RESUMO
Objective To evaluate the role of glial cell line-derived neurotrophic factor family re-ceptor alpha3(GFRα3)in the expression and membrane trafficking of transient receptor potential melasta-tin 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 intrathe-cally 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<001), and no significant change was found in the expression of TRPM8 in total and membrane proteins or m∕t ratio in group NP+siRNA(P>005). Compared with group NP+dsRNA, the number of paw lifts on the cold plate was significantly de-creased, the expression of GFRα3 and TRPM8 in total and membrane proteins was down-regulated, m∕t ra-tio was decreased(P<001), and no significant change was found in MWT or TWL in group NP+siRNA (P>005). 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.
RESUMO
Objective To evaluate the significance and expression of the GDNF/GFRα1/RET genes in distal rectum with Congenital Anorectal Malformations(ARMs) Methods Specimens were collected from resected colon which is 3 cm away to the anus and the distal of rectum in 12 ARMs patients. Haematoxylin and Eosin (HE) staining, immunohistochemical (IHC) staining and reverse transcription PCR (RT-PCR) were used to test RET, GDNF and GFRα1 expression in the differ-ent site of samples with ARMs. Results Expression of ganglia and RET, GDNF and GFRα1 were all negative in distal rec-tum in 12 ARMs patients. Ganglionic cells were found in tissues 3 cm away from the anus where RET, GDNF and GFRα1 expression were also positive in those ARMs patients. Expression of RET, GDNF and GFRα1 were strong positive in middle and low imperforate anus indicated by brown color and week positive in high imperforate anus indicated by yellow color. One case of faeces contamination and one case of loose motion without anal incontinence were found in post-op follow up of high ARMs patients. By contrast, one case of rectal mucosa prolapse and one case of occasional faeces contamination which recov-ered with hip bath were found in post op follow up in middle or low ARMs. Conclusion The unsatisfactory anorectal func-tion is possibly related to the decrease or lost of neurotrophic factors (GDNF/GFRα1/RET) and ganglia in the myenteric plex-uses post plastic surgery in ARMs patients.