Study Of Effects On TRESK In Dorsal Root Ganglion For Neuropathic Pain

BackgroundDRG(dorsal root ganglion) plays an important role in the pathogenesis of neuropathic pain. Many studies have shown that the variety of potassium channels subtype is essential for the beginning and development of neuropathic pain. TRESK(TWIK-related spinal cord potassium channels), which has been found recently, is expressed greatly in DRG. Its function is to regulate the resting potential of neurons and it is important in the process of excitability in neurons. More and more studies have proved that TRESK is likely to take part in the process of all kinds of pains. However, there aren't any studies on whether TRESK of DRG plays a role in neuropathic pain.ObjectiveThe objective of this study is to observe that how TRESK mRNA change in the DRG firstly. After setting up the recombinant adenoviral vector containing TRESK gene, we transfect the DRG neurons, to verify the efficiency of transfection and regulation, and then to observe that how TRESK genes regulation affect the release of P substance in the DRG. In addition, after intraperitoneal injecte the recombinant adenoviral vector containing TRESK gene to rats, we aim to verify the effects on regulating TRESKmRNA and protein in DRG of the recombinant adenoviral vector containing TRESK gene, and to observe that how it affect the pain threshold of rats and the activation of spinal glial cells. MethodsThe experiment include four parts.Part I:Thirty two male SD rats were randomly divided into 2 groups. They were respectively carried out shame operation(Group Shame, n=16) and spared nerve injure model (Group SNI, n=16). Eight rats in each group were sacrificed 1 day before operation, L4,5 DRG at operation side were isolated to determinate the expression of TRESKmRNAe by realtime polymerase chain reaction(realtime PCR). The remaining eight rats in each groups were measured mechanical withdrawal threshold(MWT) and thermal withdrawal latency(TWL) at 1 day before operation and 1,3,5,7,14 day after operation. The remaining rats were sacrified at 14 day after operation,L4,5 DRG at operation side were isolated to determinate the expression of TRESKmRNA by realtime PCR.PartⅡ:TRESK full-length cDNA was cloned from rat's DRG cells and confirmed by realtime PCR and DNA sequencing. Then pAd/CMV/V5-DEST-TRESK under the control of CMV promoter was constructed.Translated DH5a colibacillus and the positive recombinants were subsequently identified by PCR and enzyme cutting, then taking positive clones to DNA sequencing. The plasmid of correct sequencing were enzyme cutted and linearizated using the enzyme of pacI.293 T cells were transfected and packaged, and then we produced the recombinant adenoviral vector containing TRESK gene(pAD/CMV/V5-DEST-TRESK). The titer of adenoviral vector were verified by hole by hole dilution method.Part III:Primary cultured DRG cell from newly born SD rat were randomly divided into 6 groups with 9 wells in each group, control group (group C):it received no treatment; capsaicin group (group S):fresh culture solution with 300nmol/L capsaicin incubated 10 min; negative group(group NC):every hole added in 3x109 negative control adenovirus; negative and capsaicin group(group NCS):every hole added in 3×109 negative control adenovirus,72h later, fresh culture solution with 300nmol/L capsaicin incubated 10 min; the recombinant adenoviral vector containing TRESK gene group(group R):every hole added in 3×109 recombinant adenoviral vector containing TRESK gene (pAD/CMV/V5-DEST-TRESK); the recombinant adenoviral vector containing TRESK gene and capsaicin group(group RS):every hole added in 3×109 recombinant adenoviral vector containing TRESK gene (pAD/CMV/V5-DEST-TRESK),72h later, fresh culture solution with 300nmol/L capsaicin incubated 10 min.The expression of TRESK mRNA in three well of each group were detected by realtime PCR at 48 hours. The expression of TRESK protein in three well of each group were detected by Western blot 72 hours later. The release of SP in three well of each group were detected by radio immunoassay 72 hours later.PartⅣ:One hundred eight SD male rats were randomized into six groups (n=18):GroupⅠwas blank contrast group; GroupⅡwas sham operationgroup; GroupⅢwas sciatic nerve branch selective injury rats model(SNI) group; Group IV was prepared SNI model and intrathecal injected with physiological saline; Group V was prepared SNI model and intrathecal injected negative adenovirus; GroupⅥwas prepared SNI model and intrathecal injected with recombinant adenoviral vector containing TRESK gene (pAD/CMV/V5-DEST-TRESK).Each group was determined mechanical threshold and heat pain threshold respectively at 1 day before operation and 1,3,5,7,14 day after operation. All the rats in each group were put to execution after operations. We took the L4,5DRG in the side of operation, and assessed the expressions of DRG TRESKmRNA with PCR with six rats; Other six rats were used to assess the TRESK protein expressions. We took the L4,5 spinal cords from the remaind six rats in each group, observed the activation of spinal glial cells with the method of immunohistochemistry. ResultsPart IMechanical withdrawal threshold (MWT) were significantly lower at 1 day before operation and 1,3,5,7,14 day after operation in Group SNI than Group Shame(p<0.05).The TRESK mRNA expression in L4,5 DRG at operation side was significantly lower in Group SNI than Group Shame(p<0.05). Compare with 1 day before operation, MWT were significantly lower at 1,3,5,7,14 day after operation in Group SNI(p<0.05), the TRESK mRNA expression in L4,5 DRG at operation side at 14th after operation was significantly decreased in Group SNI(p<0.05). PartⅡ:The titer of virus was tested using hole by hole dilution titer method. The full length of TRESK from rat DRG cells is 781bp,the DNA sequencing demonstrated that the DNA sequencing was completely consistent with TRESK sequencing of rat recorded in Genebank. To compared with pAD-GFP blank vector, the PCR amplification of the pAD-TRESK gDNA was match to the anticipation.The titer of concentrated recombinant adenoviral vector containing TRESK gene (pAD/CMV/V5-DEST-TRESK) was 1.31×109 TU/ml.PartⅢ:Compared with Group C, the expression of TRESKmRNA in rat DRG cells were significantly heightened in Group R and RS(p<0.05); the expression of TRESKmRNA in rat DRG cells were significantly decreased in Group S and NCS(p<0.05).Compared with Group C, the expression of TRESK protein in rat DRG cells were significantly increased in Group R and RS(p<0.05), the expression of TRESK protein in rat DRG cells were significantly decreased in Group S and NCS(p<0.05),.Compared with Group C, the level of SP in rat DRG cells were significantly heightened in Group S,NCS and RS(p<0.05), Compared with Group S and NCS, the level of SP in rat DRG cells were significantly higher in Group S and NCS than in Group RS(p<0.05).PartⅣ:The mechanical pain threshold at 1 day before operation and 1,3,5,7,14 day after operation were lower significantly in GroupⅢ, 3,ⅤandⅥthan in GroupⅠ(p<0.05); The mechanical pain threshold at 5,7,14 day after operation were lower significantly in GroupⅢ,ⅣandⅤthan in GroupⅥ(p<0.05). The mechanical pain threshold in GroupⅢ,Ⅳ,ⅤandⅥat 1,3,5,7,14 day after operation were lower significantly than at 1 day before operation(p<0.05).The expression of TRESKmRNA in DRG in GroupⅢ,Ⅳ,ⅤandⅥwere significantly lower than in Group I(p<0.05); The expression of TRESKmRNA in DRG in GroupⅥwere significantly higher than in GroupⅢ,ⅣandⅤ(p<0.05).The expression of TRESK protein in DRG in GroupⅢ,Ⅳ,ⅤandⅥwere significantly lower than in GroupⅠ(p<0.05); The expression of TRESK protein in DRG in GroupⅥwere significantly higher than in GroupⅢ,ⅣandⅤ(p<0.05).The activation of glial cells in spinal in GroupⅢ,Ⅳ,ⅤandⅥwere significantly higher than GroupⅠ(p<0.05); the activation of glial cells in spinal in GroupⅥwere significantly lower than in GroupⅢ,ⅣandⅤ(p<0.05)ConclusionPartⅠNP may lead to down regulation of TRESKmRNA in DRG.The occurrence and development of NP may be closely related with TRESK in DRG.PartⅡ:The rat TRESK full-length cDNA was cloned and recombinant adenovirus vector containing TRESK gene (pAD/CMV/V5-DEST-TRESK) was constructed successfully. The titer of concentrated recombinant adenoviral vector containing TRESK gene (pAD/CMV/V5-DEST-TRESK) was 1.31x109 TU/ml. PartⅢ:The recombinant adenovirus vector containing TRESK gene (pAD/CMV/V5-DEST-TRESK) could up regulate the expression of TRESK gene and protein in dorsal root ganglion of rats. The up regulation of TRESK geng could inhibited the release of substance P in DRG which was provocated from capsaicin.PartⅣ:Intrathecal injection with the recombinant adenovirus vector containing TRESK gene (pAD/CMV/V5-DEST-TRESK) to rats plays a cure role for neuropathic pain. This way can up regulation the expression of TRESKmRNA and TRESK protein in DRG, and then inhibit the activation of glial cells in spinal, therefore, to achieve the purpose of cure neuropathic pain.