Questo lavoro è stato sostenuto da OTKA (Fondo di ricerca scientifica ungherese) D45933, T049621, TET (ungherese Fondazione Scienza e Tecnica) A4/04, Arg-17/2006 e SIN, Bolyai, borse di studio e Öveges TÁMOP 4.2.2-08 / 1 / KMR-2008-0004 e 4.2.1 / B 09/1/KMR-2010-0001. OTKA 83803. Vorremmo ringraziare Gesztes William per la fornitura del voice-over.

1. Preparazione H9c2 cellule cardiomyoblast H9c2 cardiomyoblasts ratto sono stati ottenuti da ATCC (Wesel, Germania) e ampliato nel glucosio (4,5 g / L) DMEM contenente 10% di siero fetale bovino, 4 mM L-glutammina, 100 U / ml di penicillina e 100 mg / ml di streptomicina. La linea cellulare H9c2 mioblasti è derivato da cuore di ratto embrionale, è usato come un modello in vitro sia per muscolo scheletrico e cardiaco 8,9. Preparare 12 pozzetti di H9c2 celle: <ol><li> Togliere terreno di coltura da piastra di Petri di H9c2 cellule. </li><li> Lavare le cellule due volte con 5 ml di PBS e digerire con 2-3 ml 0,05% tripsina / EDTA. </li><li> Mettere le capsule di Petri per 5 minuti nel 37 ° C incubatore. </li><li> Inibire la tripsina con 10 ml di mezzo di coltura DMEM. </li><li> Spin down delle celle a 1200 rpm per 8 minuti. </li><li> Dopo aver rimosso il surnatante, risospendere le cellule in 1 ml di mezzo di coltura DMEM e contare le cellule con emocitometro utilizzando tripan colorazione blu.</li><li> Semi di 30.000 H9c2 cellule in 2 ml di DMEM per bene in un piatto ben 12. </li><li> Collocare la piastra nel 37 ° C CO 2 incubatore per 24 ore. </li></ol> 2. Ischemia simulata / riperfusione Ischemia / riperfusione è stato simulato in vitro effettuando glucosio privazione di ossigeno (OGD) H9c2 su colture cellulari. Questa procedura è stata eseguita sul palco del microscopio confocale Zeiss con il sistema di incubazione delle cellule Pecon (Erbach, Germania) che consente l&#39;osservazione delle cellule durante OGD. <ol><li> Rimuovere medio di aspirazione da 12 pozzetti di H9c2 cellule. </li><li> Lavare le cellule due volte con PBS. </li><li> Aggiungi 3 medio glucosio libero ml per pozzetto. </li><li> Posizionare la piastra nel sistema di incubazione delle cellule. </li><li> Avvio di gas azoto per eliminare l&#39;ossigeno dal sistema di incubazione. </li><li> Attendere che il livello di ossigeno raggiunge il 0,5% poi avviare il timer. 0,5% di ossigeno significa circa 3 mmHg tensione parziale. Cellule soggette a 150 minuti di ischemia simulata. </li><li> Alla fine di ischemia simulata, rimuovere il supporto di glucosio libero dalle cellule. </li><li> Pipettare 2 ml di terreno di coltura normale ai pozzetti. </li><li> Posizionare i 12 pozzetti per 30 minuti nel 37 ° C CO 2 incubatore, questo è il &quot;periodo di riperfusione&quot;. </li></ol> 3. Preparazione salvataggio cellule staminali mesenchimali Midollo osseo di origine umana è stata presa in T75 palloni e diluito con Media Dulbecco Modified Eagle (DMEM) terreno di coltura contenente il 10% di siero fetale bovino (FCS), 100 U / ml di penicillina e 100 mg / ml di streptomicina, 4 mM L-glutamina e 1 g / l di glucosio. I palloni sono state incubate a 37 ° C in un ambiente completamente umidificata al 5% di CO 2 per 3 giorni. Dopo il periodo di incubazione della BMSCs aderito alla superficie dei palloni e gli altri componenti del midollo osseo sono stati eliminati mediante lavaggio con PBS. Il BMSCs utilizzati sono stati tra 1 e 5 passaggi negli esperimenti. Identitità di cellule è stata confermata dalla presenza di marcatori cellulari lineage-specifica superficie con citometria a flusso. Ematopoietiche linage specifici marcatori di superficie (CD34, CD45) e marcatori di superficie mesenchimali (CD73, CD90, CD105 e CD166) sono stati studiati. Durante l&#39;ischemia simulata, preparare salvataggio cellule staminali mesenchimali per il trapianto. <ol><li> Diluire il colorante fluorescente Vybrant DiD in rapporto 1:200 nel terreno di coltura DMEM. </li><li> Aspirare il terreno dalle cellule di soccorso e aggiungere 300 ml con DMEM Vybrant DiD. </li><li> Mettere le capsule di Petri per 30 minuti nel 37 ° C CO 2 incubatore. </li><li> Togliere terreno di coltura da piastra di Petri di cellule staminali mesenchimali. </li><li> Lavare le cellule due volte con 5 ml di PBS e digerire con 2-3 ml 0,05% tripsina / EDTA. </li><li> Mettere le capsule di Petri per 5 minuti nel 37 ° C incubatore. </li><li> Inibire la tripsina con 10 ml di mezzo di coltura DMEM. </li><li> Spin down delle celle a 1200 rpm per 8 minuti. </li><li> Dopo aver rimosso il Supernatant, risospendere le cellule in 1 ml di mezzo di coltura DMEM e contare le cellule con emocitometro utilizzando tripan colorazione blu. </li><li> Dopo 30 minuti alla fine della riperfusione aggiungere 20.000 cellule staminali mesenchimali per bene al postischemica H9c2 mioblasti cardiaci. </li><li> Luogo di co-coltura di cellule in 37 ° C CO 2 incubatore per 24 ore. </li></ol> 4. Analisi con microscopia confocale Dopo la simulazione di ischemia / riperfusione, le cellule vengono incubate in terreno di coltura normale per 24 ore. <ol><li> Dopo 24 ore lavare le cellule due volte con PBS. </li><li> Macchiare le cellule in 500 microlitri dal vivo / morto soluzione colorante per 30 minuti (5 calceina nM e 400 nM di etidio-omodimero-2 in PBS) (5x10 4 cellule / ml). </li></ol> La valutazione delle immagini confocale per le cellule vive e morte selezionati dalla morfologia e dalla fluorescenza può essere effettuata con il software ImageJ (National Institutes of Health, USA). In caso di co-culture, MSC può essere distinto dal H9c2 cellule a causa della loro etichettatura delle cellule Vybrant DiD. Il rapporto tra le cellule morte possono essere valutati in 4 campi indipendenti di vista (obiettivo 10x) per ogni cultura in maniera cieca 10. H9c2 cardiomyoblasts coltivati ​​su coprioggetto mm 42 di vetro possono anche essere studiate con microscopia video di lasso di tempo durante e dopo ischemia / riperfusione mediante il sistema di incubazione delle cellule del microscopio confocale. Cellula-cellula interazioni, come la fusione cellulare e la formazione di nanotubi intercellulare possono essere studiati nel corso del tempo. 5. Analisi con citometria a flusso Dopo la simulazione di ischemia / riperfusione, le cellule vengono incubate in terreno di coltura normale per 24 ore. <ol><li> Dopo 24 ore, raccogliere i terreni di coltura. E &#39;molto importante raccogliere il terreno di coltura anche perché alcune cellule morte potrebbe essere staccato dalla superficie del piatto cultura durante l&#39;incubazione 24 orezione. </li><li> Lavare le cellule due volte con PBS e digerire con il 0,05% tripsina-EDTA. </li><li> Inibire la tripsina con terreno di coltura DMEM. </li><li> Spin basso le celle e il terreno di coltura raccolto a 1200 giri (circa 150-200g) per 8 minuti. </li><li> Scartare il surnatante. </li><li> Sospendere le cellule in 500 microlitri dal vivo / morto soluzione colorante per 30 minuti (5 calceina nM e 400 nM di etidio-omodimero-2 in PBS) (5x10 4 cellule / ml). </li><li> Trasferimento alle cellule di tubi di citometria a flusso. </li><li> Avviare il software Pro CellQuest. </li><li> Per impostare il citometro a flusso per la misura, preparare del campioni di cellule vive e morte. <ol class="lalpha"><li> Piastra H9c2 cellule ad una densità di 3x10 4 in 12-pozzetti, come descritto al punto 1. </li><li> Preparare vivere controlli cella con trypsinisation singolo come descritto al punto 1. </li><li> Preparare i controlli cellule morte da un trattamento con 10 mM H 2 O 2 per 1 ora. </li><li> Dopo trypsinisation, resuspend vivere e controlli cellule morte in 500 microlitri dal vivo / morto soluzione colorante (5 nM calceina-AM e 400 Nm di etidio-omodimero-2 in PBS). </li></ol></li><li> Le impostazioni dello strumento dovrebbe essere regolata in modo le cellule viventi sono calceina positive e etidio omodimero negativo, mentre le cellule morte sono calceina negativi e positivi omodimero etidio. </li><li> La percentuale di queste popolazioni può essere espresso come barre su un grafico e analisi statistiche possono essere eseguite su questi valori. </li><li> Misurare i diversi gruppi sperimentali. </li></ol> 6. Rappresentante dei risultati: I nostri risultati hanno dimostrato che le cellule staminali mesenchimali aggiunto potrebbe migliorare la sopravvivenza di postischemica cellule cardiache. Immagini di microscopia confocale hanno rivelato diretto cellula-cellula interazioni, come la connessione a membrana parziale o nanotubi intercellulare, che probabilmente giocano un ruolo importante nella protezione osservati 10. I nanotubi arco su distanze di cellule diverse diameters, e il loro diametro era tra i 200 ei 500 nm (frecce bianche). <img src="/files/ftp_upload/3575/3575fig1.jpg" alt="Figura 1" /> Figura 1. Formazione di nanotubi tra le cellule. Nanotubi di formazione (freccia bianca) è stata osservata tra Vybrant cardiomyoblasts DIO etichettati e Vybrant DiD cellule staminali mesenchimali etichettati. Analisi di citometria a flusso ha mostrato le popolazioni di sopravvivere e cardiomyoblasts necrotiche e cellule staminali mesenchimali. È interessante notare che abbiamo anche osservato un terzo, &#39;ferito&#39; cardiomyoblast popolazione cellulare che conteneva calceina ma conteneva anche l&#39;indicatore delle cellule morte, etidio omodimero (Figura 2). <img src="/files/ftp_upload/3575/3575fig2.jpg" alt="Figura 2" /> Figura 2. Rappresentante dot-plot della diverse popolazioni cellulari dopo l&#39;ischemia simulata. La valutazione di diversi esperimenti shomer che l&#39;aggiunta di cellule staminali mesenchimali per postischemica cardiomyoblast H9c2 aumentato in modo significativo la percentuale di cellule viventi rispetto ai mioblasti cardiaci colta da sola dopo ischemia (Figura 3). <img src="/files/ftp_upload/3575/3575fig3.jpg" alt="Figura 3" /> Figura 3. . Effetto del trattamento con cellule staminali su cardiomyoblasts postischemica Percentuale di cellule viventi era significativamente ridotta dopo ischemia simulata rispetto al controllo (controllo rispetto al modello IR: 90,36 ± 2,60 vs 10,52 ± 0,48, *** p&gt; 0,001), che è aumentato dopo l&#39;aggiunta di cellule staminali mesenchimali (IR modello vs IR + BMSC: 10,52 ± 0,48 vs 25,21 ± 2,13, *** p&gt; 0,001). I dati rappresentano media ± SEM. L&#39;analisi statistica dei dati è stata effettuata utilizzando una analisi della varianza con il post di comparazione multipla di Tukey hoc test.

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Nessun conflitto di interessi dichiarati.

Il protocollo è dimostrato un approccio in vitro alla questione molto più complessa di terapia con cellule staminali in infarto miocardico, con tutti i vantaggi e gli svantaggi di un tale modello. Ovviamente non può riflettere il complesso (ad esempio, immunologici) eventi che si svolgeranno durante e dopo l&#39;infarto del miocardio, ma può concentrarsi sugli effetti diretti delle cellule aggiunto sulle cellule postischemica. Gli effetti di ischemia simulata su H9c2 cardiomyoblasts molto dipende dal tempo di OGD, s...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti </td></tr><tr><td> calceina-AM </td><td> Molecular Probes </td><td> L3224, C3099 </td><td> <a href="http://www.invitrogen.com">http://www.invitrogen.com</a> </td></tr><tr><td> etidio omodimero-2 </td><td> Molecular Probes </td><td> L3224, E3599 </td><td> <a href="http://www.invitrogen.com">http://www.invitrogen.com</a> </td></tr><tr><td> Vybrant DiD </td><td> Molecular Probes </td><td> V22887 </td><td> <a href="http://www.invitrogen.com">http://www.invitrogen.com</a> </td></tr></tbody></table> Tabella 1. Reagenti. <table border="1"><tbody><tr><td> Nome del materiale </td><td> Azienda </td><td> Commenti (opzionale) </td></tr><tr><td> Pecon sistema a celle di incubazione per microscopi Zeiss </td><td> Pecon GmbH </td><td> <a href="http://www.pecon.biz/">www.pecon.biz/</a> </td></tr></tbody></table> Tabella 2. Apparecchiature.

stem cell transplantation in an in vitro simulated ischemia/reperfusion model

Protocolli di trapianto di cellule staminali stanno trovando la loro strada nella pratica clinica 1,2,3. Ottenere risultati migliori, rendendo i protocolli più robusto, e di trovare nuove fonti di cellule impiantabili sono al centro di recenti ricerche 4,5. Indagare l&#39;efficacia delle terapie cellulari non è un compito facile e nuovi strumenti sono necessari per indagare i meccanismi coinvolti nel processo di trattamento 6. Abbiamo progettato un protocollo sperimentale di ischemia / riperfusione in modo da consentire l&#39;osservazione di connessioni cellulari e dei meccanismi subcellulari anche durante l&#39;ischemia / riperfusione e dopo il trapianto di cellule staminali e per valutare l&#39;efficacia della terapia cellulare. H9c2 cellule cardiomyoblast sono stati collocati su piastre di coltura cellulare 7,8. Ischemia è stato simulato con 150 minuti in media glucosio libero con il livello di ossigeno inferiore allo 0,5%. Poi, i media normale e livelli di ossigeno sono state reintrodotte per simulare riperfusione. Dopo la deprivazione di ossigeno glucosio,le cellule danneggiate sono state trattate con il trapianto di midollo osseo umano etichettato cellule staminali mesenchimali aggiungendoli alla cultura. Cellule staminali mesenchimali sono preferiti negli studi clinici, perché sono facilmente accessibili con chirurgia mini-invasiva, facilmente espandibile e autologhe. Dopo 24 ore di co-coltura, le cellule sono state colorate con calceina e etidio-omodimero di distinguere tra cellule vive e morte. Questa configurazione ci ha permesso di indagare le connessioni intercellulari utilizzando la microscopia confocale a fluorescenza e di quantificare il tasso di sopravvivenza delle cellule postischemica mediante citometria di flusso. Microscopia confocale hanno mostrato le interazioni delle due popolazioni cellulari, come la fusione cellulare e la formazione di nanotubi intercellulari. Analisi di citometria a flusso ha rivelato 3 gruppi di cellule danneggiate, che può essere tracciata su un grafico e analizzati statisticamente. Queste popolazioni possono essere indagati separatamente e si possono trarre conclusioni su questi dati sull&#39;efficacia del simulatoapproccio terapeutico.

Watch this Scientific Journal Video about Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model at JoVE.com

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

Dimostriamo come impostare uno<em&gt; In vitro</em&gt; Ischemia / riperfusione modello e come valutare l&#39;effetto della terapia con cellule staminali su postischemica cellule cardiache.

Trapianto di cellule staminali in un In vitro Simulato ischemia / riperfusione Modello

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

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

14.0K Views.  Semmelweis University. Dimostriamo come impostare uno In vitro Ischemia / riperfusione modello e come valutare l'effetto della terapia con cellule staminali su postischemica cellule cardiache.

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

A Mouse Model of in Utero Transplantation

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus. For example, in utero transplantation (IUT) of hematopoietic stem cells has been used to successfully treat patients with severe combined immunodeficiency.1,2 In several other conditions, however, IUT has been attempted without success.3 Given these mixed results, the availability of an efficient non-human model to study the biological sequelae of stem cell transplantation and gene therapy is critical to advance this field. We and others have used the mouse model of IUT to study factors affecting successful engraftment of in utero transplanted hematopoietic stem cells in both wild-type mice4-7 and those with genetic diseases.8,9 The fetal environment also offers considerable advantages for the success of in utero gene therapy. For example, the delivery of adenoviral10, adeno-associated viral10, retroviral11, and lentiviral vectors12,13 into the fetus has resulted in the transduction of multiple organs distant from the site of injection with long-term gene expression. in utero gene therapy may therefore be considered as a possible treatment strategy for single gene disorders such as muscular dystrophy or cystic fibrosis. Another potential advantage of IUT is the ability to induce immune tolerance to a specific antigen. As seen in mice with hemophilia, the introduction of Factor IX early in development results in tolerance to this protein.14 
In addition to its use in investigating potential human therapies, the mouse model of IUT can be a powerful tool to study basic questions in developmental and stem cell biology. For example, one can deliver various small molecules to induce or inhibit specific gene expression at defined gestational stages and manipulate developmental pathways. The impact of these alterations can be assessed at various timepoints after the initial transplantation. Furthermore, one can transplant pluripotent or lineage specific progenitor cells into the fetal environment to study stem cell differentiation in a non-irradiated and unperturbed host environment.
The mouse model of IUT has already provided numerous insights within the fields of immunology, and developmental and stem cell biology. In this video-based protocol, we describe a step-by-step approach to performing IUT in mouse fetuses and outline the critical steps and potential pitfalls of this technique.

A Mouse Model of in Utero Transplantation

The mouse model of in utero transplantation is a versatile tool that can be used to study the potential clinical applications of stem cell transplantation and gene therapy in the fetus. In this protocol, we present a general approach to performing this technique

Un modello murino di Nell&#39;utero Trapianto

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus.  For example, in ...

Ricerca

Medicina

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

Utilizzando quantitativa real-time PCR per la determinazione cellulare attecchimento dei donatori in un modello murino competitivo trapianto di midollo osseo

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

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.

Ischemia-riperfusione Modello di danno renale acuta e post Infortunio fibrosi nei topi

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 ...

Neural Stem Cell Transplantation in Experimental Contusive Model of Spinal Cord Injury

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can be used both to investigate the biological responses to injury and to test potential therapies. Contusion or compression injury delivered to the surgically exposed spinal cord are the most widely used models of the pathology. In this report the experimental contusion is performed by using the Infinite Horizon (IH) Impactor device, which allows the creation of a reproducible injury animal model through definition of specific injury parameters. Stem cell transplantation is commonly considered a potentially useful strategy for curing this debilitating condition. Numerous studies have evaluated the effects of transplanting a variety of stem cells. Here we demonstrate an adapted method for spinal cord injury followed by tail vein injection of cells in CD1 mice. In short, we provide procedures for: i) cell labeling with a vital tracer, ii) pre-operative care of mice, iii) execution of a contusive spinal cord injury, and iv) intravenous administration of post mortem neural precursors. This contusion model can be utilized to evaluate the efficacy and safety of stem cell transplantation in a regenerative medicine approach.

Spinal cord injury is a traumatic condition that causes severe morbidity and high mortality. In this work we describe in detail a contusion model of spinal cord injury in mice followed by a transplantation of neural stem cells.

Neural Stem Cell Transplantation in Sperimentale Contusivi Model of Spinal Cord Injury

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can ...

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

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic cerebrovascular accidents account for 80% of all strokes. A common cause of IR injury is the rapid inflow of fluids following an acute/chronic occlusion of blood, nutrients, oxygen to the tissue triggering the formation of free radicals.
Ischemic stroke is followed by blood-brain barrier (BBB) dysfunction and vasogenic brain edema. Structurally, tight junctions (TJs) between the endothelial cells play an important role in maintaining the integrity of the blood-brain barrier (BBB). IR injury is an early secondary injury leading to a non-specific, inflammatory response. Oxidative and metabolic stress following inflammation triggers secondary brain damage including BBB permeability and disruption of tight junction (TJ) integrity.
Our protocol presents an in vitro example of oxygen-glucose deprivation and reoxygenation (OGD-R) on rat brain endothelial cell TJ integrity and stress fiber formation. Currently, several experimental in vivo models are used to study the effects of IR injury; however they have several limitations, such as the technical challenges in performing surgeries, gene dependent molecular influences and difficulty in studying mechanistic relationships. However, in vitro models may aid in overcoming many of those limitations. The presented protocol can be used to study the various molecular mechanisms and mechanistic relationships to provide potential therapeutic strategies. However, the results of in vitro studies may differ from standard in vivo studies and should be interpreted with caution.

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 ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Studiare cancro del pancreas staminali caratteristiche cellulari per lo sviluppo di nuove strategie di trattamento

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

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.

Modello murino di ischemia-riperfusione intestinale Injury

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

Creation and Transplantation of an Adipose-derived Stem Cell (ASC) Sheet in a Diabetic Wound-healing Model

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients with impeded blood flow or with large wounds might be prolonged. Cell-based therapies have appeared as a new technique for the treatment of diabetic ulcers, and cell-sheet engineering has improved the efficacy of cell transplantation. A number of reports have suggested that adipose-derived stem cells (ASCs), a type of mesenchymal stromal cell (MSC), exhibit therapeutic potential due to their relative abundance in adipose tissue and their accessibility for collection when compared to MSCs from other tissues. Therefore, ASCs appear to be a good source of stem cells for therapeutic use. In this study, ASC sheets from the epididymal adipose fat of normal Lewis rats were successfully created using temperature-responsive culture dishes and normal culture medium containing ascorbic acid. The ASC sheets were transplanted into Zucker diabetic fatty (ZDF) rats, a rat model of type 2 diabetes and obesity, that exhibit diminished wound healing. A wound was created on the posterior cranial surface, ASC sheets were transplanted into the wound, and a bilayer artificial skin was used to cover the sheets. ZDF rats that received ASC sheets had better wound healing than ZDF rats without the transplantation of ASC sheets. This approach was limited because ASC sheets are sensitive to dry conditions, requiring the maintenance of a moist wound environment. Therefore, artificial skin was used to cover the ASC sheet to prevent drying. The allogenic transplantation of ASC sheets in combination with artificial skin might also be applicable to other intractable ulcers or burns, such as those observed with peripheral arterial disease and collagen disease, and might be administered to patients who are undernourished or are using steroids. Thus, this treatment might be the first step towards improving the therapeutic options for diabetic wound healing.

Creation and Transplantation of an Adipose-derived Stem Cell ASC Sheet in a Diabetic Wound-healing Model

Adipose-derived stem cells (ASCs) are easily isolated and harvested from the fat of normal rats. ASC sheets can be created using cell-sheet engineering and can be transplanted into Zucker diabetic fatty rats exhibiting full-thickness skin defects with exposed bone and then covered with a bilayer of artificial skin.

Creazione e il trapianto di un foglio di cellule staminali di derivazione adiposa (ASC) in un modello diabetico di guarigione

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients ...

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 ...

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 modello di ratto preclinico per lo studio delle lesioni ischemia-reperfusione in microchirurgia ricostruttiva

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