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Anesth Analg 2003;96:524-531
© 2003 International Anesthesia Research Society

NEUROSURGICAL ANESTHESIA

The Effect of the {alpha}2-Agonist Dexmedetomidine and the N-Methyl-D-Aspartate Antagonist S(+)-Ketamine on the Expression of Apoptosis-Regulating Proteins After Incomplete Cerebral Ischemia and Reperfusion in Rats

Kristin Engelhard, MD*, Christian Werner, MD*, Eva Eberspächer, DVM*, Monika Bachl, MD*, Manfred Blobner, MD*, Eberhard Hildt, PhD{dagger}, Peter Hutzler, PhD{ddagger}, and Eberhard Kochs, MD*

*Klinik für Anaesthesiologie and {dagger}Experimentelle Onkologie und Therapieforschung, Technische Universität München, Klinikum Rechts der Isar; and {ddagger}Institut für Pathologie der GSF-Forschungszentrum für Umwelt und Gesundheit, Munich, Germany

Address correspondence and reprint requests to Kristin Engelhard, MD, Klinik für Anaesthesiologie der Technischen Universität München, Klinikum Rechts der Isar, Ismaninger Straße 22, 81675 München, Germany. Address e-mail to k.engelhard{at}lrz.tum.de


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
In this study, we investigated whether the neuroprotection previously seen with dexmedetomidine or S(+)-ketamine involves regulation of proapoptotic (Bax and p53) and antiapoptotic (Bcl-2 and Mdm-2) proteins. Rats were anesthetized with isoflurane. After surgical preparation of isoflurane was discontinued, animals were randomly assigned to receive fentanyl and nitrous oxide (N2O)/oxygen plus 100 µg/kg of dexmedetomidine intraperitoneally 30 min before ischemia (n = 8), 1 mg · kg-1 · min-1 of S(+)-ketamine and oxygen/air (n = 8), or fentanyl and N2O/oxygen (n = 8; control group). In all three treatment groups, incomplete cerebral ischemia (30 min) was induced by unilateral carotid artery occlusion and hemorrhagic hypotension to a mean arterial blood pressure of 30–35 mm Hg. Four hours after the start of reperfusion, the brains were removed, and the expression of apoptosis-regulating proteins was determined by using immunofluorescence and Western blot analysis. The results were compared with sham-operated animals (n = 8). After cerebral ischemia/reperfusion, the relative protein concentration of Bax was increased by 110% in control animals compared with the dexmedetomidine- and S(+)-ketamine-treated rats and by 140% compared with the sham-operated animals. In animals treated with dexmedetomidine, the expression of Bcl-2 and Mdm-2 was larger compared with control (68% and 210%, respectively) or sham-operated (110% and 180%, respectively) animals. Therefore, it is possible that the neuroprotective properties of dexmedetomidine and S(+)-ketamine seen in previous studies involve ultra-early modulation of the balance between pro- and antiapoptotic proteins.

IMPLICATIONS: This study shows that dexmedetomidine and S(+)-ketamine influence the expression of apoptosis-regulating proteins in rat brains 4 h after cerebral ischemia/reperfusion. Therefore, it is possible that the neuroprotection seen with dexmedetomidine and S(+)-ketamine might also involve antiapoptotic mechanisms in addition to reducing necrotic cell death.


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
The {alpha}2-receptor agonist dexmedetomidine and the N-methyl-D-aspartate (NMDA)-receptor antagonist S(+)-ketamine are used for patients with intracranial lesions. Studies using an animal model of incomplete cerebral ischemia and reperfusion have shown that both drugs reduce necrosis and improve neurological outcome (1–3). Decreased sympathetic tone and inhibition of NMDA receptor-mediated ion currents were believed to mediate the reduction of necrotic cell death. However, several studies have shown that activation of adrenergic receptors by catecholamines or activation of NMDA receptors induces not only necrotic, but also apoptotic, cell death (4–6). Therefore, it is possible that the {alpha}2-receptor agonist dexmedetomidine and the NMDA-receptor antagonist S(+)-ketamine also mediate neuroprotection by apoptosis-related mechanisms. This study investigated the effects of dexmedetomidine and S(+)-ketamine on the expression of apoptosis-regulating proteins in the rat brain after incomplete cerebral ischemia and reperfusion.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
After animal care committee approval, 32 male Sprague-Dawley rats (Charles River, Germany; 300–420 g) were anesthetized in a bell jar saturated with isoflurane. The trachea was intubated and the lungs mechanically ventilated (PaCO2, 38–42 mm Hg) with 1.5 vol% isoflurane in nitrous oxide (N2O) and oxygen (fraction of inspired oxygen [FIO2], 0.33). Catheters were inserted into the right femoral artery and vein and into the right jugular vein for blood withdrawal, administration of drugs, and blood sampling. A loose ligature was placed around the right common carotid artery for later clamping. The rats were then placed in a stereotactic U-frame (Model 962; David Kopf Instruments, Tujunga, CA) by using atraumatic ear bars. After incision of the scalp, nonpenetrating burr holes were drilled into the cranium 0.5 mm anterior and 1 mm lateral of the bregma for continuous measurement of red blood cell flow velocity (CBFV) with a laser Doppler flowmeter (PeriFlux System 4001 Master; Perimed, Järfälla, Sweden). Laser Doppler flowprobes (Probe 403; Perimed) were placed over both hemispheres. Care was taken to place the probes over a tissue area devoid of large blood vessels visible through the thinned bone. The correct position of the laser Doppler probes was confirmed by increased CBFV in response to transient hypoventilation. Local CBFV was expressed in arbitrary "perfusion units" sampled over 0.3 s. Pericranial temperature was measured with a 22-gauge stainless-steel needle thermistor (Model 73A, YSI temperature controller; Yellow Springs Instruments Co., Inc., Yellow Springs, OH) placed beneath the right temporal muscle and was maintained constant at 37.5°C throughout the experiment by a servomechanism using an overhead heating lamp and a heating pad. On completion of the surgical preparation, the ear bars of the stereotactic frame were slightly released, and all surgical incisions were infiltrated with 0.5% bupivacaine.

At the end of the preparation, isoflurane was discontinued, and animals were randomly assigned to one of the following treatment groups. In the dexmedetomidine group (n = 8), anesthesia was maintained with IV fentanyl (bolus, 10 µg/kg; infusion, 25 µg · kg-1 · h-1) and N2O in oxygen (FIO2, 0.33), and 100 µg/kg of dexmedetomidine was administered intraperitoneally 30 min before ischemia. In the S(+)-ketamine group (n = 8), the rats received 1 mg · kg-1 · min-1 of S(+)-ketamine IV and oxygen/air (FIO2, 0.33). In the control group (n = 8), anesthesia was maintained with IV fentanyl (bolus, 10 µg/kg; infusion, 25 µg · kg-1 · h-1) and N2O in oxygen (FIO2, 0.33). In the sham-operated group (n = 8), rats received IV fentanyl (bolus, 10 µg/kg; infusion, 25 µg · kg-1 · h-1) and N2O in oxygen (FIO2, 0.33) and sham treatment, i.e., complete instrumentation without cerebral ischemia. During the study, vecuronium was given as a continuous infusion (0.1 mg · kg-1 · min-1) to maintain neuromuscular blockade. After an equilibration period of 45 min, cerebral ischemia was induced in the dexmedetomidine, S(+)-ketamine, and control groups by hemorrhagic hypotension and clip occlusion of the right common carotid artery. The withdrawal of blood was performed within 8 min. Mean arterial blood pressure was maintained within the range of 30–35 mm Hg to reduce CBFV in the ischemic hemisphere by 70%. After 30 min of cerebral ischemia, the clip was released, and shed blood was reinfused over 15 min. Throughout the experiment, arterial pH and PaCO2 were maintained at physiologic levels by IV infusion of bicarbonate and adjustment of mechanical ventilation. Arterial blood gases and plasma glucose concentrations were analyzed at the following times: 1) baseline: before ischemia was induced; 2) ischemia: at the end of ischemia; 3) reperfusion: after reperfusion of the withdrawn blood; and 4) recovery: 90 min after the start of reperfusion. Four hours after the start of reperfusion, the brains were removed and placed in tissue-freezing medium (Tissue Freezing Medium; Jung Leica Instruments, Nussloch, Germany), frozen in isopentane on dry ice, and stored at -70°C. The brains were then cut into 7-µm slices and mounted on slides for immunofluorescence analysis. Two slices of 90 µm, separated for hemispheres, were provided for the Western blot analysis.

For immunofluorescence staining, the slides were fixed with 100% ice-cold ethanol for 10 min, followed by 30 min washing period using phosphate-buffered saline with 0.1% Tween® 20 (PBST, polyoxyethylensorbitolmonolaurat; Fulka Chemica, Buchs, Switzerland). During the following 60 min, the slides were incubated in blocking buffer (10% fetal calf serum in PBST) to prevent nonspecific binding of the antibodies. Thereafter, the slices were incubated with the first antibody (rabbit polyclonal antibodies; Santa Cruz Biotechnology, Santa Cruz, CA) for 60 min by using Bax-specific antisera (I-19 and P-19; dilution 1:80 each), Bcl-2-specific antiserum ({Delta}C21; dilution 1:60), and p53-specific antiserum (FL-393; dilution 1:80). Subsequently, the slides were washed with PBST for 40 min and incubated for 60 min with the second antibody (Alexa Fluor® 488 goat anti-rabbit immunoglobulin [Ig]G antibody; Molecular Probes, Leiden, Netherlands) in a dilution of 1:800 in PBST. After another washing procedure for 40 min with PBST, the slides were covered with mounting medium (Vecta- shield® H-1000; Vector Laboratories, Burlingame, CA) and coverslips and stored at 4°C. For each protein, negative controls were performed by omitting the first antibody. Slices of the liver were used as a positive control because the proteins Bax, Bcl-2, and p53 are always expressed in this tissue. Within the next 24 h, the immunofluorescence intensity of the proteins (two slices per protein) in the hippocampal regions of the ipsilateral and contralateral hemisphere was recorded with a confocal laserscan microscope (LSM 410; Carl Zeiss, Jena, Germany) by using 488-nm excitation and emission filter BP1 515–565 nm. The constant intensity of the laser light was controlled with a powermeter, and the sensitivity of the detector of the microscope was controlled with a fluorescence standard (InSpeck® Green I-7219 Microscope Image Intensity Calibration Kit; Molecular Probes). Images were evaluated with the KS400 software (Carl Zeiss Vision, Hallbergmoos, Germany), which determined the mean intensity of immunofluorescence (gray levels) in the hippocampal neurons; this is proportional to the concentration of the fluorescence marker and therefore is proportional to the mean protein concentration in the hippocampal cells.

Two 90-µm slices (separated for hemispheres) were homogenized in lysis buffer containing protease inhibitors (Complete® Mini; Boehringer, Mannheim, Germany) for Western blot analysis. After sonification and centrifugation at 4°C, 250 µL of the lysate was collected. The protein concentrations were measured by using the method of Bradford (7) and equalized. Subsequently, the lysates were boiled at 95°C for 5 min in sodium dodecyl sulfate loading buffer. Twenty-microgram protein samples were loaded on polyacrylamide gels. To enable comparison between different blots, a custom-made standard was loaded three times in each blot. The standard was made of three rat brains that were processed in a manner similar to the 90-µm brain slices, providing sufficient standard for the entire investigation. The Rainbow RPN 756 protein molecular weight marker (Amersham Life Science Inc., Piscataway, NJ) was used to facilitate the identification of protein bands. After electrophoresis (200 V for 3.5 h), the proteins were transferred from the gel to a polyvinylidene fluoride membrane (0.8 mA/cm2). After membranes were washed in PBST for 30 min they were immersed in blocking buffer (10% fetal calf serum in PBST for analysis of Bax and p53 and 10% nonfat dry milk in PBST for analysis of Bcl-2 and Mdm-2) and then incubated with the first antibody (Santa Cruz Biotechnology; diluted 1:200 in blocking buffer) for 2 h at room temperature (Bax: P-19 rabbit polyclonal IgG; Bcl-2: C-2 mouse monoclonal IgG; p53: DO-1 mouse monoclonal IgG; Mdm-2: C-18 rabbit polyclonal IgG). After incubation, the membranes were washed with PBST for 45 min. For the next 90 min, the membranes were incubated with the second antibody and conjugated with horseradish peroxidase (Amersham Life Science Inc., dilution 1:2000). Membranes for Bcl-2 and p53 analysis were incubated with the anti-mouse antibody (NA 931), and membranes for Bax and Mdm-2 analysis were incubated with the anti-rabbit antibody (NA 934). Subsequently, the membranes were washed with PBST for 45 min. Detection of the proteins was performed by chemoluminescence by using the ECL® Western blotting detection system (Amersham Life Science Inc.), which darkens a photosensible film (Hyperfilm ECL; Amersham Life Science Inc.). The grayscales are directly proportional to the concentration of the conjugated peroxidase and, therefore, to the protein concentration in the Western blot membrane of each band. Grayscale quantification in the single bands was performed with SigmaScan software (Jandel Scientific Software, San Rafael, CA). For comparison between different blots, protein band intensities are presented as the percentage change from a custom standard for each blot. To control the protein transfer from the polyacrylamide gel to the polyvinylidene fluoride membrane, the membranes were stained with Coomassie Brilliant Blue R 250 (Fluka, Neu-Ulm, Germany). In control experiments, Bax and Mdm-2 primary antibodies were preabsorbed by specific blocking peptides (Santa Cruz Biotechnology) to prove the specificity of the antibodies. For Bcl-2 and p53, these blocking agents were not available. Therefore, purified proteins of Bcl-2 and p53 (Santa Cruz Biotechnology) were loaded to control the antibody.

To address the multiple comparisons, a hierarchical statistical model based on consecutively calculated repeated-measurement analyses of variance and Student’s t-tests was chosen. Post hoc analyses were restricted on special hypotheses. Animals treated with dexmedetomidine or S(+)-ketamine were separately compared with control and sham-operated animals. Therefore, the level of significance was corrected (P < 0.05/2 = 0.025). All variables are presented as mean ± SD. Statistical analyses were performed with SPSS 10.0 for Windows (SPSS Inc., Chicago, IL).


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Table 1 shows the physiological variables before, during, and after ischemia. There were no differences in mean arterial blood pressure, PaO2, PaCO2, and plasma glucose concentration between control animals and animals treated with dexmedetomidine or S(+)-ketamine. In sham-operated animals, physiological variables did not change over time.


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  		Table 1. Physiologic Variables
 
Figure 1 shows cortical CBFV in the ipsilateral (Fig. 1a) and the contralateral (Fig. 1b) hemisphere. There were no differences in CBFV between control animals and animals treated with dexmedetomidine or S(+)-ketamine in both hemispheres. During cerebral ischemia, CBFV was reduced to 30% of baseline in the ischemic hemisphere and to 40%–60% of baseline in the contralateral hemisphere. CBFV in sham-operated animals was constant over time.



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  		Figure 1. Cortical cerebral blood flow velocity measured by laser Doppler flowmetry over the ipsilateral (a) and the contralateral (b) hemisphere. In both hemispheres, cerebral blood flow was not different between animals of the control group and animals treated with dexmedetomidine or S(+)-ketamine; *P < 0.05 compared with the control group during ischemia; §P < 0.05 compared with sham-operated animals during ischemia; #P < 0.05, baseline versus ischemia within the control group, dexmedetomidine group, and S(+)-ketamine group.

 
Figure 2 shows the results of the immunofluorescence analysis. In the ipsilateral hemisphere of control animals, Bax (Fig. 2a) was increased by 110% compared with dexmedetomidine- and S(+)-ketamine-treated rats and was 140% larger compared with sham-operated animals. The concentration of p53 (Fig. 2b) was not different among groups. In the ipsilateral hemisphere of dexmedetomidine-treated animals, the concentration of Bcl-2 (Fig. 2c) was 68% larger compared with control rats and 110% larger compared with sham-operated animals. For all proteins, there were no differences between hemispheres.



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  		Figure 2. Concentration of the proapoptotic proteins Bax (a) and p53 (b) and the antiapoptotic protein Bcl-2 (c) in the hippocampus of the ipsilateral and contralateral hemisphere evaluated by immunofluorescence analysis 4 h after the onset of reperfusion (*P < 0.05 versus the control group; §P < 0.05 versus sham-operated animals).

 
Figure 3 shows the results of the Western blot analysis. In control animals, the concentration of Bax (Fig. 3a) was 150% larger compared with dexmedetomidine, 250% larger compared with S(+)-ketamine, and 74% larger compared with sham-operated animals. With dexmedetomidine, the intensity of Mdm-2 (Fig. 3d) was 210% and 180% larger compared with control and sham-operated animals, respectively. For the proteins p53 (Fig. 3b) and Bcl-2 (Fig. 3c), no differences between groups were found. There were no differences between hemispheres for all proteins. During the Western blot analysis of p53 and Mdm-2 in the group treated with S(+)-ketamine, technical problems occurred, resulting in a poor quality of the blots. We repeated the blots several times until the protein lysate of the brain was used up. Therefore, the concentration of p53 and Mdm-2 was not measured by Western blot analyses in this group.



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  		Figure 3. Concentration of the proapoptotic proteins Bax (a) and p53 (b) and the antiapoptotic proteins Bcl-2 (c) and Mdm-2 (d) in the ipsilateral and contralateral hemisphere evaluated by Western blot analysis 4 h after the onset of reperfusion (*P < 0.05 versus control animals; §P < 0.05 versus sham-operated animals). Analysis of p53 and Mdm-2 was not performed in animals treated with S(+)-ketamine.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
These data show that incomplete cerebral ischemia/reperfusion induces changes in apoptosis-regulating proteins as early as four hours after the start of reperfusion. In control animals, which were unprotected against the ischemic insult, the proapoptotic protein Bax was expressed in a larger concentration compared with the nonischemic, sham-treated animals. In contrast, the neuroprotective drugs dexmedetomidine and S(+)-ketamine inhibited this increase of Bax. Additionally, dexmedetomidine increased the concentration of the antiapoptotic proteins Bcl-2 and Mdm-2 compared with control animals. Because dexmedetomidine and S(+)-ketamine have been reported to reduce the extent of necrosis and improve neurological outcome (1–3), these data suggest that the neuroprotective effects of dexmedetomidine and S(+)-ketamine may be related to both antinecrotic and antiapoptotic mechanisms.

The severity of cerebral ischemia determines whether neurons die of necrosis or maintain sufficient energy to induce apoptosis. Several proteins have been identified that regulate apoptosis. One of these proteins is the 21-kd protein Bax, a promoter of apoptosis. In contrast, Bcl-2, a protein that was first isolated in B-cell lymphomas (8), acts as a repressor of programmed cell death (apoptosis). In vitro and in vivo studies have shown that Bax and Bcl-2 homodimerize or build heterodimers with each other (9). When Bax homodimers are the dominating protein cluster, they can counter the death repressor activity of Bcl-2, and apoptotic cell death is accelerated. When Bcl-2 homodimers are in excess, cells are protected (resistant to stress) (9). Bax and Bcl-2 are localized on the outer mitochondrial membrane, and both of these proteins regulate the permeability transitions of mitochondrial membranes, which leads to a release of proapoptotic protease activators such as cytochrome c or the apoptosis-inducing factor from the mitochondria into the cytosol (10,11). Overexpression of Bax induces mitochondrial permeability, whereas Bcl-2 overexpression inhibits mitochondrial permeability (12,13). These observations were confirmed by experiments showing that overexpression of Bcl-2 protects primary murine astrocyte cultures against glucose deprivation (14). Likewise, transduction of Bcl-2 by a herpes simplex virus amplicon into the hippocampus of gerbils 24 hours before transient global cerebral ischemia reduces hippocampal neuronal damage (15). In this study, Bax was increased at four hours after the start of reperfusion from incomplete cerebral ischemia in control animals. This is consistent with experiments in gerbils in which Bax was increased six hours after transient global ischemia while Bcl-2 protein levels remained constant (16). Likewise, rats subjected to permanent middle cerebral artery occlusion developed increased concentrations of the death-effector Bax in parallel with a reduction of the death-repressor Bcl-2 (17). In this study, dexmedetomidine and S(+)-ketamine were able to suppress the increase of Bax after cerebral ischemia/reperfusion. Additionally, dexmedetomidine, but not S(+)-ketamine, increased the concentration of the death-suppressing protein Bcl-2. These data suggest that the neuroprotection previously seen with dexmedetomidine and S(+)-ketamine at three days from ischemia/reperfusion may be mediated by changes in the balance of these apoptosis-regulating proteins.

Both cerebral ischemia excitotoxicity and DNA damage caused by oxygen free radicals can induce the tumor suppressor gene p53. A study in M1 myeloid leukemia cells has shown that the p53 protein, which normally regulates cell-cycle and DNA repair, upregulates Bax expression and downregulates Bcl-2 expression (18,19). This suggests that p53-induced alterations of Bax and Bcl-2 expression may be a major factor in p53-induced apoptosis. In support, experiments in rats subjected to focal cerebral ischemia have shown an overexpression of p53 in apoptotic cells (20,21). Likewise, studies in p53 knockout mice revealed decreased histopathological damage after methamphetamine-induced neurotoxicity (22), and selective inhibition of gene expression of p53 with antisense oligonucleotides protects neurons from excitotoxic death (23). In this study, the p53 protein concentration was not affected in any group at four hours from injury. This may be related to the short postischemic observation period. In contrast, Mdm-2 was increased in animals treated with dexmedetomidine. Mdm-2 inhibits p53 by concealing the activation domain of p53 and thereby directly controlling the p53-regulated gene expression (24,25). Therefore, modulation of p53 activity by Mdm-2 may be a cofactor in dexmedetomidine-induced neuroprotection.

The immunofluorescence and Western blot analyses showed consistent results for changes in the Bax protein. However, the immunofluorescence analysis detected changes in Bcl-2, whereas the Western blot analysis did not. This may be related to different regional specificities of the investigated brain-tissue samples. The immunofluorescence analysis evaluates the protein intensity in the hippocampal region, which is very sensitive to ischemic damage and therefore shows a strong reaction of apoptosis-regulating proteins. In contrast, brain slices prepared for Western blot analysis additionally contained cortex and basal ganglia, both of which are less sensitive to ischemic damage. Thus, changes of hippocampal apoptosis-regulating proteins might be veiled by proteins from regions that are less sensitive to ischemia-induced changes in apoptotic proteins.

In this model of incomplete cerebral ischemia, reperfusion changes in the concentration of apoptosis-regulating proteins were also evident in the contralateral hemisphere. The absence of differences between the hemispheres relates to the fact that hemorrhagic hypotension (as part of this ischemia model) reduced CBFV in the contralateral hemisphere to at least 40%–60% of baseline values. Although this magnitude of ischemia has not been intense enough to cause necrosis in the contralateral hemisphere three days after the insult (26), it apparently induces changes in the expression pattern of pro- and antiapoptotic proteins.

The purpose of this study was to investigate whether the neuroprotection observed with dexmedetomidine and S(+)-ketamine in previous investigations (1–3) using the same ischemia model involves changes in apoptosis-related proteins. To enable comparison between this investigation and previous investigations, we repeated the study protocol with respect to the background anesthetic technique (fentanyl/N2O) and the dosages of dexmedetomidine and S(+)-ketamine.

In conclusion, incomplete cerebral ischemia/reperfusion induces apoptotic processes at four hours after the start of reperfusion. This effect can be prevented by dexmedetomidine and S(+)-ketamine. Additionally, dexmedetomidine increased the concentration of the anti-apoptotic proteins Bcl-2 and Mdm-2. Although the neuroprotection seen with dexmedetomidine and S(+)-ketamine has traditionally been attributed to a reduction in necrotic cell death due to decreased sympathetic tone or NMDA-receptor antagonism, this study shows, for the first time, that neuroprotection mediated by the {alpha}2-agonist dexmedetomidine and the NMDA-antagonist S(+)-ketamine may also involve apoptosis-regulating proteins.


   Acknowledgments
 
Supported by a grant from Else Kröner-Fresenius-Stiftung, Bad Homburg v.d. Höhe, Germany, and by a grant from Abbott GmbH, Wiesbaden, Germany.

The authors would like to thank Doris Droese (medical technician, Klinik für Anaesthesiologie, Technische Universität München, Munich, Germany), Anne Frye (medical technician, Klinik für Anaesthesiologie, Technische Universität München, Munich, Germany), Ernst Mannweiler, PhD (computer scientist, Institut für Pathologie der GSF, Munich, Germany), and Ute Reuning, PhD (biologist, Frauenklinik, Technische Universität München, Munich, Germany) for their expertise and technical assistance.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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Accepted for publication October 9, 2002.




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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