With the increasing sophistication of surgical and anesthetic techniques, procedures on patients who are pregnant and fetal procedures are increasingly being performed.1,2 A study3 that found that the inhaled anesthetic isoflurane causes neurotoxicity in the immature brain aroused widespread social concern. Several experiments suggested that volatile anesthetics cause excitotoxicity in a concentration- and time-dependent manner in neuronal models in vitro.3,4 Neurons in the developing brain are specifically vulnerable to isoflurane hyperexcitability5,6; however, the mechanisms for isoflurane cytotoxicity remain enigmatic. Because most fetal procedures in humans are performed during midgestation, it is important and urgent to know whether the anesthetics used cause damage to the developing brain and subsequent postnatal memory problems and learning disabilities.1,4,7
Developing neurons exhibit some characteristics differently from those of adult neurons. One of the most prominent features of the immature brain is γ-aminobutyric acid (GABA)-evoked excitability.8–10 In contrast to its inhibitory action on adult neurons, GABA acts as an excitatory neurotransmitter and regulates brain morphogenesis during neuronal development, such as changes in neuronal growth, neuronal differentiation, proliferation and migration, growth rates of neuronal processes, and synapse formation and clustering.11–14 GABA mediates these processes by activation of traditional ionotropic (GABAA) and metabotropic (GABAB) receptors, and probably by both synaptic and nonsynaptic mechanisms.15 Activation of GABAergic synapses generates action potentials, directly activates voltage-dependent calcium channels (VDCCs), and increases the [Ca2+]i in the hippocampus and in other brain structures.8,16 These effects may underlie the well-characterized modulation by GABA of activity-dependent developmental processes. These highly correlated calcium signals are thought to be essential for consolidation of synaptic connections and development of the adult neuronal network. This suggests that at an early developmental stage, GABAergic synapses may provide all the information received by immature pyramidal neurons.9,17 However, overactivation of the GABAA receptor effects activation of VDCCs and produces large Ca2+ increases throughout neurons. Excessive Ca2+ influx is a critical factor in excitotoxic cell damage.18,19 Overactivation of the GABAA receptor may potentially exacerbate damage caused by neuronal swelling in this stage of neurodevelopment.
Isoflurane, one of the most widely used inhaled anesthetics for fetal procedures or procedures in pregnant patients, has been shown to induce caspase activation and apoptosis when administered in a clinically relevant concentration in the immature brain.6,20,21 It has been reported that isoflurane might induce apoptosis by increasing [Ca2+]i. Isoflurane has been shown to enhance GABAA receptor current and directly open GABAA receptor channels even in the absence of agonist.22 Isoflurane-mediated activation of GABAA receptors depolarizes the cells, opens VDCCs, increases [Ca2+]i, and promotes the activation of caspase-3.23 However, until recently, the exact mechanism to increase cytosolic-free calcium levels was unknown. In the present research, we evaluated the effect of clinically relevant concentrations of the volatile anesthetic isoflurane on the free calcium level in developing rat hippocampal neurons in vitro (5-day culture).
The use of Sprague-Dawley pregnant rats for primary hippocampal neuronal culture was approved by the Animal Welfare Committee of Huazhong University of Science and Technology. Primary cultures of hippocampal neurons were prepared as described in our previous studies.6,24 Hippocampi were dissected from the rat fetus brain at embryonic day 18. Blood vessels and meninges were removed from the tissues. The cells were dissociated by 0.125% trypsinization and trituration and resuspended in attachment medium (Dulbecco's modified Eagle medium [Invitrogen, Carlsbad, CA] with 10% heat-inactivated fetal bovine serum, 25 mM glutamate, 0.5 mM glutamine, penicillin [100 U/mL], streptomycin [0.1 mg/mL], and amphotericin B [0.1 mg/mL]). The day after plating, the culture medium was replaced by a serum-free neurobasal medium containing the B27 supplement, 2 mM glutamine, penicillin (100 U/mL), streptomycin (0.2 mg/mL), and amphotericin B (0.2 mg/mL), and 48 hours after plating, cytarabine (5 mM) was added to the medium to inhibit glial growth. Neurons grew in a humidified atmosphere with 5% CO2, 95% air at 37°C, and half of the volume of the culture medium was replaced with a fresh neurobasal medium every 2 days. Experiments were performed on day 5 of culture.
The procedure for calcium imaging was as described in our previous studies with minor modification.6,25 For [Ca2+]i measurements, 5-day–cultured rat hippocampal neurons were rinsed 3 times with artificial cerebrospinal fluid containing the following (in mM): 150 NaCl, 5 KCl, 2 CaCl2, 0.4 MgCl2, 25 D-glucose, and 25 HEPES pH 7.4. Neurons were preloaded for 30 minutes at 37°C with 4 mM Fluo-4 AM in the buffer, followed by 3 washes and a 15-minute incubation period for further deesterification of Fluo-4 AM before calcium imaging. Cells were placed in a microscope chamber for fluorescence measurements with a laser scanning confocal microscope FV500 system (Olympus, Tokyo, Japan). Fluoview tiempo time course software (Olympus) was used for hardware control, image acquisition, and image analysis. Isoflurane, GABA, nicardipine, and dantrolene were dissolved in the buffer. Isoflurane was introduced into the perfusion buffers (500 mL/min) by prebubbling the buffer for 20 minutes with an air stream (95% air, 5% CO2) containing the desired anesthetic concentration using calibrated vaporizers (Dräger, Lübeck, Germany). The concentration of the anesthetic in the buffer was quantified by gas chromatography (Agilent 6890N, HP; Santa Clara, CA), which, in this study, was subject to subclinical and clinically relevant concentrations (0.25–1 minimum alveolar concentration [MAC]), assuming that 1 MAC isoflurane for rats corresponded to 1.46 vol% or an aqueous concentration of 350 mM.26 Fluorescence signals were plotted as F/F0, with F as fluorescence during the experiment, and F0 the initial level of fluorescence.
The procedure for whole-cell patch-clamp recording was as described in previous studies.27,28 The bath solution for recording IVDCC contained the following (in mM): choline-Cl 110, MgCl2 2, CaCl2 10, TEA-Cl 20, HEPES 10, glucose 20, and the pH was adjusted to 7.4 with CsOH. The composition of the internal solution contained the following (in mM): CsF 64, CsCl 64, CaCl2 0.1, MgCl2 2, EGTA 10.0, HEPES 10.0, Tris-ATP 5.0, and the pH was adjusted to 7.2 with CsOH. The osmolality was adjusted to approximately 300 mOsm. Glass pipettes were used with a resistance of approximately 2 to 5 MΩ. The current signals were acquired at a sampling rate of 10 kHz and filtered at 2 kHz. Whole-cell recordings were performed using an EPC-10 amplifier (HEKA, Lambrecht/Pfalz, Germany) driven by Pulse/PulseFit software (HEKA).
Measurements of Caspase-3 mRNA Level with Quantitative Real-Time Polymerase Chain Reaction
Total RNA from control or isoflurane-treated, 5-day–cultured neurons was extracted with Trizol™ (Takara, Shiga, Japan) following the manufacturer's instructions. Two micrograms RNA was reversely transcribed using random primers and Superscript II Reverse Transcriptase (Takara) following the supplier's instructions. The resulting cDNA was diluted 1:40 and used as a quantitative polymerase chain reaction (qPCR) template. qPCRs were conducted in an ABI Stepone version 2.0 (Applied Biosystems, Foster City, CA) using Power SYBR™ Green PCR Master Mix (Takara) and specific primers for caspase-3 (forward: 5′-GCAGCAGCCTCAAATTGTTGACTA-3′; and reverse: 5′-TGCTCCGGCTCAAACCATC-3′) and for the normalizing control GAPDH (forward: 5′-GGCACAGTCAAGGCTGAGAATG-3′; and reverse: 5′-ATGGTGGTGAAGACGCCAGTA-3′). The amplification efficiency of caspase-3 primers was similar to GAPDH primers. Each sample was run in triplicate and data were analyzed using Sequence Detection Software version 3.0 (Applied Biosystems). Data are expressed as a normalized percentage of control groups.
Western Blot Analysis
To analyze the variation in protein levels, cell total proteins were obtained as described in previous studies.29 Proteins from the 5-day–cultured rat hippocampal neurons were separated by 12% gel electrophoresis, and proteins were transferred to a nitrocellulose membrane. The blots were incubated with a monoclonal antibody against cleaved caspase-3 (Cell Signaling Technology, Danvers, MA) at 1:1000 dilution and β-actin (Cell Signaling Technology) at 1:1000 dilution and then probed with horseradish peroxidase–conjugated secondary antibody at 1:8000 dilution, followed by enhanced chemiluminescent detection to determine cell apoptosis in hippocampus after anesthesia treatments. Detection was performed by using the ECL-PLUS system and photographed. Western blots were from 3 different animals, and the density was measured using Quantity One 4.62 software (Bio-Rad, Ontario, Canada) and a GS-800 Densitometer (Bio-Rad).
Data from at least 3 dishes from different batches of cultured rat hippocampal neurons were pooled together and analyzed for statistical differences using the Student t test or 1-way analysis of variance followed by the Tukey post hoc test. Compiled data are expressed and graphed as mean ± SEM, with n denoting the number of neurons studied for each treatment. Differences were considered significant if a P value was <0.05.
Isoflurane Enhances GABA-Triggered [Ca2+]i Transient in Developing Rat Hippocampal Neurons
To determine the effects of isoflurane on [Ca2+]i, we first characterized the [Ca2+]i, responses triggered by the GABA depolarization as control. A rapid change in the concentration of 50 μM GABA (approximately 2 seconds) with triggered transient [Ca2+]i increased in the majority of tested neurons (n = 31). Repeated GABA stimulation produced equal [Ca2+]i responses and these responses were fully reversed after 300-second washout with normal buffer (Fig. 1A). GABA and isoflurane were added to the cells with a linear barrel array made of fused silica tubes. On average, 1 MAC isoflurane enhanced the GABA-triggered [Ca2+]i transients to 142.33% ± 13.19% of control (P < 0.001, n = 11). After a 300-second washout period, the third GABA-mediated [Ca2+]i response was equal to the first (Fig. 1B). The enhancement effect of isoflurane was in a concentration-dependent manner (Fig. 1C). The 50% effective concentration for enhancement effect was 0.61 ± 0.05 MAC.
The Isoflurane-Mediated Enhancement of GABA-Evoked [Ca2+]i Response Is Mediated by Calcium-Induced Calcium Release via Ryanodine Receptors
We used dantrolene to evaluate whether intracellular calcium release was involved in the enhancement effect of isoflurane. Consistent with previous reports, dantrolene significantly decreased the GABA-triggered [Ca2+]i responses to 67.43% ± 7.74% of control (P < 0.001, n = 11). Furthermore, preconditioning with dantrolene significantly decreased the enhancement of GABA-induced [Ca2+]i transients mediated by 1 MAC isoflurane in developing hippocampal neurons (Fig. 2, A and B).
Caffeine was used to further investigate the effect of isoflurane on calcium-induced calcium release (CICR) in cultured rat hippocampal neurons on day 5 of culture in a Ca2+-free extracellular solution by external application of 3 mM EGTA. In the absence of extracellular Ca2+ medium, repeated stimulations with caffeine triggered equal [Ca2+]i responses when the interval between applications was >300 seconds (Fig. 3A). As shown in Figure 3B, in the Ca2+-free medium, treatment with 1 MAC isoflurane had no significant effect on the caffeine-mediated [Ca2+]i transients of control (P = 0.77, n = 7). After a 300-second washout period, the third caffeine-induced [Ca2+]i transient was equal to the first.
The Isoflurane-Mediated Enhancement of GABA-Evoked [Ca2+]i Increase Is Attributed to the Influx of Extracellular Calcium via Opening of VDCCs in Response to Membrane Depolarization
To evaluate whether isoflurane-mediated enhancement of the GABA-triggered [Ca2+]i increase is dependent on extracellular Ca2+, recording neurons were exposed to the Ca2+-free medium. As shown in Figure 4, A and B, this resulted in complete suppression of the isoflurane-mediated enhancement of the GABA-evoked [Ca2+]i increase to 7.83% ± 1.02% of control (P < 0.001, n = 9). The suppression phenomenon rapidly recovered on returning the neurons to the standard extracellular solution.
To further evaluate whether isoflurane mediated enhancement of the GABA-evoked [Ca2+]i increase via VDCCs, nicardipine was used to block L-type VDCCs. As shown in Figure 4B, preapplication of nicardipine partially inhibited GABA-triggered [Ca2+]i transients to 72.22% ± 8.41% of control (P < 0.001, n = 9) and isoflurane-mediated enhancement of the GABA-evoked [Ca2+]i increase to 80.67% ± 7.92% of GABA (P = 0.001, n = 9).
To confirm the role of extracellular Ca2+ influx via VDCCs in the enhancement effect of isoflurane, we used whole-cell patch-clamp recoding technology to assess the effect of isoflurane on IVDCC elicited by a depolarizing pulse in cultured rat hippocampal neurons. As illustrated in Figure 5, A and D, preconditioning the cell with 1 MAC isoflurane showed significant enhancement on the amplitude of IVDCC. As shown in Figure 5, B and C, preconditioning with isoflurane increased the amplitude of the I-V curve for IVDCC in the isolated neurons from rat hippocampus by a series of depolarization pulses. Isoflurane (0.25, 0.5, 0.75, and 1 MAC) enhanced IVDCC peak current amplitude by 109.11% ± 9.03%, 120.56% ± 11.46%, 141.33% ± 13.87%, and 146.78% ± 15.87%, respectively.
Nicardipine and bicuculline were used to further explore the mechanism of the enhancing effect of isoflurane on IVDCC. As shown in Figure 6, A to C, application of nicardipine partially inhibited the amplitude of IVDCC to 63.11% ± 8.48% of control (P < 0.001, n = 9) and application of nicardipine inhibited the amplitude of isoflurane-mediated IVDCC to 79.33% ± 7.57% of control. However, application of bicuculline had no effect on the amplitude of IVDCC (P = 0.88, n = 9) and application of bicuculline inhibited the amplitude of isoflurane-mediated IVDCC from 146.78% ± 15.87% to 116.56% ± 10.31% of control (P < 0.001, n = 9).
Effect of Isoflurane on Hippocampal Caspase-3 Levels
Considering that activation of caspase-3 is regulated by high [Ca2+]i, the possibility of increased caspase-3 levels after isoflurane in a dose- and time-dependent manner was evaluated. Western blot analysis, using anticaspase-3–specific antibody, revealed 0.25, 0.5, 0.75, and 1 MAC after 6-hour isoflurane treatment. As shown in Figure 7, A and B, the effect of treatments with isoflurane on caspase-3 activity was dose- and time-dependent, reaching maximal caspase activity after 1 MAC of 6-hour stimulation (P < 0.001, n = 3).
The importance of caspase-3 to the plasticity of developing neurons is well documented. Interestingly, independent studies have demonstrated that massive numbers of neurons may die during development when the nervous system is growing and progressing toward its mature state. Apoptosis has a central role in developing brain plasticity.30,31 For this reason, we also used real-time qPCR to determine the expression of caspase-3 mRNA after application of 0.25, 0.5, 0.75, and 1 MAC isoflurane for 6 hours. As shown in Figure 7C, the caspase-3 mRNA levels increased significantly in 5-day–cultured rat hippocampal neurons after 0.25 MAC isoflurane of 6-hour stimulation (P < 0.001, n = 3), which was also confirmed by Western blot analysis (Fig. 7, A and B).
To further explore the mechanism of isoflurane-induced neurotoxicity, we used bicuculline, nicardipine, and dantrolene. As illustrated in Figure 8, A and B, 1 MAC isoflurane-induced apoptosis could be significantly inhibited by bicuculline, nicardipine, and dantrolene. Our results further confirm isoflurane's enhancement on a GABA-triggered [Ca2+]i increase by activation of the GABAA receptor, depolarization of plasma membrane, and subsequent activation of VDCCs and CICR through ryanodine receptors. As shown in Figure 8C, hippocampal caspase-3 mRNA levels were significantly increased in preapplication of bicuculline in developing rat hippocampal neurons after 6 hours of exposure to 1 MAC isoflurane (P = 0.004, n = 3), an effect that agrees with the view that volatile anesthetics produce immobility via a multitude of ion channels.
The results described herein strongly suggest that isoflurane through GABAA receptor-mediated depolarizing stimuli activates Ca2+ entry through VDCCs. Ca2+ entry also stimulates Ca2+ release from intracellular stores, resulting in amplification and propagation of the initial Ca2+ entry signal via CICR.27,28 An increase in [Ca2+]i is a critical factor in excitotoxic cell damage, even leading to caspase activation and apoptosis.19,23 This response was partially attenuated by dantrolene and nicardipine, whereas exposure of recording neurons to a Ca2+-free extracellular solution completely prevented this [Ca2+]i response, suggesting that the enhancement was attributed to the enhancement of Ca2+ entry via VDCCs. However, our results showed that the inhibition of the ryanodine receptor by dantrolene and inhibition of L-type VDCCs by nicardipine did not completely abolish the [Ca2+]i increase induced by GABA and isoflurane (Figs. 2 and 4). In addition to GABAA receptors, L-type VDCCs, and ryanodine receptors as demonstrated in our study, the roles of the glycine receptors, the N-methyl-D-aspartate receptors, and the InsP3 receptors must also be considered in determining the precise mechanism underlying the [Ca2+]i increase induced by GABA and isoflurane. Our results are in line with the view that volatile anesthetics produce immobility via a multitude of ion channels including GABAA, glycine and glutamate receptors, and VDCCs.6,32–36
In parallel with these findings, isoflurane failed to affect the [Ca2+]i response mediated by a lower concentration of caffeine in Ca2+-free extracellular solution. Caffeine is a specific agent that acts on the ryanodine receptor and could directly induce [Ca2+]i release from the ryanodine-sensitizing Ca2+ store in the absence of Ca2+ medium.28,37 Therefore, this provides further possible evidence for the VDCC-facilitating action of isoflurane. It is widely accepted that VDCCs have a pivotal role in regulating various neuronal functions. Because calcium entry through VDCCs is essential for control of membrane excitability, synaptic function, cellular homeostasis, gene transcription, and development and growth, isoflurane-sensitive stimulation of VDCCs would be responsible for the increased excitability in developing neurons after isoflurane application.27,38 We found that isoflurane increases IVDCC in cultured rat hippocampal neurons, which, at least in part, contributed to the regulation of neuron activity by isoflurane, which may have a more significant role in the modulation of membrane potential and firing rates.
Moreover, using whole-cell patch-clamp recording, isoflurane enhanced calcium currents generated by a depolarization pulse. This result further confirmed that VDCCs had a pivotal role in the enhancing effect of isoflurane. In this study, isoflurane directly increased the amplitude of IVDCC of developing rat hippocampal neurons in a dose-dependent manner. Our work provided direct electrophysiological evidence for mechanisms underlying the effects of isoflurane on cultured rat hippocampal neurons. Although Study39 found that isoflurane inhibits multiple voltage-gated calcium currents in hippocampal pyramidal neurons, including IHVA and ILVA, the reason may be that the neurons were prepared from postnatal day 13 to 18 rats, which are physiologically more mature22 than the neurons from the embryonic day 18 rats we used. The properties of immature synapses can be very different from those of adult synapses. One of the most prominent features of the immature neurons was that the GABAA receptors, activated by their agonist, depolarize membrane voltage, activate VDCCs, and increase [Ca2+]i in neonatal neurons compared with the hyperpolarizing response in adult neurons.8,40 In our study, the application of the GABAA receptor antagonist bicuculline led to a significant decrease in the amplitude of IVDCC. This finding indicates a major role for isoflurane-mediated GABAA receptors in the increase of [Ca2+]i in 5-day–cultured rat hippocampal neurons. Tampo et al.33 demonstrated that anesthetic-induced preconditioning triggered persistent changes in the inactivation of cardiac L-type calcium channels, which was different from our findings. Previous studies in in vitro rats have shown that at low and moderate levels of anesthesia with isoflurane (1.0%–1.4%), VDCCs are still functionally active and capable of producing burst firing.41 In parallel with our results, at subanesthetic concentrations, activation of GABAA receptors would sufficiently depolarize neurons, which would relieve the state-dependent inhibition of VDCCs by isoflurane.
According to the suggestion that the increase of cytosolic-free calcium levels is the major risk factor for isoflurane-induced apoptosis in neonatal brain, we presume isoflurane, when applied in the neonatal brain, may induce apoptosis in a concentration-dependent manner.9,18,23,42 It has been estimated that a prolonged increase in [Ca2+]i and alterations in calcium homeostasis initiate the mitochondrial apoptotic pathway and induce endoplasmic reticulum stress that, in turn, leads to apoptosis.19,43 In particular, caspases-3 has a relevant role in apoptosis, which is an executioner caspase activated by multiple pathways.44,45 Animal studies have shown that apoptotic neurodegeneration triggered by isoflurane in the developing rodent brain is associated with an increase of cleaved caspase-3.46 To further investigate the neurotoxicity of high cytosolic free calcium after isoflurane in a dose-dependent manner, the possibility of increased caspase-3 levels was evaluated. We present evidence that exposure to the inhaled anesthetic isoflurane resulted in a dose-dependent enhancement of mRNA caspase-3 expression. Analysis of protein levels for cleaved caspase-3 provided evidence that only exposure to 1 MAC isoflurane leads to upregulation of cleaved caspase-3 in areas with pronounced apoptotic cell death, which could be significantly inhibited by bicuculline, nicardipine, and dantrolene. These results are clinically relevant because they show that exposure to isoflurane in a high-dose and long-term manner is potentially dangerous during brain development.
In summary, isoflurane facilitates the GABA-evoked [Ca2+]i increase attributed to the enhancement of Ca2+ entry via VDCCs in hippocampal neurons. Furthermore, Ca2+ entry stimulates Ca2+ release from intracellular stores, resulting in amplification and propagation of the initial Ca2+ entry signal via CICR. An increase in [Ca2+]i is a critical factor in excitotoxic cell damage, even leading to caspase activation and apoptosis. The regulating action of isoflurane on high [Ca2+]i has a crucial role in the regulation of caspase-3 transcription. However, our data suggest that only high-dose and long-term exposure to isoflurane results in potential dangers during brain development, which provides a rationale for further investigations into its mechanism of inhaled anesthetic excitotoxicity in embryo surgery and early postnatal life surgery.
Name: Y. L. Zhao, MD.
Contribution: Data collection and the study records.
Name: Q. Xiang, MD.
Contribution: Data collection and data analysis.
Name: Q. Y. Shi, MD.
Contribution: Data collection and manuscript preparation.
Name: S. Y. Li, MD.
Contribution: Data analysis and manuscript preparation.
Name: L. Tan, MD.
Contribution: Data analysis and manuscript preparation.
Name: J. T. Wang, MD.
Contribution: Data collection and data analysis.
Name: X. G. Jin, MD.
Contribution: Data analysis and manuscript preparation.
Name: A. L. Luo, MD, PhD.
Contribution: Study design, conduct of the study, and manuscript preparation.
This manuscript was handled by: Gregory J. Crosby, MD.
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