Effect Of Propofol At Uptake Equilibrium On Excitatory Amino Acid Transporter-2mRNA Expression In Different Brain Regions In Dogs

Propofol (2,6-diisopropylphenol) has been the most common general anesthesia as an induction and maintenance agent and is widely used as a sedative agent in the intensive care unit (ICU). Many researches have been done to investigate the anesthetic mechanism of propofol, but the mechanism underlying propofol anesthesia on human is not well understood until today.The releasing and transmission of both inhibitory and excitatory neurotransmitters in central nervous system (CNS) play a key role in the function of CNS. So the study of propofol on neurotransmitter in different brain regions will help to understand and reveal the anesthesia mechanism.Glutamate (Glu) is the main excitatory neurotransmitter in the central nervous system, which has important relationships with transmission of nerves that sense pain, allergy, learning and memory and nerve growth. Glutamate, the most abundant excitatory transmitter in the brain can lead to neurotoxicity when not properly regulated. Glutamate receptors are widely distributed incerebral cortex, hippocampus, striatum, cerebellum and so on. The effects of propofol on glutamate are as follows: inhibition of presynaptic releasing for glutamate, promoting the reuptake of glutamate, blocking glutamate receptors of the postsynaptic membrane. The uptake of glutamate into astroglia is the predominant mechanism to terminate glutamatergic neurotransmission and to prevent neurotoxic extracellular glutamate concentrations. But tert-butyl hydroperoxide inhibited glutamate uptake. Propofol prevents and reverses the inhibition of excitatory amino acid uptake in astrocytes exposed to tert-butyl hydroperoxide, and thus delay or prevent the onset of excitotoxic neuronal death. The glutamate transporters serve as the critical regulators of glutamate concentration, and these transporters are of two main types, namely, excitatory amino acid transporters (EAATs) and vesicular glutamate transporters (VGLUTs). EAAT2can uptake glutamate against the concentration gradient. In pathological conditions, many kinds of nervous disease is related with abnormal expression of EEAT2gene in astrocytes. EAAT2is the most important one in all glutamate transporters and occupies an important place in the central nervous system. Intrathecal injection of dihydrokainate, a selective inhibitor of EAAT type2, in Sprague-Dawley rats could increase the minimum alveolar concentration for isoflurane. Isoflurane at clinically relevant concentrations (1-3%) caused a time-, sodium-and concentration-dependent increase of both Vmax and Km of transporter-mediated glutamate uptake of GLAST and GLT-1in primary cultures of rat cerebral mixed glial cells. Lidocaine and bupivacaine did not change glutamate-induced inward currents at the tested concentrations (1～10000μmol). Thiopental and ketamine also did not affect the activity of EAAT2at the tested concentrations (0.3-300μmol). In cerebral ischemia/anoxia, the glutamate transporter runs in reverse and releases glutamate into the extracellular space. But midazolam and ketamine, but not thiopental and propofol, have a capacity to inhibit glutamate release via GLT-1. At clinically relevant concentrations (0.05-10μ), propofol offered protection of OGD equivalent to that of MK-801. The inhibition of the glial GLT1transporter by3-methyl-glutamate did not further modify the effect of propofol on glutamate uptake, suggesting that GLT-1was not the major target of propofol. GLT-1expression is dependent on gap junctions and the gap junction blocker, propofol, will inhibit the expression of GLT-1. These results suggest that multiple anesthetic agents reacted on the central neural nervous system and the effect is closely connected to EAAT2.Imaging technology revealed that the anesthesia effects of propofol may be closely related to special regions of brain. We found that propofol was distributed almost evenly among regional brain tissues in dogs after50minutes infusion at a constant rate. The propofol at different anesthesia depth could react on expression of glutamate in different cerebral regions. The concentration of glutamate in brain regions declined obviously at deep anesthesia, and the variation of glutamate in hypothalamus is highest.The aim of this study is to elucidate the effects of propofol on the amino acid transporters, we investigated the effect of propofol on the mRNA expression of EAAT2in different brain regions (the hypothalamus, subthalamus, dorsal thalamus, hippocampus, pons, parietal lobe and frontal lobe) in dogs.Material and methods1Animal preparation and groupingEighteen healthy mongrel dogs of male or female, aged12-18months and weighed10-12kg, were divided randomly into high dose group (group H), low dose group (group L) and control group (group C), each group with6dogs. The experiment were scheduled from8:00am to12:00am and raised in diet for12-hour were prior to experiment. The venous channel was established in the great saphenous vein of the right posterior limb.2Animal anesthesia and managementDogs in group C were sacrificed by intravenous injection of10%KCl2mg/kg without any previous intervention. Dogs in group L and group H were anesthetized with propofol at a single bolus (5.5and7.0mg/kg, respectively) in15seconds followed by propofol infusion at a constant rate (55and70mg/kg/h, respectively). After reached the appropriate depth of anesthesia which means their eyelid reflex and pedal reflex disappeared, animals were fixed supinely on the platform and inserted endotracheal tube. End-tidal partial pressure of carbon dioxide (PETCO2) was maintained at30～38mmHg by mechanical ventilation (respiratory rate15～20breaths/min, tidal volume15ml/kg). The MAP and PR was monitored by multi-parameter patient monitor (BeneView T8) and the pulse oxygen saturation (SpO2) was maintained over95%during the whole process of this test.3Sample collectionBlood samples were as placed in an Eppendorf (EP) tube containing heparin (50 I.U.), and then stored in-4℃refrigerator for measuring plasma propofol concentrations. Simultaneously, dogs in group L and H were sacrificed by rapid intravenous injection of10%KCl2mg/kg. The hypothalamus, subthalamus, dorsal thalamus, hippocampus, pons, parietal lobe and frontal lobe were then dissected and stored in-80℃to determine EAAT2mRNA levels by Quantitative Real-time PCR (qRT-PCR).4Measurement of propofol plasma concentrationsAfter anticoagulant, each blood sample was centrifuged for10minutes (3500rpm,4℃). The supernatant (200μl) was taken and placed in EP tube, then added acetonitrile (400μl). The sample was shocked in vortex device for2minutes (1000rpm), then centrifuged for10minutes again (10000rpm). The supernatant was taken to analyzed using high-performance liquid chromatography ultra-violet spectroscopy (HPLC-UV).The chromatogram column was Shim-pack VP-ODS (250mm×4.6mm) and the guard column was Shim-pack GVP-ODS (10mm×4.6mm). The mobile phase was15%pure water and85%methanol and the automatic injection volume was20μl. Both columns were kept at4℃with a mobile phase flow rate of1ml/min, with the detection wavelength was270nm.5Brain region selection schemeTake the tissues from the same brain regions of the6dogs in each group, and the quality of each pooled tissue is0.1gram. There were seven areas in each group, then the twenty-one samples were dissected to determine EAAT2mRNA levels by Quantitative Real-time PCR (qRT-PCR). The significant changed brain areas will take for further testing.6RNA isolation and qRT-PCR analysisThe EAAT2mRNA expression levels were confirmed by qRT-PCR.6.1Total RNA isolation and reverse transcriptionTotal RNA was isolated from brain tissues, followed by Trizol reagent method. Each reaction tube contained:5×Buffer2μl+reverse transcriptase1μl+dNTP0.5μl+DEPC H2O10.5μl+RNA1μl. Amplification of cDNA by PCR using each of the appropriate primers was performed for30minutes at42℃,3minutes at100℃.6.2qRT-PCR analysisSequences of the primers used for RT-PCR analysis are supplied by GenBank. Primers were designed by Primer Premier5.0and synthesized by Invitrogen (Shanghai), China. Each reaction tube contained:primer FP10μl+primer RP0.5μl+SYBR12.5μl+cDNA1μl+DEPC H2O10.5μl. The PCR reaction consisted of stage1,95℃for10minutes; stage2,40cycles of95℃for15seconds and60℃for30seconds. Each sample was placed in three different wells.6.3Data processingRelative expression levels of EAAT2mRNA was analyzed using the2-△△CT method. Relative expression of mRNA in different groups was determined as follows:△△CT=△CT of gene of interest (group L or group H)-△CT of beta actin (group C).7Statistical analysisAll data were expressed as the means±standard deviation and were analyzed with the Statistics Package for Social Sciences (SPSS, version13.0for windows; SPSS Inc, Chicago, IL, USA). Statistical analysis was performed using paired-samples t-est, an independent-samples t-test and a one-way ANOVA. Values of P<0.05were considered to be statistically significant. Multiple comparisons were analyzed by LSD test when the variances were homogeneous and Tambane’s test when the variances were unequal.Results1General conditionThe sex, age, and weight of the dogs were similar among the three groups. All animals reached objective anesthesia depth safely and quickly and there were no vomiting, asphyxia, apnea and other adverse reactions. The mean arterial pressure (MAP) and pulse rate (PR) were fluctuated within the normal physiological range. When propofol continued infusion at a constant rate for50minutes, the MAP was (77.00±6.90) mmHg and the PR was (115.50±5.17) bpm in group L. The MAP was (54.17±5.98) mmHg and the PR was (89.00±4.10) bpm in group H. The difference between the two groups both of MAP and PR was of statistical significance (P<0.05). 2Plasma concentrations of propofolThe plasma concentrations of propofol in internal carotid artery and jugular vein were no statistical differences between group L (3.18±0.06μg/ml and3.12±0.09μg/ml, P>0.05) and group H (6.21±0.07μg/ml and6.14±0.11μg/ml, P>0.05).The plasma propofol concentration in group H were higher than those in group L between internal carotid artery and jugular vein (P<0.01).3Brain regions screening resultsEAAT2mRNA levels in group L and group H were higher than that in group C (P<0.05) in hypothalamus, hippocampus, parietal lobe and dorsal thalamus, but had no significant difference in other brain regions (P>0.05).4The effect of propofol on the mRNA expression levels of EAAT2Quantitative real-time PCR revealed that in the low dose group the expression volume of EAAT2mRNA was (2.14±0.69) times that of the control groups (P<0.05) in hypothalamus, and in the high dose group it was (2.04±0.73) times that of the control groups (P<0.05). Analogously, the relative expression levels of EAAT2mRNA in group L and group H (1.83±0.58,2.42±0.87)were higher than that in group N (1.00±0.00, P<0.05) in hippocampus. In group L the expression volume of EAAT2mRNA was (1.60±0.41) times that of the control groups (P<0.05) in frontal lobe, and in group H it was (1.59±0.23) times that of group N (P<0.05). There was no significant difference between high-dosage group and low-dosage group (P>0.05) in hypothalamus, hippocampus and frontal lobe. The expression volume of EAAT2mRNA had no significant difference among these three groups in dorsal thalamus.Conclusions1. Brain uptake of propofol is in equilibrium after50minutes of propofol infusion at a constant rate.2. Propofol can increase EAAT2mRNA expression significantly at this time in hypothalamus, hippocampus and parietal lobe, but has no appreciable effect at different doses.