Cerebral microvascular changes in permeability and tight junctions induced by hypoxia-reoxygenation

Cerebral microvessel endothelial cells that form the blood-brain barrier (BBB) have tight junctions (TJ) that are critical for maintaining brain homeostasis and low permeability. Both integral (claudin-1 and occludin) and membrane-associated zonula occluden-1 and -2 (ZO-1 and ZO-2) proteins combine to form these TJ complexes that are anchored to the cytoskeletal architecture (actin). Disruptions of the BBB have been attributed to hypoxic conditions that occur with ischemic stroke, pathologies of decreased perfusion, and high-altitude exposure. The effects of hypoxia and posthypoxic reoxygenation in cerebral microvasculature and corresponding cellular mechanisms involved in disrupting the BBB remain unclear. This study examined hypoxia and posthypoxic reoxygenation effects on paracellular permeability and changes in actin and TJ proteins using primary bovine brain microvessel endothelial cells (BBMEC). Hypoxia induced a 2.6-fold increase in [(14)C]sucrose, a marker of paracellular permeability. This effect was significantly reduced (~58%) with posthypoxic reoxygenation. After hypoxia and posthypoxic reoxygenation, actin expression was increased (1.4- and 2.3-fold, respectively). Whereas little change was observed in TJ protein expression immediately after hypoxia, a twofold increase in expression was seen with posthypoxic reoxygenation. Furthermore, immunofluorescence studies showed alterations in occludin, ZO-1, and ZO-2 protein localization during hypoxia and posthypoxic reoxygenation that correlate with the observed changes in BBMEC permeability. The results of this study show hypoxia-induced changes in paracellular permeability may be due to perturbation of TJ complexes and that posthypoxic reoxygenation reverses these effects.     

Release of prostaglandin E-2 in bovine brain endothelial cells after exposure to three unique forms of the antifungal drug amphotericin-B: role of COX-2 in amphotericin-B induced fever

Common formulations of amphotericin-B include a deoxycholate colloidal suspension (d-Amph), an amphotericin-B lipid complex (Ablc), and a liposomal product (l-Amph). The clinical incidence of infusion related fever is highest with d-Amph, intermediate with Ablc, and lowest with l-Amph. In the present study, we measured the activation of cyclooxygenase-2 (COX-2) and subsequent release of prostaglandin E-2 (PgE-2) from brain microvessel endothelium treated with these three formulations of amphotericin-B. Primary cultured bovine brain microvessel endothelial cells (BBMEC) were exposed to d-Amph, Ablc and l-Amph at concentrations that can be achieved in the plasma of patients receiving the drug. Media samples from the cells were collected and analyzed for PgE-2. Release of PgE-2 from BBMEC monolayers treated with l-Amph was similar to cells receiving culture media alone. In contrast, Ablc and d-Amph caused significantly greater release of PgE-2 from BBMEC monolayers compared to controls receiving culture media alone. PgE-2 release after d-Amph treatment was similar in magnitude to that observed with bacterial lipopolysaccharide (LPS). Western blot analysis indicated significant induction of COX-2 expression in BBMEC following LPS, Ablc or d-Amph treatment. Furthermore, PgE-2 release following exposure of BBMEC monolayers to either LPS or the various amphotericin-B formulations was reduced by the addition of the selective COX-2 inhibitor, NS-398. These studies indicate that amphotericin-B induces COX-2 expression in brain microvessel endothelium resulting in release of fever producing PgE-2. The magnitude of PgE-2 release from BBMEC following exposure to various amphotericin-B formulations mirrors the clinical observations regarding amphotericin-B induced fever and serves as initial support for the clinical use of COX-2 inhibitors to reduce amphotericin-B fever.     

Pentosan polysulfate protects brain endothelial cells against bacterial lipopolysaccharide-induced damages

Peripheral inflammation can aggravate local brain inflammation and neuronal death. The blood-brain barrier (BBB) is a key player in the event. On a relevant in vitro model of primary rat brain endothelial cells co-cultured with primary rat astroglia cells lipopolysaccharide (LPS)-induced changes in several BBB functions have been investigated. LPS-treatment resulted in a dose- and time-dependent decrease in the integrity of endothelial monolayers: transendothelial electrical resistance dropped, while flux of permeability markers fluorescein and albumin significantly increased. Immunostaining for junctional proteins ZO-1, claudin-5 and beta-catenin was significantly weaker in LPS-treated endothelial cells than in control monolayers. LPS also reduced the intensity and changed the pattern of ZO-1 immunostaining in freshly isolated rat brain microvessels. The activity of P-glycoprotein, an important efflux pump at the BBB, was also inhibited by LPS. At the same time production of reactive oxygen species and nitric oxide was increased in brain endothelial cells treated with LPS. Pentosan polysulfate, a polyanionic polysaccharide could reduce the deleterious effects of LPS on BBB permeability, and P-glycoprotein activity. LPS-stimulated increase in the production of reactive oxygen species and nitric oxide was also decreased by pentosan treatment. The protective effect of pentosan for brain endothelium can be of therapeutical significance in bacterial infections affecting the BBB.     

Pericytes from Brain Microvessels Strengthen the Barrier Integrity in Primary Cultures of Rat Brain Endothelial Cells

(1) The blood–brain barrier (BBB) characteristics of cerebral endothelial cells are induced by organ-specific local signals. Brain endothelial cells lose their phenotype in cultures without cross-talk with neighboring cells. (2) In contrast to astrocytes, pericytes, another neighboring cell of endothelial cells in brain capillaries, are rarely used in BBB co-culture systems. (3) Seven different types of BBB models, mono-culture, double and triple co-cultures, were constructed from primary rat brain endothelial cells, astrocytes and pericytes on culture inserts. The barrier integrity of the models were compared by measurement of transendothelial electrical resistance and permeability for the small molecular weight marker fluorescein. (4) We could confirm that brain endothelial monolayers in mono-culture do not form tight barrier. Pericytes induced higher electrical resistance and lower permeability for fluorescein than type I astrocytes in co-culture conditions. In triple co-culture models the tightest barrier was observed when endothelial cells and pericytes were positioned on the two sides of the porous filter membrane of the inserts and astrocytes at the bottom of the culture dish. (5) For the first time a rat primary culture based syngeneic triple co-culture BBB model has been constructed using brain pericytes beside brain endothelial cells and astrocytes. This model, mimicking closely the anatomical position of the cells at the BBB in vivo, was superior to the other BBB models tested. (6) The influence of pericytes on the BBB properties of brain endothelial cells may be as important as that of astrocytes and could be exploited in the construction of better BBB models.     

Transendothelial permeability changes induced by free radicals in an in vitro model of the blood-brain barrier.

In the present study, we investigated the changes in blood-brain barrier (BBB) permeability following brain endothelial cell exposure to different xenobiotics able to promote free radical generation during their metabolism. Our in vitro BBB model consisted of confluent monolayers of immortalized rat brain capillary endothelial cells (RBE4) grown on collagen-coated filters in the presence of C6 glioma cells grown in the lower compartment. We have recently shown that a range of xenobiotics, including menadione, nitrofurazone, and methylviologen (paraquat) may undergo monoelectronic redox cycling in isolated brain capillaries, giving rise to reactive oxygen species. In this study, addition of 100 microM menadione to the culture medium for 30 min significantly increased the permeability of endothelial cell monolayers to radiolabeled sucrose. The effect on endothelial permeability induced by menadione was dose-dependent and reversible. These permeability changes preceded the onset of cell death, as assessed by the Trypan blue exclusion method. Pre-incubation with superoxide dismutase and catalase blocked changes in sucrose permeability to control levels in a dose-dependent manner, suggesting the involvement of reactive oxygen species in menadione-induced BBB opening.     

A new blood–brain barrier model using primary rat brain endothelial cells,pericytes and astrocytes

Blood–brain barrier (BBB) characteristics are induced and maintained by cross-talk between brain microvessel endothelial cells and neighbouring elements of the neurovascular unit. While pericytes are the cells situated closest to brain endothelial cells morphologically and share a common basement membrane, they have not been used in co-culture BBB models for testing drug permeability. We have developed and characterized a new syngeneic BBB model using primary cultures of the three main cell types of cerebral microvessels. The co-culture of endothelial cells, pericytes and astrocytes mimick the anatomical situation in vivo. In the presence of both pericytes and astrocytes rat brain endothelial cells expressed enhanced levels of tight junction (TJ) proteins occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. Further morphological evidence of the presence of interendothelial TJs was provided by electron microscopy. The transendothelial electrical resistance (TEER) of brain endothelial monolayers in triple co-culture, indicating the tightness of TJs reached 400 Ω cm² on average, while the endothelial permeability coefficients (P_e) for fluorescein was in the range of 3 × 10^?6 cm/s. Brain endothelial cells in the new model expressed glucose transporter-1, efflux transporters P-glycoprotein and multidrug resistance protein-1, and showed a polarized transport of rhodamine 123, a ligand for P-glycoprotein. To further characterize the model, drug permeability assays were performed using a set of 19 compounds with known in vivo BBB permeability. Good correlation (R² = 0.89) was found between in vitro P_e values obtained from measurements on the BBB model and in vivo BBB permeability data. The new BBB model, which is the first model to incorporate pericytes in a triple co-culture setting, can be a useful tool for research on BBB physiology and pathology and to test candidate compounds for centrally acting drugs.     

Nitric oxide mediates hypoxia-induced changes in paracellular permeability of cerebral microvasculature

Ischemic stroke from a reduction in blood flow to the brain microvasculature results in a subsequent decreased delivery of oxygen (i.e., hypoxia) and vital nutrients to endothelial, neuronal, and glial cells. Hypoxia associated with stroke has been shown to increase paracellular permeability of the blood-brain barrier, leading to the release of cellular mediators and brain tissue injury. Whereas reperfusion does not occur in all ischemic strokes, increased permeability has been seen in posthypoxic reoxygenation. Currently, it is unknown whether these deleterious effects result from cellular mechanisms stimulated by decreased oxygen during stroke or posthypoxic reoxygenation stress. This study used primary bovine brain microvessel endothelial cells (BBMECs) to examine the involvement of nitric oxide (NO) as a mediator in hypoxia-induced permeability changes. Hypoxia-induced increased transport of [14C]sucrose across BBMEC monolayers compared with normoxia was attenuated by either posthypoxic reoxygenation or inhibition of NO synthase (NOS). The hypoxia-induced permeability effect was further reduced when NOS inhibition was combined with posthypoxic reoxygenation. Additionally, a significant increase in total NO was seen in BBMECs after hypoxic exposure. This correlation was supported by the increased [14C]sucrose permeability observed when BBMECs were exposed to the NO donor diethylenetriaamine NONOate. Western blot analyses of NOS isoforms showed a significant increase in the inducible isoform after hypoxic exposure with a subsequent reduction in expression on reoxygenation. Results from this study suggest that hypoxia-induced blood-brain barrier breakdown can be diminished by inhibition of NO synthesis, decreased concentration of NO metabolites, and/or reoxygenation.     

Transforming Growth Factor-β1 Upregulates the Tight Junction and P-glycoprotein of Brain Microvascular Endothelial Cells

1. The present study was aimed at elucidating effects of transforming growth factor-beta (TGF-beta) on blood-brain barrier (BBB) functions with mouse brain capillary endothelial (MBEC4) cells. 2. The permeability coefficients of sodium fluorescein and Evans blue albumin for MBEC4 cells and the cellular accumulation of rhodamine 123 in MBEC4 cells were dose-dependently decreased after a 12-h exposure to TGF-beta1 (0.01-10 ng/mL). 3. The present study demonstrates that TGF-beta lowers the endothelial permeability and enhances the functional activity of P-gp, suggesting that cellular constituents producing TGF-beta in the brain may keep the BBB functioning.     

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.     

Fatty acid transport protein expression in human brain and potential role in fatty acid transport across human brain microvessel endothelial cells

The blood-brain barrier (BBB), formed by the brain capillary endothelial cells, provides a protective barrier between the systemic blood and the extracellular environment of the CNS. Passage of fatty acids from the blood to the brain may occur either by diffusion or by proteins that facilitate their transport. Currently several protein families have been implicated in fatty acid transport. The focus of the present study was to identify the fatty acid transport proteins (FATPs) expressed in the brain microvessel endothelial cells and characterize their involvement in fatty acid transport across an in vitro BBB model. The major fatty acid transport proteins expressed in human brain microvessel endothelial cells (HBMEC), mouse capillaries and human grey matter were FATP-1, -4 and fatty acid binding protein 5 and fatty acid translocase/CD36. The passage of various radiolabeled fatty acids across confluent HBMEC monolayers was examined over a 30-min period in the presence of fatty acid free albumin in a 1 : 1 molar ratio. The apical to basolateral permeability of radiolabeled fatty acids was dependent upon both saturation and chain length of the fatty acid. Knockdown of various fatty acid transport proteins using siRNA significantly decreased radiolabeled fatty acid transport across the HBMEC monolayer. Our findings indicate that FATP-1 and FATP-4 are the predominant fatty acid transport proteins expressed in the BBB based on human and mouse expression studies. While transport studies in HBMEC monolayers support their involvement in fatty acid permeability, fatty acid translocase/CD36 also appears to play a prominent role in transport of fatty acids across HBMEC.     

Transient forebrain ischemia increases the blood-brain barrier permeability for albumin in stroke-prone spontaneously hypertensive rats

1. The aim of the present study was to reveal the effect of transient forebrain ischemia on the regional and temporal changes in the permeability of the blood-brain barrier (BBB) permeability for sodium fluorescein (MW: 376 Da) and Evan's blue-labeled albumin (MW: 67 kDa) in stroke-prone spontaneously hypertensive rats (SHRSP).2. BBB permeability was significantly higher in the brain regions of 16-week-old control SHRSP than those in age-matched normotensive Wistar-Kyoto rats.3. Transient forebrain ischemia evoked by 10-min bilateral carotid occlusion increased the permeability of the BBB for albumin, but not for sodium fluorescein, after 6 and 24 h of reperfusion in brain regions of SHRSP.4. Extravasation of serum macromolecules may contribute to neuronal loss and development of hypertensive encephalopathy in SHRSP.     

P-Glycoprotein Mediated Efflux Limits the Transport of the Novel Anti-Parkinson's Disease Candidate Drug FLZ across the Physiological and PD Pathological In Vitro BBB Models

FLZ, a novel anti-Parkinson''s disease (PD) candidate drug, has shown poor blood-brain barrier (BBB) penetration based on the pharmacokinetic study using rat brain. P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are two important transporters obstructing substrates entry into the CNS as well as in relation to PD neuropathology. However, it is unclear whether P-gp and BCRP are involved in low BBB permeability of FLZ and what the differences of FLZ brain penetration are between normal and Parkinson''s conditions. For this purpose, in vitro BBB models mimicking physiological and PD pathological-related BBB properties were constructed by C6 astroglial cells co-cultured with primary normal or PD rat cerebral microvessel endothelial cells (rCMECs) and in vitro permeability experiments of FLZ were carried out. High transepithelial electrical resistance (TEER) and low permeability for sodium fluorescein (NaF) confirmed the BBB functionality of the two models. Significantly greater expressions of P-gp and BCRP were detected in PD rCMECs associated with the lower in vitro BBB permeability of FLZ in pathological BBB model compared with physiological model. In transport studies only P-gp blocker effectively inhibited the efflux of FLZ, which was consistent with the in vivo permeability data. This result was also confirmed by ATPase assays, suggesting FLZ is a substrate for P-gp but not BCRP. The present study first established in vitro BBB models reproducing PD-related changes of BBB functions in vivo and demonstrated that poor brain penetration of FLZ and low BBB permeability were due to the P-gp transport.     

Dexamethasone Regulation of P-Glycoprotein Activity in an Immortalized Rat Brain Endothelial Cell Line, GPNT

The blood-brain barrier (BBB) plays an important role in controlling the passage of molecules from the blood to the extracellular fluid environment of the brain. The multidrug efflux pump P-glycoprotein (P-gp) is highly expressed in the luminal membrane of brain capillary endothelial cells, thus forming a functional barrier to lipid-soluble drugs, notably, antitumor agents. It is of interest to develop an in vitro BBB model that stably expresses P-gp to investigate the mechanisms of regulation in expression and activity. The rat brain endothelial cell line, GPNT, was derived from a previously characterized rat brain endothelial cell line. A strong expression of P-gp was found in GPNT monocultures, whereas the multidrug resistance-associated pump Mrp1 was not expressed. The transendothelial permeability coefficient of the P-gp substrate vincristine across GPNT monolayers was close to the permeability coefficient of bovine brain endothelial cells cocultured with astrocytes, a previously documented in vitro BBB model. Furthermore, the P-gp blocker cyclosporin A induced a large increase in apical to basal permeability of vincristine. Thus, P-gp is highly functional in GPNT cells. A 1-h treatment of GPNT cells with dexamethasone resulted in decreased uptake of vincristine without any increase in P-gp expression. This effect could be mimicked by protein kinase C (PKC) activation and prevented by PKC inhibition, strongly suggesting that activation of P-gp function may involve a PKC-dependent pathway. These results document the GPNT cell line as a valuable in vitro model for studying drug transport and P-gp function at the BBB and suggest that activation of P-gp activity at the BBB might be considered in chemotherapeutic treatment of cancer patients.     

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.     

Tumor necrosis factor-alpha-induced leukocyte adhesion and microvessel permeability

The objective of this study was to investigate whether leukocyte adhesion and/or emigration are critical steps in increased microvessel permeability during acute inflammation. To conduct this study, we combined autologous blood perfusion with a single microvessel perfusion technique, which allows microvessel permeability to be measured precisely after the endothelium has interacted with blood-borne stimuli. Experiments were carried out in intact venular microvessels in rat mesenteries. Firm attachment of leukocytes to endothelial cells was induced by intravenous injection of TNF-alpha (3.5 microg/kg) and resuming autoperfusion in a precannulated microvessel. Leukocyte emigration was facilitated by superfusion of formyl-Met-Leu-Phe-OH. Microvessel permeability was measured as hydraulic conductivity (L(p)) or the solute permeability coefficient to tetramethylrhodamine isothiocyanate-labeled alpha-lactalbumin before and after leukocyte adhesion and emigration in individually perfused microvessels. We found that perfusion of a microvessel with TNF-alpha did not affect basal microvessel permeability, but intravenous injection of TNF-alpha caused significant leukocyte adhesion. However, the significant leukocyte adhesion and emigration did not cause corresponding increases in either L(p) or solute permeability. Thus our results suggest that leukocyte adhesion and emigration do not necessarily increase microvessel permeability and the mechanisms that regulate the adhesion process act independently from mechanisms that regulate permeability. In addition, silver staining of endothelial boundaries demonstrated that leukocytes preferentially adhere at the junctions of endothelial cells. The appearance of the silver lines indicates that the TNF-alpha-induced firm adhesion of leukocyte to microvessel walls did not involve apparent changes in the junctional structure of endothelial cells, which is consistent with the results of permeability measurements.     

Pericytes Contribute to the Disruption of the Cerebral Endothelial Barrier via Increasing VEGF Expression: Implications for Stroke

Disruption of the blood-brain barrier (BBB) integrity occurring during the early onset of stroke is not only a consequence of, but also contributes to the further progression of stroke. Although it has been well documented that brain microvascular endothelial cells and astrocytes play a critical role in the maintenance of BBB integrity, pericytes, sandwiched between endothelial cells and astrocytes, remain poorly studied in the pathogenesis of stroke. Our findings demonstrated that treatment of human brain microvascular pericytes with sodium cyanide (NaCN) and glucose deprivation resulted in increased expression of vascular endothelial growth factor (VEGF) via the activation of tyrosine kinase Src, with downstream activation of mitogen activated protein kinase and PI3K/Akt pathways and subsequent translocation of NF-κB into the nucleus. Conditioned medium from NaCN-treated pericytes led to increased permeability of endothelial cells, and this effect was significantly inhibited by VEGF-neutralizing antibody. The in vivo relevance of these findings was further corroborated in the stroke model of mice wherein the mice, demonstrated disruption of the BBB integrity and concomitant increase in the expression of VEGF in the brain tissue as well as in the isolated microvessel. These findings thus suggest the role of pericyte-derived VEGF in modulating increased permeability of BBB during stroke. Understanding the regulation of VEGF expression could open new avenues for the development of potential therapeutic targets for stroke and other neurological disease.     

Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm·cm² on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 ± 0.11 x 10^-3 cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.     

Effects of hypoxia-reoxygenation on rat blood-brain barrier permeability and tight junctional protein expression

Cerebral microvessel endothelial cells that form the blood-brain barrier (BBB) have tight junctions (TJs) that are critical for maintaining brain homeostasis. The effects of initial reoxygenation after a hypoxic insult (H/R) on functional and molecular properties of the BBB and TJs remain unclear. In situ brain perfusion and Western blot analyses were performed to assess in vivo BBB integrity on reoxygenation after a hypoxic insult of 6% O2 for 1 h. Model conditions [blood pressure, blood gas chemistries, cerebral blood flow (CBF), and brain ATP concentration] were also assessed to ensure consistent levels and criteria for insult. In situ brain perfusion revealed that initial reoxygenation (10 min) significantly increased the uptake of [14C]sucrose into brain parenchyma. Capillary depletion and CBF analyses indicated the perturbations were due to increased paracellular permeability rather than vascular volume changes. Hypoxia with reoxygenation (10 min) produced an increase in BBB permeability with associated alterations in tight junctional protein expression. These results suggest that H/R leads to reorganization of TJs and increased paracellular diffusion at the BBB, which is not a result of increased CBF, vascular volume change, or endothelial uptake of marker. Additionally, the tight junctional protein occludin had a shift in bands that correlated with functional changes (i.e., increased permeability) without significant change in expression of claudin-3, zonula occludens-1, or actin. H/R-induced changes in the BBB may result in edema and/or associated pathological outcomes.     

Potential Role of Cerebral Glutathione in the Maintenance of Blood-Brain Barrier Integrity in Rat

Using the model of glutathione (GSH) depletion, possible role of GSH in the maintenance of blood-brain barrier (BBB) integrity was evaluated in rats. Administration (ip) of GSH depletors, diethyl maleate (DEM, 1–4 mmol/kg), phorone (2–3 mmol/kg) and 2-cyclohexene-1-one (CHX, 1 mmol/kg), to male adults was found to deplete brain and liver GSH and increase the BBB permeability to micromolecular tracers (sodium fluorescein and [¹⁴C]sucrose) in a dose-dependent manner at 2h. However, BBB permeability to macromolecular tracers such as horseradish peroxidase and Evan's blue remained unaltered. It was also shown that observed BBB permeability dysfunction was associated with brain GSH depletion. A lower magnitude of BBB increase in rat neonates, as compared to adults, indicated a possible bigger role of GSH in the BBB function of mature brain. The treatment with N-acetylcysteine, methionine and GSH provided a partial to full protection against DEM-induced brain (microvessel) GSH depletion and BBB dysfunction; however, the treatment with -tocopherol, ascorbic acid and turmeric were not effective. Our studies showed that cerebral GSH plays an important role in maintaining the functional BBB integrity.     

Protection against hypoxia-induced blood-brain barrier disruption: changes in intracellular calcium

Tissue damage after stroke is partly due to disruption of the blood-brain barrier (BBB). Little is known about the role of calcium in modulating BBB disruption. We investigated the effect of hypoxic and aglycemic stress on BBB function and intracellular calcium levels. Bovine brain microvessel endothelial cells were treated with A-23187 to increase intracellular calcium without hypoxia or treated with a calcium chelator (BAPTA) or calcium channel blockers (nifedipine or SKF-96365) and 6 h of hypoxia. A-23187 alone did not increase paracellular permeability. Hypoxia increased intracellular calcium, and hypoxia or hypoxia-aglycemia increased paracellular permeability. Treatment with nifedipine and SKF-96365 increased intracellular calcium under normoglycemic conditions, instead of blocking calcium influx, and was protective against hypoxia-induced BBB disruption under normoglycemia. Protection by nifedipine and SKF-96365 was not due to antioxidant properties of these compounds. These data indicate that increased intracellular calcium alone is not enough to disrupt the BBB. However, increased intracellular calcium after drug treatment and hypoxia suggests a potential mechanism for these drugs in BBB protection; nifedipine and SKF-96365 plus hypoxic stress may trigger calcium-mediated signaling cascades, altering BBB integrity. nifedipine; SKF-96365; ischemia; permeability; fura 2