| The treatment of chronic pain has been the difficulty in clinical. Cold stimulus can alleviate chronic pain and avoid the side effects of commonly used analgesic drugs. However, excessive cold can evoke pain and induce hypercryalgesia. Transient receptor potential channels(TRPs), the research hotspot in recent years, which plays an important role in chronic pain, is an important kind of cation channel located on cell membrane. TRPM8, a member of TRP channel family, is activated by cool compounds such as menthol, icilin, and by temperature below 28℃.The identification of TRPM8 produces profound, novel methods to relieve pain. In the dissertation, a model of chronic arthritic rats was constructed. Based on this model, the effect of TRPM8 on the chronic arthritic rats was explored when it was activated by menthol or blocked by the antisense oligonucleotides.The results of this study are expected to provide important information for future treatment and studies associated with chronic arthritic pain.Part one:Alteration of TRPM8 expression in dorsal root ganglion of arthritic rats.Objective:To establish the rat model of complete Freund's adjuvant-induced arthritis, observe the changes of TRPM8 expression in dorsal root ganglion of the arthritic rats, and analyze the significance of TRPM8 on the development of hypercryalgesia. Methods:The 30 healthy male SD rats, weighed 300±20g, which were made into model of adjuvant induced arthritis, were randomized into 5 groups(n=6), as 1 day arthritis group (group CFA-1),4 day arthritis group (group CFA-4),7 day arthritis group (group CFA-7),10 day arthritis group (group CFA-10),14 day arthritis group (group CFA-14).The other 30 healthy male SD rats were made into control model without arthritis, randomized into 5 groups(n=6), as 1 day in control group (group NS-1),4 day in control group (group NS-4),7 day in control group (group NS-7),10 day in control group (group NS-10),14 day in control group (group NS-14). Behavioral indicators of rats were examined every day. Immunohistochemistry was used to detect the expression of TRPM8 in dorsal root ganglion. Results:As model established, the expression of TRPM8 was up-regulation which reached a peak level in groups CFA-7, CFA-10 and CFA-14, while the expression of TRPM8 in the control groups was not changed. Conclusion:TRPM8 expressed in DRG may be involved in the mechanism of cold allergy in arthritic rats.Part two:Effects of menthol and TRPM8-antisense oligonucleotides on hyperalgesia and the experssion of TRPM8 in dorsal root ganglion of rats with chronic arthritisObjective:To establish the rat model of complete Freund's adjuvant-induced arthritis, observe the effect of menthol and TRPM8-antisense oligonucleotides on the behavioral indicators and the expression of TRPM8 in dorsal root ganglion of chronic arthritic rats, and analyze the significance of TRPM8 in chronic inflammatory pain rats. Methods:Healthy male SD rats, weighed 300±20g, were implanted intrathecal catheters and randomized into 8 groups. Amongst, the rats in 4 groups were made into model of adjuvant-induced arthritis and administered intrathecally with menthol 100μg/kg(group CFA-Menthol), saline 20μ(group CFA-NS), antisense oligonucleotid-es 60μg/kg(group CFA-Antisense), missense oligonucleotides 60μg/kg(group CFA-Missense). The rest rats in the other 4 groups were made into control model without athritis and administered intrathecally with menthol 100μg/kg (group NS-Menthol), saline 20μl(group NS-NS), antisense oligonucleotides 60μg/kg(group NS-Antisense), missense oligonucleotides 60μg/kg(group NS-Missense). Drugs were given once in the 14th day in group CFA-Menthol, CFA-NS, NS-Menthol and NS-NS, while given twice a day for five days from the 9th day in the rest groups.The cold plate indicators, mechanical withdrawal threshold (MWT) and thermal paw withdrawal latency (PWL) of rats was examined to evaluate their behavior. Immunohistochemical stain and western blot were used to measure the expression of TRPM8 in the rats'right L5 dorsal root ganglion. Results:After applied menthol, the hypercryalgesia of rats in group CFA-Menthol was enhanced, while the hyperthermalgesia and the mechanical pain were reversed. And the hypercryalgesia of rats in group CFA-Antisense was inhibited. The expression of TRPM8 was up-regulation in ipsilateral L5 dorsal root ganglion of the rats in group CFA-Menthol, CFA-NS and CFA-Missense with athritis,while blocked in goup CFA-Antisense and NS-Antisense after applied with TRPM8-antisense oligonucleotides. Conclusion:TRPM8 in athritic rats, activated by menthol given intrathecally, enhanced the hypercryalgesia while reversed the hyperthermalgesia and the mechanical pain.TRPM8, blocked by TRPM8-antisense oligonucleotides, inhibited the hypercryalgesia in athritic rats while had no effect on normal rats.The changes of chronic arthritic rats'hypercryalgesia were concerned with alteration of TRPM8 expression and the analgesic effect of menthol was related with the up-regulation of TRPM8.Part three:Measurement of the ED50 and ED95 of intrathecal menthol and TRPM8-antisense oligonucleotides for arthritic rats.Objective:To establish the rat model of complete Freund's adjuvant-induced arthritis, measure the the ED50 and ED95 of intrathecal menthol and TRPM8-antisense oligonucleotides for arthritic rats, and analyze the dose-effect relationship of menthol and TRPM8-antisense oligonucleotides in chronic arthritic rats. Methods: 140 healthy male SD rats, weighed 300±20g, were implanted intrathecal catheters, made into model of adjuvant-induced artliritis and randomized into 14 groups(n=10). Amongst, the rats in 7 groups were applied intrathecally saline 20μl, menthol 31.62μg/kg,39.81μg/kg,50.12μg/kg,63.10μg/kg,79.43μg/kg, 100.00μg/kg in the fourteenth day. The rats in the other 7 groups were applied intrathecally saline 20μl, antisense oligonucleotides 18.97μg/kg,23.89μg/kg,30.07μg/kg,37.86μg/kg,47.66μg/kg,60.00μg/kg, twice a day for five days from the ninth day.The number of the right hind paw lifts was measured. And if the number in group menthol or antisense oligonucleotides was thirty percent higher or lower than that of group NS, it would be confirmed as effective. ED50 and ED95 values were calculated using a Probit Regression models. Results:ED50 and ED95 values of menthol were 53.36μg/kg, 88.31μg/kg. ED50 and ED95 values of antisense oligonucleotides were 29.92μg/kg, 50.36μg/kg.Conclusion:ED50 and ED95 values of menthol were 53.36μg/kg, 88.31μg/kg.ED50 and ED95 values of antisense oligonucleotides were 29.92μg/kg, 50.36μg/kg. |
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