Effect Of Alcohol Withdrawal On Pain Chronification After Surgery And Its Underlying Mechanisms

BackgroundPeople with alcohol dependence not only show symptoms such as insomnia,anxiety after alcohol withdrawal,but also have pain abnormality such as hyperalgesia.When alcohol dependent patients undergo surgery,they often follow the doctor's advice to abstinence alcohol.Therefore,these patients may experience aberrant postoperative pain associated with alcohol withdrawal.Animal models of alcohol withdrawal usually reduce or stop drinking after alcohol dependence induced by animal compulsion or voluntary alcohol consumption.The common methods of forced drinking include intragastric injection,alcoholic liquid food,tail vein injection,alcohol vapor,and intraperitoneal injection,while voluntary drinking refers to animals freely choosing to drink different concentrations of alcohol and water.The animal model of postoperative pain usually induces incision pain through plantar incision surgery.In general,postoperative pain manifests as acute pain,which results from tissue damage and related inflammatory response and only lasts for a period of time.However,about 5%to 75%of patients suffer from the chronic postoperative pain lasting more than 3 months after the operation.Even though the surgical wound heals,patients still could feel painful by painless stimulation.Pain chronification after surgery involves a variety of multiple physiological mechanisms.For now,the types and mechanisms of postoperative aberrant pain caused by alcohol withdrawal have been poorly reported.The alteration of AMPA(a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)receptors in nervous system is one of important mechanisms of aberrant pain.AMPA receptors,a non-selective cation channel,play key role in synaptic plasticity and excitatory synaptic transmission in central nervous system.Studies have shown that AMPA receptors were involved in acute postoperative pain,inflammatory pain and other processes.Our previous study suggested that AMPA receptors were associated with stress induced pain chronification.It has been reported that AMPA receptors acted as a target for regulation of nervous system activity.Therefore,we speculated that AMPA receptors may be involved in alcohol withdrawal induced pain chronification after surgery.N-cadherin and cAMP response element binding protein(CREB)are related proteins activated by AMPA receptors.N-cadherin,a family of cadherins,is the most widely distributed in the nervous system,and mediates Ca2+-dependent synaptic adhesions,which plays an important role in the structure and function of AMPA receptors.CREB is an important transcription factor in the nucleus.When cAMP-dependent protein kinase(PKA),CaMKI and CaMKII signaling pathways are activated,followed by phosphorylation of CREB at Ser-133,which indicates that CREB participates in the occurrence and maintenance of central sensitization.ObjectiveWe observed influence of alcohol withdrawal on plantar incision surgery induced postoperative pain in mice,and explored the role of spinal AMPA receptors associated with alcohol withdrawal in the pain chronification after surgery.Methods1.Grouping of animals.The 8-10 weeks old C57BL/6 male mice,weight 20-25g,were randomly divided into control group,incision group,alcohol withdrawal group and alcohol withdrawal + incision group.Mice from control group did not perform any surgery;mice from surgery group performed plantar incision on right hind foot;mice from alcohol withdrawal group took alcohol withdrawal but did not perform surgery;mice from alcohol withdrawal + surgery group took alcohol withdrawal and performed hind plantar incision.2.Alcohol withdrawal.Mice in alcohol withdrawal group and alcohol withdrawal +incision group were fed alcohol for 5 consecutive weeks and then switched to normal drinking water after withdrawal.Mice in control group and incision group were always fed normal drinking water.3.Pain behavior test.Plantar incision in mice was performed.Paw Withdrawal Thresholds were measured before operation and 1,3,5,7,10,20,40,60,and 80 days after operation.4.Calcium imaging.The embryonic spinal cord neurons of 14-16 days gestation were cultured in primary culture.In vitro cultured spinal cord neurons were incubated with 25 mM alcohol to observe effect of calcium influx.Calcium imaging was used to observe changes in calcium ion concentration.The ratios of fluorescence intensity at wavelengths of 340 nm and 380 nm(F340/F380)were calculated.5.Western blot experiments.Mice were executed at 5 and 50 days after operation and spinal cord L4-6 sections were taken to detect the protein expression of GluA1,GluA2,N-cadherin,CREB and pCREB(Ser133).6.Statistical analysis.SPSS 21.0 was used for statistical analysis.The pain behavior tests were analyzed by two-way ANOVA,calcium imaging was analyzed by paired t-test,and Western Blot was analyzed by one-way ANOVA.All data were expressed as mean ± standard deviation(x ± s).The differences founded with P<0.05 were considered statistically significant.Results1.Verify postoperative pain model induced by plantar incision.The mechanical pain behavior tests showed that compared with control group,ipsilateral PWT of incision group was significantly decreased from the first day to the 10th day after surgery(P<0.05).There was no significant difference in contralateral PWT(P>0.05).2.The effect of alcohol withdrawal on PWT.Compared with control group,PWT on both sides of alcohol withdrawal group were not statistically significant(P>0.05),suggesting that alcohol withdrawal had no significant effect on pain threshold.Compared with incision group,PWT of alcohol withdrawal + incision group was significantly decreased from the 10th to the 60th day after surgery(P<0.05),and PWT was no significant difference from the 1st to the 7th day and the 80th day after operation(P>0.05),suggesting that alcohol withdrawal led to longer duration of pain after surgery.3.Alcohol incubation increased calcium ion permeability and increased calcium influx in spinal neurons(P<0.05),whereas Ca2+-permeable AMPA receptor antagonists(NAPSM)blocked alcohol-induced calcium influx(P>0.05),suggesting that Ca2+-permeable AMPA receptors were involved in alcohol-induced calcium influx.4.Western blotting was used to measure protein expression of AMPA receptors and its related proteins.GluAl,N-cadherin and pCREB(Ser-133)of alcohol withdrawal group were not significantly different from those in control group(P>0.05).On the fifth day after operation,the protein expression of GluAl,N-cadherin,and pCREB of incision group and alcohol withdrawal + incision group in mice spinal cord was significantly increased(P<0.05),suggesting that spinal cord GluAl,N-cadheirn,and pCREB were involved in postoperative pain.However,there were no significant difference in protein expression of GluA1,N-cadherin and pCREB(Ser-133)between incision group and alcohol withdrawal+ incision group on the fifth day after operation(P>0.05).On the 50th day after operation,the levels of protein expression of GluAl,N-cadherin,and pCREB(Ser-133)of incision group significantly reduced,and there was no significant difference with control group(P>0.05).Protein expression of GluAl,N-cadherin,and pCREB(Ser-133)of alcohol withdrawal + incision group remained significantly increased(P<0.05),which were significantly higher than incision group,suggesting that GluAl,N-cadherin,and pCREB(Ser-133)in mice spinal cord involved in alcohol withdrawal-induced chronic postoperative pain.ConclusionAlcohol withdrawal can lead to pain chronification in plantar incision.AMPA receptor subunit GluA1,N-cadherin and CREB participated in this process,while subunit GluA2 did not.