The Expression Of NKCC1/KCC2 In A Rat Model Of Bone Cancer Pain And Its Influence On Pain

Part One:The expression of NKCC1 and KCC2 in bone cancer ratsObjective To observe the expression of NKCC1 and KCC2 in spinal cord and DRG of rats with bone cancer pain.Methods Twenty-seven female Sprague-Dawley(SD)rats weighing about 180 to200 g were randomly divided into three groups(n=9): normal group(C),sham operation group(S),bone cancer pain group(BCP).Rats in the BCP group were injected with 10 μl of Walker 256 tumor cell on the left tibial metaphysis,and 10 μl of PBS was injected into the S group.The rats in C group were fed normally.Mechanical withdrawal threshold test was performed one day before and from first day to ninth day every morning at 10 o’clock after surgery.Rats were sacrificed on the ninth day after surgery.The L4-L6 and L2-L5 DRG were sacrificed and the expression of NKCC1 and KCC2 in spinal cord and DRG were detected by RT-qPCR and Western Blot.Results Compared with Group C,the rats in BCP group appeared obvious mechanical hypealgesia(P<0.01),the expression of KCC2 mRNA(P <0.01)and its protein(P <0.05)in spinal cord were down-regulated;The expression of NKCC1 mRNA(P <0.01)and its protein(P <0.05)increased in DRG.There was no significant difference between the group S and C(P> 0.05).Conclusion The expression of NKCC1 in DRG of rats was up-regulated and the expression of KCC2 in spinal cord was down-regulated.Part two: The effect of intrathecal injection CLP257 in a rat model of bone cancer painObjective To investigate the effect of intrathecal CLP257 injection on pain behavior and the level KCC2 expression in spinal cord in a rat model of bone cancer pain.Methods 48 rats were randomly divided into 4 groups(n = 12),sham operation group(S),bone cancer pain group(BCP),bone cancer pain + solvent group(DMSO),bonecancer pain + CLP257 group(CLP).Rats in the BCP group were treated with Walker256 tumor cells.The rats in the CLP group were injected with CLP257 through the intrathecal catheter and the DMSO group was injected with the same amount of solvent.The rats in S group did not do special treatment.The mechanical withdrawal threshold was measured before d1 and d1 ~ 9 after surgery in each group.Mechanical withdrawal threshold was measured 0.5 h before administration and 0.5,1,2,4,6 h after administration.The expression of KCC2 in the spinal cord was detected by RT-qPCR,immunofluorescence and Western Blot.Results Compared with S group,the expression of KCC2 mRNA in the spinal in BCP group decreased(P<0.05),KCC2 protein decreased at the same time(P<0.05).Compared with BCP group,the expression of KCC2 mRNA in the spinal in CLP group increased(P<0.05),KCC2 protein increased at the same time(P<0.05).Compared with BCP group,The MWT of CLP group increased at 2nd hour and 4th hour after administration CLP257(P<0.05).There was no significant difference between the DMSO group(P>0.05).Conclusion CLP257 intrathecal injection can increase the expression of KCC2 in the spinal and it has an analgesic effect of bone cancer pain rats.Part three: The effect of intrathecal injection bumetanide in a rat model of bone cancer painObjective To observe the effect of intrathecal bumetanide injection on pain behavior and the level NKCC1 expression in DRG in a rat model of bone cancer pain.Methods 48 rats were randomly divided into 4 groups(n = 12),sham operation group(S),bone cancer pain group(BCP),bone cancer pain + solvent group(DMSO),bone cancer pain + Bumetanide group(BUM).Rats in the BCP group were treated with Walker256 tumor cells.The rats in the BUM group were injected with BUM through the intrathecal catheter and the DMSO group was injected with the same amount of solvent.The rats in S group did not do special treatment.The mechanical withdrawal threshold was measured before d1 and d1 ~ 9 after surgery in each group.Mechanical withdrawal threshold was measured 0.5 h before administration and 0.5,1,2,3,4,5,6 h after administration.The expression of NKCC1 in the DRG was detected by RT-qPCR,immunofluorescence and Western Blot.Results Compared with S group,the expression of NKCC1 mRNA in DRG in BCPgroup increased(P<0.01),NKCC1 protein increased at the same time(P<0.05).Compared with BCP group,the expression of NKCC1 mRNA in DRG in BUM group decreased(P<0.01),NKCC1 protein decreased at the same time(P<0.05).Compared with BCP group,the MWT of BUM group increased at 1h、2h、3h、4h after administration BUM(P<0.05).There was no significant difference between the DMSO group(P>0.05).Conclusion Bumetanide intrathecal injection can decrease the expression of NKCC1 in the DRG and it has an analgesic effect of bone cancer pain rats.