The use of anesthetics can lead to changes of the permeability of the blood-brain barrier (BBB). To eliminate those factors, such as varying hemodynamic effects that are associated with anesthesia, an in vitro model of the BBB consisting of brain microvascular endothelial cells (BMEC) was used to study the direct effects of the opiate, fentanyl, and the barbiturates methohexital and thiopental, which are widely used in the clinical setting, on the permeability of confluent monolayers.
BMEC isolated from porcine brains were grown to confluence on collagen-coated polycarbonate membranes, which were placed into 24 well dishes, thus forming a two-compartment chamber. The permeability of the BMEC monolayer to ions--determined by measurements of the transendothelial resistance (TER)--the passage of sucrose, Evans Blue albumin (EBA), and alpha-aminoisobutyric acid (AIB) across the BMEC monolayer were assessed in the presence and absence of fentanyl (25-100 ng/ml), methohexital (10-50 micrograms/ml), and thiopental (25-100 micrograms/ml).
The permeability of cultured BMEC to the tracers used increased significantly after exposure of the monolayer to arabinose and after removal of calcium ions. Fentanyl, methohexital, and thiopental did not change the permeability of the cell monolayer to ions, sucrose, albumin, and AIB. Only thiopental at the concentration of 100 micrograms/ml increased the flux of AIB.
At the concentrations tested, there is little evidence of changes in the permeability of the in vitro BBB caused by fentanyl, methohexital, and thiopental regarding the para- and transcellular route of ions, sucrose, and albumin. Only thiopental at a concentration of 100 micrograms/ml increased the passage of AIB across the BMEC monolayer.
Key words: Anesthetics, intravenous: fentanyl; methohexital; thiopental; Brain: blood-brain barrier; brain microvascular endothelial cells; permeability.
THE blood-brain barrier (BBB) is required to protect the brain against various circulating molecules and neurotoxins in the plasma. [1,2] The BBB consists of brain microvascular endothelial cells (BMEC), which possess few pinocytotic vesicles and are jointed together by tight intercellular junctions with high electrical resistance values. [3,4] These properties limit the amount of para- and transcellular flux across the BBB and regulate the passage of most substances and drugs into the brain. [5–7] There exist data from in vivo studies describing the influence of anesthetics on permeability properties of the BBB. For example, pentobarbital and alcohol can enhance the passage of macromolecules across the BBB.  Fentanyl has been shown to decrease the transfer of small hydrophilic molecules such as alpha-aminoisobutyric acid across the BBB in rats, as demonstrated by a decreased BBB coefficient and permeability-surface area product.  Barbiturates have been shown to protect the brain under clinical circumstances such as vasogenic brain edema, focal ischemia, or inflammation.  However, it is still unknown if the described effects are due to the direct action of anesthetics on the endothelial cell monolayer forming the BBB in vivo or if there are secondary effects that are usually present after anesthesia, such as alterations in cardiovascular parameters or hemodynamics. Therefore, we studied the influence of fentanyl and of the barbiturates methohexital and thiopental on permeability properties of the BBB using an in vitro model of cultured BMEC seeded onto polycarbonate membranes. This model has been widely used for estimating the passage of a solute through the BBB in vivo and for conducting studies of the transport of very hydrophilic to very lipophilic solutes and proteins.  For the permeability studies the transendothelial electrical resistance across the endothelial cell monolayer was determined, which provides a method for the estimation of the ion permeability.  Furthermore, the transport of the metabolically inert tracer [sup 3 Hydrogen]-sucrose, the macromolecule albumin—compounds that do not readily cross the BBB in vivo—and of alpha-aminoisobutyric acid were measured in absence and presence of fentanyl, methohexital, and thiopental.
Materials and Methods
Capillary endothelial cells were isolated from porcine brains, which were collected from a local slaughter house and transported in cold 70% isopropanol to the laboratory. After removal of white matter and meninges, the gray matter was digested with dispase (0.5 mg/100 ml) for 3 h at 37 degrees Celsius. Capillaries were isolated after centrifugation with 18% dextran. Pellets were resuspended in medium M199 and treated with collagenase/dispase (0.1 mg/ml) for 5 h at 37 degrees Celsius. Endothelial cells were obtained after Percoll gradient centrifugation as described by Mischek et al.  Cells were seeded onto rat tail collagen (Sigma, Munchen, Germany) coated polycarbonate Transwell filter inserts with a pore size of 0.4 micro meter and a diameter of 12 mm (Millipore, Eschborn, Germany), placed into 24 well dishes and incubated at 37 degrees Celsius in a 5% carbon dioxide-humidified incubator. The cells were grown in medium M199 supplemented with 15%(v/v) horse serum, 5%(v/v) fetal calf serum (FCS), 0.68 mM glutamine, 200 U/ml penicillin, 200 U/ml streptomycin, and 2.5 micro gram/ml amphotericin B. All ingredients were purchased from Life Technologies (Eggenstein, Germany). Four hours after seeding, the medium was changed. Formation of the BMEC monolayer was monitored by using light microscopy and TER measurements.
Characterization of BMEC
The incorporation of acetylated low density lipoprotein (Dil-Ac-LDL; Paesel & Lorei, Frankfurt, Germany) was used as marker for cells of endothelial origin as described by Voyta et al.  The marker enzymes, alkaline phosphatase gamma-glutamyl transferase, were determined using reaction mixture kits from Sigma. Absence of pericytes and astrocytes was confirmed by immunofluorescence staining with anti alpha-smooth muscle actin (Sigma) and antiglial fibrillary acidic protein antibodies (Boehringer, Mannheim, Germany). Cultures used for our experiments contained less than 5% pericytes. Cells of astroglial origin could only be detected in some cultures, but their content was always less than 1%.
Six to eight days after seeding BMEC onto polycarbonate filter membranes, measurements of the transendothelial resistance and permeability were completed as described below. The chambers, consisting of the apical part containing the filter membrane inserts with the cell monolayer and the basolateral part, were washed three times with phosphate buffered saline, pH 7.4 (PBS), and 400 micro liter medium M199 without FCS or the same amount of transendothelial buffer (122 mM NaCl, 3 mM KCL, 1.4 mM CaCl2, 1.2 mM MgSO4, 10 mM N-2 hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES), pH 7.4, 25 mM NaHCO3, 10 mM glucose and 0.4 mM K2HPO4) were added into the apical and 600 micro liter of medium or transendothelial buffer into the basolateral chamber. The TER across BMEC monolayers was measured using an assembly containing current passing and voltage-measuring electrodes (Millicell-ERS, Millipore). TER values measured in medium M199 were not significantly different from those determined in transendothelial buffer. Maximal resistance values across the BMEC monolayer were determined at the seeding density of 0.5–0.6 x 106cells/cm2, whereas at higher cell density, tube formation of the cells occurred resulting in lower resistance values.
Measurements of the TER in presence of arabinose and in absence of calcium ions were performed in the following manner: 400 micro liter of transendothelial buffer was placed into the apical chamber and 600 micro liter of the same buffer into the basolateral chamber. Arabinose to the final concentration of 1.6 M was added into the apical chamber or, for the measurements in absence of calcium ions, the transendothelial buffer in the upper and lower chamber was replaced by the same buffer without CaCl2. After 1 h incubation at 37 degrees Celsius in a 5% CO2humidified incubator, TER was measured. For the determination of the TER in the presence of anesthetics, different concentrations of fentanyl (Janssen GmbH, Neuss, Germany), meythohexital (Lilly, Giessen, Germany), or thiopental (Byk Gulden, Konstanz, Germany) dissolved in medium M199 were added into the apical chamber. The chambers were incubated at 37 degrees Celsius in a 5% CO sub 2 humidified incubator, and TER was measured after different periods. TER values are expressed as the difference of the TER measured across the endothelial cell monolayer (TERtotal) and a background resistance measured across cell-free filters coated only with rat tail collagen (TER0)(TER = TERtotal- TER0; TER0= 73 plus/minus 7 Omega cm2, n = 10).
For flux measurements, chambers showing TER values of more than 80 Omega cm2were used. They were washed three times with PBS, and the flux of [sup 3 Hydrogen] sucrose (Amersham, Buchler, Germany) across the BMEC monolayer was measured after adding 0.146 nmole (= 0.8 micro Ci)[sup 3 Hydrogen] sucrose in 400 micro liter transendothelial buffer in the apical chamber and 600 micro liter of the same buffer in the basolateral chamber. The appearance of [sup 3 Hydrogen] sucrose in the basolateral chamber was measured at various times thereafter by scintillation counting of small aliquots of the basolateral buffer. Flux rates across the BMEC monolayer and across the cell-free membrane were measured in the absence and presence of test solutions, which were added into the apical chamber. Measurements in presence of arabinose and in absence of calcium ions were performed as described for resistance measurements. Results were expressed as the ratio of the concentration in the lower chamber and the total concentration of [sup 3 Hydrogen] sucrose added (CR/Ctotal). During the course of the experiment, chambers were kept at 37 degrees Celsius in a 5% CO2humidified incubator, and care was taken to ensure that fluid levels in the apical and basolateral chambers were equal.
The transport of bovine serum albumin (BSA)(Sigma) across the BMEC monolayer was measured as described by Patterson et al.  by binding of BSA to Evans blue dye (EBD). To determine whether EBD binding to albumin was tight the dye at a concentration of 0.67 mg/ml in 4%(w/v) BSA was placed in dialysis tubing (12,000 cut off, Sigma) and dialyzed at room temperature against water. After 24 h, no measurable leakage of the dye had occurred. For the permeability studies, 0.67 mg EBD/ml medium M199 containing 4%(w/v) BSA was placed in the apical chamber (final volume 400 micro liter) and 600 micro liter of culture medium was placed in the basolateral chamber. After different time points, aliquots of the basolateral chamber were taken and placed into 96 well plates. Absorbances were measured by using a spectrophotometer (Biomek, Beckman, Munchen, Germany) equipped with a 600-nm filter. The relationship of dye concentration to absorbance was determined to be linear in the range of concentrations used. The experiments were performed and results were calculated in the same way as described for the flux of [sup 3 Hydrogen] sucrose.
For the measurement of the flux of [sup 3 Hydrogen] alpha-aminoisobutyric acid (AIB)(Biotrend, Koln, Germany), 0.8 nmole (= 0.8 micro Ci) AIB in 400 micro liter transendothelial buffer was added in the apical chamber and 600 micro liter of the same buffer in the basolateral chamber. After different periods of incubation at 37 degrees Celsius in a 5% CO2humidified incubator, aliquots of the basolateral chamber were removed and the concentration of AIB was determined by scintillation counting. Results were expressed as described for the other tracers used.
Results are presented as the arithmetic mean with standard error of the mean (SEM). Analysis of variance and subsequent multiple comparisons were performed using Scheffe's method. Absolute values determined for the TER and flux rates were used for statistical analysis. For significance, P < 0.05 was required.
Characterization of BMEC
Cells were characterized by their morphology, which is more fibroblastlike, as described by Mischek et al.,  and the uptake of low density lipoprotein, which shows their endothelial origin. The primary cultures also contain high levels of the alkaline phosphatase- and gamma-glutamyltransferase activity, typical marker enzymes for the brain endothelium.
Measurements of the Transendothelial Resistance
The following experiments were performed to determine whether fentanyl, methohexital, and thiopental change the TER across the cell monolayer. Measurements of the TER represent a method to determine the ionic permeability across a cell monolayer that decreases with increased formation of tight junctions between endothelial cells. The TER values reached a maximum 8 days after seeding, and after 9 days, values started to decrease again, which has been described by others. [16,17] First, we performed experiments that are known to cause permeability changes by opening the tight junctions between the cells to test the availability of the in vitro system. The addition of 1.6 M arabinose into the upper chamber and the removal of calcium ions by using transendothelial buffer without CaCl2caused a reversible decrease of the TER after 1 h incubation at 37 degrees Celsius (Table 1). The anesthetics fentanyl (0–100 ng/ml), methohexital (0–50 micro gram/ml), and thiopental (0–100 micro gram/ml) did not change the TER measured for up to 24 h (Table 2).
Passage of Sucrose, Albumin, and alpha-Aminoisobutyric Acid across the BMEC Monolayer
To determine the effects of the anesthetics on the permeability characteristics of BMECs, the flux of sucrose, albumin, and AIB were assessed. The flux of each tracer across the BMEC monolayer increased significantly in a reversible manner in presence of 1.6 M arabinose and after removal of calcium ions from the transendothelial buffer (Table 3). Figure 1shows the time dependency of the passage of each tracer across the BMEC monolayer and across the cell-free membrane. The in vitro system of cultured BMEC showed some passage of the small molecule sucrose across the BMEC monolayer. Because there was only little passage of albumin across the cell monolayer, we performed the corresponding experiments in absence and presence of anesthetics for longer periods.
(Table 4) shows that fentanyl at concentrations up to 100 ng/ml and methohexital at concentrations up to 50 micro gram/ml did not change the flux rate of sucrose, albumin, and AIB across the BMEC monolayer. Thiopental at the concentration of 100 micro gram/ml increased the flux of AIB significantly, whereas the permeability to ions, sucrose, and albumin was unchanged.
The current study shows that permeability to ions, sucrose, and albumin of primary cultures of capillary endothelial cells isolated from porcine brain were not changed by fentanyl, methohexital, and thiopental. Only the largest concentration of thiopental increased the permeability toward AIB. Higher concentrations of thiopental compared to methohexital were used because methohexital as an anesthetic is two to three times more potent than thiopental. Cerebral microvascular endothelial cells are joined by tight intercellular junctions or zonulac occludens, which normally excludes the paracellular passage of substances across the BBB. Transcellular passage is possible through the cytosol, pores, channels, and vesicles.  Measurements of the transendothelial resistance across a cell monolayer generally is considered to be a measure of the tightness of intercellular junctions to ions.  Eight days after seeding porcine BMEC onto collagen-coated polycarbonate membranes, we measured the TER of 98.4 plus/minus 2.0 ohm cm2, which is in agreement with values determined by others. [2,18] It has been shown that the addition of arabinose and a decrease in the extracellular calcium ion concentration will cause reversible increases in the ionic and nonelectrolyte fluxes across endothelial tissues with changes in tight junctional structure. [19–21] Accordingly, both conditions decreased the TER and increased the passage of sucrose, albumin, and AIB across the BMEC monolayer significantly.
The addition of fentanyl, methohexital, and thiopental did not change the TER suggesting that the ionic composition across the in vitro BBB will not be changed by the concentrations of the anesthetics used in this study.
The in vitro system of cultured BMEC showed some leakiness to the small membrane-impermeant molecule sucrose, which has been described by Rim et al.  The difference between the in vitro and the in vivo model regarding the lower resistance values and the higher permeability to molecules, which in vivo do not pass the BBB, could be due to the less complex tight junctions observed in primary cultures of BMEC.  It also might be due to the absence of required factors in the artificial culture environment because it has been supposed that many of the features that are characteristic for brain capillary endothelial cells are in some way induced by surrounding cells such as astrocytes. [21–25] Raub et al.  suggested that the predominant pathway for diffusion of sucrose across the BBB is through the paracellular route. All anesthetics used did not change the flux of sucrose across the BMEC monolayer suggesting that they have no effect on the paracellular route of sucrose across the in vitro BBB.
We used bovine serum albumin as another tracer because it has been assumed to cross the BBB by the transcellular route.  BSA is a large water-soluble, roughly spherically shaped protein with a molecular weight of 66,500. The transcellular flux and the binding properties of BSA to cultured brain endothelial cells were consistent with a fluid-phase pinocytic mechanism.  Albumin is known to leak across the BBB in certain pathologic conditions. [27,28] Fentanyl, methohexital, and thiopental did not change the passage of EBA across the BMEC monolayer, suggesting that these anesthetics have no effect on the transcellular route of BSA.
The transport of AIB across the in vitro BBB was measured because AIB is assumed to be a substrate for the large neutral amino acid (LNAA) carrier.  which operates in the luminal membrane of the BBB.  Brain uptake by this mechanism in vivo is negligible due to competition by other amino acids in the plasma.  This explains why AIB commonly is used as a passive permeability marker. [31,32] Our system did not contain other amino acids, suggesting that AIB is transported across the cell monolayer by the LNNA carrier. The different transport mechanism for AIB across the BBB probably explains the smaller effects of arabinose and calcium ions on the flux rate across the BMEC monolayer compared to the other tracer used. Nevertheless, further experiments are necessary to evaluate kinetics of AIB transport across the endothelial cell monolayer. It has been reported that the transport of the small, inert neutral amino acid alpha-aminoisobutyric acid across the in vivo BBB in rats was decreased after the intravenous injection of fentanyl.  However, our results show that the transport of AIB across the in vitro BBB was not changed by fentanyl. Probably, the effect of fentanyl described by Chi et al.  was not caused by an effect on the permeability of the BBB but by the reduction of the surface area of perfused cerebral capillaries. Only thiopental at the concentration of 100 micro gram/ml increased the flux of AIB across the BBB. It is improbable that disruption of the monolayer is responsible for this effect because, under these conditions, transport properties of ions, sucrose, and albumin were unaltered. Thiopental shows a high lipid solubility that is higher than that of methohexital, which may alter membrane fluidity. There also might be some interactions with the transport carrier that may lead to the higher permeability toward AIB.
In conclusion, we could show that fentanyl, methohexital, and thiopental even at high concentrations showed no effects on permeability properties of the in vitro BBB regarding the para- and transcellular route of ions, sucrose, and albumin. The passage of AIB was increased by thiopental only slightly. An advantage of the in vitro system is that many factors that modulate solute exchange (pressure, flow, blood elements) are controlled. Moreover, it is likely that many of these parameters are altered during anesthesia, which complicates conclusions about permeability. In vivo, the formation and maintenance of the BBB is induced by surrounding astrocytes, and other brain-derived factors and interactions of anesthetics with these brain-derived factors are not detectable using the in vitro system. Although the permeability of cultured brain microvascular endothelial cells is higher than that of the vessels from which BMEC were derived, the monolayer provides a convenient method to get more knowledge about effects of anesthetics on permeability properties of the BBB.
The authors thank M. Granz, for technical assistance, G. Stammler, for statistical analysis, and Dr. W. C. Chilian, for critical review of the manuscript.