Abstract

Background:

Anesthetic neurotoxicity in the developing brain of rodents and primates has raised concern. Xenon may be a nonneurotoxic alternative to halogenated anesthetics, but its toxicity has only been studied at low concentrations, where neuroprotective effects predominate in animal models. An equipotent comparison of xenon and halogenated anesthetics with respect to neurotoxicity in developing neurons has not been made.

Methods:

Organotypic hippocampal cultures from 7-day-old rats were exposed to 0.75, 1, and 2 minimum alveolar concentrations (MAC) partial pressures (60% xenon at 1.2, 2.67, and 3.67 atm; isoflurane at 1.4, 1.9, and 3.8%; and sevoflurane at 3.4 and 6.8%) for 6 h, at atmospheric pressure or in a pressure chamber. Cell death was assessed 24 h later with fluorojade and fluorescent dye exclusion techniques.

Results:

Xenon caused death of hippocampal neurons in CA1, CA3, and dentate regions after 1 and 2 MAC exposures, but not at 0.75 MAC. At 1 MAC, xenon increased cell death 40% above baseline (P < 0.01; ANOVA with Dunnett test). Both isoflurane and sevoflurane increased neuron death at 1 but not 2 MAC. At 1 MAC, the increase in cell death compared with controls was 63% with isoflurane and 90% with sevoflurane (both P < 0.001). Pretreatment of cultures with isoflurane (0.75 MAC) reduced neuron death after 1 MAC xenon, isoflurane, and sevoflurane.

Conclusion:

Xenon causes neuronal cell death in an in vitro model of the developing rodent brain at 1 MAC, as does isoflurane and sevoflurane at similarly potent concentrations. Preconditioning with a subtoxic dose of isoflurane eliminates this toxicity.

What We Already Know about This Topic
  • Xenon exhibits experimental neuroprotective properties at subanesthetic concentrations in neonatal rats

  • However, whether it causes apoptotic cell death at clinically relevant concentrations (as volatile anesthetics do) remains unknown

What This Article Tells Us That Is New
  • Using postnatal rat hippocampal slice cultures, the authors demonstrated that xenon used at 1 minimum alveolar concentration-equivalent concentration, increased apoptosis similar to sevoflurane and isoflurane at equipotent concentrations, and that this effect was abolished by preconditioning the tissue with a subtoxic concentration of isoflurane

A LINK between halogenated inhalational anesthetics and neuronal apoptosis in the developing brain has raised concerns about the safety of anesthesia in young patients.1,2  Clinically relevant doses of isoflurane and sevoflurane have both been shown to cause neuronal cell death in developing rats and mice, in both in vivo and in vitro models.3–5  However, there is also strong evidence that isoflurane and sevoflurane can be neuroprotective in the developing or mature brain, when administered before or during damaging insults involving stresses of oxygen and glucose deprivation,6  hypoxia,7  profound hypothermia,8  and even toxic anesthetic exposures.9 

The neuroprotective properties of xenon, another inhalational anesthetic, have also been widely studied in experiments involving stresses such as oxygen–glucose deprivation,10,11  hypothermia,8  and neuroapoptosis-inducing exposures to isoflurane.12  In these, and other similar studies in rodents, xenon was administered at doses between 10 and 75% of an atmosphere, equivalent to a minimum alveolar concentration (MAC) of approximately 0.06–0.79 MAC, based on xenon MAC values of 1.6 atm in rats, and 0.95 atm in mice.13,14  The neuroprotective properties of xenon at 1 MAC or greater in rodents have not been evaluated.

Although xenon clearly possesses neuroprotective qualities at sub-MAC concentrations (as do most halogenated anesthetics),15  it is unknown whether xenon is similar to halogenated anesthetics with respect to causing neuroapoptosis at 1 MAC or greater concentrations in the developing rodent brain. The question, therefore, remains whether xenon has the same potential for neurotoxicity as isoflurane and sevoflurane in the developing brain at equipotent MAC concentrations.

The MAC of xenon in humans is approximately 70%.16  Although studies in developing rats involving xenon at concentrations approximately 70% (only ~0.44 MAC)13,14  have not found significant neuron death, in vitro neuron survival studies with xenon at MAC and above have not been done. The lack of data on the potential neurotoxicity of xenon at MAC and above, and how xenon compares with other inhalational anesthetics at equipotent doses, was the impetus for this study.

In this study, we performed dose–effect experiments assessing anesthetic-induced cell death after exposure to xenon, isoflurane, and sevoflurane in hippocampal slice cultures prepared from 7-day-old Sprague–Dawley rats. This model is widely used for in vitro anesthetic neuroprotection and neurotoxicity studies. Hippocampal deficits are an important feature of the behavioral effects of halogenated anesthetic neurotoxicity in the developing brain.4  Additionally, isoflurane was employed as a preconditioning agent to determine if the neurotoxicity of xenon is mitigated by pretreatment with a lower concentration of another inhalational anesthetics, as it is known that sub-MAC levels of xenon can be neuroprotective, when administered simultaneously12  or before9  a neurotoxic concentration of isoflurane.

Materials and Methods

The studies were approved by the University of California San Francisco Committee on Animal Research and conform to relevant National Institutes of Health guidelines for the use of animals in research.

Preparation of Hippocampal Slice Cultures

Organotypic cultures of the hippocampus were prepared by standard methods.17  Seven-day-old male and female Sprague–Dawley rats (Charles River Laboratories, Hollister, CA) were decapitated without anesthesia, per University of California, San Francisco animal care guidelines. After decapitation, the hippocampi were quickly removed and placed in 4°C Gey Balanced Salt Solution with 20 mm glucose. The isolated hippocampi were transversely sliced (300–400-µm thick) with a tissue slicer (Siskiyou Design Instruments, Grants Pass, OR), and returned to Gey’s Balanced Salt Solution (University of California at San Francisco Cell Culture Facility, San Francisco, CA). Hippocampal slice cultures were maintained in a slice culture media, which contained 50% Eagle’s Minimum Essential Media, 25% Earle’s Balanced Salt Solution, and 25% horse serum. Twelve to 24 h before the experiments, the horse serum was either dropped down to 5% or replaced entirely with Eagle’s Minimum Essential Media so that experiments were done with low or no serum. Bickler et al.18  described further preparation and handling of cultures.

Approximately 16 slices were harvested from each of the 50 animals sacrificed for the study. Slices were preselected before plating so that only those with normal morphology in the CA1, CA3, and dentate neuron cell body regions were plated. Before the experiments, the slices were shuffled and selected at random for each treatment group to ensure that every group had a selection of slices from multiple animals. This process also ensured a random sample of slices from both males and females in each treatment group. Slices from males and females were mixed and analyzed together because an unpublished study from our laboratory (October 11, 2012, “Impact of gender on hippocampal neurotoxicity caused by isoflurane and sevoflurane”, Heather Brosnan, B.S. and Jeffrey Sall, M.D., Ph.D., San Francisco, CA, poster presentation at The Society for Neuroscience in Anesthesia and Critical Care meeting) investigating cell death after sevoflurane or isoflurane at 1 MAC, found no effects of gender in the hippocampus.

Anesthetic Exposure at 1 atm Total Pressure

Slice cultures were exposed to 0.75, 1, and 2 MAC isoflurane (1.4, 1.9, and 3.8% isoflurane, respectively) and 1 and 2 MAC sevoflurane (3.4 and 6.8% sevoflurane, respectively) by placing them in a 2.0 l Billups-Rothenberg modular incubator chamber (Del Mar, CA). The Billups-Rothenberg chamber was flushed for 5 min by passing humidified 5% CO2/balance air with anesthetic through the opened chamber, at 5.0 l/min for 5 min and then sealed. The concentrations of isoflurane, carbon dioxide, and oxygen in the Billups-Rothenberg chamber were measured using a Datex Ohmeda Gas Analyzer (Datex Instrumentarium Corp., Helsinki, Finland) over the course of 6 h, to confirm that the concentration of gases remained constant in the chamber. Billups-Rothenberg chambers were only used when the concentration of anesthetic did not require pressurization.

Anesthetic Exposures at Greater Than 1 atm Total Pressure

To expose the slice cultures to concentrations of xenon that exceed 1 atm, a 1.55 l hyperbaric chamber was constructed which housed two slice culture dishes. The chamber was built out of polyvinyl chloride, which was permanently sealed at one end and had a removable acrylic plate with a rubber gasket on the other. Once the tissue culture plates were inserted into the chamber, the removable acrylic plate was sealed tightly. A pressure gauge along with two valves allowed the chamber to be flushed with gas and pressurized, as required. At the end of the experiments, the pressure was slowly released from the chamber, to prevent the formation of gas bubbles or damage to the tissue by rapid decompression.

Gas Mixtures Preparation

For all experiments, the pressures of oxygen and carbon dioxide were maintained at 0.21 atm O2 and 0.05 atm CO2. To create the desired concentration of gases, each gas was measured using a glass syringe and its contents were then injected into a 3.0 l calibration syringe (Hans Rudolph, Inc., Shawnee, KS) to create a homogeneous mixture, which could be added to the pressure chamber. The contents of the 3.0 l syringe were first pushed through the pressure chamber with both valves open, to flush the chamber with the desired gas mixture. The pressure chamber was then sealed and pressurized by pumping the contents of the 3.0 l syringe into the chamber via a smaller 60-ml syringe, while monitoring the chamber pressure with a gauge. Pressure-resistant tubing was used to transfer the gas from the syringe to the chamber.

Pressure Controls

Controls were conducted using the pressure chamber to account for any potential damage to the cells caused by total pressures of 2.0, 2.67, and 3.67 atm. A slice culture tray was placed into the chamber and the chamber was pressurized with gas containing, 0.05 atm CO2, 0.21 atm O2, and balance nitrogen at final pressure. The chamber was then placed in a 37°C incubator for 6 h, and the total pressure within the chamber was monitored. For a 1-atm control, a slice culture tray was placed in a Billups-Rothenberg chamber that was flushed with 5% CO2 in air. This chamber was also placed in a 37°C incubator for the duration of the experiment.

Anesthetic Pressure Controls

Although previous research has shown that very high pressures change the potency of volatile anesthetics (in the range of 100 atm,19  25–50 times higher than the pressures in these experiments), we nonetheless, included a control experiment with isoflurane to ensure that the pressures used in this experiment did not affect potential neurotoxicity. To assess whether increased pressure affects cell survival after exposure to isoflurane, slice cultures were exposed to 1.6 MAC (3%) isoflurane at 1 atm and under total pressures of 2.0, 2.67, and 3.67 atm. Pure nitrogen was run through a calibrated isoflurane vaporizer, collected in a 3.0 l calibrating syringe and mixed with oxygen and carbon dioxide, to create a mixture equivalent to 0.03 atm isoflurane, 0.20 atm O2, and 0.05 atm CO2, when pressurized. After pressurization, the hyperbaric chamber was placed in a 37°C incubator for 6 h. The air in the hyperbaric chamber was analyzed with a Datex Ohmeda Gas Analyzer at the end of the 6-h treatment to ensure the concentrations of isoflurane, oxygen, and carbon dioxide had been maintained throughout the duration of the experiment. Another plate of slice cultures was simultaneously placed in a Billups-Rothenberg chamber that was flushed for 5 min with 3% isoflurane and humidified 5% CO2/95% air. The chamber was sealed and placed in a 37°C incubator at atmospheric pressure for 6 h. The slices from both groups were removed from their respective chambers after the 6-h exposure, and placed in a 37°C incubator with 5% CO2 in air (standard culture conditions). Twenty-four hours later, cell death was assessed.

Xenon Treatment Protocol

In all of the xenon toxicity experiments, gas mixtures were made with 60% xenon at 1 atm and the percentage of oxygen, carbon dioxide, and nitrogen were adjusted to maintain the equivalent of 5% CO2 and 20% O2, when pressurized. Multiples of MAC were obtained by increasing the pressure inside the hyperbaric chamber with the gas mixtures. The concentration of xenon was as follows: 0.75 MAC at 1.2 atm, 1 MAC at 2.67 atm, and 2 MAC at 3.67 atm.

As described above, the pressure chamber was flushed with 3 l of the desired gas concentrations and then pressurized. It was then placed in a 37°C incubator for 6 h and monitored to maintain pressure. The chamber was depressurized slowly, and the slice culture dishes were removed and placed in a humidified 37°C incubator, with 5% CO2 for 24 h. Cell death was then measured.

Preconditioning with Isoflurane

The tissue culture media was changed to 75% Eagle’s Minimum Essential Media and 25% Earle’s Balanced Salt Solution 12 h before preconditioning. A random sample of the tissue culture inserts were then preconditioned with 1.4% isoflurane (0.75 MAC) at normobaric conditions in a Billups-Rothenberg chamber for 2 h, and then returned to a humidified 37°C incubator with 5% CO2. Twenty-six hours later, all of the cultures were exposed to the equivalent of 1.0 MAC of xenon, isoflurane, or sevoflurane for 6 h. Cell death was measured 24 h later, and average cell death from the pretreated groups was compared with tissue slices that were exposed to 1.0 MAC of anesthetic without pretreatment, as well as a 37°C control group.

Assessment of Cell Death

Cell death in live tissue slices was measured by SYTOX® (Life Technologies, Eugene, OR) fluorescence. SYTOX® is an unsymmetrical cyanine dye compound with three positive charges that penetrates only damaged plasma membranes and binds to DNA, with an excitation wavelength at 488 nm and an emission peak at 530 nm. Briefly, 1.2 ml of warmed slice culture media containing 0.5 µm SYTOX® was added to the wells of culture trays 24 h after anesthetic exposure. After 10 min, the dye was removed, and the wells were rinsed three times with Earle’s Balanced Salt Solution. Images of SYTOX® fluorescence were obtained using a SPOT Jr. Digital camera (Diagnostic Instruments, Sterling Heights, MI) and an inverted microscope. The fluorescence intensity was analyzed with Image J software by a blinded observer, and measured in the CA1, CA3, and dentate. A cell body region average was calculated by averaging total fluorescence from these regions. The background fluorescent signal from nonslice regions of the images was subtracted from the total fluorescent signal in the slice region; fluorescence of native slices was negligible. The expression of SYTOX® fluorescence in hippocampal slice cultures exposed to anesthetics is highly confined to the neuron cell body regions in the CA1, CA3, and dentate regions, indicating that the method is primarily measuring neuron cell damage in these studies. We measured cell death with SYTOX® at the 24-h time point after anesthetic exposure because in preliminary experiments in which we examined cell death 24–60 h after exposure, the peak cell death was seen in the 24- to 36-h time frame.

Assessment of Cell Death in Fixed Tissue Slices

Cell death in fixed hippocampal slice cultures was measured using fluorojade, a polyanionic fluorescein derivative that binds to degenerating neurons, and has an excitation wavelength at 480 nm and emission peak at 525 nm (Millipore, Billerica, MA). Staining was done, as previously described by Shih et al.,20  with the following modifications. Tissue slices were left on membrane inserts, and 36 h after anesthetic application, were fixed at room temperature for 2 h with 4% paraformaldehyde in phosphate-buffered saline. The inserts were then immersed in 0.06% potassium permanganate for 12 min, and rinsed twice for 2 min each, in distilled water. The slices were mounted on glass slides and left to dry for 15 min. The slides were incubated at 4°C with fluorojade for 30 min, rinsed three times with distilled water for 1 min each, and then left in the dark to dry for 24 h. A drop of DePeX Mounting Solution (Electron Microscopy Sciences, Hatfield, PA) was added and a glass coverslip was then applied. Fluorescent digital images were obtained using an E400, Nikon (Tokyo, Japan) fluorescent microscope. The fluorescence intensity was analyzed using Fiji software§ by a blinded observer, by the same method described for SYTOX®, except background fluorescence was subtracted using the “Background Subtraction” tool set to 45.

Dye penetration depth for SYTOX® and fluorojade was determined using a Nikon C1Si Confocal Laser-Scanning Microscope at the University of California, San Francisco Biological Imaging Development Center.

Statistical Analysis

The cell death and fluorescent values for each treatment group were assessed for normality using a D’Agostino–Pearson normality test. All the experiments were designed to produce a normally distributed pattern of cell death as assessed by SYTOX® or fluorojade fluorescence. Therefore, a one-way between-groups ANOVA was used to compare the means of these data and corrections were made for multiple comparisons with a post hoc Tukey–Kramer or Dunnett multiple comparisons correction procedure. All statistical comparisons involve a two-tailed hypothesis of either an increase, or a decrease in the measured variable (cell death) as a result of treatment. Differences were considered significant when P was less than 0.05. The GraphPad Prism software package was used (GraphPad, Inc., La Jolla, CA). To combine data from repeated, identical experiments, and to generate dose–effect graphs, each numerical fluorescence value from each treatment group was normalized to the average fluorescent value of its respective control group for that experiment.

Results

SYTOX® and Fluorojade Provide Similar Estimates of Anesthetic-induced Cell Death in Hippocampal Slice Cultures

Measurements of hippocampal neuron death with the SYTOX® and fluorojade methods, two standard neuron death assessment techniques, after 1.9 or 3.8% isoflurane (1 or 2 MAC) yielded similar estimates (ANOVA with a post hoc Dunnett test) of fold-increases in death in the neuron regions of the cultures (fig. 1, A–B). The penetration of these dyes into the slice cultures was assessed using confocal microscopy (fig. 1C). SYTOX® penetrated live tissue slices by approximately 40 µm, whereas fluorojade penetrated fixed slices only by approximately 10 µm. For this reason SYTOX® was chosen as the cell death indicator for the remainder of the study.

Fig. 1.

Cell death assessment by SYTOX® (Life Technologies, Eugene, OR) or fluorojade after exposure to 1 or 2 minimum alveolar concentration (MAC) isoflurane. Cell death in treatment groups was compared with baseline control, using an ANOVA with a post hoc Dunnett test. To combine data from multiple experiments, fluorescent values of the treatment groups were normalized to their respective control groups. One and 2 MAC isoflurane is equivalent to 1.9 and 3.8%, respectively. (A) SYTOX®, P = 0.0006. (B) Fluorojade, P = 0.0181. Number of asterisks indicates the degree of significance as follows: * P = 0.01–0.05, *** P < 0.001. All data bars represent the mean and SEM of SYTOX® or fluorojade fluorescence in average of the hippocampal cell body regions: CA1, CA3, and dentate. Numbers above the SEM bars indicate the number of tissue slices in each treatment group. (C) Images of SYTOX® and fluorojade fluorescence overlaid on brightfield images of control and treatment slices.

Fig. 1.

Cell death assessment by SYTOX® (Life Technologies, Eugene, OR) or fluorojade after exposure to 1 or 2 minimum alveolar concentration (MAC) isoflurane. Cell death in treatment groups was compared with baseline control, using an ANOVA with a post hoc Dunnett test. To combine data from multiple experiments, fluorescent values of the treatment groups were normalized to their respective control groups. One and 2 MAC isoflurane is equivalent to 1.9 and 3.8%, respectively. (A) SYTOX®, P = 0.0006. (B) Fluorojade, P = 0.0181. Number of asterisks indicates the degree of significance as follows: * P = 0.01–0.05, *** P < 0.001. All data bars represent the mean and SEM of SYTOX® or fluorojade fluorescence in average of the hippocampal cell body regions: CA1, CA3, and dentate. Numbers above the SEM bars indicate the number of tissue slices in each treatment group. (C) Images of SYTOX® and fluorojade fluorescence overlaid on brightfield images of control and treatment slices.

Xenon Neurotoxicity in Hippocampal Neurons Is Dose-dependent

Xenon neurotoxicity in hippocampal slice cultures depended on the xenon concentration. Cell death after 0.75 MAC xenon was not statistically greater than baseline (P > 0.05; ANOVA with a post hoc Dunnett test). However, cultures exposed to xenon at 1 MAC (at a total pressure of 2.67 atm) and 2 MAC (total pressure 3.67 atm) for 6 h had a statistically significant increase in cell death over baseline in the CA1 (32% increase in cell death at 1 MAC; 36% increase at 2 MAC), CA3 (55% increase in cell death at 1 MAC; 60% increase at 2 MAC), and dentate (36% increase in cell death at 1 MAC; 64% increase at 2 MAC) regions (fig. 2, A–D; ANOVA with a post hoc Dunnett test, P < 0.0001 in the average of the cell body regions, CA1, CA3, and dentate). Representative images are shown in figure 2E.

Fig. 2.

Cell death in hippocampal slice cultures after exposure to 0.75, 1, and 2 minimum alveolar concentration (MAC) xenon. Total cell death assessed by SYTOX® (Life Technologies, Eugene, OR) fluorescence was analyzed using an ANOVA with a post hoc Dunnett test; 0.75, 1, and 2 MAC xenon is equivalent to 60% xenon at 1.2, 2.67, and 3.67 atm, respectively. (A) Average cell death in the hippocampal cell body regions, P = <0.0001. (B) Cell death in CA1, P = <0.0001. (C) Cell death in CA3, P = <0.0001. (D) Cell death in dentate gyrus (DG), P = <0.0001. To combine data from multiple experiments, fluorescent values of the treatment groups were normalized to their respective control groups. Number of asterisks indicates the degree of significance as follows: ** P = 0.001–0.01, *** P < 0.001. All data bars represent the mean and SEM of SYTOX® or fluorojade fluorescence in average of the hippocampal cell body regions: CA1, CA3, and dentate. Numbers above the SEM bars indicate the number of tissue slices in each treatment group. (E) Images of SYTOX® fluorescence overlaid on brightfield images of control and treatment slices.

Fig. 2.

Cell death in hippocampal slice cultures after exposure to 0.75, 1, and 2 minimum alveolar concentration (MAC) xenon. Total cell death assessed by SYTOX® (Life Technologies, Eugene, OR) fluorescence was analyzed using an ANOVA with a post hoc Dunnett test; 0.75, 1, and 2 MAC xenon is equivalent to 60% xenon at 1.2, 2.67, and 3.67 atm, respectively. (A) Average cell death in the hippocampal cell body regions, P = <0.0001. (B) Cell death in CA1, P = <0.0001. (C) Cell death in CA3, P = <0.0001. (D) Cell death in dentate gyrus (DG), P = <0.0001. To combine data from multiple experiments, fluorescent values of the treatment groups were normalized to their respective control groups. Number of asterisks indicates the degree of significance as follows: ** P = 0.001–0.01, *** P < 0.001. All data bars represent the mean and SEM of SYTOX® or fluorojade fluorescence in average of the hippocampal cell body regions: CA1, CA3, and dentate. Numbers above the SEM bars indicate the number of tissue slices in each treatment group. (E) Images of SYTOX® fluorescence overlaid on brightfield images of control and treatment slices.

Isoflurane and Sevoflurane Neurotoxicity

In all the three of the cell body regions, both isoflurane and sevoflurane caused a statistically significant (P < 0.05) increase in cell death over baseline 24 h after 1 MAC, but not 2 MAC (fig. 3, A–B; ANOVA with a post hoc Dunnett test). After exposure to isoflurane, cell death increased by 53% at 0.75 MAC, 63% at 1 MAC, and 13% at 2 MAC in the average of the cell body regions. After exposure to sevoflurane, cell death increased by 90% at 1 MAC and by 36% at 2 MAC. Similarly, statistically significant increases in cell death (P < 0.05) were seen in the CA1 and CA3, after exposure to both isoflurane and sevoflurane at 1 MAC, but only in the dentate, after exposure to sevoflurane. There was no statistically significant increase in cell death in the dentate after isoflurane treatment at 1 or 2 MAC (P ≥ 0.05).

Fig. 3.

Cell death in hippocampal slice cultures after exposure to 0.75, 1, and 2 minimum alveolar concentration (MAC) of isoflurane or sevoflurane. Total cell death assessed by SYTOX® (Life Technologies, Eugene, OR) fluorescence was analyzed using an ANOVA with a post hoc Dunnett test. For isoflurane, 0.75, 1 and 2 MAC are equivalent to 1.4, 1.9, and 3.8%, respectively; for sevoflurane, 1 and 2 MAC are equivalent to 3.4 and 6.8%. (A) Average cell death in the hippocampal cell body regions after exposure to a range of isoflurane doses, P = 0.0016. (B) Average cell death in the cell body regions from sevoflurane, P = 0.0009. To combine data from multiple experiments, fluorescent values of the treatment groups were normalized to their respective control groups. Number of asterisks indicates the degree of significance as follows: * P = 0.01–0.05, ** P = 0.001–0.01, *** P < 0.001. All data bars represent the mean and SEM of SYTOX® or fluorojade fluorescence in average of the hippocampal cell body regions: CA1, CA3, and dentate. Numbers above the SEM bars indicate the number of tissue slices in each treatment group.

Fig. 3.

Cell death in hippocampal slice cultures after exposure to 0.75, 1, and 2 minimum alveolar concentration (MAC) of isoflurane or sevoflurane. Total cell death assessed by SYTOX® (Life Technologies, Eugene, OR) fluorescence was analyzed using an ANOVA with a post hoc Dunnett test. For isoflurane, 0.75, 1 and 2 MAC are equivalent to 1.4, 1.9, and 3.8%, respectively; for sevoflurane, 1 and 2 MAC are equivalent to 3.4 and 6.8%. (A) Average cell death in the hippocampal cell body regions after exposure to a range of isoflurane doses, P = 0.0016. (B) Average cell death in the cell body regions from sevoflurane, P = 0.0009. To combine data from multiple experiments, fluorescent values of the treatment groups were normalized to their respective control groups. Number of asterisks indicates the degree of significance as follows: * P = 0.01–0.05, ** P = 0.001–0.01, *** P < 0.001. All data bars represent the mean and SEM of SYTOX® or fluorojade fluorescence in average of the hippocampal cell body regions: CA1, CA3, and dentate. Numbers above the SEM bars indicate the number of tissue slices in each treatment group.

Increased Pressure Is Neither Neurotoxic, Nor Does It Change Total Cell Death during Exposure to Isoflurane

The study examined treatments involving greater than 1 atm total pressure. To determine if increased total pressure causes neuron death independent of anesthetics, cultures were maintained at 1 atm or pressurized to 2, 2.67, or 3.67 atm for 6 h. Twenty-four hours later there were no statistically significant differences in cell death between any of these groups for any of the hippocampal cell body regions (fig. 4; ANOVA with a post hoc Dunnett test). Furthermore, to examine a possible synergy between anesthetic-induced neuron death and total pressure, experiments were performed, in which the anesthetic pressure was constant (in this case isoflurane, 0.03 atm, ~1.6 MAC) whereas the total pressure varied from 1 to 3.67 atm. Increasing total pressure did not increase the total cell death caused by 0.03 atm isoflurane.

Fig. 4.

Hippocampal slice culture exposure to pressures ranging from 1.0 to 3.67 atm did not cause increased cell death. Average cell death in the hippocampal cell body regions assessed with SYTOX® (Life Technologies, Eugene, OR) was compared using an ANOVA with a post hoc Dunnett, P = 0.2066. No significant differences occurred between any of the groups (P > 0.05). To combine data from multiple experiments, fluorescent values of the treatment groups were normalized to their respective control groups. All data bars represent the mean and SEM of SYTOX® or fluorojade fluorescence in average of the hippocampal cell body regions: CA1, CA3, and dentate. Numbers above the SEM bars indicate the number of tissue slices in each treatment group.

Fig. 4.

Hippocampal slice culture exposure to pressures ranging from 1.0 to 3.67 atm did not cause increased cell death. Average cell death in the hippocampal cell body regions assessed with SYTOX® (Life Technologies, Eugene, OR) was compared using an ANOVA with a post hoc Dunnett, P = 0.2066. No significant differences occurred between any of the groups (P > 0.05). To combine data from multiple experiments, fluorescent values of the treatment groups were normalized to their respective control groups. All data bars represent the mean and SEM of SYTOX® or fluorojade fluorescence in average of the hippocampal cell body regions: CA1, CA3, and dentate. Numbers above the SEM bars indicate the number of tissue slices in each treatment group.

Preconditioning Prevents Xenon Toxicity

Cultures were “preconditioned” with a nonneurotoxic concentration of isoflurane (1.4%, equivalent to 0.75 MAC) for 2 h, followed by return to standard culture conditions. Twenty-six hours later, the cultures were exposed to 1 MAC equivalent of isoflurane, sevoflurane, and xenon for 6 h. The cultures were then returned to standard culture conditions and cell death was assessed a day later. Cultures preconditioned with isoflurane had a statistically significant reduction (P < 0.05) in cell death compared with sham preconditioned controls for all three anesthetics, all of which had increases in cell death. In this experiment, xenon without preconditioning increased cell death by 84% over baseline, isoflurane caused an 88% increase, and sevoflurane caused a 92% increase compared with controls (fig. 5; all P value less than 0.05, ANOVA with a post hoc Dunnett test).

Fig. 5.

Cell death in hippocampal slice cultures with and without pretreatment (PT) with 0.75 minimum alveolar concentration (MAC) isoflurane before 1 MAC xenon exposure. (A) Average cell death of cell body regions assessed by SYTOX® (Life Technologies, Eugene, OR) and analyzed using an ANOVA with a post hoc Dunnett test, P = 0.0039. A single asterisk indicates: * P = 0.01–0.05. All data bars represent the mean and SEM of SYTOX® or fluorojade fluorescence in average of the hippocampal cell body regions: CA1, CA3, and dentate. Numbers above the SEM bars indicate the number of tissue slices in each treatment group. (B) Images of SYTOX® fluorescence overlaid on brightfield images of control, pretreated, and nonpretreated slices.

Fig. 5.

Cell death in hippocampal slice cultures with and without pretreatment (PT) with 0.75 minimum alveolar concentration (MAC) isoflurane before 1 MAC xenon exposure. (A) Average cell death of cell body regions assessed by SYTOX® (Life Technologies, Eugene, OR) and analyzed using an ANOVA with a post hoc Dunnett test, P = 0.0039. A single asterisk indicates: * P = 0.01–0.05. All data bars represent the mean and SEM of SYTOX® or fluorojade fluorescence in average of the hippocampal cell body regions: CA1, CA3, and dentate. Numbers above the SEM bars indicate the number of tissue slices in each treatment group. (B) Images of SYTOX® fluorescence overlaid on brightfield images of control, pretreated, and nonpretreated slices.

Discussion

The main findings of the study were that like isoflurane and sevoflurane, xenon causes neurotoxicity in hippocampal slice cultures from 7-day-old rats when studied at concentrations of MAC and above. We also found that neuron death caused by xenon, isoflurane, and sevoflurane was prevented by pretreatment with a nonneurotoxic exposure to 0.75 MAC (1.4%) isoflurane. One implication of these results was that anesthetic potency, at least near 1 MAC, rather than type of anesthetic, predicts neurotoxicity in the developing brain of rodents.

MAC, Pressure, and Assessment of Cell Death

The anesthetics studied in this experiment were compared at equipotent concentrations using the principle of MAC.21,22  This method of comparison has previously been employed to test the relative toxicity of isoflurane, sevoflurane, and desflurane in vivo.3,23  This is the first study to compare the toxicities of xenon, isoflurane, and sevoflurane at 1 MAC and greater levels. Although there are multiple studies showing the neuroprotective properties of xenon in rats at concentrations below 1 MAC either before, or during exposure to other neurotoxic stimuli (N-methyl-d-aspartate or glutamate, hypoxia, nitrous oxide, or isoflurane),9–12,20,24  nothing has been known about potential for neurotoxicity at 1 MAC or greater.

To compare equipotent concentrations of these inhalational anesthetics in a 7-day-old, in vitro brain model, steady state MAC values were based on previous work. Stratmann et al.25  previously reported the MAC of isoflurane in 7-day-old rat pups. For isoflurane, the MAC for slice cultures was estimated as the value of “brain MAC” (the concentration of anesthetic in the brain tissue of 7-day-old rat pups at 1 MAC), as determined by Stratmann et al.,25  which is 1.9% and represents a value approximately at the mean of brain MAC over a duration of 1–3 h. The brain MAC for sevoflurane and xenon in P7 rats has not been determined, so the MAC for sevoflurane was extrapolated from a time–dose-dependent curve created for sevoflurane (unpublished data: October 10, 2010, Greg Stratmann, M.D., Ph.D., Associate Professor, University of California, San Francisco, San Francisco, CA, verbal communication). The time point where 1.9% appears on the isoflurane curve was compared with the sevoflurane curve, and the steady state sevoflurane MAC was thus estimated to be 3.5%. There is no similar curve for xenon, so the MAC value published by Koblin et al.13  of 1.6 atm was used; however, this is the adult MAC, and it may be an under normal estimate value for P7 rat pups.

Xenon MAC values are much higher than the other inhalational anesthetics: 1 MAC for a rat is 1.6 atm, and 0.95 atm for a mouse.13,14  Because the MAC of xenon is so high in rodents, studies at 1 MAC or greater require a pressure chamber, if it is to be administered with physiologically appropriate concentrations of oxygen and carbon dioxide. The finding that increased pressure up to 3.75 atm did not cause cell death fits with previously published data showing that CA1 neurons tolerate steady state pressures up to 4.5 atmospheres absolute.26  We also found that increased pressures did not add to anesthetic toxicity (fig. 4), at least for isoflurane, suggesting that the toxicity at 1 and 2 MAC for xenon is the intrinsic toxicity of this anesthetic, not an interaction of the agent with increased pressure.

Comparing the Neurotoxic Potential of Xenon, Isoflurane, and Sevoflurane

Studies comparing neurotoxic profiles of the different anesthetics have produced inconsistent results, making it difficult to ascertain whether one anesthetic is more toxic than another.3,23  This inconsistency has been suggested to be the result of studying different rodent models under varying conditions.2  In this study, three different volatile anesthetics were studied so that they could then be compared at a range of approximately equipotent concentrations. Although xenon significantly (P < 0.05) increased cell death over baseline at 1 MAC, it resulted in the least amount of cell death compared with isoflurane and sevoflurane (fig. 6A; ANOVA with a post hoc Tukey test). At 2 MAC, however, cell death caused by isoflurane and sevoflurane was less, and xenon had the largest (50%) increase in cell death (fig. 6B; ANOVA with a post hoc Tukey test). Stratmann et al.27  demonstrated a link between neuronal cell death and neurocognitive dysfunction after 1 MAC isoflurane exposure in 7-day-old rats, suggesting that behavioral studies after this concentration of xenon may be worthwhile.

Fig. 6.

Comparison of cell death in hippocampal slice cultures after exposure to equipotent doses (1 or 2 minimum alveolar concentration [MAC]) of xenon, isoflurane, or sevoflurane. Average cell death of cell body regions assessed by SYTOX® (Life Technologies, Eugene, OR) and analyzed using an ANOVA with a post hoc Tukey test. (A) Cell death after exposure to 1 MAC, P = <0.0001. (B) Cell death after exposure to 2 MAC, P = <0.0001. To combine data from multiple experiments, fluorescent values of the treatment groups were normalized to their respective control groups. Number of asterisks indicates the degree of significance as follows: * P = 0.01–0.05, *** P < 0.001. All data bars represent the mean and SEM of SYTOX® or fluorojade fluorescence in average of the hippocampal cell body regions: CA1, CA3, and dentate. Numbers above the SEM bars indicate the number of tissue slices in each treatment group.

Fig. 6.

Comparison of cell death in hippocampal slice cultures after exposure to equipotent doses (1 or 2 minimum alveolar concentration [MAC]) of xenon, isoflurane, or sevoflurane. Average cell death of cell body regions assessed by SYTOX® (Life Technologies, Eugene, OR) and analyzed using an ANOVA with a post hoc Tukey test. (A) Cell death after exposure to 1 MAC, P = <0.0001. (B) Cell death after exposure to 2 MAC, P = <0.0001. To combine data from multiple experiments, fluorescent values of the treatment groups were normalized to their respective control groups. Number of asterisks indicates the degree of significance as follows: * P = 0.01–0.05, *** P < 0.001. All data bars represent the mean and SEM of SYTOX® or fluorojade fluorescence in average of the hippocampal cell body regions: CA1, CA3, and dentate. Numbers above the SEM bars indicate the number of tissue slices in each treatment group.

The unexpected decrease in cell death seen at 2 MAC versus 1 MAC from isoflurane and sevoflurane might be a feature of the 24-h interval between anesthetic exposure and cell death measurement. In a preliminary experiment (unpublished data, Heather Brosnan, University of California, San Francisco, 2012), cell death was analyzed with SYTOX® fluorescence 36 h after a 6-h exposure to either 1 or 2 MAC of isoflurane. With a 36-h delay between anesthetic exposure and cell death assessment, greater cell death was observed in the 2 MAC group, indicating that timing of cell death may change depending on the anesthetic concentration. Indeed, choice of cell death marker is also important because decreased cell death at 2 MAC was shown with fluorojade, when assessed 36 h after anesthetic insult. These issues do not alter our basic conclusion that isoflurane, sevoflurane, and xenon share neurotoxic potential, depending on dosage, exposure duration, and time of cell death assessment.

Preconditioning with Isoflurane

Shu et al.9  previously demonstrated that 7-day-old rats pretreated with 70% xenon (0.44 MAC) for 2 h, followed by a 24-h recovery, and then a 6-h exposure to 70% N2O + 0.75% isoflurane had less cell death over baseline compared with animals that were pretreated with nitrous oxide or hypoxia, or were not pretreated at all. Ma et al.12  described similar protection when 30 or 60% xenon (0.18 and 0.38 MAC, respectively) was coadministered with 0.75% isoflurane in 7-day-old rats. Additionally, this lab has previously shown that isoflurane can be neuroprotective when used as a preconditioning agent before oxygen–glucose deprivation at concentrations from 0.5 to 1.5%, and also when coadministered at 1 to 2% during hypothermia or rewarming in the hippocampal slice culture model.6,8  Given this data, a concentration of 1.4% (0.75 MAC) isoflurane was used to precondition the slices for 2 h, followed by a 26-h recovery, and then a 6-h exposure to 1 MAC doses of xenon, isoflurane, or sevoflurane. The statistically insignificant increase in cell death over baseline in pretreated hippocampal slice cultures, and the statistically significant increase in groups that were not pretreated, supports the hypothesis that isoflurane pretreatment at a low concentration can afford protection against anesthetic toxicity caused by xenon, isoflurane, and sevoflurane at 1 MAC.

Limitations of This Study

This study revealed xenon toxicity in a cell culture model and so the validity of an extrapolation to humans is uncertain. Studies of xenon toxicity in intact animals are warranted but require studies in a pressure chamber because the MAC for xenon in rodents exceeds 1 atm. Anesthetic toxicity in intact animals involves long-lasting hippocampal-based learning and memory deficits,4  which is consistent with the loss of specific cells in the hippocampus. However, it is unclear if in vitro results exactly predict cell loss or deficits in intact animals. Furthermore, although we studied slices from 7-day-old rats, the developmental equivalent of our cultures to intact animals is uncertain. This is important, because both anesthetic neurotoxicity and cognitive deficit after anesthesia are age-dependent, with maximal impact on animals less than 2 weeks of age.27–29 

We only measured cell death after 6-h anesthetic insults at given MAC values; however, it is possible that similar death could be seen after shorter or longer exposure times over a given range of anesthetic concentrations. As mentioned above, the MAC for isoflurane, sevoflurane, and xenon in 7-day-old rats (or hippocampal cultures) is not known with certainty, but we believe that this uncertainty is not of sufficient magnitude to alter our basic conclusions. Another potential limitation is that we restricted our cell death analysis time point to 24 h with SYTOX® because we consistently saw cell death at this interval, with only modestly increased cell death observable at longer time points of assessment. Further studies looking at different exposure times, concentrations, and cell death time points may yield further information. In addition, including other methods for identifying dead neurons, such as other histologic staining techniques, would also be indicated.

We did not investigate the mechanisms involved in xenon neurotoxicity. Because xenon has a different spectrum of pharmacologic action on γ-amino butyric acid receptors and N-methyl-d-aspartate receptors compared with halogenated general anesthetics, it is possible that the basis of anesthetic toxicity from xenon is different as well. This too requires further study.

Conclusions

This study reveals increased cell death in developing hippocampal neurons 24 h after exposure to xenon at 1 MAC and above. It also demonstrates that xenon neurotoxicity is similar to that caused by isoflurane and sevoflurane when compared at 1 MAC and that the toxicity can be prevented by earlier exposure to a nonneurotoxic concentration of isoflurane.

Available at: http://rsb.info.nih.gov/ij. Accessed September 3, 2012.

§

Available at: http://rsbweb.nih.gov/ij/docs/guide/146-2.html. Accessed October 1, 2012.

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