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.

ملاحظة: تأكد دائما أن موافقة لجنة IACUC أو الأخلاق هي في مكان قبل البدء في أي إجراء التجارب باستخدام الزرد. 1. إعداد الكواشف والإعداد <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إعداد الحلول التالية. العثور على وصفات لجميع هذه الحلول في كتيبات الزرد القياسية 10. <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> 500 مل من البيض المياه </li><li style=";text-align:right;direction:rtl"> 200 مل من 0.03٪ تريكين في البيض المياه </li><li style=";text-align:right;direction:rtl"> 100 مل من 1X الفوسفات مخزنة المالحة (PBS) </li><li style=";text-align:right;direction:rtl"> RBC الاحتياطي تحلل (اختياري) </li><li style=";text-align:right;direction:rtl"> عازلة الوجهة المفضلة (تثبيتي، Trizol، PBS، الخ) في أنبوب 1.5 مل microfuge على الجليد ملاحظة: تثبيتي، وTrizol سامة إذا كانت تتلامس مع الجلد. ارتداء القفازات عند التعامل مع هذه المواد. </li></ul></li><li style=";text-align:right;direction:rtl"> إعداد الإعداد تشريح: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ضع مصدر الضوء معقوفة على المجهر تشريح. وضع غطاء من 9 سم طبق بتري على المسرح المجهر تشريح، وتصب طبقة رقيقة من برنامج تلفزيوني 1X إلىأنه (يتم استخدام غطاء لأن الطبق نفسه هو عميق جدا لتشريح السهل). </li><li style=";text-align:right;direction:rtl"> وضع طبق بتري نفسها (وليس غطاء) على كونترتوب بالقرب من نطاق تشريح وتصب حوالي 15 مل من برنامج تلفزيوني 1X في ذلك. استخدام هذا لغسل القلب بعد تشريح. بدلا من ذلك، استخدم RBC الاحتياطي تحلل. </li><li style=";text-align:right;direction:rtl"> مكان شفرة حلاقة، واثنين من أزواج من ملقط، microscissors (اختياري)، وماصة نقل بجانب المجهر. الحصول على وعاء من الجليد إذا ما تم اختيار القتل الرحيم التي كتبها التبريد السريع. </li><li style=";text-align:right;direction:rtl"> صب 200 مل من 0.03٪ تريكين في البيض الماء في كوب 250 مل دورق إذا ما تم اختيار القتل الرحيم التي كتبها تريكين. إعداد حقيبة واقية صغيرة للتخلص من جثث الزرد. </li></ol></li></ol> 2. إعداد الزرد <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> وبالنسبة للأسماك ثابتة، وجلب الأسماك ثابتة، تشطف في برنامج تلفزيوني، إلى الإعداد المجهر. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> الموت ببطء الزرد الكبار كله التبريد السريع مع الثلج ل&gt; 10 دقيقة. </li><li style=";text-align:right;direction:rtl"> استخدام ملقط لإحداث ثقب في الجلد فوق الصفاق(بعيدا عن القلب) للمساعدة في اختراق مثبت، ومن ثم وضعها في بارافورمالدهيد 4٪ في فيكس العازلة 10 لمدة 2 ساعة على RT أو O / N عند 4 درجات مئوية. </li></ol></li><li style=";text-align:right;direction:rtl"> بدلا من ذلك، للأسماك الطازجة والأسماك المكان المراد الموت الرحيم في خزان صغير وتقديمهم إلى الإعداد المجهر. اعتمادا على بروتوكول الأسماك المنشأة الفردية والاستخدامات المصب لقلوب الأسماك تشريح، تخدير الأسماك مع تريكين أو الموت ببطء عن طريق التبريد السريع قبل قطع الرأس. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> استخدام الثلج، والتقاط الأسماك واحد مع السمك الصافي وضعها في الماء المثلج حتى توقف الأسماك تتحرك، ما يقرب من 5 دقائق. </li><li style=";text-align:right;direction:rtl"> بدلا من ذلك، لاستخدام تريكين، والتقاط الأسماك واحد مع السمك الصافي ومكان في الدورق من الحل حتى تتوقف الحركات الخيشومية. </li></ol></li><li style=";text-align:right;direction:rtl"> بسرعة التقاط الأسماك الموت الرحيم من زعنفة الذيل ووضع على جانبها في الغطاء طبق بيتري التي كانت مليئة برنامج تلفزيوني 1X. استخدام ملقط لرفع زعنفة الصدرية بيد واحدة، في حين باستخدام razoص شفرة لقطع رأس السمكة مع جهة أخرى، الخلفي عادل إلى المرفق من الزعنفة الصدرية (الشكل 1A). تنفيذ هذه الخطوة سواء من خلال النظر من خلال المجهر أو مجرد تصور مباشرة الأسماك على المسرح المجهر. ملاحظة: سوف الأسماك طازجة الموت الرحيم لا يزال ينزف بعد قطع الرأس. </li></ol><img alt="الشكل (1)" src="/files/ftp_upload/52248/52248fig1highres.jpg" /> الشكل 1. الزرد تشريح القلب الكبار يستخدم المعالم التشريحية الزرد. (A) لقطع رأس السمكة، ورفع الزعنفة الصدرية مع ملقط واستخدام شفرة حلاقة حادة نظيفة على طول خط منقط أحمر كما هو مبين. (B) لثابت رئيس الأسماك، ووضع طينة واحدة من ملقط في حين فم السمكة من طينة أخرى يكمن عبر العين، ومن ثم تحويل رئيس الأسماك بحيث السطح البطني هو ما يصل، وكلا أسنان من ملقط مستقرة ضد الجزء السفلي من طبق بيتري. (C) استخدم forc مجاناالعائد على السهم لقطع الحجز على الغطاء الخيشومي (السهم). (D) رفع هذا الشريان الأبهر الظهري مرئيا كهيكل الأبيض مع شريط وردي تدل الدم اللمعية (السهم). <a href="https://www.jove.com/files/ftp_upload/52248/52248fig1large.jpg" target="_blank">الرجاء انقر هنا لمشاهدة نسخة أكبر من هذا الرقم.</a> 3. تشريح القلب <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تصور رئيس الأسماك تحت المجهر. مع واحد ملقط، ثابت رئيس الأسماك عن طريق وضع طينة واحدة من ملقط في فم السمكة، من خلال الدماغ، في حين أن طينة الآخر هو خارج الرأس، عبر العين (الشكل 1B). ضمان كلا من أسنان ملقط هي ثابتة أمام السفلي من طبق بتري. عقد رئيس الأسماك بهذه الطريقة، سطحه البطني يجب متجهة لأعلى. </li><li style=";text-align:right;direction:rtl"> مع جهة أخرى، استخدم ملقط الثانية لقطع التعلق بطني منطقة الغطاء الخيشومي للجسم (السهم، الشكل 1C). رفع هذا قليلا مع ملقط، دستظهر orsal الشريان الأورطي تحت كهيكل الأبيض مع شريط أحمر من الدم في التجويف الأبهر (السهم، 1D الشكل). قطع الشريان الأبهر الظهري مع ملقط عن طريق معسر الشريان الأورطي وسحب صعودا. بدلا من ذلك، استخدم microscissors لقطع الشريان الأبهر الظهري. </li><li style=";text-align:right;direction:rtl"> الآن، استخدم ملقط الثانية لفهم الزعنفة الصدرية من قاعدتها، بما في ذلك الغضروف الجسم عند قاعدة الزعنفة، ورفع هذا الخروج. كرر مع غيرها من الزعنفة الصدرية. أحيانا، والقلب يخرج مع الزعانف الصدرية، وذلك بفحص هذه القطع للتأكد من عدم ويرد القلب قبل التخلص منها. ملاحظة: عند هذه النقطة، يجب أن يكون القلب مرئية، لا تزال سليمة ومتصلة إلى رئيس الأسماك المتبقية (على الرغم من أن في بعض الأحيان يتعلق الأمر قبالة مع الزعانف الصدرية). يمكن التعرف على القلب من قبل شكله، لونه وردي، وكونها محاطة التامور المصطبغة. لأنه يتم تنفيذ القتل الرحيم من الأسماك بسرعة، قد تكون قلوب الأسماك الطازجة الموت الرحيم لا يزال الضرب سلوفاكياwly. </li><li style=";text-align:right;direction:rtl"> ترك للرئيس الأسماك مع ملقط الأولى، بحيث أن كلا يديه تمسك ملقط ومجانية. استخدام كل ملقط لندف قلب بعيدا عن التامور. فهم القلب من قبل ما تبقى من الشريان الأبهر الظهري بينما يتم سحبها التامور المتبقية بعيدا. ملاحظة: سواء الثابتة أو الطازجة، والقلب هو قوي لطيف سحب في حين أن الأنسجة الضامة المحيطة بها أكثر قابلة للتفتيت. ولذلك، أي نوع من الأنسجة المحيطة التامور يمكن تشريح بعيدا مع قلب المتبقية سليمة. </li><li style=";text-align:right;direction:rtl"> تجاهل الذبيحة الأسماك متبقية في حقيبة واقية. </li></ol> 4. إعداد القلب للتطبيقات المصب <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> باستخدام ملقط، فهم قلب على حافة ظهري الشريان الأورطي / بصلة شريانية ومكان القلب في طبق جديد من برنامج تلفزيوني 1X. بدلا من ذلك، استخدم ماصة نقل. نقل القلب ذهابا وإيابا في برنامج تلفزيوني حول 10X ليغسل الكثير من الدم ممكن. </li><li style=";text-align:right;direction:rtl"> وبالنسبة للتطبيقات التي من المهم لإزالة كافة possibخلايا الدم جنيه، ويغسل في 9 سم طبق بتري مع 15 مل RBC الاحتياطي تحلل بدلا من برنامج تلفزيوني، باستخدام نفس ذهابا وإيابا الحركة. إذا الحفاظ على هيكل القلب ليس مهما لتطبيق المصب (على سبيل المثال، مما يجعل RNA)، واستخدام الملقط أو microscissors لفتح تجويف القلب وتسهيل الشطف بعيدا من خلايا الدم. </li><li style=";text-align:right;direction:rtl"> وبالنسبة للتطبيقات التي يتم المطلوب حجرات القلب منفصلة، ​​استخدم ملقط أو microscissors لتشريح بصلة شريانية، الأذين، والبطين بعيدا عن بعضها البعض. </li><li style=";text-align:right;direction:rtl"> نقل القلب، أو غرف منفصلة، ​​إلى المخزن المؤقت الوجهة على الجليد. ملاحظة: تشمل الوجهات الشائعة مخازن لتفارق الخلية، 4٪ امتصاص العرق، أو Trizol. </li></ol>

<ol>
	<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.

في حين أساليب لتشريح القلب الزرد الكبار وقد وصفت، كانت هذه الأساليب تستغرق وقتا طويلا، وتسبب عادة الأضرار التي لحقت القلب أثناء تشريح. لإجراء التجارب حيث قد تكون هناك حاجة لعدد كبير من قلوب الكبار، و / أو عندما تجنب تدهور أنسجة القلب من المهم للتطبيقات المصب، والوقت ?...

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

استخدام في النظام النموذجي الزرد لدراسة التنمية والتجديد، والمرض يتوسع نحو استخدام قلوب الكبار لتفارق الخلية وتنقية RNA، DNA، والبروتينات. كل من هذه التطبيقات مطالبة الانتعاش السريع لأعداد كبيرة من قلوب الزرد لتجنب الجينات التنظيمية والتمثيل الغذائي، والتغيرات الأخرى التي تبدأ بعد الموت. كما يطلب قلوب الزرد الكبار لدراسة بنية القلب لمجموعة متنوعة من المسوخ وللدراسة تجديد القلب. ومع ذلك، فإن تشريح القلب الزرد التقليدي بطيء وصعب ويتطلب الأدوات المتخصصة، مما يجعل نطاق واسع تشريح قلوب الزرد الكبار مملة. الطرق التقليدية أيضا المرفأ من مخاطر الإضرار القلب أثناء تشريح. هنا، نحن تصف طريقة لتشريح قلوب الزرد الكبار التي هي سريعة، استنساخه، ويحافظ على بنية القلب. وعلاوة على ذلك، لا يتطلب هذا الأسلوب الأدوات المتخصصة، وغير مؤلم لالزرد،لا يمكن أن يؤديها على عينات جديدة أو ثابتة، ويمكن أن يؤديها على الزرد الشباب مثل شهر واحد من العمر. النهج وصف يوسع استخدام الزرد الكبار للأبحاث القلب والأوعية الدموية.

باستخدام هذه الطريقة، قلب الزرد البالغين يمكن تشريح في أقل من 1 دقيقة، مقارنة مع أكثر من 5 دقيقة باستخدام الطرق التقليدية 8. قلوب تشريح باستخدام هذه الطريقة سليمة موثوق (الشكل 2A)، في حين أن الطرق التقليدية تتطلب 8 قطع عمياء في التامور وبالتالي تسبب ...

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.

التعافي من اسماك الزرد الكبار قلوب للتطبيقات عالية الإنتاجية

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 Views.  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.

Video: 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.

على نطاق واسع اسماك الزرد الجنينية القلب تشريح لتحليل الترانسكربتي

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 ...

Developmental Biology

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.

الاستقراء من احتشاء عضلة القلب في الزرد الكبار باستخدام Cryoinjury

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

Medicine

Nanoparticle-mediated siRNA Gene-silencing in Adult Zebrafish Heart

Mammals have a very limited capacity to regenerate the heart after myocardial infarction. On the other hand, the adult zebrafish regenerates its heart after apex resection or cryoinjury, making it an important model organism for heart regeneration study. However, the lack of loss-of-function methods for adult organs has restricted insights into the mechanisms underlying heart regeneration. RNA interference via different delivery systems is a powerful tool for silencing genes in mammalian cells and model organisms. We have previously reported that siRNA-encapsulated nanoparticles successfully enter cells and result in a remarkable gene-specific knockdown in the regenerating adult zebrafish heart. Here, we present a simple, rapid, and efficient protocol for the dendrimer-mediated siRNA delivery and gene-silencing in the regenerating adult zebrafish heart. This method provides an alternative approach for determining gene functions in adult organs in zebrafish and can be extended to other model organisms as well.

It remains a major challenge to develop conditional gene-knockout or effective gene-knockdown in adult zebrafish organs. Here we report a protocol for performing nanoparticle-mediated siRNA gene-silencing in adult zebrafish heart, thus providing a new loss-of-function method for studying adult organs in zebrafish and other model organisms.

SiRNA نانوحبيبات بوساطة إسكات الجينات في قلب الزرد الكبار

Mammals have a very limited capacity to regenerate the heart after myocardial infarction. On the other hand, the adult zebrafish regenerates its heart ...

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.

تصوير صدى القلب بالموجات فوق الصوتية عالي التردد لتقييم وظيفة القلب في زيبرافيش

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.

تشريح القلب في اليرقات ، اسماك الزرد الأحداث والكبار ، دانيو rerio</em

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

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.

Immunostaining تشريح القلب الجنينية اسماك الزرد

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

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.

تقييم القدرة على التحمل على السباحة وسلوك السباحة في أسماك الزرد البالغة

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

Neuroscience

Anesthesia-free Heartbeat Measurements in Freely Moving Zebrafish

Zebrafish (Danio rerio) is a widely used model organism in physiological, pharmacological, and toxicological research due to its genetic similarity to humans and transparent embryonic stage, which facilitates non-invasive cardiovascular studies. However, current methods for heart rate assessment in zebrafish often rely on anesthesia to immobilize the subject, introducing physiological alterations that compromise data accuracy and reproducibility. This study presents a novel, anesthesia-free technique for measuring heartbeat in freely moving zebrafish larvae, addressing a critical limitation in cardiovascular research. The proposed approach integrates shortwave-infrared imaging with machine-learning-based heart tracking, allowing for precise and continuous cardiac activity monitoring in non-immobilized specimens. A convolutional neural network was trained to detect the heart region, and a photoplethysmographic signal was extracted from image sequences to determine heart rate. Experimental validation demonstrated the method's reliability and consistency across multiple test conditions. A key benefit of the methodology is its ability to preserve the natural physiological state of zebrafish, minimizing stress-induced artifacts. This non-invasive, label-free technique offers significant advantages for studying cardiovascular physiology, drug cardiotoxicity, and environmental toxicology, expanding the potential applications of zebrafish as a model for biomedical research.

The study aims to develop technology for anesthesia-free heartbeat measurements in moving zebrafish. Our approach combines shortwave-infrared imaging and machine-learning-based tracking of the heart. It is a non-invasive, label-free, and user-friendly technique that suits a wide range of studies on the zebrafish model.

Zebrafish (Danio rerio) is a widely used model organism in physiological, pharmacological, and toxicological research due to its genetic similarity to ...

Bioengineering

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.

حقن داخل الصفاق لدراسة القلب البالغ زبرافيش

The adult zebrafish heart provides a powerful model in cardiac regeneration research. Although the strength of this system is based on transgenic ...

In Vivo Surface Electrocardiography for Adult Zebrafish

The electrocardiogram waveforms of adult zebrafish and those of humans are remarkably similar. These electrocardiogram similarities enhance the value of zebrafish not only as a research model for human cardiac electrophysiology and myopathies but also as a surrogate model in high throughput pharmaceutical screening for potential cardiotoxicities to humans, such as QT prolongation. As such, in vivo electrocardiography for adult zebrafish is an electrical phenotyping tool that is necessary, if not indispensable, for cross-sectional or longitudinal in vivo electrophysiological characterizations. However, too often, the lack of a reliable, practical, and cost-effective recording method remains a major challenge preventing this in vivo diagnostic tool from becoming more readily accessible. Here, we describe a practical, straightforward approach to in vivo electrocardiography for adult zebrafish using a low-maintenance, cost-effective, and comprehensive system that yields consistent, reliable recordings. We illustrate our protocol using healthy adult male zebrafish of 12-18 months of age. We also introduce a rapid real-time interpretation strategy for quality validation to ensure data accuracy and robustness early in the electrocardiogram recording process.

Here, we present a reliable, minimally invasive, and cost-effective method to record and interpret electrocardiograms in live anesthetized adult zebrafish.

في فيفو سطح الكهربائي لحمار وحشي الكبار

The electrocardiogram waveforms of adult zebrafish and those of humans are remarkably similar. These electrocardiogram similarities enhance the value of ...