Sodium Fluorescein Permeability Research Articles

Cilostazol strengthens barrier integrity in brain endothelial cells.

We studied the effect of cilostazol, a selective inhibitor of phosphodiesterase 3, on barrier functions of blood-brain barrier (BBB)-related endothelial cells, primary rat brain capillary endothelial cells (RBEC), and the immortalized human brain endothelial cell line hCMEC/D3. The pharmacological potency of cilostazol was also evaluated on ischemia-related BBB dysfunction using a triple co-culture BBB model (BBB Kit™) subjected to 6-h oxygen glucose deprivation (OGD) and 3-h reoxygenation. There was expression of phosphodiesterase 3B mRNA in RBEC, and a significant increase in intracellular cyclic AMP (cAMP) content was detected in RBEC treated with both 1 and 10 μM cilostazol. Cilostazol increased the transendothelial electrical resistance (TEER), an index of barrier tightness of interendothelial tight junctions (TJs), and decreased the endothelial permeability of sodium fluorescein through the RBEC monolayer. The effects on these barrier functions were significantly reduced in the presence of protein kinase A (PKA) inhibitor H-89. Microscopic observation revealed smooth and even localization of occludin immunostaining at TJs and F-actin fibers at the cell borders in cilostazol-treated RBEC. In hCMEC/D3 cells treated with 1 and 10 μM cilostazol for 24 and 96 h, P-glycoprotein transporter activity was increased, as assessed by rhodamine 123 accumulation. Cilostazol improved the TEER in our triple co-culture BBB model with 6-h OGD and 3-h reoxygenation. As cilostazol stabilized barrier integrity in BBB-related endothelial cells, probably via cAMP/PKA signaling, the possibility that cilostazol acts as a BBB-protective drug against cerebral ischemic insults to neurons has to be considered.

Membrane configuration optimization for a murine in vitro blood–brain barrier model

A powerful experimental tool used to study the dynamic functions of the blood–brain barrier (BBB) is an in vitro cellular based system utilizing cell culture inserts in multi-well plates. Currently, usage of divergent model configurations without explanation of selected variable set points renders data comparisons difficult and limits widespread understanding. This work presents for the first time in literature a comprehensive screening study to optimize membrane configuration, with aims to unveil influential membrane effects on the ability of cerebral endothelial cells to form a tight monolayer. First, primary murine brain endothelial cells and astrocytes were co-cultured in contact and non-contact orientations on membranes of pore diameter sizes ranging from 0.4μm to 8.0μm, and the non-contact orientation and smallest pore diameter size were shown to support a significantly tighter monolayer formation. Then, membranes made from polyethylene terephthalate (PET) and polycarbonate (PC) purchased from three different commercial sources were compared, and PET membranes purchased from two manufacturers facilitated a significantly tighter monolayer formation. Models were characterized by transendothelial electrical resistance (TEER), sodium fluorescein permeability, and immunocytochemical labeling of tight junction proteins. Finally, a murine brain endothelial cell line, bEnd.3, was grown on the different membranes, and similar results were obtained with respect to optimal membrane configuration selection. The results and methodology presented here on high throughput 24-well plate inserts can be translated to other BBB systems to advance model understanding.

Expression of Tight Junction Proteins by Umbilical Vein Epithelial Cells Co-Cultured with Allogenic Astrocytes

We studied expression of tight junction proteins and formation of the barrier properties in the culture of umbilical vein endothelial cells under conditions of co-culturing with allogenic GFAP-positive astrocytes. This culturing significantly increases of expression of tight junction proteins (claudin-5, occludin, and ZO-1). The formation of tight junctions significantly increased transendothelial resistance and reduced permeability for sodium fluorescein. Thus, reproducible in vitro model for the study of endothelial barrier properties was created based on co-culturing of umbilical endothelial cells and human astrocytes. This model can be used for evaluation of the permeability of the tissue-blood barriers for substances of different chemical structure and for studies of factors modulating the state of cell-cell contacts.

Melatonin inhibits IL1β-induced MMP9 expression and activity in human umbilical vein endothelial cells by suppressing NF-κB activation

Matrix metalloproteinases (MMPs) have been involved in inflammatory and degradative processes in pathologic conditions. The purpose of this study was to investigate the protective effect of melatonin in human umbilical vein endothelial cell (HUVEC) monolayer permeability and the regulation of MMP9 induced by interleukin 1β (IL1β (IL1B)) in HUVECs. Protection studies were carried out with melatonin, a well-known antioxidant and antiinflammatory molecule. MMP9 expression was increased with IL1β induction in HUVECs. Melatonin showed a barrier-protective role by downregulation of MMP9 and upregulation of tissue inhibitor of metalloproteinase-1 expression in HUVECs. Meanwhile, melatonin also decreased sodium fluorescein permeability and counteracted the downregulation of vascular endothelial cadherin and occludin expression in HUVECs. During inflammatory stimulus, nuclear factor-κB (NF-κB) plays a significant role in regulating MMP genes expression, thus the function of NF-κB in HUVECs' barrier disruption was investigated. IL1β induced nuclear translocation of NF-κB in HUVECs and regulated MMP9 expression. However, NF-κB translocation into the nucleus was inhibited significantly by melatonin. Our results show that melatonin decreases the permeability of monolayer endothelial cell induced by IL1β. At the same time, melatonin decreased the expression and activity of MMP9 by a NF-κB-dependent pathway in HUVECs induced by IL1β.

In-vitro and in-vivo evaluation of carrageenan/methylcellulose polymeric systems for transscleral delivery of macromolecules

In this study, polymeric dispersions composed of methylcellulose (MC) and either kappa carrageenan (KC) or iota carrageenan (IC) were proposed as a platform for transscleral delivery of macromolecules. The additive effects of the two polymers were investigated using oscillatory rheometer and FT-IR spectroscopy. Mechanical spectra demonstrated a conformation dependent association of the two polymers at 37 °C in the presence of selected counter ions. The polymer association was also confirmed by the shifts in MC peaks at 1049.5, 1114 and 1132.9 cm −1 in the presence of carrageenans, which corresponds to the stretching vibrations of C–O–C bonds of the polysaccharides. The MC–IC polymeric system displayed the highest bio-adhesion, owing to the relatively high negative charge. However, the MC–IC system did not affect the in-vitro scleral permeability of sodium fluorescein and 10 kDa FITC-dextran. Nonetheless, the formulation properties had a substantial impact on the results of the in-vivo studies. The efficacy of transscleral drug delivery was determined using rats with altered connexin 43 (Cx43) levels, a gap junction protein, in the choroid. Periocular injection of Cx43 antisense oligonucleotides (AsODN) incorporated in the MC–IC system lead to a significant reduction in the Cx43 levels in the choroid of rats at 24 h of treatment. AsODN incorporated in phosphate buffered saline (PBS) also demonstrated a trend towards reduced Cx43 levels; however this was not statistically significant owing to great variability between treated animals. Consequently the in-vivo data suggests the transscleral route to be of value in delivering therapeutics to the choroid. Moreover this study identified a new polymeric system based on MC and IC which provides aqueous loading of therapeutics and prolonged retention at the site of administration.

Inhibitory effects of alcohol on glucose transport across the blood–brain barrier leads to neurodegeneration: preventive role of acetyl-l-carnitine

Evidence shows that alcohol intake causes oxidative neuronal injury and neurocognitive deficits that are distinct from the classical Wernicke-Korsakoff neuropathy. Our previous findings indicated that alcohol-elicited blood-brain barrier (BBB) damage leads to neuroinflammation and neuronal loss. The dynamic function of the BBB requires a constant supply and utilization of glucose. Here we examined whether interference of glucose uptake and transport at the endothelium by alcohol leads to BBB dysfunction and neuronal degeneration. We tested the hypothesis in cell culture of human brain endothelial cells, neurons and alcohol intake in animal by immunofluorescence, Western blotting and glucose uptake assay methods. We found that decrease in glucose uptake correlates the reduction of glucose transporter protein 1 (GLUT1) in cell culture after 50 mM ethanol exposure. Decrease in GLUT1 protein levels was regulated at the translation process. In animal, chronic alcohol intake suppresses the transport of glucose into the frontal and occipital regions of the brain. This finding is validated by a marked decrease in GLUT1 protein expression in brain microvessel (the BBB). In parallel, alcohol intake impairs the BBB tight junction proteins occludin, zonula occludens-1, and claudin-5 in the brain microvessel. Permeability of sodium fluorescein and Evans Blue confirms the leakiness of the BBB. Further, depletion of trans-endothelial electrical resistance of the cell monolayer supports the disruption of BBB integrity. Administration of acetyl-L: -carnitine (a neuroprotective agent) significantly prevents the adverse effects of alcohol on glucose uptake, BBB damage and neuronal degeneration. These findings suggest that alcohol-elicited inhibition of glucose transport at the blood-brain interface leads to BBB malfunction and neurological complications.

Murine in vitro model of the blood–brain barrier for evaluating drug transport

In vitro blood–brain barrier (BBB) models help predict brain uptake of potential central nervous system drug candidates. Current in vitro models are composed of brain microvascular endothelial cells (BMEC) that are isolated from rat, bovine, or porcine. However, most in vivo studies on drug transport through the BBB are performed in small laboratory animals, specially mouse and thus murine in vitro BBB models serve as better surrogates to correlate with these studies. Here we describe the functional characterization of a reproducible in vitro model composed of murine BMEC co-cultured with rat primary astrocytes in the presence of biochemical inducing agents. The co-cultures presented high TEER and low sodium fluorescein permeability. Expression of specific BBB tight junction proteins (occludin, claudin-5, ZO-1) and the functionality of transporters (Pgp, GLUT1) were detected by immunocytochemistry and Western blotting. These results indicated a 2.5-fold increase in the expression levels of these proteins in the presence of astrocytes. In addition, a high correlation coefficient (0.98) was obtained between the permeability of a series of hydrophobic and hydrophilic drugs and their corresponding in vivo values. These results together establish the utility of this murine model for future drug transport, pathological, and pharmacological characterizations of the BBB.

Free Radical Scavenger Edaravone Administration Protects against Tissue Plasminogen Activator Induced Oxidative Stress and Blood Brain Barrier Damage

One of the therapeutics for acute cerebral ischemia is tissue plasminogen activator (t-PA). Using t-PA after 3 hour time window increases the chances of hemorrhage, involving multiple mechanisms. In order to show possible mechanisms of t-PA toxicity and the effect of the free radical scavenger edaravone, we administered vehicle, plasmin, and t-PA into intact rat cortex, and edaravone intravenously. Plasmin and t-PA damaged rat brain with the most prominent injury in t-PA group on 4-HNE, HEL, and 8-OHdG immunostainings. Such brain damage was strongly decreased in t-PA plus edaravone group. For the neurovascular unit immunostainings, occludin and collagen IV expression was decreased in single plasmin or t-PA group, which was recovered in t-PA plus edaravone group. In contrast, matrix metalloproteinase-9 intensity was the strongest in t-PA group, less in plasmin, and was the least prominent in t-PA plus edaravone group. In vitro data showed a strong damage to tight junctions for occludin and claudin 5 in both administration groups, while there were no changes for endothelial (NAGO) and perivascular (GFAP) stainings. Such damage to tight junctions was recovered in t-PA plus edaravone group with similar recovery in Sodium-Fluorescein permeability assay. Administration of t-PA caused oxidative stress damage to lipids, proteins and DNA, and led to disruption of outer parts of neurovascular unit, greater than the effect in plasmin administration. Additive edaravone ameliorated such an oxidative damage by t-PA with protecting outer layers of blood-brain barrier (in vivo) and tight junctions (in vitro).

The Effects of a High-Energy Diet on Hippocampal Function and Blood-Brain Barrier Integrity in the Rat

Cognitive impairment and Alzheimer's disease are linked with intake of a Western diet, characterized by high levels of saturated fats and simple carbohydrates. In rats, these dietary components have been shown to disrupt hippocampal-dependent learning and memory processes, particularly those involving spatial memory. Using a rat model, the present research assessed the degree to which consumption of a high-energy (HE) diet, similar to those found in modern Western cultures, produces a selective impairment in hippocampal function as opposed to a more global cognitive disruption. Learning and memory performance was examined following 90-day consumption of an HE-diet in three nonspatial discrimination learning problems that differed with respect to their dependence on the integrity of the hippocampus. The results showed that consumption of the HE-diet impaired performance in a hippocampal-dependent feature negative discrimination problem relative to chow-fed controls, whereas performance was spared on two discrimination problems that do not rely on the hippocampus. To explore the mechanism whereby consuming HE-diets impairs cognitive function, we investigated the effect of HE-diets on the integrity of the blood-brain barrier (BBB). We found that HE-diet consumption produced a decrease in mRNA expression of tight junction proteins, particularly Claudin-5 and -12, in the choroid plexus and the BBB. Consequently, an increased blood-to-brain permeability of sodium fluorescein was observed in the hippocampus, but not in the striatum and prefrontal cortex following HE-diet access. These results indicate that hippocampal function may be particularly vulnerable to disruption by HE-diets, and this disruption may be related to impaired BBB integrity.

Cyclosporin A induces hyperpermeability of the blood–brain barrier by inhibiting autocrine adrenomedullin-mediated up-regulation of endothelial barrier function

Cyclosporin A, a potent immunosuppressant, can often produce neurotoxicity in patients, although its penetration into the brain is restricted by the blood–brain barrier (BBB). Brain pericytes and astrocytes, which are periendothelial accessory structures of the BBB, can be involved in cyclosporin A-induced BBB disruption. However, the mechanism by which cyclosporin A causes BBB dysfunction remains unknown. Here, we show that in rodent brain endothelial cells, cyclosporin A decreased transendothelial electrical resistance (TEER) by inhibiting intracellular signal transduction downstream of adrenomedullin, an autocrine regulator of BBB function. Cyclosporin A stimulated adrenomedullin release from brain endothelial cells, but did not affect binding of adrenomedullin to its receptors. This cyclosporin A-induced decrease in TEER was attenuated by exogenous addition of adrenomedullin. Cyclosporin A dose-dependently decreased the total cAMP concentration in brain endothelial cells. A combination of cyclosporin A (1μM) with an adenylyl cyclase inhibitor, 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ22536; 10μM), or a protein kinase A (PKA) inhibitor, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H89; 1μM), markedly increased sodium fluorescein permeability in brain endothelial cells, whereas each drug alone had no effect. Thus, these data suggest that cyclosporin A inhibits the adenylyl cyclase/cyclic AMP/PKA signaling pathway activated by adrenomedullin, leading to impairment of brain endothelial barrier function.

Online monitoring of transepithelial electrical resistance (TEER) in an apparatus for combined dissolution and permeation testing

The aim of this study was to evaluate a newly implemented feature for the online monitoring of transepithelial electrical resistance (TEER) in an apparatus for combined in vitro dissolution and permeation testing. In a first step, the course of TEER was analyzed simultaneously to the permeability of sodium fluorescein, and a time frame of cell monolayer integrity inside the apparatus of approximately 3 h was found. In successive experiments cell monolayer integrity was challenged by application of EDTA (8, 6, 3 and 2 mM) in the apical compartment. After application of high EDTA concentrations of 8 and 6 mM for 45 min TEER did not recover, and permeability of the monolayer was steadily increasing. For the lower concentrations TEER recovered again while permeability of sodium fluorescein remained at an elevated level. This suggests that the EDTA induced opening of the tight junctions was preserved during the period of TEER recovery and did not change within the lifespan of the cell monolayer inside the apparatus. Online monitoring of TEER appeared to be a suitable method for real-time control of barrier integrity throughout each experiment. Moreover, this feature is intended to be used to analyze formulation approaches aiming for an improved oral drug bioavailability by application of excipients that increase the paracellular permeability of the intestinal epithelial barrier.

Pitavastatin strengthens the barrier integrity in primary cultures of rat brain endothelial cells.

Statins have a neuroprotective effect in neurological diseases, a pleiotropic effect possibly related to blood-brain barrier (BBB) function. We investigated the effect of pitavastatin on barrier functions of an in vitro BBB model with primary cultures of rat brain capillary endothelial cells (RBEC). Pitavastatin increased the transendothelial electrical resistance (TEER), an index of barrier tightness of interendothelial tight junctions (TJs), at a concentration of 10(-8) M, and decreased the endothelial permeability for sodium fluorescein through the RBEC monolayer. The increase in TEER was significantly reduced in the presence of isoprenoid geranylgeranyl pyrophosphate, whereas farnesyl pyrophosphate had no effect on TEER. Our immunocytochemical and Western blot analyses revealed that treatment with pitavastatin enhanced the expression of claudin-5, a main functional protein of TJs. Our data indicate that pitavastatin strengthens the barrier integrity in primary cultures of RBEC. The BBB-stabilizing effect of pitavastatin may be mediated partly through inhibition of the mevalonate pathway and subsequent up-regulation of claudin-5 expression.

Polychlorinated Biphenyls Disrupt Blood–Brain Barrier Integrity and Promote Brain Metastasis Formation

BackgroundPolychlorinated biphenyls (PCBs) comprise a ubiquitous class of toxic substances associated with carcinogenic and tumor-promoting effects as well as neurotoxic properties in the brain. However, the effects of PCBs on the development of tumor metastases are not fully understood.ObjectiveWe evaluated the hypothesis that exposure to individual PCB congeners can facilitate the development of brain metastases in immunocompetent mice via the disruption of the integrity of the blood–brain barrier (BBB).MethodsC57/Bl6 mice were exposed to individual PCBs by oral gavage, and 48 hr later they were injected with luciferase-labeled K1735 M2 melanoma cells into the internal carotid artery. The development of metastatic nodules was monitored by bioluminescent imaging. In addition, we evaluated the functional permeability of the BBB by measuring permeability of sodium fluorescein across the brain microvessels. Expression and colocalization of tight junction (TJ) proteins were studied by Western blotting and immunofluorescence microscopy.ResultsOral administration of coplanar PCB126, mono-ortho-substituted PCB118, and non-coplanar PCB153 (each at 150 μmol/kg body weight) differentially altered expression of the TJ proteins claudin-5, occludin, and zonula occludens-1 in brain capillaries. These alterations were associated with increased permeability of the BBB. Most importantly, exposure to individual PCB congeners enhanced the rate of formation and progression of brain metastases of luciferase-tagged melanoma cells.ConclusionsOur results show for the first time that exposure to individual PCBs can facilitate the formation of bloodborne metastases via alterations of the integrity of the brain capillary endothelium.

Tissue plasminogen activator enhances the hypoxia/reoxygenation-induced impairment of the blood-brain barrier in a primary culture of rat brain endothelial cells.

Hemorrhagic transformation is a major complication associated with tissue plasminogen activator (tPA) therapy for ischemic stroke. We studied the effect of tPA on the blood-brain barrier (BBB) function with our in vitro monolayer model generated using rat brain microvascular endothelial cells subjected either to normoxia or to hypoxia/reoxygenation (H/R) with or without the administration of tPA. The barrier function was evaluated by the transendothelial electrical resistance (TEER), the permeability of sodium fluorescein and Evans' blue-albumin (EBA), and the uptake of lucifer yellow (LY). The permeability of sodium fluorescein and EBA was used as an index of paracellular and transcellular transport, respectively. The administration of tPA increased the permeability of EBA and the uptake of LY under normoxia. It enhanced the increase in the permeability of both sodium fluorescein and EBA, the decrease in the TEER, and the disruption in the expression of ZO-1 under H/R conditions. Administration of tPA could cause an increase in the transcellular transport under normoxia, and both the transcellular and paracellular transport of the BBB under H/R conditions in vitro. Even in humans, tPA may lead to an opening of the BBB under non-ischemic conditions and have an additional effect on the ischemia-induced BBB disruption.

Use of a Human Corneal Epithelial Cell Line for Screening the Safety of Contact Lens Care Solutions In Vitro

Sodium fluorescein permeability assay, alamarBlue assay, and scanning electron microscopy were performed to study the effects of various contact lens disinfecting multipurpose solutions (MPS) on the integrity of the ocular surface epithelium by using corneal epithelial cells. The sodium fluorescein permeability and alamarBlue activity of monolayer cultures of human corneal epithelial cells were compared after exposure to ReNu MultiPlus, OPTI-FREE Express, AQuify 5 Minute, SOLO-care Plus With Aqualube, and Complete Moisture Plus contact lens care solutions for 15 minutes. Additional cell monolayers were prepared for each treatment and were analyzed with a scanning electron microscope after a 10-minute exposure. The sodium fluorescein permeability assay, alamarBlue assay, and scanning electron microscopy showed that OPTI-FREE Express was significantly more damaging to the human corneal epithelial cell monolayer than ReNu MultiPlus, SOLO-care Plus With Aqualube, Complete Moisture Plus, and AQuify 5 Minute contact lens solutions. Cell monolayers treated with OPTI-FREE Express were more permeable to sodium fluorescein and showed lower metabolic activity than cell monolayers treated with the other multipurpose solutions. This experiment shows that ReNu MultiPlus, SOLO-care Plus With Aqualube, Complete Moisture Plus, and AQuify 5 Minute contact lens solutions have a minimal effect on human corneal epithelial cells in culture, whereas OPTI-FREE Express has a higher negative effect on tight junctions, cell membranes, and overall metabolism of these human corneal epithelial cells.

Adverse effect of cyclosporin A on barrier functions of cerebral microvascular endothelial cells after hypoxia-reoxygenation damage in vitro.

Hypoxia and post-hypoxic reoxygenation induces disruption of the blood-brain barrier (BBB). Alterations of the BBB function after hypoxia/reoxygenation (H/R) injury remain unclear. Cyclosporin A (CsA), a potent immunosuppressant, induces neurotoxic effects by entering the brain, although the transport of CsA across the BBB is restricted by P-glycoprotein (P-gp), a multidrug efflux pump, and tight junctions of the brain capillary endothelial cells. The aim of this study was to evaluate whether the BBB after H/R damage is vulnerable to CsA-induced BBB dysfunction. We attempted to establish a pathophysiological BBB model with immortalized mouse brain capillary endothelial (MBEC4) cells. The effects of CsA on permeability and P-gp activity of the MBEC4 cells were then examined. Exposure to hypoxia for 4 h and reoxygenation for 1 h (H/R (4 h/1 h)) produced a significant decrease in P-gp function of MBEC4 cells, without changing cell viability and permeability for sodium fluorescein and Evan's blue-albumin at 7 days after H/R (4 h/1 h). CsA-induced hyperpermeability and P-gp dysfunction in MBEC4 monolayers at 7 days after H/R (4 h/1 h) were exacerbated. The possibility that CsA penetrates the BBB with incomplete functions in the vicinity of cerebral infarcts to induce neurotoxicity has to be considered.

In Vivo Fluorescence Microscopic Imaging for Dynamic Quantitative Assessment of Intestinal Mucosa Permeability in Mice

Herein, we introduce a novel intravital microscopic method to quantitatively assess mucosal permeability in mouse intestine. To this end, the time course of changes in the accumulation of fluorescent markers (sodium fluorescein [MW 376] and fluorescent dextran [FD4000, MW 4000; Merck, Darmstadt, Germany]) was examined in the perivascular interstitium of the mesentery of sham-operated animals and animals undergoing 30 min intestinal ischemia and 90 min postischemic reperfusion. An increased permeation of intraluminally installed sodium fluorescein was found only in the late reperfusion period, while permeability was enhanced during the entire postischemic examination period when renal excretion of the fluorescent marker was excluded by kidney ligation. Similarly, the late reperfusion phase was associated with enhanced tissue fluorescence to FD 4000 when the kidneys were ligated. Fluorescence values in the plasma, as measured by standard fluorimetry, showed a significant correlation with tissue fluorescence determined by intravital microscopy. Further, villus tip denudation at 90 min reperfusion correlated with increased permeability of sodium fluorescein in the early (10-20 min) and FD 4000 in the late reperfusion phase (90 min). Thus, intravital microscopic measurement of mesenteric perivascular fluorescence provides a simple and easily applicable on-line method to quantitatively assess epithelial permeability, allowing to detect altered mucosal barrier function and to evaluate salvage therapies which target mucosal integrity.

Adrenomedullin improves the blood-brain barrier function through the expression of claudin-5.

Brain vascular endothelial cells secret Adrenomedullin (AM) has multifunctional biological properties. AM affects cerebral blood flow and blood-brain barrier (BBB) function. We studied the role of AM on the permeability and tight junction proteins of brain microvascular endothelial cells (BMEC). BMEC were isolated from rats and a BBB in vitro model was generated. The barrier functions were studied by measuring the transendothelial electrical resistance (TEER) and the permeability of sodium fluorescein and Evans' blue albumin. The expressions of tight junction proteins were analyzed using immunocytochemistry and immunoblotting. AM increased TEER of BMEC monolayer dose-dependently. Immunocytochemistry revealed that AM enhanced the claudin-5 expression at a cell-cell contact site in a dose-dependent manner. Immunoblotting also showed an overexpression of claudin-5 in AM exposure. AM therefore inhibits the paracellular transport in a BBB in vitro model through claudin-5 overexpression.

Effect of photodynamic therapy on the function of the outer blood-retinal barrier in an in vitro model

Photodynamic therapy (PDT) is a well established clinical treatment for age-related macular degeneration (AMD), and comprises intravenous injection of verteporfin and subsequent application of a non-thermal laser beam to the area of AMD to induce selective vascular occlusion. Since there is evidence that PDT may cause outer blood-retinal barrier (BRB) breakdown and possibly RPE cell alteration, we investigated the effect of PDT on the BRB function of the RPE in an in vitro model. Twenty-one monolayers of human RPE cells were cultured on semipermeable membranes until a stable barrier function was achieved as determined by transepithelial electrical resistance (TER) and sodium fluorescein permeability. To test the effect of PDT on the outer BRB function, non-thermal laser (692 nm), verteporfin or a combination of both were applied. TER assessment prior to and after PDT was utilized to identify changes in barrier function of the RPE in this in vitro model. Finally, monolayers of RPE cells were evaluated by transmission electron microscopy (TEM). No significant TER decrease was observed after application of non-thermal laser alone or after administration of verteporfin in therapeutic concentrations, but combination of these modalities resulted in significantly decreased TER within 4 h. Except for intercellular blisters, no damage to the RPE was evident in TEM. Verteporfin added at concentrations higher than therapeutic doses (2 mg/ml) resulted in an immediate decrease in TER and damage to the RPE cells. The combination of a therapeutic concentration of verteporfin and application of non-thermal laser resulted in a morphologically and functionally detectable breakdown of the outer BRB function of the RPE without any damage to the RPE cells themselves in vitro. However, increasing the concentration of verteporfin can result in RPE cell damage.

Transport Kinetics of Iron Chelators and Their Chelates in Caco-2 Cells

Caco-2 monolayers were used to contrast the bidirectional transport of iron chelators and their chelates and to estimate fundamental kinetics associated with their intestinal absorption. Bidirectional transport was studied at 37 degrees C and pH 7.4 using 500-microM concentrations. Monolayer integrity was tested via transepithelial electrical resistance and sodium fluorescein permeability. Apical and basolateral analysis provided mass balance evidence. Apparent permeability coefficient (P(app)) served to rank and compare molecules and estimate in vivo bioavailability. Model-dependent rate constants defined cellular influx and efflux. 1) P(app) ranked in decreasing order for chelators from directional transport studies were CP363 > deferiprone> ICL670 > CP502 > deferoxamine (DFO). 2) Fe(CP502)(3), Fe(ICL670)(2), and FeDFO were not measurable in receiving chambers, whereas Fe(deferiprone)(3) and Fe(CP363)(3) were detected in both directions. 3) CP363 was transported significantly faster from the basolateral to the apical direction than the converse. 4) Mass balance of donor and receiver chambers gave approximately 100% recovery in all cases. 5) Kinetic analysis supports the view that the Caco-2 chelator efflux constants are generally greater than their influx constants. Caco-2 cells are useful in screening iron chelators and chelates and estimating bioavailabilities. Structure and distribution coefficients partially predict passive transport through Caco-2 monolayers.