Morphological and functional characterization of an in vitro blood-brain barrier model

Cell culture models have been extensively used for studies of blood-brain barrier (BBB) function. However, several in vitro models fail to reproduce some, if not most, of the physiological and morphological properties of in situ brain microvascular endothelial cells. We have recently developed a dynamic, tridimensional BBB model where endothelial cells exposed to intraluminal flow form a barrier to ions and proteins following prolonged co-culturing with glia. We have further characterized this cell culture model to determine whether these barrier properties were due to expression of a BBB phenotype. Endothelial cells of human, bovine or rodent origin were used. When co-cultured with glia, intraluminally grown endothelial cells developed features similar to in vivo endothelial cells, including tight junctional contacts at interdigitating processes and a high transendothelial resistance. This in vitro BBB was characterized by the expression of an abluminal, ouabain-sensitive Na/K pump, and thus favored passage of potassium ions towards the lumen while preventing K+ extravasation. Similarly, the in vitro BBB prevented the passage of blood-brain barrier-impermeant drugs (such as morphine, sucrose and mannitol) while allowing extraluminal accumulation of lipophylic substances such as theophylline. Finally, expression of stereo-selective transporters for Aspartate was revealed by tracer studies. We conclude that the in vitro dynamic BBB model may become an useful tool for the studies of BBB-function and for the testing of drug passage across the brain endothelial monolayer.     

An in vitro blood-brain barrier model: cocultures between endothelial cells and organotypic brain slice cultures

This communication describes a novel in vitro blood-brain barrier (BBB) model: organotypic slice cultures from the central nervous system were overlaid on endothelial cell monolayers grown on permeable membranes. Morphological, electrophysiological, and microdialysis approaches were carried out to characterize and validate this model. After 10 days in coculture, morphological studies reveal the presence of tight junctions. Electrophysiological recordings of neuronal activity performed on organotypic cultures with or without an endothelial cell monolayer show that amplitude of evoked responses were comparable, indicating good viability of cocultures after 2 weeks. Perfusion of known BBB permeable or nonpermeable molecules was used to test the coculture tightness in conjunction with electrophysiological or microdialysis approaches: application of glutamate (Glu), which doesn't easily cross the BBB, triggers off rhythmic activity only in control cultures, whereas epileptogenic activity was observed in both control cultures and cocultures during perfusions with picrotoxin, a molecule that can diffuse through the BBB. Finally, the microdialysis technique was used to determine the permeability of molecules coming from the perfusion chamber: L-dopa, dopamine, and Glu were employed to assess the selective permeability of the coculture model. Thus, these results indicate that the in vitro model described possesses characteristics similar to those of the BBB in situ and that cocultures of organotypic slices and endothelial cell monolayers have potential as a powerful tool for studying biochemical mechanisms regulating BBB function and drug delivery to the central nervous system.     

Astrocytic contributions to blood-brain barrier (BBB) formation by endothelial cells: a possible use of aortic endothelial cell for in vitro BBB model

Astrocytic contribution of endothelial cell monolayer permeability was examined in two blood-brain barrier (BBB) models, using the coculture in a double chamber system: rat astrocytes and bovine aortic endothelial cells (BAECs) or bovine brain endothelial cells (BBECs). In system 1, where astrocytes were separated from endothelial cells, a 40% reduction in L-glucose permeability of the BBEC monolayer, but not the BAEC monolayer, was observed by cocultivation with astrocytes. Although several passages of BBEC in culture elicited morphological transformation from spindle-shapes to cobblestone-like features, the passaged BBECs remained responsive to astrocytes in coculture in system 1 (37% reduction of the L-glucose permeability). By contrast, in system 2, where respective endothelial cells and astrocytes layered on the upper and lower surfaces of a membrane, the permeability of both BAEC and BBEC monolayers was reduced by cocultivation with astrocytes (75% reduction for BAEC and 40% reduction for BBEC). BAECs in this contiguous coculture (system 2) with astrocytes showed numerous tight junction-like structures characteristic of the BBB in vivo. These results suggest that primary cultured BBECs, which had been primed by astrocytes in vivo, retain a higher sensitivity to astrocytes possibly through an astrocytic soluble factor (s) to exhibit BBB-specific phenotypes, and that even BAEC from extra-neural tissues, when cultured with astrocytes in close proximity in vitro, may acquire the similar phenotypes and serve for an extensive use of BBB model in vitro.     

Monoacylation of ribonuclease A enables its transport across an in vitro model of the blood-brain barrier

A major challenge in correcting disorders affecting the central nervous system is to induce blood-brain barrier (BBB) crossing of exogenous biological compounds such as proteins or specific nucleic acid sequences. Fatty acids, due to their high membrane affinity and low toxicity, are good potential candidates to promote this barrier crossing when covalently bound to proteins. In this paper, we report that regiospecific monoacylation of ribonuclease A (RNase A) enables its transport across an in vitro model of the BBB. Myristoylated, palmitoylated and stearoylated RNases A were prepared using reversed micelles as microreactors. All the purified acylated RNases A kept their original enzymatic activity. A single fatty acid moiety was linked to RNase A through the alpha-amino group of its N-terminal lysine as shown by powerful analytical techniques. The ability of monoacylated RNases A to cross an in vitro model of the BBB is strictly dependent on the acyl chain length, which must be at least 16 carbon atoms long.     

Modulation of retinal endothelial barrier in an in vitro model of the retinal microvasculature

The prediabetic/diabetic condition functionally alters the microvascular bed of the eye and the breakdown in the transvascular barrier may be produced by changes in the retinal endothelial barrier. To better understand how retinal microvessel barrier is maintained and is altered in vivo this study applies and extends our previously described in vitro permeability technique to study retinal endothelial monolayers. The model of the retinal microvasculature consists of retinal capillary endothelial cells cultured on porous microcarrier beads and perfused in chromatographic 'cell-columns'. This model design relies on indicator-dilution techniques to measure the permeability of the retinal endothelial monolayer and detects small changes in retinal endothelial permeability produced by treatments. Bovine retinal capillary endothelial cells (RCE) were obtained using an endothelial selective media. RCE were seeded at 3 x 10(4) cells cm-2 of fibronectin-coated gelatin microcarriers. After 7 days of microcarrier culture, microcarriers were poured to form columns 0.66 cm in diameter and 1.6 cm in length. The cell-column elution patterns of coinjected optically absorbing tracers (blue dextran 2 x 10(6) Da; cyanocobalamin 1355 Da; sodium fluorescein 376 Da) were analysed to estimate the permeability of the RCE monolayers covering the microcarriers. Scanning electron microscopic examination showed complete monolayer formation on the surface of the microcarriers. We found that baseline monolayer permeability averaged 7.57 +/- 0.57 x 10(-5) cm sec-1 for cyanocobalamin and 9.29 +/- 0.78 x 10(-5) cm sec-1 for sodium fluorescein (mean +/- S.E.M., n = 39). Permeability did not increase over 2 hr of cell-column perfusion. Permeability was decreased by 1 micron isoproterenol (n = 3) and increased by 1 microgram ml-1 cytochalasin D (n = 5). This is one of the first reports of in vitro permeability values for the transport barrier formed by retinal microvascular endothelial cells. Furthermore, the endothelial component of the retinal barrier is dynamic, and is enhanced by isoproterenol and diminished by cytochalasin D.     

Functional properties of a new line of immortalized human endothelial cells

An immortalized human endothelial cell line was obtained by transfecting umbilical vein endothelial cells in primary culture with plasmid pMK16 containing SV40 replicated origin defective gene. The essential functional properties demonstrated in these immortalized human endothelial cells also retaining the classical phenotypical characteristics of endothelial cells in primary culture are: (1) endothelin-1 secretion; (2) capacity to convert big endothelin-1 into endothelin-1; (3) the capacity to secrete IL1 beta and IL6 interleukins both spontaneously and after lipopolysaccharide (LPS) stimulation; (4) arginine transfer from the extracellular to the intracellular medium. Such stable cell line could facilitate studies of regulation of endothelin-1 production; (5) No-synthase activity; (6) binding and metabolisation of acetylated low-density lipoproteins.     

Transport of GM1 and GM1 inner ester across an in vitro model of the blood-brain barrier

Gangliosides, especially GM1, attenuate the in vivo damage caused by various neurotoxins. The chemically neutral inner ester of GM1 may be a better cytoprotective agent against some neurotoxins than the parent GM1 compound, because it may cross the blood-brain barrier (BBB) more easily than the anionic GM1. Using an in vitro bovine brain endothelial cell model of the BBB, we show the inner ester more readily transverse the tight junction barrier of this model than does GM1. Further, it is demonstrated that the GM1 inner ester is stable for several hours at pH values between 7.0 and 8.2 at 37 degrees C. Finally, the results illustrate that the BBB model may be useful for testing other gangliosides and their various derivatives for increased ability to cross the BBB.     

In vitro induction of apoptosis or differentiation by dopamine in an immortalized olfactory neuronal cell line

A new neuronal cell line was generated by transfection of rat olfactory epithelium with immortalizing recombinant oncogene E1A of adenovirus-2. The resulting 13.S.1.24 line of transformed cells expressed an antigenic phenotype of olfactory neuronal progenitors. Addition of dopamine to 13.S.1.24 cultures induced reduction of cell number within 2 days. Two hallmarks of apoptosis were detected in dopamine-treated cultures: internucleosomal DNA fragmentation and nuclear condensation. Dopamine did not alter the cell proliferation rate, as assessed by [3H]thymidine incorporation. Dopamine also stimulated differentiation of surviving 13.S.1.24 cells into bipolar olfactory marker protein-immunoreactive neurons. Time-dependency assessments over 1 week of treatment indicated that apoptosis and differentiation induced by dopamine were concomitant. Both apoptosis and differentiation triggered by dopamine were dose-dependent, half-maximal effects being obtained with approximately 10 microM dopamine. Mediation of both effects by dopaminergic D2 receptors was supported by several observations: active dopamine doses in micromolar ranges, quinpirole agonism and eticlopride antagonism, D2-characteristic rank order of potency among the three agonists tested, and specific binding of a selective D2-like radioligand to 13.S.1.24 cells. The present data altogether indicated that dopamine commits immortalized olfactory neuronal cells in vitro either to apoptosis or to olfactory-like differentiation via D2 dopaminergic receptors.     

Role of P-glycoprotein in colchicine and vinblastine cellular kinetics in an immortalized rat brain microvessel endothelial cell line

Uptake and efflux of colchicine and vinblastine, whose effects are related to their high-affinity binding to tubulin, were studied in the immortalized rat brain microvessel endothelial cell line RBE4. At 10 nM extracellular drug concentration, uptake equilibrium was approached at 45 hr for colchicine, but at only 3.5 hr for vinblastine. After 1 hr preincubation with 200 nM colchicine or vinblastine, drug efflux fitted biexponential kinetics with an initial fast phase (half-life = 2.2 min and 9.6 min, respectively) and a later slow phase (half-life = 3.6 hr and 1.8 hr, respectively). After 6 hr preincubation with 200 nM colchicine, only the slow phase (half-life = 3.6 hr) could be observed. The colchicine and vinblastine uptake rate was increased by cyclosporin A, an inhibitor of the drug efflux pump P-glycoprotein, which is expressed at the blood-brain barrier. Whereas cyclosporin A decreased vinblastine efflux, its effect on colchicine efflux was apparent after only 13 hr washout and was associated with the re-uptake by cells of colchicine molecules. Differences in uptake kinetics of colchicine and vinblastine could be related to differences in their lipid solubility, and mainly in their binding affinities to tubulin. Differences in efflux kinetics could in addition be explained by the involvement of P-glycoprotein in the efflux of vinblastine, whereas efflux of colchicine was not influenced by this pump. Indeed, binding of colchicine to tubulin would imply that most intracellular colchicine may be inaccessible to P-glycoprotein. In the case of a cytotoxic drug such as colchicine, which is tightly bound to intracellular receptors, the role of P-glycoprotein within the blood-brain barrier would be more to protect the brain against entry of this drug than to detoxify the brain by its extraction.     

Interactions of orthopaedic metals with an immortalized rat osteoblast cell line

The toxicity of nickel, chromium (III) and (VI), vanadium and aluminium was compared in an immortalized neonatal rat osteoblast cell line using the MTT assay and a novel index of cytotoxicity, alkaline phosphatase (ALP) activity. Where toxicity was observed, ALP was a consistently more sensitive detection method than the MTT assay. The toxicity of the metals increased in the order aluminium < chromium (III) < vanadium < nickel < chromium (VI). alpha-Tocopherol partially prevented nickel-induced toxicity (as assessed by ALP activity), whereas ascorbic acid had no protective effect. Chromium (VI) was more toxic than (III), with significant toxicity observed at 0.5 microM. It is thought that Cr (III) cannot readily penetrate the cell membrane and this may account for the lower toxicity. Aluminium had a stimulatory effect on cell growth at low concentrations (0.5 microM). The combination of immortalized rat osteoblasts and the ALP activity test provides a powerful tool for in vitro testing of orthopaedic materials.     

Expression of glucocorticoid and mineralocorticoid receptors in an immortalized hippocampal neuronal cell line

A clonal cell line of rat embryonic hippocampal origin (H19-7) has been examined for the expression of glucocorticoid receptors (GR) and mineralocorticoid receptors (MR). H19-7 cells grown at 33 degrees C continue to divide, however when grown at 39 degrees C in reduced levels of serum the cells undergo morphological differentiation and express neuronal properties. Immunocytochemistry demonstrated that H19-7 cells express both MR and GR when grown at either 33 degrees C or 39 degrees C. GR mRNA is readily detected in H19-7 cells by RNase protection assay. MR mRNA levels in H19-7 cells are too low to detect by RNase protection, but can be detected by RT-PCR. RT-PCR also demonstrated that H19-7 cells express more GR mRNA than primary hippocampal neurons. Since previous studies have shown that the level of MR mRNA is higher than that of GR mRNA in hippocampal neurons, these studies suggest that H19-7 cells represent hippocampal neurons immortalized at an early stage when the MR system is not yet fully differentiated.     

Application of a human immortalized fibroblast cell line in laboratory diagnosis of autoimmune diseases

Many autoantibodies reacting with cellular and nuclear components have been described in sera of patients with autoimmune diseases. The most important diagnostic markers for those diseases are antinuclear antibodies (ANA). The first choice for laboratory diagnosis of autoimmune diseases is to use cultured monolayer cells as a nuclear substrate. Up to now the HEp-2 cell line derived from a human carcinoma of the larynx, appears to be the most sensitive and specific nuclear substrate. The cultured fibroblast monolayer cells have also been applied to detect the ANA, although the application was not recommended by one study. Thus to evaluate the applicability of our immortalized human fibroblast cell line (CCFS-1/KMC) as a nuclear substrate, commercial HEp-2 MBL monolayer cells was used as the standard substrate. The results of this report showed the applicability of the CCFS-1/KMC cell line as a nuclear substrate to detect the ANA of autoimmune diseases. The sensitivity of this fibroblast cell line was the same as both of the HEp-2 nuclear substrates (HEp-2 and HEp-2 MBL). The specificity of the CCFS-1/KMC cell line was similar to the HEp-2 substrate. Since the specificity of both of the above substrates were lower than the standard nuclear substrate HEp-2 MBL, therefore, if the specificity can be improved by changing the protocols of the substrate preparation, the CCFS-1/KMC cell line will be a good nuclear substrate for detecting the ANA of autoimmune diseases.     

Astrocyte-derived monocyte-chemoattractant protein-1 directs the transmigration of leukocytes across a model of the human blood-brain barrier

The migration of leukocytes across the blood-brain barrier (BBB) into the central nervous system is critical in the pathogenesis of central nervous system inflammatory diseases. The production of chemokines, such as monocyte-chemoattractant protein-1 (MCP-1), by endothelial cells (EC) and astrocytes may initiate and amplify this process. Using a coculture of human EC and astrocytes to model the BBB, we demonstrated that exogenous MCP-1 induces the transmigration of monocytes in a dose-dependent manner. TNF-alpha, IFN-gamma, or IL-1beta treatment of cocultures also induced significant migration of monocytes that correlates with the induction of MCP-1 protein. TGF-beta, previously shown to induce MCP-1 expression in astrocytes, but not in EC, caused migration of monocytes across cocultures, but not across EC grown alone. Monocytes and lymphocytes transmigrated across cytokine-treated cocultures in greater numbers than across EC alone. Astrocytes were the main source of cytokine-induced MCP-1, supporting a role for astrocytes in facilitating leukocyte transmigration. A blocking Ab to MCP-1 inhibited MCP-1- and cytokine-induced transmigration of monocytes by 85-90%. Cytokine treatment of cocultures also resulted in the transmigration of activated, CD69-positive lymphocytes. The MCP-1-mediated transmigration of monocytes across cocultures was blocked using an Ab to ICAM-1 and inhibited by 55% using an Ab to E-selectin. These data suggest a central role for astrocyte-derived MCP-1 in directing the migration of monocytes and lymphocytes across the BBB.     

Preliminary characterization of glial-secreted factors responsible for the induction of high electrical resistances across endothelial monolayers in a blood-brain barrier model

Low levels of sex hormone-binding globulin (SHBG) are considered to be an indirect index of hyperinsulinemia, predicting the later onset of diabetes mellitus type 2. In the insulin resistance state and in the presence of an increased pancreatic beta-cell demand (e.g. obesity) both absolute and relative increases in proinsulin secretion occur. In the present study we investigated the correlation between SHBG and pancreatic beta-cell secretion in men with different body compositions. Eighteen young men (30.0 +/- 2.4 years) with normal glucose tolerance and body mass indexes (BMI) ranging from 22.6 to 43.2 kg/m2 were submitted to an oral glucose tolerance test (75 g) and baseline and 120-min blood samples were used to determine insulin, proinsulin and C-peptide by specific immunoassays. Baseline SHBG values were significantly correlated with baseline insulin (r = -0.58, P < 0.05), proinsulin (r = -0.47, P < 0.05), C-peptide (r = -0.55, P < 0.05) and also with proinsulin at 120 min after glucose load (r = -0.58, P < 0.05). Stepwise regression analysis revealed that proinsulin values at 120 min were the strongest predictor of SHBG (r = -0.58, P < 0.05). When subjects were divided into obese (BMI > 28 kg/m2, N = 8) and nonobese (BMI < or = 25 kg/m2, N = 10) groups, significantly lower levels of SHBG were found in the obese subjects. The obese group had significantly higher baseline proinsulin, C-peptide and 120-min proinsulin and insulin levels. For the first time using a specific assay for insulin determination, a strong inverse correlation between insulinemia and SHBG levels was confirmed. The finding of a strong negative correlation between SHBG levels and pancreatic beta-cell secretion, mainly for the 120-min post-glucose load proinsulin levels, reinforces the concept that low SHBG levels are a suitable marker of increased pancreatic beta-cell demand.     

Culture of endothelial cells as an in vitro model for experimental and clinical research]

Culture of endothelial cell is gotten from human umbilical cord by enzymatic digestion. For the growth of cells in culture, medial RPMI 1640 with 20% mixed human serum (NHS) or 20% fetal calf serum (FCS), endothelial cell growth factor (ECGF) and heparin have been used. Plastic, 0.1% gelatin and fibronectin have been used as a fundament. Immune identification of endothelial cells was culture is performed by monoclonal antibodies on vWF:Ag. Homogeneous cell line in culture might be used as in vitro model in both experimental and practice work.     

Involvement of platelet-activating factor in cell death induced under ischemia/postischemia-like conditions in an immortalized hippocampal cell line

The involvement of platelet-activating factor (PAF) in cell damage induced by ischemia/postischemia-like conditions was studied in a hippocampus-derived cell line, HN33.11. Cells exposed to N2-saturated glucose-free HEPES-buffered saline (ischemia) for 5 h followed by 18 h of incubation in serum-free control medium (postischemia reincubation) remained 67.4 +/- 2.4% viable in comparison with sham-treated cells. Analysis of DNA fragmentation in combination with Hoechst 33258 staining indicates that apoptosis is the dominant mode of cell death in the present model. PAF level during 10 h of ischemia was unchanged. However, an increase in PAF accumulation was found early during the reincubation period that followed 5 h of ischemia. Peak PAF concentrations were noted at 2 h after initiation of reincubation and rapidly declined to control level after 7 h of reincubation. Consistent with a role of PAF in mediating cell death under ischemia/postischemia reincubation in this model, the PAF antagonist BN 50739 exerted a dose-dependent protective effect. Maximal protection (85.7 +/- 5.4%) of the cells from ischemia/reincubation-induced cell damage was achieved at 0.1 microM BN 50739. The PAF antagonist lacked any protective effect against ischemia-induced cell death. On the other hand, the addition of the stable PAF analogue 1-O-hexadecyl-2-N-methylcarbamyl-sn-glycero-3-phosphocholine (MC-PAF) at the onset of ischemia potentiated ischemia/reincubation-induced apoptosis--an effect that was blocked by BN 50739. Pretreatment of HN33.11 cells with the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid acetoxymethyl ester (BAPTA-AM) also provided a protective effect against ischemia/reincubation-induced cell damage. BAPTA-AM increased cell viability by 50%. Pretreatment with BAPTA-AM also decreased ischemia/reincubation-induced PAF accumulation in HN33.11 cells. The results suggest that PAF, acting via a PAF receptor, is at least in part mediating apoptosis under ischemia/postischemia-like conditions in HN33.11 cells.     

BG-1 ovarian cell line: an alternative model for examining estrogen-dependent growth in vitro

Between 1.4.96 and 1.3.97 27 patients with acute infections of bone and soft tissues (n = 13), chronic osteomyelitis (n = 8), and chronic wounds (n = 6) were treated by using Instillation-Vacuum-Sealing. Polyvinylalcohol sponges with drainage tubes were used to cover the internal or external wound surfaces which resulted from surgical debridement. Having hermetically covered the wound with a transparent film dressing a vacuum source generated a partial vacuum in the sponge which was modified according to the type of wound between 20 and 80 kPa. Several times daily, the vacuum line was blocked and, in an alternating fashion, antiseptic or antibiotic solution instilled for 30 minutes. Then, the vacuum was reestablished and the fluids drained from the wound. Seven days later, intermittent drug instillation was stopped and there was either immediate or delayed wound closure by secondary suturing (n = 22), skin grafting (n = 3) or spontaneous epithelialization (n = 2). During a follow-up from the beginning of the instillation treatment of 4.2 (3-14) months there was one recurrency of infection in a patient with chronic osteomyelitis.     

Quantification of different transferrin receptor pools in primary cultures of porcine blood-brain barrier endothelial cells

Distribution of iron in the brain varies with region, cell type, and age. Furthermore, some neurological diseases are accompanied by an abnormal accumulation of iron in specific areas of the CNS. These findings implicate a mobile intracerebral iron pool; however, transport of iron across the blood-brain barrier and its regulation are largely unknown. In an extensive series of experiments in primary cultures of porcine blood-brain barrier endothelial cells, we separately quantified surface-bound and total cellular transferrin receptor pools. Although 90% of all transferrin receptors were located inside the cell, only 10% of these intracellular receptors actively took part in the endocytic cycle. This large "inactive" intracellular transferrin receptor pool could either function as a storage site for spare receptors or be activated by the cell to increase its capacity for iron transport. Data were corrected for nonspecific binding by a separate biochemical assessment using a 100-fold excess of unlabeled ligand. Data were also analyzed in a nonlinear curve-fit program. This resulted in a less elaborate and less biased estimate of nonspecific binding.     

Electron microscopy of the blood-brain barrier in disease

The anatomical site of the blood-brain barrier (BBB) is at the capillary endothelium mainly, with some contribution from astrocytes. Electron microscopic observations of endothelial cells and perivascular astrocytes comprising the BBB in brain edema and other pathological conditions are reviewed in this article. The tight junctions of cerebral endothelial cells open under several conditions such as infusion of hyperosmolar solutions. Pinocytotic vesicles increase under various pathological conditions and fenestrae appear in blood vessels of certain brain tumors and several non-neoplastic lesions. Inflammatory cells penetrate between or through endothelial cells. In long standing lesions, endothelial cells containing various tubular structures such as Weibel-Palade bodies proliferate. Other alterations include surface infoldings of endothelial cells and fluid diffusion through damaged endothelium. Astrocytic alterations include abnormal junctions between astrocytic processes in certain gliomas. In vivo and in vitro studies suggest that astrocytes maintain or develop certain functions of BBB. As the BBB is disrupted, edema fluid infiltrates the brain parenchyma. Because the white matter consists of nerve fibers without demonstrable junctions, it invades between nerve fibers. In the gray matter, expansion of the fluid is limited by complicated anatomical structures. In myelinated nerve fibers, edema fluid accumulates in five separate compartments of extracellular space.