Orexinergic Neuron Is Involved In The Regulation Of Anesthesia-arousal Of Propofol Anesthesia In The Rat

Although general anesthesia has been widely used in clinic for more than onehundred and sixty years, its mechanism is still not well understood. To explorethe mechanism of general anesthesia is helpful to understand the formation ofconscious. Thus the study of general anesthesia mechanism had been list as oneof important questions of neuroscience. Recently, neuronal network regulationtheory is proved to be the most popular theory of general anesthesia mechanism.However, is still unknown which neuronal signaling system plays a crucial role inregulating the anesthesia-arousal cycle. In previous studies, Orexinegic neuronshave been found to regulate sleep-arousal cycle as well as inhalationanesthesia-arousal cycle. The aim of current study is to test whether this novelneurotransmitter system is also involved in the anesthesia-arousal regulation inintravenous anesthesia induced by propofol.Objective To evaluate the role of Orexinergic neurons in the anesthesia-arousalregulation of propofol anesthesia.Methods Forty-eight male SD rats weighing230²50g used in this study.Experiment1：Thirty SD rats were randomly divided into5groups （n=6）：Animals in Group I were sacrificed before the propofol injection. Animals in groups II, III, and IV were intravenous injected with propofol (60mg·kg^-1·h^-1) for30,60and120min respectively via jugular vein. Animals in Group V weresacrificed at the emergence time when propofol terminated after120min infusion.Blood samples were taken from femoral vein for determination of plasmicconcentrations of Orexin-A by radioimmunoassay. Rats were sacrificed byinfused10%Kcl via jugular vein and the hypothalamus was immediatelyremoved after perfusion for immunofluorescent staining to elucidate activities ofOrexinergic neurons. The activation of Orexin neuron was determined by theco-expression of Orexin-A and c-Fos.Experiment2：Eighteen SD rats were randomly divided into3groups （n=6）. Theanimals were anesthetized with intraperitoneal2%pentobarbital40-60mg/kg andplaced in a stereotactic apparatus to locate a guide cannula into the basalforebrain. After3-5days recovery from the surgery, rats in groups A,B, and Cwere administered Ringer’s solution30or100pmol of Orexin-A respectively viamicroinjection into basal forebrain （the microinjection time was5min）. Propofolwas infused at the dose of60mg·kg^-1·h^-1at10min after BF microinjection. Theloss of righting reflex time was recorded as the induction time. The other day, ratsin groups A, B, and C received infusion of propofol and15min later the Ringer’ssolution,30or100pmol of Orexin-A were microinjected into BF respectively（the time of microinjection was5min）. The propofol was terminated at30minafter infusion. The return of righting reflex was recorded as emergence time. EEGwas recorded during whole procedure of propofol anesthesia. Rats in groups A, B,and C received Ringer’s solution,5or20μg of SB-334867respectively after7days. The induction time and emergence time were recorded as above describe.ResultsExperiment1：The results showed that in group I, c-Fos positive Orexinergic neurons were （36.8±1.0）%. When propofol pumping30,60min, c-Fos positiveOrexinergic neurons were （7.3±0.4）%and （2.3±0.3）%, respectively. Especially,there were no c-Fos positive Orexinergic neurons exist when propofol infused120min. However, the number of c-Fos positive Orexinergic neurons wassignificantly recovered at （25.0±0.6）%after the emergence from propofolanesthesia. The plasmic Orexin-A concentrations were （24.96±0.64） pg/ml ingroup Ⅰ. When propofol pumping30min （group Ⅱ）,60min （group Ⅲ） and120min （group Ⅳ）, the lever of Orexin-A was （19.25±0.56） pg/ml,（12.31±0.29）pg/ml and （12.33±0.45） pg/ml, respectively （P<0.05）. But Orexin-Aconcentration increased at （39.67±0.89） pg/ml in group V （P<0.05vs Ⅰ-Ⅳgroup）.Experiment2：For induction time, there was no significant difference amonggroups A, B, and C （P>0.05）. The emergence time was significantly shorten ingroup C than in group A （P<0.05）, while no significant change was found ingroup B （P>0.05）. There was no significant difference in the emergence timebetween groups B and C （P>0.05）. For SB-334867, there was no significantdifference among groups A, B, and C in induction time; the emergence time wassignificantly delayed in group C than in groups A and B （P<0.01vs A, B）. Theresult of EEG showed that microinjection of Orexin-A changed EEG from a burstand suppression pattern to an arousal pattern.Conclusion Our findings indicate that the mechanism of propofol at least partlyeffect Orexinegic neurons in hypothalamus and the Orexinergic signals may playa promote role in emergence of propofol. In addition, the basal forebrain may beone of import targets of Orexin-A to regulate the anesthesia-arousal of propofolanesthesia.