1 Department of Anesthesiology, University of Wisconsin, Madison, WI 53706, USA. 2 Department of Anesthesia, Stanford University, California, USA
* Corresponding author. E-mail: rapearce{at}wisc.edu
Accepted for publication July 22, 2004.
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Abstract |
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Methods. Rat brain slices 300-µm thick were placed in a solution of 100 µM propofol in artificial cerebrospinal fluid for times ranging from 7.5 to 480 min. Concentrations in these slices were measured by HPLC to determine diffusion and partition coefficients. Electrophysiological measurements of the rate at which effects of 5 µM propofol developed were compared with the calculated rate of increase in tissue concentration.
Results. The diffusion coefficient was approximately 0.02x106 cm2 s1, and the brain:artificial cerebrospinal fluid partition coefficient was 36. Diffusion times in brain slices agreed well with time course measurements of propofol-induced depression of synaptic responses, which continued to increase over 5 h. This depression was reversed by blocking GABA inhibition with picrotoxin (100 µM).
Conclusions. Propofol does enhance inhibition in brain slices at a concentration of 0.63 µM in the superfusate, which produces brain concentrations corresponding with those achieved in vivo, but equilibration requires several hours. It is likely that slow diffusion to GABA receptors accounts for the high concentrations (>10 µM) that were needed to depress evoked responses in previous investigations.
Keywords: anaesthetics, i.v., propofol ; pharmacokinetics, propofol ; brain, GABA ; brain, hippocampus ; measurement techniques, electrophysiology
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Introduction |
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To clarify the relative roles of different putative drug targets that are identified using biochemical techniques or reduced preparations it is useful to study drug effects in more intact systems. Towards this end, brain slice preparations have been developed for anaesthetic studies.1618 In addition to the mechanical stability and intact circuitry that such preparations afford, they offer a number of other advantages over in vivo electrophysiological techniques. These include the ability to precisely control the ionic milieu and to deliver pharmacological agents at known concentrations. Using brain slice recordings to assess the effects of propofol in the hippocampus, concentrations between 7 and 28 µM were required to enhance GABAA receptor-mediated recurrent inhibition,19 and the apparent EC50 for slowing inhibitory postsynaptic current decay was 11.4 µM.20 In contrast, a widely quoted estimate for the free concentration that produces anaesthesia is only 0.4 µM,21 and effects on GABAA receptors expressed in cultured cells are observed at comparably low concentrations.4 22 23 A possible explanation for this discrepancy, and for the generally high concentrations required in other brain slice studies,24 is that the tissue concentrations that are achieved within the time frame of typical experiments may be lower than equilibrium concentrations because of slow equilibration. For most drugs that are applied via superfusion, steady-state effects are achieved over several minutes. However, for some drugs, particularly highly lipid-soluble and aqueous-insoluble agents, equilibration times can be substantially longer.25 26 Propofol may fit into this latter category. Indeed, it was reported that suppression of stimulated dopamine release in brain slices continued to increase without reaching a plateau over a 2-h period,24 and its effect on recurrent inhibition recovered by only 10% during a 40-min wash period.19
Therefore, to determine the rate at which propofol equilibrates with brain slice tissue in vitro, and the concentration that must be delivered to achieve concentrations that correspond to those produced by in vivo administration, we applied propofol to brain slices for varying durations up to 480 min and measured the net uptake of drug into the tissue by extraction and HPLC analysis. Application of a one-dimensional diffusion model27 allowed us to estimate the diffusion and partition coefficients of the drug and to determine the drug concentrations within the tissue as a function of depth and time. This analysis showed that depending on the tissue depth at which physiological responses are tested it can take several hours to reach equilibrium. Physiological tests of the effect of propofol on evoked population activity were found to be compatible with such slow equilibration, and further suggested that free concentrations below 1 µM do indeed substantially enhance recurrent inhibition in the hippocampal circuit. Thus, our results indicate that when the rate of equilibration of propofol with brain tissue is taken into account, propofol does alter GABAA receptor-mediated inhibition in brain slices at clinically observed concentrations.
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Methods |
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Propofol uptake experiments
Single 300-µm-thick brain slices were placed in 200-ml flasks containing ACSF plus 100 µM propofol and 1:1000 dimethyl sulfoxide (DMSO) that had been pre-equilibrated with carbogen gas. Flasks were placed in a covered container with a humidified carbogen atmosphere, and the assembly was agitated gently using a clinical rotator (Fisher Scientific, Pittsburgh, PA) to ensure that the slices remained suspended in solution so that drug diffused into both tissue surfaces. Slices remained in solution for time intervals ranging from 7.5 to 480 min, at which time they were removed from solution, blotted to remove excess ACSF, weighed, and transferred to a test tube for extraction and analysis.
Extraction
250 µl of HPLC-grade water and 20 µl of HPLC-grade methanol were added to the brain slice. The sample was vortexed for 30 s, then homogenized for 5 s using a hand-held variable speed homogenizer (Tissue Tearor, Biospec Products, Inc.). 500 µl of HPLC-grade acetonitrile was added, the sample was vortexed for 1 min, then transferred to a 1.5 ml centrifuge tube and centrifuged for 30 min at 4000 g. The supernatant was transferred to a clean 1.5 ml centrifuge tube and stored covered at 20°C overnight before analysis.
Chromatographic instrumentation and sample analysis
Propofol concentrations were determined using an adaptation of the method described by Knibbe and colleagues.29 The HPLC system consisted of a Waters 600 system controller and a Waters 2487 Dual wavelength absorbance detector set at 276 nm (Waters Corporation, Milford, MA). Separation was achieved using a 125x4.0 mm Waters spherisorb column at room temperature, with a mobile phase consisting of acetonitrile, water, and trifluoroacetic acid (60:40:0.1, v/v/v) eluted at 1.5 ml min1. Samples were thawed at room temperature, and then injected manually with a 50-µl syringe through a Rheodyne 7725i injector port fitted with a 20-µl sample loop. The syringe was cleaned between injections by three rinses with acetone followed by three rinses with the mobile phase. Data were recorded and processed using Waters Millennium32 (version 3.005) software. Each sample was measured in duplicate, and the concentration was taken as the average of the two measurements. The mass of drug in the brain slice was computed from the measured concentration in the injected sample by comparing the propofol peak area from the injected sample to that of a standard curve prepared by adding a known amount of propofol to brain slices of known mass, then subjecting the samples to extraction and analysis. Comparison of propofol concentrations in standard samples vs calibration solutions showed that 61±1.4% of propofol was recovered by the extraction procedure (n=15 samples at three different concentrations).
Data were analysed using MS Excel (Microsoft, Redmond, WA) and Microcal Origin (Microcal Software, Northampton, MA).
Modeling concentrationdepth profiles
The concentrationdepth profile for diffusion into a brain slice was modelled using a one-dimensional diffusion equation.27 This model was chosen because of the geometry of the brain slices, which were approximately 10x10x0.3 mm thick. The model is given in dimensionless terms by the equation:
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Electrophysiological experiments
Brain slices were prepared as described above, except that ACSF for incubation and superfusion contained (in mM): NaCl 124, NaH2PO4 1.25, KCl 3.5, NaHCO3 26, CaCl2 2, MgSO4 2, glucose 10, saturated with carbogen gas, pH 7.4. Hemisected brain slices were transferred to a recording chamber and equilibrated for an additional 10 min before electrophysiological recording. ACSF flowed through the recording chamber at a rate of 3.0 ml min1.
To evoke population responses, Schaffer collateral inputs to hippocampal CA1 pyramidal neurons were activated using bipolar tungsten microelectrodes placed in the stratum radiatum. Glass recording electrodes were used to record evoked field potentials. They were filled with ACSF (25 M), and placed at a depth of approximately 125250 µm in the stratum pyramidale to record population spikes, or in the stratum radiatum to record excitatory postsynaptic potentials (EPSPs). Single stimulus pulses (0.010.05 ms duration; 1080 µA at 1.05.0 V) were delivered via constant current stimulus isolation units (Grass Instruments, SIU 6D) connected to a Grass S8800 two channel stimulator, at a stimulus rate of 0.05 Hz. Field potential signals were amplified (x1000), filtered (1 Hz to 10 kHz, bandpass), and digitally stored for off-line analysis on a Pentium microcomputer using DataWave (DataWave Technologies, Longmont, CO) or Strathclyde (kindly provided by J. Dempster) electrophysiological analysis software.
Solutions and drugs
Propofol-containing solutions were prepared by diluting a stock solution of 100 mM propofol in DMSO into ACSF to achieve the desired concentration. HPLC calibration solutions were prepared by diluting a solution of 5 mg ml1 propofol in methanol into mobile phase to yield concentrations of 0.001, 0.01, 0.1, and 0.25 mg ml1. Glass flasks were used for propofol application to minimize adsorption of propofol during extraction and analysis.3 Salts and reagents were obtained from Sigma/Aldrich, and were of HPLC-grade purity. Propofol was obtained from RBI/Sigma or was a kind gift from Zeneca Pharmaceuticals.
Data analysis
Data were analysed and plotted using Origin (Microcal) and Excel (Microsoft Corporation, Redmond, WA). Fits to mono- or bi-exponential functions were calculated using non-linear least square minimization.
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Results |
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Discussion |
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Although the diffusion coefficient of propofol in brain tissue is low enough that equilibration is very slow when the drug is applied to the surface of a tissue slice in vitro, when the drug is delivered to the living brain in vivo via intravascular injection then movement into brain tissue can still occur quite rapidly, as evidenced by the rapid onset of action when propofol is used for the induction of anaesthesia. This is due at least in part to the short distances between the microvasculature and tissue sites of action, as the equilibration rate varies with the square of the distance (equation 1). Administration of doses that produce blood levels higher than the EC50 equilibrium concentration may also contribute to a rapid onset of clinical effects.
To estimate the diffusion coefficient of propofol we used 300-µm-thick brain slices and we exposed both surfaces to drug-containing ACSF. These conditions are not typical for physiological experiments, but we did this so that nearly complete equilibration would be achieved before the health of these acutely prepared slices began to deteriorate. Typically, brain slices remain viable for several hours. It would have taken substantially longer than this for complete equilibration had only one surface of a 400-µm-thick slice been exposed. Full equilibration was considered desirable so that we could gauge as accurately as possible the brain:ACSF partition coefficient and the diffusion coefficient. Despite these measures, maximum or near-maximum values were still not reached until drug had been applied for 240 min (Fig. 1). Although we do not have any direct evidence, a probable explanation for the slight reduction between 240 and 480 min is that during this interval the tissue properties changed and tissue integrity was reduced, so that when the brain slice was transferred to the extraction vial for quantitative analysis less that the full amount of tissue (and thus propofol) was included. Indeed, in pilot experiments we found that after 480 min of exposure brain slices began to deteriorate visibly. Therefore, to calculate the partition coefficient we considered the values at 360 min to represent the fully equilibrated condition (Fig. 1), and we used values only up to 360 min to estimate the diffusion coefficient (Fig. 2). If undetected loss of tissue integrity occurred before the 360-min time point, this would have caused us to underestimate the brain:ACSF partition coefficient and overestimate the diffusion coefficient. As we saw no physical evidence of degradation during this time, and in physiological experiments tissue did remain healthy for this duration, we expect any such error to be small.
Drug concentrations that correspond to behavioural effects
Extensive in vivo pharmacokinetic studies have been performed to define the distribution and disposition of propofol in humans and animals. The free concentration that corresponds to the specific end-point of awakening in humans, or recovery of the righting reflex in animals, may be estimated from concentrations in blood or plasma together with measurements of partition coefficients and protein binding. The concentration of propofol in whole blood at which rats recover the righting reflex was reported to be 2.832 or 3.5 µg ml1.33 Utilizing an average awakening blood concentration of 3.2 µg ml1, a whole blood:plasma partition coefficient for rats of 2.3,3436 a fraction of drug bound to plasma proteins of 97.5%,33 and a molecular weight of 178, this corresponds to a free concentration of 0.20 µM. Similar concentrations have been derived from human studies. Spontaneous eye opening occurs at a blood concentration of 2.2 µg ml1,37 so for a free fraction in human blood of 1.5%,38 this corresponds to a free concentration of 0.19 µM.
How does this estimate of free propofol concentration correspond to brain concentrations that have been measured at different behaviourally or physiologically derived end-points, and to concentrations that we achieved in our experiments?
In sheep, loss of response to a noxious stimulus occurs at brain concentrations of 69 µg g1 brain;39 tail flick latency in rats is greater than 30 s at a concentration of 15.7 µg g1 brain;36 and in humans, brain concentrations of approximately 17 µg g1 produce deep anaesthesia and burst suppression.40 Brain concentrations that correspond to awakening or loss of righting reflex have not been measured directly, but may be calculated using previous measurements of blood concentration at awakening and appropriate partition coefficients. Using a value for brain:water of 113,30 and a brain density of 1.04, the brain concentration that corresponds to a free concentration of 0.20 µM would be 3.9 µg g1 brain. This value is reasonable given the concentrations (616 µg g1) that lead to loss of reflex movement. As the partition coefficient that we measured (
brain:ACSF=36) is only about one-third of the value for
brain:water estimated by Weaver and colleagues,30 perhaps as a result of a higher solubility of propofol in ACSF plus DMSO than in water, we calculate that superfusion with a solution containing 0.63 µM propofol would lead to an equilibrium brain concentration in vitro of 3.9 µg g1.
Electrophysiologic effects of propofol in the brain slice
The amplitude of the first evoked population spike was reduced by approximately 50% after propofol had been applied for 300 min (Fig. 4A). At that time, average free concentration within the top 250 µm of the slice would have been approximately 3 µM (Fig. 3). The second population spike was essentially eliminated by this concentration of propofol (Fig. 4B). Previous studies have attributed paired-pulse depression of the population spike to GABAA receptor-mediated recurrent inhibition.17 41 Thus, the transformation from facilitation to depression may be caused by an enhancement of GABAA receptor function by propofol. Consistent with this hypothesis, subsequent application of picrotoxin caused the response pattern to revert to facilitation (Fig. 4). In addition, the first response returned to its control amplitude, suggesting that its depression by propofol was also mediated by GABAA receptors.
If the inhibitory receptors that underlie propofol effects on the first and second paired responses are present at similar depths within the tissue, a possible explanation for the more rapid effect on the second response is that this effect occurs at a lower concentration than is required for depression of the first response. Examination of Figure 2 reveals that after 10 min of drug application, at which time the first spike was reduced by only 10% and the second spike was reduced by approximately 50%, propofol has reached only 8% of its equilibrium concentration. Thus, it appears that GABAA receptor-mediated recurrent inhibition is substantially enhanced by 0.4 µM propofol (8% of 5 µM). The difference in sensitivity of the first and second responses may reflect differences in the sensitivity of different types of receptors that control the responses, or a difference in the degree of enhancement required to alter the responses. Inhibitory synapses are physiologically heterogeneous in the CA1 region,42 and in addition extrasynaptic receptors that produce a tonic inhibitory current exist.43 These different types of receptors may respond differently to propofol.44 Also, as noted previously, low concentrations of propofol augment responses to GABA but higher concentrations are required to directly activate receptors.3 Thus, it is also possible that the different sensitivity of the two responses depends on the mode of activation of receptors. Further studies to define the molecular receptor-based mechanisms by which propofol alters integrated neuronal responses will be required to address these questions. It will be necessary to take into account the slow equilibration of propofol with brain tissue when designing and interpreting such experiments. These considerations may also apply to other highly lipophilic drugs such as thiopentone and steroid anaesthetics.
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Acknowledgments |
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