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Anesth Analg 2001;93:1281-1287
© 2001 International Anesthesia Research Society

NEUROSURGICAL ANESTHESIA

Oxygen and Glucose Deprivation-Induced Neuronal Apoptosis is Attenuated by Halothane and Isoflurane

Lisa Wise-Faberowski, MD*, Mohan K. Raizada, PhD{dagger}, and Colin Sumners, PhD{dagger}

*Department of Anesthesiology, Childrens Hospital and Harvard Medical School, Boston, Massachusetts; and {dagger}Department of Physiology, University of Florida College of Medicine, and the University of Florida Brain Institute, Gainesville, Florida

Address correspondence and reprint requests to Lisa Wise-Faberowski, MD, Department of Anesthesiology, Children’s Hospital, 300 Longwood Ave., Boston, MA 02460. Address e-mail to Faberowski{at}tch.harvard.edu


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Both in vitroand in vivo evidence supports the reduction of early ischemic, both global and focal, brain injury by volatile anesthetics. However, the protection afforded by volatile anesthetics in later neuronal death, i.e., apoptosis, caused by global ischemia has not been investigated. We induced oxygen and glucose deprivation in neuronal cortical cell cultures prepared from newborn rats on in vitro Days 10–14. This hypoxic (PO2 <50 mm Hg) condition was maintained continuously (30, 60, and 90 min). In a separate experiment, the neuronal cell cultures were exposed to isoflurane (1.13%, 2.3%, or 3.3%) or halothane (1.7%, 3.4%, or 5.1%) before oxygen and glucose deprivation, with continued exposure to isoflurane or halothane during oxygen and glucose deprivation. After 48 h, neuronal apoptosis was assessed with terminal deoxynucleotidyl transferase-mediated in situ nick-end labeling and DNA gel electrophoresis. Oxygen and glucose deprivation (30, 60, and 90 min) caused significant apoptosis of cerebral cortical cultured neurons. However, pretreatment and continued treatment during the period of oxygen and glucose deprivation with halothane or isoflurane resulted in a concentration-dependent attenuation of oxygen and glucose deprivation-induced neuronal apoptosis.

IMPLICATIONS: This is the first investigation to evaluate the effect of volatile anesthetics on oxygen and glucose deprivation-induced neuronal apoptosis. Oxygen and glucose deprivation-induced neuronal apoptosis can be decreased by prior and continued administration of halothane or isoflurane.


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Neuronal cell death after ischemia occurs via two distinct processes, necrosis and apoptosis (1,2). Apoptosis, programmed cell death, is denoted by chromatin condensation, nuclear blebbing, cellular shrinkage, and DNA fragmentation. Unlike apoptosis, cellular swelling and lysis is suggestive of necrosis. However, these two processes are related not only to the severity but also to the duration of ischemia. Infarction after severe ischemia (90 min) occurs within 6 h and is fully developed in 24 h. However, after mild ischemia (30 min), no infarction is present after 24 h but subsequently develops over 78 h (3). The neurons bordering the infarct area show evidence of neuronal apoptosis. This suggests that apoptosis may contribute to delayed infarction after mild temporary focal ischemia and that cell death is a continuum from necrosis to apoptosis (1–4).

Isoflurane has been examined at 1–2 minimum alveolar anesthetic concentrations (MAC) at varying levels of ischemia, comparing temporary focal ischemia with near-complete global ischemia, with evidence of decreased infarct volume (5–7). Halothane at 1–1.4 MAC demonstrates a beneficial effect during focal and incomplete cerebral ischemia (7,8). The early neuroprotective effects of volatile anesthetics are known, but the delayed effects are unclear (5–8). Thus, we performed an in vitroinvestigation to determine whether prior exposure before oxygen and glucose deprivation and continued exposure during oxygen and glucose deprivation to three concentrations each of halothane or isoflurane offered late neuroprotection, as determined by examination for neuronal apoptosis rather than necrosis.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
This study was approved by the University of Florida Animal Care and Use committee. For all experiments we used an in vitrosystem of primary cultured cortical neurons prepared from newborn rat cerebral cortex. Briefly, cerebral cortices were removed from newborn Sprague-Dawley rats (Charles River Laboratories, Worchester, MA) and placed in an isotonic solution containing penicillin, streptomycin, and amphotericin B. The brains were stripped of meninges and blood vessels, minced, and dissociated. Cells dissociated from the cortex were pooled and resuspended in Dulbecco’s modified Eagle medium (DMEM) containing 10% plasma-derived horse serum (Sigma, St. Louis, MO) (9). The cells were seeded in poly L-lysine-coated, 35-mm tissue culture dishes and maintained in a humidified incubator (37°C with air and 6% CO2). At 3 days the cells were dispersed with trypsin and treated with 1 µM cytosine arabinoside for 2 days. The neuronal cell cultures were used from in vitro Days 10 to 14, with a neuronal cell count of approximately 10 x 106 (9). The neuronal cultures consist of 90% neurons and approximately 10% astroglia and microglia, as estimated by immunocytochemical staining with antibodies against neurofilament proteins and glial fibrillary acidic protein (9).

The cultures were exposed to glucose deprivation and hypoxia (Table 1). The original glucose-containing medium was removed and replaced with a glucose-free phosphate-buffered saline (PBS) (pH 7.4). All media changes were followed by a wash with PBS. No serum was included in the glucose-free PBS. Dishes exposed to hypoxia (PO2 <50 mm Hg) were placed in a small, 3-L, airtight experimental hypoxia chamber (Billups-Rothenberg, San Diego, CA) with inflow and outflow connectors (10–12). The experiments were conducted in a constant 37°C environment by placing the chambers in a water-jacketed incubator. The gaseous environment was controlled by the delivery of all gas via a heater-humidifier (Fisher-Paykel, Laguna Hills, CA) servo-controlled to 37°C via the inflow adapter of the chamber. Using a portable blood gas analyzer (I-Stat, East Windsor, NJ) the oxygen, CO2, and pH of the media covering the cells were monitored. In the oxygen and glucose-deprivation phase, the media were washed with PBS and changed to hypoxic, glucose-free PBS.


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  		Table 1. Experimental Group: Halothane or Isoflurane at Three Separate Concentrations
 
The glucose-free, hypoxic PBS was prepared by passing the PBS through a microbubbler apparatus containing the hypoxic (94% N2/6% CO2, pH 7.4) gas mixture. The hypoxic, glucose-free PBS was then applied in a thin layer (enough to cover the cells) to the cell culture dishes. The cultures were then placed in the sealed chambers as described previously and exposed to the hypoxic gas mixture (12,13). The outflow adapter of the experimental chamber was connected to an exhaust tubing connected to an underwater seal. Cultures were exposed to conditions of oxygen and glucose deprivation for 30, 60, or 90 min. This exposure was terminated by the removal of the PBS, addition of the original media for 48 h, and then analysis of apoptosis as described below.

Cultures were exposed to volatile anesthetics (Table 1). For cultures receiving pretreatment with halothane or isoflurane, an in-line agent-specific calibrated Fluotec (Cyprane, England) gas vaporizer was used. A sampling port was connected to an end-tidal gas monitor (Datex Instruments Corporation, Tewksbury, MA) that was interposed between the outflow adapter and the gas exhaust system to measure oxygen and CO2 in addition to the volatile anesthetic administered. Fresh gas consisting of 20% oxygen, 69% nitrogen, and 6% CO2, in addition to the concentration of the selected volatile anesthetic (1.0, 2.0, and 3.0 MAC, respectively: halothane, 0.77%, 1.54%, and 2.31% ± 0.2%; and isoflurane, 1.13%, 2.26%, and 3.33% ± 0.3%) (12,13), was administered at 3 L/min until the desired end-tidal volatile anesthetic concentration was obtained as measured by the end-tidal gas analyzer. After the desired end-tidal concentration of anesthetic was achieved, approximately 10 min, the gas flow was decreased and maintained at 700 mL/min for 30 min. After 30 min, the inflow fresh gas mixture was changed to the hypoxic gas mixture while the previous end-tidal gas concentration was maintained. The volatile anesthetic gas concentration of the PBS was confirmed with a gas chromatograph (within 5%–10% of the end-tidal gas concentration) (14). Cultures were exposed to halothane or isoflurane, in addition to oxygen and glucose deprivation, for 30, 60, or 90 min. Removing the PBS and returning the original media to the cultures terminated the period of anesthetic exposure. The cells were then placed back in the original incubator for 48 h, after which time the neurons were examined for apoptosis. All experiments were performed in triplicate.

Two approaches were used to assess neuronal apoptosis in these studies: terminal deoxynucleotidyl transferase-mediated in situ nick-end labeling (TUNEL) and DNA fragmentation. The cultures were analyzed for apoptotic DNA fragmentation by TUNEL by using the In Situ Cell Death Detection Kit, AP® (Boehringer Mannheim, Eugene, OR) (15,16). Terminal deoxynucleotidyl transferase labels the 3'-OH terminal of the DNA strand breaks. Antifluorescein antibody Fab from sheep conjugated with alkaline phosphatase detects the incorporated fluorescein. After substrate reaction with Fast Red® (Boehringer Mannheim), the cells were examined under light microscopy, with blinding to treatment group. Cell morphology was also examined, distinguishing between cell lysis and apoptosis (17). All stained neurons, from those colored light pink, which may be an early apoptotic cell, to dark pink, which may represent a cell in the late stages of apoptosis or an apoptotic body, were considered as apoptotic without further classification. In addition, as neurons were located on top of the glia within the culture dish, we were able to focus the microscope away from the glia and include only the examination of stained neurons (18) (Fig. 1). The number of apoptotic neurons from each area (area = 0.09 mm2) were combined, and an average value was obtained for each treatment group. For each culture dish, the total number of neurons was counted with a Nikon Eclipse E-400 microscope (Nikon, Melville, NY) hooked up to a Sony color video camera (model DXC-970; Sony, Tokyo, Japan), and the image was analyzed with a computer program (microcomputer imaging device program, MCID-M4-3.0; Imaging Research, Inc., Ontario, Canada). Ten to 20 fields were examined in each dish and expressed per 10,000 total neurons. The total number of cells in each dish was analyzed across various treatments within each experiment, as well as across various experiments, to detect dish-to-dish variation and variation within different batches of cells (17).



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  		Figure 1. Cortical neuronal cultures were exposed to oxygen and glucose deprivation as described in Methods. This was followed by analysis of apoptosis by using the terminal deoxynucleotidyl transferase-mediated in situ nick-end labeling (TUNEL) method. TUNEL positive-staining cells were examined for morphology. Those cells demonstrating nuclear condensation, cellular shrinkage, and pyknotic nuclei were counted positive for apoptosis (arrow), and those with evidence of cellular swelling and lysis (not demonstrated) were excluded.

 
The detection of DNA fragmentation in cultured neurons was performed essentially according to previously published procedures (19). For DNA gel electrophoresis, cellular DNA was harvested from the cultures at 48 h. After treating the samples with RNase A (Sigma) and protein kinase (Sigma), the protein was precipitated with trichloracetic acid and washed with ethanol. The DNA was electrophoresed on a 1.2% agarose gel by using ethidium bromide staining (Nusieve®, Seakem, 3:1; FMC Bioproducts, Rockland, ME) for 5 h at 50 V. The ethidium bromide gels were examined and photographed under ultraviolet light to visualize the DNA bands.

All results are expressed as the mean ± SEM and were obtained by combining individual experiments. Multiple means were compared by using one- or two-way analysis of variance followed by the Newman-Keuls test to assess statistical significance (P < 0.05). Statistical analyses were performed by using SigmaStat Software (Jandel Scientific, San Rafael, CA).


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
In the first series of experiments, TUNEL staining demonstrated that exposure of cerebral cortical neurons to oxygen and glucose deprivation caused a significant increase in neuronal apoptosis as compared with control cells. This apoptotic effect is directly related to the time of exposure to oxygen and glucose deprivation (Figs. 2–4). Exposure to PBS alone, glucose deprivation, did not result in significant apoptosis (Figs. 2–4; Time 0), nor did exposure to halothane or isoflurane alone (data not displayed).



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  		Figure 2. Cortical neurons were pretreated with halothane or isoflurane (1.0 minimum alveolar anesthetic concentration [MAC]) for 30 min with continued exposure to the same concentration of volatile anesthetic during the designated time period of oxygen and glucose deprivation (30, 60, or 90 min). The cells were examined for neuronal apoptosis 48 h after exposure to oxygen and glucose deprivation. Determination of neuronal apoptosis was by terminal deoxynucleotidyl transferase-mediated in situ nick-end labeling staining, as previously described; 1.0 MAC concentration of halothane ({blacksquare}); 1.0 MAC concentration of isoflurane ({blacktriangleup}); oxygen and glucose deprivation ({diamond}). The controls were those neurons exposed to media change alone, with no exposure to oxygen and glucose deprivation or volatile anesthetic. Data are represented as mean ± 95% confidence interval.

 


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  		Figure 3. Cortical neurons were pretreated with halothane or isoflurane (2.0 minimum alveolar anesthetic concentration [MAC]) for 30 min, with continued exposure to the same concentration of volatile anesthetic during the designated time period of oxygen and glucose deprivation (30, 60, or 90 min). The cells were examined for neuronal apoptosis 48 h after exposure to oxygen and glucose deprivation. Determination of neuronal apoptosis was by terminal deoxynucleotidyl transferase-mediated in situ nick-end labeling staining, as previously described; 2.0 MAC concentration of halothane ({blacksquare}); 2.0 MAC concentration of isoflurane ({blacktriangleup}); oxygen and glucose deprivation ({diamond}). The controls were those neurons exposed to media change alone, with no exposure to oxygen and glucose deprivation or volatile anesthetic. Data are represented as mean ± 95% confidence interval.

 


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  		Figure 4. Cortical neurons were pretreated with halothane or isoflurane (3.0 minimum alveolar anesthetic concentration [MAC]) for 30 min with continued exposure to the same concentration of volatile anesthetic during the designated time period of oxygen and glucose deprivation (30, 60, or 90 min). The cells were examined for neuronal apoptosis 48 h after exposure to oxygen and glucose deprivation. Determination of neuronal apoptosis was by terminal deoxynucleotidyl transferase-mediated in situ nick-end labeling staining, as previously described; 3.0 MAC concentration of halothane ({blacksquare}); 3.0 MAC concentration of isoflurane ({blacktriangleup}); oxygen and glucose deprivation ({diamond}). The controls were those neurons exposed to media change alone, with no exposure to oxygen and glucose deprivation or volatile anesthetic. Data are represented as mean ± 95% confidence interval.

 
In the second series of experiments, exposure of cultured neurons to halothane significantly attenuated oxygen and glucose deprivation-induced neuronal apoptosis. This neuroprotective effect of halothane was demonstrated at concentrations as small as 1.6% (65% attenuation); moreover, at 3.2% and 5.4% concentrations of halothane, this effect of attenuation of oxygen and glucose-induced neuronal apoptosis was further enhanced (~100%) (Figs. 2–4). The effects produced by the various concentrations of halothane used were not statistically significant from one another (P < 0.05). The attenuation of cortical neuronal oxygen and glucose deprivation-induced neuronal apoptosis by halothane was independent of the time period of exposure to oxygen and glucose deprivation.

Exposure of cortical neurons to isoflurane showed a similar but less pronounced reduction in neuronal apoptosis as compared with halothane (Figs. 2–4). Treatment with 1.13% isoflurane elicited no effect in the number of apoptotic neurons as compared with oxygen and glucose deprivation alone at the time periods of lesser exposure to oxygen and glucose deprivation (30 min). Attenuation of neuronal apoptosis by 1.13% isoflurane was not demonstrated until 60 and 90 min of exposure to oxygen and glucose deprivation (Fig. 2). Attenuation of oxygen glucose deprivation-induced neuronal apoptosis by isoflurane was similar to halothane and statistically significant from exposure to oxygen and glucose deprivation alone at 2.2% and 3.3% concentrations. Oxygen glucose deprivation neuronal apoptosis was attenuated by >50% at 2.2% and by ~100% at 3.3%. This effect of isoflurane on hypoxia- and ischemia-induced neuronal apoptosis was independent of the time period of exposure to oxygen and glucose deprivation (Figs. 3 and 4).

The number of apoptotic neurons in the treated versus nontreated groups was statistically significant at all concentrations of halothane used (P < 0.05). The treatment effect, attenuation of neuronal apoptosis, was noted more at larger concentrations of isoflurane. Statistical differences of treatment with halothane versus isoflurane were noted more at the smaller concentration of halothane and isoflurane (P < 0.05). No statistical difference was seen between the two volatile anesthetics at larger concentrations (4.8% halothane and 3.3% isoflurane). When combining the independent factors, the duration of exposure to oxygen and glucose deprivation and the concentration of the administered volatile anesthetic were interrelated variables (P < 0.05). In addition, the effect of the administration of the volatile anesthetic and the concentration administered were covariables (P < 0.05).

These effects of halothane and isoflurane on hypoxia- and ischemia-induced neuronal apoptosis were confirmed by analyzing DNA fragmentation via gel electrophoresis. Exposure of cultured neurons to oxygen and glucose deprivation resulted in significant DNA fragmentation, as demonstrated by laddering, an effect that was attenuated by isoflurane and halothane.


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
This is the first study to investigate the effects of volatile anesthetics on neuronal apoptosis in the presence of oxygen and glucose deprivation. We describe a system for exposing cortical neuronal cultures to oxygen and glucose deprivation and have investigated the effects of oxygen and glucose deprivation on neuronal apoptosis in these cell cultures. Our data indicate that exposure of cultured neurons to oxygen and glucose deprivation for 30–90 minutes results in neuronal apoptosis. We have expanded this investigation to include the effect of pretreatment and continued treatment of these cell cultures during oxygen and glucose deprivation with halothane or isoflurane at three incremental concentrations each. Neuronal apoptosis is attenuated by both volatile anesthetics equally and significantly at the two larger concentrations. At the smallest concentration, oxygen and glucose deprivation was attenuated with halothane.

Cell cultures provide a simple, straightforward system for examining the direct effects of volatile anesthetics on cellular response to oxygen and glucose deprivation, outside the complex physiologic milieu of the intact brain (10,11,20). This is the first logical step to establish the importance of volatile anesthetics on neuronal apoptosis after oxygen and glucose deprivation at the cellular level and will help to establish variables for future study. This system allows for control of variables, such as CO2, oxygen, pH, and temperature, that cannot be easily obtained in an in vivo model (10,21,22). In addition, anesthetic administration in a live rodent model to allow for the experimental procedure in and of itself allows for only the relative effects of the volatile anesthetics to be examined. This in vitromodel also eliminates the variability observed in cerebral blood flow in treatment groups in an in vivo model. However, anesthetic variability in cerebral blood flow and mean arterial blood pressure, with resultant effects on neuronal apoptosis, is an important aspect to examine and cannot be determined from this model. However, future investigation in an intact system would be useful to further establish the effect of volatile anesthetics on oxygen and glucose deprivation-induced neuronal apoptosis.

Previous in vitromodels of cerebral ischemia to evaluate the neuroprotective effects of volatile anesthetics have not used cell survival as a mechanism to measure efficacy (23). Examinations for cell death, i.e., with necrosis as an end point, have used cultured neurons as in this investigation (10,11,20). Neurons and astrocytes demonstrate distinct patterns of vulnerability. Pure cortical neuronal cultures show the greatest injury after three hours of oxygen and glucose deprivation as compared with striatal and hippocampal neurons (24), thus the use of pure neuronal cultures for this investigation.

The method of oxygen and glucose deprivation is similar to that used in other models of in vitroinvestigation (10–12). The use of PBS pH 7.4, our artificial cerebrospinal fluid (aCSF), allowed for us to eliminate glucose and serum, which could serve as a source of lactate and thus confuse the results obtained from the study. All cell cultures had their original media removed and replaced with PBS regardless of exposure to oxygen and glucose deprivation. The glucose deprivation alone (control: media change but no exposure to hypoxia), was, in and of itself, not a cause for neuronal apoptosis. Most likely, the change in millimolar concentration of glucose was not significant enough, in and of itself, to cause neuronal apoptosis. Previous preparation for this experiment demonstrates that the cell culture media, DMEM, contains 15 mM glucose that decreases 1 mM/d (unpublished data). Thus, the glucose concentration on Day 10–14 is approximately 5–9 mM. This may not represent a significant change in glucose concentration when removed and replaced with PBS. It is also important to note that the original medium was returned to all the cell cultures, including the control. Variability in glucose concentration should not detract from the results of this experiment and can be demonstrated by the limited variability in the results (95% confidence limits).

The hypoxic conditions were created with a glucose-free aCSF bubbled with 95% nitrogen and 5% CO2 (10–12). Determination of oxygen content is not as previously described, but was consistently <50 mm Hg, because this is the lower limit of the I-Stat monitor. The hypoxic conditions were sustained with airtight plastic containers (Billups-Rothenberg) to create a hypoxic chamber housed within an incubator to maintain a consistent temperature of 37°C. The methodology could have been improved with the use of a Clark oxygen electrode. Regardless, the conditions provided by the methodology were able to initiate neuronal apoptosis during the time periods examined consistently, as demonstrated by the narrow confidence intervals.

The administration of volatile anesthetics was as described by Popovik et al. (14). End-tidal gas concentration was confirmed with gas chromatography and noted to be within 5% of the end-tidal gas concentration as displayed on the Datex monitor. Preparative work for this investigation by using gas chromatography demonstrated that a minimum of three hours at high flows was necessary to achieve the desired gas concentration in the cell media (DMEM) preparation. At lower flows, the desired gas concentration in the cell media (DMEM) could be achieved at 12 hours (unpublished data). Complications of this preliminary investigation were evaporative losses and changes in the pH of the media based on duration of exposure. This prompted the use of PBS, pH 7.4, as our aCSF in this investigation. The desired gas concentration could be achieved within 10 minutes by using high flow and maintained at low flows for the time periods examined.

Neuronal necrosis occurs within two to six hours in the presence of prolonged severe oxygen and glucose deprivation (90 minutes). However, rodents subjected to mild temporary focal ischemia (30 minutes) and global ischemia exhibit delayed neuronal death via apoptosis within 48–72 hours (2,16). Neuronal cell death is a continuum from necrosis to apoptosis. Neuronal apoptosis is a process denoted by endonuclease cleavage into 180-kd base pair fragments that can be detected by TUNEL staining (15,16). Some argue that TUNEL staining alone cannot differentiate the etiology of broken DNA, whether via apoptosis or necrosis (1). However, examination of cell morphology, such as cellular shrinkage and chromatin condensation, in addition to gel electrophoresis, further substantiates the presence of apoptosis (1,11). It is difficult to compare the results of our study with those of previous investigations for several reasons. Few investigations used cell cultures to look at neuronal injury (7,11,12). It is more important to note that that necrosis or infarct volume, a marker for early neuronal death, is the usual end point examined (1–7,10,11,20). Trypan blue staining or lactic dehydrogenase analysis would help to quantify neuronal necrosis in cell culture preparations, such as in this experiment (11,20). However, the end point for this investigation was neuronal apoptosis, which has not been investigated by others.

In a model of severe global ischemia, isoflurane at clinical concentrations, as used in this experiment, showed major decreases in histiologic damage, i.e., necrosis (6,7). Previous studies have shown selective delayed neuronal death with early attenuation (two days) by 1.5% isoflurane (5). However, when examined at two weeks, there was no difference between the two study groups examined. This model of forebrain ischemia would suggest that isoflurane does not attenuate neuronal apoptosis, but this is speculative. Our study, although in vitro, suggests differently. Furthermore, it is known that halothane offers protection when given during ischemia, but this is not true when halothane is administered after the insult (25). This investigation did not examine the effect of anesthetic neuroprotection in terms of neuronal apoptosis when the volatile anesthetic is administered after the onset of injury.

In conclusion, our investigation suggests that in the presence of oxygen and glucose deprivation, isoflurane and halothane attenuate neuronal apoptosis in a concentration-dependent manner. Protection is afforded at concentrations that are considered clinically relevant. Furthermore, the protective effects of volatile anesthetics, such as isoflurane and halothane, may extend beyond the initial interval of the original insult.


   Acknowledgments
 
This work was supported by a grant from the I. Heermann Anesthesia Foundation, Gainesville, FL.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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Accepted for publication July 20, 2001.




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A. W. Loepke, J. C. McCann, C. D. Kurth, and J. J. McAuliffe
The Physiologic Effects of Isoflurane Anesthesia in Neonatal Mice
Anesth. Analg., January 1, 2006; 102(1): 75 - 80.
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L. Wise-Faberowski, H. Zhang, R. Ing, R. D. Pearlstein, and D. S. Warner
Isoflurane-Induced Neuronal Degeneration: An Evaluation in Organotypic Hippocampal Slice Cultures
Anesth. Analg., September 1, 2005; 101(3): 651 - 657.
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L. Xiong, Y. Zheng, M. Wu, L. Hou, Z. Zhu, X. Zhang, and Z. Lu
Preconditioning with Isoflurane Produces Dose-Dependent Neuroprotection via Activation of Adenosine Triphosphate-Regulated Potassium Channels After Focal Cerebral Ischemia in Rats
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Lippincott, Williams & Wilkins Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins with the assistance of Stanford University Libraries' HighWire Press®. Copyright 2006 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press