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>

<ol>
	<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. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+biology+of+ischemia/reperfusion+injury.">Cell biology of ischemia/reperfusion injury.</a> Int Rev Cell Mol Biol. 298, 229-317 (2012).</li><li>Yang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lycium+barbarum+polysaccharides+reduce+intestinal+ischemia/reperfusion+injuries+in+rats.">Lycium barbarum polysaccharides reduce intestinal ischemia/reperfusion injuries in rats.</a> Chem Biol Interact. 204 (3), 166-172 (2013).</li><li>Kaur, C., Ling, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+brain+barrier+in+hypoxic-ischemic+conditions.">Blood brain barrier in hypoxic-ischemic conditions.</a> Curr Neurovasc Res. 5 (1), 71-81 (2008).</li><li>Khatri, R., McKinney, A. M., Swenson, B., Janardhan, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood-brain+barrier,+reperfusion+injury,+and+hemorrhagic+transformation+in+acute+ischemic+stroke.">Blood-brain barrier, reperfusion injury, and hemorrhagic transformation in acute ischemic stroke.</a> Neurology. 79 (13), S52-S57 (2012).</li><li>Hossmann, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+models+for+the+investigation+of+brain+ischemia.">Experimental models for the investigation of brain ischemia.</a> Cardiovasc Res. 39, 106-120 (1998).</li><li>Ogawa, S., Gerlach, H., Esposito, C., Pasagian-Macaulay, A., Brett, J., Stern, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+modulates+the+barrier+and+coagulant+function+of+cultured+bovineendothelium.+Increased+monolayer+permeability+and+induction+of+procoagulant+properties.">Hypoxia modulates the barrier and coagulant function of cultured bovineendothelium. Increased monolayer permeability and induction of procoagulant properties.</a> J Clin Invest. 85 (4), 1090-108 (1990).</li><li>Zulueta, J. J., Sawhney, R., Yu, F. S., Cote, C. C., Hassoun, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+generation+of+reactive+oxygen+species+in+endothelial+cellsexposed+to+anoxia-reoxygenation.">Intracellular generation of reactive oxygen species in endothelial cellsexposed to anoxia-reoxygenation.</a> Am J Physiol. 272 (5 Pt 1), L897-L902 (1997).</li><li>Zhu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Baicalin+reduces+the+permeability+of+the+blood-brain+barrier+during+hypoxia+in+vitro+by+increasing+the+expression+of+tight+junction+proteins+in+brain+microvascular+endothelial+cells.">Baicalin reduces the permeability of the blood-brain barrier during hypoxia in vitro by increasing the expression of tight junction proteins in brain microvascular endothelial cells.</a> J Ethnopharmacol. 141 (2), 714-720 (2012).</li><li>Abramov, A. Y., Scorziello, A., Duchen, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three+distinct+mechanisms+generate+oxygen+free+radicals+in+neurons+and+contribute+to+cell+death+during+anoxia+and+reoxygenation.">Three distinct mechanisms generate oxygen free radicals in neurons and contribute to cell death during anoxia and reoxygenation.</a> J Neurosci. 27 (5), 1129-1138 (2007).</li><li>Gundimeda, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Green+tea+polyphenols+precondition+against+cell+death+induced+by+oxygen-glucose+deprivation+via+stimulation+of+laminin+receptor,+generation+of+reactive+oxygen+species,+and+activation+of+protein+kinase+Cepsilon.">Green tea polyphenols precondition against cell death induced by oxygen-glucose deprivation via stimulation of laminin receptor, generation of reactive oxygen species, and activation of protein kinase Cepsilon.</a> J Biol Chem. 287 (41), 34694-34708 (2012).</li><li>Mehta, S. L., Manhas, N., Raghubir, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+targets+in+cerebral+ischemia+for+developing+novel+therapeutics.">Molecular targets in cerebral ischemia for developing novel therapeutics.</a> Brain Res Rev. 54 (1), 34-66 (2007).</li><li>Alluri, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+Oxygen+Species-Caspase-3+Relationship+in+Mediating+Blood-Brain+Barrier+Endothelial+Cell+Hyperpermeability+Following+Oxygen-Glucose+Deprivation+and+Reoxygenation.">Reactive Oxygen Species-Caspase-3 Relationship in Mediating Blood-Brain Barrier Endothelial Cell Hyperpermeability Following Oxygen-Glucose Deprivation and Reoxygenation.</a> Microcirculation. 21 (2), 187-195 (1111).</li><li>Sun, H., Breslin, J. W., Zhu, J., Yuan, S. Y., Wu, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rho+and+ROCK+signaling+in+VEGF-induced+microvascular+endothelial+hyperpermeability.">Rho and ROCK signaling in VEGF-induced microvascular endothelial hyperpermeability.</a> Microcirculation. 13 (3), 237-247 (2006).</li><li>Doggett, T. M., Breslin, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+of+the+actin+cytoskeleton+in+live+endothelial+cells+expressing+GFP+actin.">Study of the actin cytoskeleton in live endothelial cells expressing GFP actin.</a> J Vis Exp. (57), (2011).</li></ol>

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 border="0" cellpadding="0" cellspacing="0" width="917">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Name of Material/ Equipment</td>
			<td style="width:167px;">Company</td>
			<td style="width:129px;">Catalog Number</td>
			<td style="width:251px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Proox model 110</td>
			<td style="width:167px;">Biospherix</td>
			<td style="width:129px;">Model 110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">DMEM, no glucose</td>
			<td style="width:167px;">Gibco, Life technologies</td>
			<td style="width:129px;">11966-025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Rhodamine Phalloidin</td>
			<td style="width:167px;">Life technologies</td>
			<td style="width:129px;">R415</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">ZO-1 Rabbit Polyclonal Antibody</td>
			<td style="width:167px;">Life technologies</td>
			<td align="right" style="width:129px;">617300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nunc Lab Tek II-CC 8&#160;well sterile, glass&#160;slides&#160;</td>
			<td style="width:167px;">Thermo scientific</td>
			<td align="right" style="width:129px;">177402</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FITC-tagged anti-rabbit secondary antibody&#160;</td>
			<td style="width:167px;">Santa cruz</td>
			<td style="width:129px;">sc-2090</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">DPBS 1X</td>
			<td style="width:167px;">Thermo scientific</td>
			<td style="width:129px;">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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Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

17.8K 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

Normothermic Cardiac Arrest and Cardiopulmonary Resuscitation: A Mouse Model of Ischemia-Reperfusion Injury

Acute Kidney Injury (AKI) is a common, highly lethal, complication of critical illness which has a high mortality1-4 and which is most 
frequently caused by whole-body hypoperfusion.5,6 Successful reproduction of whole-body hypoperfusion in rodent models has been fraught with 
difficulty.7-9,9,10 Models which employ focal ischemia have repeatedly demonstrated results which do not translate to the clinical setting, and larger 
animal models which allow for whole body hypoperfusion lack access to the full toolset of genetic manipulation possible in the mouse.11,12 However, in recent 
years a mouse model of cardiac arrest and cardiopulmonary resuscitation has emerged which can be adapted to model AKI.13 This model reliably reproduces 
physiologic, functional, anatomic, and histologic outcomes seen in clinical AKI, is rapidly repeatable, and offers all of the significant advantages of a murine surgical 
model, including access to genetic manipulative techniques, low cost relative to large animals, and ease of use. Our group has developed extensive experience 
with use of this model to assess a number of organ-specific outcomes in AKI.14,15

A powerful model for perioperative and critical care related acute kidney injury is presented. Using whole body hypoperfusion induced by cardiac arrest it is possible to nearly replicate the histologic and functional changes of clinical AKI.

Normothermen Herzstillstand und Herz-Lungen: Ein Maus-Modell der Ischämie-Reperfusionsschaden

Acute Kidney Injury (AKI) is a common, highly lethal, complication of critical illness which has a high mortality1-4 and which is most 
frequently caused ...

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Medizin

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and finding new sources for implantable cells are the focus of recent research4,5. Investigating the effectiveness of cell therapies is not an easy task and new tools are needed to investigate the mechanisms involved in the treatment process6. We designed an experimental protocol of ischemia/reperfusion in order to allow the observation of cellular connections and even subcellular mechanisms during ischemia/reperfusion injury and after stem cell transplantation and to evaluate the efficacy of cell therapy. H9c2 cardiomyoblast cells were placed onto cell culture plates7,8. Ischemia was simulated with 150 minutes in a glucose free medium with oxygen level below 0.5%. Then, normal media and oxygen levels were reintroduced to simulate reperfusion. After oxygen glucose deprivation, the damaged cells were treated with transplantation of labeled human bone marrow derived mesenchymal stem cells by adding them to the culture. Mesenchymal stem cells are preferred in clinical trials because they are easily accessible with minimal invasive surgery, easily expandable and autologous. After 24 hours of co-cultivation, cells were stained with calcein and ethidium-homodimer to differentiate between live and dead cells. This setup allowed us to investigate the intercellular connections using confocal fluorescent microscopy and to quantify the survival rate of postischemic cells by flow cytometry. Confocal microscopy showed the interactions of the two cell populations such as cell fusion and formation of intercellular nanotubes. Flow cytometry analysis revealed 3 clusters of damaged cells which can be plotted on a graph and analyzed statistically. These populations can be investigated separately and conclusions can be drawn on these data on the effectiveness of the simulated therapeutical approach.

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

We demonstrate how to set up an in vitro ischemia/reperfusion model and how to evaluate the effect of stem cell therapy on postischemic cardiac cells.

Stem Cell Transplantation in einem In-vitro- Simulierte Ischämie / Reperfusion-Modell

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and ...

Ischemia-reperfusion Model of Acute Kidney Injury and Post Injury Fibrosis in Mice

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often unreported mortality rates that may confound analyses. Bilateral renal pedicle clamping is commonly used to induce IR-AKI, but differences between effective clamp pressures and/or renal responses to ischemia between kidneys often lead to more variable results. In addition, shorter clamp times are known to induce more variable tubular injury, and while mice undergoing bilateral injury with longer clamp times develop more consistent tubular injury, they often die within the first 3 days after injury due to severe renal insufficiency. To improve post-injury survival and obtain more consistent and predictable results, we have developed two models of unilateral ischemia-reperfusion injury followed by contralateral nephrectomy. Both surgeries are performed using a dorsal approach, reducing surgical stress resulting from ventral laparotomy, commonly used for mouse IR-AKI surgeries. For induction of moderate injury BALB/c mice undergo unilateral clamping of the renal pedicle for 26 min and also undergo simultaneous contralateral nephrectomy. Using this approach, 50-60% of mice develop moderate AKI 24 hr after injury but 90-100% of mice survive. To induce more severe AKI, BALB/c mice undergo renal pedicle clamping for 30 min followed by contralateral nephrectomy 8 days after injury. This allows functional assessment of renal recovery after injury with 90-100% survival. Early post-injury tubular damage as well as post injury fibrosis are highly consistent using this model.

We describe models of moderate and severe ischemia-reperfusion-induced kidney injury in which the mice undergo unilateral renal pedicle clamping followed by simultaneous or delayed contralateral nephrectomy, respectively. These models consistently give rise to renal dysfunction and post-injury fibrosis, but injury severity and survival are dependent on mouse background, age and surgical equipment.

Ischämie-Reperfusion-Modell des akuten Nierenschädigung und nach der Verletzung Fibrose bei Mäusen

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often ...

Stretch in Brain Microvascular Endothelial Cells (cEND) as an In Vitro Traumatic Brain Injury Model of the Blood Brain Barrier

Due to the high mortality incident brought about by traumatic brain injury (TBI), methods that would enable one to better understand the underlying mechanisms involved in it are useful for treatment. There are both in vivo and in vitro methods available for this purpose. In vivo models can mimic actual head injury as it occurs during TBI. However, in vivo techniques may not be exploited for studies at the cell physiology level. Hence, in vitro methods are more advantageous for this purpose since they provide easier access to the cells and the extracellular environment for manipulation.
Our protocol presents an in vitro model of TBI using stretch injury in brain microvascular endothelial cells. It utilizes pressure applied to the cells cultured in flexible-bottomed wells. The pressure applied may easily be controlled and can produce injury that ranges from low to severe. The murine brain microvascular endothelial cells (cEND) generated in our laboratory is a well-suited model for the blood brain barrier (BBB) thus providing an advantage to other systems that employ a similar technique. In addition, due to the simplicity of the method, experimental set-ups are easily duplicated. Thus, this model can be used in studying the cellular and molecular mechanisms involved in TBI at the BBB.

Stretch in Brain Microvascular Endothelial Cells cEND as an In Vitro Traumatic Brain Injury Model of the Blood Brain Barrier

In vitro traumatic brain injury models are being developed to reproduce in vivo brain deformation. Stretch-induced injury has been employed for astrocytes, neurons, glial cells, aortic, and brain endothelial cells. However, our system uses a blood brain barrier (BBB) model that possesses properties constituting a legitimate model of the BBB to establish an in vitro&#160;TBI model.

Stretchlimousine in Gehirn mikrovaskulären Endothelzellen (CEND) als<em&gt; In Vitro</em&gt; Traumatische Hirnverletzung Modell der Blut-Hirn-Schranke

Due to the high mortality incident brought about by traumatic brain injury (TBI), methods that would enable one to better understand the underlying ...

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 ...

Murine Model of Intestinal Ischemia-reperfusion Injury

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, hypotension, necrotizing enterocolitis, bowel transplantation, trauma and chronic inflammation. Intestinal ischemia-reperfusion (IR) injury is a consequence of acute mesenteric ischemia, caused by inadequate blood flow through the mesenteric vessels, resulting in intestinal damage. Reperfusion following ischemia can further exacerbate damage of the intestine. The mechanisms of IR injury are complex and poorly understood. Therefore, experimental small animal models are critical for understanding the pathophysiology of IR injury and the development of novel therapies.
Here we describe a mouse model of acute intestinal IR injury that provides reproducible injury of the small intestine without mortality. This is achieved by inducing ischemia in the region of the distal ileum by temporally occluding the peripheral and terminal collateral branches of the superior mesenteric artery for 60 min using microvascular clips. Reperfusion for 1 hr, or 2 hr after injury results in reproducible injury of the intestine examined by histological analysis. Proper position of the microvascular clips is critical for the procedure. Therefore the video clip provides a detailed visual step-by-step description of this technique. This model of intestinal IR injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Here we describe the detailed procedure of intestinal ischemia-reperfusion in mice which results in reproducible injury without mortality to encourage the standardization of this technique across the field. This model of intestinal ischemia-reperfusion injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Murine Modell der intestinalen Ischämie-Reperfusionsschaden

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, ...

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) ...

Evaluation of Coronary Flow Reserve After Myocardial Ischemia Reperfusion in Rats

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via recanalization of the coronary artery is the most effective strategy for reducing the size of ischemic myocardium. The coronary microvasculature cannot be visualized and imaged&#160;in vivo, but there are several invasive and noninvasive techniques that can be used to assess parameters which depend directly on coronary microvascular function. The endothelial function after ischemia reperfusion can be assessed also at the level of the coronary circulation via the coronary flow reserve (CFR).&#160;In this study, peak velocity of left anterior descending (LAD) coronary arteries was measured in rats in vivo via Transthoracic Doppler Echocardiography during resting and stress challenge (induced by Dobutamine). A normal heart can increase its coronary blood flow up to four times above the resting values during stress induction. Following ischemia reperfusion, we found a significantly diminished CFR, which can be used as a marker of coronary microvascular dysfunction. CFR has opened a window on the importance of microvascular dysfunction and has been shown to predict cardiovascular risk independent of whether the severe obstructive disease is present.

Coronary flow reserve (CFR), is defined as the ratio of maximal coronary blood flow to the resting coronary blood flow. We present a protocol for evaluating CFR in rats via ultrasound, which offers the opportunity to predict cardiovascular risk factors in the absence of obstructive coronary disease.

Bewertung des koronaren Flussreservats nach Myokard-Ischämie-Reperfusion bei Ratten

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via ...

A Pre-clinical Rat Model for the Study of Ischemia-reperfusion Injury in Reconstructive Microsurgery

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many areas of biomedical research due to its cost-effectiveness and its translation to humans. This protocol describes a method to create a preclinical free skin flap model in rats with ischemia-reperfusion injury. The described 3 cm x 6 cm rat free skin flap model is easily obtained after the placement of several vascular ligatures and the section of the vascular pedicle. Then, 8 h after the ischemic insult and completion of the microsurgical anastomosis, the free skin flap develops the tissue damage. These ischemia-reperfusion injury-related damages can be studied in this model, making it a suitable model for evaluating therapeutic agents to address this pathophysiological process. Furthermore, two main monitoring techniques are described in the protocol for the assessment of this animal model: transit-time ultrasound technology and laser speckle contrast analysis.

Here, we describe a pre-clinical animal model for studying the pathophysiology of ischemia-reperfusion injury in reconstructive microsurgery. This free skin flap model based on the superficial caudal epigastric vessels in the rat may also allow for the evaluation of different therapies and compounds to counteract ischemia-reperfusion injury-related damage.

Ein präklinisches Rattenmodell zur Untersuchung von Ischämie-Reperfusionsverletzungen in der rekonstruktiven Mikrochirurgie

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many ...

Delayed Intramyocardial Delivery of Stem Cells after Ischemia Reperfusion Injury in a Murine Model

There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, cardiac stem cell therapy is studied by delivering SCs concurrently with the induction of myocardial injury. However, this approach presents two significant limitations: the early hostile pro-inflammatory ischemic environment may affect the survival of transplanted SCs, and it does not represent the subacute infarction scenario where SCs will likely be used. Here we describe a two-part series of surgical procedures for the induction of ischemia-reperfusion injury and delivery of mesenchymal stem cells (MSCs). This method of stem cell administration may allow for the longer viability and retention around damaged tissue by circumventing the initial immune response. A model of ischemia reperfusion injury was induced in mice accompanied by the delivery of mesenchymal stem cells (3.0 x 105), stably expressing the reporter gene firefly luciferase under the constitutively expressed CMV promoter, intramyocardially 7 days later. The animals were imaged via ultrasound and bioluminescent imaging for confirmation of injury and injection of cells, respectively. Importantly, there was no added complication rate when performing this two-procedure approach for SC delivery. This method of stem cell administration, collectively with the utilization of state-of-the-art reporter genes, may allow for the in vivo study of viability and retention of transplanted SCs in a situation of chronic ischemia commonly seen clinically, while also circumventing the initial pro-inflammatory response. In summary, we established a protocol for the delayed delivery of stem cells into the myocardium, which can be used as a potential new approach in promoting regeneration of the damaged tissue.

Stems cells are continuously investigated as potential treatments for individuals with myocardial damage, however, their decreased viability and retention within injured tissue can impact their long-term efficacy. In this manuscript we describe an alternative method for stem cell delivery in a murine model of ischemia reperfusion injury.

Verzögerte intramyokardiale Lieferung von Stammzellen nach Ischämie-Reperfusionsverletzung in einem Murine-Modell

There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, ...