Effects Of Amputation Of Extremity Of The Tail In Mice On Their Pain Responses,Serum Melatonin Contents And Their Mechanisms

Peripheral tissue or nerve injury often causes the persistent pain which last for an extended period of time after the injury, from days to months. Various diseases and accidents may bring about amputation of the part limb, which will often induce the persistent pain in the remaining part of the limb and phantom limb pain (PLP) lasting for months or even years. PLP is experienced in a limb that is no longer present with different properties and intensities of pains. It is reported that 60% to 70% of amputees suffer from PLP. Many studies show that PLP is caused by a series of peripheral and central mechanisms, and may be related to the pain before the operation, the nociceptive stimulation during the operation, the pain after the operation and the patients'mental state. PLP also may be related to the change of each component of sensory afferent pathway, for example, the functional reorganization of cerebral cortex. At present, the precise reasons for PLP remain unclear, and there is no effective treatment method for PLP.In the study of PLP, tail amputation may serve as a mouse model for studying long-term plastic change in central nervous system after amputation. Glutamic acid is the main excitatory neurotransmitter of brain and spinal cord, and displays postsynaptic effects through different receptors. Studies have discovered that noxious afferent signals cause increase in release of excitatory amino acids, especially glutamic acid of nerve centers and abnormal activation of their receptors. All types of glutamic acid receptors are involved in pain formation. But different glutamic acid receptors have different effects and mechanisms in formation of algesia. Many studies shows that the melatonin (MLT) secreted by the pineal body of mammals has obvious analgesic effect. Pain sensibility and release of pain mediators, and their effects are regulated by MLT. However, little is available with respect to research on whether MLT and glutamic acid acceptors participate in the pain-changing process 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 their pain responses,serum melatonin contents, and to examine whether glutamic acid receptors were involved in these changes after the amputation, which could provide new theoretical evidences for clinical treating and preventing from PLP.In the present study, the hot plate experiment method was used to investigate effect of amputation of extremity of the tail in mice on their pain responses. 260 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. In order to study the change of the mice'pain threshold in different period after amputation of extremity of the tail in mice, the distal 2.5 cm length of the tails in 90 mice was removed. Gossypium absorbens was used to stop bleeding. The 90 mice were divided into 6 groups randomly and each group was subjected to hot plate experiment at 0.5h, 1h, 2h, 3h, 24h, 168h after the amputation, respectively. Mice were removed from the chamber if they did not respond within 60s, and the response latency was 60s recorded. Following each experimental group, each blank control group was subjected to hot plate experiment. The experimental results showed that pain threshold was no significant changes between 0.5h or 1h groups after amputation and their control groups or before amputation(P> 0.05). But compared with pain threshold of their control groups or before amputation, pain threshold of 2h groups after amputation was significantly increased(P<0.01),one of 3h groups after amputation was significantly increased(P<0.05),too. Pain threshold was no significant changes between 24h or 168h groups after amputation and their control groups or before amputation(P>0.05). The result indicated that there were effects of amputation of extremity of the tail in mice on their pain responses, and the mice's pain threshold of 2h-3h after amputation was significantly increased.Because pain threshold change is maximal at 2h after amputation, effect of glutamic acid receptors antagonists on the mice's pain threshold change of 2h after amputation was studied. The results showed that the intravenous glutamic acid receptors antagonists, MK-801 or CNQX antagonized the increase in mice's pain threshold of 2h after amputation, which suggested that the NMDA receptors, the AMPA/Kainate receptors are involved in the process of increase in pain threshold induced by the amputation.Moreover, in the present study, the blood of mice of each control group and each amputated experimental group was obtained by cutting the mice heads after finishing the hot plate experiment mentioned above. The serum melatonin contents were measured by Elisa method. The results showed that compared with serum melatonin contents of their control groups, serum melatonin content of 2h group after amputation was significantly decreased(P<0.05) , and the intravenous glutamic acid receptors antagonists, MK-801 or CNQX antagonized the decrease in mice's serum melatonin contents of 2h after amputation, which suggested that the NMDA receptors, the AMPA/Kainate receptors are involved in the process of decrease in serum melatonin contents induced by the amputation.