The Effect of Propofol on Increased Superoxide Concentration in Cultured Rat Cerebrocortical Neurons After Stimulation of N-Methyl-d-Aspartate Receptors
2002-10-01

Neuronal cell death after hypoxia/ischemia, status epilepticus, or traumatic brain injury is at least, in part, because of over-activation of glutamate receptors and subsequent excessive calcium loading (1). This calcium deregulation activates a multitude of pathways involved in neuronal cell death, among which the stimulation of reactive oxygen species (ROS) production seems to be a key event. In neurons, mitochondria are a major source of ROS production and, at the same time, seem to be damaged by ROS. Thus, ROS-induced mitochondrial dysfunction seems to be implicated in glutamate neurotoxicity (2,3). ROS can be investigated in living neurons by using redox sensitive fluorochromes that are initially nonfluorescent but exhibit increasing fluorescence upon oxidation. In this study, we used the fluorochrome dihydroethidium acetoxymethylester (hydroethidin [HET]) for monitoring the superoxide anion concentration in cerebrocortical neurons. HET is specifically oxidized by superoxide anions to ethidium and thereby exhibits fluorescence that can be measured by confocal microscopy. The aim of this study was to investigate whether propofol reduces the net superoxide anion concentration in cultured rat forebrain neurons after sustained stimulation of N-methyl-d-aspartate (NMDA)-type glutamate receptors. Materials and Methods Primary neuronal cultures were obtained from the forebrains of embryonic Day-18 Sprague-Dawley rat embryos (Charles River, Sulzfeld, Germany). Cells were maintained at 37°C in 95% air/5% CO2 in a culture medium (vol/vol solution of 79% Dulbecco’s Modified Eagles Medium, 10% fetal calf serum, 10% horse serum, and 2 mM of L-glutamine) without antibiotics. Glia cell proliferation was stopped by adding 5 μM of cytosinarabinoside to the culture medium at Day in vitr. (DIV) 4. Experiments were performed between DIV 13 and DIV 22. At the day of the experiment, neurons were rinsed with HEPES-buffered salt solution ([HBSS]; 143 mM of NaCl, 3 mM of KCl, 2 mM of MgCl2, 4 mM of NaHC03, 0.5 mM of NaH2PO4, 15 mM of glucose, and 10 mM of HEPES, with a pH value of 7.4) before each experiment. HET was purchased from Molecular Probes (Leiden, the Netherlands), propofol from RBI (Deisenhofen, Germany), and Mn (III)tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP) from Calbiochem (Bad Soden, Germany). All other chemicals were obtained in the highest purity available. Confocal imaging experiments were conducted with a Biorad MRC 1024 laser scanning confocal microscope with a 100 mW argon ion laser (Biorad, Hemel Hempstead, Great Britain) coupled to an Olympus BX-50 fixed stage microscope (Olympus, Hamburg, Germany). Laser power was attenuated to 1% of maximal output power to prevent photobleaching and phototoxic damage to neurons under study. In each experiment, baseline fluorescence was assessed during the initial 3 min. Confocal images were obtained from areas of 512 × 512 pixels at 0.516 μm pixel size without any electronic zoom or digital filters. Typically, three to five individual neurons were studied from one coverslip in each experiment. Data from at least three different preparations were analyzed for controls and each propofol concentration. Individual recordings were corrected for background arising mainly from underlying glial cells by background subtraction and thresholding. Cells were loaded with the redox-sensitive nonfluorescent HET (1 μM) for 15 min at 37°C in HBSS and subsequently equilibrated for 10 min at room temperature. Within intracellular compartments, HET is oxidized specifically by superoxide, resulting in the strongly fluorescing dye ethidium. Coverslips were continuously superfused with HBSS in a perfusion chamber at a flow of 1.0 mL/min. HET (1 μM) was added to all superfusion solutions. Monochromatic excitation was performed at 514 nm and emitted fluorescence passed a 585-nm-long pass filter before photodetection at a sampling interval of 20 s. Because propofol is insoluble in water, a 10 mM stock solution of propofol in dimethyl sulfoxide was prepared before further dilution to appropriate concentrations (0.1–100 μM) in HBSS. Equivalent volumes of dimethyl sulfoxide were added to all controls. Furthermore, tetrodotoxine (0.5 μM) was added to the HBSS to exclude network-mediated excitation. For stimulation of NMDA-type glutamate receptors, 300 μM of NMDA and 10 μM of glycine were co-administered to the superfusion buffer from 180 to 360 s. All fluorescence intensities were normalized to baseline fluorescence. Baseline fluorescence was determined as the mean value of five consecutive recordings starting at 20 s after the beginning of the control period. For each experiment, data are presented as mean ± sem from 12–52 neurons from at least 3 different preparations. The effect of different propofol concentrations on superoxide concentration was calculated at 1200 s. Groups were compared using analysis of variance and post hoc Bonferroni test for multiple comparisons. Results Control experiments showed a slight increase in normalized ethidium fluorescence to 1.22 ± 0.02 at 1200 s (n = 34;Fig. 1). Neurons were exposed to an excitotoxic concentration of NMDA (300 μM) for 180 s. Exposure to NMDA (180–360 s) induced a marked fluorescence increase to 1.80 ± 0.05 (n = 52;P < 0.001).Figure 1: Effect of propofol on N-methyl-d-aspartate (NMDA)-induced dihydroethidium acetoxymethylester (hydroethidin [HET]) oxidation as an indicator for cellular superoxide concentration. Neurons were loaded with 1 μM of HET (see Materials and Methods). Propofol was added to the superfusion buffer from the beginning of the experiment. After the initial 180-s baseline recording, 300 μM of NMDA and 10 μM of glycine were co-administered for 180 s (see bar). (A) Timecourse of ethidium fluorescence. (1) Control (hairline), (2) 300 μM of NMDA and 10 μM of glycine (hairline, intersected), and (3) effect of propofol (10 μM) on NMDA-induced fluorescence increase (bold line). Data are given as mean traces. (B) Concentration dependence of propofol-mediated effect on NMDA-induced fluorescence increase at 1200 s. Data are given as mean ± sem.Figure 1A shows the effect of propofol (10 μM) on the timecourse of NMDA-induced ethidium fluorescence increase. Attenuation of NMDA-induced ethidium fluorescence increase by propofol is shown in Figure 1B. Propofol (0.1–100 μM) attenuated the NMDA-induced fluorescence increase concentration dependently at 1200 s: 1.63 ± 0.06 (propofol 0.1 μM;n = 22; not significant), 1.50 ± 0.05 (propofol 1 μM;n = 12;P < 0.05), 1.37 ± 0.09 (propofol 10 μM;n = 13;P < 0.001), and 1.30 ± 0.05 (propofol 100 μM;n = 29;P < 0.001). To confirm that NMDA-induced HET oxidation was indeed induced by enhanced superoxide formation, superoxide dismutation was increased by MnTBAP (50 μM). Addition of this membrane-permeable superoxide dismutase mimetic resulted in a reduction of NMDA-induced fluorescence increase to 1.40 ± 0.07 (n = 27;P < 0.01). Discussion In neurons, mitochondria are a major source of superoxide anions (4) that originate from the one-electron reduction of molecular oxygen within the respiratory chain (5). Because propofol exerts neuroprotective properties in different experimental settings (6,7), the aim of this study was to investigate whether propofol reduces the net superoxide anion concentration in cultured rat forebrain neurons after sustained stimulation of ionotropic glutamate receptors. In our experiments, the superoxide-specific dye HET was added to all solutions at a small concentration (1 μM) to prevent artificial fluorescence changes caused by increasing quantum efficiency by potential-dependent dye release from mitochondria into the cytosol and subsequent binding to cytosolic and nuclear polynucleotides (8). We confirmed that NMDA-induced HET oxidation was indeed caused by enhanced superoxide formation by increasing superoxide dismutation with MnTBAP. Addition of this membrane-permeant manganic porphyrine resulted in attenuation of NMDA-induced HET oxidation, revealing clearly that the fluorescence increase was caused by superoxide anions. Control experiments showed a slight increase in ethidium fluorescence, reflecting HET oxidation by superoxide produced under physiological conditions. Exposure to NMDA for 180 s induced a pronounced increase in ethidium fluorescence, which is indicative of superoxide formation. This corresponds to previous experiments on hippocampal neurons (9). Propofol reduced the increase of superoxide anion concentration dose dependently when neurons were exposed to NMDA for 180 s. Even small concentrations of propofol (1 μM) attenuated the NMDA-induced increase in superoxide anions significantly. This seems to be important under clinical conditions because the concentration range of propofol used in this study (0.1–100 μM) covers pharmacologically relevant concentrations for the induction and maintenance of anesthesia (10). Propofol exerts neuroprotective effects through several mechanisms, e.g., activation of γ-aminobutyric acidA receptors (7) or a reduction of intracellular Ca2+ concentration (11). Propofol has antioxidative properties (12) and can scavenge free radical species by a process of hydrogen abstraction (13). This report shows that propofol additionally reduces cellular superoxide concentration after stimulation of NMDA receptors and thereby interacts with cellular ROS production at an early stage. The authors wish to thank Anneliese Benker, Kati Breiter, Judit Finn, and Franz Tokai for excellent technical assistance.