The Role Of Midbrain VTA Dopamine Transporter (DAT) In Propofol General Anesthesia

Objective: To explore the role of dopamine transporter(DAT)in the ventral tegmental area(VTA)of the midbrain in the awakening of rats under general anesthesia with propofol.Methods: Male SD rats,weighing 250-300 g.In order to observe the effect of VTA in propofol anesthesia,20 rats were randomly divided into 2 groups: Control group(Negative control,NC),6-OHDA damage group(6-OHDA),10 rats in each group.In order to observe the effect of propofol anesthesia on DAT in VTA,20 rats were randomly divided into 2 groups: saline group(Saline,S)and propofol anesthesia group(Propofol,P),with 10 rats in each group.In the rest of the experiment,90 rats were randomly divided into3 groups: control group(Negative control,NC),AAV empty group(AAV-NC),AAV2-DAT-RNAi group(DAT-RNAi),30 rats in each group.In order to observe the effect of propofol on the expression of c-fos protein in the prefrontal cortex(PFC),the immunofluorescence and Western blot experiments were added with saline injection group(Saline,S)and propofol anesthesia group(Propofol,P).There are 10 subgroups with 10 animals in each group.Among them,the NC group was a normal rat;the AAV-NC group and the DAT-RNAi group were injected with a DAT-knockdown adeno-associated virus(GV480)and a negative control virus(CON304)on both sides of the VTA via a stereotaxic instrument.For 3ul,injection for 10 min.In group P,propofol was slowly injected via the tail vein at a dose of 70 mg/kg until the loss of the righting reflex(LORR)disappeared for 30 minutes,and the brain was perfused for 10 minutes after the drug was stopped;The same dose of normal saline was infused intravenously at the same speed,and the brain was perfused 10 minutes after stopping the infusion.The behavioral experiment compares the induction and recovery time of propofol anesthesia in rats by LORR time and the recovery of righting reflex(RORR)time;compares the rats’ performance during propofol anesthesia and recovery by the depth of anesthesia score table Depth of anesthesia score;in vivo EEG recording to observe the changes of the rat’s EEG during propofol anesthesia and awakening;immunofluorescence staining to observe the expression of DAT protein and prefrontal cortex(PFC)c-fos protein in VTA;Western blot Method to detect the protein content of c-fos in PFC.Results: Compared with the NC group,the LORR time of the 6-OHDA group was shortened,while the RORR time was prolonged(P<0.05);compared with the S group,the DAT expression in the VTA of the P group was reduced(P<0.05);compared with the NC group,DAT-RNAi There was no significant change in LORR time in the group(P>0.05),and RORR time was shortened(P<0.05);compared with the NC group,there was no significant difference in the depth of anesthesia in the DAT-RNAi group during propofol anesthesia(P>0.05).During the awakening period from propofol anesthesia,rats in the DAT-RNAi group had lower anesthesia depth scores(P<0.05);during propofol anesthesia,the frequency of alpha waves and beta waves increased(P<0.05).During the phenol recovery period,the frequency of β wave increased and the frequency of theta wave decreased.During the recovery period from propofol anesthesia,the expression of c-fos protein in PFC increased(P<0.05);compared with the NC group,the LORR time and RORR time in the AAV-NC group,propofol There was no significant difference in the depth of anesthesia score during phenol anesthesia and recovery,EEG during propofol anesthesia and recovery,and c-fos protein expression in PFC during recovery from propofol anesthesia(P>0.05).Conclusion: Knockdown of DAT in VTA can promote the recovery of rats from propofol anesthesia.