Role Of TRPM8 On Primary Afferent Neuron In Rats With Chronic Neuropathic Pain

Part I Alteration of TRPM8 in dorsal root ganglion in rat model of neuropathic painObjective:To investigate the change of the expression of transient receptor potential melastatin 8 (TRPM8) in dorsal root ganglion (DRG) in the rat model of chronic constriction injury (CCI) of the siatic nerve.Methods:Seventy-two male SD rats weighing 250-280g were randomly divided into 2 groups(n=36 each):group I (CCI) and groupⅡ(sham operation).The thresholds of cold hyperalgesia,mechanical hyperalgesia and heat hyperalgesia were measured before operation(baseline) and on 1,4,7,10 and 14d after operation.Six rats were killed at each time point in each group.The L5 DRGs ipsilateral to nerve injury were dissected out for deternination of TRPM8 by immunohistochemical assay.Results:The thresholds of cold, mechanical and thermal stimuli started to decrease at 4d after CCI in operation group and maintained at a relatively low level until the end of experiment.The cold and thermal hyperalgesia peaked at 10d after operation and mechanical hyperalgesia at 14d. Immunohistochemical assay demonstrated that expression of TRPM8 protein in L5DRG on the operated side was increased significantly at 4d after CCI and reached the peak at 10d and was maintained at a high level until the end of experiment.Conclusion:The upregulaion of TRPM8 in DRG is involved in the mechanism of neuropathic pain.PartⅡEffects of menthol and TRPM8 antisense oligonucleotide on hyperalgesia and the experssion of TRPM8 in dorsal root ganglion of rats with chronic neuropathic painObjective:To investigate the effects of intrathecal menthol and TRPM8 antisense oligonucleotide(ASODN) on hyperalgesia and the experssion of TRPM8 in DRG of rats with chronic constriction injury of the sciatic nerve,and try to elucidate the role of TRPM8 channel in the mechanism of neuropathic pain.Methods:Part one:Thirty-two male SD rats weighing 300±10g were randomly divided into 4 groups(n=8 each):group sham operation+normal saline (group A),group sham operation+menthol (group B),group CCI+normal saline (group C) and group CCI+menthol (group D).Intrathecal catheter was placed according to the technique described by Yaksh et al.Five days after intrathecal catheter placement, the constriction injury of the sciatic nerve was performed in group C and group D. In group A and group B, the sciatic nerve was exposed but not ligated.Forteen days after CCI or sham operation,all four groups were administered intrathecally normal saline 20μl or menthol 100μg/kg.The number of brisk lifts of the treated hindpaw using cold plate,mechanical withdrawal threshold(MWT) using von Frey filaments and paw withdrawal latency(PWL) using hot plate were measured prior to CCI or sham operation (baseline) and before and after intrathecal injection on 14d following operation.The rats were killed after behavioral tests on 14d and L5 DRGs ipsilateral to nerve injury were dissected out for deternination of TRPM8 by immunohistochemical assay and western blot analysis. Part two:Thirty-two male SD rats weighing 300±10g were randomly divided into 4 groups(n=8 each):group sham operation+TRPM8 mismatch oligonucleotide(MSODN) (group E),group sham operation+ASODN(group F),group CCI+MSODN (group G) and group CCI+ASODN(group H).Intrathecal catheter was placed according to the technique described by Yaksh et al.Five days after intrathecal catheter placement,CCI operation was performed in group G and H and sham operation in group E and F.9-13d after CCI or sham operation,all four groups were administered intrathecally TRPM8 ASODN or MSODN 60μg/kg, twice a day.The number of brisk lifts of the treated hindpaw, MWT and PWL were measured prior to CCI or sham operation (baseline),8d and 14d after operation.The rats were killed after behavioral tests on 14d following operation and L5 DRGs ipsilateral to nerve injury were dissected out for deternination of TRPM8 by immunohistochemical assay and western blot analysis.Results:Part one:Intrathecal menthol attenuated mechanical and thermal hyperalgesia and enhanced cold hyperalgesia of CCI rats.Menthol had no effect on hyperalgesia of sham operation rats. Expressions of TRPM8 in group C and D were obviously higher than those in group A and B.Intrathecal menthol had no effect on the expression of TRPM8 protein in CCI rats,and so as the case in sham operation rats. Part two:TRPM8 ASODN attenuated cold hyperalgesia of CCI rats but had no effect on mechanical and thermal hyperalgesia of CCI rats.ASOND had no effect on hyperalgesia of sham operation rats.ASODN significantly decreased the expression of TRPM8 protein both in CCI and sham operation rats, but there was no significant difference in the level of TRPM8 protein between group F and H. MSODN had no effect on the expression of TRPM8 protein in CCI and sham operation rats.Conclusion:Part one:In rats with neuropathic pain,menthol exerts its effect via the activation of TRPM8 mediated signal transduction pathway.The activation of TRPM8 has no effect on hyperalgesia of sham operation rats. Part two:TRPM8 ASODN can reduce cold hyperalgesia of CCI rats by down-regulating the expression of TRPM8 protein.PartⅢDose-response relationship of menthol and TRPM8 antisense oligonucleotide on neuropathic painObjective:To establish the dose-response curves of menthol and TRPM8 ASODN and to evaluate the 50% effective dose (ED50) and 95% effective dose (ED95) of menthol and TRPM8 ASODN.Methods:Part one:Seventy male SD rats weighing 300±10g were randomly divided into 7 groups(n=10 each):normal saline group and 6 menthol groups of different doses.Intrathecal catheter was placed according to the technique described by Yaksh et al.Five days after intrathecal catheter placement,CCI operation was performed in all seven groups. Forteen days after CCI, normal saline group was administered intrathecally normal saline 20μl and 6 menthol groups were administered different doses of menthol.The dose-response curve constructed by probit regression analysis was established to calculate ED50 and ED95 of menthol.Part two:Seventy male SD rats weighing 300±10g were randomly divided into 7 groups(n=10 each):normal saline group and 6 TRPM8 ASODN groups of different doses. Intrathecal catheter was placed according to the technique described by Yaksh et al.Five days after intrathecal catheter placement,CCI operation was performed in all seven groups.9-13d after CCI, normal saline group was administered intrathecally normal saline 20μl and 6 TRPM8 ASODN groups were administered different doses of ASODN,twice a day. The dose-response curve constructed by probit regression analysis was established to calculate ED50 and ED95 of TRPM8 ASODN.Results:The ED50 of menthol was 56.24μg/kg(95% CI was 49.66-63.68μg/kg) and ED95 was 86.09μg/kg (95% CI was 73.43-121.18μg/kg).The ED50 of TRPM8 ASODN was 31.37μg/kg(95% CI was 27.85-35.37μg/kg) and ED95 was 46.34μg/kg (95% CI was 39.87-64.65μg/kg).Conclusion:The method of probit regression analysis is used to calculate ED50 and ED95 of menthol and TRPM8 ASODN.