저자는 박사 감사합니다. 제임스 호킨스, 국립 심장, 폐에서 즈 - 사이 유와 법사 숨어 및 마우스 수술 준비와 파라핀 섹션의 염색과의 도움을 혈액 연구소 (NHLBI). 저자는 편집 지원에 대한 NIH 휄로우 편집위원회에 감사드립니다.

모든 마우스 실험 프로토콜 번호 H0083R3와 국립 보건원 (NIH)의 동물 관리 및 사용 프로그램에 의해 승인되었다. NHLBI IACUC는 진통제없이 프로토콜을 승인했다. 신생아 마우스 1. 저체온 마취 <ol><li> 수술 전에 오토 클레이브 스폰지 및 수술 장비 소독. 모든 수술 재료를 준비하고 270 ° C에 240 ° C에 도달하기 위해 사전에 뜨거운 구슬 살균기 15 ~ 20 분에 전환합니다. </li><li> 신선한 침구와 중첩 재료와 깨끗한 마우스 케이지에 자신의 간호 어머니의 모든 C57BL / 6 새끼 (연령 P1)를 전송합니다. 새끼는 수술 방으로 촬영하면, 자신의 시간은 어머니로부터 분리 된 시간에 산모 잠식의 위험을 줄이기 위해 최소화하기 위해 신속하게 혀끝 수술을 수행합니다. </li><li> 얼음 침대에 스폰지를 넣고 2 ~ 3 분 저체온 마취를 달성하기 위해 다음 스펀지에 한 강아지를 놓습니다. AP를 관찰하여 마취를 확인NEA와 무 운동과 후방 발을 곤란. 너무 적은 시간이 강아지의 무 운동성 및 apneic 및 생존율 (12)을 낮출 수 마취의 과도 기간을 확인하지 않습니다 자주하기 때문에 신생아의 상태를 확인합니다. </li></ol> 2. 개흉술 <ol><li> 수술 벤치 탑 지역에 얼음 침대에서 강아지를 전송하고 앙와위에서의 팔, 다리, 꼬리를 고정하는 테이프를 사용합니다. </li><li> betadine을 사용하여 가슴을 소독하고 부드럽게 70 % 알코올 준비 패드를 사용하여 청소합니다. </li><li> 스프링 Vannas 가위를 사용 흉강의 항 늑간 영역을 따라 횡 피부 절개를하고 심장에 대한 액세스를 용이하게하기 위해 기재된 늑간 근육을 무디게 절개. 참고 : 혈액 손실이 수술 과정에서 최소화 될 때 생존율을 향상시킬 수 있습니다. </li></ol> 심장 심실 에이펙스 3. 수술 절단 수술 <ol><li> 손으로 부드럽게를 외면 화하다하기 위해 복부에 압력을가마음의 정점. 명확한 시각화를위한 멸균 면봉 어플리케이터와 외과 영역의 주위에 피를 흡수한다. 가짜 운영 제어 신생아의 경우, 4 (봉합 및 마우스의 복구) 단계로 직접 진행합니다. </li><li> 돋보기 램프와 가위를 사용하여 홍채 절제술에서 부드럽게 LV 실이 노출 될 때까지 좌심실 (LV)의 단편적인 절제술을 수행합니다. LV의 절제 부분을 최소화하기 위해주의해야합니다. 약 15 %의 절제는 LV 챔버의 최적의 노출을 달성 할 필요가있다. </li><li> LV 실이 노출되면 심장이 흉강에 반환해야합니다. </li></ol> 4. 봉합 및 마우스의 복구 <ol><li> 멸균 프롤린 6-0 봉합사를 사용 흉강을 함께 밀봉 립과 근육을 봉합 한 후 조심스럽게 피부 접착제를 이용하여 피부 절개 부위를 닫는다. </li><li> 복구 ~ 3 분 동안 가열 램프하에 신생아를 따뜻하게 한 후 70 % 알코올을 준비 사이클로 작업하기 전에 패드를 사용하여 혈액과 접착제 흔적을 깨끗하게그 한배 새끼에 재 소개 재. 어머니로부터 분리 소요되는 시간을 최소화하는 것이 치근단 절제술 후 강아지의 생존을 향상하기 때문에 10 분 이내에 전체 수술 절차를 완료하려고합니다. </li><li> 각각의 수술 후, 완전한 살균 약 20 초 동안 뜨거운 구슬 살균기의 구슬과 접촉 수술 도구를 배치합니다. 수술 도구는 각각의 수술 전에 실온으로 냉각 할 수 있습니다. </li><li> 쓰레기의 모든 새끼에 대한 수술을 완료 한 후, 어머니의 둥지로 돌아 오기 전에 어머니의 침구와 배설물로 새끼를 섞는다. 참고 : 일반적으로, ICR / CD-1 마우스는 더 나은 위탁 C57BL / 6보다 어머니,하지만 심지어 같은 유전 적 배경의 간호 어머니 사이는, 육성 본능에 따라 다릅니다. 간호 어머니를 변경하면, 산모의 잠식에 의해 사망​​률을 감소 간호 어머니 P0 새끼를 제거한 다음 (P1)에 혀끝의 절제술을 수행 할 필요가있는 경우. 만 허위 조작이나 혀끝의 연구를 수행새끼의 한 쓰레기에서 esection, 혼합 가짜 수술 새끼가 새끼 6 수술 군의 생존율을 감소 할 수 있기 때문이다. </li></ol> 5. 수술 후 분석 <ol><li> 하루 수술 후, 새끼를 모니터링하고 가짜 수술 군 간의 차이가 없다는 것을 확인하고 생존율을 측정하는 새끼의 개수를 카운트. 수술이 적절하게 수행되는 경우, 생존율은 가짜 수술 군 모두에서 유사한 60 % 이상이어야한다. </li><li> 심장 격리 및 고정. <ol><li> 일 1에서 새끼 자궁 전위에 의해 사망​​ 보장과 이산화탄소에 의해 참수하고 21 일 후 수술을 통해 2 후 수술을 안락사, 다음 70 % 알코올 준비를 사용하여 가슴을 청소합니다. </li><li> 가슴의 중간 선 피부와 근육을 절개 한 후 가슴을 엽니 다. </li><li> 흉강에서 전체의 마음을 절제하고 4 % 파라 포름 알데히드의 O / RT에서 N 5ml에 각 샘플의 전체 마음을 고정한다. </li></ol></li><li> 다음날, 70 % 에탄올로 샘플들을 전달. 샘플은 파라핀 삽입하기 전에 일주까지 저장할 수 있습니다. </li><li> 슬라이스는 5 μm의 두께의 파라핀 전체 심실을 통해 섹션과 회생 응답 5, 7을 검토 표준 헤 마톡 실린 및 에오신 (H &amp; E)과 메이슨의 트리 크롬 염색을 수행한다. 특히, H &amp; E 염색은 근육의 교체를 검토하는 데 사용되며 메이슨의 트리 크롬 염색이 사용됩니다 섬유 성 반응 (5)을 조사한다. </li><li> 관심의 영역을 선택하여 수동으로 슬라이드 스캐너를 사용하여 샘​​플 당 적어도 세 초점을 설정합니다. 이미지 및 디폴트 매개 변수와 제조자의 지시에 따라 40 × 배율에서 염색 슬라이드를 분석한다. </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>

이 기사에 대한 출판 비용은 박사 지앤 허우에 파인 과학 도구의 선물 후원했다.

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

심혈관 질환으로 인해 집중적 인 생활 습관의 변화로 전 세계를 재앙. 심장 재생은 수리 인한 부상과 질병에 손실 심근 세포를 복원하는 큰 약속을 보유하고있다. 특정 낮은 척추 동물의 강력한 심장 재생과는 달리, 성인 포유류의 마음은 일반적으로 심장 재생 및 수리를위한 최소한의 용량을 보여줍니다. 그러나, 최근의 연구 (P7에 P1) 1-7 출생 후의 일 사이에, 신생아 마우스 마음이 혀끝의 절제 (즉, 수술 절단 및 좌심실 정점의 노출) 후 상당한 회생 가능성을 유지, 연구 결과와 상당한 과학적 관심을 촉발했다. 이 발견을 통해 하나의 주요 논쟁은 복제하거나이 중요한 발견에 확장하기위한 노력에 사용되는 다양한 수술 관련 절차로 인해 수 있습니다. 이 지침은 동적으로 마우스 모델에서 혀끝의 절제에 대한 자료와 방법론을 제시한다. 이 설치류 surviva의 두드러진 단계L 수술은 저체온 마취, 개흉술, 심장 심실 정점의 수술 절단 및 봉합와 마우스의 회복을 포함한다. 심장 혈관 연구를위한 치근단 절제술 마우스 모델의 응용 프로그램을 확장 할 수 설명하는 방법.

마우스 새끼는 1, 2, 이십일일 후 치근단 절제술을 안락사시키고, 그들의 마음을 H &amp; E 및 메이슨 트리 크롬 염색의 수집 하였다. 메이슨의 트리 크롬 염색 블루 색상은 심 외막 세포 외 기질 (5)의 증착을 나타냅니다. 도 1a에 도시 된 바와 같이 성공적인 치근단 절제술, 혈병을 효과적으로, LV 일일 후 치근단 절제술을 밀봉하도록 형성된다. 혈액 응고 초기 심장 섬유화의 점진?...

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.

치근단 절제술 마우스 모델은 초기 포유류의 심장 재생을 연구하는

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 조회수.  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.

비디오: 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.

영상은 단일 셀 해상도에서 배아 및 출생 후의 마음을 해제

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

연구

발생학

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.

신생아 마우스 심장 대식세포를 성인 마우스로 이식

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

생화학

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.

클리어 심장 3 차원 이미징을 통해 표적 세포 집단의 심장 손상 반응을 캡처

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.

생쥐 배아에서 초기 심장 전구체의 생생한 영상

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

Electroporation-based transgenesis of Cardiac cells in mouse hearts(생쥐 심장의 심장 세포의 전기천공법 기반 형질전환)

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.

저자 스포트라이트: 신속한 유전자 스크리닝을 위한 심장 세포 형질전환(Cardiac Cell Transgenesis)

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.

마우스 배아 심장에서 유전자 전달을 위한 효율적인 transgenesis 접근법

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.

신생아 쥐의 심근 경색, 심장 재생의 모델

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 ...

의학

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.

심근 경색의 뮤린 모델에서 3D 생체 인쇄 패치의 이식

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

치근단 절제술 마우스 모델은 초기 포유류의 심장 재생을 연구하는

요약

더 많은 비디오 탐색

이 비디오의 챕터

영상은 단일 셀 해상도에서 배아 및 출생 후의 마음을 해제

신생아 마우스 심장 대식세포를 성인 마우스로 이식

클리어 심장 3 차원 이미징을 통해 표적 세포 집단의 심장 손상 반응을 캡처

Clonal Analysis of the Neonatal Mouse Heart using Nearest Neighbor Modeling

생쥐 배아에서 초기 심장 전구체의 생생한 영상

마우스 배아 심장에서 유전자 전달을 위한 효율적인 transgenesis 접근법

마우스 배아 심장에서 유전자 전달을 위한 효율적인 transgenesis 접근법

마우스 배아 심장에서 유전자 전달을 위한 효율적인 transgenesis 접근법

신생아 쥐의 심근 경색, 심장 재생의 모델

심근 경색의 뮤린 모델에서 3D 생체 인쇄 패치의 이식