| Objective:Propofol has become one of the most widely used intravenous anesthetics due to its unique pharmacokinetic properties of short-acting,and rapid but complete recovery as well,but there are reports of euphoria and addictive potential of propofol in some case reports and findings that have been obtained from experimental animals.Indeed,several articles have highlighted the abuse potential of propofol,particularly among health professionals.Clinical study observed and evaluated the postoperative experience of 82 patients undergoing gastroenteroscopy under propofol anesthesia and found that 43.9%of the patients experience obvious euphoria during the period of anesthesia recovery.Another a questionnaire was sent to 48 forensic medicine departments in Germany,Austria and Switzerland concerning the number of autopsies carried out on medical personnel with the suspicion of abuse of analgesics.It was found that propofol was the major cause of death（85%）.In view of this,some scholars suggested that propofol should be listed as a psychostimulant substance in2010.And propofol has already been controlled as a psychostimulant properties of a drug in South Korea on February 1,2011.However,There is little known about the neurobiological mechanism of hedonic effects of propofol.The major objective of the present study was to observe whether propofol can induce euphoria and addiction in rats,and to explore the optimal dose of propofol.We also aimed to explain its possible mechanisms of action of effects of propofol on euphoria and addictive potential.It was expected to provide a theoretical explanation for the euphoria and addiction potential of propofol and provide a theoretical basis for the detoxification of addiction of propofol by this study.Materials and Methods:1 Hedonic facial responses score in rat Propofol was delivered via tail-vein bolus injection at five different doses:0,2,4,6,8 mg/kg.According to the dose of propofol,80 SD rats were randomly assigned to five groups（n=16）.After propofol bolus injection,10-min period of facial reactions were recorded by a digital camera.The facial and body responses of the rats in videos were analyzed and scored.The frequency and duration of paw licks,rhythmic midline tongue protrusions,and lateral tongue protrusions,regular mouth movement,ordinary grooming,head shakes and face washes were observed in rats within 0-5min and 5-10min after propofol injection,and the scores of hedonic response,neutral response and aversion response were compared.The dosage of propofol which can make the rats have the highest score of hedonic facial reaction was determined.2 Locomotor activity response One weeks after the hedonic responses test,the rats in five groups carried out the locomotor activity procedure.It took two days for each group to complete the experiment.According to the grouping,the rats were injected with NS of the same volume as propofol by tail vein on the first day,and then injected with propofol on the next day.The locomotor activity of the rats was recorded with Smart 3.0 behavioral test system.The changes of movement distance,speed and trajectory of the rats in 0-5min and 5-10min after injection of propofol or NS were recorded.And the dosage of propofol that can make rats produce the maximum locomotor activity distance and speed were determined.At the same time,the correlation analysis was made between the score of hedonic response and the distance and speed of locomotor activity.3 Conditioned place preference（CPP）test One weeks after the locomotor activity test,the rats in five groups carried out conditioned place preference.The CPP test consisted of three consecutive phases and conducted between 12:00 and6:00 PM.The first phase is pre-conditioning and rat without congenital preference were excluded from the study.The second phase is conditioning.Each rat in five groups was injected with saline before being confined to the vehicle paired compartment for 30 min and,after an interval of 4 h,received propofol before being confined to the drug-paired compartment for 30 min.The third phase is post-conditioning,each rat was placed into the gray partition,the time spent by the rats in white compartment during a 15-min period was recorded and CPP index were calculated.The dosage of propofol was recorded,which could make the rats have the most favorable position preference behavior.The correlation between the hedonic response score and CPP,the distance in locomotor activity and CPP were analyzed.4 The fiber photometry signals of GCa MPs recording According to the location of neurons,30 rats were randomly assigned to two groups（n=15）.AVV-h Syn-GCa MP6s（500 n L）was injected in the right VTA or the right LC and allowed 3 weeks for sufficient virus expression.Optic fibers were implanted targeting the right VTA or LC to transmit signals for fiber photometry.The recording protocol lasted for 150 s,and consisted of 50 s pre-propofol and 100s post-propofol periods.During each period,GCa MP signals of spontaneous activity was recorded.Propofol was delivered via tail-vein bolus injection（i.v.）at 4 mg/kg.Brain tissue was harvested after recordings for validation of virus expression and optic fiber locations.rat with incorrect position were excluded from the study.5 Chemogenetic inhibition neuron and behavioral test 30 SD rats were randomly divided to two groups（n=15）.AAV vectors containing the Gi-coupled inhibitory human M4 muscarinic receptor（r AAV-h Syn-h M4D（Gi）-EGFP）（n=10）or controlled AAV which was not contained h M4D（Gi）（n=5）were injected in the LC and allowed 3 weeks for sufficient virus expression.Then we conducted hedonic facial responses scoring and CPP test in two groups of rats.In group 1,15 rats expressing h M4D（Gi）（n=10）or not expressing h M4D（Gi）（n=5）in LC were intraperitoneally injected with clozapine N-oxide（CNO,1 mg/kg）,which allowed specific inhibition of h M4D（Gi）expressing neurons.60 min after the injection of CNO,hedonic responses scoring of propofol administration were conducted as mentioned above.In group 2,new 15 rats expressing h M4D（Gi）（n=10）or not expressing h M4D（Gi）（n=5）in LC were intraperitoneally injected with CNO.After60 min,CPP test were conducted as mentioned above.The dose of propofol that induced the most obvious positive reaction in previous hedonic response scoring and CPP test was chosen for the chemogenetic inhibition experiments.Brain tissue was harvested after tests for validation of virus expression.6 The concentrations of plasma noradrenaline and dopamine 8 SD rats were randomly selected as control.After anesthesia,the thoracic cavity was opened to expose the heart.Anticoagulant blood collection vessels were used to collect 3-5ml of blood and centrifuged.The upper serum was taken and stored in the refrigerator at-80℃.In addition,the blood sample of the rat with 4mg/kg propofol injection were collected when validation test was doing.the concentrations of plasma NE and DA were measured by enzyme-linked immunosorbent assay（ELISA）.Results:1 Hedonic facial response score of rats Compared with 0 mg/kg groups,the score of hedonic facial responses was not significantly different in the 2 and 8 mg/kg groups(P2mg/kg=0.495,P8mg/kg=0.175).The score of hedonic facial responses was significantly increased in 4 mg/kg groups（2.38±0.41 vs 21.25±1.43,P=0.000）and6 mg/kg groups（2.38±0.41 vs 7.06±1.19,P=0.013）.It was different in the duration of euphoria after propofol injection.Compared with 0-5min after propofol injection,the score of euphoria response in 4mg/kg group（21.25±1.43 vs 5.88±0.34,P=0.000）and 6mg/kg group（7.06±1.19 vs 4.38±0.51,P=0.029）decreased significantly within 5-10min after propofol injection.There was no significant difference in the neutral reaction score and aversion reaction score in the other groups except for the8mg/kg group.The neutral response score and aversion response score of 8 mg/kg group were significantly lower than those of 0 mg/kg group(Pneutral=0.000,Paversion=0.000).Compared with 0-5min after propofol injection,The neutral response score and aversion response score of 8 mg/kg group during 5-10min after propofol injection were significantly increased(Pneutral=0.000,Paversion=0.000).Additionally,8/12 rats in 8 mg/kg group were anesthetized and show no autonomous activities,the rats gradually woke up after about 5min and start to move.Collectively,hedonic facial responses can be induced by low dose of propofol,not anesthetic dose of propofol.2 Locomotor activity response in rats Locomotor activity in the NS injection group and in the propofol injection group was recorded.There was no significant difference in the locomotor activity distance(P 0-5 min=0.973,P 5-10 min=0.496)and locomotor activity speed(P 0-5 min=0.975,P 5-10 min=0.484)in the groups injected NS.Compared with 0 mg/kg propofol group,there was no significant difference in locomotor activity distance and speed in the 2 mg/kg group(P2mg/kg=0.563)and 6mg/kg group(P6mg/kg=0.078)within 0-5 min after injection,but the locomotor activity distance and speed of 4 mg/kg group were significantly increased（58.3±1.7 vs 101.5±2.5,0.194±0.005 vs 0.339±0.008）,and the difference was statistically significant(P distance=0.000,P speed=0.000);the distance and speed of 8mg/kg group were significantly reduced(P distance=0.000,P speed=0.000).Compared with NS group of the same volume,there was no significant difference in locomotor activity distance(P2mg/kg=0.656,P6mg/kg=0.329)and speed(P2mg/kg=0.661,P6mg/kg=0.334)in the 2mg/kg group and 6mg/kg group at 0-5min after injection,while the locomotor activity distance and speed of 4mg/kg group were significantly increased（58.6±1.7 vs 101.5±2.5）,The difference was statistically significant(P distance=0.000,P speed=0.000);the distance and speed of 8 mg/kg group were significantly reduced(P distance=0.000,P speed=0.000);Compared with0-5min after propofol injection,there was no significant difference in locomotor activity distance(P2mg/kg=0.587,P6mg/kg=0.628)and speed(P2mg/kg=0.573,P6mg/kg=0.638)in 2mg/kg and 6mg/kg group within 5-10min after propofol injection,but the locomotor activity distance and speed of 4mg/kg rats decreased significantly（101.5±2.5 vs 60.4±2.6）,The difference was statistically significant(P distance=0.000,P speed=0.000);the distance and speed of 8 mg/kg group were significantly increased(P distance=0.000,P speed=0.000).For the 8 mg/kg dose,a transient hypnosis occurred in 8 of the 16 rats after propofol bolus injection and the rats gradually woke up after about 5min and start to move.3 CPP experimental results Preliminary experiments were performed to assess the stimulus inconsistency of the apparatus.The apparatus was considered“biased”as the animals preferred black box.As a result,There were 16 rats were excluded because they did not meet the requirements.The remaining rats were followed up.Conditioned place preference test were performed in the five groups.The rats who entered the experiment had obvious preference for the preference box,and the time of staying in the black box increased significantly（129.52±2.57 vs 677.66±9.110,P=0.000）.After propofol conditioning training,compared with before conditioning training,there was no significant change in the residence time of rats in white box in0 mg/kg,2 mg/kg and 8 mg/kg groups(P0 mg/kg=0.628,P2 mg/kg=0.266,P8 mg/kg=0.062),while 4 mg/kg and 6 mg/kg groups could induce CPP behavior,and the residence time of rats in white box was significantly prolonged（130.67±6.37 vs418.42±17.39,126.17±4.77 vs 244.92±13.50）,the difference was statistically significant(P4mg/kg=0.000,P6mg/kg=0.000).Compared with 0 mg/kg group,there was no significant difference in preference index in the 2 mg/kg and 8 mg/kg groups(P2mg/kg=0.769,P8mg/kg=0.820),but the preference index of 4 mg/kg and6 mg/kg groups increased significantly,especially in the 4 mg/kg group（0.138±0.005 vs 0.466±0.019）,the difference was statistically significant(P4mg/kg=0.000,P6mg/kg=0.000).Additionally,9/12 rats in 8 mg/kg group were anesthetized and show no autonomous activities,the rats gradually woke up after about 5min and start to move.4 The result of the correlation between some indicators Pearson correlation analysis showed that there was a positive correlation between hedonic facial expression score and the locomotor movement distance（r=0.754,P=0.000）;there was a positive correlation between hedonic facial expression score and the locomotor movement speed（r=0.791,P=0.000）;there was a positive correlation between hedonic facial expression score and conditional position preference time（r=0.869,P=0.000）;and there was a positive correlation between the locomotor movement distance and conditional position preference time（r=0.699,P=0.000）It was positively correlated with the locomotor movement speed and conditional position preference time（r=0.772,P=0.000）.The results suggest that propofol may activate a certain neural pathway through common mechanism,which makes three kinds of behavioral phenomena of euphoria and addiction appear at the same time.5 Experiment of recording calcium signal in neurons by optical fiber GCa MP was expressed on the LC and VTA neurons as a calcium indicator to examine real-time neuronal activity.The AAV-h Syn-Gca MP6s virus was injected into the LC area or the VTA area.We analyzed Gca MP signals in two periods:50 s pre-propofol and 100 s post propofol tail-vein bolus injection.As result,5 rats had died,and optical fiber was fell off in 2 rats,and the optical fiber position were verified were incorrect after the experiment in 3 rats.Finally,there were 20 SD rats were randomly assigned to two groups（n=10）.Based on the results of hedonic facial response test and CPP experiments,4 mg/kg of propofol induced the most obvious euphoric effects and,therefore,was chosen for the fiber photometry experiment.In LC Gca MP expressing rats,we observed that administration of 4 mg/kg propofol trigger reliable increases in Gca MP fluorescence（27.90±2.34 vs 6.90±1.20,P=0.000）.However,in VTA Gca MP expressing rats,administration of 4 mg/kg propofol did not induce any change of Gca MP signals（7.70±1.48 vs 8.31±1.67,P=0.260）.Our results suggested that the euphoric effects of low dose of propofol are mediated by the activation of LC neurons,not VTA neurons.6 The result of chemical genetics inhibition experiment As result,4 rats were died during the model making process,the virus injection site verified in 3 rats was incorrect after the experiment,and 1 rat had no obvious preference for preference box or obvious preference for white box.So the experimental results of these rats were removed,and finally,12 rats injected with h M4D（Gi）（n=6）and 10 rats injected with controlled AAV（n=5）enter the follow-up experiment.The chemogenetic inhibition of neuronal activity was verified by a c-Fos staining in the LC area.The score of hedonic facial responses was not significantly different between 0 mg/kg propofol group and CNO group which was injected control AAV（2.20±0.37 vs2.38±0.41,P=0.822）.And the time of CPP was not significantly different between 0mg/kg propofol group and CNO group which was injected control AAV（118.00±6.96vs 124.08±4.25,P=0.456）.When compared to 4 mg/kg propofol group,the facial score was significantly inhibited in CNO+4 mg/kg propofol group（21.25±1.43 vs1.33±0.49,P=0.000）.Another 6 rats were used for CPP test,the place preference increased by 4 mg/kg propofol was abolished by CNO administration（418.42±17.40vs 128.17±9.07,P=0.000）.CNO was intraperitoneally injected to the rats that received the control r AAV-h Syn-EGFP injection in the LC area to exclude the influence of viral vector and CNO on the results.Such results suggest that inhibition of the neuronal activity in the LC area could inhibit the putative pleasant affective state induced by low dose of propofol.7 The results of serum NE and DA contents Compared with normal rats without propofol injection,the content of NE in serum increased significantly（7.46±0.59 vs 9.55±0.70,P=0.042）,but the content of DA did not change significantly（4.67±0.60 vs 5.08±0.51,P=0.609）.The content of NE in serum was positively correlated with the fluorescence intensity of calcium signal（r=0.723,P=0.018）.The results suggest that it is possible that propofol can activate noradrenergic neurons in LC neurons.Conclusion:1 Low dose of propofol can induce hedonic effects and addictive issue in rats.2 The mechanism may be related to the activation of neurons in locus coeruleus,especially the activity of noradrenergic neurons in locus coeruleus. |
PDF Full Text Request