Name: oxygen-glucose deprivation and reoxygenation as an in vitro ischemia-reperfusion injury model for studying blood-brain barrier dysfunction
Uploaded: 2015-05-07T15:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction at JoVE.com

Wir erkennen an, Scott and White Hospital Research Grants Program für die finanzielle Unterstützung und die Texas A &amp; M Health Science Center College of Medicine Integrated Imaging Laboratory für die Verwendung des konfokalen Lasermikroskop. Wir erkennen an, Mr. Glen Cryer für die Hilfe bei Manuskriptbearbeitung.

1. Seeding von Endothelzellen <ol><li> Erhalten Primärkulturen von RBMEC die von erwachsenen Sprague Dawley Ratten (oder erhalten sie im Handel). </li><li> Kulti RBMECs in 100 cm Fibronectin (50 ug / ml) beschichtete Petrischalen unter Verwendung des Rattenhirn endothelialen Zellwachstumsmedium. Ändern Sie das Medium alle zwei Tage, bis Konfluenz erreicht ist. </li><li> Bei Erreichen von 80 bis 90% Konfluenz, vorsichtig waschen Sie die Zellen in 5 ml phosphatgepufferter Kochsalzlösung (PBS) durch Schwenken. Die Zellen werden dann, indem man sie 1 ml warmem 0,25% Trypsin-Ethylendiamintetraessigsäure (EDTA) -Lösung, auf 37 ° C äquilibriert abgelöst. </li><li> Inkubieren der Zellen bei 37 ° C für 2-5 min, bis die Zellen abgelöst und dispergiert. HINWEIS: Tippen Sie auf die Kulturschale, um die Zellen zu lösen. Ansicht Zellen unter dem Mikroskop, um eine vollständige Ablösung der Zellen von der Oberfläche der Schale zu bestätigen. </li><li> 5 ml vollständigem Medium in eine Petrischale, um Trypsin zu neutralisieren.Pipettieren Sie das Medium, das abgelösten Zellen und sammeln sie in ein 15 ml Zentrifugenröhrchen. . </li><li> Zentrifugieren Sie die Medien, die Endothelzellen bei 220 xg für 5 min. </li><li> Saugen Sie den Überstand und die Erhaltung der Pellet, das cells.Suspend das Pellet in 3-5 ml frischem Rattenhirn endothelialen Zellwachstumsmedium durch sanftes Mischen nach oben und unten mit Pipette Zellen werden dann unter Verwendung von automatisierten Zellzählers oder einer Zählkammer gezählt werden. </li><li> Übertragen Sie die Zellsuspension in Fibronektin (50 ug / ml) vorbeschichtet 8 auch sterile Kammer Schlittensystem, 0,7 cm 2 / gut mit einer Aussaatdichte im Bereich von 10.000-15.000 Zellen pro Vertiefung. Wachsen die Zellen bei 37 ° C bis zur Konfluenz erreicht ist HINWEIS: Für die Durchführung Western Blots oder anderen Experimenten Zellen in 10 cm Zellkulturschalen oder spezielle Gerichte wie durch das Experiment erforderlich gehalten werden. </li></ol> 2. Sauerstoff und Glukose-Entzug-Reoxygenierung In-vitro-Modell HINWEIS: Das folgende Protokoll wurde von Zulueta et al angepasst. 1997; Zhu H et al. 2012 8,9. <ol><li> Verwenden Sie die Hypoxie Zellkultursystem, um die Auswirkungen der OGD-R auf RBMECs in (siehe Abbildung 1) zu studieren. Eingestellt und justiert die Hypoxie Zellkultursystem vor Beginn des Experiments gemß den Anweisungen des Herstellers. </li><li> Entfernen der zusammenlaufenden Kammerobjektträgern (Schritt 1.8) von dem 37 ° C Inkubator. Ersetzen die Vollmedium in der Kammer Schlitten mit desoxygeniertem, keine Glucose, Dulbeccos modifiziertes Eagle-Medium (DMEM) und Hypoxie Kammer mit 95% N 2 und 5% CO 2 für 2 h bei 37 ° C vorgelegt, auf OGD Zustand darzustellen. </li><li> Verschieben Sie die Zellen zurück in den Inkubator mit 95% O 2, 5% CO 2 bei 37 ° C und mit frischem Rattenhirn Endothelzellenkomplettmedium vorgesehen und inkubiere für 1 weitere Stunde bei 37 ° C. Hinweis: Dieser Schritt stellt eine Reoxygenierung Situation. </li><li> Mithilfe der Kammer-Objektträgern für Immunfluoreszenzlokalisierung und Rhodamin-Phalloidin Kennzeichnung (siehe Kapitel 3). </li></ol> 3. Immunfluoreszenz Lokalisierung von ZO-1 und f-Actin-Labeling Mit Rhodamin Phalloidin <ol><li> Setzen Sie das Kammer-Objektträgern enthalten RBMEC Monoschichten zu 100 ul opti-MEM / reduzierter Serum Medien / well 1 h. 3-mal in 100 ul phosphatgepufferter Kochsalzlösung (PBS, pH 7,0 bis 7,2) Waschen Sie die Kammer-Objektträgern. </li><li> Fixieren die Zellen unter Verwendung von 100 ul 4% Paraformaldehyd in PBS (pH 7,0-7,2) für 15 min und Waschen der Kammer-Objektträgern für 3 weitere Male in PBS (pH 7,0-7,2). </li><li> Permeabilisierung der Zellen unter Verwendung von 100 ul 0,5% Triton X-100 in PBS (pH 7,0-7,2) für weitere 15 min. Blockieren mit 100 ul 2% Rinderserumalbumin (BSA) in PBS für eine Stunde. Nach diesem Schritt entweder Fleck / Label Zellen entweder für ZO-1 oder F-Actin. <ol><li> Für die Immunfluoreszenz staining Inkubieren der Zellen mit einem Anti-Kaninchen primären Antikörper gegen ZO-1 in 1: 150 in 2% BSA-PBS für O bei 4 ° C hergestellt / N. In PBS Waschen Sie die Zellen 3-mal, (pH 7,0 bis 7,2). Inkubieren mit 100 &amp; mgr; l Fluorescein-Isothiocyanat (FITC) -markierten anti-Kaninchen Sekundär-Antikörper für 1 h bei RT. </li><li> Rhodamin Phalloidin Kennzeichnung, nach Blockierung, setzen die Zellen zu 100 ul von Rhodamin-Phalloidin 1:50 Verdünnung in 2% BSA-PBS für 20 min. </li><li> Waschen Sie die Zellen von Immunfluoreszenzfärbung und Rhodamin-Phalloidin Kennzeichnung in PBS (pH 7,0 bis 7,2). Montieren Sie die Kammern mit Montage Medien, die anti-fade-Reagens mit DAPI. </li></ol></li><li> Visualisierung der Zellen unter Verwendung eines 60 X Wasser-Immersionslinse und die Zellen werden in einer einzigen optischen Ebene unter einem konfokalen Mikroskop gescannt. </li></ol>

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	<li>Eltzschig, H. K., Eckle, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ischemia+and+reperfusion--from+mechanism+to+translation.">Ischemia and reperfusion--from mechanism to translation.</a> Nat Med. 17 (11), 1391-1401 (2011).</li><li>Kalogeris, T., Baines, C. P., Krenz, M., Korthuis, R. 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Y., Scorziello, A., Duchen, M. 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The authors have nothing to disclose.

OGD-R als ein in vitro Modell für Ischämie-Reperfusion wurde auch für die Untersuchung Neuronen 10,11 hergestellt. Es gibt auch Studien, die die Wirkung auf OGD Hirnendothelzellen und Veränderungen der Permeabilität und TJ Integrität 9. Unsere Studie zeigt jedoch, die Wirkung von OGD und Reoxygenierung, die eine nähere Darstellung des ischämischen Reperfusionsschaden in In-vivo-Bedingungen, die folgende ischämischen Schlaganfalls auftreten. Hy...

Ischämie-Reperfusions (IR) Verletzung festgestellt wird, das häufige Ursache verschiedener schwächende Komplikationen und Todesfälle im Zusammenhang mit Schlaganfall, Myokardinfarkt, Trauma, periphere Gefäßkrankheit und traumatischer Hirnverletzung 1,2 zugeordnet werden. IR Verletzungen in Hirngefäße ist eine frühe sekundäre Verletzungen, die zu Entzündungen und Ödeme 3. Einer der schwerwiegenden Komplikationen, die als Folge der oxidativen und metabolischen Stress folgenden Entzündung auftritt ist der Verlust der homöostatischen Gleichgewicht, was zu Bildung von freien Radikalen, Veränderungen in der Blut-Hirn-Schranke (BHS) Tight Junctions (TJs) und mikrovaskuläre Permeabilität 4,5. Derzeit werden in-vivo-Modellen verwendet werden, um die Auswirkungen von IR Verletzung am BBB Studie sind mittleren Hirnarterie (MCAO), Mikroembolien und transgenen oder Knockout-Tieren. Jedoch hat jede ihre Nachteile und Einschränkungen durch Hossmann 6 diskutiert. MCAO-Modell verwendet die effec zu studierents von Redox-Stress, Veränderungen in der Junction-Kommunikation des BBB und die Interaktionen zwischen Gehirn und Immunzellen. Jedoch weisen sie verschiedene technische Herausforderungen, wie die Notwendigkeit für eine präzise Mikrochirurgie und die Schwierigkeiten darin. Mikroembolie sofort bricht die BBB, während die Verwendung von transgenen oder Knockout-Tieren zu zerebraler Ischämie studieren können Herausforderungen wie Gen-abhängige Molekular Einflüsse auf Infarktbildung, Veränderungen in der Gefäßanatomie und unterschiedlichen Körpergewichte 6 haben. Daher wird in vitro-Modellen von Ischämie haben gefunden zunehmendes Interesse in der letzten Zeit vor allem aufgrund ihrer Eignung bei der Durchführung mechanistische Studien für Medikamente. Allerdings können die Ergebnisse der in-vitro-Studien nicht vollständig repräsentieren eine in-vivo-Studie und müssen mit Vorsicht interpretiert werden 6. Gegenläufig wirkte der geringe Sauerstoffkonzentrationen auf die endotheliale Zellmonoschichten und mikrovaskuläre Permeabilität wurdenstudierte von Ogawa 7. Rattenhirn-mikrovaskulären Endothelzellen (RBMECs) wurden verwendet, um die in vitro BBB entwickeln. Die Sauerstoff-Glukose Deprivation und Reoxygenierung (OGD-R-Technik) in diesem Protokoll vorgestellt wurde von Studien, die von Zulueta et al und Zhu et al 8,9 angepasst. Wir exponierten Hirnendothelzellen auf OGD-R, indem sie in einer Hypoxie / Anoxie Raum mit 0% O 2, 5% CO 2 und 95% N 2. Die Zellen wurden später für die Veränderungen in TJ Integrität und Stressfaserbildung mittels Immunfluoreszenz-Lokalisierung und Rhodamin-Phalloidin Kennzeichnung bzw. beurteilt. Immunfluoreszenzfärbung für das Zonula-occludens-1 (ZO-1) durchgeführt, um festzustellen, TJ Integrität als ZO-1 eine wichtige Gerüst Membran gebunden TJ Protein. Rhodamin-Phalloidin Kennzeichnung bestimmt die filamentöse Aktin (F-Actin) in der Zelle Zytoskeletts und ist ein deutliches Zeichen von Aktin-Stressfaserbildung in Endothelzellen. &lt;p class = &quot;jove_content&quot;&gt; Das Ziel dieses Verfahrens ist, um einen Einblick in die Entwicklung OGD-R als in vitro-Modell zum Studium IR BBB Endothelzellen TJ Integrität und F-Actin Spannungsfaserbildung bereitzustellen. Die Ergebnisse werden Informationen über das Schicksal der TJ-Protein liefern, ZO-1 und Stressfaserbildung nach OGD-R. Das Verständnis dieser Zusammenhänge wird die Möglichkeit, die zugrunde liegenden molekularen Mechanismen, die ausgelöst folgenden OGD-R sind zu ermitteln und entwickeln mögliche therapeutische Strategien, um die Störung BBB folgenden OGD-R Behandlung zu verbessern.

<table><tbody><tr><td>Proox model 110</td><td>Biospherix</td><td>Model 110</td><td></td></tr><tr><td>DMEM, no glucose</td><td>Gibco, Life technologies</td><td>11966-025</td><td></td></tr><tr><td>Rhodamine Phalloidin</td><td>Life technologies</td><td>R415</td><td></td></tr><tr><td>ZO-1 Rabbit Polyclonal Antibody</td><td>Life technologies</td><td>617300</td><td></td></tr><tr><td>Nunc Lab Tek II-CC 8&amp;nbsp;well sterile, glass&amp;nbsp;slides&amp;nbsp;</td><td>Thermo scientific</td><td>177402</td><td></td></tr><tr><td>FITC-tagged anti-rabbit secondary antibody&amp;nbsp;</td><td>Santa cruz</td><td>sc-2090</td><td></td></tr><tr><td>DPBS 1X</td><td>Thermo scientific</td><td>SH 30028.03</td><td>Any other PBS available can be used</td></tr></tbody></table>

oxygen-glucose deprivation and reoxygenation as an in vitro ischemia-reperfusion injury model for studying blood-brain barrier dysfunction

Ischämie-Reperfusion (IR) Verletzung bekannt ist, wesentlich zur Morbidität und Mortalität bei ischämischen Schlaganfällen assoziiert beitragen. Ischämische Schlaganfälle machen 80% aller Schlaganfälle. Eine häufige Ursache für IR Verletzungen ist der schnelle Zustrom von Flüssigkeiten nach einer akuten / chronischen Verschluss von Blut, Nährstoffe, Sauerstoff zum Auslösen der Bildung von freien Radikalen Gewebe. Ischämischen Schlaganfall wird durch Blut-Hirn-Schranke (BBB) ​​Dysfunktion und vasogenic Hirnödem gefolgt. Strukturell Tight Junctions (TJs) zwischen den Endothelzellen spielen eine wichtige Rolle bei der Aufrechterhaltung der Integrität der Blut-Hirn-Schranke (BHS). IR Verletzungen ist eine frühe Sekundärverletzung führt zu einer unspezifischen Entzündungsreaktion. Oxidativen und metabolischen Stress folgenden Entzündung auslöst sekundären Hirnschäden einschließlich BBB Lässigkeit und Unterbrechung der Tight Junction (TJ) Integrität. Unser Protokoll stellt ein in vitro &lt;/ Em&gt; Beispiel von Sauerstoff-Glukose Deprivation und Reoxygenierung (OGD-R) auf Rattenhirn Endothelzellen TJ Integrität und Stressfaserbildung. Derzeit mehreren experimentellen in vivo-Modelle werden benutzt, um die Effekte von IR Schädigung zu untersuchen; sie haben jedoch mehrere Einschränkungen auf, wie die technischen Herausforderungen bei der Durchführung von Operationen, Gens abhängige Molekular Einflüsse und Schwierigkeiten bei der Untersuchung mechanistische Beziehungen. Jedoch kann in vitro Modellen in der Überwindung vieler dieser Einschränkungen zu unterstützen. Das vorgestellte Protokoll verwendet, um die verschiedenen molekularen Mechanismen und mechanistische Beziehungen zu studieren, um potentielle therapeutische Strategien bereitzustellen. Allerdings können die Ergebnisse der in-vitro-Studien von Standard-in-vivo-Studien unterscheiden und sollten mit Vorsicht interpretiert werden.

Zellen auf Fibronektin vorbeschichteten Nunc II Kammer-Objektträgern kultiviert wurden OGD-R, indem Sie in einem Biospherix ProOx Modell 110 Kammer unterzogen. Nach Aussetzen Zellen OGD-R, die sie für ZO-1 junctional Färbung mit Immunfluoreszenztechnik verarbeitet wurden, wie in Abbildung 2 und Zytoskelett Anordnung gezeigt anzeigt F-Aktin Bildung von Stressfasern mit Rhodamin-Phalloidin-Färbung Etikett, wie in Fig. 3 gezeigt, die Kontrollzellen waren nicht OGD-R unterzogen zeigte k...

Watch this Scientific Journal Video about Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction at JoVE.com

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is associated with a high rate of morbidity and mortality. The goal of the in vitro model of oxygen-glucose deprivation and reoxygenation (OGD-R) described here is to assess the effects of ischemia reperfusion injury on a variety of cells, particularly in blood-brain barrier (BBB) endothelial cells.

Sauerstoff-Glukose Deprivation und Reoxygenierung als<em&gt; In-vitro-</em&gt; Ischämie-Reperfusion Injury Modell zum Studium der Blut-Hirnschranke Dysfunction

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic ...

oxygen-glucose-deprivation-reoxygenation-as-an-vitro-ischemia

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Medicine

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

17.9K Aufrufe.  Texas A&M University Health Science Center College of Medicine. Ischemia-Reperfusion (IR) injury is associated with a high rate of morbidity and mortality. The goal of the in vitro model of oxygen-glucose deprivation and reoxygenation (OGD-R) described here is to assess the effects of ischemia reperfusion injury on a variety of cells, particularly in blood-brain barrier (BBB) endothelial cells. 

Serie: Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

2-Vessel Occlusion/Hypotension: A Rat Model of Global Brain Ischemia

Cardiac arrest followed by resuscitation often results in dramatic brain damage caused by ischemia and subsequent reperfusion of the brain. Global brain ischemia produces damage to specific brain regions shown to be highly sensitive to ischemia 1. Hippocampal neurons have higher sensitivity to ischemic insults compared to other cell populations, and specifically, the CA1 region of the hippocampus is particularly vulnerable to ischemia/reperfusion 2.
The design of therapeutic interventions, or study of mechanisms involved in cerebral damage, requires a model that produces damage similar to the clinical condition and in a reproducible manner. Bilateral carotid vessel occlusion with hypotension (2VOH) is a model that produces reversible forebrain ischemia, emulating the cerebral events that can occur during cardiac arrest and resuscitation. We describe a model modified from Smith et al. (1984) 2, as first presented in its current form in Sanderson, et al. (2008) 3, which produces reproducible injury to selectively vulnerable brain regions 3-6. The reliability of this model is dictated by precise control of systemic blood pressure during applied hypotension, the duration of ischemia, close temperature control, a specific anesthesia regimen, and diligent post-operative care. An 8-minute ischemic insult produces cell death of CA1 hippocampal neurons that progresses over the course of 6 to 24 hr of reperfusion, while less vulnerable brain regions are spared. This progressive cell death is easily quantified after 7-14 days of reperfusion, as a near complete loss of CA1 neurons is evident at this time.
In addition to this brain injury model, we present a method for CA1 damage quantification using a simple, yet thorough, methodology. Importantly, quantification can be accomplished using a simple camera-mounted microscope, and a free ImageJ (NIH) software plugin, obviating the need for cost-prohibitive stereology software programs and a motorized microscopic stage for damage assessment.

Bilateral carotid occlusion coupled with systemic hypotension produces global brain ischemia in the rat, resulting in damage to the hippocampus with reproducible severity. Animal subjects are impaired with predictable patterns of brain damage, they recover expediently, and mortality rates are comparatively low.

2-Gefäßverschluss / Hypotonie: Eine Ratte Modell der globalen Hirnischämie

Cardiac arrest followed by resuscitation often results in dramatic brain damage caused by ischemia and subsequent reperfusion of the brain. Global brain ...

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Medizin

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&#183;cm2 on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 &#177;&#160;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.

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

The aim of the present study was to validate the reproducibility of an in vitro BBB model involving a rat syngeneic co-culture of endothelial cells and astrocytes. The endothelial cell monolayer presented high TEER and low LY permeability. Expression of specific TJ proteins, functional responses to inflammation and functionality of transporters and receptors were assessed.

Einrichten einer<em&gt; In-vitro-</em&gt; Modell der Ratte Blut-Hirn-Schranke (BBB): Ein Fokus auf BBB-Undurchlässigkeit und Rezeptor-vermittelten 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 ...

A Triple Primary Cell Culture Model of the Human Blood-Brain Barrier for Studying Ischemic Stroke In Vitro

Ischemic stroke is a major cause of death and disability worldwide with limited therapeutic options. The neuropathology of ischemic stroke is characterized by an interruption in blood supply to the brain leading to cell death and cognitive dysfunction. During and after ischemic stroke, blood-brain barrier (BBB) dysfunction facilitates injury progression and contributes to poor patient recovery. Current BBB models primarily include endothelial monocultures and double co-cultures with either astrocytes or pericytes.
Such models lack the ability to fully imitate a dynamic brain microenvironment, which is essential for cell-to-cell communication. Additionally, commonly used BBB models often contain immortalized human endothelial cells or animal-derived (rodent, porcine, or bovine) cell cultures that pose translational limitations. This paper describes a novel well-insert-based BBB model containing only primary human cells (brain microvascular endothelial cells, astrocytes, and brain vascular pericytes) enabling the investigation of ischemic brain injury in vitro. 
The effects of oxygen-glucose deprivation (OGD) on barrier integrity were assessed by passive permeability, transendothelial electrical resistance (TEER) measurements,and direct visualization of hypoxic cells. The presented protocol offers a distinct advantage inmimicking the intercellular environment of the BBB in vivo, serving as a more realistic in vitro BBB model for developing new therapeutic strategies in the setting of ischemic brain injury.

A Triple Primary Cell Culture Model of the Human Blood-Brain Barrier for Studying Ischemic Stroke In Vitro

Here, we describe the method for establishing a triple cell culture model of the blood-brain barrier based on primary human brain microvascular endothelial cells, astrocytes, and pericytes. This multicellular model is suitable for studies of neurovascular unit dysfunction during ischemic stroke in vitro or for the screening of drug candidates.

Ein dreifaches primäres Zellkulturmodell der menschlichen Blut-Hirn-Schranke zur Untersuchung des ischämischen Schlaganfalls in vitro

Ischemic stroke is a major cause of death and disability worldwide with limited therapeutic options. The neuropathology of ischemic stroke is ...

Biochemie

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). This process is characterized by the transmigration of activated T cells across brain endothelial cells (ECs), the main constituents of the BBB. However, the consequences on brain EC function upon interaction with such T cells are largely unknown. Here we describe an assay that allows for the evaluation of primary mouse brain microvascular EC (MBMEC) function and barrier integrity during the interaction with T cells over time. The assay makes use of impedance cell spectroscopy, a powerful tool for studying EC monolayer integrity and permeability, by measuring changes in transendothelial electrical resistance (TEER) and cell layer capacitance (Ccl). In direct contact with ECs, stimulated but not na&#239;ve T cells are capable of inducing EC monolayer dysfunction, as visualized by a decrease in TEER and an increase in Ccl. The assay records changes in EC monolayer integrity in a continuous and automated fashion. It is sensitive enough to distinguish between different strengths of stimuli and levels of T cell activation and it enables the investigation of the consequences of a targeted modulation of T cell-EC interaction using a wide range of substances such as antibodies, pharmacological reagents and cytokines. The technique can also be used as a quality control for EC integrity in in vitro T-cell transmigration assays. These applications make it a versatile tool for studying BBB properties under physiological and pathophysiological conditions.

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Here, we describe an in vitro murine model of the blood-brain barrier that makes use of impedance cell spectroscopy, with a focus on the consequences on endothelial cell integrity and permeability upon interaction with activated T cells.

Ein<em&gt; In Vitro</em&gt; Modell der Blut-Hirn-Schranke Mit Impedanzspektroskopie: Ein Fokus auf T-Zell-Endothelzellinteraktion

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal ...

Immunologie und Infektion

Quantification of Neurovascular Protection Following Repetitive Hypoxic Preconditioning and Transient Middle Cerebral Artery Occlusion in Mice

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week protocol for repetitive hypoxic preconditioning (RHP) induces long-term protection against central nervous system (CNS) injury in a mouse model of focal ischemic stroke. RHP consists of 9 stochastic exposures to hypoxia that vary in both duration (2 or 4 hr) and intensity (8% and 11% O2). RHP reduces infarct volumes, blood-brain barrier (BBB) disruption, and the post-stroke inflammatory response for weeks following the last exposure to hypoxia, suggesting a long-term induction of an endogenous CNS-protective phenotype. The methodology for the dual quantification of infarct volume and BBB disruption is effective in assessing neurovascular protection in mice with RHP or other putative neuroprotectants. Adult male Swiss Webster mice were preconditioned by RHP or duration-equivalent exposures to 21% O2 (i.e. room air). A 60 min transient middle cerebral artery occlusion (tMCAo) was induced 2 weeks following the last hypoxic exposure. Both the occlusion and reperfusion were confirmed by transcranial laser Doppler flowmetry. Twenty-two hr after reperfusion, Evans Blue (EB) was intravenously administered through a tail vein injection. 2 hr later, animals were sacrificed by isoflurane overdose and brain sections were stained with 2,3,5- triphenyltetrazolium chloride (TTC). Infarcts volumes were then quantified. Next, EB was extracted from the tissue over 48 hr to determine BBB disruption after tMCAo. In summary, RHP is a simple protocol that can be replicated, with minimal cost, to induce long-term endogenous neurovascular protection from stroke injury in mice, with the translational potential for other CNS-based and systemic pro-inflammatory disease states.

This protocol describes repetitive hypoxic preconditioning, or brief exposures to systemic hypoxia that reduce infarct volumes and blood-brain barrier disruption following transient middle cerebral artery occlusion in mice. It also details dual quantification of infarct volume and blood-brain barrier disruption after stroke to assess the efficacy of neurovascular protection.

Die Quantifizierung der neurovaskulären Schutz Nach Repetitive hypoxische Präkonditionierung und Transient mittleren Hirnarterie Occlusion in Mice

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week ...

An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke

Ischemic stroke leads to vasogenic cerebral edema and subsequent primary brain injury, which is mediated through destruction of the blood-brain barrier (BBB). Rats with induced ischemic stroke were established and used as in vivo models to investigate the functional integrity of the BBB. Spectrophotometric detection of Evans blue (EB) in the brain samples with ischemic injury could provide reliable justification for the research and development of novel therapeutic modalities. This method generates reproducible results, and is applicable in any laboratory without a need for special equipment. Here, we present a visualized and technical guideline on the detection of the extravasation of EB following induction of ischemic stroke in rats.

An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke

The overall goal of this procedure is to provide a highly reproducible technique for in vivo assessment of the blood-brain barrier disruption in rat models of ischemic stroke.

Eine In-Vivo -Bewertung der Störung der Blut - Hirn-Schranke in einem Rattenmodell des ischämischen Schlaganfalls

Ischemic stroke leads to vasogenic cerebral edema and subsequent primary brain injury, which is mediated through destruction of the blood-brain barrier ...

Neurowissenschaften

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 &#177; 80 &#937; cm2, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10-6 &#177; 0.13 10-6 cm sec-1 (mean &#177; SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery.

Verbesserte Methode für die Einrichtung einer In-vitro- Blut - Hirn-Schranke Modell auf porcinen Endothelzellen Gehirnzellen basiert

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...

Two-vessel Occlusion Mouse Model of Cerebral Ischemia-reperfusion

In this study, a middle cerebral artery (MCA) occlusion mouse model is employed to study cerebral ischemia-reperfusion. A reproducible and reliable mouse model is useful for investigating the pathophysiology of cerebral ischemia-reperfusion and determining potential therapeutic strategies for patients with stroke. Variations in the anatomy of the circle of Willis of C57BL/6 mice affects their infarct volume after cerebral-ischemia-induced injury. Studies have indicated that distal MCA occlusion (MCAO) can overcome this problem and result in a stable infarct size. In this study, we establish a two-vessel occlusion mouse model of cerebral ischemia-reperfusion through the interruption of the blood flow to the right MCA. We distally ligate the right MCA and right common carotid artery (CCA) and restore blood flow after a certain period of ischemia. This ischemia-reperfusion injury induces an infarct of stable size and a behavioral deficit. Peripheral immune cells infiltrate the ischemic brain within the 24 h infiltration period. Additionally, the neuronal loss in the cortical area is less for a longer reperfusion duration. Therefore, this two-vessel occlusion model is suitable for investigating the immune response and neuronal recovery during the reperfusion period after cerebral ischemia.

A mouse model of cerebral ischemia-reperfusion is established to investigate the pathophysiology of stroke. We distally ligate the right middle cerebral artery and right common carotid artery and restore blood flow after 10 or 40 min of ischemia.

Zwei-Schiff Okklusion Mausmodell der zerebralen Ischämie-reperfusion

In this study, a middle cerebral artery (MCA) occlusion mouse model is employed to study cerebral ischemia-reperfusion. A reproducible and reliable mouse ...

Ein 3D-Blut-Hirn-Schranken-Modell, das aus menschlichen pluripotenten Stammzellen gewonnen wurde

Reconstruction of the Blood-Brain Barrier In Vitro to Model and Therapeutically Target Neurological Disease

The blood-brain barrier (BBB) is a key physiological component of the central nervous system (CNS), maintaining nutrients, clearing waste, and protecting the brain from pathogens. The inherent barrier properties of the BBB pose a challenge for therapeutic drug delivery into the CNS to treat neurological diseases. Impaired BBB function has been related to neurological disease. Cerebral amyloid angiopathy (CAA), the deposition of amyloid in the cerebral vasculature leading to a compromised BBB, is a co-morbidity in most cases of Alzheimer&#39;s disease (AD), suggesting that BBB dysfunction or breakdown may be involved in neurodegeneration. Due to limited access to human BBB tissue, the mechanisms that contribute to proper BBB function and BBB degeneration remain unknown. To address these limitations, we have developed a human pluripotent stem cell-derived BBB (iBBB) by incorporating endothelial cells, pericytes, and astrocytes in a 3D matrix. The iBBB self-assembles to recapitulate the anatomy and cellular interactions present in the BBB. Seeding iBBBs with amyloid captures key aspects of CAA. Additionally, the iBBB offers a flexible platform to modulate genetic and environmental factors implicated in cerebrovascular disease and neurodegeneration, to investigate how genetics and lifestyle affect disease risk. Finally, the iBBB can be used for drug screening and medicinal chemistry studies to optimize therapeutic&#160;delivery to the CNS. In this protocol, we describe the differentiation of the three types of cells (endothelial cells, pericytes, and astrocytes) arising from human pluripotent stem cells, how to assemble the differentiated cells into the iBBB, and how to model CAA in vitro using exogenous amyloid. This model overcomes the challenge of studying live human brain tissue with a system that has both biological fidelity and experimental flexibility, and enables the interrogation of the human BBB and its role in neurodegeneration.

Autor im Rampenlicht: Ein personalisierter Ansatz zur Erforschung der Alzheimer-Krankheit unter Verwendung eines In-vitro-Blut-Hirn-Schrankenmodells 

The blood-brain barrier (BBB) has a crucial role in sustaining a stable and healthy brain environment. BBB dysfunction is associated with many neurological diseases. We have developed a 3D, stem-cell-derived model of the BBB to investigate cerebrovascular pathology, BBB integrity, and how the BBB is altered by genetics and disease.

Rekonstruktion der Blut-Hirn-Schranke in vitro zur Modellierung und therapeutischen Behandlung neurologischer Erkrankungen

The blood-brain barrier (BBB) is a key physiological component of the central nervous system (CNS), maintaining nutrients, clearing waste, and protecting ...

Sauerstoff-Glukose Deprivation und Reoxygenierung als

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Rekonstruktion der Blut-Hirn-Schranke in vitro zur Modellierung und therapeutischen Behandlung neurologischer Erkrankungen

Rekonstruktion der Blut-Hirn-Schranke in vitro zur Modellierung und therapeutischen Behandlung neurologischer Erkrankungen