Yazarlar Dr teşekkür. James Hawkins, Ulusal Kalp, Akciğer gelen Zu-Xi Yu ve Xuan Qu ve fare ameliyat hazırlanması ve parafin kesitlerin boyanması ile yardım için Kan Enstitüsü (NHLBI). Yazarlar editoryal yardım için NIH Fellows Yayın Kurulu minnettarız.

Tüm fare deneyleri protokol numarası H0083R3 ile Ulusal Sağlık Enstitüleri (NIH) de Hayvan Bakımı ve Kullanımı Programı tarafından kabul edildi. NHLBI IACUC analjezik olmadan protokolü onayladı. Yenidoğan Farelerde 1. Hipotermi Anestezi <ol><li> Ameliyat öncesi bir otoklav içinde sünger ve cerrahi ekipman sterilize edin. Tüm cerrahi malzemelerin hazırlayın ve 270 ° C 240 ° C&#39;ye ulaşması önceden sıcak bir boncuk sterilizatör 15-20 dk açın. </li><li> Taze yatak ve yuvalama materyalleri ile temiz fare kafesine kendi hemşirelik anneden tüm C57BL / 6 yavrular (yaş P1) aktarın. Yavruların bir ameliyat odasına alınır sonra, onların zaman anneden ayrılmış geçirdi ve anne markada riskini azaltmak için en aza indirmek için derhal apikal ameliyat. </li><li> Bir buz yatakta sünger koyun ve ~ 3 dk hipotermi anestezi elde etmek için daha sonra sünger tek yavru yerleştirin. Ap gözlemleyerek anestezi onaylayınnea ve akinezi ve arka ayak kısma. Çok az zaman yavru akinetik ve apne ve hayatta kalma oranını 12 düşürebilirsiniz anestezi aşırı süresini yapmaz nedeniyle sık sık yenidoğanların durumunu kontrol edin. </li></ol> 2. Torakotomi <ol><li> Cerrahi Benchtop alanı üzerine buz yataktan yavru aktarın ve bir yatar pozisyonda kolları, bacakları ve kuyruk hareketsiz hale getirmek için bandı kullanın. </li><li> Betadin kullanarak göğüs dezenfekte ve yavaşça bir% 70 alkol hazırlık takımını kullanarak temizleyin. </li><li> Bir Vannas Bahar Makas kullanarak göğüs boşluğunun ortaya interkostal alanı boyunca enine cilt kesisi yapın ve sonra kalp erişimi kolaylaştırmak için ileri interkostal kasları disseke künt. NOT: kan kaybı, cerrahi işlem sırasında minimize edildiğinde sağkalım oranları artırmak. </li></ol> Kalp Ventriküler Apex 3. Cerrahi Amputasyon <ol><li> Elle nazikçe exteriorize karın üzerinde baskı uygulamakkalbin apeksi. Net görüntüleme için steril pamuk uçlu aplikatörler cerrahi alanı çevresinde kan emer. Sahte çalışan kumanda yenidoğanlarda için, 4 (Sütür ve Farelerin Kurtarma) Adım doğrudan devam. </li><li> Bir büyüteç lamba ve makas iridektomi kullanarak altında yavaşça LV odası açığa çıkana kadar sol ventrikül (LV) parça parça rezeksiyonu gerçekleştirin. LV rezeksiyon bölümlerini en aza indirmek için dikkatli olun. Yaklaşık% 15 rezeksiyon LV odasının optimum pozlama sağlamak için gereklidir. </li><li> LV odası maruz kez kalp göğüs boşluğu döner emin olun. </li></ol> 4. Sütür ve Farelerin Kurtarma <ol><li> Steril Proline 6-0 sütür kullanarak göğüs boşluğuna mühür birlikte kaburga ve kasları sütür ve daha sonra dikkatlice cilt yapıştırıcı kullanarak cilt kesi siteyi kapatın. </li><li> Kurtarma için ~ 3 dakika boyunca bir ısı lambası altında yenidoğan ısıtın ve bir% 70 alkol Hazırlık pedi befo kullanılarak, kan ve tutkal izlerini temizlemekonun littermates onu reintroducing yeniden. Anneden ayrı harcanan zamanı minimize apikal rezeksiyon sonrası yavru hayatta kalma artırır, çünkü 10 dakika içinde tüm cerrahi prosedür tamamlamaya çalışın. </li><li> Her Ameliyatı takiben, tam sterilizasyon yaklaşık olarak 20 saniye boyunca sıcak boncuk sterilizatör taneleri ile temas cerrahi aletler yerleştirin. Cerrahi aletler her ameliyat öncesi oda sıcaklığına soğumasını bekleyin. </li><li> Bir çöp tüm yavruların için ameliyat tamamladıktan sonra, annenin yuvaya onları dönmeden önce annenin yatak ve dışkı ile yavrular karıştırın. NOT: Genellikle ICR / CD-1 fareler daha bakıcı C57BL / 6 den anne, hatta aynı genetik kökenli emziren anneler arasında, teşvik içgüdüleri değişir. Hemşirelik anne, değişen anne markada tarafından ölümlerini azaltmak hemşirelik anneye P0 yavrular kaldırmak ve daha sonra P1 apikal rezeksiyon gerçekleştirmek için gerekirse. Sadece sahte işlemi veya apikal r ya gerçekleştirmekYavruların bir çöp içinde esection, karıştırma sahte ve cerrahi yavrular yavruların 6 cerrahi grubunun sağkalım oranlarını azaltmak çünkü. </li></ol> 5. Post-Cerrahi Analizi <ol><li> Bir gün ameliyat sonrası, yavrular izlemek sham ve cerrahi gruplar arasında hiçbir fark yoktur emin olun ve hayatta kalma oranını ölçmek için yavruların sayısı. Ameliyatlar düzgün yapılırsa, sağkalım oranları sham ve cerrahi her iki grupta da benzer ve daha fazla% 60 olması gerekmektedir. </li><li> Kalp izolasyonu ve tespit. <ol><li> Günlerde 1 de yavrular ve servikal dislokasyon ile ölüm güvencesi ile CO2 ile Dekapitasyon ve gündüz 21 sonrası ameliyat yoluyla 2 sonrası ameliyat Euthanize, daha sonra% 70 alkol hazırlık kullanarak göğüs temizleyin. </li><li> Göğüs orta hat cilt ve kas insizyon ve sonra kutuyu açın. </li><li> Göğüs boşluğundan tüm kalpleri tüketim ve% 4 paraformaldehid O / RT&#39;de N 5 ml Her numunenin tüm kalbini düzeltmek. </li></ol></li><li> Bir sonraki gün,% 70 etanol örnekleri aktarın. Numuneler parafine önce bir haftaya kadar saklanabilir. </li><li> Dilim 5 mikron kalınlığında parafin tüm ventrikül ile bölümleri ve rejeneratif yanıtı 5, 7 incelemek için standart hematoksilen ve eozin (H &amp; E) ve Masson trikrom gerçekleştirin. Özellikle, H &amp; E boyama kas değiştirme incelemek için kullanılır ve Masson Triclırome boya kullanılır fibrotik yanıtları 5 incelemek için. </li><li> Ilgi alanı seçin ve elle bir slayt tarayıcı kullanılarak örnek başına en az üç odaklar kurdu. Görüntü ve varsayılan parametrelerle üreticinin talimatlarına göre 40 × büyütmede boyama slaytlar analiz. </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>

Bu yazı için Yayın ücretleri Dr. Jian Hou Güzel Bilim Araçlar hediye sponsorluğunda bulundu.

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 mammalian animal models is a relatively new but burgeoning research field for the therapeutics of heart failure3-6. 
Recently, two surgical approaches, apical resection and myocardial infarction, have been established in the study of heart regeneration in neonatal mice6. Although apical resection provides a relatively easy and direct model for loss and/or injury of cardiomyocytes, the main limitation of apical resection compared to myocardial infarction is that this surgical method cannot be performed beyond P7 due to high mortality6. There are, however, a number of ways to reduce mortality with this method. For apical surgery on P7 pups, the pups are cooled to 16&#176;C for optimum hypothermia anesthesia. Generally, sterile conditions and surgical proficiency are critical for successful apical resection surgery. To improve survival, the surgery should be performed as quickly and gently as possible and a minimally invasive operation will reduce bleeding. Additionally, ICR/CD-1 mice are thought as good foster mothers and can effectively avoid of severe maternal cannibalization. For example, placing the P0 C57BL/6 pups with an ICR/CD-1 mother increases the survival rates of P1 pups with apical resection approximately from 60% to 80%ref.6. In addition, cryoinjury is an alternative procedure for study of cardiac regeneration. Compared to apical resection, cryoinjury model represents a different regenerative response and may be a more reproducible model15, 16. Thus far, a comparison of apical resection with other models warrants further investigations. 
Using phospho-histone H3 and 5-bromo-2-deoxyuridine staining, Porrello and colleagues showed that cardiomyocyte proliferation contributes to the heart regeneration of neonatal P1 mice after apical resection response. Moreover, no significant cardiac hypertrophy and increase of cell size have been observed in mice with apical resection relative to sham-operated controls. After apical resection of 7-day-old mice, there is no regenerative response for myocardium5. A more detailed time course analysis of morphology and cardiac fibrosis after apical resection has been demonstrated5. Of note, the usefulness of apical resection in neonatal mouse models for heart regeneration research has been questioned by Anderson and colleagues8. Although many independent groups have demonstrated the reproducibility of apical resection, variations in the details of surgical procedures and different amounts of resected myocardium might be responsible for confusing results10. Quantification of the genomic DNA in resected heart is helpful for control of minimal chamber exposition and reproducibility of this technique. Additional research will be needed to fully understand neomyogenesis and complete vs. incomplete heart regeneration in the apical resection model8-11.
The main aim of this study is to present the procedures of apical resection in a visualized way. This video of apical resection in the neonatal mouse will be helpful to increase the use of this method and teach it to heart regeneration researchers. In addition, it may promote the pursuit of better approaches for mammalian heart regeneration in animal models and facilitate the discovery and translation of the molecular and cellular mechanisms responsible for heart regeneration in humans.

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

Kardiyovasküler hastalık nedeniyle yoğun yaşam tarzı değişiklikleri tüm dünyayı veba. Kalp rejenerasyon tamir nedeniyle yaralanma ve hastalık kaybetti kardiyomiyositlerde geri yükleme için büyük umut vaat ediyor. Bazı alt omurgalıların sağlam kardiyak rejenerasyon aksine, erişkin memeli kalpler tipik kalp rejenerasyon ve tamir için asgari kapasiteyi gösterir. Ancak son çalışmalar (P7 P1) 7-1 doğum sonrası gün arasında, yenidoğan fare kalp apikal rezeksiyon (yani cerrahi amputasyon ve sol ventrikül apeks maruz sonra) önemli rejeneratif potansiyelini korur, bulgu ile hatırı sayılır bir bilimsel ilgi uyandırmıştır. Bu bulgu üzerine bir büyük tartışma çoğaltmak veya bu önemli bulgu üzerine genişletmek çabalarında kullanılan çeşitli cerrahi ile ilgili prosedürlere bağlı olabilir. Bu talimatlar dinamik bir fare modelinde apikal rezeksiyon için malzeme ve metodoloji sunuyoruz. Bu kemirgen surviva belirgin adımlarıl cerrahi hipotermi anestezi, torakotomi, kalp ventrikül cerrahi amputasyon ve dikiş ve farelerin kurtarma içerir. Kardiyovasküler araştırmalar için apikal rezeksiyon fare modelinin uygulanmasını genişletmek olabilir açıklanan yaklaşım.

Fare yavrular 1, 2, ve 21 gün sonrası apikal rezeksiyon ötenazi ve kalpleri H &amp; E ve Masson Trikrom leke toplandı. Masson trikrom boyamada Mavi renk epikardiyal ekstraselüler matriks 5 birikimini gösterir. Şekil 1A gösterildiği gibi başarılı apikal rezeksiyon, bir kan pıhtısı etkili LV, bir gün sonrası apikal rezeksiyon kapamak için oluşturulmuştur. Kan pıhtısı ve erken kalp fibrozis kademeli erimesi gözlenir ve miyokardın tam rejenerasyon 21 gün apikal rezeksiyon sonrası (Şekil 1B ve C) gerçekleşir kadar miyokard dokusu tarafından yerine oluşur. Morfolojik analiz 21 gün cerrahi operasyon sonrası (Şekil 1C ve D) rezeke ve sham kalpleri arasında bir fark gösterir. <img alt="figür 1" src="/files/ftp_upload/53488/53488fig1.jpg" /> Şekil 1. Temsilcisi H &amp; E ve Masson enTrichrome leke. Yenidoğan fare kalpleri 1 (A), 2 (B) veya apikal rezeksiyon sonrasında 21 (C) gün ve Sham (D) sonra 21 gün Ventriküler miyokard. Her alt çizim için, sol panelin Ölçek çubuğu 1 mm ve sağ panelin ölçek çubuğu 100 mikron olduğunu. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53488/53488fig1large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a>

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.

Apikal Rezeksiyon Fare Modeli Erken Memeli Kalp Rejenerasyon Eğitim

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

Nanyang Technological University

Research

JoVE Journal

Medicine

11.2K Görüntüleme.  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

Imaging Cleared Embryonic and Postnatal Hearts at Single-cell Resolution

Single clonal tracing and analysis at the whole-heart level can determine cardiac progenitor cell behavior and differentiation during cardiac development, and allow for the study of the cellular and molecular basis of normal and abnormal cardiac morphogenesis. Recent emerging technologies of retrospective single clonal analyses make the study of cardiac morphogenesis at single cell resolution feasible. However, tissue opacity and light scattering of the heart as imaging depth is increased hinder whole-heart imaging at single cell resolution. To overcome these obstacles, a whole-embryo clearing system that can render the heart highly transparent for both illumination and detection must be developed. Fortunately, in the last several years, many methodologies for whole-organism clearing systems such as CLARITY, Scale, SeeDB, ClearT, 3DISCO, CUBIC, DBE, BABB and PACT have been reported. This lab is interested in the cellular and molecular mechanisms of cardiac morphogenesis. Recently, we established single cell lineage tracing via the ROSA26-CreERT2; ROSA26-Confetti system to sparsely label cells during cardiac development. We adapted several whole embryo-clearing methodologies including Scale and CUBIC (clear, unobstructed brain imaging cocktails and computational analysis) to clear the embryo in combination with whole mount staining to image single clones inside the heart. The heart was successfully imaged at single cell resolution. We found that Scale can clear the embryonic heart, but cannot effectively clear the postnatal heart, while CUBIC can clear the postnatal heart, but damages the embryonic heart by dissolving the tissue. The methods described here will permit the study of gene function at a single clone resolution during cardiac morphogenesis, which, in turn, can reveal the cellular and molecular basis of congenital heart defects.

We describe a protocol to volumetrically image fluorescent protein labeled cells deep inside intact embryonic and postnatal hearts. Utilizing tissue-clearing methods in combination with whole mount staining, single fluorescent protein-labeled cells inside an embryonic or postnatal heart can be imaged clearly and accurately.

Görüntüleme Tek hücreli Çözünürlük embriyonik ve Postnatal Kalpleri Temizlendi

Single clonal tracing and analysis at the whole-heart level can determine cardiac progenitor cell behavior and differentiation during cardiac development, ...

Gelişim Biyolojisi

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.

Yenidoğan Fare Kardiyak Makrofajların Yetişkin Farelere Nakli

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

Biyokimya

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.

Temizlenmiş Kalp Üç Boyutlu Görüntüleme ile Hedefhücre Popülasyonlarının Kardiyak Yaralanma Yanıtının Yakalanması

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

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 ...

Live Imaging of Early Cardiac Progenitors in the Mouse Embryo

The first steps of heart development imply drastic changes in cell behavior and differentiation. While analysis of fixed embryos allows studying in detail specific developmental stages in a still snapshot, live imaging captures dynamic morphogenetic events, such as cell migration, shape changes, and differentiation, by imaging the embryo as it develops. This complements fixed analysis and expands the understanding of how organs develop during embryogenesis. Despite its advantages, live imaging is rarely used in mouse models because of its technical challenges. Early mouse embryos are sensitive when cultured ex vivo and require efficient handling. To facilitate a broader use of live imaging in mouse developmental research, this paper presents a detailed protocol for two-photon live microscopy that allows long-term acquisition in mouse embryos. In addition to the protocol, tips are provided on embryo handling and culture optimization. This will help understand key events in early mouse organogenesis, enhancing the understanding of cardiovascular progenitor biology.

We present a detailed protocol for mouse embryo culture and imaging that enables 3D + time imaging of cardiac progenitor cells. This video-toolkit addresses the key skills required for successful live imaging otherwise hard to acquire from text-only publications.

Fare Embriyosundaki Erken Kardiyak Progenitörlerin Canlı Görüntülenmesi

The first steps of heart development imply drastic changes in cell behavior and differentiation. While analysis of fixed embryos allows studying in detail ...

Fare Kalplerinde Kardiyak Hücrelerin Elektroporasyona Dayalı Transgenezi

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.

Yazar Spotlight: Hızlı Gen Taraması için Kardiyak Hücre Transgenezi

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.

Fare Embriyonik Kalbinde Gen İletimi için Etkili Bir Transgenez Yaklaşımı

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.

Yenidoğan Farelerde Miyokard Enfarktüsü, Kalp Rejenerasyon Bir Model

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 ...

Tıp

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.

Miyokard Enfarktüsü'nün Murine Modelinde 3Boyutlu Biyobaskılı Yama Transplantasyonu

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

Apikal Rezeksiyon Fare Modeli Erken Memeli Kalp Rejenerasyon Eğitim

Özet

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Bu videodaki bölümler

Görüntüleme Tek hücreli Çözünürlük embriyonik ve Postnatal Kalpleri Temizlendi

Yenidoğan Fare Kardiyak Makrofajların Yetişkin Farelere Nakli

Temizlenmiş Kalp Üç Boyutlu Görüntüleme ile Hedefhücre Popülasyonlarının Kardiyak Yaralanma Yanıtının Yakalanması

Clonal Analysis of the Neonatal Mouse Heart using Nearest Neighbor Modeling

Fare Embriyosundaki Erken Kardiyak Progenitörlerin Canlı Görüntülenmesi

Fare Embriyonik Kalbinde Gen İletimi için Etkili Bir Transgenez Yaklaşımı

Fare Embriyonik Kalbinde Gen İletimi için Etkili Bir Transgenez Yaklaşımı

Fare Embriyonik Kalbinde Gen İletimi için Etkili Bir Transgenez Yaklaşımı

Yenidoğan Farelerde Miyokard Enfarktüsü, Kalp Rejenerasyon Bir Model

Miyokard Enfarktüsü'nün Murine Modelinde 3Boyutlu Biyobaskılı Yama Transplantasyonu