Name: apical resection mouse model to study early mammalian heart regeneration
Uploaded: 2016-01-23T15:00:00.000+00:00
Duration: 6 min 8 s
Description: Watch this Scientific Journal Video about Apical Resection Mouse Model to Study Early Mammalian Heart Regeneration at JoVE.com

Die Autoren danken Drs. James Hawkins, Zu-Xi Yu Xuan und Qu aus dem National Heart, Lung, and Blood Institute (NHLBI) für ihre Unterstützung bei der Maus-Operation und Vorbereitung und Färbung von Paraffinschnitten. Die Autoren sind dankbar, dass die NIH Fellows Editorial Board für die redaktionelle Unterstützung.

Alle Maus-Experimente wurden von der Animal Care und Verwenden Programm an der National Institutes of Health (NIH) mit Protokollnummer H0083R3 genehmigt. Das NHLBI IACUC genehmigt das Protokoll ohne Analgetika. 1. Hypothermie Anästhesie in neugeborenen Mäusen <ol><li> Sterilisieren, Schwämme und chirurgische Geräte in einem Autoklaven vor der Operation. Bereiten Sie alle chirurgischen Materialien und schalten auf eine heiße Perle Sterilisator 15-20 Minuten vorher auf 240 ° C bis 270 ° C zu erreichen. </li><li> Übertragen Sie alle C57BL / 6 Welpen (Alters P1) von ihren stillenden Mutter in eine saubere Maus Käfig mit frischer Bettwäsche und Nistmaterial. Sobald die Welpen werden in einen Operationssaal genommen, führen Sie die Apikalchirurgie unverzüglich zu minimieren ihre Zeit damit verbracht von der Mutter getrennt und das Risiko der Mütter Kannibalisierung zu reduzieren. </li><li> Setzen Schwämme auf einem Eis-Bett und legen Sie dann einen Welpen auf dem Schwamm für ~ 3 min zu Unterkühlung Anästhesie zu erreichen. Anästhesie Bestätigen Sie durch Beobachtung apnea und Akinese und Kneifen einen hinteren Fuß. Überprüfen Sie den Status des Neugeborenen häufig, weil zu wenig Zeit nicht den Welpen akinetischen und Apnoen, und eine übermäßige Dauer der Anästhesie kann die Überlebensrate 12 niedriger zu machen. </li></ol> 2. Thorakotomie <ol><li> Bringen Sie den Welpen aus dem Eis Bett auf eine chirurgische Tischbereich und verwenden Sie Klebeband um seine Arme, Beine und Schwanz in Rückenlage zu immobilisieren. </li><li> Desinfizieren Sie die Brust mit Betadin und reinigen Sie sie mit einem 70% igem Alkohol prep Pad sanft. </li><li> Einen Querhautschnitt entlang des hervorZwischenRippenBereich des Brusthöhle mit einem Vannas Frühling Scissor und dann stumpf sezieren die hervorZwischenRippenMuskeln, um Zugang zum Herzen zu erleichtern. HINWEIS: Die Überlebensraten zu verbessern, wenn der Blutverlust wird während des chirurgischen Prozesses minimiert. </li></ol> 3. Chirurgische Amputation des Herzens ventrikuläre Apex <ol><li> Von Hand, vorsichtig anwenden Druck auf den Bauch, um die exteriorisierenApex des Herzens. Blut aufnehmen um den OP-Bereich mit einem sterilen Wattestäbchen für klare Visualisierung. Für scheinoperierten Kontroll Neugeborenen, gehen Sie direkt zu Schritt 4 (Naht und Wiederherstellung von Mäusen). </li><li> Unter einem Vergrößerungslampe und mit Iridektomie Schere vorsichtig auszuführen stück Resektion des linken Ventrikels (LV), bis der LV Kammer ausgesetzt ist. Seien Sie vorsichtig, um die reseziert Teile des LV minimieren. Etwa 15% Resektion ist notwendig, um eine optimale Belichtung der LV Kammer zu erreichen. </li><li> Sicherzustellen, dass das Herz wieder in den Brustraum, sobald der LV Kammer ausgesetzt ist. </li></ol> 4. Naht und Wiederherstellung von Mäusen <ol><li> Naht der Rippen und Muskeln zusammen, um die Brusthöhle zu versiegeln mit sterilen Proline 6-0 Nahtmaterial, und dann sorgfältig schließen Sie die Hautschnitt Site mit Hautkleber. </li><li> Erwärmen Sie die Neugeborenen unter einer Wärmelampe für ca. 3 min für die Wiederherstellung, und klicken Sie dann reinigen das Blut und Leimspuren mit einem 70% igem Alkohol prep Pad before Wiedereinführung seine Wurfgeschwister. Versuchen Sie, den gesamten chirurgischen Eingriffs innerhalb von 10 Minuten abgeschlossen werden, da die Minimierung der Zeitaufwand von der Mutter getrennt verbessert pup Überleben nach Wurzelspitzenresektion. </li><li> Nach jeder Operation, legen die chirurgischen Werkzeuge in Kontakt mit den Wülsten des heißen Wulst Sterilisator für etwa 20 sec für die vollständige Sterilisation. Lassen Sie die chirurgische Instrumente auf RT vor jeder Operation zu kühlen. </li><li> Nach Abschluss der Operationen für alle Welpen in einem Wurf, mischen Sie die Welpen mit Bettwäsche und Kot der Mutter, bevor sie dann wieder in Nest der Mutter. HINWEIS: Im Allgemeinen sind ICR / CD-1-Mäusen besser Pflegemütter als C57BL / 6, aber auch bei stillenden Müttern mit dem gleichen genetischen Hintergrund, variieren Förderung Instinkte. Wenn Ändern einer stillenden Mutter ist notwendig, um die Sterblichkeit durch mütterliche Kannibalisierung zu reduzieren, entfernen Sie die P0 Welpen auf eine stillende Mutter und führen Sie dann Wurzelspitzenresektion an P1. Entweder den Schein-Operation oder die apikale r nur durchführenesection in einem Wurf von Welpen, weil das Mischen Schein und chirurgische Jungtieren konnte die Überlebensraten der chirurgischen Gruppe von Welpen 6 zu verringern. </li></ol> 5. Post-OP-Analyse <ol><li> Einen Tag nach der Operation, die Welpen zu überwachen, stellen Sie sicher, dass es keinen Unterschied zwischen Schein und chirurgische Gruppen und zählen die Anzahl der Welpen, um die Überlebensrate zu messen. Wenn die Operationen richtig durchgeführt wird, sollte die Überlebensraten ähnlich und mehr als 60% sowohl in der Schein-und chirurgischen Gruppen sein. </li><li> Herz Isolierung und Fixierung. <ol><li> Euthanize die Welpen an den Tagen 1 und 2 nach der Operation über die Enthauptung und Tag 21 nach der Operation von CO2 mit der Versicherung des Todes durch Genickbruch, dann reinigen Sie die Brust mit einem 70% igem Alkohol prep. </li><li> Einzuschneiden die Mittellinie Haut und Muskeln der Brust und öffnen Sie die Brust. </li><li> Verbrauchsteuern Das gesamte Herz aus dem Brustraum und befestigen Sie das gesamte Herz einer jeden Probe in 5 ml 4% Paraformaldehyd O / N bei RT. </li></ol></li><li> Am nächsten Tag, übertragen Sie die Proben auf 70% Ethanol. Die Proben können bis zu einer Woche vor dem Paraffineinbettung gespeichert werden. </li><li> Slice 5 um dicke Paraffinschnitte durch das gesamte Herzkammer und führen Sie Standard Hämatoxylin und Eosin (H &amp; E) und Massons Trichrom-Färbung, um die regenerative Antwort 5, 7 zu untersuchen. Insbesondere H &amp; E-Färbung wird verwendet, um Muskelersatz zu prüfen und Massons Trichrom-Färbung verwendet um fibrotische Reaktionen 5 zu untersuchen. </li><li> Wählen Sie den Bereich von Interesse und mindestens drei Foci pro Probe manuell mit einem Dia-Scanner eingestellt. Bild und analysieren die Färbung Folien bei 40-facher Vergrößerung nach den Anweisungen des Herstellers mit Standardparametern. </li></ol>

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	<li>Lin, Z., Pu, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+for+cardiac+regeneration+and+repair.">Strategies for cardiac regeneration and repair.</a> Sci Transl Med. 6, 231-239 (2014).</li><li>Xin, M., Olson, E. N., Bassel-Duby, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mending+broken+hearts:+cardiac+development+as+a+basis+for+adult+heart+regeneration+and+repair.">Mending broken hearts: cardiac development as a basis for adult heart regeneration and repair.</a> Nat Rev Mol Cell Biol. 14, 529-541 (2013).</li><li>Mahmoud, A. I., Porrello, E. 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I., Porrello, E. R., Kimura, W., Olson, E. N., Sadek, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+models+for+cardiac+regeneration+in+neonatal+mice.">Surgical models for cardiac regeneration in neonatal mice.</a> Nat Protoc. 9, 305-311 (2014).</li><li>Allen, T. C., Prophet, E. B., Mills, B., Arrington, J. B., Sobin, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematoxylin+and+eosin.">Hematoxylin and eosin.</a> Laboratory methods in Histotechnology. , 53-58 (1992).</li><li>Andersen, D. C., Ganesalingam, S., Jensen, C. H., Sheikh, S. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+hypothermia+for+general+anesthesia+in+preweanling+rodents.">Use of hypothermia for general anesthesia in preweanling rodents.</a> Physiol Behav. 38, 887-890 (1986).</li><li>Laflamme, M. A., Murry, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerating+the+heart.">Regenerating the heart.</a> Nat Biotechnol. 23, 845-856 (2005).</li><li>Laflamme, M. A., Murry, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+regeneration.">Heart regeneration.</a> Nature. , 326-335 (2011).</li><li>Jesty, S. 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Publikationskosten für diesen Artikel wurden von einem Geschenk von Fine Science Tools, um Dr. Jian Hou gesponsert.

Cardiac regeneration shows potential for the treatment and prevention of heart failure1, 2. Animal models are indispensable and play a critical role in understanding how cardiac regeneration occurs3-6. Many amphibians and fish regenerate heart tissue in response to injury, providing insight into our understanding of human cardiovascular disease13, 14. In terms of evolution, however, the pathophysiology should be more conserved between a mouse model and humans. Heart regeneration in mammal...

Prolonged human life span leads to various aging- and lifestyle-related diseases, including heart failure, a leading cause of mortality. However, without replacement of lost or dysfunctional cardiac muscle cells, current therapeutics can only transiently improve cardiac function1, 2. Thus, it is necessary to discover and develop innovative strategies for cardiac regeneration and repair. The adult mammalian heart has limited regenerative potential. Studies from lower vertebrates, such as urodele amphibians and teleost fish, have provided unprecedented insights into the molecular and cellular mechanisms underlying heart regeneration3, 4. Recently, a neonatal mouse model of heart regeneration has emerged that might enable identification and characterization of more evolutionarily conserved pathophysiological events required for human heart regeneration5. 
Apical resection refers to the surgical removal of left ventricular apex. This procedure is restricted to 1- to 7-day-old (P1 to P7) mice due to its high lethality in older mice6. The cardiac regeneration process in neonatal mice after apical resection is expected to be as follows: (I) rapid and effective formation of a hematoma to seal the apex and prevent exsanguination; (II) cardiomyocyte regeneration and restoration of systolic function5, 7. Recent work has stimulated debate on the significance and efficiency of this model8-11. Thus, it is important to present the apical resection clearly and in detail. To this end, this protocol vividly and specifically describes a video of how I did apical resection based on a previous protocol6. 
Understanding the molecular mechanisms underlying cardiac regeneration is of importance for treatment of heart disease characterized by loss and/or injured cardiomyocytes, such as heart failure1, 2. Given the current and promising progress of apical resection in the research of cardiac regeneration, this study could promote the use of this technique and its uses in cardiac regeneration research.

<table><tbody><tr><td>Olsen-Hegar Needle Holders with Scissors, 1.5 mm</td><td>Fine Science Tools</td><td>12002-12</td><td></td></tr><tr><td>Vannas Spring Scissors - 2 mm Cutting Edge</td><td>Fine Science Tools</td><td>15000-03</td><td>Iridectomy scissors</td></tr><tr><td>Hot Bead Sterilizer</td><td>Fine Science Tools</td><td>18000-45</td><td></td></tr><tr><td>Iris Forceps, Straight, Serrated</td><td>Fine Science Tools</td><td>11064-07</td><td></td></tr><tr><td>Iris Forceps, Curved, Serrated</td><td>Fine Science Tools</td><td>11065-07</td><td></td></tr><tr><td>Shea Scissors - Curved/Blunt-Blunt/12cm</td><td>Fine Science Tools</td><td>14105-12</td><td></td></tr><tr><td>Round Handled Suture Tying Forceps, Straight</td><td>Fine Science Tools</td><td>18025-12</td><td></td></tr><tr><td>Round Handled Vannas Spring Scissors</td><td>Fine Science Tools</td><td>15400-12</td><td></td></tr><tr><td>Fine Hemostat</td><td>Fine Science Tools</td><td>13007-12</td><td></td></tr><tr><td>Magnifying Lamp</td><td>Luxolamp Corp</td><td>IM120&amp;nbsp;</td><td></td></tr><tr><td>Heating lamp</td><td>Brandt Equipment llc</td><td>51152/3</td><td></td></tr><tr><td>6-0 Prolene sutures</td><td>Ethicon</td><td>8889H</td><td></td></tr><tr><td>Skin glue-Vetbond Tissue Adhesive&amp;nbsp;</td><td>3M&amp;nbsp;</td><td>1469</td><td></td></tr><tr><td>Sterile Cotton Tipped Applicators</td><td>Dynarex</td><td>4305</td><td></td></tr><tr><td>WEBCOL Alcohol Prep Pad</td><td>Covidien</td><td>6818</td><td>Medium 2 PLY, 200/BOX, Satured with 70% Isopropyl Alcohol</td></tr><tr><td>Curity All Purpose Sponges</td><td>Covidien</td><td>9024</td><td>Non-woven 4 PLY, 4&quot;x4&quot; (10.2cm&amp;times;10.2cm)</td></tr><tr><td>Bench top protector sheet</td><td>KIMTECH SCIENCE</td><td>7546</td><td>18&quot; x 19.5&quot; (45.72cm x 49.53cm) x 50</td></tr><tr><td>0.9% Sodium Chloride, 250ml</td><td>Hospira Inc.</td><td>NDC 0409-6138-22</td><td></td></tr><tr><td>Betadine Solution Swabsticks</td><td>Purdue Products L.P.</td><td>NDC 67618-153-03</td><td></td></tr><tr><td>Autoclave</td><td>TOMY Digital Biology</td><td>SX-700</td><td>HIGH-PRESSURE STEAM STERILIZER</td></tr><tr><td>Slide scanner</td><td>HAMAMATSU</td><td>NanoZoomer 2.0-RS</td><td></td></tr></tbody></table>

apical resection mouse model to study early mammalian heart regeneration

Herz-Kreislauf-Krankheit plagt die ganze Welt durch intensive Lifestyle-Änderungen. Herzregeneration birgt ein großes Versprechen für die Reparatur und Wiederherstellung der Herzmuskelzellen wegen Verletzung und Krankheit verloren. Im Gegensatz zu der robusten kardialen Regeneration von bestimmten niederen Wirbeltieren, erwachsenen Säugetier-Herzen zeigen in der Regel eine minimale Kapazität für Herz Regeneration und Reparatur. Allerdings haben jüngste Studien beträchtliche wissenschaftliche Interesse mit der Feststellung, dass zwischen postnatalen Tag 1-7 (P1 bis P7), behält der neonatalen Mäuseherz signifikante Regenerationspotential nach Wurzelspitzenresektion (dh Amputationen und Belichtung des linken ventrikulären Apex) ausgelöst. Eine wichtige Kontroverse über diesen Befund könnte durch die vielfältigen Operation bezogene Verfahren bei den Bemühungen zu replizieren oder zu erweitern auf diese wichtige Erkenntnis verwendet wird. Diese Anweisungen dynamisch für Wurzelspitzenresektion in einem Mausmodell zeigen die Materialien und Methodik. Die wesentlichen Schritte dieses Nagetier Survival Chirurgie beinhalten Hypothermie Anästhesie, Thorakotomie, chirurgische Amputation der Herzkammerspitze, und das Nahtmaterial und Rückgewinnung von Mäusen. Die beschriebene könnte die Anwendung der Wurzelspitzenresektion Mausmodells für die kardiovaskuläre Forschung zu erweitern Ansatz.

Maus Welpen eingeschläfert 1, 2, und 21 Tage nach der Wurzelspitzenresektion, und ihre Herzen wurden für die H &amp; E und Massons Trichrom gesammelt. Blaue Farbe in Massons Trichrom-Färbung zeigt die Ablagerung von epikardialen extrazellulären Matrix 5. Mit erfolgreichen Wurzelspitzenresektion, ist ein Blutgerinnsel effektiv gebildet, um die LV 1 Tag nach Wurzelspitzenresektion abzudichten, wie in 1A gezeigt. Eine allmähliche Resorption des Blutgerinnsels und frühen Herzfibrose beobach...

Watch this Scientific Journal Video about Apical Resection Mouse Model to Study Early Mammalian Heart Regeneration at JoVE.com

Apical Resection Mouse Model to Study Early Mammalian Heart Regeneration

A step-by-step video protocol of apical resection is demonstrated in this study. Apical resection is a recently highlighted surgical approach in mammalian heart regeneration research. This study may promote the application of apical resection as a standard methodology in research into the mechanism underlying heart regeneration.

Wurzelspitzenresektion Mausmodell zur frühen Säugetierherzregeneration Studieren

Cardiovascular disease plagues the whole world due to intensive lifestyle changes. Heart regeneration holds great promise for repairing and restoring ...

apical-resection-mouse-model-to-study-early-mammalian-heart

Research

JoVE Journal

Medicine

11.2K Aufrufe.  National Institutes of Health. A step-by-step video protocol of apical resection is demonstrated in this study. Apical resection is a recently highlighted surgical approach in mammalian heart regeneration research. This study may promote the application of apical resection as a standard methodology in research into the mechanism underlying heart regeneration.

Serie: Apical Resection Mouse Model to Study Early Mammalian Heart Regeneration

Transplantation of Neonatal Mouse Cardiac Macrophages into Adult Mice

In an injured neonatal myocardium, macrophages facilitate cardiomyocyte proliferation and angiogenesis and promote heart regeneration. The present study reveals that transplantation of neonatal cardiac macrophages recruited by injury promotes adult heart regeneration after myocardial infarction with improvement of cardiac function and cardiomyocyte proliferation. The results indicate that neonatal cardiac macrophage transplantation could be a promising strategy for cardiac injury treatment. Here, we provide the technical details, including the isolation of neonatal cardiac macrophages from apical resection-injured neonatal mouse hearts, the transplantation of macrophages into myocardial-infarcted adult mice, and the estimation of heart regeneration after a macrophage graft.

We provide a protocol for neonatal cardiac macrophage separation and transplantation into an adult mouse heart, which could be a promising way to promote cardiac repair.

Transplantation von neonatalen Maus-Herzmakrophagen in erwachsene Mäuse

In an injured neonatal myocardium, macrophages facilitate cardiomyocyte proliferation and angiogenesis and promote heart regeneration. The present study ...

Forschung

Biochemie

Capturing the Cardiac Injury Response of Targeted Cell Populations via Cleared Heart Three-Dimensional Imaging

Cardiovascular disease outranks all other causes of death and is responsible for a staggering 31% of mortalities worldwide. This disease manifests in cardiac injury, primarily in the form of an acute myocardial infarction. With little resilience following injury, the once healthy cardiac tissue will be replaced by fibrous, non-contractile scar tissue and often be a prelude to heart failure. To identify novel treatment options in regenerative medicine, research has focused on vertebrates with innate regenerative capabilities. One such model organism is the neonatal mouse, which responds to cardiac injury with robust myocardial regeneration. In order to induce an injury in the neonatal mouse that is clinically relevant, we have developed a surgery to occlude the left anterior descending artery (LAD), mirroring a myocardial infarction triggered by atherosclerosis in the human heart. When matched with the technology to track changes both within cardiomyocytes and non-myocyte populations, this model provides us with a platform to identify the mechanisms that guide heart regeneration. Gaining insight into changes in cardiac cell populations following injury once relied heavily on methods such as tissue sectioning and histological examination, which are limited to two-dimensional analysis and often damage the tissue in the process. Moreover, these methods lack the ability to trace changes in cell lineages, instead providing merely a snapshot of the injury response. Here, we describe how technologically advanced methods in lineage tracing models, whole organ clearing, and three-dimensional (3D) whole-mount microscopy can be used to elucidate mechanisms of cardiac repair. With our protocol for neonatal mouse myocardial infarction surgery, tissue clearing, and 3D whole organ imaging, the complex pathways that induce cardiomyocyte proliferation can be unraveled, revealing novel therapeutic targets for cardiac regeneration.

Cardiomyocyte proliferation following injury is a dynamic process that requires a symphony of extracellular cues from non-myocyte cell populations. Utilizing lineage tracing, passive CLARITY, and three-dimensional whole-mount confocal microscopy techniques, we can analyze the influence of a variety of cell types on cardiac repair and regeneration.

Erfassung der Herzverletzungsreaktion von gezielten Zellpopulationen über cleared Heart Three-Dimensional Imaging

Cardiovascular disease outranks all other causes of death and is responsible for a staggering 31% of mortalities worldwide. This disease manifests in ...

Entwicklungsbiologie

Permanent Ligation of the Left Anterior Descending Coronary Artery in Mice: A Model of Post-myocardial Infarction Remodelling and Heart Failure

Heart failure is a syndrome in which the heart fails to pump blood at a rate commensurate with cellular oxygen requirements at rest or during stress. It is characterized by fluid retention, shortness of breath, and fatigue, in particular on exertion. Heart failure is a growing public health problem, the leading cause of hospitalization, and a major cause of mortality. Ischemic heart disease is the main cause of heart failure.
Ventricular remodelling refers to changes in structure, size, and shape of the left ventricle. This architectural remodelling of the left ventricle is induced by injury (e.g., myocardial infarction), by pressure overload (e.g., systemic arterial hypertension or aortic stenosis), or by volume overload. Since ventricular remodelling affects wall stress, it has a profound impact on cardiac function and on the development of heart failure. A model of permanent ligation of the left anterior descending coronary artery in mice is used to investigate ventricular remodelling and cardiac function post-myocardial infarction. This model is fundamentally different in terms of objectives and pathophysiological relevance compared to the model of transient ligation of the left anterior descending coronary artery. In this latter model of ischemia/reperfusion injury, the initial extent of the infarct may be modulated by factors that affect myocardial salvage following reperfusion. In contrast, the infarct area at 24 hr after permanent ligation of the left anterior descending coronary artery is fixed. Cardiac function in this model will be affected by 1) the process of infarct expansion, infarct healing, and scar formation; and 2) the concomitant development of left ventricular dilatation, cardiac hypertrophy, and ventricular remodelling.
Besides the model of permanent ligation of the left anterior descending coronary artery, the technique of invasive hemodynamic measurements in mice is presented in detail.

Heart failure is the leading cause of hospitalization and a major cause of mortality. A model of permanent ligation of the left anterior descending coronary artery in mice is applied to investigate ventricular remodelling and cardiac dysfunction post-myocardial infarction. The technique of invasive hemodynamic measurements in mice is presented.

Permanent Ligation des linken vorderen absteigenden Koronararterie bei Mäusen: Ein Modell der Post-Myokardinfarkt Umbau und Herzinsuffizienz

Heart failure is a syndrome in which the heart fails to pump blood at a rate commensurate with cellular oxygen requirements at rest or during stress. It ...

Medizin

Clonal Analysis of the Neonatal Mouse Heart using Nearest Neighbor Modeling

By replacing lost or dysfunctional myocardium, tissue regeneration is a promising approach to treat heart failure. However, the challenge of detecting bona fide heart regeneration limits the validation of potential regenerative factors. One method to detect new cardiomyocytes is multicolor lineage tracing with clonal analysis. Clonal analysis experiments can be difficult to undertake, because labeling conditions that are too sparse lack sensitivity for rare events such as cardiomyocyte proliferation, and diffuse labeling limits the ability to resolve clones. Presented here is a protocol to undertake clonal analysis of the neonatal mouse heart by using statistical modeling of nearest neighbor distributions to resolve cardiomyocyte clones. This approach enables resolution of clones over a range of labeling conditions and provides a robust analytical approach for quantifying cardiomyocyte proliferation and regeneration. This protocol can be adapted to other tissues and can be broadly used to study tissue regeneration.

Presented here is a protocol for using multicolor lineage tracing and nearest-neighbor modeling to identify clonally derived cardiomyocytes during growth and regeneration in mice. This approach is objective, works across different labeling conditions, and can be adapted to incorporate a variety of image analysis pipelines.

By replacing lost or dysfunctional myocardium, tissue regeneration is a promising approach to treat heart failure. However, the challenge of detecting ...

Myocardial Infarction in Neonatal Mice, A Model of Cardiac Regeneration

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and regeneration, and to define new targets for therapeutics. For decades, models of complete heart regeneration existed in amphibians and fish, but a mammalian counterpart was not available. The recent discovery of a postnatal window during which mice possess regenerative capabilities has led to the establishment of a mammalian model of cardiac regeneration. A surgical model of mammalian cardiac regeneration in the neonatal mouse is presented herein. Briefly, postnatal day 1 (P1) mice are anesthetized by isoflurane and placed on an ice pad to induce hypothermia. After the chest is opened, and the left anterior descending coronary artery (LAD) is visualized, a suture is placed around the LAD to inflict myocardial ischemia in the left ventricle. The surgical procedure takes 10-15 min. Visualizing the coronary artery is crucial for accurate suture placement and reproducibility. Myocardial infarction and cardiac dysfunction are confirmed by triphenyl-tetrazolium chloride (TTC) staining and echocardiography, respectively. Complete regeneration 21 days post myocardial infarction is verified by histology. This protocol can be used to as a tool to elucidate mechanisms of mammalian cardiac regeneration after myocardial infarction.

This protocol describes a highly reproducible model of cardiac regeneration by surgical induction of myocardial infarction in the left ventricle of postnatal day 1 mice. The method involves induction of hypothermic anesthesia and ligation of the left anterior descending coronary artery.

Myokardinfarkts bei neugeborenen Mäusen, ein Modell von Cardiac Regeneration

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and ...

Transplantation of a 3D Bioprinted Patch in a Murine Model of Myocardial Infarction

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ligation is a challenging procedure and has a high mortality rate due to its nature. We developed a method to consistently transplant bioprinted patches of cells and hydrogels onto the epicardium of an infarcted mouse heart to test their regenerative properties in a robust and feasible way. First, a deeply anesthetized mouse is carefully intubated and ventilated. Following left lateral thoracotomy (surgical opening of the chest), the exposed LAD is permanently ligated and the bioprinted patch transplanted onto the epicardium. The mouse quickly recovers from the procedure after chest closure. The advantages of this robust and quick approach include a predicted 28-day mortality rate of up to 30% (lower than the 44% reported by other studies using a similar model of permanent LAD ligation in mice). Moreover, the approach described in this protocol is versatile and could be adapted to test bioprinted patches using different cell types or hydrogels where high numbers of animals are needed to optimally power studies. Overall, we present this as an advantageous approach which may change preclinical testing in future studies for the field of cardiac regeneration and tissue engineering.

This protocol aims to transplant a 3D bioprinted patch onto the epicardium of infarcted mice modeling heart failure. It includes details regarding anesthesia, the surgical chest opening, permanent ligation of the left anterior descending (LAD) coronary artery and application of a bioprinted patch onto the infarcted area of the heart.

Transplantation eines biobedruckten 3D-Patches in einem Murine-Modell des Myokardinfarkts

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ...

Biotechnik

Blood Circuit Reconstruction in an Abdominal Mouse Heart Transplantation Model

The surgical technique of heterotopic abdominal heart transplantation in mice is a standard model for research in transplantation immunology. Here, the established technique for a modified blood circuit reconstruction in a heterotopic abdominal heart transplantation model is presented. This method uses the intrathoracic inferior vena cava (IIVC) instead of the pulmonary artery of the donor heart for the anastomosis to the inferior vena cava of the recipient. It is facilitating and improving success rates for abdominal heart transplantation in mice.

A novel technique for blood circuit reconstruction in a heterotopic abdominal mouse heart transplantation model is demonstrated.

Rekonstruktion des Blutkreislaufs in einem abdominalen Mausherztransplantationsmodell

The surgical technique of heterotopic abdominal heart transplantation in mice is a standard model for research in transplantation immunology. Here, the ...

A Modified Cuff Technique for Mouse Cervical Heterotopic Heart Transplantation Model

Cardiac allograft rejection limits the long-term survival of patients after heart transplantation. A mouse heart transplantation model is ideal for investigating the mechanism of cardiac allograft rejection in preclinical studies because of their high homology with human genes. This understanding would help develop unique approaches to improving patients&#39; long-term survival treated with cardiac allografts. In a mouse model, abdominal donor heart implantation is commonly performed with an end-to-end&#160;anastomosis to the recipient&#39;s aorta and inferior vena cava using stitches. In this model, the donor&#39;s heart is implanted by end-to-end anastomosis to the recipient&#39;s carotid artery and jugular vein by the modified-Cuff technique. The transplantation surgery is performed without stitching and thus may increase the survival of the recipient since there is no interference with the blood supply and venous reflux of the lower body. This mouse model would help investigate the mechanisms underlying the immunological and pathological (acute/chronic) rejection of cardiac allografts.

In the present protocol, a mouse heart transplantation model is used for investigating the mechanism of cardiac allograft rejection. In this heterotopic heart transplantation model, operation efficiency is improved, and the survival of cardiac grafts is ensured by a cervical end-to-end anastomosis of heart implantation using a modified Cuff technique.

Eine modifizierte Manschettentechnik für das zervikale heterotope Herztransplantationsmodell der Maus

Cardiac allograft rejection limits the long-term survival of patients after heart transplantation. A mouse heart transplantation model is ideal for ...

An Apical Resection Model in the Adult Xenopus tropicalis Heart

It is known that in adult mammals, the heart has lost its regenerative capacity, making heart failure one of the leading causes of death worldwide. Previous research has demonstrated the regenerative ability of the heart of the adult Xenopus tropicalis, an anuran amphibian with a diploid genome and a close evolutionary relationship with mammals. Additionally, studies have shown that following ventricular apex resection, the heart can regenerate without scarring in X. tropicalis. Consequently, these previous results suggest that X. tropicalis is an appropriate alternative vertebrate model for the study of adult heart regeneration. A surgical model of cardiac regeneration in the adult X. tropicalis is presented herein. Briefly, the frogs were anesthetized and fixed; then, a small incision was made with iridectomy scissors, penetrating the skin and pericardium. Gentle pressure was applied to the ventricle, and the apex of the ventricle was then cut out with scissors. Cardiac injury and regeneration were confirmed by histology at 7-30 days post resection (dpr). This protocol established an apical resection model in adult X. tropicalis, which&#160;can be employed to elucidate the mechanisms of adult heart regeneration.

An Apical Resection Model in the Adult Xenopus tropicalis Heart

Xenopus tropicalis is an ideal model for regenerative research as many of its organs possess a remarkable regenerative capacity. Here, we present a method for constructing a heart injury model in X. tropicalis via apex resection.

Ein apikales Resektionsmodell im adulten Xenopus tropicalis-Herzen

It is known that in adult mammals, the heart has lost its regenerative capacity, making heart failure one of the leading causes of death worldwide. ...

Preparation of a Non-Cardiomyocyte Cell Suspension for Single-Cell RNA Sequencing from a Post-Myocardial Infarction Adult Mouse Heart

Myocardial infarction (MI) is one of the most common cardiovascular diseases, with increasing mortality worldwide. Non-cardiomyocytes account for more than half of the total cardiac cell population, and they contribute to adaptive compensations upon myocardial injury, including inflammatory responses, tissue repair, and scar formation. To study the post-MI cardiac microenvironment, single-cell RNA sequencing (scRNA-seq) is widely used to identify different cardiac cell types and intercellular communications. Among the procedures of scRNA-seq sample preparation, preparing the cell suspension is one of the most critical steps, because the cell viability can affect the quality of the scRNA-seq results. Therefore, we designed an experimental protocol for preparing a non-cardiomyocyte cell suspension from post-MI mouse hearts with an extra focus on improving the cell viability by choosing mild digestive enzymes, controlling the digestion time, and applying fluorescence-activated cell sorting (FACS). Finally, we isolated CD45+ cells from the non-cardiomyocyte cell suspension obtained through this protocol, and then we performed scRNA-seq.

Here, we describe a protocol to isolate a sufficient amount of single non-cardiomyocytes with high viability from a post-myocardial infarction (MI) mouse heart. This can be used for subsequent single-cell sequencing, flow cytometry analysis, and primary cell culture.

Herstellung einer Nicht-Kardiomyozyten-Zellsuspension für die Einzelzell-RNA-Sequenzierung aus einem erwachsenen Mausherzen nach Myokardinfarkt

Myocardial infarction (MI) is one of the most common cardiovascular diseases, with increasing mortality worldwide. Non-cardiomyocytes account for more ...