Role of GFRα3 in expression and membrane trafficking of TRPM8 in dorsal root ganglion during cold hyperalgesia in rats with neuropathic pain

Abstract

Objective To evaluate the role of glial cell line-derived neurotrophic factor family receptor alpha3(GFRα3)in the expression and membrane trafficking of transient receptor potential melastatin 8(TRPM8)in the dorsal root ganglion(DRG)during cold hyperalgesia in rats with neuropathic pain(NP). Methods Thirty-two healthy adult male Sprague-Dawley rats, aged 10-12 weeks, weighing 250-280 g, in which intrathecal catheters were successfully implanted, were divided into 4 groups(n=8 each)using a random number table: sham operation plus GFRα3 dsRNA group(Sham+ dsRNA group), sham operation plus GFRα3 siRNA group(group Sham+ siRNA), NP plus GFRα3 dsRNA group(group NP+ dsRNA)and NP plus GFRα3 siRNA group(group NP+ siRNA). NP was produced by chronic constriction injury to the sciatic nerve.At 10-30 days after operation, GFRα3 dsRNA 10 μg/20 μl was intrathecally injected once a day for 4 consecutive days in Sham+ dsRNA and NP+ dsRNA groups, and 10 μg/20 μl GFRα3 siRNA, of which the sense strand was modified with 2′-O-methyl and 5′-cholesterol, was intrathecally injected once a day for 4 consecutive days in Sham+ siRNA and NP+ siRNA groups.The number of paw lifts on the cold plate, mechanical paw withdrawal threshold(MWT)and thermal paw withdrawal latency(TWL)were measured on 1 day before operation and 10, 11, 12, 13(before intrathecal injection)and 14 days after operation.The rats were sacrificed after the last behavioral testing, and ipsilateral DRGs of the lumbar segment(L4-6)were dissected for detection of the expression of GFRα3 and TRPM8 in total and membrane proteins by Western blot, and the ratio of TRPM8 expression in the membrane protein to that in the total protein(m/t ratio)was calculated. Results Compared with group Sham+ dsRNA, the number of paw lifts on the cold plate was significantly increased, the MWT was decreased, and TWL was shortened after operation in NP+ dsRNA and NP+ siRNA groups, the expression of GFRα3 and TRPM8 in total and membrane proteins was significantly up-regulated, and m/t ratio was increased in group NP+ dsRNA, and the expression of GFRα3 in DRGs was significantly down-regulated(P 0.05). Compared with group NP+ dsRNA, the number of paw lifts on the cold plate was significantly decreased, the expression of GFRα3 and TRPM8 in total and membrane proteins was down-regulated, m/t ratio was decreased(P 0.05). Conclusion GFRα3 in DRGs can up-regulate the expression of TRPM8 and enhance the membrane trafficking of TRPM8, which may be involved in the maintenance mechanism of cold hyperalgesia in rats with NP. Key words: Glial cell line-derived neurotrophic factor receptors; TRPM cation channels; Neuralgia; Hyperalgesia, cold; Ganglia, spinal