Study On Effect Of Tuberomammilary Nucleus In The Propofol-induced Loss And Recovery Of Consciousness

Objective:Propofol is a widely used clinical intravenous anesthetic.It is a GABA_A receptor agonist,however,the mechanism that leads to the loss of consciousness remains unclear.Studies have found that propofol-induced anesthesia has many similarities to sleep.Therefore,we hypothesized that the process of propofol-induced loss and recovery of consciousness may be achieved through the sleep-wake pathways.The Tuberomammillary nucleus(TMN)is the main component of the wake pathways and the only source of histamine in the brain.Histamine secretion is regulated by GABA and contrarily,it inhibits the release of GABA.Therefore,this study aims to investigate the role of TMN in the loss and recovery of consciousness caused by propofol.Method:1.Select out the best dose of propofol injected intraperitoneally:42wild-type C57 mice were randomly selected out.Propofol was intraperitoneally injected at dose of 50,60,70,80,90,and 100(mg/kg).Selecting out the best dose of propofol intraperitoneal injection of mice induced of Loss of right reflex(LORR).2.Testing the LORR and RORR and recording electroencephalogram after selective lesion of TMN by drug:Mice were randomly divided into three groups,n=14,saporin,saline and control.Orexin2R-Saporin(250 ng/ul),saline and nothing respectively were microinjected to TMN on both sides of the mice.Then the EEG recording electrodes were inserted.After two weeks adopted in isolated cages,the mice were measured the time of LORR and RORR and EEG under propofol anesthesia.3.Fiber Photometry:Six mice were chosen randomly and microinjected with 0.3μl of virus(rAAV-hSyn-Gcamp6s-WPRE-pA).The optical fiber was implanted at 200μm above the injection site.After reaching the point,the calcium-sensitive protein on TMN neurons was activated with a 488-nm laser light source,and the fluorescence signal of TMN neurons under propofol anesthesia was detected in real-time using fiber photometry to determine the effect of propofol on neuronal activity.4.Immunofluorescence:The mice above were perfused and the brains were removed.After fixed dehydration,histamine-specific antibody(anti-histidine decarboxylase antibody)was used for immunofluorescence staining and histological examination was performed.Results:1.The dose of LORR produced by intraperitoneal injection of propofol in mice was 90 mg/kg,and the ED50 was 69.21 mg/kg after the dose-response curve was given.2.After the selective drug lesion(lesion rate:69.57%),there was no significant difference in the time of occurrence of LORR and RORR and the features of EEG(P>0.05)between groups.3.The fiber photometry recordings showed that the fluorescence signal significantly decreased when LORR occurred under propofol anesthesia(P<0.05),and the fluorescence signal increased when RORR occurred(P<0.05),but it could not quickly recover to the level before anesthesia.Conclusion:1.Propofol significantly inhibits the activities of histaminergic neurons in the TMN during the propofol-induced loss of consciousness;2.There are no significant changes in behavioral and electroencephalographic features of mice under propofol anesthesia which suggest that TMN may not be the major target for loss and recovery of consciousness induced by propofol.