Roles Of Astrocyte Activity And PCREB In Dorsal Horn In Gene Therapy Of Neuropathic Pain Of Rats Suffered From CCI Mediated By DREAM SiRNA

Objective: To detect the application of DREAM silencing through siRNA mediated by lentiviral vector in gene therapy of rats neuropathic pain due to chronic constriction injury of the sciatic nerve (CCI), and to observe the changes of astrocyte activity and phosphorylation of cyclic AMP response element binding protein (pCREB) under the control of RNA interference(RNAi). And to find out the roles of astrocyte activity and pCREB in dorsal horn in gene therapy of neuropathic pain of rats suffered from CCI mediated by DREAM siRNA.Method: To select 24 rats which were male and healthy, then to divide them randomly into 4 groups(n=6 each), that is, Group RNAi, Group blank vector, Group NS and Group sham. To measure the basic pain threshold before building the model(TO), including paw withdraw thermal latency(PWTL) and paw withdraw mechanical threshold(PWMT), then to repeat the operations once a day until 14th day(T14) after CCI. And we had continuously given pKCSHR-Puro/GFP- DREAM- LV (1×10~9 IU/ml) 5μl + NS 5μl or blank vector 5μl+ NS5μl or NS 10μl by intrathecal injection once a day from 8th day after CCI(T8) to 14th day(T14). At last, we took out the spinal cords(L4-5) and made them into paraffin sections, then through immunofluorescence method, we could observe the expressions of green fluorescent protein(GFP), glial fibrillary acidic protein (GFAP) and pCREB. Result: Pain threshold: there were no differences among 4 groups with basic pain threshold(P>0.05); PWTL of left paw on T7-T10 and T12 in Group RNAi and T7-T10 in Group sham, and on T7-T14 in Group blank vector and NS were much lower than the respective basic pain threshold(P<0.05 or P<0.01); and those on T10,T11,T13 and T14 in Group RNAi were much higher than that on T7 in the group (P<0.01); And those on T9-T14 in blank vector and NS were much lower(P<0.05 or P<0.01 vs T7); PWTL of left paw on T13-T14 in Group blank vector and Group NS were lower than that in Group sham; and PWTL of left paw on T11,T13 and T14 in Group RNAi were higher than those in Group blank vector and NS (P<0.05). There were no differences among 4 groups with PWTL of right paw on all points(P>0.05). PWMT of left paw: all values noting post-CCI were lower than the level of preoperation. T8 in Group RNAi, T8 and T12-T14 in Group blank vector and Group NS were much lower than those on each point in Group sham(P<0.05 or P<0.01). T14 in Group RNAi was higher than the Group sham's(P<0.01). And T12-T14 in Group RNAi were also higher than that in Group blank vector and NS(P<0.05 or P<0.01). And the case with PWMT of right paw was the same as PWTL of right paw.GFP: positive expression was only observed in dorsal horn in Group RNAi, and dark was in the views of other groups.GFAP(average of fluorescence intensity): Group blank vector and Group NS were the highest. Also Group RNAi and Group sham were similar with each other(P>0.05), and both of which were lower than the groups above(P<0.01).pCREB(average of fluorescence intensity) in dorsal horn: Group RNAi was significantly reduced(P<0.01 vs Group blank vector and NS), but higher than Group sham (P<0.05).pCREB(average of fluorescence intensity) in astrocytes of dorsal horn: Group blank vector and Group NS were the highest. Also Group RNAi and Group sham were similar with each other(P>0.05), and both of which were lower than the groups above(P<0.01).Conclusion: 1. The continuous-intrathecal injection of pKCSHR-Puro/GFP-DREAM-LV could evidently relieve pain of rats suffered from CCI in behavioristics.2.The low expression of DREAM could inhibit the activation of astrocyte in dorsal horn due to nociceptive stimulus with sciatic nerve.3.The low expression of DREAM could inhibit the total expression of pCREB in dorsal horn, especially in astrocyte.4.The inhibition of astrocyte's activating and the expression of pCREB in dorsal horn may play an important part in gene therapy of rats with neuropathic pain through DREAM siRNA.