The intravenous anaesthetic 2,6-diisopropylphenol (propofol) has been shown to exert neuroprotective effects with in vivo [1,2] and in vitro [3-5] studies. Regarding the mechanisms of neuroprotective action, it was recently reported that propofol can reduce intracellular superoxide concentration after sustained stimulation of N-methyl-D-aspartate (NMDA) receptors in rat cerebrocortical neurons . The occurrence of superoxide anions under pathophysiological conditions is a consequence of intracellular Ca2+ influx following sustained activation of NMDA receptors . Since the elevation of intracellular Ca2+ is a key event in the excitotoxic cell death cascade, this study was designed to investigate the effects of propofol on NMDA-induced increase of intracellular Ca2+.
Primary neuronal cultures were obtained from the forebrains of embryonic day-18 Sprague-Dawley rat embryos (Charles River, Sulzfeld, Germany) as previously described . Cortical lobes were incubated in 0.05% trypsin in phosphate-buffered saline, triturated and passed through a 40 μm cell mesh. Cells were plated at a density of 1.8 × 105 cells/cm2 onto poly-D-lysine (100 μg mL−1) and laminine (1.1 μg mL−1) coated coverslips. Cells were maintained at 37°C in 95% air/5% CO2 in culture medium (vol/vol solution of 79% Dulbecco's Modified Eagles Medium, 10% fetal calf serum, 10% horse serum, 2 mmol L-glutamine) without antibiotics. Glia cell proliferation was stopped by adding of 5 μmol cytosinarabinoside to the culture medium after 4 days in vitro. Experiments were performed on neurons between 13 and 22 days in vitro. At the day of experiment, neurons were rinsed with N-2-hydroxyethylpiperazine-N-2-ethane-sufonic acid (HEPES)-buffered salt solution (143 mmol NaCl, 3 mmol KCl, 2 mmol MgCl2, 4 mmol NaHCO3, 0.5 mmol NaH2PO4, 15 mmol glucose, 10 mmol HEPES and pH 7.4) before each experiment.
Calcium green-5N-acetoxymethylester (CG-5N) was purchased from Molecular Probes (Leiden, Netherlands) and propofol from RBI (Deisenhofen, Germany). All other chemicals were acquired commercially in highest purity available. Since propofol is insoluble in water, a 10 mmol stock solution of propofol in dimethyl sulfoxide was prepared before further dilution to appropriate concentrations (0.1-100 μmol) in HEPES-buffered salt solution. Equivalent volumes of dimethyl sulfoxide were added to all controls. Furthermore, tetrodotoxine (0.5 μmol) was added to the HEPES-buffered salt solution to exclude network-mediated neuronal excitation . NMDA-induced responses were pharmacologically isolated by blocking several postsynaptic receptor types. Neurons were exposed to NMDA (300 μmol) and glycine (10 μmol) in the presence of 6-cyano-7-nitroquinoxaline-2,3-dione (10 μmol) to exclude activation of alpha-amino-3-hydroxy-5-methyl-4 isoxazole propionic acid-type glutamate receptors . Bicuculline (10 μmol) was added to the superfusion solution to prevent effects of propofol exerted through gamma-amino-butyric acid (GABAA) receptors .
Confocal imaging experiments were conducted with a Biorad MRC 1024 laser scanning confocal microscope with an 100 mW argon ion laser (Biorad, Hemel Hempstead, Great Britain) coupled to an Olympus BX-50 fixed stage microscope (Olympus, Hamburg, Germany). Samples were recorded at 2 s intervals. 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 analysed for controls and each propofol concentration. Individual recordings were corrected for background arising mainly from underlying glial cells by background subtraction and thresholding. For sustained stimulation of NMDA-type glutamate receptors, 300 μmol NMDA and 10 μmol glycine were co-administered to the superfusion buffer from 180 to 360 s. Data acquisition was stopped at 900 s.
Cytosolic calcium changes were monitored using the low affinity calcium probe CG-5N (Kd = 4 μmol), which has been proven useful for measuring large changes in intraneuronal calcium changes as they occur following sustained agonist stimulation of NMDA receptors. Cells were loaded with 5 μmol CG-5N for 45 min at 37°C in HEPES-buffered salt solution. After dye loading, fluorochrome concentration was allowed to equilibrate at room temperature for 30 min. Monochromatic excitation was performed at 488 nm, emitted fluorescence passed a 540/30 nm bandpass filter before photodetection. Neurons were visually identified as previously described .
All fluorescence intensities were normalized to baseline fluorescence, F0. F0 was determined as the mean value of five consecutive recordings starting at 20 s after beginning of the control period. For each experiment, data are presented as mean ± standard error (SE) from 10-35 neurons of at least three different preparations of mother rat animals. The effects of different propofol concentrations on cytoplasmic calcium level was calculated at the peak CG-5N fluorescence, reflecting peak calcium concentration (400 s) after stimulation with NMDA. Groups were compared using analysis of variance (ANOVA) and post hoc Bonferroni's test for multiple comparisons.
Figure 1 shows the increase in CG-5N fluorescence caused by application of 300 μmol NMDA. For better clarity, the data are given as the mean trace from 35 recordings. The increase of CG-5N fluorescence is indicative of an increase in intracellular calcium. Exposure to NMDA induced a fast fluorescence increase to 2.71 ± 0.14 (n = 35) at 400 s followed by a slow fluorescence decay. Preincubation with the non-competitive NMDA receptor antagonist mitogen-activated protein kinase (MK) 801 (10 μmol) completely blocked the increase in fluorescence (1.07 ± 0.02, P < 0.001, n = 13).
In Figure 2, we evaluated the implications of the extracellular Ca2+ concentration on the increase of intracellular Ca2+ as a consequence of exposure to 300 μmol NMDA. Reducing the extracellular calcium concentration from 3 mmol to 10 nmol resulted in a concentration-dependent decrease of CG-5N fluorescence: 2.22 ± 0.07 (200 μmol Ca2+, P < 0.05, n = 15), 1.85 ± 0.04 (10 μmol Ca2+, P < 0.01, n = 8), 1.24 ± 0.04 (1 μmol Ca2+, P < 0.001, n = 14), 1.12 ± 0.03 (100 nmol Ca2+, P < 0.001, n = 15) and 1.09 ± 0.05 (10 nmol Ca2+, P < 0.001, n = 15).
The concentration-dependent effects of propofol on the increase in CG-5N fluorescence in cultured neurons are presented in Figure 3. Addition of propofol to the superfusion buffer resulted in a concentration-dependent reduction of NMDA-induced fluorescence increase: 2.71 ± 0.15 (0.1 μmol propofol, not significant (n.s.), n = 18), 2.68 ± 0.12 (1 μmol propofol, n.s., n = 15), 2.57 ± 0.13 (3 μmol propofol, n.s., n = 10), 2.15 ± 0.09 (10 μmol propofol, P < 0.05, n = 15), 1.84 ± 0.12 (30 μmol propofol, P < 0.01, n = 10) 1.82 ± 0.10 (100 μmol propofol, P < 0.001, n = 15). Analysis of the concentration-response relationship revealed an IC50 value for propofol of 9.0 μmol (R2 = 0.988). The capacity of propofol was limited, since even high concentrations of propofol (100 μmol) diminished calcium rise by only 50%.
Excitotoxic cell death by overactivation of NMDA-type glutamate receptors has been implicated in the death of neurons in a variety of pathological conditions including stroke, cerebral trauma and epileptic seizures. Overactivation of the NMDA receptor has been shown to play a pivotal role in cultured neurons by substantially increasing the cytosolic Ca2+ concentration , leading to a rise in superoxide anion concentration . Propofol has been reported to exert neuroprotective effects through several mechanisms, e.g. activation of GABAA receptors  or scavenging free radical species by a process of hydrogen abstraction . Furthermore, propofol is known to improve electrophysiological recovery from anoxia during hyperthermia by attenuating changes in Na+, K+ and Ca2+ during hyperthermic anoxia in rat hippocampal slices , and to protect cultured rat hippocampal neurons against NMDA receptor-mediated glutamate toxicity , leaving the mechanisms of protection unclear. Our study was designed to investigate the concentration-dependent effects of propofol on reduction of intracellular calcium rise in rat cerebrocortical neurons after sustained stimulation with cytotoxic concentrations of NMDA. The effects of propofol on NMDA-mediated calcium increase have been separated from other ion channel effects of propofol contributing to its overall neuroprotective actions, e.g. GABAergic effects or inhibition of voltage-operated sodium channels . However, a limitation of the study is that effects of propofol on voltage-operated calcium channels have not been clearly separated from NMDA-mediated effects.
Control experiments revealed a fast increase in CG-5N fluorescence induced by overactivation of NMDA receptors, indicative of a fast increase of intracellular calcium. The intracellular calcium rise reached a maximum at 400 s followed by a slow exponential decay. This timecourse corresponds to data previously published by Nicholls and Budd . Propofol reduced the increase of NMDA receptor-induced intraneuronal calcium dose dependently with an EC50 of 9.0 μmol, a concentration that was also shown to be effective against NMDA receptor-mediated rise of intraneuronal superoxide anion concentration . The threshold concentration for significant effects of propofol on NMDA-induced increase of intraneuronal calcium turned out to be 10 μmol. A concentration of 10 μmol is in the upper limit of propofol concentrations that are considered to be clinically relevant [16-18].
Since propofol is known to reduce intraneuronal rise of superoxide anions after sustained stimulation of NMDA receptors , this study indicates that this might appear as a result of reduction of intraneuronal calcium by propofol. Although the threshold concentration in our experimental setting demonstrated to be effective in reducing intraneuronal calcium increase induced by NMDA is in the upper limit of clinically relevant concentrations, inhibition of NMDA receptor-mediated calcium increase by high propofol concentrations might contribute to neuroprotective effects observed with propofol.
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