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 - 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 -