Winfried Neuhaus

Validation of in vitro cell culture models of the blood–brain barrier: Tightness characterization of two promising cell lines

In the course of the validation of blood-brain barrier in vitro models the aim of this work was to characterize two promising continuous cell lines with regard to their tightness properties. PBMEC/C1-2 and ECV304 cells were cultured in several media with different compositions on Transwell inserts. Inducibility and functionality of tightness were investigated by transendothelial electrical resistance (TEER) and by transport studies with transcellular marker diazepam, glycine antagonist Bu101 and paracellular marker APTS-dextran. Inducibility, expression and localization of tight junctional proteins were assessed by western blotting and immunofluorescence microscopy. Presence of factors derived from glioma cell line C6 resulted in increased TEER in both cell lines. Comparison to APTS-dextran data across Caco-2 layers emphasized that correlations between permeability of the paracellular marker and TEER are dependent on each investigated cell line and the corresponding growth medium. Presence and inducibility of tight junctional proteins ZO-1 and Occludin were proven for ECV304 layers. Cell line ECV304 seemed to be suitable for TEER dependent transport studies, whereas PBMEC/C1-2 showed higher potential for P-gP studies.

Effects of NMDA receptor modulators on a blood-brain barrier in vitro model

Changes of the functionality of the blood-brain barrier (BBB) have been reported in the context of several brain related diseases such as multiple sclerosis, epilepsy, Alzheimers disease and stroke. Several publications indicated the presence and functionality of the NMDA receptor (NMDAR) at the brain endothelium and a possible involvement of the NMDAR in the above-mentioned diseases. Recently, it was shown that the application of the NMDAR antagonist MK801 can block several adverse effects at the BBB in vitro, but also that MK801 can significantly change the proteome of brain endothelial cells without simultaneous stimulation of NMDAR by glutamate. Based on these reports we investigated if NMDAR antagonists MK801 and D-APV can affect the intracellular calcium level (Ca²⁺i) of an in vitro BBB model based on human cell line ECV304 on their own and compared these results to effects mediated by NMDAR agonists glutamate and NMDA. Treatment of ECV304 cells for 30 min with glutamate resulted in no significant change of Ca²⁺i. On the contrary, application of NMDA and NMDAR antagonists D-APV and MK801 led to a significant and concentration dependent decrease of Ca²⁺i. Further studies revealed that glutamate was able to decrease the transendothelial electrical resistance (TEER) of the BBB in vitro model, whereas NMDA and D-APV were able to increase TEER. Analysis of the protein expression levels of tight junctional molecules ZO-1 and occludin showed a complex regulation after application of NMDAR modulators. In summary, it was shown that NMDAR antagonists can alter BBB key properties in vitro on their own. Moreover, although qPCR results confirmed the presence of NMDA receptor subunits NR1, NR2A, NR2B and NR2C, membrane binding studies failed to prove the typical plasma membrane localization and functionality in human BBB cell line ECV304.

The pivotal role of astrocytes in an in vitro stroke model of the blood-brain barrier

Stabilization of the blood-brain barrier during and after stroke can lead to less adverse outcome. For elucidation of underlying mechanisms and development of novel therapeutic strategies validated in vitro disease models of the blood-brain barrier could be very helpful. To mimic in vitro stroke conditions we have established a blood-brain barrier in vitro model based on mouse cell line cerebEND and applied oxygen/glucose deprivation (OGD). The role of astrocytes in this disease model was investigated by using cell line C6. Transwell studies pointed out that addition of astrocytes during OGD increased the barrier damage significantly in comparison to the endothelial monoculture shown by changes of transendothelial electrical resistance as well as fluorescein permeability data. Analysis on mRNA and protein levels by qPCR, western blotting and immunofluorescence microscopy of tight junction molecules claudin-3,-5,-12, occludin and ZO-1 revealed that their regulation and localisation is associated with the functional barrier breakdown. Furthermore, soluble factors of astrocytes, OGD and their combination were able to induce changes of functionality and expression of ABC-transporters Abcb1a (P-gp), Abcg2 (bcrp), and Abcc4 (mrp4). Moreover, the expression of proteases (matrixmetalloproteinases MMP-2, MMP-3, MMP-9, and t-PA) as well as of their endogenous inhibitors (TIMP-1, TIMP-3, PAI-1) was altered by astrocyte factors and OGD which resulted in significant changes of total MMP and t-PA activity. Morphological rearrangements induced by OGD and treatment with astrocyte factors were confirmed at a nanometer scale using atomic force microscopy. In conclusion, astrocytes play a major role in blood-brain barrier breakdown during OGD in vitro.

A Novel Tool to Characterize Paracellular Transport: The APTS-Dextran Ladder

PurposeThe aim of this work was to develop an easy, manageable, and precise analytic tool to describe the tightness of cell layers by a molecular weight ladder.MethodsDextrans were labeled by reductive amination with fluorescent 8-aminopyrene-1,3,6-trisulfonate (APTS). This mixture, including the internal standard diazepam, was used for transport studies in Transwell models using Caco-2, ECV304, and PBMEC/C1–2 cell lines. Samples were analyzed by fluorimetry, capillary electrophoresis, and reverse-phase high-performance liquid chromatography.ResultsFollowing this approach, a logarithm correlation of R2 = 0.8958 between transepithelial electrical resistance (TEER) and APTS–dextran permeability was shown. In addition, a TEER-dependent permeability pattern could be observed including each single fraction from free APTS, APTS–glucose up to APTS–dextran consisting of 35 glucose units. The TEER-independent permeability coefficients of diazepam and confocal laser scanning microscopy images confirmed the paracellular transport of APTS–dextran.ConclusionsAll in all, the developed APTS–dextran ladder is a useful tool to characterize cell layer tightness and especially to describe paracellular transport ways and the extent of leakiness of cell layers (for blood–brain barrier or intestinal studies) over time—applying a wide array from smaller to larger molecules at the same time to refine TEER, sucrose, or Evans blue measurements.

Transport Rankings of Non-Steroidal Antiinflammatory Drugs across Blood-Brain Barrier In Vitro Models

The aim of this work was to conduct a comprehensive study about the transport properties of NSAIDs across the blood-brain barrier (BBB) in vitro. Transport studies with celecoxib, diclofenac, ibuprofen, meloxicam, piroxicam and tenoxicam were accomplished across Transwell models based on cell line PBMEC/C1-2, ECV304 or primary rat brain endothelial cells. Single as well as group substance studies were carried out. In group studies substance group compositions, transport medium and serum content were varied, transport inhibitors verapamil and probenecid were added. Resulted permeability coefficients were compared and normalized to internal standards diazepam and carboxyfluorescein. Transport rankings of NSAIDs across each model were obtained. Single substance studies showed similar rankings as corresponding group studies across PBMEC/C1-2 or ECV304 cell layers. Serum content, glioma conditioned medium and inhibitors probenecid and verapamil influenced resulted permeability significantly. Basic differences of transport properties of the investigated NSAIDs were similar comparing all three in vitro BBB models. Different substance combinations in the group studies and addition of probenecid and verapamil suggested that transporter proteins are involved in the transport of every tested NSAID. Results especially underlined the importance of same experimental conditions (transport medium, serum content, species origin, cell line) for proper data comparison.

Inhibition of Proteasomal Glucocorticoid Receptor Degradation Restores Dexamethasone-mediated Stabilization of the Blood–brain Barrier After Traumatic Brain Injury*

Objectives:To establish the molecular background for glucocorticoid insensitivity, that is, failure to reduce edema formation and to protect blood–brain barrier integrity after acute traumatic brain injury. Design:Controlled animal study. Setting:University research laboratory. Subjects:Male C57Bl/6N mice. Interventions:Mechanical brain lesion by controlled cortical impact. Measurements and Main Results:Our study demonstrates that 1) proteasomal glucocorticoid receptor degradation is established in brain endothelial cells after traumatic brain injury as a form of posttranslational glucocorticoid receptor modification; 2) inhibition of the proteasomal degradation pathway with bortezomib (0.2 mg/kg) in combination with the glucocorticoid dexamethasone (10 mg/kg) by subcutaneous injection 30 minutes postinjury restores levels of barrier sealing glucocorticoid receptor target occludin in brain endothelial cells, improves blood–brain barrier integrity, reduces edema formation, and limits neuronal damage after brain trauma. Conclusions:The results indicate that the stabilizing effect of glucocorticoids on the blood–brain barrier is hampered after cerebral lesions by proteasomal glucocorticoid receptor degradation in brain endothelial cells and restored by inhibition of proteasomal degradation pathways. The results provide underlying mechanisms for the clinically observed inefficacy of glucocorticoids. The novel combined treatment strategy might help to attenuate trauma-induced brain edema formation and neuronal damage as secondary effects of brain trauma.

Establishment of a Human Blood-Brain Barrier Co-culture Model Mimicking the Neurovascular Unit Using Induced Pluri- and Multipotent Stem Cells

Summary In vitro models of the human blood-brain barrier (BBB) are highly desirable for drug development. This study aims to analyze a set of ten different BBB culture models based on primary cells, human induced pluripotent stem cells (hiPSCs), and multipotent fetal neural stem cells (fNSCs). We systematically investigated the impact of astrocytes, pericytes, and NSCs on hiPSC-derived BBB endothelial cell function and gene expression. The quadruple culture models, based on these four cell types, achieved BBB characteristics including transendothelial electrical resistance (TEER) up to 2,500 Ω cm2 and distinct upregulation of typical BBB genes. A complex in vivo-like tight junction (TJ) network was detected by freeze-fracture and transmission electron microscopy. Treatment with claudin-specific TJ modulators caused TEER decrease, confirming the relevant role of claudin subtypes for paracellular tightness. Drug permeability tests with reference substances were performed and confirmed the suitability of the models for drug transport studies.

The effects of colloid solutions on renal proximal tubular cells in vitro.

Renal failure is a common complication of critically ill patients. Colloids such as hydroxyethyl starch (HES), gelatin, or albumin are regularly used for intravascular volume resuscitation, but there are increasing reports about the nephrotoxic side effects of synthetic colloids in septic patients. Therefore, we investigated the influence of colloids (HES130/0.4 (Voluven®), gelatin (Gelafundin®), human albumin, and the crystalloid Sterofundin® ISO on cell viability of human proximal tubular (HK-2) cells. HK-2 cells were incubated with colloids (0.1%–4%) and with equivalent volumes of the crystalloid solution Sterofundin ISO. After 21 hours, cell viability of HK-2 cells was measured by EZ4U assay (dye XTT). Application of HES130/0.4 decreased cell viability significantly in a concentration-dependent manner (86.80% ± 10.79% by 0.5% HES down to 24.02% ± 4.27% by 4% HES). Human albumin (>1.25%) as well as gelatin (>1%) also showed deleterious effects on HK-2 cells. Interestingly, in lower concentrations, human albumin and the crystalloid solution Sterofundin ISO were cytoprotective in comparison with the NaCl control. In conclusion, synthetic and natural colloids showed a harmful impact on HK-2 cells in higher concentrations without any prior proinflammatory stimulus. HES130/0.4 exhibited the most distinctive harmful impact, whereas the application of crystalloid Sterofundin ISO revealed cytoprotective effects.

Transport of a GABAA receptor modulator and its derivatives from Valeriana officinalis L. s. l. across an in vitro cell culture model of the blood-brain barrier.

The roots and rhizome of Valeriana officinalis L . s. l. are therapeutically used for their sedative and sleep-enhancing effects. Some of the active compounds found in commonly used extracts are the sesquiterpenic acids, especially valerenic acid, which was recently identified as a GABA (A) receptor modulator. To interact with this receptor in the brain, substances such as valerenic acid and its derivatives acetoxyvalerenic acid and hydroxyvalerenic acid have to cross the blood-brain barrier (BBB). The aim of our study was to obtain BBB permeability data of these compounds for the first time and to elucidate possible transport pathways across our BBB in vitro model. Transport of valerenic acid, acetoxyvalerenic acid and hydroxyvalerenic acid was compared with the permeability of the GABA (A) modulator diazepam, which is known to penetrate into the central nervous system transcellularly by passive diffusion. Experiments were carried out with an established Transwell in vitro model based on the human cell line ECV304. Results indicated clearly that all three acids permeated significantly slower than diazepam. The ranking was confirmed in group studies as well as in single-substance studies after normalization to diazepam. Valerenic acid (1.06 +/- 0.29 microm/min, factor 0.03 related to diazepam) was the slowest to permeate in the group study, followed by hydroxyvalerenic acid (2.72 +/- 0.63 microm/min, factor 0.07 related to diazepam) and acetoxyvalerenic acid (3.54 +/- 0.58 microm/min, factor 0.09 related to diazepam). To elucidate the contribution of the paracellular transport, studies were performed at different tightness status of the cell layers reflected by different transendothelial electrical resistance (TEER) values. Results showed an exponential correlation between transport and TEER for all three acids, whereas diazepam permeated TEER independently. In summary, it is hypothesized that the investigated compounds from Valeriana officinalis L. S. L. can probably only pass through the BBB by a still unknown transport system and not transcellularly by passive diffusion.

Addition of NMDA-receptor antagonist MK801 during oxygen/glucose deprivation moderately attenuates the upregulation of glucose uptake after subsequent reoxygenation in brain endothelial cells

During stroke the blood-brain barrier (BBB) is damaged which can result in vasogenic brain edema and inflammation. The reduced blood supply leads to decreased delivery of oxygen and glucose to affected areas of the brain. Oxygen and glucose deprivation (OGD) can cause upregulation of glucose uptake of brain endothelial cells. In this letter, we investigated the influence of MK801, a non-competitive inhibitor of the NMDA-receptor, on the regulation of the glucose uptake and of the main glucose transporters glut1 and sglt1 in murine BBB cell line cerebEND during OGD. mRNA expression of glut1 was upregulated 68.7-fold after 6h OGD, which was significantly reduced by 10μM MK801 to 28.9-fold. Sglt1 mRNA expression decreased during OGD which was further reduced by MK801. Glucose uptake was significantly increased up to 907% after 6h OGD and was still higher (210%) after the 20h reoxygenation phase compared to normoxia. Ten micromolar MK801 during OGD was able to reduce upregulated glucose uptake after OGD and reoxygenation significantly. Presence of several NMDAR subunits was proven on the mRNA level in cerebEND cells. Furthermore, it was shown that NMDAR subunit NR1 was upregulated during OGD and that this was inhibitable by MK801. In conclusion, the addition of MK801 during the OGD phase reduced significantly the glucose uptake after the subsequent reoxygenation phase in brain endothelial cells.