Downregulations of TRPM8 expression and membrane trafficking in dorsal root ganglion mediate the attenuation of cold hyperalgesia in CCI rats induced by GFRα3 knockdown.

BACKGROUND

Cold hyperalgesia is an intractable sensory abnormality commonly seen in peripheral neuropathies. Although glial cell line-derived neurotrophic factor family receptor alpha3 (GFRα3) is required for the formation of pathological cold pain has been revealed, potential transduction mechanism is poorly elucidated. We have previously demonstrated the contribution of enhanced activity of transient receptor potential melastatin 8 (TRPM8) to cold hyperalgesia in neuropathic pain using a rat model of chronic constriction injury (CCI) to the sciatic nerve. Recently, the enhancement of TRPM8 activity is attributed to the increased TRPM8 plasma membrane trafficking. In addition, TRPM8 can be sensitized by the activation of GFRα3, leading to increased cold responses in vivo. The aim of this study was to investigate whether GFRα3 could influence cold hyperalgesia of CCI rats via modulating TRPM8 expression and plasma membrane trafficking in dorsal root ganglion (DRG).

METHODS

Mechanical allodynia, cold and heat hyperalgesia were measured on 1day before CCI and the 1st, 4th, 7th, 10th and 14th day after CCI. TRPM8 total expression and membrane trafficking as well as GFRα3 expression in DRG were detected by immunofluorescence and western blot. Furthermore, GFRα3 small interfering RNA (siRNA) was intrathecally administrated to reduce GFRα3 expression in DRG, and the effects of GFRα3 knockdown on CCI-induced behavioral sensitization as well as TRPM8 total expression and membrane trafficking in both mRNA and protein levels were investigated, and the change in coexpression of TRPM8 with GFRα3 was also evaluated. Then, the effect of GFRα3 activation with artemin on pain behavior of CCI rats pretreated with the selective TRPM8 antagonist RQ-00203078 was observed.

RESULTS

Here we found that TRPM8 total expression and plasma membrane trafficking as well as GFRα3 expression in DRG were initially increased on the 4th day after CCI, and maintained at the peak level from the 10th to the 14th day, which entirely conformed with the induction and maintenance of behavioral-reflex facilitation following CCI. The coexpression of TRPM8 with GFRα3, which was mainly located in peptidergic C-fibers DRG neurons, was also increased after CCI. Downregulation of GFRα3 protein in DRG attenuated CCI-induced cold hyperalgesia without affecting mechanical allodynia and heat hyperalgesia, and reduced the upregulations of TRPM8 total expression and plasma membrane trafficking as well as coexpression of TRPM8 with GFRα3 induced by CCI. Additionally, the inhibition of TRPM8 abolished the influence of GFRα3 activation on cold hyperalgesia after CCI.

CONCLUSION

Our results demonstrate that GFRα3 knockdown specially inhibits cold hyperalgesia following CCI via decreasing the expression level and plasma membrane trafficking of TRPM8 in DRG. GFRα3 and its downstream mediator, TRPM8, represent a new analgesia axis which can be further exploited in sensitized cold reflex under the condition of chronic pain.