Effect Of Inhalation Anesthetic Isoflurane On Calcium Oscillation And Axonal Development Of Hippocampal Neurons In Developing Rat

BackgroundIsoflurane (1-chloro-2,2,2-trifluoroethyl difluoromethyl ether) is one of the most widely used inhalation anesthetics in the world, and it is generally accepted that it mainly through acts at the GABAA and Gly receptors in an agonistic manner to perform its function. Developing nervous system is experiencing a peak period of neurogenesis and synapse formation, the growth and differentiation of the nervous system were very vulnerable to different kinds of interference factors during this period. Since the central nervous system is the target organ of general anesthestics, which attracted many researchers to investigate the neurotoxic effect of anesthetic drugs on the developing brain in recent years.GABA is one of the most important inhibitory neurotransmitter in central nervous system (CNS) and was attributed to mediate fast synaptic transmission via the activation of ion channel intrinsic to GABAA receptor. It is generally accepted that its receptors can be divided into GABAA, GABAB, and GABAc receptor subtype. GABAA receptors expressed at birth and have the function at that time, while GABAB and GABAc receptors matured gradually 7-14 days after birth. GABAA receptors are widely distributed in CNS such as cerebral cortex, lateral geniculate body in cerebellum, hippocampus, amygdala, thalamus, substantia nigra, hypothalamus, and periaqueductal substantia nigra as well. GABAA receptor is a ligand gated chloride ion channel receptor, and mainly guides sedative, antianxiety, anticonvulsant and anesthesia effects by postsynaptic inhibition. After activation by native agonist GABA, chloride ion channel is opened and the Cl- can influx or efflux through it by facilitated diffusion. Previous researches have confirmed that during the brain growth-spurt period of nervous system (from the late embryonic stage to postnatal 2 weeks), intracellular chloride concentration in immature neurons was higher than that in mature. The reason for this can be attributed to the delayed expression of the Cl" extruders KCC2 and the early over-expression of Cl- accumulating NKCC in the cytomembrane. Previous researches have confirmed that the glutamatergic synaptic transmission in adult involves the combined effect of both the AMPA and NMDA types of receptors. While in immature brain, glutamatergic synaptic responses seem to be devoid of an AMPA receptor-mediated component for its dysfunction. Transformation of these immature synapses into the classical AMPA/NMDA receptors' functional synapses occurs during the first week after birth. During this period, GABAA receptor plays the analogical role similar to the role of AMPA receptor in the mature neurons in hippocampus by acting in cooperation with the NMDA receptor, which offering the depolarization required to remove the voltage-dependent Mg2+ block. Therefore, contrast to its function in mature, during the period from late embryonic stage to early postnatal, GABAA receptor mediated excitatory postsynaptic effects. This may play an important role in axon growth, synapse formation and neuronal plasticity.Hippocampal neuronal circuits exhibit a very strong plasticity through the long-term potentiation or long-term depression. The formation of neural circuits in hippocampus depends on the development of neurites and the formation of synapses. There are spontaneous calcium oscillations existing in developing hippocampal neurons, which play an important role in the outgrowth of growth cone, development and differentiation of neurite, and the formation of synaptic connections between neurons and so on. The mechanism of calcium oscillation in neuron is very complex and still need further investigation. Previous studies confirmed that spontaneous Ca2+-oscillations may be a possible mechanism for Ca2+-mediated signal transduction in neurons. In hippocampal neurons, they exhibited by a periodical increase and decrease of cytosolic Ca2+, which enters the cytoplasm from the extracellular medium via voltage-gated or receptor-operated Ca2+ channels, then a Ca2+ ions induced Ca2+ release from the endoplasmic reticulum (ER) take place. And the increased Ca2+ was rapidly returned to baseline level either by reuptake into endoplasmic reticulum or being pumped out of cells by various pumps and exchangers. The frequencies and amplitudes of calcium oscillations in neurons are inversely related to the rates of axon outgrowth. The material basis of growth cone migration is the reconstruction of the cytoskeleton, and the transient elevation of cytosolic Ca2+ plays an important role in the reconstruction of the cytoskeleton. When growth cone suspended its migration, it forms a large microtubule loop. As the microtubule loop undergoes splaying and fragmentation, the growth cone restart its migration again. Restart the outgrowth of axonal growth cone need the interaction between dynamic microtubules and actin filaments. Enhancement of calcium oscillations can slow the outgrowth of growth one by acting on the calcineurin to depolymerize actin filament and maintain the stability of microtubule loop. Previous study confirmed that the use of L-type Ca2+ channel blockers to eliminate neuronal calcium oscillations can promote axon growth and the migration of growth cone; while the increase of amplitude and frequency of calcium oscillation may slow the rates of growth cone migration.Therefore, the present study was designed to investigate the neurotoxicity of inhaled anesthetics isoflurane on developing hippocampal neuron through 3 aspects as follow:(1) effect of isoflurane on calcium oscillation in developing hippocampal neuron in vitro; (2) effect of isoflurane on neurons survival; (3) effect of isoflurane on growth and differentiation of axon in developing hippocampal neurons. This study tested the following hypotheses:(1) isoflurane enhances the calcium oscillations of developing hippocampal neurons; (2) the clinical and safe concentration of isoflurane used within a safe period does not lead the developing hippocampal neuron to apoptosis; (3) clinically relevant concentration of isoflurane can inhibit the migration of growth cone by enhancing calcium oscillation in developing hippocampal neurons.Methods and Results1. Establishing a model for observing the effects of isoflurane on culture cells in vitroMethods:Petri dishes with 8.6cm diameter were exposed to isoflurane in a tight gas chamber, which was placed inside a cell incubator, and the gas chamber has an inlet and an outlet on it.80 ml medium composed of Neurobasel+B27 (98:2) was infused into the dish. Isoflurane was continuously delivered into chamber through inlet with an air stream (95% air+5% CO2) containing desired anesthetic concentration using a calibrated vaporizer. According to the different concentrations of the isoflurane in the air stream, the medium were divided into ten groups as follows:(1)Test groups:0.25%(groupⅠ),0.5% (groupⅡ),1.0%(groupⅢ),1.5%(groupⅣ),2%(groupⅤ),2.5%(groupⅥ), 3%(groupⅦ),4%(groupⅧ), (2) Control groups:air stream composed only of 95% air and 5% CO2 (groupⅨ), and medium without air stream bubbling (group X). A tube was used to connect the outlet to an exhaust outlet (before each test, the mixture gas carrying with desired concentration of isoflurane was flow through the chamber 20 min). Medium samples of each test group were taken at 5min, 10min,15min,30min,60min,120min and 180min after isoflurane intervention. Isoflurane concentrations in the medium were measure by double balanced headspace gas chromatography method. The samples of both the test groups and the control groups were taken at the time of 60min or 120min after intervention to measure the values of PH, PO2, PCO2 and HCO3-.Results:In each of test groups, isoflurane concentrations achieved equilibrium between intervention gas and medium at 15 min after intervention. Afterwards the isoflurane concentrations in them became linearly correlated, the linear correlation equation of them is Y=1.521X-0.0368. The values of pH, PO2, PCO2 and HCO3- between test groups and control groups had no significant difference at each time points.2. The characteristics and mechanism of calcium oscillation in developing hippocampal neurons in vitroMethods:Primary cultures of hippocampal neurons were prepared from the dissociated hippocampus of rat fetus at embryonic day 18 or of rat at postnatal day 0.2-5 days after plating, hippocampal neurons were loaded with Fluo-4 AM for Ca2+ measurements with a Laser scanning confocal microscope. Through removing of Mg2+ or Ca2+ in the medium or application of NMDA, NMDA receptor antagonist MK 801, GABAA receptor agonist muscimol and (or) antagonist bicuculline, AMPA receptor antagonist (CNQX), and L-type calcium channel blocker nicadipine on the developing hippocampal neurons to investigate the characteristics and mechanism of calcium oscillation in developing hippocampal neurons in vitro.Results:Application of NMDA (100μM) or removal of extracellular Mg2+ within the medium can enhance calcium oscillations in neurons, and the calcium oscillation would gradually returned to normal when the extracellular solutions returned to previous medium. Non-competitive NMDA receptor antagonist MK-801 (40μM) inhibited the amplitude and frequency of calcium oscillation in hippocampal neurons. While application of non-competitive AMPA receptor antagonist CNQX (100μM) did not show any significant effect on the amplitude and frequency of calcium oscillation. Exposure of oscillating hippocampal neurons to a Ca2+-free medium by application of 3mM of EGTA resulted in a complete inhibition of calcium oscillations, and the calcium oscillations rapidly recovered into normal on returning the neurons to the standard extracellular KB's solution. Muscimol (30μM), a competitive GABAA receptor agonist enhanced the amplitude and frequency of calcium oscillation significantly. In contrast, application of bicuculline (50mM), a GABAA receptor antagonist, resulted in a significant depression in the amplitude and frequency. When nicardipine (10μM), a L-type Ca2+channel blocker was used, the amplitude and frequency of calcium oscillation in hippocampal neurons were decreased significantly.3. Effects of inhalation anesthetic isoflurane on calcium oscillation in developing hippocampal neurons in vitroMethod:Primary cultures of hippocampal neurons were prepared from the dissociated hippocampus from rat fetus at embryonic day 18 or of rat at postnatal day 0.2-5 days after plating, hippocampal neurons were loaded with Fluo-4 AM for Ca2+measurements with a Laser scanning confocal microscope. Perfusion buffer which was pre-bubbled 20 minutes by an air stream (95% air+5%CO2) containing desired anesthetic concentration (from 0 to 1 MAC, assuming that 1 MAC equal 1.46 vol%) were used to interfere with it. Drugs such as bicuculline, MK801 and (or) nicardipine were applied in combination with 1MAC isoflurane to observe their combined effects on the calcium oscillations in developing hippocampal neurons, and to analyze the exact mechanism of isoflurane on calcium oscillation in developing hippocampal neurons.Results:At different concentration ranging from 0.25MAC to 1MAC, isoflurane dose-dependently enhanced the amplitude and frequency of calcium oscillations in neurons. 1MAC isoflurane increased the oscillation amplitude (F/Fo) from 1.67±0.17 to 2.11±0.27, and the oscillation frequency from 0.03±0.005 Hz to 0.04±0.007 Hz. The increased amplitude and frequency of calcium oscillations by isoflurane (1MAC) could be completely reversed in the presence of 50μM bicuculline. Nicardipine (10μM) applied alone can suppress the spontaneous calcium oscillations significantly but not completely, but when co-application with MK801 the spontaneous Ca2+oscillations could be suppressed completely. 4. Interventional effects of isoflurane on the apoptosis rate of developing hippocampal neurons in vitroMethods:Primary cultures of hippocampal neurons were prepared from the dissociated hippocampus of rat fetus at embryonic day 18 or of rat at postnatal day 0.48 h after plating, the cultured hippocampal neurons were randomly divided into isoflurane (0.25,0.5,0.75, 1.0 MAC) intervention group and control group. After intervention of isoflurane, each group of test neurons was used for MTT assay together with a group of control. In each culture hole, MTT (5mg/ml) 20μl was added, then the cells was placed into 37℃incubator for incubating 4 hours, Then the culture medium with MTT was removed and 150μl dimethyl sulfoxide was added to each well to dissolve the formazan. Absorbance was measured at 490 mn with an ELISA detector. And Cell viability was expressed as a percentage of the value in control cultures. Meanwhile, western blot technique was applied to detect the expression of cleaved caspase-3.Results:Through application of MTT assay to detect the survival rate of hippocampal neurons after intervention of isoflurane we could found that 0.25,0.5, and 0.75 MAC of isoflurane interfered with developing hippocampal neurons 2 to 8 hours did not play any role on the survival rate of them, and 1MAC isoflurane intervention of 2 to 6 hours also had no significant effect on it. But when the neurons were interfered with 1MAC isoflurane 8 hours, the survival rate of them should be decreased significantly (P<0.05). The application of western blot detection of activated caspase-3 expression also show that exposure to 1MAC isoflurane for 8h resulted in an over expression of activated caspase-3 of developing hippocampal neurons, which means that the low survival rates was attributed to the activation of neuronal apoptosis pathway induced by the over expression of caspase-3. 5. Effects of isoflurane on the outgrowth and differentiation of developing hippocampal neurons in vitroMethods:After plating on the etched grid glass coverslips, the hippocampal neurons were cultured in the cell incubator. Images of neurons were acquired at 48h after culture with a 20×magnification,0.7Neofluor CF160 objective in a Nikon TE2000 inverted epifluorescence microscope. The hippocampal neurons were then placed into a sealed gas chamber and were exposed to isoflurane (0,0.25,0.5,0.75,1MAC) with a different intervention time (from 2 hours to 6 hours). At the end of intervention, the culture mediums were exchanged to fresh medium immediately. Then the images of the neurons were acquired at the 54h,96h, and 144h after plating. The neuronal photographs were processed with Image-Pro Plus 6.0 software, and the measurement tool of its was used for measuring the length and branch of axon.Results:48h after plating, primary culture hippocampal neuron exhibited an immature morphology with a pyramidal body, four dendrites and a long axon. An obvious growth cone was show at the axonal terminal. After cultured 96-144h, the dendrites grow slightly, while and axons grow significantly and gradually grow more branches. Exposure of hippocampal neurons to clinically relevant concentration of isoflurane (0.25-1 MAC) showed that exposed to isoflurane at concentrations ranging from 0.25MAC to 0.5MAC for 2-6h or exposed to isoflurane at concentrations ranging from 0.75MAC to 1.0MAC for 2-4h did not play any role on the developmental morphology of hippocampal axon growth. In contrast, a 6-h-long exposure of neurons to isoflurane at concentration of 0.75MAC or1.0 MAC led to a significant inhibition of the outgrowth of axon (P<0.05), which included slowing down the axon growth rate, reducing the number of axon branches, and reducing the total length of axon. 6. Statistical analysisData from calcium fluorescence assay, MTT colorimetric test, Western blot analysis of expression of cleaved caspase-3 were pooled together and analyzed for statistical differences using the paired Student's t-test. Other results were analyzed using ANOVA. Compiled data are expressed and graphed as mean±SEM. SPSS 12.0 software was used for statistic analysis, and differences were considered to be significant if a P value was less than 0.05.SUMMARY1. Gas chromatography was applied to determin the concentration of isoflurane in the medium to construct an in vitro intervention model for isoflurane on the primary cultured hippocampal neurons, and to confirm its safety and efficacy in cell culture.2. Through analyzing the characteristics and mechanisms of calcium oscillation in cultured primary hippocampal neurons in vitro by dying the neurons with calcium fluorescent dye Fluo-4 AM and detecting it under a laser confocal microscope, we confirmed that inhaled anesthetics isoflurane enhanced calcium oscillation in developing hippocampal neurons by exciting the GABAA receptor.3. From investigating the effect of isoflurane on the outgrowth of axon in developing hippocampal neurons, we can draw a conclusion that exposed to clinically revelent concentrations of isoflurane within a safe period did not result in apoptosis of hippocampal neurons, but inhibition in outgrowth and differentiation of axon.Conclusion:Clinically relevant concentrations of inhaled anesthetics isoflurane inhibit the outgrowth and differentiation of axon by enhancing the calcium oscillation in developing hippocampal neurons in vitro.