Nous reconnaissons Scott et le Programme de subventions de recherche Hôpital Blanche pour un soutien financier et Texas A &amp; M Health Science Center College de médecine de laboratoire intégré Imaging pour l&#39;utilisation du microscope confocal à balayage laser. Nous reconnaissons M. Glen Cryer de l&#39;aide pour l&#39;édition de manuscrits.

1. L&#39;ensemencement des cellules endothéliales <ol><li> Obtenir des cultures primaires de RBMEC de partir adultes rats Sprague-Dawley (ou de les obtenir dans le commerce). </li><li> Cultiver RBMECs à 100 cm de la fibronectine (50 ug / ml) des boîtes de Petri revêtues en utilisant le milieu de croissance des cellules endothéliales de cerveau de rat. Changer le milieu tous les deux jours, jusqu&#39;à confluence est atteint. </li><li> En arrivant à 80-90% de confluence, laver délicatement les cellules dans 5 ml tampon phosphate salin (PBS) par tourbillonnement. Les cellules sont ensuite détachés en les exposant à 1 ml de trypsine acide éthylènediaminetétraacétique (EDTA) solution chaude de 0,25%, équilibrée à 37 ° C. </li><li> Incuber les cellules à 37 ° C pendant 2-5 min jusqu&#39;à ce que les cellules sont décollées et dispersés. REMARQUE: Appuyez sur la boîte de culture pour détacher les cellules. Voir les cellules sous le microscope pour confirmer le détachement complet de cellules de la surface de la boîte. </li><li> Ajouter 5 ml médias complet à la boîte de Pétri afin de neutraliser la trypsine.Pipeter le milieu contenant les cellules détachées et les recueillir dans un tube de centrifugeuse de 15 ml. . </li><li> Centrifuger les médias contenant des cellules endothéliales à 220 g pendant 5 min. </li><li> Aspirer le surnageant et de préserver le culot contenant cells.Suspend le culot dans 3-5 milieu de croissance des cellules endothéliales de cerveau de rat ml fraîche en mélangeant doucement de haut en bas avec des cellules de pipettes seront alors comptés en utilisant un compteur de cellules automatisé ou un hémocytomètre. </li><li> Transférer la suspension cellulaire dans la fibronectine (50 ug / ml) prérevêtue 8 système chambre de coulissement puits stérile, 0,7 cm 2 / puits avec une densité de semis comprise entre 10.000-15.000 cellules par puits. Cultiver les cellules à 37 ° C jusqu&#39;à ce que la confluence est atteinte REMARQUE: Pour effectuer des transferts Western ou d&#39;autres expériences, les cellules peuvent être cultivées dans 10 cm des boîtes de culture cellulaire ou des plats spéciaux tel que requis par l&#39;expérience. </li></ol> 2. oxygène et de glucose Privation-réoxygénation In VitroModèle REMARQUE: Le protocole suivant a été adapté à partir de Zulueta et al. 1997; Zhu H et al. 2012 8,9. <ol><li> Utilisez le système de culture cellulaire à l&#39;hypoxie pour étudier les effets de d&#39;autres ministères-R sur RBMECs dans (voir Figure 1). Le programme d&#39;installation et de calibrer le système de culture cellulaire à l&#39;hypoxie avant de commencer l&#39;expérience, selon les instructions du fabricant. </li><li> Retirez les diapositives de la chambre de confluence (étape 1.8) à partir de la 37 ° C incubateur. Remplacer le milieu complet dans la glissière de la chambre avec désoxygénée, aucun glucose, milieu de Eagle modifié par Dulbecco (DMEM) et placé dans la chambre de l&#39;hypoxie avec 95% de N 2 et 5% de CO 2 pendant 2 h à 37 ° C, pour représenter l&#39;état OGD. </li><li> Déplacez les cellules de retour à l&#39;incubateur avec 95% de O 2, 5% de CO 2 à 37 ° C et muni d&#39;un cerveau de rat cellules endothéliales milieu complet frais et incuber pendant 1 heure à 37 ° C. NOTE: Cette étape représente une situation de réoxygénation. </li><li> Les diapositives de la chambre pour immunofluorescence localisation et l&#39;étiquetage de la rhodamine phalloïdine (voir section 3). </li></ol> 3. immunofluorescence localisation de ZO-1 et f étiquetage actine Utilisation rhodamine phalloïdine <ol><li> Exposer les diapositives de la chambre contenant des monocouches RBMEC 100 pi de milieu Opti-MEM / sérique réduite / puits pendant 1 heure. Laver les lames de la chambre 3 fois dans 100 ul de solution saline tamponnée au phosphate (PBS, pH 7,0 à 7,2). </li><li> Fixer les cellules en utilisant 100 pi de paraformaldehyde à 4% dans du PBS (pH 7,0 à 7,2) pendant 15 min et laver les lames creuses pour 3 fois dans du PBS (pH 7,0 à 7,2). </li><li> Perméabiliser les cellules en utilisant 100 ul de 0,5% de Triton X-100 dans du PBS, pH (7,0 à 7,2) pendant 15 min. Bloquer avec 100 ul de 2% d&#39;albumine de sérum bovin (BSA) dans du PBS pendant une heure. Après cette étape soit des cellules tache / étiquette soit pour ZO-1 ou f- actine. <ol><li> Pour immunofluorescence staining incuber les cellules avec un anticorps primaire anti-lapin contre ZO-1 en 1: 150, établi en 2% de BSA-PBS pour O / N à 4 ° C. Laver les cellules trois fois dans du PBS (pH 7,0-7,2). Incuber avec 100 ul de la fluorescéine isothiocyanate (FITC) -tagged anticorps secondaire anti-lapin pendant 1 heure à température ambiante. </li><li> Pour le marquage rhodamine phalloïdine, après blocage, exposer les cellules à 100 pi de la rhodamine phalloïdine en dilution 1:50, préparé dans 2% de BSA-PBS, pendant 20 min. </li><li> Laver les cellules d&#39;immunofluorescence et la rhodamine étiquetage phalloïdine dans du PBS (pH 7,0-7,2). Montez les chambres en utilisant les médias de montage contenant un réactif anti-fade avec DAPI. </li></ol></li><li> Visualisez les cellules en utilisant un 60 X eau lentille à immersion et les cellules sont analysés dans un plan optique unique sous un microscope confocal. </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 comme un modèle in vitro pour les lésions d&#39;ischémie-reperfusion a été bien établi pour étudier les neurones 10,11. Il existe également des études montrant l&#39;effet de l&#39;OGD sur les cellules et les modifications endothéliales cérébrales de la perméabilité et l&#39;intégrité TJ 9. Cependant, notre étude montre l&#39;effet de l&#39;OGD ainsi que réoxygénation, qui est une représentation plus proche de lésions de reperfusion ischémique dans des ...

Ischémie-reperfusion (IR) des blessures se trouve être la cause fréquente de diverses complications et de décès débilitants associés à l&#39;AVC, infarctus du myocarde, un traumatisme, une maladie vasculaire périphérique et une lésion cérébrale traumatique 1,2. Blessures IR dans les vaisseaux cérébraux est une blessure secondaire au début conduisant à une inflammation et un oedème 3. Une des complications graves qui se produisent à la suite de stress oxydatif et l&#39;inflammation métabolique qui suit est la perte de l&#39;équilibre homéostatique conduisant à la formation de radicaux libres, des altérations de la barrière hémato-encéphalique (BHE) jonctions serrées (TJ) et la perméabilité microvasculaire 4,5. Actuellement, des modèles in vivo utilisées pour étudier les effets des blessures IR sur le BBB inclure une occlusion de l&#39;artère cérébrale moyenne (MCAO), microembolies et transgéniques ou knock-out animaux. Cependant, chacun a ses inconvénients et limitations comme indiqué par Hossmann 6. Modèle MCAO est utilisée pour étudier l&#39;effits de stress redox, changements dans les communications de jonction de la BHE et les interactions entre le cerveau et les cellules immunitaires. Cependant, ils présentent divers défis techniques tels que la nécessité de procédures de microchirurgie précis et les difficultés qui y sont. Microembolies rompt instantanément sur ​​le BBB tandis que l&#39;utilisation de transgéniques ou knock-out animaux pour étudier l&#39;ischémie cérébrale peut avoir des défis tels que les influences génétiques moléculaires dépendant sur ​​la formation de l&#39;infarctus, les changements dans l&#39;anatomie vasculaire et variabilité de poids corporel 6. Ainsi, des modèles in vitro d&#39;ischémie ont trouvé un intérêt croissant ces derniers temps principalement en raison de leur applicabilité dans la réalisation d&#39;études mécanistes pour les médicaments. Cependant, les résultats des études in vitro peuvent ne pas représenter pleinement une étude in vivo et doivent être interprétées avec prudence 6. Réactives effet de faibles concentrations d&#39;oxygène sur des monocouches de cellules endotheliales et la perméabilité microvasculaire étéétudié par Ogawa 7. Les cellules endothéliales microvasculaires du cerveau de rat (RBMECs) ont été utilisés pour développer in vitro du Bureau. La privation et la réoxygénation oxygène-glucose (OGD-R) technique présentée dans ce protocole a été adapté à partir d&#39;études de Zulueta et al et Zhu et al 8,9. Nous avons exposé les cellules endotheliales du cerveau à OGD-R en les plaçant dans une chambre de l&#39;hypoxie / anoxie contenant 0% O 2, 5% CO 2 et 95% de N 2. Les cellules ont ensuite été évalués pour des altérations de l&#39;intégrité et le stress TJ formation de fibres par immunofluorescence localisation et la rhodamine étiquetage phalloidin respectivement. Immunofluorescence pour zonula occludens-1 (ZO-1) est effectuée pour déterminer l&#39;intégrité TJ, comme ZO-1 est une membrane d&#39;échafaudage importante protéine liée à TJ. Étiquetage rhodamine phalloïdine détermine l&#39;actine filamenteuse (f actine) dans le cytosquelette de la cellule et est une indication claire de l&#39;actine la formation de fibres de stress dans les cellules endothéliales. &lt;p class = &quot;jove_content&quot;&gt; Le but de cette méthode est de donner un aperçu de l&#39;élaboration OGD-R comme un modèle in vitro de IR pour étudier BBB intégrité de TJ de cellules endothéliales et la formation de fibres de stress f-actine. Les résultats fourniront des informations sur le sort de la protéine TJ, ZO-1 et la formation de fibres de stress suivant OGD-R. La compréhension de ces relations sera l&#39;occasion de déterminer les mécanismes moléculaires sous-jacents qui sont déclenchés suivent OGD-R et développer des stratégies thérapeutiques potentielles pour améliorer la perturbation BBB après le traitement AMG-R.

<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

Ischémie-reperfusion (IR) des blessures est connu pour contribuer de manière significative à la morbidité et de la mortalité associée à un accident ischémique cérébral. Accidents vasculaires cérébraux ischémiques représentent 80% de tous les accidents vasculaires cérébraux. Une cause fréquente de blessures IR est l&#39;afflux rapide de liquides suite à une occlusion chronique / aiguë de sang, les nutriments, oxygène vers les tissus déclenchant la formation de radicaux libres. L&#39;AVC ischémique est suivie par barrière hémato-encéphalique (BHE) et la dysfonction vasogénique oedème cérébral. Structurellement, les jonctions serrées (TJS) entre les cellules endotheliales jouent un rôle important dans le maintien de l&#39;intégrité de la barrière hémato-encéphalique (BHE). IR blessure est une blessure secondaire au début conduisant à une réponse inflammatoire non spécifique. Le stress oxydatif et l&#39;inflammation métabolique suivante déclenche des dommages au cerveau secondaire comprenant perméabilité de la BHE et la perturbation des jonctions serrées (TJ) intégrité. Notre protocole présente in vitro &lt;/ Em&gt; exemple de la privation d&#39;oxygène-glucose et réoxygénation (AM-R) sur des cellules de cerveau de rat endothéliale TJ intégrité et la formation de fibres de stress. Actuellement, plusieurs expérimentale dans des modèles in vivo sont utilisés pour étudier les effets des blessures IR; mais ils ont plusieurs limitations, telles que les difficultés techniques liées à des opérations chirurgicales, les influences moléculaires dépendantes de gènes et la difficulté à étudier les relations mécanistes. Cependant, les modèles in vitro peuvent aider à surmonter bon nombre de ces limitations. Le protocole présenté peut être utilisé pour étudier les différents mécanismes moléculaires et les relations mécaniques à fournir des stratégies thérapeutiques potentielles. Cependant, les résultats des études in vitro peuvent différer de la norme des études in vivo et doivent être interprétées avec prudence.

Les cellules cultivées sur des lames fibronectine de chambre Nunc II prérevêtus ont été soumis à d&#39;autres ministères-R en plaçant dans un modèle 110 chambre Biospherix ProOx. Après avoir soumis les cellules à OGD-R, ils ont été traités pour ZO-1 jonctionnelle coloration en utilisant une technique d&#39;immunofluorescence comme représenté sur la figure 2 et l&#39;assemblage du cytosquelette indiquant la F-actine la formation de fibres de stress en utilisant la rhodamine phalloïdine ?...

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.

Oxygène-glucose privation et réoxygénation comme un<em&gt; In Vitro</em&gt; Ischémie-reperfusion modèle pour étudier la dysfonction barrière hémato-encéphalique

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 vues.  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. 

Vidéo: 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.

Normothermique arrêt cardiaque et la réanimation cardiorespiratoire: un modèle murin de l&#39;ischémie-reperfusion

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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Médecine

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.

Transplantation de cellules souches dans un In vitro Simulation d&#39;ischémie / reperfusion modèle

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.

Ischémie-reperfusion modèle de lésions rénales aiguës et post blessures fibrose chez la souris

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.

Étirer dans le cerveau cellules endothéliales microvasculaires (CEND) comme<em&gt; In Vitro</em&gt; Traumatisme modèle du cerveau de la barrière hémato-encéphalique

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.

Mise en place d&#39;un<em&gt; In Vitro</em&gt; Modèle de Rat barrière hémato-encéphalique (BHE): un regard sur le Bureau imperméabilité et de transport médié par le récepteur

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.

Modèle murin de Intestinal Ischémie-reperfusion

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.

Procédé amélioré pour la création d’un In Vitro barrière hémato - encéphalique modèle basé sur les cellules endothéliales du cerveau porcin

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.

Évaluation de la réserve de flux coronaire après la réperfusion d'ischémie myocardique chez les rats

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.

Un modèle préclinique de rat pour l'étude des dommages d'ischémie-réperfusion dans la microchirurgie reconstructive

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.

Retard de l’accouchement intramyocardique des cellules souches après une blessure à l’Ischémie Reperfusion dans un modèle murine

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