained as long as 92 days along with morphine administrations for 62 times.
According to the recent studies on the roles of neuroinflammation, oxidative stress and the activation of glutamate NMDA receptors in central nervous system involved in the development of morphine tolerance [10-14], we have tried in this study the effects of Green Tea Polyphenols (GTP) and memantine either alone or in combination (GTPm) on the analgesic effects of morphine and the morphine tolerance. This research approach was based on our previous findings that GTP possessed the pleiotropic effects including antioxidant, anti-inflammatory and neuroprotective effects [15][16][17][18][19], especially the potentiating effects of GTP on memantine (an uncompetitive antagonist of NMDA receptors) on neuroexcitability [20]. The results obtained in this study revealed that GTP and particularly GTPm alone exhibited not only analgesic effect but also enhanced that of morphine.

Animals
Adult male ICR (Institute for Cancer Research) mice 8-weeks old were used in this study. All mice were housed in groups of 5-7 in a cage with the same strain mates, in the animal faculty of the Chung Shan Medical University. Mice were allowed free access to food and water in a temperature controlled (22±1.5 O C) and relative humidity 50-70% environment maintained on a 12/12 hours light/dark cycle (light on 07:00 to 19:00). The experiment protocols were approved by the Animal Care Committee of Chung Shan Medical University (Approval No.1777).

Preparation of Green Tea Polyphenols (GTP)
One hundred grams of Chinese green tea, Longjing tea (produced by Wangs' Tea Enterprise Co., Ltd., Taipei, Taiwan), was suspended in 1 L of distilled water at 75°C for 30 min; then the supernatant was collected. This step was repeated three times. The supernatant was filtered to eliminate chlorophylls and undissolved particles. The total aqueous layers were concentrated to 0.5 L under reduced pressure using a rotary vacuum evaporator. The concentrated solution was extracted with an equal volume of chloroform three times to eliminate caffeine and pigments. The remaining aqueous phase was then extracted with an equal volume of ethyl acetate three times to extract tea polyphenols. The ethyl acetate was combined and evaporated in vacuum. The residue was dissolved in a small volume of distilled water and freeze-dried. This golden brown solid matter was called green tea polyphenols.
Green tea polyphenols (GTP 60 mg/Kg), memantine (Mem 10 mg/Kg), GTPm (GTP 30 mg/Kg plus Mem 3 mg/Kg) were dissolved in distilled water, which were orally administered to mice once per day and five times per week consecutively for 16 weeks. Morphine dissolved in saline was intraperitoneally injected (10 mg/kg/day) for consecutive five days and weekly off two days.

Nociceptive Assay
Orofacial pain was induced by injecting capsaicin (10 μg/5 μl) subcutaneously in vibrissa pad of mice and induced spontaneous orofacial pain response included 3 distinct patterns of acute grooming behaviors: fore-paw rubbing, lower lip skin/cheek rubbing against enclosure floor and hind paw scratching [21]. We record the total frequencies of orofacial pain response in 20 minutes after receiving capsaicin to quantify orofacial nociceptive level.

TruScan Photobeam Tracking
TruScan photobeam Tracking are used to record behavior (walking distance in margin and center area, number of times for jumping, rest time and total time of walking) of mice to compute emotional alteration. The tracking activity of depressed mice exhibits limited margin area walking distance; while normal mice distributes equally in margin and center area walking distance. Additionally, the tally of jumping and standing show the exploration and curious behavior of normal mice. One of the side effects of morphine is depressed mood, which causes reduction in locomotor activity and exploratory behavior. If memantine and GTPm can improve this side effect, locomotor activity and exploratory behavior will increase and the tracking in margin and center area will be equally distributed. TruScan photobeam Tracking will be measured 20 minutes after the injection of morphine. Volume 2018 Issue 01

Elevated Plus Maze Test
The elevated plus-maze test is one of the most widely used assessments to evaluate the anxiogenic-like behavior in rodents. The mouse was placed individually in the center of the maze (40 cm above the floor) facing an enclosed arm. The time spent in and number of entries into (with all 4 paws) both the open and enclosed arms (60 × 12 cm) were recorded for 5 min. The percentage of time spent in and the entries into the open arms was calculated by dividing the time spent in and the entries into the open arms by the total time spent in and the total arm entries into the both arms, respectively.

Statistics
Results for each experiment were represented as mean ± SEM. One-way ANOVA followed by a post-hoc t test was used to evaluate differences between the groups. The level of significance was defined as p<0.05.

Effects of Drugs on Capsaicin-induced Orofacial Pain
As shown in Figure 1, the responses (frequencies of face rubbing or scratching) to orofacial pain could be reduced by GTP and memantine either alone or in combination. After the long term oral administration (92 days), GTPm exhibited not only a better analgesic by itself but also enhanced that of morphine. Memantine alone apparently exerted analgesic effects at earlier stage but gradually attenuated this effect. GTP, memantine and GTPm alone were not only effective in attenuating orofacial pain induced by capsaicin but also enhanced analgesic effects of morphine.

Changes of Locomotor Activities of mice after Chronic Administrations of the drugs
As shown in Figure 2, GTP, memantine and GTPm had little effects on the locomotor activities (total distance) and the rest time (Figure 2A and Figure 2C). Memantine appeared to increase jump after 90 days administrations ( Figure 2B). Morphine alone increased locomotor activities and decreased in jump activities and rest time after long term administrations. Memantine but not GTP nor GTPm enhanced morphine in increasing locomotor activities of the mice after 90 days (Figure 2A). GTP, memantine and GTPm have little effect on locomotor activities but morphine markedly increased locomotor activities and decreased jump activities and rest time after 90 days. Memantine exacerbated but GTPm attenuated morphine in increasing locomotor activities.

Effects of Long Term Administrations of Drugs on the Exploratory Activities in the Elevated Plus Maze
The exploratory activities of the mice would increase its activities in the open field of the elevated plus maze apparatus, whereas the depressive mice would prefer to stay in the close field. As shown in Figure   After prolonged treatment, morphine decreased the exploratory activities (both frequencies and time) in open field and increased time duration in close field, which could be attenuated by both GTP and GTPm.

Discussion
Morphine is an effective analgesic frequently used in dental orofacial pain therapy. The severe problem of morphine tolerance awaited to be solved. Based on the recent studies 4-8 of neuroinflammation, increased oxidative stress and the activation of NMDA receptors involved in the induction of morphine tolerance, we have designed a novel regimen of a composite (GTPm) containing GTP and memantine which was shown to be a promising enhancer of morphine analgesic effect on orofacial pain induced by capsaicin in mice.
The signaling pathways of μ-opioid receptors in central nervous system play important roles in morphine analgesic effects. It has been found that phosphorylation of μ-opioid receptors, coupling of G-protein with adenylate cyclase-cAMP system, receptor desensitization and receptor internalization, all were continuously increased which may account for the development of morphine tolerance. Based on these findings, we suggested that morphine holidays may be able to let the central nervous system to have repairing opportunities and restored to the normalized condition, so that morphine analgesic effect could be normalized. In this study, we have successfully demonstrated that the week off two days model effectively delayed morphine tolerance in mice, chronically treated with low dose (10mg/kg/day) of morphine for 3 months. Since GTPm by itself is not only effective analgesic but also enhanced morphine analgesic effects, it is suggested that GTPm may be a promising adjunctive to morphine (low dose) in management of orofacial pain. Moreover, alternative application of nonopioid analgesic agents during morphine holidays and the combination use of morphine with other antioxidants (phytopolyphenols) with another NMDA receptor antagonist could be an ideal strategy to combat clinical chronic pain. This proposal is hopefully validated in the near future.

Conclusion
We have demonstrated that week off two days model and the combination use with GTPm could be successfully in maintenance of morphine analgesic effect on orofacial pain as long as for 3 months. The implication of this finding is that low dose of morphine in combination of antioxidants (such as GTP or phytopolyphenols) with NMDA receptor antagonists (memantine or others) and week off two day model (alternative use of non-opioid analgesic during morphine holidays) could possibly delay the development of morphine analgesic tolerance in clinical patients