Correlation Between Orexin A And Withdrawal Symptoms During Different Withdrawal Periods In Alcohol-dependent Rats

Objective: In a previous clinical study by our project team,we found that plasma Orexin A(OXA)levels correlated with withdrawal response and craving during acute withdrawal in male alcohol-dependent patients.The present experiment was designed to further verify the correlation between plasma OXA levels and the severity of withdrawal symptoms at different withdrawal periods by establishing an alcohol-dependent rat model,so as to further clarify the relationship between plasma OXA levels and alcohol withdrawal response.Methods: Twenty-four SPF-grade SD(Sprague-Dawley,SD)male rats [body mass(250±10)g,age(6±1)weeks] were numbered,weighed,and randomized into three groups(n=8 for each group),A,B,and C.Group A served as a blank control group and was given normal drinking water for 28 d;group B served as a 6-h withdrawal group and group C as an 18-h withdrawal group.Group A was fed with 6%(v/v)alcoholic aqueous solution as the only source of water for 28 d.At the29 th day,group A was not treated for withdrawal symptoms;groups B and C were stopped from supplying alcoholic aqueous solution and replaced with normal drinking water,and their withdrawal symptoms were assessed at 6 h and 18 h after alcohol withdrawal,respectively.The severity of withdrawal symptoms was evaluated by two trained laboratory staff members,and the arithmetic mean of the two evaluations was taken.Blood samples from all three groups were collected immediately after assessment using abdominal aortic blood sampling,preserved by EDTA anticoagulation and centrifuged(3000 r/min,10 min).500 ul of the upper plasma layer was frozen at-70°C in centrifuge tubes.The concentration of OXA was determined using an enzyme-linked immunosorbent assay(Elisa): a double antibody sandwich method was used,and the arithmetic mean of the two determinations was taken as the final determined concentration of OXA.Results: 1.The alcohol withdrawal scores of rats in each group were compared with the total score and each score,and the results showed that the total withdrawal score and each score of rats in group B were statistically different from those in group A(P < 0.05);the total withdrawal score of group C was statistically different from those in group A(P < 0.05),while the two scores of rats’ agitation and tail tonicity were statistically different from those in group A in the comparison of each score(P < There was no statistically significant difference between group B and group A(P>0.05).2.The results of plasma OXA levels in the three groups showed that the plasma OXA levels in group B were significantly higher than those in groups A and C,and there was a statistically significant difference(P < 0.05);the OXA levels in group C were also higher than those in group A,but not statistically significant(P = 0.57).3.Correlation analysis between OXA levels and withdrawal symptom scores of rats in groups B and C revealed that there was a statistically positive correlation between OXA levels and severity of withdrawal symptoms in alcohol-dependent model rats(r=0.70,P<0.05),while there was no correlation with weight change and alcohol consumption of rats during drinking(P>0.05).Conclusion: The symptom scores of alcohol-dependent rats at 6h and 18 h of withdrawal were overall higher than those of normal healthy rats.The OXA levels at 6h and 18 h post-withdrawal in alcohol-dependent rats were generally higher than those in normal healthy rats.OXA may play an important role in the alcohol withdrawal process and may be a potential target for the treatment of alcohol withdrawal syndrome.