Rat Brain Microvascular Endothelial Cells Research Articles

μ-Opioid agonists inhibit the enhanced intracellular Ca 2+ responses in inflammatory activated astrocytes co-cultured with brain endothelial cells

In order to imitate the in vivo situation with constituents from the blood–brain barrier, astrocytes from newborn rat cerebral cortex were co-cultured with adult rat brain microvascular endothelial cells. These astrocytes exhibited a morphologically differentiated appearance with long processes. 5-HT, synthetic μ-, δ- or κ-opioid agonists, and the endogenous opioids endomorphin-1, β-endorphin, and dynorphin induced higher Ca 2+ amplitudes and/or more Ca 2+ transients in these cells than in astrocytes in monoculture, as a sign of more developed signal transduction systems. Furthermore, stimulation of the co-cultured astrocytes with 5-HT generated a pronounced increase in intracellular Ca 2+ release in the presence of the inflammatory or pain mediating activators substance P, calcitonin gene-related peptide (CGRP), lipopolysaccharide (LPS), or leptin. These Ca 2+ responses were restored by opioids so that the δ- and κ-opioid receptor agonists reduced the number of Ca 2+ transients elicited after incubation in substance P+CGRP or leptin, while the μ- and δ-opioid receptor agonists attenuated the Ca 2+ amplitudes elicited in the presence of LPS or leptin. In LPS treated co-cultured astrocytes the μ-opioid receptor antagonist naloxone attenuated not only the endomorphin-1, but also the 5-HT evoked Ca 2+ transients. These results suggest that opioids, especially μ-opioid agonists, play a role in the control of neuroinflammatory activity in astrocytes and that naloxone, in addition to its interaction with μ-opioid receptors, also may act through some binding site on astrocytes, other than the classical opioid receptor.

Increased P-glycoprotein function and level after long-term exposure of four antiepileptic drugs to rat brain microvascular endothelial cells in vitro

To investigate whether long-term exposure to four typical antiepileptic drugs (AEDs): phenobarbital (PB), phenytoin (PHT), carbamazepine (CBZ) and valproic acid (VPA) can increase P-glycoprotein (P-gp) level and function in primary cultured rat brain microvascular endothelial cells (rBMECs) in vitro, the rBMECs were incubated in culture medium containing indicated drugs (PB, PHT, CBZ, VPA and rifampin) for 60 days in a gradient concentration manner. Age-matched cells were incubated in normal culture medium. After a 60-day exposure to the indicated drugs, P-gp function and level in cells were measured using rhodamine 123 (Rho123) accumulations and Western blot analysis, respectively. Lower Rho123 accumulation in drug-treated cells was found than that in age-matched cells. Cyclosporin A (CsA) and verapamil (Ver) increased Rho123 accumulation both in drug-treated cells and age-matched cells. The magnitude of increased Rho123 accumulation in drug-treated cells was larger than that in age-matched cells. Higher P-gp levels were found to be consistent with decrease of Rho123 accumulation in drug-treated cells. The results verified the hypothesis that long-term exposure to the four antiepileptic drugs can induce P-gp function and level in rBMECs.

Transport activities involved in intracellular pH recovery following acid and alkali challenges in rat brain microvascular endothelial cells

Transport activities involved in intracellular pH (pH(i)) recovery after acid or alkali challenge were investigated in cultured rat brain microvascular endothelial cells by monitoring pH(i) using a pH-sensitive dye. Following relatively small acid loads with pH(i) approximately 6.5, HCO(-)(3) influx accounted for most of the acid extrusion from the cell with both Cl(-)-independent and Cl(-)-dependent, Na(+)-dependent transporters involved. The Cl(-)-independent component has the same properties as the NBC-like transporter previously shown to account for most of the acid extrusion near the resting pH(i). Following large acid loads with pH(i) < 6.5, most of the acid extrusion was mediated by Na(+)/H(+) exchange, the rate of which was steeply dependent on pH(i). Concanamycin A, an inhibitor of V-type ATPase, had no effect on the rates of acid extrusion. Following an alkali challenge, the major component of the acid loading leading to recovery of pH(i) occurred by Cl(-)/HCO(-)(3) exchange. This exchange had the same properties as the AE-like transporter previously identified as a major acid loader near resting pH(i). These acid-loading and acid-extruding transport mechanisms together with the Na(+), K(+), ATPase may be sufficient to account not only for pH(i) regulation in brain endothelial cells but also for the net secretion of HCO(-)(3) across the blood-brain barrier.

Repetitive/temporal hypoxia increased P-glycoprotein expression in cultured rat brain microvascular endothelial cells in vitro

The aim of the study was to investigate whether repetitive/temporal hypoxia up-regulated P-glycoprotein (P-gp) in cultured rat brain microvascular endothelial cells (rBMECs). Cultured rBMECs were used as in vitro blood brain barrier (BBB) model. Cells reached confluence were subjected to temporal hypoxic exposure. Under free-glucose cultured medium, the cells were covered by sterile paraffin oil for 15 min, inducing temporal hypoxic exposure. The hypoxic-exposure was carried out once every day up to 8 days, leading to the repetitive/temporal hypoxia in rBMECs. The cell viability was tested using CCK-8 kit, function and levels of P-gp in the cells were measured using rhodamine 123 uptake and western blot, respectively. It was found that 8-temporal hypoxic exposure induced 1.6-fold increase of P-gp level in cells, accompanied by decrease of cellular accumulation of rhodamine 123. Cellular accumulation of phenobarbital was also decreased. These findings indicated that repetitive/temporal hypoxia may be one of the factors resulting in P-gp overexpression in refractory epilepsy.

P-glycoprotein-mediated efflux of phenobarbital at the blood-brain barrier evidence from transport experiments in vitro

The purpose of the present studies was to investigate characteristics of phenobarbital (PB) transport across the blood-brain barrier (BBB). Cultured rat brain microvascular endothelial cells (rBMECs) were used as an in vitro BBB model. Experiments were conducted to examine time-, concentration- and temperature-dependent elements of PB uptake, and the effect of tested agents on the steady-state uptake of PB. PB efflux from rBMECs and the polarised transport of PB were examined to evaluate whether P-glycoprotein (P-gp) was involved in the PB efflux transport across BBB. The results demonstrated that the uptake of PB by rBMECs was in a time-, concentration- and temperature-dependent manner. P-gp modulators, cyclosporin A (CsA), ketoconazole and metabolic inhibitor dinitrophenol, increased the PB steady-state uptake by more than 50% (p < 0.01), and decreased the PB efflux by more than 50% (p < 0.01). Similar results were observed in the uptake and efflux studies of rhodamine-123 by co-administration of CsA. Further results were obtained in the polarised transendothelial transport of PB in the apical to basolateral (A-B) and basolateral to apical (B-A) directions either with or without CsA. The result showed that transport of PB in the B-A direction was significantly greater than the transport in the A-B direction (p < 0.05), and the co-administration of 50microM CsA almost inhibited P-gp-involved efflux in the rBMECs. Overall, these findings suggest that P-gp may contribute to the efflux transport of PB across BBB.

Inhibition of glioblastoma growth and angiogenesis by gambogic acid: An in vitro and in vivo study

Gambogic acid (GA) is the major active ingredient of gamboge, a brownish to orange resin exuded from Garcinia hanburryi tree in Southeast Asia. The present study aims to demonstrate that gambogic acid (GA) has potent anticancer activity for glioblastoma by in vitro and in vivo study. Rat brain microvascular endothelial cells (rBMEC) were used as an in vitro model of the blood–brain barrier (BBB). To reveal an involvement of the intrinsic mitochondrial pathway of apoptosis, the mitochondrial membrane potential and the western blot evaluation of Bax, Bcl-2, Caspase-3, caspase-9 and cytochrome c released from mitochondria were performed. Angiogenesis was detected by CD31 immunochemical study. The results showed that the uptake of GA by rBMEC was time-dependent, which indicated that it could pass BBB and represent a possible new target in glioma therapy. GA could cause apoptosis of rat C6 glioma cells in vitro in a concentration-dependent manner by triggering the intrinsic mitochondrial pathway of apoptosis. In vivo study also revealed that i.v. injection of GA once a day for two weeks could significantly reduce tumor volumes by antiangiogenesis and apoptotic induction of glioma cells. Collectively, the current data indicated that GA may be of potential use in treatment of glioblastoma by apoptotic induction and antiangiogenic effects.

Tanshinone IIA inhibits endothelin-1 production in TNF-?-induced brain microvascular endothelial cells through suppression of endothelin-converting enzyme-1 synthesis

To investigate the effects of tanshinone IIA (Tan IIA) on the regulation of the production of endothelin (ET)-1 (including large ET-1), mRNA levels of ET-1, endothelin-converting enzyme-1 (ECE-1), endothelin-A receptor (ETA) and endothelin-B receptor (ETB) induced by TNF-alpha in rat brain microvascular endothelial cells (BMVEC). The ET-1 release (including large ET-1) into the culture medium was determined by enzyme immunoassay. The levels of ET-1, ECE-1, ETA, and ETB mRNA were measured by RT-PCR. Endothelin receptor binding was also tested. The induction of ET-1 release by TNF-alpha from cultured BMVEC was dose-dependently reduced by Tan IIA, but large ET-1 levels progressively increased in response to Tan IIA; the mRNA expression of ET-1 was unaffected. Tan IIA also caused a decrease in ETA receptor mRNA and ECE-1 expression in a dose-dependent manner. Endothelin receptor binding was unaltered in BMVEC stimulated with TNF-alpha alone or a combination of TNF-alpha and Tan IIA. These findings suggest that Tan IIA may inhibit ET-1 production in TNF-alpha-induced BMVEC through the suppression of ECE-1 synthesis.

Interaction between nicotine and MPTP/MPP + in rat brain endothelial cells

To examine the interaction between nicotine and MPTP/MPP + in the blood–brain barrier, cellular uptake of MPTP and MPP + was studied in the presence of nicotine and several compounds, including MPTP/MPP + analogs and a specific inhibitor of organic cation transporter (OCT) in an adult rat brain microvascular endothelial cell line (ARBEC). The kinetic properties of the uptake of MPTP, MPP +, and nicotine were also examined. In addition, a microdialysis study was performed to evaluate the in vivo effect of nicotine (i.p.) on extracellular levels of MPTP and MPP + in the brain after intravenous administration of MPTP. The results showed that uptake of MPTP, MPP +, and nicotine was partly mediated by a carrier system that was sensitive to decynium22, a specific OCT inhibitor. RT-PCR showed the presence of OCT1 mRNA in ARBEC. Capacity for uptake of MPTP and nicotine was much higher than that for MPP + ( K m and V m values of 10.94 ± 1.44 μM and 0.049 ± 0.007 pmol/mg s, respectively, for MPP +, compared to values of 35.75 ± 0.85 μM and 40.95 ± 3.56 pmol/mg s for MPTP and 25.29 ± 6.44 μM and 51.15 ± 14.18 pmol/mg s for nicotine). In addition, nicotine competitively inhibited the uptake of both MPTP and MPP +, with inhibition constants ( K i) of 328 μM and 210 μM, respectively. In vivo microdialysis results showed that nicotine significantly reduced brain extracellular levels of MPTP in the first 30min (507.4 ± 8.5 ng/ml vs. 637.9 ± 30.8 ng/ml with and without nicotine pre-treatment, respectively), but did not have significant effect on those of MPP +. In conclusion, nicotine can inhibit in vitro cellular uptake and in vivo transfer of MPTP across the blood–brain barrier, which can be mediated by multiple pathways including OCT1.

Role of P-glycoprotein in Limiting the Brain Penetration of Glabridin, An Active Isoflavan from the Root of Glycyrrhiza glabra

Glabridin is a major active constituent of Glycyrrhiza glabra which is commonly used in the treatment of cardiovascular and central nervous system (CNS) diseases. Recently, we have found that glabridin is a substrate of P-glycoprotein (PgP/MDR1). This study aimed to investigate the role of PgP in glabridin penetration across the blood-brain barrier (BBB) using several in vitro and in vivo models. Cultured primary rat brain microvascular endothelial cells (RBMVECs) were used in the uptake, efflux and transcellular transport studies. A rat bilateral in situ brain perfusion model was used to investigate the brain distribution of glabridin. The brain and tissue distribution of glabridin in rats with or without coadministered verapamil or quinidine were examined with correction for the tissue residual blood. In addition, the brain distribution of glabridin in mdr1a(-/-) mice was compared with the wild-type mice. Glabridin in various biological matrices was determined by a validated liquid chromatography mass spectrometric method. The uptake and efflux of glabridin in cultured RBMVECs were ATP-dependent and significantly altered in the presence of a PgP or multi-drug resistance protein (Mrp1/2) inhibitor (e.g. verapamil or MK-571). A polarized transport of glabridin was found in RBMVEC monolayers with facilitated efflux from the abluminal (BL) to luminal (AP) side. Addition of a PgP or Mrp1/2 inhibitor in both luminal and abluminal sides attenuated the polarized transport across RBMVECs. In a bilateral in situ brain perfusion model, the uptake of glabridin into the cerebrum increased from 0.42 +/- 0.09% at 1 min to 9.27 +/- 1.69% (ml/100 g tissue) at 30 min and was significantly greater than that for sucrose. Co-perfusion of a PgP or Mrp1/2 inhibitor significantly increased the brain distribution of glabridin by 33.6-142.9%. The rat brain levels of glabridin were only about 27% of plasma levels when corrected by tissue residual blood and it was increased to up to 44% when verapamil or quinidine was coadministered. The area under the brain concentration-time curve (AUC) of glabridin in mdr1a(-/-) mice was 6.0-fold higher than the wild-type mice. These findings indicate that PgP limits the brain penetration of glabridin through the BBB and PgP may cause drug resistance to glabridin (licorice) therapy for CNS diseases and potential drug-glabridin interactions. However, further studies are needed to explore the role of other drug transporters (e.g. Mrp1-4) in restricting the brain penetration of glabridin.

Role of P-glycoprotein in restricting the brain penetration of tanshinone IIA, a major active constituent from the root of Salvia miltiorrhiza Bunge, across the blood–brain barrier

Tanshinone IIA (TSA) is a major constituent of Salvia miltiorrhiza Bunge widely used in the treatment of stroke. This current study aimed to investigate the nature of brain penetration of TSA using several in vitro and in vivo models. The uptake and efflux of TSA in primary rat brain microvascular endothelial cells (RBMVECs) were altered in the presence of a PgP inhibitor or multidrug-resistance-associated protein (Mrp1/2) inhibitor. A polarized transport of TSA was found in RBMVEC monolayers with facilitated efflux from the abluminal to the luminal side. The polarized transport of TSA was attenuated by PgP or Mrp1/2 inhibitors. In an in situ rat brain perfusion model, TSA crossed the blood–brain barrier at a greater rate than that for sucrose, and the brain penetration was increased in the presence of a PgP or Mrp1/2 inhibitor. The brain levels of TSA were only about 31% of that in the plasma and it increased to 74–77% of plasma levels when verapamil or quinidine was coadministered in rats. The entry of TSA to the central nervous system (CNS) significantly increased in rats subjected to middle cerebral artery occlusion or treatment with quinolinic acid. The normalized brain penetration of TSA in mdr1a(−/−) mice was much higher than the wild-type mice. Taken collectively, these findings provide evidence that TSA has limited brain penetration through the blood–brain barrier owing to the contribution of PgP and possibly Mrp1/2.

Effect of baicalin and berberine on transport of nimodipine on primary-cultured, rat brain microvascular endothelial cells

To investigate whether baicalin and berberine affects the transport of nimodipine (NMD) across the blood-brain barrier (BBB). Primary-cultured, rat brain microvascular endothelial cells (rBMEC) were used as an in vitro model of the BBB. When cells became confluent, the steady-state uptake of NMD by rBMEC with or without baicalin and berberine was measured. The effects of baicalin and berberine on the efflux of NMD from rBMEC were also studied. Baicalin (2-5 microg/mL) increased the uptake of NMD, and baicalin (10-20 microg/mL) decreased the uptake. The steady-state uptake of NMD was higher than that of control group in the presence of 0.01-1 microg/mL berberine, but was lower in the presence of 2-10 microg/mL berberine. The bidirectional effect of baicalin and berberine on the uptake of NMD by rBMEC was found. Higher concentration showed an inhibitory effect, and lower concentration demonstrated an increasing effect.

Low dose of bradykinin selectively increases intracellular calcium in glioma cells

To investigate the underlying basis for the selective modulation of the permeability of the blood–brain barrier by small doses of bradykinin, we first established cell lines of rat brain microvascular endothelial cells (BMECs) and astrocytes by primary cultures from neonatal rats. BMECs, astrocytes and C6 glioma cells were treated with different concentrations of BK (range from 10 – 8 M to 10 – 4 M), and changes of intracellular calcium levels were measured by fluorescence spectrophotometry. Expression levels of B2 receptors were analyzed by Western blot analysis. We found that a low dose (10 – 6 M) of BK could trigger an elevation of intracellular calcium level in C6 glioma cells, whereas astrocytes responded to a higher concentration of BK (10 – 5 M), and the BMECs remained unresponsive to BK. Western blot results showed that C6 glioma cells expressed the highest level of B2 receptors compared with primary astrocytes or BMECs. B2 receptors are highly expressed in glioma cells and a low dose of BK selectively increases the intracellular calcium level in tumor cells which, in turn, could contribute to the selective increase in the permeability of the blood–tumor barrier by small doses of BK.

Comparative proteome analysis of rat brain and coronary microvascular endothelial cells

The endothelium of different organs displays a remarkable heterogeneity, although it presents many common functional and morphological features. However, despite our knowledge of heterogeneity among endothelial cells from different sites, the differences between brain microvascular endothelial cells (BMEC) and coronary microvascular endothelial cells (CMEC) are poorly defined. The aim of this study was to investigate whether BMEC are distinct from CMEC at the protein level. Using the proteomic approach, we comparatively analyzed the proteome of cultured BMEC and CMEC. We reproducibly separated over 2000 polypeptides by using two-dimensional electrophoresis (2-DE) at pH range of 3-10. Using PDQuest software to process the 2-DE gel images, forty-seven protein spots were differentially expressed in the two-endothelial cells. Of these, thirty-five proteins are highly expressed in BMEC, whereas twelve proteins are highly expressed in CMEC. Fifteen proteins in BMEC and seven proteins in CMEC were identified with high confidence by matrix-associated laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF-MS). Our data suggested that BMEC and CMEC were different in several aspects including cytokine and growth-related molecules, stress-related proteins, metabolic enzymes, signal transduction proteins and others. The identification of a set of proteins preferentially expressed in BMEC and CMEC provided new data on the heterogeneity of the endothelium.

Induction of aquaporin 1 by dexamethasone in lipid rafts in immortalized brain microvascular endothelial cells

Water homeostasis in the brain is essential for brain function. We have studied how aquaporin (AQP) 1 expression in GP8 immortalized rat brain microvascular endothelial cells is regulated by glucocorticoid. AQP1 protein level was raised by dexamethasone treatment in a time- and concentration-dependent manner. The up-regulation of AQP1 protein by dexamethasone was associated with an increase of AQP1 mRNA level, with no change in the degradation rate of AQP1 mRNA. AQP1 was concentrated in detergent-insoluble fractions in the cells treated with or without dexamethasone, suggesting that function/trafficking of AQP1 may be regulated via the interaction with lipid rafts. Since glucocorticoid therapy has well known beneficial effects in the treatment of brain edema, the induction of AQP1 by dexamethasone raises a possibility that AQP1 plays a role in ameliorating brain edema.

One-step synthesis of magnetic hollow silica and their application for nanomedicine

Magnetic nanoparticles are usually present in the form of magnetic carriers and used in nanomedicine and biosystem. In this paper, magnetic hollow silica (MHS) nanoparticles were fabricated by a one-step synthesis of Fe3O4 nanoparticles and then coating of silica on nanosized spherical calcium carbonate under alkaline conditions, in which nanosized calcium carbonate (CaCO3, 25–60nm) was used as a scarified template, tetraethoxysilane as a precursor, and Fe3O4 nanoparticles (∼5nm), formed in the initial reaction stage, as magnetic agents. The as-synthesized nanoparticles were immersed in a weak acetic acidic solution to remove CaCO3, forming MHS carriers. The nanostructures of the MHS carriers were characterized by scanning electron microscope, transmission electron microscope, and x-ray diffraction. Superconducting quantum interference device measurement exhibited that the MHS nanoparticles were superparamagnetic. Toxicity was tested for MHS carriers using rat brain microvascular endothelial cells. The cells treated with concentration lower than 50μg∕ml of the MHS nanoparticles showed no significant toxicity. After modification by silane coupling agent, the MHS carriers have strong absorption for ibuprofen in nanomedicine field.

Puromycin‐purified rat brain microvascular endothelial cell cultures exhibit improved barrier properties in response to glucocorticoid induction

In vitro blood-brain barrier (BBB) models using primary rat brain microvessel endothelial cells (BMEC) are often hampered by a lack of culture purity and poor barrier properties. To address these problems, the translation inhibitor puromycin was used to purify rat BMEC cultures. BMEC purities of 99.8% were routinely attained using puromycin treatment, and this technique proved to be far superior to other purification methods of similar difficulty. In contrast to cultures without puromycin treatment, purity of puromycin-treated cultures was unaffected by initial seeding density. Next, rat BMEC monolayer transendothelial electrical resistance (TEER) was increased by glucocorticoid treatment with either corticosterone (CORT) or hydrocortisone (HC), and a corresponding decrease in monolayer permeability to small molecules was observed. Importantly, cultures treated with both puromycin and glucocorticoid attained significantly higher TEER values (CORT 168 +/- 13 Omega x cm2; HC 218 +/- 66 Omega x cm2) than those treated by the glucocorticoid alone (CORT 57 +/- 5 Omega x cm2; HC 70 +/- 2 Omega x cm2). Glucocorticoid induction resulted in BMEC morphological changes that accompanied the increases in TEER, and BMEC tight junctions exhibited improved integrity as visualized by the localization of tight junction proteins zonula occluden-1, occludin and claudin-5. The combined use of puromycin and glucocorticoid therefore provides an in vitro system that is well suited for molecular level BBB investigations.

Molecular mechanisms of radiation‐induced brain injury

The present study was designed to address potential cellular and molecular mechanisms that contribute to brain injury after irradiation. Primary rat astrocytes and rat brain microvascular endothelial cells (RBE4), which are major components of the blood-brain barrier (BBB), were exposed to a single dose of 5 Gy and incubated for up to 48 h following irradiation. To investigate the pro-inflammatory mechanisms of brain injury by irradiation, expression of a variety of pro-inflammatory mediators was assessed by quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR). A marked increase in expression of interleukin-6 (IL-6) and matrix metalloproteinases (MMP-2 and MMP-9) was observed in astrocytes after irradiation. Irradiation also up-regulated expression of tumor necrosis factor-α (TNF-α) and IL-6 in RBE4 cells. In addition, expression levels of vascular endothelial growth factor (VEGF) were determined in astrocytes and RBE4 cells to examine the effects of radiation on angiogenic factors. Exposure of both astrocytes and brain endothelial cells to radiation significantly down-regulated VEGF expression. These data suggest that radiation-induced brain injury may be mediated through two independent mechanisms: the pro-inflammatory pathways by up-regulation of inflammatory mediators and the cerebrovascular rarefaction by down-regulation of VEGF. (This work was supported by CA112593 and VMRCVM New Initiative Grants)

Caspase-8 activation and oxidative stress are involved in the cytotoxic effect of β-amyloid on rat brain microvascular endothelial cells

Several studies have demonstrated that cerebrovascular dysfunction and damage play a significant role in the pathogenesis of Alzheimer disease (AD). In fact, beta-amyloid peptides (Abetas), the major component of the senile plaques and cerebrovascular amyloid deposits in AD, were shown to be cytotoxic to endothelial cells. We have recently observed that Abetas exert a toxic effect on neuromicrovascular endothelial cells (NECs) in a time- and concentration-dependent manner, apoptosis playing a pivotal role in this process. Hence, it seemed worthwhile to investigate the Abeta-mediated apoptosis mechanism in NECs. Abetas were found to induce, after a short incubation period, apoptosis throughout caspase-8 activation. Moreover, Abetas elicited a highly significant (p < 0.001) increase in superoxide dismutase (SOD) levels after a 3-h exposure period, while SOD concentration was not affected after a 24-h incubation. The time-dependent increase in SOD concentration is probably correlated with the production of an excess of reactive oxygen species. Collectively, our findings allow us to conclude that: i) Abetas may induce apoptosis via the activation of caspase-8, presumably by cross-linking and activating receptors of the death-receptor family; ii) oxidative stress is possibly involved in the Abeta-induced cytotoxic effect; and iii) these two mechanisms do not act sequentially but, probably, are independent of each other.

Transport of carbamazepine and drug interactions at blood-brain barrier1

To investigate the characteristics of carbamazepine (CBZ) transport and drug interactions at the blood-brain barrier. Cultured rat brain microvascular endothelial cells (rBMEC) were used as an in vitro model of the blood-brain barrier (BBB). When cells became confluent, CBZ uptake over time was recorded by incubation of the cells in a medium containing 10 mg/L CBZ at 37 degree. The steady-state uptake of CBZ by rBMEC was tested for different CBZ concentrations at 37 degree. The effects of various agents on the steady-state uptake of CBZ and efflux of CBZ from rBMEC were also studied. The uptake of CBZ by rBMEC was time- and concentration-dependent. The steady-state uptake occurred at 30 min for incubation. The steady-state uptake was significantly increased (P<0.01) by treatment with dinitrophenol. The co-administration of cyclosporine A significantly increased the steady-state uptake of CBZ by the rBMEC, whereas co-administration of olanzapine significantly decreased the uptake in a concentration- and temperature-dependent manner. The efflux of CBZ from rBMEC was inhibited by CsA. The transport of CBZ at the BBB is mediated by many transporters. Some specific ABC (ATP-binding cassette, ABC) efflux transporters may be involved in the transport of CBZ. Drugs influence the transport of CBZ at the BBB in different ways.

Pericyte abundance affects sucrose permeability in cultures of rat brain microvascular endothelial cells

The blood–brain barrier is a physical and metabolic barrier that restricts diffusion of blood-borne substances into brain. In vitro models of the blood–brain barrier are used to characterize this structure, examine mechanisms of damage and repair and measure permeability of test substances. The core component of in vitro models of the blood–brain barrier is brain microvascular endothelial cells. We cultured rat brain microvascular endothelial cells (RBMEC) from isolated rat cortex microvessels. After 2–14 days in vitro (DIV), immunohistochemistry of these cells showed strong labeling for zona occludens 1 (ZO-1), a tight junction protein expressed in endothelial cells. Pericytes were also present in these cultures, as determined by expression of α-actin. The present study was performed to test different cell isolation methods and to compare the resulting cell cultures for abundance of pericytes and for blood–brain barrier function, as assessed by 14C-sucrose flux. Two purification strategies were used. First, microvessels were preabsorbed onto uncoated plastic for 4 h, then unattached microvessels were transferred to coated culture ware. Second, microvessels were incubated with an antibody to platelet-endothelial cell adhesion molecule 1 (PECAM-1; CD31) precoupled to magnetic beads, and a magnetic separation procedure was performed. Our results indicate that immunopurification, but not preadsorption, was an effective method to purify microvessels and reduce pericyte abundance in the resulting cultures. This purification significantly reduced 14C-sucrose fluxes across cell monolayers. These data indicate that pericytes can interfere with the development of blood–brain barrier properties in in vitro models that utilize primary cultures of RBMECs.