Ce travail a été soutenu par OTKA (Fonds hongrois de la Recherche Scientifique) D45933, T049621, TET (Hongrie Fondation Science et Technologie) A4/04, Arg-17/2006 et le NAS, Bolyai, bourses et Öveges TÁMOP 4.2.2-08 / 1 / KMR-2008-0004 et 4.2.1 / B 09/1/KMR-2010-0001. OTKA 83803. Nous tenons à remercier William Gesztes pour fournir la voix-off.

1. Préparation H9c2 cellules cardiomyoblast H9c2 cardiomyoblasts rat ont été obtenus auprès de l&#39;ATCC (Wesel, Allemagne) et élargi en glucose (4,5 g / L) DMEM contenant 10% de sérum fœtal bovin, 4 mM de L-glutamine, 100 U / ml de pénicilline et 100 ug / ml de streptomycine. La ligne de cellules myoblastes H9c2 est dérivé de coeur de rat embryonnaire, il est utilisé comme un modèle in vitro pour les deux muscles squelettiques et cardiaques 8,9. Préparer la plaque 12 puits de cellules H9c2: <ol><li> Enlever un milieu de culture à partir de la boîte de Pétri H9c2 cellules. </li><li> Laver les cellules deux fois avec 5 ml de PBS et de digérer avec 2-3 ml de 0,05% de trypsine / EDTA. </li><li> Placez une boîte de Pétri pendant 5 minutes dans l&#39;étuve à 37 ° C. </li><li> Inhibition de la trypsine avec 10 ml de milieu de culture DMEM. </li><li> Isoler les cellules à 1200 RPM pendant 8 minutes. </li><li> Après élimination du surnageant, remettre les cellules en 1 ml de milieu de culture DMEM et les cellules compter avec l&#39;aide hémocytomètre coloration au bleu trypan.</li><li> Graine 30000 H9c2 cellules dans 2 ml de DMEM par puits dans une plaque de 12 puits. </li><li> Placer la plaque dans l&#39;incubateur à 37 ° C CO 2 pendant 24 heures. </li></ol> 2. L&#39;ischémie simulée / reperfusion Ischémie / reperfusion a été simulé in vitro, en effectuant privation de glucose d&#39;oxygène (AMG) sur H9c2 cultures cellulaires. Cette procédure a été effectuée sur la scène du microscope confocal Zeiss avec le système PeCon incubation cellulaire (Erbach, Allemagne) permettant l&#39;observation des cellules au cours des AMG. <ol><li> Retirer moyennes par aspiration à partir de 12 plaques à puits d&#39;H9c2 cellules. </li><li> Laver les cellules deux fois avec du PBS. </li><li> Ajouter 3 ml de glucose moyennes gratuitement par puits. </li><li> Placer la plaque dans le système d&#39;incubation des cellules. </li><li> Démarrer l&#39;azote gazeux à purger l&#39;oxygène du système d&#39;incubation. </li><li> Attendez que le niveau d&#39;oxygène atteint 0,5% puis démarrer le chronomètre. D&#39;oxygène de 0,5% signifie environ 3 mmHg de tension partielle. Sous réserve de cellules 150 minutes d&#39;ischémie simulée. </li><li> A la fin de l&#39;ischémie simulée, éliminer le milieu glucose libre à partir des cellules. </li><li> Pipeter 2 ml de milieu de culture normales dans les puits. </li><li> Placer la plaque de 12 puits pendant 30 minutes dans les 37 ° C de CO 2 incubateur, ce qui est de la &quot;période de reperfusion». </li></ol> 3. Préparation des cellules souches mésenchymateuses de secours La moelle osseuse d&#39;origine humaine a été prise en flacons T75 et dilué avec de milieu Dulbecco modifié Eagle (DMEM) milieu de culture contenant 10% de sérum de veau foetal (FCS), 100 U / ml de pénicilline et 100 ug / ml de streptomycine, 4 mM de L-glutamine et 1 g / l de glucose. Les fioles ont été incubées à 37 ° C dans une atmosphère totalement humidifiée de 5% de CO 2 pendant 3 jours. Après la période d&#39;incubation de la BMSCs collé à la surface des fioles et les autres composantes de la moelle osseuse ont été éliminés par un lavage avec du PBS. Le BMSCs utilisées étaient entre 1 et 5 passages dans les expériences. L&#39;identité de cellules a été confirmé par la présence de la lignée des marqueurs spécifiques de surface cellulaire avec la cytométrie de flux. Marqueurs de surface spécifiques lignée hématopoïétiques (CD34, CD45) et des marqueurs de surface mésenchymateuses (CD73, CD90, CD105 et CD166) ont été étudiés. Durant l&#39;ischémie simulée, de préparer le sauvetage des cellules souches mésenchymateuses pour la transplantation. <ol><li> Diluer le colorant fluorescent Vybrant DID en 1:200 ratio en milieu de culture DMEM. </li><li> Aspirer moyennes à partir des cellules de sauvetage et ajouter 300 ul DMEM avec Vybrant DID. </li><li> Placez une boîte de Petri pendant 30 minutes dans les 37 ° C incubateur à CO 2. </li><li> Retirer milieu de culture de boîte de Pétri de cellules souches mésenchymateuses. </li><li> Laver les cellules deux fois avec 5 ml de PBS et de digérer avec 2-3 ml de 0,05% de trypsine / EDTA. </li><li> Placez une boîte de Pétri pendant 5 minutes dans l&#39;étuve à 37 ° C. </li><li> Inhibition de la trypsine avec 10 ml de milieu de culture DMEM. </li><li> Isoler les cellules à 1200 RPM pendant 8 minutes. </li><li> Après avoir retiré la REMPrnatant, remettre les cellules en 1 ml de milieu de culture DMEM et les cellules compter avec l&#39;aide hémocytomètre coloration au bleu trypan. </li><li> Après 30 minutes à la fin de la reperfusion ajouter 20 000 cellules souches mésenchymateuses par puits à l&#39;H9c2 postischémique myoblastes cardiaques. </li><li> Placez la co-culture de cellules dans les 37 ° C incubateur à CO 2 pendant 24 heures. </li></ol> 4. Analyse par microscopie confocale Après la simulation de l&#39;ischémie / reperfusion, les cellules sont incubées dans un milieu de culture normal pour 24 heures. <ol><li> Après 24 heures laver les cellules deux fois avec du PBS. </li><li> Colorer les cellules dans 500 ul vivants / morts solution de colorant pendant 30 minutes (5 nM et 400 nM calcéine éthidium homodimère-2-en PBS) (5x10 4 cellules / ml). </li></ol> L&#39;évaluation des images confocal pour les cellules vivantes et mortes sélectionné par la morphologie et la fluorescence peut être effectuée avec le logiciel ImageJ (National Institutes of Health, Etats-Unis). En cas de co-cultures, les CSM peuvent être distinguées des cellules H9c2 en raison de leur marquage cellulaire Vybrant DID. Le ratio de cellules mortes peuvent être évalués dans 4 champs de vue indépendant (objectif 10x) pour chaque culture d&#39;une manière aveugle, 10. H9c2 cardiomyoblasts cultivés sur des lamelles en verre 42 mm peuvent également être étudiés par microscopie vidéo laps de temps pendant et après l&#39;ischémie / reperfusion en utilisant le système d&#39;incubation des cellules du microscope confocal. De cellule à cellule des interactions telles que la fusion cellulaire et la formation des nanotubes intercellulaires peuvent être étudiés au cours du temps. 5. Analyse avec la cytométrie en flux Après la simulation de l&#39;ischémie / reperfusion, les cellules sont incubées dans un milieu de culture normal pour 24 heures. <ol><li> Après 24 heures, la récolte des milieux de culture. Il est très important de recueillir le milieu de culture aussi parce que certaines cellules mortes pourraient avoir détaché de la surface boîte de culture au cours des 24 heures d&#39;incubationtion. </li><li> Laver les cellules deux fois avec du PBS et digérer avec 0,05% de trypsine-EDTA. </li><li> Inhibition de la trypsine avec milieu de culture DMEM. </li><li> Isoler les cellules et le milieu de culture récoltée à 1200 RPM (environ 150-200g) pendant 8 minutes. </li><li> Jeter le surnageant. </li><li> Suspendre les cellules dans 500 ul vivants / morts solution de colorant pendant 30 minutes (5 nM et 400 nM calcéine éthidium homodimère-2-en PBS) (5x10 4 cellules / ml). </li><li> Transfert aux cellules de flux de tubes de cytométrie. </li><li> Démarrez le logiciel Pro CellQuest. </li><li> Pour configurer le cytomètre en flux pour la mesure, de contrôle de préparer des échantillons de cellules vivantes et mortes. <ol class="lalpha"><li> Plaque H9c2 cellules à une densité de 3x10 4 en plaques de 12 puits comme décrit au point 1. </li><li> Préparer contrôle direct cellule avec trypsinisation unique comme décrit au point 1. </li><li> Préparer les contrôles de cellules mortes par un traitement avec 10 uM H 2 O 2 pendant 1 heure. </li><li> Après trypsinisation, resuspend vivre et de contrôle de cellules mortes dans 500 ul solution de colorant vivants / morts (5 nM calcéine-AM et 400 nM éthidium homodimère-2-en PBS). </li></ol></li><li> Les réglages de l&#39;instrument doit être réglé afin que les cellules vivantes sont calcéine positive et éthidium homodimère négative, tandis que les cellules mortes sont calcéine négatifs et positifs éthidium homodimère. </li><li> Le pourcentage de ces populations peut être exprimé comme barres sur un graphique et l&#39;analyse statistique peuvent être effectuées sur ces valeurs. </li><li> Mesurer les différents groupes expérimentaux. </li></ol> 6. Les résultats représentatifs: Nos résultats ont montré que les cellules souches mésenchymateuses ajoutée pourrait améliorer la survie des cellules cardiaques post-ischémique. Images de microscopie confocale a révélé direct de cellule à cellule des interactions, comme la connexion à membrane partielle ou nanotubes intercellulaires, qui jouent probablement un rôle important dans la protection observée 10. Ces nanotubes s&#39;étendent sur des distances de plusieurs cellules diameters, et leurs diamètres sont compris entre 200 et 500 nm (flèches blanches). <img src="/files/ftp_upload/3575/3575fig1.jpg" alt="Figure 1" /> Figure 1. Formation de nanotubes entre les cellules. Formation de nanotubes (flèche blanche) a été observée entre cardiomyoblasts DiO Vybrant étiquetés et Vybrant DiD étiquetés cellules souches mésenchymateuses. L&#39;analyse par cytométrie en flux a montré les populations de survivre et de cardiomyoblasts nécrotiques et les cellules souches mésenchymateuses. Fait intéressant, nous avons également observé une troisième population, «blessés» cardiomyoblast cellule qui contenait calcéine mais contenait aussi l&#39;indicateur des cellules mortes, d&#39;éthidium homodimère (figure 2). <img src="/files/ftp_upload/3575/3575fig2.jpg" alt="Figure 2" /> Figure 2. Représentant dot-plot des populations cellulaires différentes après ischémie simulée. L&#39;évaluation de plusieurs expériences shoMer que l&#39;ajout de cellules souches mésenchymateuses à H9c2 cardiomyoblast postischémique augmenté de manière significative le pourcentage de cellules vivantes par rapport à des myoblastes cardiaques cultivées seules après ischémie (figure 3). <img src="/files/ftp_upload/3575/3575fig3.jpg" alt="Figure 3" /> Figure 3. . Effet du traitement sur ​​les cellules souches cardiomyoblasts postischémique pourcentage de cellules vivantes a été significativement réduite après une ischémie simulée par rapport au contrôle (contrôle vs IR modèle: 90,36 ± 2,60 vs 10,52 ± 0,48, *** p&gt; 0,001), qui a augmenté après l&#39;addition des cellules souches mésenchymateuses (IR modèle vs IR + BMSC: 10,52 ± 0,48 vs 25,21 ± 2,13, *** p&gt; 0,001). Les données représentent la moyenne ± SEM. L&#39;analyse statistique des données a été réalisée en utilisant une analyse de variance avec post comparaison multiple de Tukey hoc test.

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Pas de conflits d&#39;intérêt déclarés.

Le protocole a démontré une approche in vitro à la question beaucoup plus complexe de la thérapie de cellules souches dans l&#39;infarctus du myocarde, avec tous les avantages et les inconvénients d&#39;un tel modèle. Évidemment, il ne peut pas refléter la complexité (immunologiques, par exemple) des événements qui ont lieu pendant et après l&#39;infarctus du myocarde, mais peuvent se concentrer sur les effets directs des cellules ajoutés sur les cellules post-ischémique. Les effets de l&#39;ischémie simu...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Société </td><td> Numéro de catalogue </td><td> Commentaires </td></tr><tr><td> calcéine-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> éthidium homodimère-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> Tableau 1. Réactifs. <table border="1"><tbody><tr><td> Nom de l&#39;équipement </td><td> Société </td><td> Commentaires (optionnel) </td></tr><tr><td> Système de PeCon incubation des cellules pour les microscopes Zeiss </td><td> PeCon GmbH </td><td> <a href="http://www.pecon.biz/">www.pecon.biz/</a> </td></tr></tbody></table> Tableau 2. Équipement.

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

Protocoles greffe de cellules souches trouvent leur chemin dans la pratique clinique 1,2,3. Obtenir de meilleurs résultats, ce qui rend les protocoles plus robustes, et de trouver de nouvelles sources de cellules implantables font l&#39;objet de recherches récentes 4,5. Étude de l&#39;efficacité des thérapies cellulaires n&#39;est pas une tâche facile et de nouveaux outils sont nécessaires pour étudier les mécanismes impliqués dans le processus de traitement 6. Nous avons conçu un protocole expérimental d&#39;ischémie / reperfusion en vue de permettre l&#39;observation des connexions cellulaires et des mécanismes subcellulaires même durant l&#39;ischémie / reperfusion et après la transplantation de cellules souches et d&#39;évaluer l&#39;efficacité de la thérapie cellulaire. H9c2 cellules cardiomyoblast ont été placés sur des plaques de culture cellulaire 7,8. L&#39;ischémie a été simulé avec 150 minutes dans un milieu glucose libre avec le niveau d&#39;oxygène inférieur à 0,5%. Ensuite, les médias normaux et les niveaux d&#39;oxygène ont été réintroduits afin de simuler la reperfusion. Après privation de glucose d&#39;oxygène,les cellules endommagées ont été traités avec la transplantation de moelle osseuse humaine marquée dérivées des cellules souches mésenchymateuses en les ajoutant à la culture. Les cellules souches mésenchymateuses sont préférés dans les essais cliniques, car ils sont facilement accessibles grâce à la chirurgie invasive minimale, facilement extensible et autologues. Après 24 heures de co-culture, les cellules ont été colorées avec calcéine et éthidium homodimère-à différencier entre les cellules vivantes et mortes. Cette configuration nous a permis d&#39;étudier les connexions intercellulaires utilisant microscopie confocale à fluorescence et de quantifier le taux de survie des cellules post-ischémique par cytométrie en flux. La microscopie confocale a montré les interactions entre les deux populations cellulaires telles que la fusion cellulaire et la formation des nanotubes intercellulaires. L&#39;analyse par cytométrie en flux a révélé trois groupes de cellules endommagées qui peuvent être reportés sur un graphique et analysées statistiquement. Ces populations peuvent être étudiés séparément et conclusions peuvent être tirées de ces données sur l&#39;efficacité de la simulationapproche thérapeutique.

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

Nous démontrons comment mettre en place une<em&gt; In vitro</em&gt; Ischémie / reperfusion modèle et la façon d&#39;évaluer l&#39;effet du traitement sur les cellules souches des cellules cardiaques post-ischémique.

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 ...

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

14.0K vues.  Semmelweis University. Nous démontrons comment mettre en place une In vitro Ischémie / reperfusion modèle et la façon d'évaluer l'effet du traitement sur les cellules souches des cellules cardiaques post-ischémique.

Vidéo: 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 modèle murin de 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 ...

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Médecine

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.

Utilisation quantitative PCR en temps réel pour déterminer prise de greffe de donneurs de cellules dans un modèle murin concurrentiel greffe de moelle osseuse

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.

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 ...

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 Transplantation de cellules souches dans la partie expérimentale Modèle contusion de la moelle épinière des blessures

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.

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 ...

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.

Etudier le cancer du pancréas Stem Cell Caractéristiques pour le développement de nouvelles stratégies de traitement

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.

Modèle murin de Intestinal Ischémie-reperfusion

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.

Création et la Transplantation d’une feuille adipeux dérivées de cellules souches (ASC) dans un modèle de cicatrisation diabétique

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.

Modèle de souris de deux vaisseaux Occlusion d’ischémie-reperfusion cérébrale

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 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 ...