Ultra-rapid Detoxification Under General Anesthesia On Morphine Addiction Withdrawal Symptoms And Plasma Morphine And Central Endorphin In Rats

Part One: Effects of ultra-rapid opiate detoxification under general anesthesia on the withdrawal signs in morphine addicted ratsObjective Evidence from the animal researches concerning the technique of ultra-rapid opiate detoxification (UROD) is lacking, which has been a hindrance for its clinical popularization. The present study investigated the effects of UROD under general anesthesia on the withdrawal signs in morphine addicted rats, with an aim to evaluate the feasibility of this technique and to explore the optimal use of adjuvant drugs during this process.Methods Forty-two Wistar rats were divided into five groups: normal group (non-addiction) (n=6), naturally withdrawal group (n=6), anesthesia only group (n=10), anesthesia with naloxone group (n=10) and clonidine pretreatment group (clonidine with anesthesia and naloxone) (n=10). After addiction, each group received corresponding treatments. The occurrences of withdrawal signs precipitated by naloxone were observed and scored during detoxification and in each of the following 3 days after detoxification. Times of diarrhea and the loss of body weight during and after detoxification were also recorded.Results Wet dog shaking was one of the most obvious withdrawal signs observed. It was most obvious in the anesthesia with naloxone group (7.7+ 0.7 times), severer than the clonidine pretreatment group (1.5 ±0.7 times). In the first day after detoxification, the occurrences of wet dog shaking increased in all addiction groups, with naturally withdrawal group andanesthesia only group most significant. However, this syndrome relieved gradually in the following two days, with clonidine pretreatment group remaining one of the least suffered groups.The occurrence of wet dog shaking was consistent with the scaling of withdrawal score, and the clonidine pretreatment group was also the group having the lowest withdrawal score in the subsequent 1 ~ 3 days after detoxification, followed by anesthesia with naloxone group. No significant difference was found between the anesthesia only group and the naturally withdrawal group. Diarrhea and weight loss occurred in all the four addiction groups in the first day after treatment. Compared to the normal group and naturally withdrawal group, they were more significant in the anesthesia with naloxone group and clonidine pretreatment group. Whereas, these symptoms alleviated in the following two days.Conclusion Ultra-rapid detoxification under anesthesia with naloxone is feasible and effective for the treatment of morphine addicted rats, and the adjuvant use of clonidine may increase the efficiency of this technique. However, aggravation of diarrhea and body weight loss could be expected shortly after the detoxification, which implies that complicated mechanisms might exist during the process of detoxification. Objective This part was aimed to establish a sensitive method for detectingmorphine concentration in the plasma by gas chromatography/massspectrometry (GC-MS), thus to provide a useful tool for the evaluation of theeffects of UROD on the plasma morphine in addicted rats.Methods GC-MS was employed in this study with a combination use ofsolid-phase extraction (SPE) and derivatisation reaction withpentafluoropropionic anhydride (PFPA). Nalorphine served as an internalstandard. The ion m/z 414 and 440 were chosen as the qualifying ion formorphine and nalorphine, with their retention time by 5.3 min and 6.1 min,respectively.Results The linear relationship was excellent within the range of 0.022Hg-ml'1 to 2.2 |o.g#ml"1 (R2>0.99, n=5), and the limit of detection was 1 ng*ml"1. The relative recovery rate of morphine was 91 - 98%. Within day andday-to-day coefficient of variation were 2.4 - 6.0% and 2.3 - 7.0%,respectively.Conclusion GC-MS is an accurate, sensitive and convenient method for thequantitative analysis of morphine concentration in the plasma.Part Three: Effects of ultra-rapid opiate detoxification on the plasma morphine and brain /3-endorphin in addicted ratsObjective This part was to investigate the effects of UROD on the concentration of plasma morphine and the content of ft — endorphin concentration in the brain in addicted rats.Methods Ninety Wistar rats were randomized into six groups: normal group (non-addiction) (n=5), addicted group (n=5), naturally withdrawal group (n=20), anesthesia only group (n=20), naloxone with anesthesia group (n=20) and clonidine pretreatment group (clonidine with anesthesia and naloxone) (n=20). Each group received corresponding treatments. Samples of the blood and the brain tissue were collected before detoxification in normal and addicted group, and in the other groups, every five rats were sacrificed immediately after the treatment and in each of the following three days. The concentration of plasma morphine was determined by GC-MS, and the content of /3-endorphin in brain tissue was quantified by radioimmunoassay method.Results Shortly after the treatment, morphine concentration in the plasma decreased stiffly in all the four withdrawal groups. It was relatively higher in the clonidine pretreatment group (105.9 + 29 ng/ml) than that of the other three groups, which ranged from 36.6 + 9.5 to 70.1 + 15.3 ng/ml. However, the difference disappeared gradually in the following days. Brain /3-endorphin in the normal group and addicted group was 33.5 + 3.8 ng/mg and 18.7 + 4.2 ng/mg, respectively. Detoxification with naloxone and/or clonidine resulted in a quick temporary increase of its level. Immediately after the detoxification, the content of brain /3-endorphin in the naloxone with anesthesia group and the clonidine pretreatment group was 103.3±39.7 ng/mg and 123.0±99.3 ng/mg respectively, which decreased to 20.0 ± 7.9ng/ml and19.7±11.7 ng/mg in the first day after detoxification. However, a slow increase of its level was observed in the following two and three days, although it's remaining in a lower level compared to the normal group. Compared to the naloxone with anesthesia group and clonidine pretreatment group, brain content of /3-endorphin in anesthesia only group didn't show such a significant increase shortly after treatment, and remained in relatively lower level in the following days.Conclusion Although the procedure of anesthesia and the use of naloxone have no obvious effects on the concentration of plasma morphine, pretreatment of clonidine can temporally increase its level. In contrast, the use of clonidine and/or naloxone can significantly increase /3-endorphin content in the brain. These observations imply that there remains a considerable quantity of morphine in the plasma after UROD treatment, and the relief of withdrawal signs might be contributed to the increase of /3-endorphin in the brain.