N-methyl-d-aspartate (NMDA) receptor antagonists such as MK-801 (1–3) cause neurotoxicity in perinatal rat brain in both in vitro and in vivo models. Ethanol is both a γ-amino butyric acid (GABA)A agonist and NMDA antagonist and causes neurotoxicity in perinatal brain (4–8). Volatile anesthetics also have affinity for NMDA and GABAA receptors (8). Jevtovic-Todorovic et al. (9) exposed normal postnatal day (PND) 7 rat pups to combinations of isoflurane, midazolam, and nitrous oxide (N2O). Exposure to isoflurane (0.75%, 1.0%, and 1.5%) for 6 h caused neuronal apoptosis with 1.5% isoflurane having the greatest effect on neurodegeneration. Addition of midazolam and N2O to isoflurane accentuated neuronal apoptosis, necrosis, and learning and memory deficits measured 19 weeks after anesthetic exposure.
The results of the in vivo investigation by Jevtovic-Todorovic et al. (9) may have implications for both pediatric anesthetic practice and sedation in neonatal intensive care units (10,11). The possibility that a routine pediatric anesthetic or sedation practice would promote neuronal degeneration has promoted anxiety in many individuals involved in the care of young children and neonates. Although countless numbers of pediatric anesthetics have been administered without apparent (albeit unstudied) neurologic or neurocognitive injury, neurologic injury in infants having anesthesia for cardiac surgery is recognized (12). However, the possibility that anesthetics are a contributing factor has not been addressed.
Unmonitored hemodynamic variables, lack of repeated blood gas analysis, and questions concerning the duration of fasting and interrupted fostering of the pup by the dam lend question to anesthetics being the etiology for the neurotoxicity observed in rat pups exposed to isoflurane for prolonged durations (13). Furthermore, shorter durations of anesthetic exposure (14) and rat pups of other ages were not investigated. Thus, it is not known if there is a specific stage in brain ontogeny at which vulnerability to anesthetic exposure is prominent. Such information might allow later definition of the mechanistic basis of anesthetic neurotoxicity allowing a rational extrapolation to the human condition and focused clinical studies.
Using organotypic hippocampal slices (OHSs), we evaluated whether the duration of 1.5% isoflurane exposure and age of the rat pup have an impact on neuronal degeneration. OHSs offer a model of intact neural circuits and avoid some of the variables inherent in an in vivo investigation in small rodents.
All studies were approved by the Duke University Institutional Animal Care and Use Committee. OHS cultures were prepared according to the methods described by Stoppini et al. (15,16) with some modification. PNDs 4, 7, and 14 Sprague-Dawley rat pups (Zivic Laboratories, Pittsburgh, PA) were anesthetized using an intraperitoneal injection of ketamine (10 mg/kg) and diazepam (0.2 mg/kg). The pups were decapitated and the hippocampi were removed and placed in 4°C Gey’s balanced solution (Sigma-Aldrich, St. Louis, MO) with 100 μM adenosine. Using an MX-TS brain slicer (Siskiyou Design Instruments, Grants Pass, OR), the hippocampi were cut transversely (400-μm thickness) and transferred to 30-mm-diameter membrane inserts (Millicell-CM; Millipore, Bedford, MA). Approximately 3–5 slices were placed within each well of a 6-well culture tray with media for 7 or 14 days before study. The culture media consisted of 50% minimal essential media (Invitrogen, Carlsbad, CA), 25% Earle’s balanced salt solution (Invitrogen), and 25% Hyclone heat-inactivated horse serum (Perbio, Cell Culture Division, South Logan, UT) with 6.5 mg/mL glucose and 5 mM KCL. The media was exchanged after the second day in culture and twice a week thereafter. OHSs were cultivated in a humidified atmosphere at 37°C and 5% CO2. No antibiotics or antimycotics were used.
During isoflurane exposure, the media was unchanged. OHSs were placed within a 3-L air-tight chamber (Billup’s Rothenberg, San Diego, CA) housed within a water jacketed incubator maintained at 37°C. An in-line calibrated anesthetic agent vaporizer was used to deliver isoflurane to the gas phase of the culture wells (16,17). Fresh gas (21% O2, 5% CO2, 69% N2) was heated and humidified to 37°C via a heated humidifier (Fisher-Paykel, Laguna Hills, CA) and was administered at a flow rate of 3 L/min, until the appropriate effluent concentration of the anesthetic was achieved. This required approximately 5–10 min. The flow rate was then decreased and maintained at 700 mL/min for a period of 1, 3, or 5 h. Effluent isoflurane, O2, and CO2 concentrations were continuously monitored via a sampling port connected to an anesthetic agent analyzer (Datex Instruments Corporation, Tewksbury, MA). The pH of the media before and after exposure was 7.4. Control slices were OHSs exposed to fresh gas only for 5 h. After exposure to isoflurane (or fresh gas only), the slices were removed and returned to the incubation chamber for 3 days. Neuronal viability in slices exposed to fresh gas only was compared with that in a sham control condition, not exposed to fresh gas. No difference was observed (data not presented) and thus sham data were not used for further analysis.
Evaluation of Cell Death
Twenty-four hours after isoflurane or fresh gas exposure, the media was exchanged with media containing 5 μM Sytox and was left unchanged for the remainder of the experiment. Sytox (Molecular Probes, Eugene, OR) is a high-affinity nucleic acid stain specific for cells with compromised plasma membranes and does not penetrate live cells (18,19). The slices were imaged 3 days after isoflurane or fresh gas exposure using a Leica inverted microscope (2.5×) (Wetzlar, Germany). Fluorescent digital images were taken using a CoolSnap digital camera (Image Processing Solutions, North Reading, MA), excitation wavelength 490 nm and emission 590 nm. The parameters for imaging were standardized for all slices.
Both light and fluorescent microscopic images were made simultaneously for each slice and stored for later analysis. CA1, CA2, CA3, and dentate gyrus were manually outlined on images obtained by light microscopy using MCID software (Imaging Research, Inc., St. Catherines, Ontario). The outlines were then superimposed on the fluorescent images. The mean optical density (fluorescence intensity) was then measured for each hippocampal region. Slices with intense fluorescence in hippocampal CA2, in either fresh gas or isoflurane groups, were excluded from analysis. These represented nonviable slices, which constituted approximately 5%–10% of the OHS population (16,17,20). Analysis of the images was performed by an investigator blinded to condition assignment and after completion of each experiment for consistency in measurement.
Experiments 1 and 2: Isoflurane Exposure and OHSs
Three isoflurane exposure chambers were stacked with fresh gas introduced into the bottom chamber and that was allowed to flow through the middle chamber to the top chamber. Culture wells containing OHSs from each age group (PNDs 4, 7, and 14) were randomly placed in the different chambers. OHSs were exposed to 1.5% isoflurane for 1, 3, or 5 h. OHSs were removed from the top chamber first (1-h exposure), and the bottom chamber last (5-h exposure) to eliminate changes in gas concentration in the remaining chambers. Control slices were exposed to fresh gas for 5 h in the absence of isoflurane in each trial. For each experimental condition, a minimum of 12 slices were assessed for CA1, CA3, and dentate gyrus fluorescence intensity.
In experiment one, OHSs were maintained in culture for 7 days before exposure to isoflurane/fresh gas. In experiment two, OHSs were maintained in culture for 14 days after isoflurane/fresh gas exposure. These experiments were performed to determine whether the number of days in vitro affected the response to isoflurane to control for possible changes in GABAA and NMDA receptor subunit composition known to be a function of development in vivo (21).
Experiment 3: In Situ Hybridization in Whole Brain Slices
PNDs 4, 7, and 14 rat pups were euthanized as previously described for the hippocampal slice preparation and the brains were freshly frozen in liquid nitrogen. Coronal (20 μm) sections were cut by cryostat. Slices were stored at −80°C immediately after sectioning until further processing.
Rat GABAA receptor α-1 subunit cDNA nucleotides 419–1200 (GeneBank accession no. L08490) and α-2 subunit cDNA nucleotides 387–1078 (GeneBank accession no. L08491) were cloned into TOPO TA cloning vector (Invitrogen). Both [35S] dUTP labeled antisense and sense RNA probes were synthesized by in vitro transcription using either T7 or SP6 RNA polymerase (Roche Molecular Biochemicals, Indianapolis, IN). Brain sections were fixed in 4% paraformaldehyde for 20 min and treated with 20 μg/mL proteinase K in 50 mM Tris-HCl pH 7.6 with 5 mM EDTA. Sections were acetylated in 0.3% acetic anhydride in 0.1 M triethanolamine solution, then washed in phosphate-buffered saline and dehydrated for hybridization. In situ hybridization was achieved in an incubator oven at 62°C in hybridization solution consisting of 50% formamide, 10% dextran sulfate, 0.3 M NaCl, 20 mM Tris-HCl pH 8, 5 mM EDTA, 0.02% filcoll, 0.02% Rnase-free bovine serum albumin, 0.02% polyvinylpyrrolidone, 0.5 mg/mL tRNA and RNA probes at 500,000 cpm per slide. After incubation for 16 h, slides were washed in 5× sacrosyl (N-lauroyl) sacrosine solution (SSC) for 15 min at 50°C. Stringent washes were performed in 50% formamide and 2× SSC at 65°C for 30 min. Unbound probes were removed by incubation with 20 mg/mL RNaseA, 0.5 M NaCl, 10 mM Tris-HCl pH 8.0, 1 mM EDTA for 30 min at 37°C. Slides were then washed once again in stringent washing solution at 65°C. β-Mercaptoethanol (20 mM) was present in all washing solutions. Slides were then desalted through 2× SSC, 0.1% SSC, and dehydrated through ascending graded ethanol solution. Slides were allowed to air dry for autoradiography.
Slides were exposed to autoradiography film (Eastman Kodak, Rochester, NY) for 3 days. The intensity of the hippocampal CA1 hybridization signal in autoradiographs was quantified by measuring the relative optical density (ROD) values using the MCID image analysis system.
Experiments one and two were not performed concurrently and therefore were analyzed separately with identical procedures. Within each experiment, fluorescence intensity was compared initially with two-way analysis of variance (ANOVA) (PND X duration of isoflurane exposure). Each hippocampal region (CA1, CA3, and dentate gyrus) was analyzed independently. Based on the data from Jevtovic-Todorovic et al. (9), we assumed that 5 h of isoflurane exposure would yield the greatest effect on cell viability. Therefore, one-way ANOVA was used to compare fluorescence intensity values among PND groups exposed to isoflurane for 5 h. The results of this analysis caused us to then focus on PND 7 OHSs. Values from these slices were compared as a function of duration of isoflurane exposure using one-way ANOVA and Scheffé test. For in situ hybridization, the ratio of α-2 to α-1 ROD was determined for the CA1 region of the hippocampus and compared using one-way ANOVA for the three PND ages studied. When indicated by a significant F value, post hoc testing was performed using Scheffé test. Values are presented as mean ± sd. Significance was assumed when P ≤ 0.05.
Experiment 1 (OHSs Cultured 7 Days Before Isoflurane Exposure)
In CA1, an effect of isoflurane on cell death, as measured by Sytox (green) fluorescence was significant. The amount of neuronal death depended on PND (P < 0.0001) and exposure duration (P = 0.002) without an interaction between factors (P = 0.16). Neuronal death was most frequent among PND ages for slices exposed to isoflurane for 5 h (P < 0.0001). Differences in the amount of neuronal death were observed between PND 4 versus PND 7 (P = 0.0003) and PND 7 versus PND 14 (P = 0.0009). In both cases, more cell death was observed at PND 7 (Fig. 1). Among PND 7 OHSs, the duration of isoflurane exposure (P < 0.03) was significant. Little or no effect occurred with 1 or 3 h isoflurane exposure, whereas 5 h resulted in increased Sytox fluorescence (P = 0.05) (Fig. 2). These patterns were repeated in CA3 and dentate gyrus (Figs. 1 and 2).
Experiment 2 (OHSs Cultured 14 Days Before Isoflurane Exposure)
In CA1, CA3, and dentate gyrus (Fig. 3), Sytox uptake, defined by fluorescence intensity, was greater in PND 7 versus either PND 4 or PND 14 slices. For PND 7 OHSs, maximal Sytox uptake was observed in those slices exposed to isoflurane for 5 h (Fig. 4). Data were not directly compared between OHS days in vitro (DIV) 7 versus 14 before isoflurane exposure because the experiments were not performed concurrently. However, the effect of PND and duration of isoflurane exposure seemed to be independent of this variable, at least with respect to pattern of effect.
Experiment 3 (GABAA Subunit In Situ Hybridization)
Brain slices from PNDs 4, 7, and 14 rat pups were compared. The autoradiographs showed an age-related difference in the CA1 ratio of α-2 to α-1 GABAA subunit composition (PND 4 = 5.4 ± 5.8, PND 7 = 1.8 ± 0.7, and PND 14 = 0.798 ± 0.4, P = 0.0035) (Fig. 5). The α-2 to α-1 GABAA subunit ratio was largest in the hippocampal region of slices harvested from PND 4 rat pups.
Isoflurane (1.5%) caused neurodegeneration in some OHSs. This effect was evident in OHSs derived from PND 7, but not PND 4 or PND 14 rat pups. Furthermore, this effect was only observed with an isoflurane exposure of five hours’ duration. These in vitro data confirm the findings of Jevtovic-Todorovic et al. (9), in which PND rat pups exposed to 6 hours of 1.5% isoflurane developed an apoptotic response resulting in persistent behavioral deficits. Our findings extend the work of Jevtovic-Todorovic et al. (9) by demonstrating that isoflurane neurotoxicity occurs in vitro in the presence of controlled physiologic variables. Furthermore, we have shown that isoflurane-induced neuronal degeneration is dependent on both the duration of isoflurane exposure and the age of the rat pup from which the OHS was derived.
The in vivo observations made by Jevtovic-Todorovic et al. (9) have been questioned because of lack of physiologic control during anesthetic exposure, which otherwise would accompany anesthetic administration in humans (13). However, their observations are consistent with other studies of neurotoxicity in the developing brain. Using phenobarbital and other anticonvulsants, Bittigau et al. (22,23) have shown an age-related vulnerability to neuronal cell death. Neuronal vulnerability increased as a function of postnatal age of the rat pup studied, peaking at PND 7. Older-age rat pups were not evaluated. Similarly, NMDA antagonists (MK-801) (1–3) initiate apoptosis in immature rat brain. PND 7 represents an especially vulnerable ontogenetic stage (24). Isoflurane enhances GABAergic neurotransmission and antagonizes glutamate at the NMDA receptor (25), potentially invoking mechanisms of neurotoxicity via both receptor systems.
The OHS model used in the present investigation eliminated confounding physiologic variables such as hemodynamics and blood gases while maintaining the normal relationship of neurons, glia, and other membrane structures. Therefore, our in vitro investigation lends credibility to the contention that isoflurane has potential to cause neurotoxicity in developing brain. Our results obtained at different ages suggest a developmental window of neuronal vulnerability.
Extrapolation of these findings beyond the PND 7 rat must be strictly limited at this time. The rat brain is different than the human in numerous ways, including lack of sulci and gyri, a different ratio of cortical to subcortical volume, and rate of synaptogenesis and neurotransmitter receptor development. However, because the rat pup age from which the OHSs were derived was critical in defining neurotoxicity, isolation and focus of study on events occurring during this window might give clues as to when windows of vulnerability might be present in other species.
The findings of neurodegeneration after anesthetic exposure are in contrast to many studies that have found neuroprotective effects from isoflurane and other volatile anesthetics in models of ischemia in developing brain. In a pure cortical neuronal cell culture derived from perinatal brain, isoflurane attenuated oxygen and glucose deprivation (OGD)-induced neuronal apoptosis (17). Similarly, in primary mixed neuronal glial cultures derived from prenatal rat brain, isoflurane reduced cell death resulting from NMDA exposure (26). In OHSs subjected to either OGD or glutamate excitotoxicity, isoflurane again reduced cell death, this effect being persistent over a 2-week recovery (20). Isoflurane preconditioning induced neuroprotection in a PND 7 rat-pup hypoxia-ischemia model (27). Exposure of neonatal piglets to desflurane during deep hypothermic circulatory arrest and low-flow cardiopulmonary bypass also showed a neuroprotective effect (28,29). Thus, during periods of stress, such as OGD or global ischemia, volatile anesthetics, for a limited duration, were neuroprotective in perinatal models. Others have shown that repeated, but not short-term, ketamine exposure promoted neuronal cell death in PND 7 rat pups (14). A prolonged exposure, i.e., 5 hours, to anesthetics seems to be critical in the absence of stress (9,14,22).
There may be a number of reasons for this developmental age-related vulnerability in the rat (24). A potential mechanism is ontogenetic changes in receptor subunit composition and functionality (30). Maturational changes of the GABAA receptor subunit constituents are reflected in functional changes involving chloride permeability and calcium influx (31). The maturational changes of both GABAA and chloride transporters (NCC1 and KCC2) have been evaluated as a function of age in the rat (32,33). With increasing age, the KCC2 transporter, which is responsible for moving chloride out of the neuron, increases. This allows for the previously depolarized state of the GABAA receptor to become inhibitory, i.e., hyperpolarized. This functional shift from depolarization to hyperpolarization occurs around PND 7 (30). Simultaneous with the hyperpolarizing effects of GABA, NMDA glutamatergic transmission becomes prominent at the end of the first postnatal week (34). The delayed maturation of inhibition may allow increased NMDA receptor activation, thereby opening a developmental window of enhanced plasticity in the neonatal period (35).
These maturational changes in the GABAA receptor have been shown in our investigation using acute brain slices and are consistent with the findings of others (36). The α-2/α-1 subunit ratio decreases as a function of increasing age. Although this is true in fresh brain specimens, we could find limited information regarding the impact of culturing hippocampal explants 7–14 DIV on either the rate or continuation of in vivo ontogenetic development (32,37–39). Our ability to perform this investigation in our slices was confounded by their growth into the culture membrane and thus fresh slices were used. Thus, we do not know the course of receptor subunit composition that occurred in vitro. Because the neurodegenerative response to 5-hours 1.5% isoflurane in OHSs derived from PND 7 pups occurred regardless of whether cultures were maintained for 7 or 14 days before isoflurane exposure, it is tempting to speculate that development was arrested. However, this requires specific analysis.
Our in vitro investigation supports the observation of Jevtovic-Todorovic et al. (9) that PND 7 rat brain is vulnerable to isoflurane-induced neurodegeneration. However, at least in OHSs, this vulnerability is dependent on both the age of rat pup from which the tissue was explanted and on the duration of exposure to isoflurane. There is evidence that both the NMDA and GABAA receptors are undergoing major subunit composition changes during this interval. We postulate that these changes may account for the unique window of vulnerability to isoflurane-induced neurodegeneration in the rat. Further study in other species that examines the effects of prolonged isoflurane exposure at different levels of ontogeny and correlation of those findings with ontogenetic changes in GABAA and NMDA receptor subunits would be important to determine if this window of vulnerability is unique to the rat or present in other species.
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© 2005 International Anesthesia Research Society
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