TY - JOUR A1 - Burek, Malgorzata A1 - Salvador, Ellaine A1 - Förster, Carola Y. T1 - Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model JF - Journal of Visualized Experiments N2 - Epithelial and endothelial cells (EC) are building paracellular barriers which protect the tissue from the external and internal environment. The blood-brain barrier (BBB) consisting of EC, astrocyte end-feet, pericytes and the basal membrane is responsible for the protection and homeostasis of the brain parenchyma. In vitro BBB models are common tools to study the structure and function of the BBB at the cellular level. A considerable number of different in vitro BBB models have been established for research in different laboratories to date. Usually, the cells are obtained from bovine, porcine, rat or mouse brain tissue (discussed in detail in the review by Wilhelm et al. 1). Human tissue samples are available only in a restricted number of laboratories or companies 2,3. While primary cell preparations are time consuming and the EC cultures can differ from batch to batch, the establishment of immortalized EC lines is the focus of scientific interest. Here, we present a method for establishing an immortalized brain microvascular EC line from neonatal mouse brain. We describe the procedure step-by-step listing the reagents and solutions used. The method established by our lab allows the isolation of a homogenous immortalized endothelial cell line within four to five weeks. The brain microvascular endothelial cell lines termed cEND 4 (from cerebral cortex) and cerebEND 5 (from cerebellar cortex), were isolated according to this procedure in the Förster laboratory and have been effectively used for explanation of different physiological and pathological processes at the BBB. Using cEND and cerebEND we have demonstrated that these cells respond to glucocorticoid- 4,6-9 and estrogen-treatment 10 as well as to pro-infammatory mediators, such as TNFalpha 5,8. Moreover, we have studied the pathology of multiple sclerosis 11 and hypoxia 12,13 on the EC-level. The cEND and cerebEND lines can be considered as a good tool for studying the structure and function of the BBB, cellular responses of ECs to different stimuli or interaction of the EC with lymphocytes or cancer cells. KW - in vitro cell culture models KW - blood-brain barrier KW - neuroscience KW - immunology KW - brain KW - microvascular endothelial cells KW - immortalization KW - cEND Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-126702 VL - 66 IS - e4022 ER - TY - JOUR A1 - Salvador, Ellaine A1 - Shityakov, Sergey A1 - Förster, Carola T1 - Glucocorticoids and endothelial cell barrier function JF - Cell and Tissue Research N2 - Glucocorticoids (GCs) are steroid hormones that have inflammatory and immunosuppressive effects on a wide variety of cells. They are used as therapy for inflammatory disease and as a common agent against edema. The blood brain barrier (BBB), comprising microvascular endothelial cells, serves as a permeability screen between the blood and the brain. As such, it maintains homeostasis of the central nervous system (CNS). In many CNS disorders, BBB integrity is compromised. GC treatment has been demonstrated to improve the tightness of the BBB. The responses and effects of GCs are mediated by the ubiquitous GC receptor (GR). Ligand-bound GR recognizes and binds to the GC response element located within the promoter region of target genes. Transactivation of certain target genes leads to improved barrier properties of endothelial cells. In this review, we deal with the role of GCs in endothelial cell barrier function. First, we describe the mechanisms of GC action at the molecular level. Next, we discuss the regulation of the BBB by GCs, with emphasis on genes targeted by GCs such as occludin, claudins and VE-cadherin. Finally, we present currently available GC therapeutic strategies and their limitations. KW - endothelial cells KW - glucocorticoids KW - glucocorticoid receptor KW - blood brain barrier Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-132091 VL - 355 IS - 3 ER - TY - JOUR A1 - Shityakov, Sergey A1 - Salvador, Ellaine A1 - Pastorin, Giorgia A1 - Förster, Carola T1 - Blood-brain barrier transport studies, aggregation, and molecular dynamics simulation of multiwalled carbon nanotube functionalized with fluorescein isothiocyanate JF - International Journal of Nanomedicine N2 - In this study, the ability of a multiwalled carbon nanotube functionalized with fluorescein isothiocyanate (MWCNT-FITC) was assessed as a prospective central nervous system-targeting drug delivery system to permeate the blood-brain barrier. The results indicated that the MWCNT-FITC conjugate is able to penetrate microvascular cerebral endothelial monolayers; its concentrations in the Transwell® system were fully equilibrated after 48 hours. Cell viability test, together with phase-contrast and fluorescence microscopies, did not detect any signs of MWCNT-FITC toxicity on the cerebral endothelial cells. These microscopic techniques also revealed presumably the intracellular localization of fluorescent MWCNT-FITCs apart from their massive nonfluorescent accumulation on the cellular surface due to nanotube lipophilic properties. In addition, the 1,000 ps molecular dynamics simulation in vacuo discovered the phenomenon of carbon nanotube aggregation driven by van der Waals forces via MWCN-TFITC rapid dissociation as an intermediate phase. KW - endothelial cells KW - cytotoxicity KW - blood-brain barrier KW - fluorescein isothiocyanate KW - aggregation KW - molecular dynamics KW - fluorescence microscopy KW - Transwell® system KW - multiwalled carbon nanotube KW - mice Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-149233 VL - 10 ER - TY - JOUR A1 - Salvador, Ellaine A1 - Burek, Malgorzata A1 - Förster, Carola Y. T1 - Stretch and/or oxygen glucose deprivation (OGD) in an in vitro traumatic brain injury (TBI) model induces calcium alteration and inflammatory cascade JF - Frontiers in Cellular Neuroscience N2 - The blood-brain barrier (BBB), made up of endothelial cells of capillaries in the brain, maintains the microenvironment of the central nervous system. During ischemia and traumatic brain injury (TBI), cellular disruption leading to mechanical insult results to the BBB being compromised. Oxygen glucose deprivation (OGD) is the most commonly used in vitro model for ischemia. On the other hand, stretch injury is currently being used to model TBI in vitro. In this paper, the two methods are used alone or in combination, to assess their effects on cerebrovascular endothelial cells cEND in the presence or absence of astrocytic factors. Applying severe stretch and/or OGD to cEND cells in our experiments resulted to cell swelling and distortion. Damage to the cells induced release of lactate dehydrogenase enzyme (LDH) and nitric oxide (NO) into the cell culture medium. In addition, mRNA expression of inflammatory markers interleukin (I L)-6, IL-1\(\alpha\) chemokine (C-C motif) ligand 2 (CCL2) and tumor necrosis factor (TNF)-\(\alpha\) also increased. These events could lead to the opening of calcium ion channels resulting to excitotoxicity. This could be demonstrated by increased calcium level in OGD-subjected cEND cells incubated with astrocyte-conditioned medium. Furthermore, reduction of cell membrane integrity decreased tight junction proteins claudin-5 and occludin expression. In addition, permeability of the endothelial cell monolayer increased. Also, since cell damage requires an increased uptake of glucose, expression of glucose transporter glut1 was found to increase at the mRNA level after OGD. Overall, the effects of OGD on cEND cells appear to be more prominent than that of stretch with regards to TJ proteins, NO, glutl expression, and calcium level. Astrocytes potentiate these effects on calcium level in cEND cells. Combining both methods to model TBI in vitro shows a promising improvement to currently available models. KW - receptor antagonist KW - cytokine expression KW - tight junctions KW - cell stretch KW - calcium level KW - nitric oxide KW - endothelial cells KW - necrosis factor alpha KW - barrier properties KW - cerebral ischemia KW - nervous system KW - CNS injury KW - blood brain barrier KW - cEND KW - astrocytes KW - traumatic brain injury KW - oxygen-glucose deprivation Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-148255 VL - 9 IS - 323 ER - TY - JOUR A1 - Shityakov, Sergey A1 - Puskás, István A1 - Pápai, Katalin A1 - Salvador, Ellaine A1 - Roewer, Norbert A1 - Förster, Carola A1 - Broscheit, Jens-Albert T1 - Sevoflurane-sulfobutylether-\(\beta\)-cyclodextrin complex: preparation, characterization, cellular toxicity, molecular modeling and blood-brain barrier transport studies JF - Molecules N2 - The objective of the present investigation was to study the ability of sulfobutylether-\(\beta\)-cyclodextrin (SBECD) to form an inclusion complex with sevoflurane (SEV), a volatile anesthetic with poor water solubility. The inclusion complex was prepared, characterized and its cellular toxicity and blood-brain barrier (BBB) permeation potential of the formulated SEV have also been examined for the purpose of controlled drug delivery. The SEV-SBE\(\beta\)CD complex was nontoxic to the primary brain microvascular endothelial (pEND) cells at a clinically relevant concentration of sevoflurane. The inclusion complex exhibited significantly higher BBB permeation profiles as compared with the reference substance (propranolol) concerning calculated apparent permeability values (P\(_{app}\)). In addition, SEV binding affinity to SBE\(\beta\)CD was confirmed by a minimal Gibbs free energy of binding (ΔG\(_{bind}\)) value of -1.727 ± 0.042 kcal・mol\(^{-1}\) and an average binding constant (K\(_{b}\)) of 53.66 ± 9.24 mM indicating rapid drug liberation from the cyclodextrin amphiphilic cavity. KW - pharmaceutical applications KW - in vitro KW - propranolol KW - water KW - primary microvascular endothelial cells KW - molecular liphophilicity potential KW - molecular docking KW - blood-brain barrier KW - ulfobutylether-\(\beta\)-cyclodextrin KW - sevoflurane KW - cyclodextrin formulations KW - safety KW - etomidate KW - formulations KW - hydrochloride KW - ether KW - intestinal absorption Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-148543 VL - 20 ER - TY - JOUR A1 - Reinhold, Ann Kristin A1 - Schwabe, Joachim A1 - Lux, Thomas J. A1 - Salvador, Ellaine A1 - Rittner, Heike L. T1 - Quantitative and Microstructural Changes of the Blood-Nerve Barrier in Peripheral Neuropathy JF - Frontiers in Neuroscience N2 - Peripheral neuropathy is accompanied by changes in the neuronal environment. The blood-nerve barrier (BNB) is crucial in protecting the neural homeostasis: Tight junctions (TJ) seal paracellular spaces and thus prevent external stimuli from entering. In different models of neuropathic pain, the BNB is impaired, thus contributing to local damage, immune cell invasion and, ultimately, the development of neuropathy with its symptoms. In this study, we examined changes in expression and microstructural localization of two key tight junction proteins (TJP), claudin-1 and the cytoplasmic anchoring ZO-1, in the sciatic nerve of mice subjected to chronic constriction injury (CCI). Via qPCR and analysis of fluorescence immunohistochemistry, a marked downregulation of mRNA as well as decreased fluorescence intensity were observed in the nerve for both proteins. Moreover, a distinct zig-zag structure for both proteins located at cell-cell contacts, indicative of the localization of TJs, was observed in the perineurial compartment of sham-operated animals. This microstructural location in cell-cell-contacts was lost in neuropathy as semiquantified via computational analysis, based on a novel algorithm. In summary, we provide evidence that peripheral neuropathy is not only associated with decrease in relevant TJPs but also exhibits alterations in TJP arrangement and loss in barrier tightness, presumably due to internalization. Specifically, semiquantification of TJP in cell-cell-contacts of microcompartments could be used in the future for routine clinical samples of patients with neuropathy. KW - neuropathic pain KW - chronic constriction injury KW - blood-nerve barrier KW - tight junction protein KW - claudin-1 KW - ZO-1 KW - Expression KW - Pain Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-225179 VL - 12 ER - TY - JOUR A1 - Rösing, Nils A1 - Salvador, Ellaine A1 - Güntzel, Paul A1 - Kempe, Christoph A1 - Burek, Malgorzata A1 - Holzgrabe, Ulrike A1 - Soukhoroukov, Vladimir A1 - Wunder, Christian A1 - Förster, Carola T1 - Neuroprotective Effects of Isosteviol Sodium in Murine Brain Capillary Cerebellar Endothelial Cells (cerebEND) After Hypoxia JF - Frontiers in Cellular Neuroscience N2 - Ischemic stroke is one of the leading causes of death worldwide. It damages neurons and other supporting cellular elements in the brain. However, the impairment is not only confined to the region of assault but the surrounding area as well. Besides, it also brings about damage to the blood-brain barrier (BBB) which in turn leads to microvascular failure and edema. Hence, this necessitates an on-going, continuous search for intervention strategies and effective treatment. Of late, the natural sweetener stevioside proved to exhibit neuroprotective effects and therapeutic benefits against cerebral ischemia-induced injury. Its injectable formulation, isosteviol sodium (STVNA) also demonstrated favorable results. Nonetheless, its effects on the BBB have not yet been investigated to date. As such, this present study was designed to assess the effects of STVNA in our in vitro stroke model of the BBB.The integrity and permeability of the BBB are governed and maintained by tight junction proteins (TJPs) such as claudin-5 and occludin. Our data show increased claudin-5 and occludin expression in oxygen and glucose (OGD)-deprived murine brain capillary cerebellar endothelial cells (cerebEND) after STVNa treatment. Likewise, the upregulation of the transmembrane protein integrin-αv was also observed. Finally, cell volume was reduced with the simultaneous administration of STVNA and OGD in cerebEND cells. In neuropathologies such as stroke, the failure of cell volume control is a major feature leading to loss of cells in the penumbra as well as adverse outcomes. Our initial findings, therefore, point to the neuroprotective effects of STVNA at the BBB in vitro, which warrant further investigation for a possible future clinical intervention. KW - isosteviol sodium KW - hypoxia KW - cerebEND cells KW - blood brain barrier KW - neuroprotection Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-215013 SN - 1662-5102 VL - 14 ER - TY - JOUR A1 - Feldheim, Jonas A1 - Kessler, Almuth F. A1 - Schmitt, Dominik A1 - Salvador, Ellaine A1 - Monoranu, Camelia M. A1 - Feldheim, Julia J. A1 - Ernestus, Ralf-Ingo A1 - Löhr, Mario A1 - Hagemann, Carsten T1 - Ribosomal Protein S27/Metallopanstimulin-1 (RPS27) in Glioma — A New Disease Biomarker? JF - Cancers N2 - Despite its significant overexpression in several malignant neoplasms, the expression of RPS27 in the central nervous system (CNS) is widely unknown. We identified the cell types expressing RPS27 in the CNS under normal and disease conditions. We acquired specimens of healthy brain (NB), adult pilocytic astrocytoma (PA) World Health Organization (WHO) grade I, anaplastic PA WHO grade III, gliomas WHO grade II/III with or without isocitrate dehydrogenase (IDH) mutation, and glioblastoma multiforme (GBM). RPS27 protein expression was examined by immunohistochemistry and double-fluorescence staining and its mRNA expression quantified by RT-PCR. Patients’ clinical and tumor characteristics were collected retrospectively. RPS27 protein was specifically expressed in tumor cells and neurons, but not in healthy astrocytes. In tumor tissue, most macrophages were positive, while this was rarely the case in inflamed tissue. Compared to NB, RPS27 mRNA was in mean 6.2- and 8.8-fold enhanced in gliomas WHO grade II/III with (p < 0.01) and without IDH mutation (p = 0.01), respectively. GBM displayed a 4.6-fold increased mean expression (p = 0.02). Although RPS27 expression levels did not affect the patients’ survival, their association with tumor cells and tumor-associated macrophages provides a rationale for a future investigation of a potential function during gliomagenesis and tumor immune response. KW - glioblastoma multiforme KW - low-grade glioma KW - astrocytoma KW - recurrence KW - relapse KW - mRNA KW - protein KW - brain KW - expression KW - MPS1 Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-203648 SN - 2072-6694 VL - 12 IS - 5 ER - TY - JOUR A1 - Burek, Malgorzata A1 - Burmester, Sandra A1 - Salvador, Ellaine A1 - Möller-Ehrlich, Kerstin A1 - Schneider, Reinhard A1 - Roewer, Norbert A1 - Nagai, Michiaki A1 - Förster, Carola Y. T1 - Kidney Ischemia/Reperfusion Injury Induces Changes in the Drug Transporter Expression at the Blood–Brain Barrier in vivo and in vitro JF - Frontiers in Physiology N2 - Ischemia/reperfusion injury is a major cause of acute kidney injury (AKI). AKI is characterized by a sudden decrease in kidney function, systemic inflammation, oxidative stress, and dysregulation of the sodium, potassium, and water channels. While AKI leads to uremic encephalopathy, epidemiological studies have shown that AKI is associated with a subsequent risk for developing stroke and dementia. To get more insights into kidney–brain crosstalk, we have created an in vitro co-culture model based on human kidney cells of the proximal tubule (HK-2) and brain microvascular endothelial cells (BMEC). The HK-2 cell line was grown to confluence on 6-well plates and exposed to oxygen/glucose deprivation (OGD) for 4 h. Control HK-2 cells were grown under normal conditions. The BMEC cell line cerebED was grown to confluence on transwells with 0.4 μm pores. The transwell filters seeded and grown to confluence with cereEND were inserted into the plates with HK-2 cells with or without OGD treatment. In addition, cerebEND were left untreated or treated with uremic toxins, indole-3-acetic acid (IAA) and indoxyl sulfate (IS). The protein and mRNA expression of selected BBB-typical influx transporters, efflux transporters, cellular receptors, and tight junction proteins was measured in BMECs. To validate this in vitro model of kidney–brain interaction, we isolated brain capillaries from mice exposed to bilateral renal ischemia (30 min)/reperfusion injury (24 h) and measured mRNA and protein expression as described above. Both in vitro and in vivo systems showed similar changes in the expression of drug transporters, cellular receptors, and tight junction proteins. Efflux pumps, in particular Abcb1b, Abcc1, and Abcg2, have shown increased expression in our model. Thus, our in vitro co-culture system can be used to study the cellular mechanism of kidney and brain crosstalk in renal ischemia/reperfusion injury. KW - kidney ischemia/reperfusion injury KW - brain pathology KW - blood–brain barrier KW - drug transporter KW - tight junctions Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-216413 SN - 1664-042X VL - 11 ER - TY - JOUR A1 - Salvador, Ellaine A1 - Burek, Malgorzata A1 - Löhr, Mario A1 - Nagai, Michiaki A1 - Hagemann, Carsten A1 - Förster, Carola Y. T1 - Senescence and associated blood-brain barrier alterations in vitro JF - Histochemistry and Cell Biology N2 - Progressive deterioration of the central nervous system (CNS) is commonly associated with aging. An important component of the neurovasculature is the blood-brain barrier (BBB), majorly made up of endothelial cells joined together by intercellular junctions. The relationship between senescence and changes in the BBB has not yet been thoroughly explored. Moreover, the lack of in vitro models for the study of the mechanisms involved in those changes impede further and more in-depth investigations in the field. For this reason, we herein present an in vitro model of the senescent BBB and an initial attempt to identify senescence-associated alterations within. KW - senescence KW - in vitro model KW - aging KW - CNS diseases KW - blood–brain barrier Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-267435 SN - 1432-119X VL - 156 IS - 3 ER -