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
Duration: 8 min 56 s
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

Riconosciamo Scott and White Hospital Assegni di Ricerca Programma per il sostegno finanziario e Texas A &amp; M Health Science Center College of Medicine Laboratorio Integrato Imaging per l&#39;utilizzo del microscopio laser confocale. Riconosciamo Mr. Glen Cryer per un aiuto con la modifica del manoscritto.

1. Semina delle cellule endoteliali <ol><li> Ottenere colture primarie di RBMEC di da adulti Sprague Dawley (o ottenerle in commercio). </li><li> Coltivare RBMECs a 100 centimetri fibronectina (50 mg / ml) capsule di Petri rivestita utilizzando il mezzo di crescita delle cellule endoteliali del cervello di ratto. Cambia il mezzo ogni due giorni, fino al raggiungimento di confluenza. </li><li> Giunti 80-90% di confluenza, lavare delicatamente le cellule in 5 ml di tampone fosfato salino (PBS) agitando. Le cellule sono poi staccate esponendoli a 1 ml di soluzione calda 0,25% trypsin- acido etilendiamminotetraacetico (EDTA), equilibrati a 37 ° C. </li><li> Incubare le cellule a 37 ° C per 2-5 minuti fino a quando le cellule vengono staccate e dispersi. NOTA: Toccare il piatto cultura per staccare le cellule. Vista cellule al microscopio per confermare completo distacco di cellule dalla superficie del piatto. </li><li> Aggiungere 5 ml completo supporto alla scatola Petri per neutralizzare tripsina.Pipettare il terreno contenente le cellule staccate e raccogliere in una provetta da centrifuga da 15 ml. . </li><li> Centrifugare il supporto contenente cellule endoteliali a 220 g per 5 min. </li><li> Aspirare il surnatante e conservare il pellet contenente cells.Suspend il pellet in 3-5 ml fresco cervello di ratto mezzo di crescita delle cellule endoteliali delicatamente mescolando su e giù con celle pipetta sarà quindi essere conteggiati con contatore di cellule automatico o un emocitometro. </li><li> Trasferire la sospensione cellulare in fibronectina (50 ug / ml) prerivestita 8 sistema di scorrimento camera ben sterile, 0.7 cm 2 / pozzetto con una densità di semina compreso tra 10.000-15.000 cellule per pozzetto. Far crescere le cellule a 37 ° C fino a confluenza NOTA: per l&#39;esecuzione di macchie occidentali o altri esperimenti, le cellule possono essere coltivate in 10 centimetri piatti di coltura di cellule o piatti speciali, come richiesto dal esperimento. </li></ol> 2. ossigeno e glucosio privazione-riossigenazione in vitroModello NOTA: Il seguente protocollo è stato adattato da Zulueta et al. 1997; Zhu H et al. 2012 8,9. <ol><li> Utilizza il sistema di coltura cellulare all&#39;ipossia per studiare gli effetti di di OGD-R RBMECs in (vedi Figura 1). Setup e calibrare il sistema di coltura cellulare ipossia prima di iniziare l&#39;esperimento, secondo le istruzioni del produttore. </li><li> Rimuovere i vetrini camera confluent (passo 1,8) dalla C incubatore 37 °. Sostituire il terreno completo nella diapositiva da camera con deossigenato, senza glucosio, Modified mezzo di Eagle Dulbecco (DMEM) e posto in camera di ipossia con il 95%, N 2 e 5% di CO 2 per 2 ore a 37 ° C, per rappresentare condizioni OGD. </li><li> Spostare le cellule indietro per l&#39;incubatore con 95% O 2, 5% CO 2 a 37 ° C e provvisto di fresco cervello di ratto cellule endoteliali terreno completo e incubare per un altro 1 ora a 37 ° C. NOTA: Questo passo rappresenta una situazione riossigenazione. </li><li> Utilizzare le diapositive da camera per la localizzazione e l&#39;etichettatura immunofluorescenza rodamina phalloidin (vedi sezione 3). </li></ol> 3. immunofluorescenza Localizzazione di ZO-1 e f Etichettatura actina Utilizzando Rhodamine falloidina <ol><li> Esporre le diapositive da camera contenenti monostrati RBMEC a 100 ml di Opti-MEM / siero ridotta media / bene per 1 ora. Lavare le diapositive camera 3 volte con 100 pl di tampone fosfato salino (PBS, pH 7.0-7.2). </li><li> Fissare le cellule con 100 ml di 4% paraformaldeide in PBS (pH 7.0-7.2) per 15 minuti e lavare i vetrini camera per 3 volte in PBS (pH 7.0-7.2). </li><li> Permeabilize le cellule utilizzando 100 ml di 0,5% Triton X-100 in PBS, pH (7.0-7.2) per altri 15 min. Blocco con 100 ml di 2% di albumina sierica bovina (BSA) in PBS per un&#39;ora. Dopo questo passaggio sia cellule macchia / etichetta sia per ZO-1 o f- actina. <ol><li> Per immunofluorescenza staining Incubare le cellule con un anticorpo anti-coniglio primaria contro ZO-1 a 1: 150, preparata in 2% BSA-PBS per O / N a 4 ° C. Lavare le cellule per 3 volte in PBS, (pH 7.0-7.2). Incubare con 100 ml di fluoresceina isotiocianato (FITC) -tagged anticorpo secondario anti-coniglio per 1 ora a temperatura ambiente. </li><li> Per l&#39;etichettatura Rodamina falloidina, dopo il blocco, esporre le cellule a 100 ml di rhodamine falloidina in diluizione 1:50, preparato nel 2% BSA-PBS, per 20 min. </li><li> Lavare le cellule da immunofluorescenza e l&#39;etichettatura rodamina falloidina in PBS, (pH 7.0-7.2). Montare le camere di utilizzo dei supporti di montaggio contenenti reagenti anti-dissolvenza con DAPI. </li></ol></li><li> Visualizzare le cellule utilizzando una lente immersione in acqua 60 X e le cellule sono scansionati in un unico piano ottico al microscopio confocale. </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. 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 come un modello in vitro per danno da ischemia-riperfusione è stato ben definito per studiare i neuroni 10,11. Ci sono anche studi che dimostrano l&#39;effetto di OGD sulle cellule endoteliali cerebrali e alterazioni nella permeabilità e TJ integrità 9. Tuttavia, il nostro studio mostra l&#39;effetto di OGD nonché reoxygenation, che è una rappresentazione più stretta di riperfusione ischemica in condizioni in vivo che si verificano ictus ischemico seguente. Condizioni di ipossia ischemica sono noti per indurre l&#39;infiammazione nel sistema nervoso centrale, che porta a BBB perturbazione aumentando permeabilità paracellular e vasogenico 4. La riperfusione è molto complesso e dinamico processo, in cui vi è un&#39;eccessiva produzione di specie reattive dell&#39;ossigeno, l&#39;esaurimento di ATP, aumento di potassio extracellulare, il rilascio di neurotrasmettitori eccitatori, endoteliale e gonfiore neuronale, l&#39;attivazione delle cellule immunitarie. Questo, a sua volta, causes attivazione di varie vie infiammatorie, portando alla produzione di citochine, l&#39;induzione di nucleasi e proteasi, portando a edema, che sono tutti dimostrato di attivare diverse caspasi, che porta alla rottura dell&#39;integrità BBB 5,12. Le cellule endoteliali sono particolarmente soggetti a danno ischemico, per la presenza di un gran numero di mitocondri. Le cellule endoteliali adiacenti sono collegate tra loro da giunzioni strette mantenuti da proteine ​​di giunzione stretti. ZO-1 è indicata a svolgere un ruolo importante nel mantenimento dell&#39;integrità TJ dalla sua interazione con le altre proteine ​​di giunzione stretto e &#160; il gruppo actina citoscheletro. Inoltre, la regolazione precisa della actina citoscheletro è essenziale per molti processi di sviluppo e fisiologici tra cui adesioni cellula-cellula. Nelle cellule endoteliali, fibra formazione di stress actina è risultato essere associati a disfunzione barriera e iperpermeabilità 13,14. Rhodamine phalloidin tecnica di marcatura utilizzato in questo studio per osservare la formazione di fibre di stress f-actina è uno studio punto finale. Tuttavia, imaging dinamico e vivo della fibra formazione stress può anche essere eseguita come dimostrato da Doggett e Breslin 15. L&#39;attuale studio sottolinea majorly il contributo di ZO-1 verso l&#39;integrità bivio stretto BBB. Tuttavia, altre molecole di derivazione strette come claudina-5, occludina, molecole di adesione giunzionali (JAM), ecc possono anche essere utilizzati come marcatori per studiare l&#39;integrità di giunzione a tenuta BBB seguente OGD-R. In questo studio, abbiamo impiegato localizzazione immunofluorescenza ed etichettatura f-actina come tecnica qualitativa per determinare l&#39;integrità tight junction. Tuttavia, possiamo anche studiare le altre caratteristiche importanti del BBB, come misurazione quantitativa della permeabilità utilizzando marcatori fluorescenti e resistenza elettrica transendoteliale (TEER). La tecnica OGD-R presentato in questo studio utilizzando laSistema Biospherix può essere utilizzato solo per gli studi ipossia / anossia ad una concentrazione fissa di ossigeno; tuttavia, non possiamo utilizzare questo sistema per studiare l&#39;effetto della variazione concentrazioni di ossigeno sulle cellule endoteliali. Per studiare gli effetti di diverse concentrazioni di ossigeno sulle cellule endoteliali dobbiamo utilizzare altri modelli disponibili adatti allo scopo. Questa tecnica può essere utilizzata per studiare le relazioni meccanicistici tra vari eventi cellulari e molecolari che regolano BBB iperpermeabilità e integrità TJ. La comprensione si forniscono informazioni per lo sviluppo di varie strategie molecolari per attenuare disagi BBB e microvascolare permeabilità.

Ischemia-riperfusione (IR) ferita si trova ad essere la causa frequente di varie complicazioni debilitanti e decessi associati a ictus, infarto miocardico, trauma, malattia vascolare periferica e traumi cerebrali 1,2. Lesioni IR in vasi cerebrali è uno dei primi lesione secondaria che porta a infiammazione ed edema 3. Una delle complicazioni gravi che si verifica a causa di stress ossidativo e metabolico seguito infiammazione è la perdita di equilibrio omeostatico che porta alla formazione di radicali liberi, alterazioni della barriera emato-encefalica (BBB) ​​giunzioni strette (TJs) e la permeabilità microvascolare 4,5. Attualmente, in modelli in vivo utilizzati per studiare gli effetti di lesioni IR sul BBB includere mezzo occlusione dell&#39;arteria cerebrale (MCAO), microembolia, e transgenici o knockout animali. Tuttavia, ognuno ha i suoi svantaggi e limitazioni come discusso da Hossmann 6. Modello MCAO viene utilizzato per studiare il effects di stress redox, i cambiamenti nella comunicazione giunzionale della BBB e le interazioni tra il cervello e le cellule del sistema immunitario. Tuttavia, essi presentano vari problemi tecnici quali la necessità di precise procedure microchirurgia e le difficoltà in esso. Microembolia rompe istantaneamente lungo la BBB, mentre l&#39;uso di transgenici o knockout animali per studiare ischemia cerebrale può avere sfide come influenze gene-dipendente molecolari sulla formazione infarto, cambiamenti di anatomia vascolare e vari peso corporeo 6. Quindi, modelli in vitro di ischemia hanno trovato un crescente interesse negli ultimi tempi soprattutto a causa della loro applicabilità in esecuzione di studi meccanicistici per i farmaci. Tuttavia, i risultati degli studi in vitro non possono rappresentare appieno uno studio in vivo e devono essere interpretati con cautela 6. Effetto counteractive di basse concentrazioni di ossigeno su monostrati di cellule endoteliali e la permeabilità microvascolare sono statistudiato da Ogawa 7. Cellule endoteliali Rat microvascolari cerebrali (RBMECs) sono stati utilizzati per sviluppare in vitro BBB. La deprivazione di ossigeno e glucosio e riossigenazione (OGD-R) tecnica presentata in questo protocollo è stato adattato da studi di Zulueta et al e Zhu et al 8,9. Abbiamo esposto le cellule endoteliali cerebrali a OGD-R mettendoli in una camera di ipossia / anossia contenente 0% O 2, 5% di CO 2 e il 95% N 2. Le cellule sono state poi valutate per alterazioni TJ integrità e lo stress formazione fibra con localizzazione immunofluorescenza e l&#39;etichettatura falloidina rodamina rispettivamente. Immunofluorescenza colorazione per zonula occludere-1 (ZO-1) viene eseguita per determinare l&#39;integrità TJ, come ZO-1 è un importante membrana ponteggio legato proteina TJ. Etichettatura Rhodamine falloidina determina l&#39;actina filamentosa (f actina) nel citoscheletro cellulare ed è una chiara indicazione di formazione fibra sforzo actina in cellule endoteliali. &lt;p class = &quot;jove_content&quot;&gt; L&#39;obiettivo di questo metodo è quello di fornire una conoscenza di sviluppo OGD-R come un modello in vitro IR per lo studio BBB cellule endoteliali integrità TJ e la formazione di fibre di stress f-actina. I risultati forniranno informazioni sulla sorte di proteine ​​TJ, ZO-1 e la formazione di fibre di stress dopo OGD-R. La comprensione di queste relazioni sarà l&#39;occasione per determinare i meccanismi molecolari alla base che vengono attivati ​​seguendo OGD-R e sviluppare potenziali strategie terapeutiche per migliorare la perturbazione BBB in seguito al trattamento OGD-R.

<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

Ischemia-riperfusione (IR) infortunio è noto per contribuire in modo significativo la morbilità e la mortalità associata con ictus ischemico. Accidenti cerebrovascolari ischemiche rappresentano l&#39;80% di tutti i colpi. Una causa comune di lesioni IR è il rapido afflusso di liquidi dopo occlusione acuta / cronica di sangue, nutrienti, ossigeno al tessuto innescando la formazione di radicali liberi. Ictus ischemico è seguito da barriera emato-encefalica (BBB) ​​erettile e edema cerebrale vasogenico. Strutturalmente, giunzioni strette (TJS) tra le cellule endoteliali svolgono un ruolo importante nel mantenimento dell&#39;integrità della barriera ematoencefalica (BBB). Lesioni IR è uno dei primi lesione secondaria che conduce a un non specifico, la risposta infiammatoria. Ossidativo e stress metabolico seguente infiammazione innesca un danno cerebrale secondario compresi BBB permeabilità e la rottura di giunzione a tenuta (TJ) integrità. Il nostro protocollo presenta in vitro &lt;/ Em&gt; esempio di deprivazione di ossigeno e glucosio e riossigenazione (OGD-R) su cellule endoteliali cervello di ratto TJ integrità e la formazione di fibre di stress. Attualmente, molti sperimentale in modelli in vivo vengono utilizzati per studiare gli effetti di lesioni IR; ma hanno alcune limitazioni, come le difficoltà tecniche per l&#39;esecuzione di interventi chirurgici, le influenze genetiche e molecolari dipendenti difficoltà di studiare le relazioni meccanicistiche. Tuttavia, i modelli in vitro possono aiutare a superare molti di questi limiti. Il protocollo presentato può essere usato per studiare i vari meccanismi molecolari e relazioni meccanicistici di fornire potenziali strategie terapeutiche. Tuttavia, i risultati degli studi in vitro possono essere diverse dallo standard studi in vivo e devono essere interpretati con cautela.

Le cellule coltivate su fibronectina pretrattata camera di diapositive Nunc II sono stati sottoposti ad OGD-R mettendo in un modello Biospherix ProOx 110 camera. Dopo aver sottoposto celle a OGD-R, sono stati trattati per ZO-1 giunzionale colorazione utilizzando la tecnica di immunofluorescenza come mostrato in figura 2 e l&#39;assemblaggio del citoscheletro indicando fibra formazione di stress F-actina usando label macchia rodamina falloidina come mostrato in figura 3. Le cellule di controllo che sono stati non sottoposto a OGD-R ha mostrato integrità delle giunzioni continuo mentre le cellule endoteliali sottoposte a OGD-R hanno mostrato giunzioni discontinui, che indica la perdita di TJ integrità figura 2. Le cellule di controllo che non sono stati sottoposti a trattamento OGD-R hanno mostrato nessuna o una minima f fibra di stress actina formazione mentre le cellule endoteliali sottoposte a OGD-R hanno dimostrato un aumento della formazione f fibra sforzo actina indicando i cambiamenti nel citoscheletro actina assemblaggio figura 3; Rappresentante<html> Rint con il permesso di riferimento # 13 <img alt="Figura 1" src="/files/ftp_upload/52699/52699fig1.jpg" /> Figura 1. Tipica configurazione del modello di ipossia sistema ProOx 110. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52536/52536fig1large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 2" src="/files/ftp_upload/52699/52699fig2.jpg" /> Figura 2. immunofluorescenza Localizzazione di stretta Junction Protein (TJP) ZO-1 Dimostrare Turbativa delle giunzioni strette seguenti OGD-R in RBMECs, come mostrato da bianchi Arrows. Scala bar = 10 micron. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52536/52536fig2large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questo figura.</a> &quot;&gt; <img alt="Figura 3" src="/files/ftp_upload/52699/52699fig3.jpg" /> Figura 3. Rhodamine falloidina etichettatura per f actina stress fibra Formazione Dimostrare Variazioni citoscheletro Assemblea Dopo OGD-R in RBMECs, come mostrato da bianchi Arrows. Barra di scala = 10 micron. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52536/52536fig3large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a>

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.

L&#39;ossigeno-glucosio Deprivazione e riossigenazione come<em&gt; In Vitro</em&gt; Ischemia-riperfusione Injury modello per lo studio barriera sanguigna del cervello Erettile

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

Universidad San Sebastian

Research

JoVE Journal

Medicine

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

17.9K Views.  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. 

Video: 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-occlusione di vasi / Ipotensione: un modello di ratto di Global Ischemia cerebrale

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

Ricerca

Medicina

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.

Istituzione di un<em&gt; In Vitro</em&gt; Modello di Rat barriera emato-encefalica (BBB): A Focus on BBB impermeabilità e Trasporti recettore-mediata

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.

Un triplo modello di coltura cellulare primaria della barriera emato-encefalica umana per lo studio dell'ictus ischemico in vitro

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

Biochimica

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.

Un<em&gt; In Vitro</em&gt; Modello della barriera emato-encefalica Utilizzando spettroscopia di impedenza: un focus sui T Cell-endoteliale cellulare Interazione

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 ...

Immunologia e infezioni

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.

Quantificazione della Protezione Neurovascular seguito ripetitivo ipossico precondizionamento e transitoria occlusione dell&#39;arteria cerebrale media nei topi

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.

Una valutazione In Vivo di rottura della barriera emato - encefalica in un modello del ratto del colpo ischemico

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

Neuroscienze

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.

Migliorato il metodo per la creazione di un In Vitro barriera emato - encefalica modello basato sulle cellule endoteliali Porcine cervello

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.

Occlusione del vaso due Mouse modello di Ischemia-riperfusione cerebrale

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

Un modello 3D di barriera emato-encefalica derivato da cellule staminali pluripotenti umane

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.

Autore in primo piano: Un approccio personalizzato verso lo studio della malattia di Alzheimer utilizzando un modello di barriera emato-encefalica in vitro 

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.

Ricostruzione della barriera emato-encefalica in vitro per modellare e colpire terapeuticamente la malattia neurologica

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

L'ossigeno-glucosio Deprivazione e riossigenazione come

Riepilogo

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Capitoli in questo video

2-occlusione di vasi / Ipotensione: un modello di ratto di Global Ischemia cerebrale

Istituzione di un

Un triplo modello di coltura cellulare primaria della barriera emato-encefalica umana per lo studio dell'ictus ischemico in vitro

Un

Quantificazione della Protezione Neurovascular seguito ripetitivo ipossico precondizionamento e transitoria occlusione dell'arteria cerebrale media nei topi

Una valutazione In Vivo di rottura della barriera emato - encefalica in un modello del ratto del colpo ischemico

Migliorato il metodo per la creazione di un In Vitro barriera emato - encefalica modello basato sulle cellule endoteliali Porcine cervello

Occlusione del vaso due Mouse modello di Ischemia-riperfusione cerebrale

Ricostruzione della barriera emato-encefalica in vitro per modellare e colpire terapeuticamente la malattia neurologica

Ricostruzione della barriera emato-encefalica in vitro per modellare e colpire terapeuticamente la malattia neurologica