Berndt, Philipp

Blasig, Ingolf

Brodin, Birger

Helms, Hans Christian Cederberg

Pedersen, Stine Helene Falsig

Tornabene, Erica

Waagepetersen, Helle S.

English

Copenhagen University Research Information System

u n i ve r s i t y  o f  co pe n h ag e n  Københavns UniversitetA bovine in vitro blood-brain barrier model under oxygenglucose deprivation (OGD)conditionTornabene, Erica; Helms, Hans Christian Cederberg; Berndt, Philipp; Blasig, Ingolf;Pedersen, Stine Helene Falsig; Waagepetersen, Helle S.; Brodin, BirgerPublication date:2016Document versionPublisher's PDF, also known as Version of recordCitation for published version (APA):Tornabene, E., Helms, H. C. C., Berndt, P., Blasig, I., Pedersen, S. H. F., Waagepetersen, H. S., & Brodin, B.(2016). A bovine in vitro blood-brain barrier model under oxygenglucose deprivation (OGD) condition. Postersession presented at 19th Symposium in Signal Transduction, Copenhagen, Denmark.Download date: 03. Feb. 2020U N I V E R S I T Y  O F  C O P E N H A G E N  F A C U LT Y  O F  H E A LT H  A N D  M E D I C A L  S C I E N C E S     A bovine in vitro blood-brain barrier model under oxygen-glucose deprivation (OGD) condition     Erica Tornabene1, Hans Christian Cederberg Helms2, Philipp Berndt3, Ingolf Blasig3, Stine Falsig Pedersen4,  Helle S. Waagepetersen5 and Birger Brodin1   1Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark   2Novo Nordisk A/S, Denmark 3Leibniz Institut für Molekulare Physiogie, Campus Berlin-Buch, Berlin, Germany 4Department of Biology, Faculty of Science, University of Copenhagen, Denmark  5 Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark   Aim  During stroke, the brain endothelium experiences low glucose and oxygen. We therefore wish to investigate the effects of low glucose and oxygen in cultured brain  capillary endothelial cells, focusing particularly on barrier properties and transport proteins.                    Background Figure 4. Confocal laser scanning microscopy images of brain endothelial cells co-cultured with rat astrocytes . Cells were fixed, permeabilized and immunostained with  Claudin-5, ZO-2 or GLUT-1 antibody (green), cell nuclei were counterstained with propidium iodide (red). n=4. Permeability  increased during OGD and recovered  during “reperfusion” Figure 3. TEER was measured at various time points, before, under and after the OGD protocol. Measurements were performed with an Endohm cup electrode. N=3-6, n= 6 (left figure). Paracellular tightness was also evaluated by determining the passive permeability of 3 marker molecules: 14C-mannitol, FITC-dextran4 and FITC-dextran40. N=3-4, n=3 (right figures)   Ischemia is a devastating disease which affects million of people every year. During ischemia, the loss of regional cerebral blood-flow and the subsequent reperfusion induce significant changes in in the transport pathways and barrier properties of the blood-brain barrier (BBB). Conclusions  •    The brain endothelial cells, co-cultured with astrocytes, showed a decrease in tightness during OGD, an effect which was reversible upon reperfusion.  •    The tight junction proteins Claudin-5 and ZO-2 translocated from the junction complex to the cytosol during OGD, and relocalized to junctions during reperfusion. Their protein level decreased during OGD and recovered upon reperfusion. •    The transporter GLUT1 migrated to the cell border during reperfusion. •    Pgp protein expression decreased during reperfusion. The protein level of HBEGF, LRP1 and InsR increased in the reperfusion phase.  Future experiments  • Evaluating the degree of Pgp activity reduction after 24h of reperfusion by testing the permeation of known Pgp substrates across the endothelial cell monolayer. • Investigating the possibility that the receptors HBEGF, LRP1 and InsR may mediate the delivery of drugs across the ischemic BBB by transport experiments. • To examine other brain cell types influence on barrier properties during and after the OGD treatment. Acknowledgement  The Lundbeck Foundation is acknowledged for support via the RIBBDD project grant.   Oxygen-glucose deprivation (OGD) protocol in a bovine blood-brain barrier in vitro model  Figure 2. Bovine brain endothelial cells were grown from capillary fragments in culture flasks for 5 days. They were then trypzinized and co-cultured with rat astrocytes in coated filter inserts for additional 6 days. Thus, they were subjected to oxygen-glucose deprivation (OGD) conditions for 4 hours in a hypoxia workbench and a subsequent ”reperfusion” for 24-48h.  CLDN-5 and ZO-1 translocated from the cell junctions during OGD. GLUT1 localized in the cell mebrane during reperfusion  Figure 1. Brain affected by ischemic stroke.  Abbreviations  BBB: Blood-brain barrier; OGD: Oxygen-glucose deprivation; PCR: Polymerase chain reaction; R: reperfusion; TEER: Transendothelial electrical resistance.    References          Figure 1: From S.M. Allan & N.J. Rothwell, 2001  Normal Incubator O2: 19% Glucose: 5.5 mM  Hypoxia Workbench O2: <1% Glucose: 0 mM  End of the  experiment OGD (4h) Reperfusion (24h-48h) Bovine BCECs Rat Astrocytes mRNA and protein expression level of selected proteins before,  during and after OGD Figure 5. mRNA expression of  target genes was investigated by RealTime-PCR using primers designed for bovine. n=4, (upper figures). Proteins were isolated from bovine endothelial cell/rat astrocyte in non contact co-cultures. An equal amount of proteins was loaded in each lane of the gel for western blot analisys. (n=2). (lower figures). 14C-mannitol FITC- dextran4 FITC- dextran40 CLDN5Control4h OGD24h R48h R05101520nsExpression level normalized to HPRT1ns  Protein  Control  4h OGD  24h R      CLDN-5       ZO-2        GLUT1 

A bovine in vitro blood-brain barrier model under oxygenglucose deprivation (OGD) condition

https://curis.ku.dk/portal/files/168929905/Erica_Poster_IBBS_2016.pdf

A bovine in vitro blood-brain barrier model under oxygenglucose deprivation (OGD) condition

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