Gli autori ringraziano Drs. James Hawkins, Zu-Xi Yu e Xuan Qu dal National Heart, Lung, and Blood Institute (NHLBI) per la loro assistenza con la chirurgia del mouse e la preparazione e la colorazione delle sezioni di paraffina. Gli autori sono grati al NIH Fellows Comitato Editoriale per l&#39;assistenza editoriale.

Tutti gli esperimenti di topo sono stati approvati dalla cura degli animali e Use Program presso il National Institutes of Health (NIH) con il numero di protocollo H0083R3. Il NHLBI IACUC ha approvato il protocollo, senza analgesici. 1. L&#39;ipotermia Anestesia nei topi neonatale <ol><li> Sterilizzare spugne e apparecchi chirurgici in autoclave prima dell&#39;intervento chirurgico. Preparare tutti i materiali chirurgici e accendere un caldo tallone sterilizzatrice 15-20 minuti in anticipo per raggiungere 240 ° C a 270 ° C. </li><li> Trasferire tutte C57BL / 6 cuccioli (età P1) dalla loro madre durante l&#39;allattamento per una gabbia pulita mouse con biancheria fresca e materiali per i nidi. Una volta che i cuccioli sono presi in una sala operatoria, eseguire la chirurgia apicale prontamente per minimizzare il tempo trascorso separato dalla madre e per ridurre il rischio di cannibalizzazione materna. </li><li> Mettere spugne su un letto di ghiaccio e poi mettere un cucciolo sulla spugna per ~ 3 min per raggiungere l&#39;anestesia ipotermia. Confermare anestesia osservando apnea e acinesia e pizzicando un piede posteriore. Controllare lo stato dei neonati spesso perché troppo poco tempo non farà il akinetic cucciolo e apnea, e una durata eccessiva di anestesia può abbassare il tasso di sopravvivenza 12. </li></ol> 2. Toracotomia <ol><li> Trasferire il cucciolo dal letto di ghiaccio su una zona banco chirurgica e l&#39;uso del nastro di immobilizzare le braccia, gambe e la coda in posizione supina. </li><li> Disinfettare il petto con betadine e delicatamente pulire con un tampone imbevuto di alcool al 70%. </li><li> Fare un incisione cutanea trasversale lungo la zona via intercostale della cavità toracica con una Vannas primavera forbice, e quindi smussare sezionare i muscoli intercostali avanti per facilitare l&#39;accesso al cuore. NOTA: I tassi di sopravvivenza migliorano quando la perdita di sangue è ridotto al minimo durante il processo chirurgico. </li></ol> 3. amputazione chirurgica del Cuore ventricolare Apex <ol><li> A mano, applicare delicatamente pressione sull&#39;addome per esteriorizzare laapice del cuore. Assorbire sangue intorno l&#39;area chirurgica con applicatori con punta in cotone sterili per la visualizzazione chiara. Per i neonati di controllo sham, procedere direttamente al punto 4 (Sutura e recupero dei Topi). </li><li> Sotto una lampada di ingrandimento e con iridectomia forbici, eseguire delicatamente la resezione frammentaria del ventricolo sinistro (LV) finché la camera LV è esposto. Fare attenzione a ridurre al minimo le parti asportate del LV. Circa il 15% la resezione è necessaria per conseguire esposizione ottimale della camera di LV. </li><li> Assicurarsi che il cuore ritorna alla cavità toracica appena la camera di LV è esposto. </li></ol> 4. Sutura e recupero dei topi <ol><li> Suturare le costole e muscoli insieme per sigillare la cavità toracica utilizzando sterili suture Proline 6-0, e quindi chiudere con cura il sito di incisione cutanea con colla pelle. </li><li> Scaldare il neonato sotto una lampada di calore per ~ 3 minuti di recupero, e poi pulire il sangue e tracce di colla utilizzando un preparato alcolico pad 70% before reintroducendo ai suoi fratellini. Prova a completare l&#39;intera procedura chirurgica entro 10 minuti perché riducendo al minimo il tempo trascorso separato dalla madre migliora la sopravvivenza dei cuccioli dopo resezione apicale. </li><li> Dopo ogni intervento chirurgico, collocare gli strumenti chirurgici in contatto con i talloni del tallone sterilizzatrice a caldo per circa 20 sec per sterilizzazione completa. Lasciare gli strumenti chirurgici per raffreddare a TI prima di ogni intervento chirurgico. </li><li> Dopo aver completato interventi chirurgici per tutti i cuccioli in una cucciolata, mescolare i cuccioli con biancheria da letto e gli escrementi della madre prima di tornare al nido della madre. NOTA: In genere, ICR / CD-1 topi sono madri adottive meglio di C57BL / 6, ma anche tra le madri che allattano di uno stesso background genetico, gli istinti che favoriscono variano. In caso di sostituzione di una madre che allatta è necessario per ridurre la mortalità materna entro cannibalizzazione, rimuovere i cuccioli P0 ad una madre che allatta e quindi eseguire la resezione apicale in P1. Solo eseguire sia l&#39;operazione simulata o r apicaleesection in una cucciolata di cuccioli, perché mescolando finzione e cuccioli chirurgiche potrebbero diminuire il tasso di sopravvivenza del gruppo chirurgico di cuccioli 6. </li></ol> Analisi 5. post-chirurgica <ol><li> Un giorno dopo l&#39;intervento chirurgico, monitorare i cuccioli, garantire che non vi è alcuna differenza tra finzione e gruppi chirurgici, e contare il numero di cuccioli per misurare il tasso di sopravvivenza. Se gli interventi vengono eseguiti correttamente, i tassi di sopravvivenza dovrebbero essere simili e superiore al 60% sia finto e gruppi chirurgici. </li><li> Isolamento Cuore e fissazione. <ol><li> Eutanasia i cuccioli a giorni 1 e 2 post-operatorio tramite decapitazione e il giorno 21 post-operatorio da CO2, con una garanzia di morte da dislocazione cervicale, quindi pulire il petto con un preparato alcolico del 70%. </li><li> Incidere la pelle linea mediana e muscoli del torace e poi aprire il torace. </li><li> Asportare l&#39;intero cuori dalla cavità toracica e fissare l&#39;intero cuore di ogni campione in 5 ml di 4% paraformaldeide O / N a RT. </li></ol></li><li> Il giorno successivo, trasferire i campioni al 70% di etanolo. I campioni possono essere conservati fino a una settimana prima della paraffina. </li><li> Slice sezioni di paraffina a 5 micron di spessore attraverso l&#39;intero ventricolo ed eseguire ematossilina standard ed eosina (H &amp; E) e la colorazione tricromica di Masson per esaminare la risposta rigenerativa 5, 7. In particolare, H &amp; E macchia è utilizzato per esaminare la sostituzione del muscolo e macchia tricromica di Masson è utilizzato esaminare le risposte fibrotiche 5. </li><li> Selezionare l&#39;area di interesse e creare almeno tre focolai per campione manualmente utilizzando uno scanner di diapositive. Immagine e analizzare i vetrini di colorazione a 40 × ingrandimenti secondo le istruzioni del produttore con i parametri di default. </li></ol>

<ol>
	<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. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Turning+back+the+cardiac+regenerative+clock:+lessons+from+the+neonate.">Turning back the cardiac regenerative clock: lessons from the neonate.</a> Trends Cardiovasc Med. 22, 128-133 (2012).</li><li>Porrello, E. R., Olson, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+neonatal+blueprint+for+cardiac+regeneration.">A neonatal blueprint for cardiac regeneration.</a> Stem Cell Res. 13, 556-570 (2014).</li><li>Porrello, E. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+regenerative+potential+of+the+neonatal+mouse+heart.">Transient regenerative potential of the neonatal mouse heart.</a> Science. 331, 1078-1080 (2011).</li><li>Mahmoud, A. 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. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Do+neonatal+mouse+hearts+regenerate+following+heart+apex+resection?.">Do neonatal mouse hearts regenerate following heart apex resection?.</a> Stem cell reports. 2, 406-413 (2014).</li><li>Kotlikoff, M. I., Hesse, M., Fleischmann, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comment+on+'Do+neonatal+mouse+hearts+regenerate+following+heart+apex+resection?'.">Comment on 'Do neonatal mouse hearts regenerate following heart apex resection?'.</a> Stem cell reports. 3, 2 (2014).</li><li>Sadek, H. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-investigator+letter+on+reproducibility+of+neonatal+heart+regeneration+following+apical+resection.">Multi-investigator letter on reproducibility of neonatal heart regeneration following apical resection.</a> Stem cell reports. 3, 1 (2014).</li><li>Andersen, D. C., Jensen, C. H., Sheikh, S. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+to+Sadek+et+al.+and+Kotlikoff+et+al.">Response to Sadek et al. and Kotlikoff et al.</a> Stem cell reports. 3, 3-4 (2014).</li><li>Phifer, C. B., Terry, L. 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. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=c-kit++precursors+support+postinfarction+myogenesis+in+the+neonatal,+but+not+adult,+heart.">c-kit+ precursors support postinfarction myogenesis in the neonatal, but not adult, heart.</a> Proc Natl Acad Sci U S A. 109, 13380-13385 (2012).</li><li>Gonzalez-Rosa, J. M., Martin, V., Peralta, M., Torres, M., Mercader, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extensive+scar+formation+and+regression+during+heart+regeneration+after+cryoinjury+in+zebrafish.">Extensive scar formation and regression during heart regeneration after cryoinjury in zebrafish.</a> Development. 138, 1663-1674 (2011).</li></ol>

Spese di pubblicazione per questo articolo sono stati sponsorizzati da un dono da Strumenti Belle Science il dottor Jian Hou.

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

Le malattie cardiovascolari affligge tutto il mondo a causa di cambiamenti di lifestyle intensive. Rigenerazione del cuore una grande promessa per riparazione e ripristino cardiomiociti persi a causa di infortunio e malattia. In contrasto con la rigenerazione cardiaca robusta di alcuni vertebrati inferiori, cuori mammiferi adulti mostrano tipicamente una capacità minima di rigenerazione cuore e riparazione. Tuttavia, studi recenti hanno suscitato un notevole interesse scientifico con la constatazione che, tra il giorno postnatale 1-7 (P1 a P7), il cuore neonatale del mouse mantiene notevole potenziale rigenerativo dopo la resezione apicale (cioè, amputazione chirurgica e l&#39;esposizione di sinistra apice ventricolare). Una polemica importante su questo risultato potrebbe essere dovuto alle diverse procedure di chirurgia legati utilizzati negli sforzi per replicare o espandere su questo importante risultato. Queste istruzioni presentano dinamicamente i materiali e la metodologia per la resezione apicale in un modello di topo. Le fasi salienti di questo roditore Survivachirurgia l coinvolge ipotermia anestesia, toracotomia, amputazione chirurgica del ventricolo cuore apice, e sutura e il recupero di topi. L&#39;approccio descritto potrebbe espandere l&#39;applicazione del modello di topo di resezione apicale per la ricerca cardiovascolare.

Cuccioli di topo sono stati sacrificati 1, 2, e 21 giorni dopo la resezione-apicale, e il loro cuore sono stati raccolti per H &amp; E e macchia Trichrome di Masson. Colore blu a macchia tricromica di Masson indica la deposizione di matrice extracellulare epicardico 5. Con successo resezione apicale, un coagulo di sangue è effettivamente formata per sigillare il LV uno giorno post-resezione apicale, come mostrato nella Figura 1A. Un graduale riassorbimento del coagulo di san...

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.

Modello apicale resezione mouse per primo studio Mammalian rigenerazione cardiaca

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 Views.  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.

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

Murine Cervical Heart Transplantation Model Using a Modified Cuff Technique

Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are available to perform mechanistic in vivo studies. While heart transplantation models using a suture technique were first successfully developed in rats, the translation into an equally widespread used murine equivalent was never achieved due the technical complexity of the microsurgical procedure. In contrast, non-suture cuff techniques, also developed initially in rats, were successfully adapted for use in mice1-3. This technique for revascularization involves two major steps I) everting the recipient vessel over a polyethylene cuff; II) pulling the donor vessel over the formerly everted recipient vessel and holding it in place with a circumferential tie. This ensures a continuity of the endothelial layer, short operating time and very high patency rates4.
Using this technique for vascular anastomosis we performed more than 1,000 cervical heart transplants with an overall success rate of 95%. For arterial inflow the common carotid artery and the proximal aortic arch were anastomosed resulting in a retrograde perfusion of the transplanted heart. For venous drainage the pulmonary artery of the graft was anastomosed with the external jugular vein of the recipient5.
Herein, we provide additional details of this technique to supplement the video.

The murine cervical heart transplantation model is well suited for immunological as well as ischemia reperfusion injury studies. We modified the procedure using a non-suture cuff technique and performed more than 1,000 successful transplants with this approach.
Herein, we provide additional details of this technique to supplement the video.

Murino cervicale Cuore trapianto Modello utilizzando una tecnica Cuff Modificato

Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are ...

Ricerca

Medicina

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.

Trapianto di macrofagi cardiaci di topo neonatale in topi adulti

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

Biochimica

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.

Catturare la risposta alle lesioni cardiache delle popolazioni cellulari mirate tramite l'imaging tridimensionale del cuore autorizzato

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

Biologia dello sviluppo

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.

Legatura permanente dell&#39;arteria discendente anteriore sinistra coronarica nei topi: un modello di post-infarto rimodellante miocardico e insufficienza cardiaca

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 ...

Transgenesi basata sull'elettroporazione di cellule cardiache in cuori di topo

An Efficient Transgenesis Approach for Gene Delivery in the Mouse Embryonic Heart

The mammalian heart is a complex organ formed during development via highly diverse populations of progenitor cells. The origin, timing of recruitment, and fate of these progenitors are vital for the proper development of this organ. The molecular mechanisms that govern the morphogenesis of the heart are essential for understanding the pathogenesis of congenital heart diseases and embryonic cardiac regeneration. Classical approaches to investigate these mechanisms employed the generation of transgenic mice to assess the function of specific genes during cardiac development. However, mouse transgenesis is a complex, time-consuming process that often cannot be performed to assess the role of specific genes during heart development. To address this, we have developed a protocol for efficient electroporation and culture of mouse embryonic hearts, enabling transient transgenesis to rapidly assess the effect of gain- or loss-of-function of genes involved in cardiac development. Using this methodology, we successfully overexpressed Meis1 in the embryonic heart, with a preference for epicardial cell transfection, demonstrating the capabilities of the technique.

Autore in primo piano: Transgenesi delle cellule cardiache per lo screening genico rapido

This protocol presents a detailed methodological framework for electroporation-based transgenesis of cardiac cells in developing mouse hearts. The video assets provided here will facilitate learning of this versatile technique.

Un approccio efficiente alla transgenesi per la consegna genica nel cuore embrionale di topo

The mammalian heart is a complex organ formed during development via highly diverse populations of progenitor cells. The origin, timing of recruitment, ...

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.

Infarto del miocardio in topi neonati, un modello di rigenerazione cardiaca

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.

Trapianto di una patch biostampa 3D in un modello Murine di infarto miocardico

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

Bioingegneria

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.

Ricostruzione del circuito sanguigno in un modello di trapianto di cuore di topo addominale

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

Modello apicale resezione mouse per primo studio Mammalian rigenerazione cardiaca

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Legatura permanente dell'arteria discendente anteriore sinistra coronarica nei topi: un modello di post-infarto rimodellante miocardico e insufficienza cardiaca

Un approccio efficiente alla transgenesi per la consegna genica nel cuore embrionale di topo

Un approccio efficiente alla transgenesi per la consegna genica nel cuore embrionale di topo

Un approccio efficiente alla transgenesi per la consegna genica nel cuore embrionale di topo

Infarto del miocardio in topi neonati, un modello di rigenerazione cardiaca

Trapianto di una patch biostampa 3D in un modello Murine di infarto miocardico

Ricostruzione del circuito sanguigno in un modello di trapianto di cuore di topo addominale