The brain slice method for studying drug distribution in the CNS
© Loryan et al.; licensee BioMed Central Ltd. 2013
Received: 7 November 2012
Accepted: 2 January 2013
Published: 21 January 2013
The high-throughput brain slice method is a precise and robust technique for estimating the overall uptake of drugs into brain tissue through determination of the unbound volume of distribution in the brain (Vu,brain; ml·g brain-1). Vu,brain describes the relationship between the total drug concentration in the brain and the concentration of unbound drug in the brain interstitial fluid, regardless of blood–brain barrier function. The brain slice method is more physiologically based than the brain homogenate method with respect to the assessment of drug distribution in the brain because the cell-cell interactions, pH gradients and active transport systems are all conserved. The method provides information that is directly relevant to issues such as nonspecific binding to brain tissue, lysosomal trapping, and active uptake into the cells. For these reasons, the brain slice method is recommended for estimation of target-site pharmacokinetics in the early drug discovery process and fundamental pharmacological studies. This article provides a detailed protocol for the rat and mouse brain slice methods, with the aim of enabling simple, cost-effective profiling of compounds with diverse physicochemical properties. The procedure for assessing the viability of the brain slices after the 5 h incubation period is also described. The results are interpreted for a set of compounds covering a wide range of physicochemical properties and various pharmacological targets. Application of the method for evaluating the unbound intracellular-to-extracellular concentration ratio (Kp,uu,cell) and the unbound brain-to-plasma concentration ratio (Kp,uu,brain) is discussed.
KeywordsBrain slice method Unbound volume of distribution in the brain Neuropharmacokinetics Drug discovery High-throughput screening
It is generally accepted that the cerebral concentration of unbound drug is the main pharmacokinetic determinant of CNS activity for neurotherapeutics [1–3]. Preliminary assessment of the clinically relevant pharmacokinetic parameters required for approximation of the unbound-drug concentration in the brain interstitial fluid is thus pivotal in guiding early drug discovery research . Because of the cost and complexity of the methodology, many of the available “gold standard” pharmacokinetic methods are not appropriate for use in the early stages of drug discovery. Consequently, there is an urgent need for adequate high-throughput in vitro systems and methods for CNS drug development programs.
The implementation of the high-throughput equilibrium dialysis-based assay for estimation of the fraction of unbound drug in the brain tissue (fu,brain), combined with measurement of whole brain concentrations in vivo, was groundbreaking for the field . However, homogenization of the brain as used in this method changes the brain tissue binding properties, leading to implicit errors in the readouts .
In contrast, the brain slice method has a more physiological basis and has several benefits over the brain homogenate method. The brain slice preparation method was implemented by Henry McIlwain and is now extensively used in neurobiology, biophysics and quantitative pharmacology [7–9]. It has the advantage of offering a strongly regulated in vitro environment, while preserving much of the complex cellular integrity, including cellular barriers and intact circuitry, and as a result conserving functionality – resulting in an in vitro environment more comparable to the in vivo brain than seen in the homogenate method.
Several research groups have used the method to estimate the uptake of exogenous compounds into the brain [10–15]. Moreover, studies have investigated mechanistic pharmacokinetic/pharmacodynamic links using brain slice methodology [13, 16].
Measurements obtained from in vivo microdialysis have also been compared to those from the in vitro brain slice and homogenate methods . The reasonable correspondence (within a 3-fold range) between the cerebral microdialysis and brain slice method results in this study indicates that the brain slice method is the choice of preference .
The brain slice method has recently been further developed for high-throughput, making it more accessible for use by pharmaceutical companies . It is now a precise, robust technique for estimating the overall uptake of drugs into brain tissue through determination of the unbound volume of distribution in the brain (Vu,brain; ml·g brain-1). Vu,brain describes the relationship between the total drug concentration in the brain and the unbound-drug concentration in the brain interstitial fluid, regardless of blood–brain barrier function. The key assumption of the experiment is that, at equilibrium, the unbound-drug concentration in the brain slice interstitial fluid (ISF) or extracellular fluid (ECF) is equal to the drug concentration in the buffer in the beaker.
This article provides a detailed protocol for the rat and mouse brain slice methods, with the aim of encouraging simple, cost-effective profiling of compounds with diverse physicochemical properties and unifying procedures among laboratories in order to aid the achievement of comparable results.
Methods and Design
The protocols presented below are based on animal experiments approved by the Animal Ethics Committee of Uppsala, Sweden (C21/9 and C351/11). Drug-naïve male Sprague–Dawley 250–300 g rats and Naval Medical Research Institute (NMRI) 25–30 g mice were used (Taconic, Lille Skensved, Denmark). The fresh brain slices can be prepared from various strains of wild type and genetically modified mice and rats, depending on the purpose of the study and the respective laboratory traditions. The brain slices could as well be genetically manipulated using methods such as viral infection , biolistics , etc.
Artificial extracellular fluid
To ensure maintenance of the brain slices in a healthy state, the artificial settings should mimic the in vivo cellular environment. The composition of artificial cerebrospinal fluid or extracellular fluid (aECF) is crucial. A large number of formulations for these artificial fluids can be found in the literature. In the experimental settings used in the studies underlying this paper, the HEPES-buffered aECF contained 129 mM NaCl, 3 mM KCl, 1.4 mM CaCl2, 1.2 mM MgSO4, 0.4 mM K2HPO4, 25 mM HEPES, 10 mM glucose and 0.4 mM ascorbic acid . Ascorbic acid is used as a natural free-radical scavenger to protect the cell membranes from lipid peroxidation and the brain slices from swelling .
Prior to starting an experiment, a stock solution of aECF (1290 mM NaCl, 30 mM KCl, 12 mM MgSO4, 4 mM K2HPO4, 250 mM HEPES) is prepared and stored at room temperature. The 400 mM stock solution of ascorbic acid should be stored at +4°C.
Composition of the working aECF solution
Weight (g)/volume (ml)
280 mM CaCl2
400 mM Ascorbic acid
Adjust pH to 7.6 (up to 1.5 ml of 10 M NaOH)
Adjust volume to 1000 ml
Critical steps in the brain slice experiment
Control the pH, osmolarity and oxygenation of the aECF
Take into account the pKa values of the compounds when selecting the drugs to be investigated in one cassette
Do not take longer than 1 minute to extract the brain
Keep cold-chain during the brain slicing procedure
Preserve the brain slices in ice-cold oxygenated aECF before starting the incubation
Keep oxygenation, temperature and stirring constant during the incubation
Preparation of the samples for bioanalysis
Make sure all minor debris from the brain slices has sedimented before taking the aECF samples after the 5 h incubation
Preparation of cassettes
This protocol allows simultaneous investigation of a selection of up to ten compounds within the same experiment, allowing coverage of a wide range of physicochemical properties and various pharmacological targets in the same cassette.
When deciding on the compounds of each cassette, the pKa values of the compounds should be taken into account. Because high concentrations of weak bases can increase the pH of the acidic intracellular compartments, the extent of lysosomal trapping of a weak base could be affected by the existence of another weak base. The interaction between two weak bases is mainly regulated by the concentrations of the free compounds in the cassette and their potency in increasing intralysosomal pH . Consequently, it is recommended that the final aECF concentration of each studied compound in the cassette should be 100–200 nM and the total concentration of the studied compounds should not exceed 1 μM .
Each cassette of compounds is prepared individually, ex tempore, in scintillation vials (20 ml glass vials with screw lids; one vial per rat or mouse brain). Initially, the required volume of stock drug solution is added to an empty scintillation vial. To reduce possible toxic effects of the solvents (methanol, acetonitrile, etc.) on the brain slices, the solvents are evaporated under a gentle stream of nitrogen before diluting the sample with aECF. When using dimethyl sulfoxide (DMSO) to dissolve the compounds, it is strongly recommended that the final concentration of DMSO is kept as low as possible (no higher than 1%). Subsequently, 20 ml of ice-cold aECF, pre-equilibrated with 100% oxygen, is added to each scintillation vial and ultrasonicated for 10 minutes to facilitate the dissolution of the compounds. These ready-to-use solutions are maintained at 37°C until incubation.
Preparation of slices
The glassware and tools are set up for the dissection prior to preparing the brain slices. The vibrating blade microtome (e.g. Leica VT1200 (Leica Microsystems AB, Sweden)) is prepared for slicing and the chambers are chilled.
Drug-naïve rats/mice are anesthetized with inhalation anesthesia using 5% isoflurane. When deep anesthesia is reached, up to 10 ml of blood (rats) is collected intracardially. The animal is then decapitated and the skull quickly opened. The isolated brain is immediately placed into blank ice-cold aECF saturated with oxygen. In our experience, the brains should be sliced within 15–20 minutes of harvesting to retain their viability. Slices from three rat/mouse brains are generally equilibrated during the same day with one cassette of drugs.
The pre-chilled chamber of the vibratome is filled with ice-cold oxygenated aECF just before use and is then placed in the ice-filled tray of the vibratome.
It is advantageous to put one or two drops of cyanoacrylate glue on the cutting platform one minute before mounting the brain, to allow the glue to dry slightly.
After discarding the first one or two brain slices, 6 (rat brain) or 10 (mouse brain) consecutive 300 μm brain slices are cut on a coronal plane, starting approximately 1.7 mm anterior to the bregma (rostral striatum).
The 300 μm thickness provides good cell preservation without compromising the diffusion of oxygen into the core of the slice. The equilibration time during incubation is inversely related to the square of the brain slice thickness .
The slices are moved, using a micro spatula, to the brain slice storage beaker filled with oxygenated blank aECF which is kept in an ice bucket before the incubation. It is recommended that only brain slices with intact edges be used for the experiment, to reduce the amount of debris detaching from the brain tissue during incubation.
The slicing platform should be scrubbed to remove the brain and glue remnants, and the chamber refilled with fresh ice-cold oxygenated aECF, before proceeding with the next brain. The vibratome chambers should be cleaned, disinfected and dried at the end of each experimental day.
The incubation-equilibration process is started by gently transferring the 6 (rat) or 10 (mouse) brain slices from the storage beaker (using a micro double-ended spatula) into one 45 mm high, 80 mm diameter, flat-bottomed glass beaker (Duran Group, VWR, Sweden) containing 15 ml (rat) or 10 ml (mouse) of the aECF containing the selection of drugs to be investigated (Figure 1C). The beaker is then filled with humidified 100% oxygen over the aECF and covered with a custom-fabricated lid (Figure 1D) composed of a Teflon fluorinated ethylene-propylene (FEP) film (50 Å, 12.7 μm thick; DuPont, Katco Ltd, UK) as designed by Potter and DeMarse with minor modifications . A “blank” beaker is also incubated in parallel to check stability of the compounds added into the buffer.
The transparent Teflon FEP film is used in preference to a glass lid because it is selectively permeable to gases (e.g. oxygen) while remaining relatively impermeable to water vapor. This significantly decreases evaporation (allowing improved control of osmolarity and pH), thus permitting the use of a non-humidified incubator.
Finally, the beaker is placed inside the small plastic box in the incubated shaker (e.g. MaxQ4450 Thermo Fisher Scientific, NinoLab, Sweden) for 5 h at 37°C (Figure 1E). Control of the temperature by an external thermometer is recommended. A rotation speed of 45 rpm and oxygen flow of about 75–80 ml per minute through a glass frit apparatus appear sufficient.
The pH of the aECF should be measured at 37°C immediately after the 5 h incubation. A reduction of more than 0.15 pH units over the 5 hours indicates more than acceptable acidification of the buffer.
Preparation of the samples for bioanalysis
for thermostability testing of the compounds studied (sampled before and after incubation in aECF without the brain slices)
for measurement of Cbuffer – the final concentrations of the unbound compounds in aECF (sampled at the end of the 5 h incubation with the brain slices)
Brain slices samples
for measurement of Abrain – the amount of drug in the brain slices (sampled at the end of the 5 h incubation)
Assessment of the thermostability of the compounds provides valuable information and is recommended for each drug selection tested. A 200 μl sample of aECF is taken directly from the beaker at the beginning of the study and at the end of 5 h incubation without brain slices for analysis of initial and final concentrations of the drugs in aECF. The aECF sample is transferred into an Eppendorf tube containing 200 μl blank brain homogenate that has been prepared beforehand with 4 volumes of blank aECF. The brain homogenate is included to prevent nonspecific binding of drugs to the plastic as well as to match the matrix of the slice homogenates, as required for the following LC-MS-MS analysis.
Since the unbound drug concentration in the brain slice interstitial fluid at equilibrium is taken to be equal to the drug concentration in the aECF in the beaker, the presence of any minor debris from the brain tissue should be avoided in the sampled aECF after the 5 h incubation. To achieve this, the beaker needs to sit still for 5 minutes after finishing the incubation before sampling the aECF. To sample, 200 μl of the aECF is aspirated from just below the surface (without wetting the tip before sampling) and dispensed into an Eppendorf tube containing the 200 μl of blank brain homogenate, as described in Ia. It is recommended that duplicate samples of the aECF be taken at this stage. Filtration of aECF, which is recommended by several authors, is not suitable for drug distribution studies because of the possible loss of the compounds in the filter.
Brain slice samples
After sampling the aECF, the brain slices in the aECF are individually removed, dried on filter paper (about 30 s), weighed (~33 mg per rat brain slice and ~13 mg per mouse brain slice) and homogenized separately in 9 volumes (w/v) of aECF with an ultrasonic processor (e.g. VCX-130; Sonics, Chemical Instruments AB, Sweden).
The samples are then stored at −20°C pending bioanalysis (e.g. LC-MS-MS).
Assessment of viability of brain slices
The brain slices must remain viable during the experiment. There are several methods of assessing viability based on biochemical and/or electrophysiological parameters. In our laboratory, the viability of the brain slices is assessed by measuring the relative activity of released lactate dehydrogenase (LDH) using a cytotoxicity detection kit  according to the manufacturer's instructions (Roche Diagnostics GmbH, Germany), with some modifications.
Brief description of assessment of viability of brain slices based on the activity of released lactate dehydrogenase
Preparation of Background absorbance control. Take 200 μl of aECF buffer from the scintillation vial containing the drug cassette before incubation and mix with 200 μl of blank aECF (store at +4°C pending analysis)
Preparation of Low control. Take 200 μl of aECF buffer from the beaker containing the drug cassette five minutes after transferring the freshly prepared brain slices into the beaker and mix with 200 μl of blank aECF buffer (store at +4°C pending analysis)
Preparation of Samples. Take 200 μl of aECF buffer from the beaker containing the drug cassette and brain slices after 5 hours of incubation and mix with 200 μl of blank aECF buffer (store at +4°C pending analysis)
Preparation of High control. Place one (rat) or 3 (mouse) weighed brain slices in an Eppendorf tube after 5 hours of incubation with the drug cassette, and add 9 volumes (w/v) of 2% Triton X-100 solution in aECF. Put Eppendorf tubes in ultrasound bath for 1 hour at +4°C. Then incubate the tubes for 30 minutes in a water bath at 37°C. Centrifuge at 10000 rpm for 5 minutes at +4°C Take supernatant and store at +4°C pending analysis
Preparation of the samples for analysis, see Table 4. Perform all tests in triplicate Protect the plate from light after addition of freshly prepared reaction mixture
Incubate the plate for up to 25 minutes at room temperature
Measure the absorbance of the samples at 492 nm (use 690 nm as a reference wavelength)
Preparation of samples for assessment of viability of brain slices based on the activity of released lactate dehydrogenase
To calculate the viability of the brain slices (as a percentage) after the 5 h incubation-equilibration period, the following steps are taken for each experimental setup:
Background absorbance control - provides information about the background absorbance of the assay medium (aECF). For this purpose, 200 μl sample of aECF is taken at the beginning of the experiment from the scintillation vial and mixed with 200 μl blank aECF. The obtained absorbance value is then subtracted from all other values.
Low control - provides information about the activity of LDH released from the brain slices as a result of damage to the membranes caused by slicing the brain. A 200 μl aECF sample is taken 5 minutes after transferring the brain slices into the beaker for incubation and is then mixed with 200 μl blank aECF.
High control - provides information about the maximum possible activity of releasable LDH in the brain slice. To achieve this, one rat or three mouse brain slices are used after the 5 h incubation for each experimental setup. After drying the brain slice(s) on filter paper and individually weighing them, 9 volumes (w/v) of 2% Triton X-100 solution in aECF is added. To facilitate the release of LDH from the brain slice(s), the Eppendorf tube is placed in an ultrasound bath for one hour at +4°C followed by 30 minutes of incubation in a water bath at 37°C. The supernatant obtained after centrifugation of the tube for 5 minutes at 10,000 rpm and +4°C is stored at +4°C pending analysis (no longer than 5 days).
For preparation of the experimental samples (experimental value), 200 μl of aECF sample is taken after the 5 h incubation-equilibration period and mixed with 200 μl blank aECF. For evaluation of the effects of changes in viability of the brain slices during the incubation, aECF samples can be taken at different time points (after 1, 2, 3, etc. hours).
In practice, it is recommended that 85-90% viability be aimed for; however, a viability of around 60% provides similar results according to our experience (data not shown). Viability values lower than 50% after the 5 h incubation period are associated with dramatic changes in the estimation of Vu,brain, especially for weak bases and results from the experiments should be discarded.
Estimation of Vu,brain
Bioanalytically determined drug concentrations in the brain slices and the 5 h aECF samples are used to estimate Vu,brain. It is critical to remember to scale back the obtained concentrations (or areas under the concentration-time peaks) to the undiluted buffer and brain slice concentrations by multiplying by the dilution factors as appropriate. The concentration in each brain slice sample is multiplied by 10 to account for the dilution during preparation of the homogenate. The concentration in aECF is multiplied by 2 to account for the dilution during 1:1 mixing of the aECF sample with blank brain homogenate (in 4 volumes (w/v) of aECF). The dilutions associated with protein precipitation are not accounted for because they are the same for all samples.
High-throughput screening capacity
Checklist before starting the experiments
280 mM CaCl2
400 mM ascorbic acid
Dispensed the day before 1 L MQ water
Orbital shaking incubator
Glass- and lab-ware
45 mm high, 80 mm diameter, flat-bottomed glass beakers
Custom-fabricated lids of Teflon FEP film for beakers
Surgical instruments for dissection of brain
Pre-labeled Eppendorf tubes
Nunc 96-well plates
Blank brain homogenate (in 4 volumes (w/v) of aECF)
Cytotoxicity detection kit
The estimated Vu,brain value can be put into context by comparison with the physiological volume of brain fluids, e.g. Vu,brain values higher than 0.8 ml · g brain-1 (the volume of total brain fluids is 0.8 ml · g brain-1) are interpreted as intracellular distribution of the drug in question [4, 18]. Values higher than this indicate that proportionally more of the drug is intracellularly distributed, e.g. into lysosomes, and/or bound to parenchymal components. A value lower than 0.8 ml · g brain-1 shows restricted distribution. The lowest volume possible in vivo is the brain interstitial fluid volume of 0.2 ml · g brain-1; however, in the slices, this is somewhat higher because of damage to the surface layer of cells.
Unbound volume of distribution in the brain (V u,brain ) determined using Sprague–Dawley (SD) rat and Naval Medical Research Institute (NMRI) mouse brain slices
Vu,brainml·g brain -1
An additional very important aspect of drug discovery is the ability to extrapolate the results to other species. Recently, it has been shown that unbound fraction of drug in brain homogenate value obtained from the Wistar Han rat brain homogenate can be used as a representative value for any preclinical species and also humans . The results in Table 6 indicate an absence of any significant dissimilarity in Vu,brain values between Sprague–Dawley rats and NMRI mice. However, a more systematic investigation is desirable before the possibility of interchangeable use of Vu,brain measurements can be supported for translational studies.
It has been proposed by Fridén et al.  that in vitro determination of Vu,brain in combination with in vivo determination of the total brain-to-plasma concentration ratio (Kp,brain) and in vitro determination of the fraction of unbound drug in plasma (fu,plasma) would allow rapid evaluation of unbound brain-to-plasma concentration ratio (Kp,uu,brain) (Figure 2). Moreover, combining Vu,brain with fu,brain would allow estimation of the unbound intracellular-to-extracellular concentration ratio (Kp,uu,cell). It is essential to emphasize that, in terms of predicting Cu,brainISF or Kp,uu,brain, rank ordering of compounds with respect to Vu,brain is futile, since there is no causal relationship .
Complex evaluation of the abovementioned neuropharmacokinetic parameters gives insight to drug distribution in the brain. For instance, analogue of γ-aminobutyric acid gabapentin has Kp,brain equal to 0.64 . However, after correction of Kp,brain for brain tissue uptake (using Vu,brain derived from the brain slice method) and plasma protein binding (using fu,plasma) the BBB net flux was estimated as a 0.14 meaning that only 14% of unbound drug in plasma is passing the BBB. Moreover, after passing BBB gabapentin (substrate to the large neutral amino acid transporter) tends to accumulate in the cells as it could be judged from Kp,uu,cell equal to 4.55 .
The brain slice method can also be used to identify suitable positron emission tomography (PET) tracers, which should have a low degree of nonspecific binding (i.e. a low Vu,brain value) to obtain higher specificity for their targets.
In summary, the brain slice method, used for assessment of the volume of distribution of unbound drug in the brain, is a useful tool for both drug discovery and fundamental pharmacology research.
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Artificial extracellular fluid
Amount of drug in brain tissue
Final drug concentration in aECF
brainISF: Concentration of unbound drug in the brain interstitial fluid
Central nervous system
brain: Unbound fraction of drug in brain homogenate
plasma: Unbound fraction of drug in plasma
brain: Total brain-to-plasma concentration ratio
uu,brain: Unbound brain-to-plasma concentration ratio
uu,cell: Unbound intracellular-to-extracellular concentration ratio
brain: Unbound volume of distribution in brain
We thank Britt Jansson and Waqas Sadiq for excellent assistance with LC-MS-MS analysis of verapamil, oxycodone, docetaxel and digoxin; the results were used for estimation of the unbound volume of distribution in the brain.
- Hammarlund-Udenaes M, Friden M, Syvanen S, Gupta A: On the rate and extent of drug delivery to the brain. Pharm Res. 2008, 25: 1737-1750. 10.1007/s11095-007-9502-2.PubMed CentralPubMedView Article
- Hammarlund-Udenaes M: Active-site concentrations of chemicals - are they a better predictor of effect than plasma/organ/tissue concentrations?. Basic Clin Pharmacol Toxicol. 2010, 106: 215-220. 10.1111/j.1742-7843.2009.00517.x.PubMedView Article
- Watson J, Wright S, Lucas A, Clarke KL, Viggers J, Cheetham S, Jeffrey P, Porter R, Read KD: Receptor occupancy and brain free fraction. Drug Metab Dispos. 2009, 37: 753-760. 10.1124/dmd.108.022814.PubMedView Article
- Hammarlund-Udenaes M, Bredberg U, Friden M: Methodologies to assess brain drug delivery in lead optimization. Curr Top Med Chem. 2009, 9: 148-162. 10.2174/156802609787521607.PubMedView Article
- Kalvass JC, Maurer TS: Influence of nonspecific brain and plasma binding on CNS exposure: implications for rational drug discovery. Biopharm Drug Dispos. 2002, 23: 327-338. 10.1002/bdd.325.PubMedView Article
- Friden M, Bergstrom F, Wan H, Rehngren M, Ahlin G, Hammarlund-Udenaes M, Bredberg U: Measurement of unbound drug exposure in brain: modeling of pH partitioning explains diverging results between the brain slice and brain homogenate methods. Drug Metab Dispos. 2011, 39: 353-362. 10.1124/dmd.110.035998.PubMedView Article
- McIlwain H: Metabolic response in vitro to electrical stimulation of sections of mammalian brain. Biochem J. 1951, 48:
- McIlwain H, Buchel L, Cheshire JD: The inorganic phosphate and phosphocreatine of Brain especially during metabolism in vitro. Biochem J. 1951, 48: 12-20.PubMed CentralPubMedView Article
- Li CL, McIlwain H: Maintenance of resting membrane potentials in slices of mammalian cerebral cortex and other tissues in vitro. J Physiol. 1957, 139: 178-190.PubMed CentralPubMedView Article
- Patlak CS, Hospod FE, Trowbridge SD, Newman GC: Diffusion of radiotracers in normal and ischemic brain slices. J Cereb Blood Flow Metab. 1998, 18: 776-802.PubMedView Article
- Becker S, Liu X: Evaluation of the utility of brain slice methods to study brain penetration. Drug Metab Dispos. 2006, 34: 855-861. 10.1124/dmd.105.007914.PubMedView Article
- Kakee A, Terasaki T, Sugiyama Y: Brain efflux index as a novel method of analyzing efflux transport at the blood–brain barrier. J Pharmacol Exp Ther. 1996, 277: 1550-1559.PubMed
- Gredell JA, Turnquist PA, Maciver MB, Pearce RA: Determination of diffusion and partition coefficients of propofol in rat brain tissue: implications for studies of drug action in vitro. Br J Anaesth. 2004, 93: 810-817. 10.1093/bja/aeh272.PubMedView Article
- Blasberg R, Levi G, Lajtha A: A comparison of inhibition of steady state, new transport, and exchange fluxes of amino acids in brain slices. Biochim Biophys Acta. 1970, 203: 464-483. 10.1016/0005-2736(70)90186-0.PubMedView Article
- Newman GC, Hospod FE, Wu P: Glucose utilization of ischemic hippocampal slices. J Neurosci Methods. 1989, 28: 23-34. 10.1016/0165-0270(89)90006-X.PubMedView Article
- Benkwitz C, Liao M, Laster MJ, Sonner JM, Eger EI: 2nd, Pearce RA: Determination of the EC50 amnesic concentration of etomidate and its diffusion profile in brain tissue: implications for in vitro studies. Anesthesiology. 2007, 106: 114-123. 10.1097/00000542-200701000-00020.PubMedView Article
- Friden M, Gupta A, Antonsson M, Bredberg U, Hammarlund-Udenaes M: In vitro methods for estimating unbound drug concentrations in the brain interstitial and intracellular fluids. Drug Metab Dispos. 2007, 35: 1711-1719. 10.1124/dmd.107.015222.PubMedView Article
- Friden M, Ducrozet F, Middleton B, Antonsson M, Bredberg U, Hammarlund-Udenaes M: Development of a high-throughput brain slice method for studying drug distribution in the central nervous system. Drug Metab Dispos. 2009, 37: 1226-1233. 10.1124/dmd.108.026377.PubMedView Article
- Stokes CE, Murphy D, Paton JF, Kasparov S: Dynamics of a transgene expression in acute rat brain slices transfected with adenoviral vectors. Exp Physiol. 2003, 88: 459-466. 10.1113/eph8802551.PubMedView Article
- Wellmann H, Kaltschmidt B, Kaltschmidt C: Optimized protocol for biolistic transfection of brain slices and dissociated cultured neurons with a hand-held gene gun. J Neurosci Methods. 1999, 92: 55-64. 10.1016/S0165-0270(99)00094-1.PubMedView Article
- Rice ME: Use of ascorbate in the preparation and maintenance of brain slices. Methods. 1999, 18: 144-149. 10.1006/meth.1999.0767.PubMedView Article
- MacIntyre AC, Cutler DJ: The potential role of lysosomes in tissue distribution of weak bases. Biopharm Drug Dispos. 1988, 9: 513-526. 10.1002/bod.2510090602.PubMedView Article
- Potter SM, DeMarse TB: A new approach to neural cell culture for long-term studies. J Neurosci Methods. 2001, 110: 17-24. 10.1016/S0165-0270(01)00412-5.PubMedView Article
- Dos-Anjos S, Martinez-Villayandre B, Montori S, Salas A, Perez-Garcia CC, Fernandez-Lopez A: Quantitative gene expression analysis in a brain slice model: influence of temperature and incubation media. Anal Biochem. 2008, 378: 99-101. 10.1016/j.ab.2008.03.048.PubMedView Article
- Di L, Umland JP, Chang G, Huang Y, Lin Z, Scott DO, Troutman MD, Liston TE: Species independence in brain tissue binding using brain homogenates. Drug Metab Dispos. 2011, 39: 1270-1277. 10.1124/dmd.111.038778.PubMedView Article
- Friden M, Winiwarter S, Jerndal G, Bengtsson O, Wan H, Bredberg U, Hammarlund-Udenaes M, Antonsson M: Structure-brain exposure relationships in rat and human using a novel data set of unbound drug concentrations in brain interstitial and cerebrospinal fluids. J Med Chem. 2009, 52: 6233-6243. 10.1021/jm901036q.PubMedView Article
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