| A biological rhythm is one or more biological events or functions that reoccur in time in a repeated order and with a repeated interval between occurrences. Biological rhythms are the ways that organisms adapt and live with the environmental rhythms around them. Studies on Chronobiology show that circadian rhythms of the pain sensibility are demonstrated in both human beings and animals. However, the reports on circadian rhythm of normal mice's pain threshold are different and contradictory. Liu Xiaoping experimented on mice with hot-plate and tail-pressing methods in the same condition, and found that the mice had the same circadian rhythm to the two painful stimuli, and the pain response was maximal in dark phase. While Ma Kongchen reported the pain threshold in mice was higher in dark phase and lower in the light phase by using the hot-plate method,which suggested that the pain sensitivity is lower in dark-phase and higher in light phase. Therefore, the present study intended to further prove the circadian rhythm of mice pain sensitivity by using hot-plate method, which could provide the scientific evidence for clinical analgesia treatment and new reference for most researchers studying pain.PLP is experienced in a limb that is no longer present with different properties and intensities of pains. According to the clinical reports, more than 50% amputees suffer from PLP. At present, the precise reasons for PLP remain unclear, and there is no effective treatment method for PLP. The research shows that human beings have no uniform circadian rhythm of the pain response. At present, the report about circadian rhythm of pain response after amputation has not been seen. Therefore, it has very important theoretical and practical value to study pain threshold changes, circadian rhythm of pain response after amputation and its mechanisms for doctors treating PLP by chosing suitable time and improving clinical curative effect.Many studies show that the melatonin (MT) secreted by the pineal body of mammals has obvious circadian rhythm. As an internal zeitgeber, MT can regulate organism's circadian rhythm activities. MT has obvious analgesic effect and is involved in the formation of circadian rhythm of pain sense. However, the report about the relationship between the serum MT level's circadian rhythm of amputated mice and circadian rhythm of pain responses of amputated mice has not been seen. In the study of PLP, tail amputation may serve as a mouse model for studying long-term plastic changes in central nervous system after amputation. Therefore, in the present study, the hot plate experiment and Elisa methods were used to investigate effects of amputation of extremity of the tail in mice on circadian rhythms of their both pain responses and serum MT contents, the relationship between both, which could offer scientific experiment evidence for doctors treating PLP by chosing suitable time and improving clinical curative effect.In the present study, 144 Kunming adult female mice were used with weighting 23±3 grams. The latencies of the mice's licking their hindpaws on the hot plate (55±1℃) were recorded and used as an index of pain threshold by hot plate method. We selected the mice as the experimental animals whose latencies for the response were within 30s.Two weeks before the experiment, mice were reared under a standard light-dark rhythm(light phase 06:00-18:00;light:dark=12:12h), fared and drank freely. The room temperature was(15±3)℃. 72 female mice were divided into 6 groups randomly and each group was subjected to hot plate experiment at 8:00, 12:00, 16:00, 20:00, 24:00 and 4:00 respectively to measure the mice's pain threshold. We researched the normal mice's circadian rhythm of the pain response to hot plate stimulation. Another 72 mice were randomly put in 6 groups. The distal 2.5 cm length of the tail in the mice was removed. Gossypium absorbens was used to stop bleeding. After two weeks, each group was subjected to hot plate experiment at 8:00, 12:00, 16:00, 20:00, 24:00 and 4:00 respectively to measure the amputated mice's pain threshold. We researched the amputated mice's circadian rhythm of the pain response to hot plate stimulation. Mice were removed from the chamber if they did not respond within 60s, and the response latency was 60s recorded. After measuring the pain threshold of normal mice and amputated mice, the mice were decollated, and the blood were picked quickly to study circadian rhythm of serum MT levels and the relationship between circadian rhythms of serum MT level and circadian rhythm of the pain response by ELISA method.The results of this experiment showed that significant circadian rhythm of normal mice's response to hot plate stimulation was found. The pain sensitivity was minimal and the pain threshold was maximal (21.6±3.88 s, n=12) at 4:00;the pain sensitivity was maximal and the pain threshold was minimal(15.13±5.55 s, n=12) at 12:00. The pain sensitivity of the amputated mice also had significant circadian rhythm. The pain sensitivity was minimal and the pain threshold was maximal (19.26±4.62 s, n=12) at 4:00, the pain sensitivity was maximal and the pain threshold was minimal (12.65±3.74 s, n=12) at 12:00. Compared with normal mice's, every pain threshold of the amputated mice was decreased,and the pain sensitivity of the amputated mice had an increasing tendency. The skin of amputated mice's normal hindpaw was easier to produce pain response when it was stimulated, which might be caused by increase in excitability of neurons of pain centers induced by amputation of a segment of the tail and might be one of the reasons of PLP.The result of this experiment showed that significant circadian rhythm of normal mice's serum MT level was found. The serum MT level was lower at day-time and higher at night.The level in the night is about 2.5 times as the one in the day-time. Significant circadian rhythm of amputated mice's serum MT level was found, too. The level was lower at day-time and higher at night.The level in the night was about 1.8 times as the one in the day-time. There were close relationship between circadian rhythm of serum MT level and circadian rhythm of the pain sensitivity. And the experiment also proved that MT has the analgesic effect.In the dark phase, serum MT level was increased and made mice's pain sensitivity decreased. In the light phase, serum MT level was decreased and made mice's pain sensitivity to increase. Compared with normal mice's, serum MT level of amputated mice was significantly increased (p<0.01) and fluctuated obviously, which might be associated with the increase of pineal body'secreting MT induced by amputation pain stimulation. This study provided new scientific experiment evidence for the further explanation of PLP and for doctors treating PLP by chosing suitable time. |
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