Name: recovery of adult zebrafish hearts for high-throughput applications
Uploaded: 2014-12-12T15:00:00.000+00:00
Duration: 6 min 51 s
Description: Watch this Scientific Journal Video about Recovery of Adult Zebrafish Hearts for High-throughput Applications at JoVE.com

The authors would like to thank Dr. Shaun Coughlin for hosting the filming of this procedure in his laboratory, and for general support. R.A. was supported by the NIH (F32HL110489) and the Sarnoff Cardiovascular Research Foundation. S.R. was supported by a Research Fellowship of the Deutsche Forschungsgemeinschaft (DFG) and the American Heart Association (AHA). D.Y.R.S was supported by the NIH (RO1HL54737), the Packard Foundation, and the Max Planck Society.

참고 : 항상 IACUC 또는 윤리위원회의 승인이 제브라 피쉬를 사용하여 실험 절차를 시작하기 전에 제자리에 있는지 확인하십시오. 1. 시약 및 설치 준비 <ol><li> 다음과 같은 솔루션을 준비합니다. 표준 제브라 피쉬 매뉴얼 (10)에서 이러한 모든 솔루션에 대한 조리법을 찾을 수 있습니다. <ul><li> 계란 물 500㎖ </li><li> 계란 물에 0.03 % Tricaine 200 ㎖ </li><li> 1X 인산염 완충 식염수 100 ㎖ (PBS) </li><li> RBC 용해 버퍼 (선택 사항) </li><li> 얼음에 1.5 ml의 미세 원심 튜브에 선택의 대상 버퍼 (정착액, 트리 졸, PBS 등) 주 : 피부와 접촉하면 정착액 및 트리 졸은 독성. 이 물질을 취급 할 때는 장갑을 착용 할 것. </li></ul></li><li> 해부 설치를 준비합니다 : <ol><li> 해부 현미경을 통해 구즈넥 광원을 배치합니다. 해부 현미경 무대에서 9cm 페트리 접시의 뚜껑을 놓고에 1X PBS의 얇은 층을 부어그것은 (요리 자체가 쉽게 해부 너무 깊은 때문에 뚜껑이 사용됩니다). </li><li> 해부 근처의 수조에 페트리 접시 자체 (안 뚜껑)을 놓고 그 안에 1X PBS의 약 15 ml에 붓는다. 절개 한 후 심장을 씻어 사용합니다. 또한, RBC 용해 버퍼를 사용합니다. </li><li> 장소 면도날, 두 집게, microscissors (옵션)의 쌍, 및 현미경 옆에 전송 피펫. 급속 냉각에 의한 안락사 선택하면 얼음의 컨테이너를 가져옵니다. </li><li> Tricaine 의해 안락사 선택하면 250 ㎖의 유리 비이커에 달걀 물 0.03 % Tricaine 200㎖를 붓고. 제브라 피쉬의 사체의 처분을위한 작은 생물 학적 가방을 준비합니다. </li></ol></li></ol> 2. Zebrafish의 준비 <ol><li> 고정 물고기를 들어, 현미경 설정에, PBS에 세척 고정 물고기를 가져온다. <ol><li> &gt; 10 분 동안 얼음과 급속 냉각하여 전체 성인 제브라 피쉬를 안락사. </li><li> 복막을 통해 피부에 구멍을 만들기 위해 집게를 사용하여(멀리 마음에서) 고정액의 침투를 돕기 다음 4 ° C에서 RT 또는 O / N에서 2 시간 동안 수정 버퍼 (10)에 4 % 파라 포름 알데히드에 넣어합니다. </li></ol></li><li> 또한, 신선한 물고기, 장소 물고기는 작은 저장 탱크에 안락사하고 현미경 설정에 가져다. 개별 시설의 물고기 프로토콜과 해부 물고기 마음의 다운 스트림 용도에 따라 Tricaine 물고기를 마취 또는 잘린 전에 급속 냉각에 의해 안락사. <ol><li> 물고기가 움직이지 않을 때까지 얼음 물에 물고기 그물과 장소에 약 5 분을 한 물고기를 선택, 얼음을 사용합니다. </li><li> 또한, 아가미의 움직임이 멈출 때까지 용액의 비커에 물고기 그물과 장소 하나 물고기를 선택, Tricaine를 사용합니다. </li></ol></li><li> 빠르게 꼬리 지느러미에 의해 안락사 물고기를 선택하고 1X PBS로 가득 차 있었다 페트리 접시 덮개에 옆으로 누워. razo를 사용하는 동안, 한 손으로 가슴 지느러미를 들어 올려 집게를 사용하여R 블레이드는 가슴 지느러미 (그림 1A)의 부착 후부, 다른 손으로 물고기를 목을 벨 수 있습니다. 현미경을 통해보고하거나 직접 현미경 무대에서 물고기를 시각화하여이 작업 단계를 수행합니다. 참고 : 갓 안락사 물고기 여전히 잘린 후 피합니다. </li></ol><img alt="그림 1" src="/files/ftp_upload/52248/52248fig1highres.jpg" /> 그림 1. Zebrafish의 성인 심장 해부는 제브라 피쉬의 해부학 적 구조를 사용한다. (A)는 물고기 목을 벨 집게와 가슴 지느러미를 들어 올려 그림과 같이 빨간색 점선을 따라 날카로운 깨끗한 면도날을 사용합니다. (B), 생선 머리를 안정 물고기 입 동안 포셉 한 가지를 배치하려면 다른 타인의 눈을 통해 거짓말을하고, 복부면이 위를하고 집게의 두 타인 페트리 접시의 바닥에 안정되도록하고 물고기 머리를 켜십시오. (C) 무료 그렇게해야 사용EPS는 아가미 (화살표). (D)이 리프팅의 첨부 파일을 잘라, 지느러미 대동맥 내강 혈액 (화살표)를 나타내는 핑크 스트라이프 흰색 구조로 볼 수있다. <a href="https://www.jove.com/files/ftp_upload/52248/52248fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> 3. 심장을 해부하다 <ol><li> 현미경으로 생선 머리를 시각화. 다른 타인이 눈 (그림 1B)를 통해, 머리 외부에있는 동안 하나의 집게로, 뇌를 통해 물고기의 입에 집게 중 하나 타인을 배치하여 물고기의 머리를 안정. 집게의 두 타인 페트리 접시의 바닥에 대한 안정세를 확인합니다. 물고기 머리를 이런 식으로 잡아서 복부 표면 위쪽으로 향해야합니다. </li><li> 다른 손으로, 몸 (그림 1C 화살표)에 아가미의 복부 첨부 파일을 잘라 두 번째 집게를 사용합니다. 집게와 약간이 리프팅, Dorsal 대동맥은 대동맥 루멘 (그림 1D 화살표)에 혈액의 빨간 줄무늬와 흰색 구조로 아래에 나타납니다. 대동맥 곤란 위쪽으로 잡아 당겨 집게 등의 대동맥을 잘라; 대안 등의 대동맥을 잘라 microscissors를 사용합니다. </li><li> 이제, 지느러미의 기지에서 몸 연골을 포함하여, 그 기지에서 가슴 지느러미를 파악하기 위해 두 번째 집게를 사용하고이 들어 올립니다. 다른 가슴 지느러미와 반복합니다. 때때로, 마음은 가슴 지느러미와 함께 나오는, 그래서 마음을 폐기하기 전에 연결되어 있지 않은 확인이 조각을 검사합니다. 참고 : (때때로는 가슴 지느러미와 벗기 있지만)이 시점에서, 마음이, 눈에 보이는 그대로 남은 생선의 머리에 연결해야합니다. 심장은 그 형상, 그 핑크 색상 및 색소 심낭에 의해 포위 됨으로써 확인할 수있다. 물고기의 안락사가 신속하게 수행되기 때문에, 갓 안락사 물고기 마음은 여전히​​ SLO를 구타 할 수있다wly. </li><li> 양손 집게를 보유하고 무료 있도록, 먼저 집게로 물고기의 머리 가자. 멀리 심낭의 마음을 애타게 두 집게를 사용합니다. 나머지 심낭 멀리 당겨 동안 지느러미 대동맥의 나머지에 의해 마음을 잡고. 참고 : 고정 또는 신선한 여부, 심장 주위의 결합 조직이 더 부서지기 쉬운 반면 당기 부드러운 강인한. 따라서, 모든 주변의 심낭 조직은 심장이 그대로 남아 멀리 해부 할 수 있습니다. </li><li> 생물 학적 가방에 남은 물고기 시체를 버리십시오. </li></ol> 4. 다운 스트림 응용 프로그램에 대한 마음을 준비 <ol><li> 집게를 사용하여, 지느러미 대동맥 / bulbus 동맥의 가장자리에 마음을 잡고 1X PBS의 신선한 접시에 마음을 놓습니다. 또한, 전송 피펫을 사용합니다. 앞뒤로 PBS의 마음을 움직 약 10 배 가능한 한 많은 피를 씻어. </li><li> 응용 프로그램의 모든 possib을 제거하는 것이 중요하다있는르 혈액 세포는 동일한 전후 모션을 사용하는 대신에 PBS 15 ml의 용해 완충액으로 RBC 9cm 페트리 접시에 씻는다. 심장의 구조를 유지하는 (예를 들어, RNA 제조), 심장 공동을 열고 멀리 혈구 린스 용이하게 집게 나 microscissors를 사용 하류 애플리케이션에 중요하지 않은 경우. </li><li> 별도의 심장 챔버가 요구되는 응용 프로그램의 경우, 서로 떨어져 bulbus 개존증, 심방 및 심실을 해부 집게 또는 microscissors를 사용합니다. </li><li> 얼음에 대상 버퍼에 심장, 또는 별도의 챔버를 전송합니다. 참고 : 일반 대상은 세포 분리, 4 % 파라 포름 알데히드, 또는 트리 졸에 대한 버퍼를 포함한다. </li></ol>

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	<li>Arnaout, 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=Zebrafish+model+for+human+long+QT+syndrome.">Zebrafish model for human long QT syndrome.</a> Proceedings of the National Academy of Sciences of the United States of America. 104 (27), 11316-11321 (2007).</li><li>Chi, N. C., 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=Genetic+and+physiologic+dissection+of+the+vertebrate+cardiac+conduction+system.">Genetic and physiologic dissection of the vertebrate cardiac conduction system.</a> PLoS Biology. 6 (5), 109 (2008).</li><li>Poss, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Getting+to+the+heart+of+regeneration+in+zebrafish.">Getting to the heart of regeneration in zebrafish.</a> Seminars in Cell &amp; Developmental Biology. 18 (1), 36-45 (2007).</li><li>Fang, Y., 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=Translational+profiling+of+cardiomyocytes+identifies+an+early+Jak1/Stat3+injury+response+required+for+zebrafish+heart+regeneration.">Translational profiling of cardiomyocytes identifies an early Jak1/Stat3 injury response required for zebrafish heart regeneration.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (33), 13416-13421 (2013).</li><li>Manoli, M., Driever, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence-activated+cell+sorting+(FACS)+of+fluorescently+tagged+cells+from+zebrafish+larvae+for+RNA+isolation.">Fluorescence-activated cell sorting (FACS) of fluorescently tagged cells from zebrafish larvae for RNA isolation.</a> Cold Spring Harbor Protocols. 2012 (8), (2012).</li><li>Cannon, J. E., 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=Global+analysis+of+the+haematopoietic+and+endothelial+transcriptome+during+zebrafish+development.">Global analysis of the haematopoietic and endothelial transcriptome during zebrafish development.</a> Mechanisms of Development. 130 (2-3), 122-131 (2013).</li><li>Wang, Z., Gerstein, M., Snyder, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-Seq:+a+revolutionary+tool+for+transcriptomics.">RNA-Seq: a revolutionary tool for transcriptomics.</a> Nature Reviews Genetics. 10 (1), 57-63 (2009).</li><li>Gupta, T., Mullins, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+organs+from+the+adult+zebrafish.">Dissection of organs from the adult zebrafish.</a> Journal of Visualized Experiments. 37, (2010).</li><li>Singleman, C., Holtzman, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+dissection+in+larval,+juvenile+and+adult+zebrafish,+Danio+rerio.">Heart dissection in larval, juvenile and adult zebrafish, Danio rerio.</a> Journal of Visualized Experiments. 55, (2011).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The zebrafish book: a guide for the laboratory use of zebrafish (Brachydanio rerio. , (1993).</li></ol>

The authors have no disclosures.

성인 zebrafish의 심장을 해부하는 방법이 설명되어 있지만,이 방법은 시간이 많이 소요되었고 일반적으로 해부하는 동안 심장에 손상을 일으키는 원인이되었다. 성인 하트 다수 필요할 수있는 실험을 수행하기 위해 및 / 또는 심장 조직의 열화를 피하는 하류 응용에 중요 할 때, 전통적인 절개 기술을 사용하여 요구되는 시간은 금지된다. 마찬가지로, 재현성 손상되지 않은 그대로의 마음을 얻는 ?...

Zebrafish are an excellent model for studying heart development and human disease1,2. Specific advantages include the translucent nature of zebrafish embryos, the availability of many genetic mutants and transgenic reporter lines, and the availability of genome editing technologies. In addition to their advantages for studying early heart development, zebrafish are an ideal system for studying vertebrate heart regeneration3.
More recently, adult zebrafish are playing an important part in bioinformatics approaches to studying cardiovascular development and disease, due to their relatively large clutch size and relatively quick and inexpensive breeding compared to other vertebrate models. Promising techniques include ribosome profiling, RNA-Seq, and cell dissociation and FACS sorting4-7. However, for these techniques the quality of the data can depend on obtaining a large number of samples in a rapid, efficient, and reproducible manner, before gene regulatory, metabolic, transcriptional, and other changes occur.
Dissection of adult zebrafish organs has been described in the past8,9. However, previous approaches to dissection of the heart were slow, ran the risk of damaging the heart during dissection, required special tools, and/or required fixation of the zebrafish prior to dissection; for these reasons, past approaches to zebrafish adult heart dissection were not optimized for high-throughput applications and/or applications requiring fresh tissue.
Here, we describe a method for adult zebrafish heart dissection that is simple, fast, efficient, and reproducible, while preserving cardiac morphology. This method does not include cutting into the pericardial space and therefore does not risk damaging the heart during dissection. Instead, this method relies on anatomical landmarks of the zebrafish, and therefore, it is highly reproducible. This dissection method is also versatile in that it can be used on fresh or fixed fish, and on zebrafish as young as one month old. Finally, this method results in minimal suffering to the zebrafish because after anesthesia and/or rapid cooling, the fish is additionally decapitated and pithed in the course of the dissection procedure.

<table><tbody><tr><td>Small tank for transporting fish</td><td>Aquaneering</td><td>ZHCT100</td><td></td></tr><tr><td>Fish net</td><td>Petsmart</td><td>36-16731</td><td></td></tr><tr><td>250 ml glass beaker</td><td>Kimble</td><td>14005-250</td><td></td></tr><tr><td>9 cm polystyrene Petri dish</td><td>Nunc</td><td>172958</td><td></td></tr><tr><td>Razor blade</td><td>Personna American Safety Razor Company</td><td>94-120-71</td><td></td></tr><tr><td>2 Dumont #5SF forceps</td><td>Fine Science Tools</td><td>11252-00</td><td></td></tr><tr><td>Dissecting microscope</td><td>Olympus</td><td>SZX16</td><td></td></tr><tr><td>Tricaine</td><td>Sigma</td><td>A-5040</td><td></td></tr><tr><td>Plastic transfer pipette</td><td>Thermo Scientific</td><td>202-20S</td><td></td></tr><tr><td>Gooseneck light source</td><td>Dolan-Jenner Industries, Inc</td><td>Fiber-Lite 180 Illuminator, 181 Dual Gooseneck System</td><td></td></tr><tr><td>Fluorescent light source</td><td>Lumen Dynamics</td><td>X-Cite 120Q</td><td>optional</td></tr><tr><td>Micro-scissors</td><td>Biomedical Research Instruments, Inc</td><td>11-1000</td><td>optional</td></tr><tr><td>RBC lysis buffer</td><td>eBioscience</td><td>00-4333-57</td><td>optional</td></tr></tbody></table>

recovery of adult zebrafish hearts for high-throughput applications

개발, 재생을 공부하고, 질병이 세포 분리 및 RNA, DNA, 단백질의 정제를위한 성인 마음의 사용으로 확대되고의 제브라 피쉬 모델 시스템의 사용. 이러한 응용 프로그램의 모든 규제 유전자, 대사, 그리고 죽음 이후 시작하는 다른 변경을 방지하기 위해 제브라 피쉬 마음의 상당한 숫자의 신속한 복구를 요구하고있다. 성인 zebrafish의 마음은 돌연변이의 다양한 심장 구조를 공부하고 심장 재생을 공부해야합니다. 그러나, 기존의 제브라 피쉬의 심장 해부는 느리고 어려운 성인 zebrafish의 마음의 대규모 해부 지루한 제작, 특수 도구가 필요합니다. 전통적인 방법은 해부 동안 심장 손상의 위험을 항구. 여기, 우리는 빠른 재현, 심장 구조를 유지 성인 zebrafish의 마음의 절개하는 방법을 설명합니다. 또한,이 방법은 특수 도구가 필요하지 않습니다, 제브라 피쉬에 대한 통증이수 신선 또는 고정 시험편상에서 수행 될, 1 개월 이전처럼 어린 지브라 피쉬 수행 가능하다. 기재된 접근 방법은 심혈관 연구 성인 지브라 피쉬의 사용을 확장한다.

이 방법을 사용하여, 성인 심장 지브라 피쉬는 전통적인 방법을 사용하여 8 분에 걸쳐 5에 비해, 1 분 이하로 해부 될 수있다. 전통적인 방법 (8) 심낭에 맹목적으로 절단하기 때문에 일반적으로 심방 또는 bulbus 동맥 (그림 2B)의 손상이나 손실이 발생할 필요로하지만이 방법을 사용하여 해부 하트 (그림 2A) 안정적으로 그대로입니다. 해부 하트 그들?...

Watch this Scientific Journal Video about Recovery of Adult Zebrafish Hearts for High-throughput Applications at JoVE.com

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

높은 처리량 응용 프로그램에 대한 성인 Zebrafish의 마음의 복구

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

recovery-of-adult-zebrafish-hearts-for-high-throughput-applications

Research

JoVE Journal

Biology

12.2K 조회수.  University of California San Francisco. Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

비디오: Recovery of Adult Zebrafish Hearts for High-throughput Applications

Large-scale Zebrafish Embryonic Heart Dissection for Transcriptional Analysis

The zebrafish embryonic heart is composed of only a few hundred cells, representing only a small fraction of the entire embryo. Therefore, to prevent the cardiac transcriptome from being masked by the global embryonic transcriptome, it is necessary to collect sufficient numbers of hearts for further analyses. Furthermore, as zebrafish cardiac development proceeds rapidly, heart collection and RNA extraction methods need to be quick in order to ensure homogeneity of the samples. Here, we present a rapid manual dissection protocol for collecting functional/beating hearts from zebrafish embryos. This is an essential prerequisite for subsequent cardiac-specific RNA extraction to determine cardiac-specific gene expression levels by transcriptome analyses, such as quantitative real-time polymerase chain reaction (RT-qPCR). The method is based on differential adhesive properties of the zebrafish embryonic heart compared with other tissues; this allows for the rapid physical separation of cardiac from extracardiac tissue by a combination of fluidic shear force disruption, stepwise filtration and manual collection of transgenic fluorescently labeled hearts.

To analyse cardiac gene expression profiles during zebrafish heart development, total RNA has to be extracted from isolated hearts. Here, we present a protocol for collecting functional/beating hearts by rapid manual dissection from zebrafish embryos to obtain cardiac-specific mRNA.

전사 분석을위한 대규모 Zebrafish의 배아 심장의 해부

The zebrafish embryonic heart is composed of only a few hundred cells, representing only a small fraction of the entire embryo. Therefore, to prevent the ...

연구

발생학

Induction of Myocardial Infarction in Adult Zebrafish Using Cryoinjury

The mammalian heart is incapable of significant regeneration following an acute injury such as myocardial infarction1. By contrast, urodele amphibians and teleost fish retain a remarkable capacity for cardiac regeneration with little or no scarring throughout life2,3. It is not known why only some non-mammalian vertebrates can recreate a complete organ from remnant tissues4,5. To understand the molecular and cellular differences between regenerative responses in different species, we need to use similar approaches for inducing acute injuries.
In mammals, the most frequently used model to study cardiac repair has been acute ischemia after a ligation of the coronary artery or tissue destruction after cryoinjury6,7. The cardiac regeneration in newts and zebrafish has been predominantly studied after a partial resection of the ventricular apex2,3. Recently, several groups have established the cryoinjury technique in adult zebrafish8-10. This method has a great potential because it allows a comparative discussion of the results obtained from the mammalian and non-mammalian species.
Here, we present a method to induce a reproducible disc-shaped infarct of the zebrafish ventricle by cryoinjury. This injury model is based on rapid freezing-thawing tissue, which results in massive cell death of about 20% of cardiomyocytes of the ventricular wall. First, a small incision was made through the chest with iridectomy scissors to access the heart. The ventricular wall was directly frozen by applying for 23-25 seconds a stainless steel cryoprobe precooled in liquid nitrogen. To stop the freezing of the heart, fish water at room temperature was dropped on the tip of the cryoprobe. The procedure is well tolerated by animals, with a survival rate of 95%.
To characterize the regenerative process, the hearts were collected and fixed at different days after cryoinjury. Subsequently, the specimen were embedded for cryosectioning. The slides with sections were processed for histological analysis, in situ hybridization and immunofluorescence. This undertaking enhances our understanding of the factors that are required for the regenerative plasticity in the zebrafish, and provide new insights into the machinery of cardiac regeneration. A conceptual and molecular understanding of heart regeneration in zebrafish will impact both developmental biology and regenerative medicine.

Zebrafish represents a valuable model to study the mechanisms of heart regeneration in vertebrates. Here, we present a protocol for induction of a heart infarct in adult zebrafish using cryoinjury. This method results in massive cell death within 20% of the ventricular wall, similar to that observed in mammalian infarcts.

성인 Zebrafish에서 심근 경색의 유도는 Cryoinjury 사용

The mammalian heart is incapable of significant regeneration following an acute injury such as myocardial infarction1. By contrast, urodele amphibians and ...

의학

High-Frequency Ultrasound Echocardiography to Assess Zebrafish Cardiac Function

The zebrafish (Danio rerio) has become a very popular model organism in cardiovascular research, including human cardiac diseases, largely due to its embryonic transparency, genetic tractability, and amenity to rapid, high-throughput studies. However, the loss of transparency limits heart function analysis at the adult stage, which complicates modeling of age-related heart conditions. To overcome such limitations, high-frequency ultrasound echocardiography in zebrafish is emerging as a viable option. Here, we present a detailed protocol to assess cardiac function in adult zebrafish by non-invasive echocardiography using high-frequency ultrasound. The method allows visualization and analysis of zebrafish heart dimension and quantification of important functional parameters, including heart rate, stroke volume, cardiac output, and ejection fraction. In this method, the fish are anesthetized and kept underwater and can be recovered after the procedure. Although high-frequency ultrasound is an expensive technology, the same imaging platform can be used for different species (e.g., murine and zebrafish) by adapting different transducers. Zebrafish echocardiography is a robust method for cardiac phenotyping, useful in the validation and characterization of disease models, particularly late-onset diseases; drug screens; and studies of heart injury, recovery, and regenerative capacity.

We describe a protocol to assess heart morphology and function in adult zebrafish using high-frequency echocardiography. The method allows visualization of the heart and subsequent quantification of functional parameters, such as heart rate (HR), cardiac output (CO), fractional area change (FAC), ejection fraction (EF), and blood inflow and outflow velocities.

제브라피시 심장 기능을 평가하는 고주파 초음파 심초음파

The zebrafish (Danio rerio) has become a very popular model organism in cardiovascular research, including human cardiac diseases, largely due to its ...

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work examining post-embryonic through adult cardiac development has been limited7-10. Examining the changing morphology of the maturing and aging heart are restricted by the lack of techniques available for staging and isolating juvenile and adult hearts. In order to analyze heart development over the fish's lifespan, we dissect zebrafish hearts at numerous stages and photograph them for further analysis11. The morphological features of the heart can easily be quantified and individual hearts can be further analyzed by a host of standard methods. Zebrafish grow at variable rates and maturation correlates better with fish size than age, thus, post-fixation, we photograph and measure fish length as a gauge of fish maturation. This protocol explains two distinct, size dependent dissection techniques for zebrafish, ranging from larvae 3.5mm standard length (SL) with hearts of 100&mu;m ventricle length (VL), to adults, with SL of 30mm and VL 1mm or larger. Larval and adult fish have quite distinct body and organ morphology. Larvae are not only significantly smaller, they have less pigment and each organ is visually very difficult to identify. For this reason, we use distinct dissection techniques.
We used pre-dissection fixation procedures, as we discovered that hearts dissected directly after euthanization have a more variable morphology, with very loose and balloon like atria compared with hearts removed following fixation. The fish fixed prior to dissection, retain in vivo morphology and chamber position (data not shown). In addition, for demonstration purposes, we take advantage of the heart (myocardial) specific GFP transgenic Tg(myl7:GFP)twu34 (12), which allows us to visualize the entire heart and is particularly useful at early stages in development when the cardiac morphology is less distinct from surrounding tissues. Dissection of the heart makes further analysis of the cell and molecular biology underlying heart development and maturation using in situ hybridization, immunohistochemistry, RNA extraction or other analytical methods easier in post-embryonic zebrafish. This protocol will provide a valuable technique for the study of cardiac development maturation and aging.

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

A clear, standardized method for dissection and isolation of the zebrafish heart at multiple developmental stages are described. Annotation and quantification techniques are also discussed.

애벌레의 심장 해부, 소년과 성인 Zebrafish, Danio rerio</em

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work ...

생물학

Immunostaining of Dissected Zebrafish Embryonic Heart 

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology and genetics 1,2. About 100-200 embryos are readily available every week from a single pair of adult fish. The transparent embryos that develop ex utero make them ideal for assessing cardiac defects 3. The expression of any gene can be manipulated via morpholino technology or RNA injection 4. Moreover, forward genetic screens have already generated a list of mutants that affect different perspectives of cardiogenesis 5. 
Whole mount immunostaining is an important technique in this animal model to reveal the expression pattern of the targeted protein to a particular tissue 6. However, high resolution images that can reveal cellular or subcellular structures have been difficult, mainly due to the physical location of the heart and the poor penetration of the antibodies. 
 Here, we present a method to address these bottlenecks by dissecting heart first and then conducting the staining process on the surface of a microscope slide. To prevent the loss of small heart samples and to facilitate solution handling, we restricted the heart samples within a circle on the surface of the microscope slides drawn by an immEdge pen. After the staining, the fluorescence signals can be directly observed by a compound microscope. 
Our new method significantly improves the penetration for antibodies, since a heart from an embryonic fish only consists of few cell layers. High quality images from intact hearts can be obtained within a much reduced procession time for zebrafish embryos aged from day 2 to day 6. Our method can be potentially extended to stain other organs dissected from either zebrafish or other small animals.

Immunostaining of Dissected Zebrafish Embryonic Heart

A rapid way to conduct immunostaining of zebrafish embryonic heart is described. Compared to the whole mount immunostaining approach, this method dramatically increases the penetration of the antibodies, which allows obtaining high resolution images that reveal cellular/subcellular structures in the heart within a much reduced processing time.

해부 Zebrafish 배아 심장의 Immunostaining

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology ...

가상 현실 상호 작용을 통한 제브라피시 심장 수축 분석

4D Light-sheet Imaging of Zebrafish Cardiac Contraction

Zebrafish is an intriguing model organism known for its remarkable cardiac regeneration capacity. Studying the contracting heart in vivo is essential for gaining insights into structural and functional changes in response to injuries. However, obtaining high-resolution and high-speed 4-dimensional (4D, 3D spatial + 1D temporal) images of the zebrafish heart to assess cardiac architecture and contractility remains challenging. In this context, an in-house light-sheet microscope (LSM) and customized computational analysis are&#160;used to overcome these technical limitations. This strategy, involving LSM system construction, retrospective synchronization, single cell tracking, and user-directed analysis, enables one to investigate the micro-structure and contractile function across the entire heart at the single-cell resolution in the transgenic Tg(myl7:nucGFP) zebrafish larvae. Additionally, we are able to further incorporate microinjection of small molecule compounds to induce cardiac injury in a precise and controlled manner. Overall, this framework allows one to track physiological and pathophysiological changes, as well as the regional mechanics at the single-cell level during cardiac morphogenesis and regeneration.

저자 스포트라이트: 심장 기능의 단일 세포 분석을 위한 제브라피시의 고해상도 4D 광시트 이미징 및 가상 현실

This protocol utilizes light-sheet imaging to investigate cardiac contractile function in zebrafish larvae and gain insights into cardiac mechanics through cell tracking and interactive analysis.

제브라피시 심장 수축의 4D 광시트 이미징

Zebrafish is an intriguing model organism known for its remarkable cardiac regeneration capacity. Studying the contracting heart in vivo is essential for ...

생체공학

Assessment of Swim Endurance and Swim Behavior in Adult Zebrafish

Due to their renowned regenerative capacity, adult zebrafish are a premier vertebrate model to interrogate mechanisms of innate spinal cord regeneration. Following complete transection of their spinal cord, zebrafish extend glial and axonal bridges across severed tissue, regenerate neurons proximal to the lesion, and regain their swim capacities within 8 weeks of injury. Recovery of swim function is thus a central readout for functional spinal cord repair. Here, we describe a set of behavioral assays to quantify zebrafish motor capacity inside an enclosed swim tunnel. The goal of these methods is to provide quantifiable measurements of swim endurance and swim behavior in adult zebrafish. For swim endurance, zebrafish are subjected to a constantly increasing water current velocity until exhaustion, and time at exhaustion is reported. For swim behavior assessment, zebrafish are subjected to low current velocities and swim videos are captured with a dorsal view of the fish. Percent activity, burst frequency, and time spent against the water current provide quantifiable readouts of swim behavior. We quantified swim endurance and swim behavior in wild-type zebrafish before injury and after spinal cord transection. We found that zebrafish lose swim function after spinal cord transection and gradually regain that capacity between 2 and 6 weeks post-injury. The methods described in this study could be applied to neurobehavioral, musculoskeletal, skeletal muscle regeneration, and neural regeneration studies in adult zebrafish.

Capable of functional recovery after spinal cord injury, adult zebrafish is a premier model system to elucidate innate mechanisms of neural regeneration. Here, we describe swim endurance and swim behavior assays as functional readouts of spinal cord regeneration.

성인 제브라 피쉬의 수영 지구력 및 수영 행동 평가

Due to their renowned regenerative capacity, adult zebrafish are a premier vertebrate model to interrogate mechanisms of innate spinal cord regeneration. ...

신경과학

Intrathoracic Injection for the Study of Adult Zebrafish Heart

The adult zebrafish heart provides a powerful model in cardiac regeneration research. Although the strength of this system is based on transgenic approaches, a rapid delivery of exogenous factors provides a complementary technique in functional studies. Here, we present a method that relies on administration of a few microliters of solution into the pericardial cavity without causing myocardial damage. Intrathoracic (IT) injections can efficiently deliver proteins and chemical compounds directly onto the heart surface. The injected substances diffuse through the epicardium into the underlying cardiac tissues. Compared to intraperitoneal (IP) injections, the main advantage of intrathoracic injections is the focal administration of the tested factors on the target organ. The delivery of molecules directly into the pericardium is a suitable strategy for studies of cardiac preconditioning and regeneration in adult zebrafish.

This method relies on the injection of 0.5&#8722;3 &#956;L of solution into the thorax of adult zebrafish. The procedure efficiently delivers proteins and chemical compounds into the proximity of the zebrafish heart without damaging the organ. The approach is suitable for testing effects of exogenous factors on various tissues of the heart.

높은 처리량 응용 프로그램에 대한 성인 Zebrafish의 마음의 복구

요약

더 많은 비디오 탐색

이 비디오의 챕터

전사 분석을위한 대규모 Zebrafish의 배아 심장의 해부

성인 Zebrafish에서 심근 경색의 유도는 Cryoinjury 사용

제브라피시 심장 기능을 평가하는 고주파 초음파 심초음파

애벌레의 심장 해부, 소년과 성인 Zebrafish, Danio rerio

해부 Zebrafish 배아 심장의 Immunostaining

제브라피시 심장 수축의 4D 광시트 이미징

제브라피시 심장 수축의 4D 광시트 이미징

제브라피시 심장 수축의 4D 광시트 이미징

성인 제브라 피쉬의 수영 지구력 및 수영 행동 평가

성인 제 브라 피시 하트 연구를 위한 흉부 내 주사