Effect Of Propofol At Different Anesthesia Depth On Glutamic Acid In Different Cerebral Regions In Dogs

Propofol has been widely used in general anesthesia,special treatment for out-patient and used as a sedative agent in intensive care unit(ICU).but the mechanism of propofol remains unclear.The releasing and transmission of neurotransmitter in central nervous system (CNS) play an important role in the function of CNS.The study of propofol on neurotransmitter in different brain regions will help to understand and reveal the anesthesia mechanism.y-aminobutyric acid (GABA) is the most important inhibitory neurotransmitter in CNS.The GABA receptors are widely distributed, but each has its own distribution, pharmacology and neural electrophysiology. GABA receptor is the main target of propofol.After it is activated, nervous conductance and depolarization increase,resulting in general anesthesia. It is the most important hypothesis that the effect of general anesthetics are related with synaptic transmission.Many neurotransmitters have an importment role in mechanism of anesthetics including propofol.GABA can inhibit the release of glutamate acid(GLU) and acetylcholine(Ach),so it is necessary to study other neurotransmitter.GLU is one of the most important excitory neurotransmitter in CNS,its form of small vesicles are widely existing in the cerebral cortex synapses in vivo,the excitatory postsynaptic potential (EPSP) can maintain the brain electrical activity under normal physiological conditions.Its receptors distribute widely in the hippocampus,cerebral cortex,cerebellum,striatum and so on,which has important relationships with learning,memory,central pain transduction and post-traumatic brain neuronal death.The effects of propofol on GLU as follow:inhibition of presynaptic action potential propagation,inhibition of presynaptic releasing for GLU,promoting the reuptake of GLU,blocking GLU receptors of the postsynaptic membrane.As the anatomical structure and distribution of CNS has a certain specificity,the effect of anesthetics are selective.The target action of propofol is in hypothalamus,but we don’t know where the bullseye is. In this study, the hypothalamus are dissected to observe the effect of propofol on GLU in different cerebral regions in dogs.The research of cerebral uptake of propofol usually bases on mass balance principles.Due to the limitation of experimental methods and the particularity of brain fuction in different brain regions, the concentrations of propofol at its site of action in the brain can’t be accurately counted and the character of regional cerebral distribution of propofol can’t be revealed by this method. To improve the research of cerebral uptake, the concentrations of propofol in different cerebral regions must be detected directly by anatomy.The methods of HPLC-UC can detect the uptake and distribution in different regions under the condition of different anesthesia depth. Our previous study found that the uptake and distribution in different regions were different at different anesthesia depth:propofol was injected with a bolus,the propofol concentration were highest in hypothalamus and brainstem and lowest in hippocampus in light anesthesia. However in deep anesthesia, highest in thalamus and lowest in hippocampus.The other study showed that plasma propofol concentrations in the internal carotid artery and internal jugular vein reach equilibrium and propofol was distributed evenly except the dorsal thalamus contaning a higher concentration at30min of propofol constant infusion70mg/(kg·h), propofol was distributed almost evenly among regional cerebral tissues at50min in dogs. The effect of propofol at different anesthesia depth on excitory or inhibitory neurotransmitter in different cerebral regions is still unknown.The aim of this study is to investigate the effect of propofol on GLU in different regions (dorsal thalamus, epithalamus, metathalamus, hypothalamus, subthalamus, frontal lobe, parietal lobe, temporal lobe, occipital lobe, hippocampus, cingulate gyrus, cerebellum, midbrain, pons, medulla oblongata, and cervical cord) when cerebral uptake and distribution of propofol reach equilibrium at different anesthesia depths.Material and methods1Animal preparation and groupingTwelve12-18-month-old healthy hybrid dogs weighing10-12kg were randomly divided into light anesthetic group(group L, n=6) and deep anesthetic group(group D, n=6).The experiment were scheduled during the day and raised in diet for10-hour were prior to experiment. The venous channel was established in the great saphenous vein of the right posterior limb.2Animal anesthesia and managementGroup L:propofol5.5mg.kg-1was given by intravenous injection(15s) and then continuously infused with a constant rate of55mg.kg-1.h-1for50min.Group D:propofol7mg.kg-1was given by intravenous injection (15s) and then continuously infused with a constant rate of70mg.kg-1.h-1for50min.After reached the appropriate depth of anesthesia, all animals were inserted endotracheal tube to keep PEYCO230-38mmHg by mechanical ventilation (respiratory rate20-25breaths/min, tidal volume15ml.kg-1),and which were monitored mean arterial pressure (MAP) and pulse rate(PR).3Sample collectionBlood samples were taken from the internal carotid artery and jugular vein respectively and were stored in4℃refrigerator for measuring plasma propofol concentrations50min after the start of the infusion in both groups. Then the dogs were sacrificed and cerebral tissues were taken from different regions (dorsal thalamus, epithalamus, metathalamus, hypothalamus, subthalamus, frontal lobe, parietal lobe, temporal lobe, occipital lobe, hippocampus, cingulate gyrus, cerebellum, midbrain, pons, medulla oblongata,and cervical cord) and were stored in-17℃refrigerator for measuring GLU concentrations by high pressure liquid chromatography (HPLC).4Sample processingAfter anticoagulant, each blood sample was centrifuged for10min at4℃centri -fuge(3500r.min-1),200ul plasma was taken and placed in EP tube, after adding acet onitrile400ul,the sample was shocked in vortex device for2min,1000r.min-1centri fuge for10min.The supernatant was taken to analyzed.The brain tissues were extracted with acetonitrile(2ml.g-1) and homogenized for15min,the homogenates were placed in EP tube for centrifugaled for10min,10000r.min-1.The supernatant(50μl and200μl) for derivatization2min to analyzed immediately.5The conditions of HPLCDetermination of propofol plasma concentrations:Column (Shim-pack VP-ODS,250nm×4.6mmID), Guard column(Shim-pack GVP-ODS,10mm×4.6mmID),mobile phase:15%pure water and85%methanol,the injection volume20ul,column temperature40℃,flow rate1ml.min-1,detection wavelength at270nm.Determining GLU in brain tissue:Column:Agilent XDB-C18(5μm,150mm×4.6mm), column temperature:35℃, mobile phase A:0.1mol.L-1sodium acetate,mobile phase B:methanol,excitation wavelength of330nm,emission wavelength, injection volume100ul.6Statistical analysisAll data were expressed as mean±standard deviation and were analyzed with the Statistics Package for Social Sciences(SPSS,version13.0for windows;SPSS Inc,Chicago,IL,USA).Differences were considered statistically significant when P was less than0.05.We used one-way ANOVA、Paired-Samples T Test and Repeated Measure to test for differences.Multiple comparisons were analyzed by LSD test.Results1All animals reached objective anesthesia depth safely and quickly and there were no vomiting,asphyxia, apnea and other adverse reactions.The MAP and PR were significantly higher in group L than in group D(105.33±4.18vs89.50±1.87mmHg,90.50±3.62vs72.83±3.31bpm, respectively,P <0.05).The PETCO2was36±1.41mmHg in group L and37±1.41mmHg in group D and there were no statistical differences(P>0.05). 2The plasma concentrations of propofol in internal carotid artery and jugular vein were no statistical differences between group L (3.00±0.31and3.10±0.51ug.ml-1respectively,t=0.335, P=0.751) and group D (6.41±0.05and6.40±0.11ug.ml-1respectively,t=0.371,P=0.726).3In group D, the concentrations of GLU in cerebral regions(dorsal thalamus, epithalamus, metathalamus, hypothalamus, subthalamus, frontal lobe, parietal lobe, temporal lobe, occipital lobe, hippocampus, cingulate gyrus,cerebellum,midbrain, pons,medulla oblongata,and cervical cord tissues) were much lower than those in group L(P<0.01).The variations of GLU (88.76±0.04%) in hypothalamus was highest among all the brain regions at different anesthesia depths (P<0.05)Conclusions1The plasma propofol concentrations in internal carotid artery and internal jugular vein reach equilibrium at50min after constant rate infusion of propofol.2GLU concentration in cerebral regions is lower at deep anesthesia than light anesthesia, and the variation of GLU in hypothalamus is highest.3Hypothalamus and it’s variation of GLU are associated with the anesthesia depth of propofol.