Diese Arbeit wurde von OTKA (Hungarian Scientific Research Fund) D45933, T049621, TET (Hungarian Science and Technology Foundation) A4/04, Arg-17/2006 und SIN, Bolyai, Öveges Stipendien und TÁMOP unterstützt 4.2.2-08 / 1 / KMR-2008-0004 und 4.2.1 / B 09/1/KMR-2010-0001. OTKA 83803. Wir möchten William Gesztes für die Bereitstellung der Voice-over danken.

1. Vorbereitung H9c2 cardiomyoblast Zellen H9c2 Ratte cardiomyoblasts wurden von ATCC (Wesel, Deutschland) erhalten und ausgebaut in hoher Glukose (4,5 g / L) DMEM mit 10% fötalem Rinderserum, 4 mM L-Glutamin, 100 U / ml Penicillin und 100 ug / ml Streptomycin. Die H9c2 Myoblasten-Zelllinie aus embryonalen Ratte Herzen abgeleitet wird, wird es als ein in vitro Modell für Skelett-und Herzmuskeln 8,9 verwendet. Bereiten Sie 12 gut Platte H9c2 Zellen: <ol><li> Entfernen Kulturmedium aus Petrischale H9c2 Zellen. </li><li> Wash Zellen zweimal mit 5 ml PBS und verdauen mit 2-3 ml 0,05% Trypsin / EDTA. </li><li> Legen Sie Petrischale für 5 Minuten in die 37 ° C Inkubator. </li><li> Inhibit Trypsin mit 10 ml DMEM Kulturmedium. </li><li> Spin down die Zellen bei 1200 RPM für 8 Minuten. </li><li> Nach Entfernen des Überstands, die Zellen in 1 ml DMEM Kulturmedium und Zählen von Zellen mit Zählkammer mit Trypan-Blau-Färbung.</li><li> Seed 30.000 H9c2 Zellen in 2 ml DMEM pro Well in einer 12-Well-Platte. </li><li> Legen Sie die Platte in die 37 ° C CO 2-Inkubator für 24 Stunden. </li></ol> 2. Simulierte Ischämie / Reperfusion Ischämie / Reperfusion simuliert wurde in vitro, indem Sauerstoff Glukose Deprivation (OGD) auf H9c2 Zellkulturen. Dieses Verfahren wurde auf der Bühne des Zeiss konfokalen Mikroskop mit dem Pecon Zellinkubation System (Erbach, Deutschland) erlaubt die Beobachtung der Zellen während der OGD durchgeführt. <ol><li> Entfernen Medium durch Absaugen von 12-Well-Platte des H9c2 Zellen. </li><li> Wash Zellen zweimal mit PBS. </li><li> Add 3 ml Glucose-freiem Medium pro Vertiefung. </li><li> Legen Sie die Platte in die Zelle Inkubation System. </li><li> Starten Stickstoff, um den Sauerstoff aus der Inkubation System zu spülen. </li><li> Warten Sie, bis der Sauerstoffgehalt erreicht 0,5% dann den Timer zu starten. 0,5% Sauerstoff bedeutet ca. 3 mmHg teilweisen Spannung. Subject Zellen bis 150 Minuten von simulierten Ischämie. </li><li> Am Ende der simulierten Ischämie, entfernen Sie die Glukose-freiem Medium von den Zellen. </li><li> 2 ml normalen Kulturmedium in die Vertiefungen. </li><li> Legen Sie die 12-Well-Platte für 30 Minuten in die 37 ° C CO 2-Inkubator, das ist der &quot;Reperfusion Zeit&quot;. </li></ol> 3. Vorbereitung Rettung mesenchymalen Stammzellen Knochenmark menschlichen Ursprungs wurde in T75 Flaschen genommen und verdünnt mit Dulbecco modifiziertem Eagle-Medium (DMEM) Kultur-Medium mit 10% fötalem Kälberserum (FCS), 100 U / ml Penicillin und 100 ug / ml Streptomycin, 4 mM L-Glutamin und 1 g / l Glukose. Die Kolben wurden bei 37 ° C in einem vollständig befeuchteten Atmosphäre von 5% CO 2 für 3 Tage inkubiert. Nach der Inkubationszeit der BMSCs haftete an der Oberfläche des Flakons und die übrigen Komponenten des Knochenmarks wurden durch Waschen mit PBS entfernt. Die verwendeten BMSCs wurden zwischen 1 und 5 Passagen in den Experimenten. Die IdenTity der Zellen wurde durch die Anwesenheit von Lineage-spezifische Zelloberflächenmarker mit Durchflusszytometrie bestätigt. Hämatopoetische Lineage-spezifische Oberflächenmarker (CD34, CD45) und mesenchymalen Oberflächenmarker (CD73, CD90, CD105 und CD166) wurden untersucht. Während simulierten Ischämie, bereiten Rettung mesenchymale Stammzellen für die Transplantation. <ol><li> Verdünnen Sie die fluoreszierenden Farbstoff Vybrant in 1:200-Verhältnis in DMEM Kulturmedium DiD. </li><li> Absaugen Medium aus dem Rettungs-Zellen und 300 ul DMEM mit Vybrant DiD. </li><li> Legen Sie Petrischale für 30 Minuten in die 37 ° C CO 2-Inkubator. </li><li> Entfernen Kulturmedium aus Petrischale von mesenchymalen Stammzellen. </li><li> Wash Zellen zweimal mit 5 ml PBS und verdauen mit 2-3 ml 0,05% Trypsin / EDTA. </li><li> Legen Sie Petrischale für 5 Minuten in die 37 ° C Inkubator. </li><li> Inhibit Trypsin mit 10 ml DMEM Kulturmedium. </li><li> Spin down die Zellen bei 1200 RPM für 8 Minuten. </li><li> Nach dem Entfernen der Supernatant, die Zellen in 1 ml DMEM Kulturmedium und Zählen von Zellen mit Zählkammer mit Trypan-Blau-Färbung. </li><li> Nach 30 Minuten am Ende der Reperfusion add 20.000 mesenchymalen Stammzellen pro Vertiefung der postischämischen H9c2 kardiale Myoblasten. </li><li> Ort Co-Kultur von Zellen in die 37 ° C CO 2-Inkubator für 24 Stunden. </li></ol> 4. Die Analyse mit der konfokalen Mikroskopie Nach der simulierten Ischämie / Reperfusion, werden die Zellen in normalen Nährmedium für 24 Stunden inkubiert. <ol><li> Nach 24 Stunden waschen Sie die Zellen zweimal mit PBS. </li><li> Stain die Zellen in 500 ul Live / Dead-Farbstoff-Lösung für 30 Minuten (5 nM Calcein und 400 Nm Ethidium-Homodimer-2 in PBS) (5x10 4 Zellen / ml). </li></ol> Die Auswertung der konfokalen Bilder für lebende und tote Zellen durch Morphologie und Fluoreszenz ausgewählt werden können mit dem ImageJ Software (National Institutes of Health, USA) durchgeführt werden. Im Falle von co-Kulturen können MSCs aus H9c2 Zellen aufgrund ihrer Vybrant DiD Zelle gekennzeichnet werden. Das Verhältnis von abgestorbenen Zellen kann in 4 unabhängigen Sehfelder (Objektiv 10x) für jede Kultur in einem blinden fashion 10 ausgewertet werden. H9c2 cardiomyoblasts auf 42 mm Deckgläschen kultiviert kann auch mit Zeitraffer-Video-Mikroskopie, während und nach Ischämie / Reperfusion mithilfe der Zell-Inkubation System der konfokalen Mikroskop untersucht werden. Zell-Zell-Interaktionen wie Zell-Fusion und die Bildung von interzellulären Nanoröhrchen können im Laufe der Zeit untersucht werden. 5. Die Analyse mit Durchflusszytometrie Nach der simulierten Ischämie / Reperfusion, werden die Zellen in normalen Nährmedium für 24 Stunden inkubiert. <ol><li> Nach 24 Stunden ernten die Kulturmedien. Es ist sehr wichtig, um das Kulturmedium sammeln auch, weil einige tote Zellen könnten von der Kulturschale Oberfläche während der 24 Stunden Inkubation abgelöst habenTION. </li><li> Wash Zellen zweimal mit PBS und verdauen mit 0,05% Trypsin-EDTA. </li><li> Inhibit Trypsin mit DMEM Kulturmedium. </li><li> Spin down der Zellen und die geernteten Kulturmedium bei 1200 RPM (ca. 150-200g) für 8 Minuten. </li><li> Überstand verwerfen. </li><li> Suspend die Zellen in 500 ul Live / Dead-Farbstoff-Lösung für 30 Minuten (5 nM Calcein und 400 Nm Ethidium-Homodimer-2 in PBS) (5x10 4 Zellen / ml). </li><li> Transfer-Zellen-Zytometrie Rohre fließen. </li><li> Starten Sie den CellQuest Pro Software. </li><li> So richten Sie das Durchflusszytometer für die Messung, bereiten Kontrollproben von lebenden und toten Zellen. <ol class="lalpha"><li> Platte H9c2 Zellen bei einer Dichte von 3x10 4 in 12-Well-Platten, wie in Punkt 1 beschrieben. </li><li> Bereiten Sie leben Zelle steuert mit einzelnen Trypsinierung wie in Punkt 1 beschrieben. </li><li> Bereiten tote Zelle steuert durch die Behandlung mit 10 uM H 2 O 2 für 1 Stunde. </li><li> Nach Trypsinierung, resuspend lebenden und toten Zellen steuert in 500 ul Live / Dead-Farbstofflösung (5 nM Calcein-AM und 400 Nm Ethidium-Homodimer-2 in PBS). </li></ol></li><li> Das Instrument Einstellungen sollten so eingestellt, die lebenden Zellen Calcein positiven und Ethidium Homodimer negativ sind, während die toten Zellen Calcein negativen und Ethidium Homodimer positive sind. </li><li> Der Anteil dieser Bevölkerungsgruppen ausgedrückt als Balken in einem Diagramm und die statistische Analyse auf diesen Werten durchgeführt werden kann. </li><li> Messen Sie die verschiedenen experimentellen Gruppen. </li></ol> 6. Repräsentative Ergebnisse: Unsere Ergebnisse zeigten, dass hinzugefügt mesenchymalen Stammzellen könnte das Überleben der postischämischen kardialen Zellen zu verbessern. Die konfokale Mikroskopie-Bilder zeigten direkte Zell-Zell-Interaktionen, wie teilweise Membran-Verbindung oder interzelluläre-Nanoröhren, die wahrscheinlich eine wichtige Rolle spielen in den beobachteten Schutz 10. Diese Nanoröhren span über Entfernungen von mehreren Zell-diameters, und ihre Durchmesser wurden zwischen 200 und 500 nm (weiße Pfeile). <img src="/files/ftp_upload/3575/3575fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Nanotube-Bildung zwischen den Zellen. Nanotube-Bildung (weißer Pfeil) wurde zwischen Vybrant DiO gekennzeichnet cardiomyoblasts und Vybrant DiD gekennzeichnet mesenchymalen Stammzellen beobachtet. Durchflusszytometrie-Analyse zeigte die Bevölkerung zu überleben und nekrotische cardiomyoblasts und mesenchymalen Stammzellen. Interessanterweise haben wir auch beobachtet, eine dritte, &#39;verletzt&#39; cardiomyoblast Zellpopulation, die Calcein enthalten, sondern enthielt auch die Anzeige der toten Zellen, Ethidium Homodimer (Abbildung 2). <img src="/files/ftp_upload/3575/3575fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Vertreter dot-plot der verschiedenen Zellpopulationen nach simulierten Ischämie. Die Auswertung mehrerer Versuche showed, dass die Zugabe von mesenchymalen Stammzellen zu postischämischen H9c2 cardiomyoblast signifikant erhöht den Anteil der lebenden Zellen im Vergleich zu den kardialen Myoblasten allein nach Ischämie kultiviert (Abbildung 3). <img src="/files/ftp_upload/3575/3575fig3.jpg" alt="Abbildung 3" /> Abbildung 3. ., Die nach Zugabe erhöht: Einfluss der Stammzell-Therapie am postischämischen cardiomyoblasts Prozentsatz der lebenden Zellen war signifikant nach simulierten Ischämie im Vergleich zur Kontrollgruppe (90,36 ± 2,60 vs 10,52 ± 0,48, *** p&gt; 0,001 Kontrolle vs IR-Modell) reduziert von mesenchymalen Stammzellen (IR-Modell vs IR + BMSC: 10,52 ± 0,48 vs 25,21 ± 2,13, *** p&gt; 0,001). Die Daten repräsentieren Mittelwert ± SEM. Die statistische Analyse der Daten erfolgte mit one-way Varianzanalyse mit mehreren Vergleich Tukey post hoc Test durchgeführt.

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Keine Interessenskonflikte erklärt.

Der Beweis-Protokoll ist ein in vitro Ansatz für die viel mehr komplexe Thema der Stammzellentherapie bei Herzinfarkt, mit allen Vor-und Nachteile eines solchen Modells. Natürlich kann es nicht spiegeln die komplexe (zB immunologische) stattfindenden Veranstaltungen während und nach Myokard-Infarkt, kann aber auf die direkten Auswirkungen der zusätzlichen Zellen auf der postischämischen Zellen konzentrieren. Die Auswirkungen der simulierten Ischämie auf H9c2 cardiomyoblasts in hohem Maße abhängig von der Zeit des OGD, auf dem Durchgang Anzahl der verwendeten Zellen und auf den O 2-Konzentration. Man muss diese Parameter exakt einzustellen und finden Sie die optimalen Bedingungen für einen bestimmten Zelltyp, um ein standardisiertes Protokoll. Danach kann das Protokoll in vielerlei Hinsicht verwendet werden (Nieren-, Leber-oder zerebraler Ischämie / Reperfusion 11,12,13,14), um die Auswirkungen verschiedener hinzugefügt Zelltypen wie embryonale Stammzellen, induzierte pluripotente Stammzellen oder adulte Stammzellen zu untersuchen 15, eIRKSAMKEIT von konditioniertem Medium 16 oder verschiedene Vorbehandlungen 17,18 oder alle Parameter in den Mittelpunkt des Interesses.

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare </td></tr><tr><td> Calcein-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> Ethidium Homodimer-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> Tabelle 1. Reagenzien. <table border="1"><tbody><tr><td> Bezeichnung des Geräts </td><td> Firma </td><td> Kommentare (optional) </td></tr><tr><td> Pecon Zellinkubation System für Zeiss-Mikroskope </td><td> Pecon GmbH </td><td> <a href="http://www.pecon.biz/">www.pecon.biz/</a> </td></tr></tbody></table> Tabelle 2. Equipment.

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

Stammzelltransplantation Protokolle finden ihren Weg in die klinische Praxis 1,2,3. Getting bessere Ergebnisse, so dass die Protokolle mehr robust, und die Suche nach neuen Quellen für implantierbare Zellen stehen im Mittelpunkt der jüngsten Forschung 4,5. Untersuchung der Wirksamkeit von Zelltherapien ist keine leichte Aufgabe, und neue Werkzeuge sind nötig, um die Mechanismen in der Behandlung Verfahren 6 zu erforschen. Wir haben ein experimentelles Protokoll der Ischämie / Reperfusion, um die Beobachtung von zellulären Verbindungen und sogar subzelluläre Mechanismen während der Ischämie / Reperfusion und nach Stammzelltransplantation zu ermöglichen und die Wirksamkeit der Zelltherapie zu bewerten. H9c2 cardiomyoblast Zellen wurden auf Zellkulturplatten 7,8 platziert. Ischämie simuliert wurde mit 150 Minuten in einer Glucose-freiem Medium mit Sauerstoff unter 0,5%. Dann wurden normale Medien-und Sauerstoff wieder zu Reperfusion zu simulieren. Nach Sauerstoff Glukose Deprivation,die geschädigten Zellen wurden mit der Transplantation von markiertem menschlichem Knochenmark abgeleiteten mesenchymalen Stammzellen, indem sie in die Kultur behandelt. Mesenchymale Stammzellen sind in klinischen Studien bevorzugt, weil sie leicht zu erreichen mit minimal-invasiven Chirurgie, leicht erweiterbar und autologe sind. Nach 24-stündiger Kokultivierung wurden die Zellen mit Calcein und Ethidium-Homodimer gefärbt, um zwischen lebenden und toten Zellen zu unterscheiden. Dieses Setup erlaubt uns, die interzellulären Verbindungen mit Hilfe der konfokalen Fluoreszenzmikroskopie zu untersuchen und die Überlebensrate von postischämischen Zellen mittels Durchflusszytometrie zu quantifizieren. Die konfokale Mikroskopie zeigte die Wechselwirkungen der beiden Zellpopulationen wie Zellfusion und die Bildung von interzellulären Nanoröhren. Durchflusszytometrie-Analyse ergab 3 Cluster von beschädigten Zellen, die in einem Diagramm dargestellt und können statistisch ausgewertet werden. Diese Populationen können separat untersucht werden und Rückschlüsse auf diese Daten über die Wirksamkeit der simulierten gezogen werdenTherapieansatz.

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

Wir zeigen, wie die Einrichtung eines<em&gt; In-vitro-</em&gt; Ischämie / Reperfusion-Modell und wie man den Effekt der Stammzelltherapie am postischämischen Herzzellen zu bewerten.

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

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

stem-cell-transplantation-an-vitro-simulated-ischemiareperfusion

Nanyang Technological University

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JoVE Journal

Medicine

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

14.0K Aufrufe.  Semmelweis University. Wir zeigen, wie die Einrichtung eines In-vitro- Ischämie / Reperfusion-Modell und wie man den Effekt der Stammzelltherapie am postischämischen Herzzellen zu bewerten.

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

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. The current protocol presents an efficient and reproducible strategy in which functional tissue engineered vessel grafts can be generated using partially induced pluripotent stem cell (PiPSC) from human fibroblasts. We designed a decellularized vessel scaffold bioreactor, which closely mimics the matrix protein structure and blood flow that exists within a native vessel, for seeding of PiPSC-endothelial cells or smooth muscle cells prior to grafting into mice. This approach was demonstrated to be advantageous because immune-deficient mice engrafted with the PiPSC-derived grafts presented with markedly increased survival rate 3 weeks after surgery. This protocol represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell-therapy in the near future.

Here, we present a protocol to generate tissue engineered vessel grafts that are functional for grafting into mice by double seeding partially induced pluripotent stem cell (PiPSC) - derived smooth muscle cells and PiPSC - derived endothelial cells on a decellularized vessel scaffold bioreactor.

Generierung und Pfropfen von Tissue Engineering Gefäße in einem Maus-Modell

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

Forschung

Biotechnik

Intestinal Stem Cell Isolation and Culture in a Porcine Model of Segmental Small Intestinal Ischemia

Intestinal ischemia remains a major cause of morbidity and mortality in human and veterinary patients. Many disease processes result in intestinal ischemia, when the blood supply and therefore oxygen is decreased to the intestine. This leads to intestinal barrier loss and damage to the underlying tissue. Intestinal stem cells reside at the base of the crypts of Lieberk&#252;hn and are responsible for intestinal renewal during homeostasis and following injury. Ex vivo cell culture techniques have allowed for the successful study of epithelial stem cell interactions by establishing culture conditions that support the growth of three-dimensional epithelial organ-like systems (termed "enteroids" and "colonoids" from the small and large intestine, respectively). These enteroids are composed of crypt and villus-like domains and mature to contain all of the cell types found within the epithelium. Historically, murine models have been utilized to study intestinal injury. However, a porcine model offers several advantages including similarity of size as well as gastrointestinal anatomy and physiology to that of humans. By utilizing a porcine model, we establish a protocol in which segmental loops of intestinal ischemia can be created within a single animal, enabling the study of differing time points of ischemic injury and repair in vivo. Additionally, we describe a method to isolate and culture the intestinal stem cells from the ischemic loops of intestine, allowing for the continued study of epithelial repair, modulated by stem cells, ex vivo.

This protocol will enable readers to successfully establish a porcine model of segmental intestinal ischemia and subsequently isolate and culture intestinal stem cells for the study of epithelial repair following injury.

Intestinale Stammzellen isoliert und Kultur in einem porcinen Modell der segmentalen kleinen intestinalen Ischämie

Intestinal ischemia remains a major cause of morbidity and mortality in human and veterinary patients. Many disease processes result in intestinal ...

Medizin

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.

Zwei-Schiff Okklusion Mausmodell der zerebralen Ischämie-reperfusion

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

A Cell Culture Model for Studying the Role of Neuron-Glia Interactions in Ischemia

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue, leading to oxygen and glucose deprivation, and the consequent neuronal loss. Numerous evidence suggests that the interaction between glial and neuronal cells exert beneficial effects after an ischemic event. Therefore, to explore potential protective mechanisms, it is important to develop models that allow studying neuron-glia interactions in an ischemic environment. Herein we present a simple approach to isolate astrocytes and neurons from the rat embryonic cortex, and that by using specific culture media, allows the establishment of neuron- or astrocyte-enriched cultures or neuron-glia cultures with high yield and reproducibility.
To study the crosstalk between astrocytes and neurons, we propose an approach based on a co-culture system in which neurons cultured in coverslips are maintained in contact with a monolayer of astrocytes plated in multiwell plates. The two cultures are maintained apart by small paraffin spheres. This approach allows the independent manipulation and the application of specific treatments to each cell type, which represents an advantage in many studies.
To simulate what occurs during an ischemic stroke, the cultures are subjected to an oxygen and glucose deprivation protocol. This protocol represents a useful tool to study the role of neuron-glia interactions in ischemic stroke.

Here, we present a simple approach using specific culture media that allows the establishment of neuron- and astrocyte-enriched cultures, or neuron-glia cultures from the embryonic cortex, with high yield and reproducibility.

Ein Zellkulturmodell zum Studium der Rolle von Neuron-Glia-Interaktionen in Ischämie

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue, leading to oxygen and glucose deprivation, and the consequent ...

Neurowissenschaften

Pre-clinical Model of Cardiac Donation after Circulatory Death

Cardiac transplantation demand is on the rise; nevertheless, organ availability is limited due to a paucity of suitable donors. Organ donation after circulatory death (DCD) is a solution to address this limited availability, but due to a period of prolonged warm ischemia and the risk of tissue injury, its routine use in cardiac transplantation is seldom seen. In this manuscript we provide a detailed protocol closely mimicking current clinical practices in the context of DCD with continuous monitoring of heart function, allowing for the evaluation of novel cardioprotective strategies and interventions to decrease ischemia-reperfusion injury.
In this model, the DCD protocol is initiated in anesthetized Lewis rats by stopping ventilation to induce circulatory death. When systolic blood pressure drops below 30 mmHg, the warm ischemic time is initiated. After a pre-set warm ischemic period, hearts are flushed with a normothermic cardioplegic solution, procured, and mounted onto a Langendorff ex vivo heart perfusion system. Following 10 min of initial reperfusion and stabilization, cardiac reconditioning is continuously evaluated for 60 min using intraventricular pressure monitoring. A heart injury is assessed by measuring cardiac troponin T and the infarct size is quantified by histological staining. The warm ischemic time can be modulated and tailored to develop the desired amount of structural and functional damage. This simple protocol allows for the evaluation of different cardioprotective conditioning strategies introduced at the moment of cardioplegia, initial reperfusion and/or during ex vivo perfusion. Findings obtained from this protocol can be reproduced in large models, facilitating clinical translation.

This protocol shows a simple and flexible approach for the evaluation of new conditioning agents or strategies to increase the feasibility of cardiac donation after circulatory death.

Präklinisches Modell der Herzspende nach Kreislauftod

Cardiac transplantation demand is on the rise; nevertheless, organ availability is limited due to a paucity of suitable donors. Organ donation after ...

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

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

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

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

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

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

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

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

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

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

Organ Ischemia-Reperfusion Injury by Simulating Hemodynamic Changes in Rat Liver Transplant Model

Orthotopic liver transplantation (OLT) in rats is a tried and proven animal model used for preoperative, intraoperative, and postoperative studies, including ischemia-reperfusion injury (IRI) of extrahepatic organs. This model requires numerous experiments and devices. The duration of anhepatic phase is closely related to the time to develop IRI after transplantation. In this experiment, we used hemodynamic changes to induce extrahepatic organ damage in rats and determined the maximum tolerance time. The time until the most severe organ injury varied for different organs. This method can easily be replicated and can also be used to study IRI of the extrahepatic organs after liver transplantation.

This paper provides a detailed description of how to build an animal model of the anhepatic phase (liver ischemia) in rats to facilitate basic research into ischemia-reperfusion injury after liver transplantation.

OrganIschemia-Reperfusion Verletzung durch Simulation hämodynamischer Veränderungen im Lebertransplantationsmodell der Ratte

Orthotopic liver transplantation (OLT) in rats is a tried and proven animal model used for preoperative, intraoperative, and postoperative studies, ...

A Heterotopic Rat Heart Transplantation Model using Circulatory Death Donor Hearts

The objective of this protocol is to set up a rat heterotopic heart transplantation model with donation after circulatory death (DCD) donor hearts. There are two setups for this protocol: heart donor setup and recipient setup. In the heart donor setup, Sprague Dawley rats are anesthetized, endotracheally intubated, and ventilated. The right carotid artery is cannulated to deliver heparin and the paralytic agent vecuronium-bromide. The DCD process is initiated by terminating the ventilation. After 20 min, the heart is exposed and the aorta distal to the brachiocephalic branch is clamped. At 25 min from terminating the ventilator, ice-cold University of Wisconsin (UW) solution is perfused through the carotid catheter to flush the heart. The heart is procured by dividing the aorta, pulmonary artery, venae cavae, and pulmonary veins and stored in UW solution for implantation. In the recipient setup, the Lewis rat is anesthetized with isoflurane. Slow-release buprenorphine is administered subcutaneously to facilitate a smooth postoperative recovery. Through a midline abdominal incision, the infra-renal aorta and the inferior vena cava are isolated and clamped with an atraumatic vascular clamp. The donor heart aorta and pulmonary artery are sutured to the recipient abdominal aorta and vena cava, respectively, with a running 8-0 Prolene. The vascular clamp is removed to reperfuse the heart. The abdominal wall is closed and the rat is recovered. After a set interval (24 h to 2 weeks), the recipient rat is anesthetized, the transplanted heart is exposed, and a balloon-tip-catheter is inserted into the left ventricle via the apex to record developed pressure and dP/dt using a data acquisition system. The heart tissue is collected for histology, immunology, or molecular analysis. A successful DCD donor rat heart transplantation model will allow further studies on the cardioprotective approaches to improve heart transplantation outcomes from DCD donors.

Here, we describe the preparation and technical details of murine heterotopic heart transplantation utilizing a circulatory death donor heart.

The objective of this protocol is to set up a rat heterotopic heart transplantation model with donation after circulatory death (DCD) donor hearts. There ...

Development of a Cell Co-Culture Model to Mimic Cardiac Ischemia/Reperfusion In Vitro

Ischemic heart disease is the leading cause of death and disability worldwide. Reperfusion causes additional injury beyond ischemia. Endothelial cells (ECs) can protect cardiomyocytes (CMs) from reperfusion injury through cell-cell interactions. Co-cultures can help investigate the role of cell-cell interactions. A mixed co-culture is the simplest approach but is limited as isolated treatments and downstream analyses of single cell types are not feasible. To investigate whether ECs can dose-dependently attenuate CM cell damage and whether this protection can be further optimized by varying the contact distance between the two cell lines, we used Mouse Primary Coronary Artery Endothelial Cells and Adult Mouse Cardiomyocytes to test three types of cell culture inserts which varied in their inter-cell layer distance at 0.5, 1.0, and 2.0 mm, respectively. In CMs-only, cellular injury as assessed by lactate dehydrogenase (LDH) release increased significantly during hypoxia and further upon reoxygenation when the distance was 2.0 mm compared to 0.5 and 1.0 mm. When ECs and CMs were in nearly direct contact (0.5 mm), there was only a mild attenuation of the reoxygenation injury of CMs following hypoxia. This attenuation was significantly increased when the spatial distance was 1.0 mm. With 2.0 mm distance, ECs attenuated CM injury during both hypoxia and hypoxia/reoxygenation, indicating that sufficient culture distancing is necessary for ECs to crosstalk with CMs, so that secreted signal molecules can circulate and fully stimulate protective pathways. Our findings suggest, for the first time, that optimizing the EC/CM co-culture spatial environment is necessary to provide a favorable in vitro model for testing the role of ECs in CM-protection against simulated ischemia/reperfusion injury. The goal of this report is to provide a step-by-step approach for investigators to use this important model to their advantage.

Development of a Cell Co-Culture Model to Mimic Cardiac Ischemia/Reperfusion In Vitro

Spatial distance is a key parameter in assessing hypoxia/reoxygenation injury in a co-culture model of separate endothelial and cardiomyocyte cell layers, suggesting, for the first time, that optimizing the co-culture spatial environment is necessary to provide a favorable in vitro model for testing the role of endothelial cells in cardiomyocyte protection.

Entwicklung eines Zell-Co-Kultur-Modells zur Nachahmung von kardialer Ischämie/Reperfusion in vitro

Ischemic heart disease is the leading cause of death and disability worldwide. Reperfusion causes additional injury beyond ischemia. Endothelial cells ...