| Objective To elucidated whether spinal CX3CL1 and CX3CR1 are involved in the development of morphine tolerance.Methods 1.Forty-two SPF male SD rats were randomly divided into three groups(n = 12): Na?ve group,Sham group and MT group.The animals in Sham group and MT group were performed with lumbosacral indwelling catheterization.A 7-day recovery period were allowed before drug administration.Rats in Sham group and MT group were intrathecally injected with 5 μL(2 μg/μL)morphine and equal volume of saline respectively.10 μL saline was used to flushing the catheter and intrathecal injection was administrated twice daily for seven consecutive days.The development of morphine tolerance was evaluated by tail-flick latency test and %MPE(Maximal Possible Antinociceptive Effect)was calculated on day 1,3,5 and 7.After behavioral tests,the lumber enlargement of spinal cord of rats were quickly removed on day 7.The expression of Iba-1 protein was evaluated by Western blot,and MOR,CX3CL1 and CX3CR1 by real-time PCR and Western blot.2.Forty-two SPF male SD rats were randomly divided into seven groups including Sham group,MT group,IgG+MT group,rrCX3CL1(100 ng)+MT group,rrCX3CL1(500 ng)+MT group,anti-CX3CR1(5 μg)+MT group and anti-CX3CR1(10 μg)+MT group,with six rats for each group.After assessing the baseline of tail-flick latency,all animals were performed with intrathecal catheter implantation.Rats in rrCX3CL1(100 ng)+MT group and rrCX3CL1(500 ng)+MT group were intrathecally injected with recombinant rat CX3CL1(100 ng or 500 ng)30 minutes before 10 μg(2 μg/μL)morphine injection,respectively.Anti-CX3CR1 neutralizing antibody(5 μg or 10 μg)or placebo IgG(10 μg)was intrathecally injected 30 min before morphine administration,respectively.The effect of recombinant rat CX3CL1 or anti-CX3CR1 neutralizing antibody on morphine tolerance development was assessed by tail-flick latency test on day 1,3,5 and 7.The lumber enlargement of spinal cord segments of rats were quickly removed after behavioral tests on day 7,which were used to investigate the activation of microglia by western blot.3.Six SPF male rats were randomly divided into two groups: Sham group and MT group.All animals were performed with intrathecal catheter implantation and rested for seven days.Rats were perfused with 4% ice-cold paraformaldehyde(PFA).Lumbar enlargements of rats were obtained and made into frozen section which were utilized to evaluate the expressions of CX3CL1 and CX3CR1 in spinal dorsal horn by immunofluorescence.Immunofluorescence double-staining was conducted to investigate the cellular expressions of CX3CL1 and CX3CR1 in neuron,astrocyte and microglia.Results 1.Rats received morphine exhibited significantly higher %MPE compared with saline-treated rats on day 1(P < 0.001)and day 3(P < 0.01).On day 5 and day 7,there was no significant difference of %MPE levels between morphine-treated and saline-treated rats(P > 0.05),suggesting that chronic morphine tolerance had been successfully established.Compared to na?ve and sham rats,the expression of Iba-1 protein was significantly increased in morphine-treated rats measured by western blots(P < 0.05).The expressions of MOR mRNA and protein were not affected by morphine treatment measured by real-time PCR and western blots(P > 0.05).Neither the levels of CX3CL1 mRNA and CX3CR1 mRNA,nor the expressions of CX3CL1 protein and CX3CR1 protein were affected by intrathecal administration of morphine when compared to saline-treated rats(P > 0.05).2.There was no significant difference of baseline levels of tail-flick latency measured prior to drug administration among all groups,indicating that intrathecal catheterization did not affect behavioral responses of rats(P > 0.05).Recombinant rat CX3CL1(100 ng or 500 ng),anti-CX3CR1 neutralizing antibody(5 μg or 10 μg)or control IgG was intrathecally injected 30 min before morphine administration,respectively.Neither 100 ng nor 500 ng rrCX3CL1 exhibited statistically significant effect on %MPE in rats treated with morphine compared with that in IgG-morphine treated rats(P > 0.05).Moreover,there was no significant effects of 5 μg or 10 μg anti-CX3CR1 neutralizing antibody on %MPE in rats treated with morphine when compared with that in IgG-morphine treated rats(P > 0.05).Western blots results showed that the expressions of Iba-1 were significantly increased in all morphine-treated rats(P < 0.05).However,neither rrCX3CL1 nor anti-CX3CR1 neutralizing antibody had statistically significant effect on morphine induced expressions of Iba-1(P > 0.05).These results suggest that both exogenous CX3CL1 stimulation and CX3CR1 inhibition could not affect the microglia activity and the development of morphine tolerance.3.In sham group,CX3CL1 expression was extensively distributed to all layers of spinal dorsal horn,and CX3CR1 was mainly expressed in laminaⅠto lamina Ⅲ of spinal cord.The results of immunofluorescence quantitative analysis showed that there was no significant difference between morphine-treated rats and saline-treated rats in the expression of CX3CL1 or CX3CR1(P > 0.05).In the spinal dorsal horn of saline-treated rats,the immunoreactivity of CX3CL1 was co-localized with the neuronal marker NeuN,while CX3CR1 was co-localized with the astrocyte marker GFAP and microglia marker Iba-1.However,in morphine-treated rats,the immunoreactivity of CX3CL1 and CX3CR1 were found to be co-localized with NeuN,GFAP and Iba-1.These results indicate the shift of CX3CL1 and CX3CR1 expressions might occur during the development of morphine tolerance.Conclusion 1.Activation of microglia was induced significantly by chronic morphine treatment while MOR expression did not changed in spinal cord of rats during morphine tolerance development.2.Chronic morphine exposure does not alter the CX3CL1 and CX3CR1 expressions in the spinal cord.3.Both exogenous CX3CL1 and CX3CR1 inhibitor have no effects on morphine analgesia and the development of drug tolerance 4.Both CX3CL1 and CX3CR1 are co-localized with neuron,astrocyte and microglia in the spinal cord of morphine-tolerant rats.In conclusion,spinal CX3CL1 and its receptor CX3CR1 may not contribute to morphine tolerance development by changing the expression quantities,but related to the connection between neuron and microglia. |
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