Effect Of Clinically Relevant Concentration Isoflurane On Electrophysiology And Neurotoxicity Of Hippocampal Neurons In The Developing Rat

BackgroundWith the increasing sophistication of surgical and anesthetic techniques, increasingly pregnant patients and fetal surgeries are being undertaken. Statistics showed that in the U.S. alone, about6million children annually undergo surgery and anesthesia, including1.5million infant patients. In this view, the study that the inhalation anesthetics isoflurane causes neurotoxicity in the immature brain arouses wide-spread social concern. Recent experiments suggest that volatile anesthetics cause excitotoxicity in a concentration-and time-dependent manner in neuronal models in vitro. And neurons in the developing brain are specifically vulnerable to isoflurane hyperexcitability. However, the mechanisms for isoflurane cytotoxicity remain enigmatic. Because most fetal surgeries in humans are performed during mid-gestation, it is important and urgent to know if the anesthetics used cause damage to the developing brain and subsequent postnatal memory problems and learning disabilities.Developing neurons exhibit some characteristics different from those of adult neurons. GAB A mediates excitability in the immature brain. In this respect, a major finding is that the GABAA receptor, activated by its agonist, induces a depolarizing membrane in neonatal neurons compared with the hyperpolarizing one in adult neurons, and results in a cytosolic Ca2+increase. The probable mechanism through which GABA elicits cytosolic Ca2+increases in the neurons seems to involve activation of the GABAa receptors; subsequent Cl-efflux attributable to a developmental depolarized Cl-reversal potential causes membrane depolarization and Ca2+entry via voltage-activated Ca2+channels (VDCC) During the early period of synapse development, the immature neuron has a relatively high intracellular cell concentration of Cl-because of developmental-specific expression of the Na+-K+-2Cl-co-transporters (NKCCs) and the K+-Cl-co-transporters (KCCs) and GABA depolarizes and excites neuronal membranes by a reverse chloride gradient. Animal experiments suggest that activation of GABAergic synapses generates action potentials, removes the voltage-dependent Mg2+block from NMDA channels in the hippocampus and in other brain structures. These effects may underlie the well-characterized modulation by GABA of activity-dependent developmental processes including neuronal growth, neuronal differentiation, proliferation and migration, growth rates of neuronal processes and synapse formation and clustering. GABA through the activation of GABAA receptors triggers spontaneous giant depolarizing potentials. These large depolarizations give rise to fast action potentials, which are synchronous over the entire hippocampus and are associated with spontaneous calcium transients. These highly correlated calcium signals are thought to be essential for consolidation of synaptic connections and development of the adult neuronal network. Fig.l The different actions of GABA and AMPA receptors in developing neurons.(A)The equilibrium between excitation (glutamate-releasing synapses) and inhibition (GABA-releasing synapses) in the adult mammalian brain (B) The disequilibrium between glutamate-mediated excitation and GABA-mediated inhibition.The activation of GABAA receptors in neonatal slices generated a depolarization that was sufficient to remove the voltage-dependent Mg2+block of NMDA receptors. Several experiments in vitro confirm that a conversion of "silent" glutamatergic synapses to conductive AMPA synapses starts gradually after P2in the CA3-CA1pyramidal neurons.Isoflurane, one of the most widely used inhalation anesthetics for various fetal surgeries or procedures in pregnant patients, has been shown previously to induce caspase activation and apoptosis when applied in concentration in the immature brain. Recently, it was reported that isoflurane might induce apoptosis by increasing [Ca2+]i. Isoflurane has been shown to potentiate GABAA receptor current and directly open GABAA receptor and glycine receptor channels even in the absence of agonist. Isoflurane-mediated activation of GABAA receptors depolarizes the cells, facilitation of NMDA receptors and opens VDCC, elevates [Ca2+]i, and promotes the activation of caspase-3. However, up until recently, the exact mechanism to induce apoptosis remains enigmatic.Fig.2During development, when the GABAA receptors are activated, there is an efflux of chloride, leading to a depolarization that can remove the voltage-dependent Mg2+block from NMDA receptors, generate sodium and calcium action potentials, directly activates VDCC and elevates the [Ca2+]i, leads further CICR from the ryanodine-sensitizing Ca2+store in the hippocampus and in other brain structures. The influx of GABA-induced Ca2+through extracellular medium linked to the binding of Ca2+to CaM and the rapid translocation of CaM to the cell nucleus. This process leads to the activation of the transcription factors, CREB. When activated, CREB promoted the transcription of a set of genes, such as the expression of c-fos, BDNF, PSD-95, synapsinl, Bcl-2and so on, which regulate expression of axon growth and synaptic plasticity in the developing hippocampal neurons and results in the stimulation of new gene expressionIn the present research, we evaluated the effect of clinically relevant concentrations of isoflurane on electrophysiological properties and gene expression in developing rat hippocampal neurons. We tested the following hypotheses:Ⅰ isoflurane could affect neuronal electrophysiological function in the immature brain; Ⅱ clinically relevant concentrations of volatile anesthetic isoflurane have no effect on the neuronal apoptosis in short-term; Ⅲ Bidirectional effect of isoflurane on regulation of neuronal development in the immature brain Methods and Results1. Isoflurane potentiates IGABA in primary rat hippocampus neuronal culture at day in vitro (DIV)5Methods:The hippocampi were dissected from newborn Sprague-Dawley rats and primary hippocampal neuronal cultures of5days in vitro were used. The peak of IGABA was recorded by means of the whole cell patch clamp technique with use of EPC10exposed to isoflurane. To further explore the mechanism of the potentiation effect of isoflurane on IGABA. bicuculline was employed.Results:GABA evoked inward current (IGABA), the EC50was23.73μmol/L, bicuculline blocked IGABA; isoflurane dose-dependent increase IGABA, the EC50was23.73μmol/L.2. Isoflurane potentiates INMDA in primary rat hippocampus neuronal culture at DIV5Methods:The hippocampi were dissected from newborn Sprague-Dawley rats and primary hippocampal neuronal cultures of5days in vitro were used. The peak of INMDA was recorded by means of the whole cell patch clamp technique with use of EPC10exposed to isoflurane. To further explore the mechanism of the potentiation effect of isoflurane on INMDA, bicuculline and MK801were employed.Results:Isoflurane (0.25,0.5,0.75and1MAC) potentiated INMDA peak current amplitude by116±8.8,122±11.7,135±14.3and132±14.6%, respectively. Application of bicuculline partially inhibited the amplitude of isoflurane-induced potentiation of INMDA (P<0.05), and application of MK801completely inhibited the amplitude of isoflurane-induced potentiation of INMDA (P<0.05)3. Isoflurane potentiates IVDCC in primary rat hippocampus neuronal culture at DIV5Methods:The hippocampi were dissected from newborn Sprague-Dawley rats and primary hippocampal neuronal cultures of5days in vitro were used. The peak of INMDA was recorded by means of the whole cell patch clamp technique with use of EPC10exposed to isoflurane. To further explore the mechanism of the potentiation effect of isoflurane on INMDA, bicuculline and nicardipine were employed.Results:Isoflurane (0.25,0.5,0.75and1MAC) potentiated IVDCC peak current amplitude by109.11±9.03,120.56±11.46,141.33±13.87and146.78±15.87%, respectively. pplication of nicadipine partially inhibited the amplitude of IVDCC to the63.11±8.48%of control (P<0.001) and application of nicadipine inhibited the amplitude of isoflurane-mediated IVDCC to the79.33±7.57%of control, but application of bicuculline had no effect on the amplitude of IVDCC (P>0.05) and application of bicuculline inhibited the amplitude of isoflurane-mediated IVDCC from146.78±15.87%to116.56±10.31%of control4. Isoflurane potentiates GABA-triggered [Ca2+]i transient in primary rat hippocampus neuronal culture at DIV5Methods:The hippocampi were dissected from newborn Sprague-Dawley rats and primary hippocampal neuronal cultures of5days in vitro were used.[Ca2+]i was monitored using Fluo-4AM fluorescence imaging. To evaluate whether intracellular calcium release was involved in the potentiation effect of isoflurane, Nicardipine and Dantrolene was employed.Results:The enhancement effect of isoflurane was in a concentration-dependent manner. The EC50for enhancement effect was0.61±0.05MAC. Consistent with previous reports, dantrolene significantly decreased the GABA-triggered [Ca2+]i responses of control (P<0.001, n=11). Furthermore, preconditioning with dantrolene significantly decreased the potentiation of GAB A induced [Ca2+]i transients mediated by1MAC isoflurane in developing hippocampal neurons. Pre-application of nicadipine partially inhibited GABA-tiggered [Ca2+]i transients of control (P<0.001) and isoflurane-mediated potentiation of the GABA-evoked [Ca2+]i rise of GABA (P=0.001).5. Effect of isoflurane on caspase-3levels in primary rat hippocampus neuronal culture at DIV5Method:The hippocampi were dissected from newborn Sprague-Dawley rats and primary hippocampal neuronal cultures of5days in vitro were used. Taking into account that activation of caspase-3is regulated by high [Ca2+]i, the possibility of increased caspase-3 levels after isoflurane in dose-and time-dependent manner was evaluated.Results:The effect of treatments with isoflurane on caspase-3activity was dose-and time-dependent, reaching a maximal caspase activity after1MAC of6-h stimulation (P <0.05). Hippocampal caspase-3mRNA levels began to be significantly increased in isoflurane-treated cultured rat hippocampal neurons after0.25MAC of6-h stimulation (P<0.001).6. Effect of isoflurane on the levels of cytochrome C in primary rat hippocampus neuronal culture at DIV5Method:The hippocampi were dissected from newborn Sprague-Dawley rats and primary hippocampal neuronal cultures of5days in vitro were used. Taking into account that activation of cytochrome C is regulated by high [Ca2+]i, the possibility of increased caspase-3levels after isoflurane in dose-and time-dependent manner was evaluated.Results:The effect of treatments with isoflurane on cytochrome C activity was dose-and time-dependent, reaching a maximal caspase activity after1MAC of6-h stimulation (P <0.05). Hippocampal cytochrome C mRNA levels began to be significantly increased in isoflurane-treated cultured rat hippocampal neurons after0.25MAC of6-h stimulation (P<0.05).7. Effect of isoflurane on the levels of transcriptional factor CREB in primary rat hippocampus neuronal culture at DIV5Method:The hippocampi were dissected from newborn Sprague-Dawley rats and primary hippocampal neuronal cultures of5days in vitro were used. Real-time-PCR was used to detect the expressions of the CREB signaling pathway (GAP-43mRNA, PSD-95mRNA, CREB mRNA, BDNF mRNA and Bcl-2mRNA) after intervention.Results:Compared with the group C, isoflurane increased in neuronal GAP-43mRNA, PSD-95mRNA, CREB mRNA, BDNF mRNA and Bcl-2mRNA expression in dose-dependent manner (P<0.05).8. Effect of isoflurane on the levels of hippocampal transcriptional factor MEF2in primary rat hippocampus neuronal culture at DIV5Method:The hippocampi were dissected from newborn Sprague-Dawley rats and primary hippocampal neuronal cultures of5days in vitro were used. Real-time-PCR was used to detect the expressions of the MEF2signaling pathway (MEF2mRNA,synGAPI mRNA and Arc mRNA) and Synapsin I mRNA after intervention. Western blot was used to detect the expressions of Synapsin I at the protein level.Results:Compared with the group C, the expressions of MEF2mRNA, synGAPI mRNA, Arc mRNA and Synapsin I mRNA of the neurons from isoflurane groups all increased statistically (P<0.05)9. Effect of Isoflurane on the development of hippocampal neurons apoptosis in vivoMethods:Forty5-day-old rats which were administrated in relatively airtight container were randomly divided into five groups, treatment groups which received1MAC isoflurane6h. And a control group which received no treatment. Pups were killed by decapitation to extract total mRNA after intervention. Real-tine PCR was used to detect the expressions of caspase-3.Results:Compared with the group C, isoflurane increased in neuronal caspase-3expression (P<0.05)10. Effect of isoflurane on the levels of hippocampal transcriptional factor MEF2in vivoMethods:Forty5-day-old rats which were administrated in relatively airtight container were randomly divided into five groups, treatment groups which received1MAC isoflurane2,4,6h, respectively. And a control group which received no treatment. Pups were killed by decapitation to extract total RNA from hippocampal neurons after intervention. Real-time-PCR was used to detect the expressions of the MEF2signaling pathway (MEF2mRNA,synGAPI mRNA and Arc mRNA) and SynapsinI mRNA after intervention. Western Blot was used to detect the expressions of synapsinl at the protein level. Results:Compared with the group C, the expressions of MEF2mRNA, synGAPI mRNA, Arc mRNA and SynapsinI mRNA of the neurons from isoflurane groups all increased statistically (P<0.05)Main Results1. Isofluane-induced developmental neurotoxicity in developing hippocampal neurons was confirmed by researches in vitro (5-day cultured primary neurons) and in vivo (whole animal experiments) through two aspects of survival and neurogenesis of neurons, neurotoxicity.2. Isoflurane-mediated activation of GABAA receptors depolarizes the cells, facilitates NMDA receptors and opens VDCC, elevates [Ca2+]i. Effects of isoflurane on MEF2and CREB signal pathway were confirmed both in vitro and in vivo.3. From the investigation of the effect of isoflurane on the synaptic plasticity in developing hippocampal neurons, we can draw a conclusion that bidirectional effects of isoflurane on regulation of neuronal development in the immature brain. ConclusionsClinical relevant concentrations of inhaled anesthetics isoflurane-mediated potentiation of GABA-triggered [Ca2+]i release results from membrane depolarization with subsequent activation of VDCC and further CICR from the ryanodine-sensitizing Ca2+store. An increase in [Ca2+]i, due to activation of GABAA receptor, facilitation of NMDA receptor and opening of VDCC, is necessary for isoflurane-induced calcium overload of immature rat hippocampal neurons, which may be involved in the mechanism of an isoflurane-induced neurotoxic effect in the developing rodent brain.