Experimental Study On The Effect Of Amitriptyline On Bone Cancer Pain Rats

Objective To observe amitriptyline（AMI）effect on mechanical withdrawal threshold（MWT）,glutamate-aspartate transporter（GLAST）of spinal cord dorsal horn and glutamate of cerebrospinal fluid（CSF）in bone cancer pain（BCP）rats,and explore its possible mechanism.Methods A total of 90 female Sprague-Dawley（SD）rats weighing 180-220g were divided into six groups randomly,and 15 rats in each group(control group,sham-operated group,BCP group,AMI _1.5 group,AMI₃ group,AMI₁₀ group).There was no any treatment in control group,sham-operated group injected 10 ul PBS into right hind tibal medullary cavity,BCP group injected 10 ul Walker 256 carcinoma cells suspended in PBS into tibal medullary cavity,and intraperitoneal injected 1ml of saline twice daily on the 5 days after the operation,AMI group intraperitoneal injected three different doses（1.5,3,10mg/kg）of AMI twice daily on the 5 days after the operation.Right hind MWT were tested at day 0 before operation and days 4,7,11,14,17 and 21 after operation;RT-PCR was used to detect the levels of GLAST mRNA;Western blot was used to detect the expression of GLAST and PKA/PKC in spinal cord dorsal horn;and the concentration of glutamate in CSF was detected by using ultraviolet spectrophotometry.The statistical significance of differences between groups was analyzed with a one-way analysis of variance（ANOVA）and the Kruskal-Wallis H test.P<0.05 was considered statistically significant.Results（1）MWT:The baselines of MWTs in rats showed no significant difference among groups（P>0.05）.No significant change in MWT was observed in sham rats compared with CONT group(P>0.05.The results of one-factor ANOVA showed that the MWT of BCP group was significantly decreased from day 7 compared with CONT group（P<0.05）.Compared with BCP group,no significant change in MWT was observed in the AMI_1.5 animals.However,increased MWT was observed in the AMI₃ and AMI₁₀ group on day 7、11、14、17、21 compared with BCP group（P<0.05）,and MWT in AMI₁₀ group was significantly increased on day 17 and 21 compared with AMI₃ group（P<0.05）.（2）RT-PCR:GLAST mRNA had no significant change between CONT and Sham group（P>0.05）.And the level of GLAST mRNA in BCP group showed remarkab decreased compared with CONT group in a time-dependent manner（P<0.05）.Compared with BCP group,no significant change in GLAST mRNA expression was observed in the AMI_1.5,AMI₃ and AMI₁₀ group animals on day7,14 and 21（P<0.05）.（3）Western blot:Compared with CONT group,no significant change in GLAST and PKA/PKC expression was observed in the Sham group animals（P>0.05）;GLAST on membrane was significantly decreased in BCP rats compared with that in CONT or Sham rats（P<0.05）,and PKA/PKC was increased（P<0.05）.All three doses of the amitriptyline（1.5,3 and 10 mg/kg）produced significant decrease of PKA/PKC in spinal cord dorsal（P<0.05）.All three doses of the amitriptyline（1.5,3 and 10 mg/kg）produced significant decrease of PKA/PKC and cytosol GLAST in spinal cord dorsal and increase of membrane GLAST（P<0.05）.（4）The result of ultraviolet spectrophotometry:The glumate concentration of CSF in CONT group have no significant differences compared with Sham group.There was a significant increase of glutamate concentration in BCP grop compared to CONT or Sham rats（P<0.05）.Intraperitoneal injection with amitriptyline produced glutamate concentration that were higher than the BCP rats（P<0.05）.Conclusion These results suggest that amitriptyline intraperitoneal injection can prevent the decrease of MWT in BCP rats;the underlying mechanisms may be associated with inhibition of PKA/PKC expression,and thus promoting the GLAST trafficking onto glial cell surface which reduces EAA concentration in the CSF.