Name: repeated blood collection for blood tests in adult zebrafish
Uploaded: 2015-08-30T14:00:00.000+00:00
Duration: 6 min 17 s
Description: Watch this Scientific Journal Video about Repeated Blood Collection for Blood Tests in Adult Zebrafish at JoVE.com

This work was supported by JSPS KAKENHI Grant Number 25860294 and 25590073. We would like to thank Ms. Yui Namie for the hand-drawn illustration, and Mr. Koshi Kataoka and Ms. Sayuri Ichikawa for assistance with the zebrafish maintenance.

وقد وافق جميع الإجراءات الحيوانية من لجنة أخلاقيات جامعة مي، وتم إجراء فقا لالياباني تنظيم الرفق بالحيوان &quot;قانون رعاية وإدارة الحيوانات&quot; (وزارة البيئة في اليابان) والامتثال للمبادئ التوجيهية الدولية. 1. إعداد إبرة ملاحظة: تم إجراء جميع التجارب تحت التخدير، وقدمت كل الجهود للحد من المعاناة. للقتل الرحيم، ومغمورة الأسماك في حمام الماء المثلج (5 أجزاء الجليد / 1 جزء ماء في ≤4 درجة مئوية) ل≥20 دقيقة. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إعداد الإبر الزجاج microcapillary عن طريق سحب الزجاج 1.0 ملم-الخارجي قطرها الشعرية مع مجتذب إبرة (الشكل 1A). </li><li style=";text-align:right;direction:rtl"> قطع نصائح من الإبر بشكل غير مباشر باستخدام مقص غرامة (الشكل 1B). يجب أن يكون قطر الحافة المثالي حوالي 100-200 ميكرون (الشكل 1C). إذا كان diamete طرفص ضيقة جدا، وسوف الدم لا يدخل الإبرة. </li><li style=";text-align:right;direction:rtl"> حل الهيبارين في المياه المالحة إلى تركيز 5 ملغ / مل. </li><li style=";text-align:right;direction:rtl"> وضع إبرة قبل القطع في نهاية الأنفية من تجميع أنبوب الشافطة وعقد لسان حال في الفم، أو ربط الإبرة إلى موزع لمبة. تزج رأس إبرة في حل الهيبارين، ويعالج بالهيبارين إبرة عن طريق الشفط والنفخ من خلال حل (1D الشكل و1E). ملاحظة: تم استخدام أنبوب التجميع الشافطة لالزرد الحيوانات المنوية الحفظ بالتبريد 16. أنبوب المطاط طويلة يمكن أن يوقف أي دم تحلق في الفم. ويمكن تحديد مرشح في منتصف الطريق من الأنبوب تجنب المخاطر. </li><li style=";text-align:right;direction:rtl"> تخزين الإبر heparinized في طبق بتري 10 سم والهواء الجاف لا يقل عن 1 ساعة. يمكن إعداد عدد كبير من الإبر مقدما. </li></ol> 2. التخدير <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تحضير محلول مخدر في حالة من البلاستيك الصغيرة عن طريق خلط 200 مل من الماء السمك مع 100 ميكرولتر من 2 فينوكسييثانول (2-PE). التركيز النهائي من التخدير هو 500 جزء في المليون. الحذر! 2-PE له تأثير سريع. </li><li style=";text-align:right;direction:rtl"> إزالة العدد المطلوب من السمك (AB سلالة) من نظام التداول. </li><li style=";text-align:right;direction:rtl"> استخدام شبكة لنقل الأسماك في التخدير ل1-2 دقيقة (الشكل 1F). مراقبة الأسماك تسبح تدريجيا، وانتشار الزعانف الصدرية أفقيا، لهاث، ويكون الغطاء الخيشومي الحركات السريعة في حدود 1 دقيقة. </li><li style=";text-align:right;direction:rtl"> كما يذهب الوقت على، ومراقبة الأسماك تقع في الجزء السفلي من هذه القضية، وأخيرا وقف السباحة. والطائرة من التخدير الجراحي تصل عند توقف الأسماك إلى اللحظات والحركات الغطاء الخيشومي بطيئة. عند هذه النقطة، الأسماك على استعداد لجمع الدم. </li><li style=";text-align:right;direction:rtl"> باستخدام مقشدة رفع الأسماك تخدير من 2-PE ووضعه برفق على منشفة ورقية مبللة مع التخدير (الشكل 1G). تغطية رأس السمكة مع ورقة الأنسجة الرخوة غارقة أيضا مع حل 2-PE لمنع جفاف العين واستخدام المناديل الورقية الناعمة الجافة أخرى لبلطفتجف من على سطح الجسم. </li></ol> 3. جمع الدم <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> وضع إبرة heparinized في نهاية الأنفية للجمعية أنبوب الشافطة (أو موزع لمبة) وعقد نهاية الناطقة باسم التجمع أنبوب الشافطة في الفم. </li><li style=";text-align:right;direction:rtl"> فهم في نهاية الأنفية والإبرة معا، وإزالة موازين التدخل بعناية مع طرف الإبرة. ادخال الإبرة في 30 - 45 درجة زاوية في موقع جمع الدم. تجنب ثقب الجهاز الهضمي (الشكل 1H). في حالة استخدام لمبة موزع، اضغط على لمبة مع الإبهام والإصبع الوسطى، ومنع ثقب في طرف لمبة مع الإصبع الأول، ثم أدخل الإبرة كما هو موضح أعلاه. ملاحظة: هذا الموقع لجمع الدم على طول محور الجسم والخلفية إلى فتحة الشرج في المنطقة من الشريان الأبهر الظهري. الشريان الأبهر الظهري (DA) والوريد الخلفي الكاردينال (PCV) ليست سوى بطني في العمود الفقري (الشكل 2). </li><li style=";text-align:right;direction:rtl"> تبدأ في مصالناطقة بلسان نهاية الأنبوب التجمع الطموح عندما يشعر الإبرة لمس العمود الفقري. إذا لم يرتفع في الدم، ونقل معلومات سرية إبرة بمهارة باليد لتشجيع تدفق الدم. لاحظ أنه بمجرد الدم يرتفع إلى الإبرة، والتوقف فورا عن الهز برفق وتمتص (الشكل 1I). في حالة استخدام لمبة موزع، والإفراج عن الضغط من لمبة لنضح الدم. </li><li style=";text-align:right;direction:rtl"> نلاحظ ان الدم ترتفع ببطء في الإبرة بطريقة نابض دون شفط، والتي من المرجح بسبب ضغط الدم الشرياني. وبالتالي، فإنه ليس من الضروري أن تمتص إذا كانت إبرة تخترق الشريان بشكل صحيح. </li><li style=";text-align:right;direction:rtl"> وقف الشفط بعد أن يتم جمع حجم مناسب من الدم. إزالة الإبرة من الأسماك والضغط على الموقع ثقب باستخدام المناديل الورقية الناعمة لوقف أي نزيف. مراقبة وقف النزيف بعد حوالي 10-20 ثانية من ضغط الإصبع (الشكل 1J). </li><li style=";text-align:right;direction:rtl"> بعد توقف النزيف، ونقل على الفور الأسماك مرة أخرى إلى شركة كليوالمياه الدافئة (~ 28 ° C) دبابة. مساعدة الأسماك للتعافي من قبل يحوم بلطف المياه نحو الخياشيم حتى يبدأ في السباحة. ملاحظة: حافظ على ما يصل الى 5 الأسماك postsampling في خزان 2 L وربط خزان لنظام التداول إلى الأوكسجين الأسماك. إضافة المضادات الحيوية إلى أسماك المياه ليست ضرورية. الحفاظ على الأسماك مع السكن العادي والتغذية. </li><li style=";text-align:right;direction:rtl"> طرد الدم من الإبرة إلى منطقة نظيفة من قطعة من parafilm (الشكل 1K). </li><li style=";text-align:right;direction:rtl"> قياس السكر في الدم باستخدام أي غلوكمتر يده التجاري (الشكل 1L). ملاحظة: يستخدم غلوكمتر الجلوكوز-فلافين نازعة الأدينين ثنائي النوكليوتيد القطب ويتطلب حجم عينة من 0.6 ميكرولتر. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إدراج شريط الاختبار تماما في متر وتلمس مباشرة قطرة دم. مراقبة سحب الدم إلى شريط الاختبار تلقائيا والحصول على نسبة الجلوكوز في الدم نتيجة 5 ثانية على منطقة العرض. سجل النتيجة وتجاهل شريط الاختبار. إستخدمجديد شريط الاختبار لكل قياس. </li></ol></li><li style=";text-align:right;direction:rtl"> (خطوة اختيارية) أخذ كميات دقيقة من الدم قبل pipetting لونقل الدم إلى أنبوب microcentrifuge لإجراء المزيد من التحليلات (خضاب الدم، الدهون الثلاثية (TG) والكوليسترول الكلي، وما إلى ذلك). إذا لزم الأمر، وتمييع الدم كله من الأسماك واحد بمحلول ملحي. الطرد المركزي عينات الدم لمدة 3 دقائق في 680 x ج في RT وحصاد البلازما. نقل البلازما في أنبوب جديد. في هذه المرحلة، وانها مستعدة لاستخدامها في التحليلات الكيميائية الحيوية. </li></ol>

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The authors have nothing to disclose.

نقدم هنا بروتوكول مفصلة للحصول على متسلسل الدم من الزرد الكبار. هذه الطريقة واضحة ومباشرة لتنفيذ ونستخدمها في المختبر على أساس يومي. ويستند هذا الأسلوب جمع الدم على إدخال إبرة الشعرية الزجاج في ظهري الشريان الأورطي الزرد ل. خلال هذا الإجراء، فمن الأهمية بمكان أن نكو?...

الزرد تكتسب شعبية متزايدة باعتبارها نموذجا قيما من الأمراض التي تصيب الإنسان بسبب أعضائهم وعلم الوراثة مماثلة لتلك التي من البشر 1،2. في مجال علم الأحياء التطوري، وقد أثبتت العديد من الدراسات أن الزرد وتظهر البشري علامة التشابه في تكون الدم 3، الارقاء 4،5، وتكون النقويات 6. وتستخدم الزرد الكبار أيضا لدراسة المناعية 7، 8 الاعصاب والأمراض المرتبطة بالسمنة 9 لأن هذا النموذج الحي سهم مسارات مشتركة مع تلك تعطلت في الأمراض التي تصيب الإنسان. للسمنة والأمراض المرتبطة بالسمنة (السكري، تشحم الكبد غير الكحولي والتهاب الكبد الدهني وتصلب الشرايين)، وقد تم التحقيق الزرد السكر في الدم ومستويات الدهون تماما في العديد من النماذج المعدلة وراثيا السمنة والنظام الغذائي الناجم عن 10-13. سوف تتكرر أخذ عينات دم من الحيوانات الفردية تقليل استخدام الحيواني وديسمبرrease الخلافات interindividual. ومع ذلك، وجمع عينة متكررة من الصعب من الناحية الفنية في الحيوانات الصغيرة مثل الزرد بسبب حجمها الصغير نسبيا في الدم وعدم وجود السفن الوصول إليها بسهولة. وقد وضعت عدة طرق لجمع الدم لمرة واحدة من الزرد، على الرغم من أن هذه الطرق لها سلبياتها الخاصة، بما في ذلك الفتك، تلف الأنسجة المرتبطة وحجم الدم محدودة. على سبيل المثال، 1-5 الدم ميكرولتر يمكن حصاده من شق الوحشي من حوالي 0.3 سم في الطول في المنطقة من الشريان الأبهر الظهري 5. قطع الرأس مع مقص من خلال قطع حزام الصدر يمكن جمع 5-10 ميكرولتر الدم 10. مريحة طريقة أخرى أخذ عينات الدم هو ذيل الاجتثاث 14. ثقب في القلب هو طريقة بديلة محتملة واحدة لجمع الدم المتكررة من نفس السمك، ولكن كمية صغيرة جدا الحصول على (ما يقرب من 50 NL) مع هذا الإجراء يحد من عدد من التحليلات التي يمكن أن تكون ادائهاميد 11. وفقا لذلك، هناك حاجة إلى بروتوكول جديد لتمكين المتكررة اخذ عينات من الدم غير المميتة، والتي ستكون تقدما بالغ الأهمية اللازمة لهذا الكائن أن يكون كائن النموذج القياسي لأمراض الإنسان. أن هذه التقنية تسمح لاختبار استجابة الدوائية، اكتشاف المؤشرات الحيوية الجزيئية للتشخيص وتحديد التشخيص، ورصد مختلف الأمراض، مثل أمراض التمثيل الغذائي والأمراض التنكسية وعدة أنواع من الأورام الخبيثة. لذا طورنا طريقة مينيملي للحصول على الدم من الزرد متسلسل 15. نحن هنا لشرح الإجراء بصريا وتوفير بروتوكول مفصلة لهذه التقنية. باستخدام هذه الطريقة، تم تقييم القيمة العادية على أساس معايير مختلفة، بما في ذلك الهيموغلوبين، وصوم الجلوكوز في الدم، ونسبة الدهون في الدم من الزرد البالغين الأصحاء. بالإضافة إلى ذلك، نحن أيضا تقييم ما إذا كانت هذه الطريقة مناسبة للدراسات التي تتطلب عينات التسلسلية التي كتبها monitoring التغيرات الزمانية في مستويات السكر في الدم أثناء التجارب فرط.

<table><tbody><tr><td>Glass capillaries with filament</td><td>Narishige</td><td>GD-1</td><td>1.0 mm outer diameter.</td></tr><tr><td>Needle puller</td><td>Narishige</td><td>PC-10</td><td>To produce the needles</td></tr><tr><td>Heparin</td><td>Wako Pure Chemical Industries</td><td>081-00136</td><td>For heparinization</td></tr><tr><td>Aspirator tube assembly</td><td>Drummond</td><td>2-040-000</td><td>For blood collection</td></tr><tr><td>Bulb dispenser</td><td>Drummond</td><td>1-000-9000</td><td>For blood collection</td></tr><tr><td>2-phenoxyethanol</td><td>Wako Pure Chemical Industries</td><td>163-12075</td><td>For anesthetizing the fish</td></tr><tr><td>DRI-CHEM3500V</td><td>Fujifilm</td><td>-</td><td>For hemoglobin measurement</td></tr><tr><td>DRI-CHEM Slides</td><td>Fujifilm</td><td>Hb-WII</td><td>For hemoglobin measurement</td></tr><tr><td>Glutest Neo Super</td><td>Sanwa Kagaku Kenkyusho</td><td>-</td><td>For bood glucose measurement</td></tr><tr><td>Wako L-type TG kit</td><td>Wako Pure Chemical Industries</td><td>464-44201</td><td>For TG measurement</td></tr><tr><td>Wako L-type CHO kit</td><td>Wako Pure Chemical Industries</td><td>460-44301</td><td>For total cholesterol measurement</td></tr><tr><td>Parafilm M</td><td>Alcan Packaging</td><td>PM996</td><td>To expel the blood on</td></tr></tbody></table>

repeated blood collection for blood tests in adult zebrafish

جمع الدم المتكررة هي واحدة من التقنيات الأكثر شيوعا التي أجريت على حيوانات المختبر. ومع ذلك، لم يثبت بروتوكول غير قاتلة لجمع الدم من الزرد. الطرق السابقة لجمع الدم من الزرد هي القاتلة، مثل شق الجانبي، وقطع الرأس والذيل الاجتثاث. وهكذا وضعنا رواية &quot;المتكررة&quot; طريقة جمع الدم، والحاضر هنا بروتوكول مفصلة توضح هذا الإجراء. هذا الأسلوب هو الغازية الحد الأدنى والنتائج في معدل وفيات منخفضة جدا (2.3٪) لالزرد، وبالتالي تمكين المتكررة اخذ عينات من الدم من نفس الفرد. الحد الأقصى لحجم اخذ عينات من الدم يعتمد على وزن جسم السمكة. يجب أن يكون حجم لأخذ عينات الدم المتكرر على فترات ≤0.4٪ من وزن الجسم كل أسبوع أو ≤1٪ كل 2 أسابيع، والتي تم تقييمها من قبل القياسات الهيموغلوبين في الدم. بالإضافة إلى ذلك، الهيموجلوبين، وصوم الجلوكوز في الدم، الدهون الثلاثية البلازما (TG) وإجمالي جتم قياس مستويات holesterol في الزرد الكبار من الذكور والإناث. علينا أيضا أن تطبق هذه الطريقة للتحقيق في ديسريغولاتيون ايض الجلوكوز في النظام الغذائي الناجم عن السمنة. هذه الطريقة سوف جمع الدم تسمح العديد من التطبيقات، بما في ذلك الجلوكوز واستقلاب الشحوم والدراسات الدموية، الأمر الذي سيزيد من استخدام الزرد كنموذج المرض كائن البشري.

يؤدي هذا الأسلوب جمع الدم الإصابة الحد الأدنى لالزرد (أ &lt;1 مم ثقب؛ الشكل 1J) وينتج معدل وفيات منخفض جدا من 2.3٪. درسنا الحد الأقصى لحجم الدم التي يمكن جمعها من سمكة واحدة وتقييم العلاقة لوزن الجسم (الشكل 3). وجدنا أن حجم الدم الأقصى التي تم جمعها مرتبطة خ...

Watch this Scientific Journal Video about Repeated Blood Collection for Blood Tests in Adult Zebrafish at JoVE.com

Repeated Blood Collection for Blood Tests in Adult Zebrafish

Repeated blood sampling is necessary and important in animal research. We developed a novel, non-lethal and reliable method for repeated blood collection from adult zebrafish, and applied this method to the study of blood biochemistry, including glucose metabolism.

كرر جمع الدم لاختبارات الدم في اسماك الزرد الكبار

Repeated blood collection is one of the most common techniques performed on laboratory animals. However, a non-lethal protocol for blood collection from ...

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Research

JoVE Journal

Biology

34.9K Views.  Mie University. Repeated blood sampling is necessary and important in animal research. We developed a novel, non-lethal and reliable method for repeated blood collection from adult zebrafish, and applied this method to the study of blood biochemistry, including glucose metabolism.

Video: Repeated Blood Collection for Blood Tests in Adult Zebrafish

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA mutation rates. Changes in replication timing occur during development and in cancer, but the role replication timing plays in development and disease is not known. Zebrafish were recently established as an in vivo model system to study replication timing. Here is detailed the protocols for using the zebrafish to determine DNA replication timing. After sorting cells from embryos and adult zebrafish, high-resolution genome-wide DNA replication timing patterns can be constructed by determining changes in DNA copy number through analysis of next generation sequencing data. The zebrafish model system allows for evaluation of the replication timing changes that occur in vivo throughout development, and can also be used to assess changes in individual cell types, disease models, or mutant lines. These methods will enable studies investigating the mechanisms and determinants of replication timing establishment and maintenance during development, the role replication timing plays in mutations and tumorigenesis, and the effects of perturbing replication timing on development and disease.

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

Zebrafish were recently used as an in vivo model system to study DNA replication timing during development. Here is detailed the protocols for using zebrafish embryos to profile replication timing. This protocol can be easily adapted to study replication timing in mutants, individual cell types, disease models, and other species.

التنميط الحمض النووي توقيت النسخ المتماثل باستخدام الزرد كنظام نموذجي في فيفو

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA ...

Genetics

Retro-orbital Injection in Adult Zebrafish

Drug treatment of whole animals is an essential tool in any model system for pharmacological and chemical genetic studies. Intravenous (IV) injection is often the most effective and noninvasive form of delivery of an agent of interest. In the zebrafish (Danio rerio), IV injection of drugs has long been a challenge because of the small vessel diameter. This has also proved a significant hurdle for the injection of cells during hematopoeitic stem cell transplantation. Historically, injections into the bloodstream were done directly through the heart. However, this intra-cardiac procedure has a very high mortality rate as the heart is often punctured during injection leaving the fish prone to infection, massive blood loss or fatal organ damage. Drawing on our experience with the mouse, we have developed a new injection procedure in the zebrafish in which the injection site is behind the eye and into the retro-orbital venous sinus. This retro-orbital (RO) injection technique has been successfully employed in both the injection of drugs in the adult fish as well as transplantation of whole kidney marrow cells. RO injection has a much lower mortality rate than traditional intra-cardiac injection. Fish that are injected retro-orbitally tend to bleed less following injection and are at a much lower risk of injury to a major organ like the heart. Further, when performed properly, injected cells and/or drugs quickly enter the bloodstream allowing compounds to exert their effect on the whole fish and kidney cells to easily home to their niche. Thus, this new injection technique minimizes mortality while allowing efficient delivery of material into the bloodstream of adult fish. Here we exemplify this technique by retro-orbital injection of Tg(globin:GFP) cells into adult casper fish as well as injection of a red fluorescent dye (dextran, Texas Red ) into adult casper fish. We then visualize successful injections by whole animal fluorescence microscopy.

Here we show how to do retro-orbital injection in adult zebrafish.

الرجعية المدارية في حقن اسماك الزرد الكبار

Drug treatment of whole animals is an essential tool in any model system for pharmacological and chemical genetic studies. Intravenous (IV) injection is ...

Using Flow Cytometry to Detect and Quantitate Altered Blood Formation in the Developing Zebrafish

The diversity of cell lineages that comprise mature blood in vertebrate animals arise from the differentiation of hematopoietic stem and progenitor cells (HSPCs). This is a critical process that occurs throughout the lifespan of organisms, and disruption of the molecular pathways involved during embryogenesis can have catastrophic long-term consequences. For a multitude of reasons, zebrafish (Danio rerio) has become a model organism to study hematopoiesis. Zebrafish embryos develop externally, and by 7 days postfertilization (dpf) have produced most of the subtypes of definitive blood cells that will persist for their lifetime. Assays to assess the number of hematopoietic cells have been developed, mainly utilizing specific histological stains, in situ hybridization techniques, and microscopy of transgenic animals that utilize blood cell-specific promoters driving the expression of fluorescent proteins. However, most staining assays and in situ hybridization techniques do not accurately quantitate the number of blood cells present; only large differences in cell numbers are easily visualized. Utilizing transgenic animals and analyzing individuals with fluorescent or confocal microscopy can be performed, but the quantitation of these assays relies on either counting manually or utilizing expensive imaging software, both of which can make errors. Development of additional methods to assess blood cell numbers would be economical, faster, and could even be automated to quickly assess the effect of CRISPR-mediated genetic modification, morpholino-mediated transcript reduction, and the effect of drug compounds that affect hematopoiesis on a large scale. This novel assay to quantitate blood cells is performed by dissociating whole zebrafish embryos and analyzing the amount of fluorescently labelled blood cells present. These assays should allow elucidation of molecular pathways responsible for blood cell generation, expansion, and regulation during embryogenesis, which will allow researchers to further discover novel factors altered during blood diseases, as well as pathways essential during the evolution of vertebrate hematopoiesis.

This assay is a simple method to quantitate hematopoietic cells in developing embryonic zebrafish. Blood cells from dissociated zebrafish are subjected to flow cytometry analysis. This allows the detection of blood defects in mutant animals and after genetic modification.

استخدام قياس التدفق الخلوي للكشف عن تكوين الدم المعدل وكميته في سمك الحمار الوحشي النامي

The diversity of cell lineages that comprise mature blood in vertebrate animals arise from the differentiation of hematopoietic stem and progenitor cells ...

Developmental Biology

Examining Muscle Regeneration in Zebrafish Models of Muscle Disease

Skeletal muscle has a remarkable ability to regenerate following injury, which is driven by obligate tissue resident muscle stem cells. Following injury, the muscle stem cell is activated and undergoes cell proliferation to generate a pool of myoblasts, which subsequently differentiate to form new muscle fibers. In many muscle wasting conditions, including muscular dystrophy and ageing, this process is impaired resulting in the inability of muscle to regenerate. The process of muscle regeneration in zebrafish is highly conserved with mammalian systems providing an excellent system to study muscle stem cell function and regeneration, in muscle wasting conditions such as muscular dystrophy. Here, we present a method to examine muscle regeneration in zebrafish models of muscle disease. The first step involves the use of a genotyping platform that allows the determination of the genotype of the larvae prior to eliciting an injury. Having determined the genotype, the muscle is injured using a needle stab, following which polarizing light microscopy is used to determine the extent of muscle regeneration. We therefore provide a high throughput pipeline which allows the examination of muscle regeneration in zebrafish models of muscle disease.

Skeletal muscle regeneration is driven by tissue resident muscle stem cells, which are impaired in many muscle diseases such as muscular dystrophy, and this results in the inability of muscle to regenerate. Here, we describe a protocol that allows the examination of muscle regeneration in zebrafish models of muscle disease.

فحص تجديد العضلات في نماذج حمار وحشي من أمراض العضلات

Skeletal muscle has a remarkable ability to regenerate following injury, which is driven by obligate tissue resident muscle stem cells. Following injury, ...

Optimized Intravenous Injection in Adult Zebrafish

Intravenous (IV) injection is widely recognized as the most effective and commonly utilized method for achieving systemic delivery of substances in mammalian research models. However, its application in adult zebrafish for drug delivery, stem cell transplantation, and regenerative and cancer studies has been limited due to the challenges posed by their small body size and intricate blood vessels. To overcome these limitations, alternative injection techniques such as intracardiac and retro-orbital (RO) injection have been explored in the past for stem cell transplantation in adult zebrafish. However, these techniques have their drawbacks, including the need for meticulous injection techniques or increased risk of mortality.
In this study, we have developed a refined and optimized IV injection procedure specifically tailored to adult zebrafish, addressing the challenges associated with their unique anatomy. To demonstrate the effectiveness of this technique, we performed successful IV injections of whole kidney marrow cells from Tg(mpo: EGFP) fish and FITC-dextran dye into adult Casper fish. The subsequent visualization of injected cells and dyes using a fluorescence microscope confirmed their successful delivery and engraftment within the zebrafish. Furthermore, we demonstrated that compared with the intracardiac and RO injections, the IV injection resulted in improved survival rates and engraftment efficiency in treated zebrafish. This approach enables precise delivery and localization of substances and holds great potential for large-scale drug and chemical screening using adult zebrafish. Additionally, the ability to visually track the injected cells and dyes provides invaluable insights into their engraftment, migration, and interactions with host tissues, enabling a more comprehensive evaluation of therapeutic effects and biological processes in zebrafish models.

Here, we present an optimized and effective protocol for the intravenous injection of cells or pharmacological agents into adult zebrafish, resulting in enhanced cell engraftment and increased survival rates of the treated zebrafish.

الحقن الأمثل في الوريد في أسماك الزرد البالغة

Intravenous (IV) injection is widely recognized as the most effective and commonly utilized method for achieving systemic delivery of substances in ...

Cancer Research

Blood Collection for Biochemical Analysis in Adult Zebrafish

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of molecular events and therapeutic strategies as well as for evaluating the physiological mechanisms of some pathologies1. 
There are relatively few publications related to adult zebrafish physiology of organs and systems2, which may lead researchers to infer that the basic techniques needed to allow the exploration of zebrafish systems are lacking3. Hematologic biochemical values of zebrafish were first reported in 2003 by Murtha and colleagues4 who employed a blood collection technique first described by Jagadeeswaran and colleagues in 1999. Briefly, blood was collected via a micropipette tip through a lateral incision, approximately 0.3 cm in length, in the region of the dorsal aorta5. Because of the minute dimensions involved, this is a high-precision technique requiring a highly skilled practitioner. The same technique was used by the same group in another publication in that same year6. In 2010, Eames and colleagues assessed whole blood glucose levels in zebrafish7. They gained access to the blood by performing decapitations with scissors and then inserting a heparinized microcapillary collection tube into the pectoral articulation. They mention difficulties with hemolysis that were solved with an appropriate storage temperature based on the work Kilpatrick et al.8. When attempting to use Jagadeeswaran's technique in our laboratory, we found that it was difficult to make the incision in precisely the right place as not to allow a significant amount of blood to be lost before collection could be started. 
Recently, Gupta et al.9 described how to dissect adult zebrafish organs, Kinkle et al.10 described how to perform intraperitoneal injections, and Pugach et al.11 described how to perform retro-orbital injections. However, more work is needed to more fully explore basic techniques for research in zebrafish.
The small size of zebrafish presents challenges for researchers using it as an experimental model. Furthermore, given this smallness of scale, it is important that simple techniques are developed to enable researchers to explore the advantages of the zebrafish model.

This paper presents a technique for collection of blood from the dorsal aorta of Zebrafish. It also provides instructions for obtaining serum for use in biochemical analyses, such as tests for determining cholesterol and triglyceride levels.

جمع الدم لتحليل الكيمياء الحيوية في الزرد الكبار

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of ...

Studying Diabetes Through the Eyes of a Fish: Microdissection, Visualization, and Analysis of the Adult tg(fli:EGFP) Zebrafish Retinal Vasculature

Diabetic retinopathy is the leading cause of blindness among middle-aged adults. The rising prevalence of diabetes worldwide will make the prevention of diabetic microvascular complications one of the key research fields of the next decades. Specialized, targeted therapy and novel therapeutic drugs are needed to manage the increasing number of patients at risk of vision-loss. The zebrafish is an established animal model for developmental research questions with increasing relevance for modeling metabolic multifactorial disease processes. The advantages of the species allow for optimal visualization and high throughput drug screening approaches, combined with the strong ability to knock out genes of interest. Here, we describe a protocol which will allow easy analysis of the adult tg(fli:EGFP) zebrafish retinal vasculature as a fast read-out in settings of long-term vascular pathologies linked to neoangiogenesis or vessel damage. This is achieved via dissection of the zebrafish retina and whole-mounting of the tissue. Visualization of the exposed vessels is then achieved via confocal microscopy of the green EGFP reporter expressed in the adult retinal vasculature. Correct handling of the tissue will lead to better outcomes and less internal vessel breakage to assure the visualization of the unaltered vascular structure. The method can be utilized in zebrafish models of retinal vasculopathy linked to changes in the vessel architecture as well as neoangiogenesis.

Studying Diabetes Through the Eyes of a Fish: Microdissection, Visualization, and Analysis of the Adult tgfli:EGFP Zebrafish Retinal Vasculature

Here, we discuss a method protocol which will allow an easy analysis of the adult tg(fli:EGFP) zebrafish retinal vasculature as a fast read-out in settings of long-term vascular pathologies linked to neoangiogenesis and structural changes.

دراسة "مرض السكر من خلال عيون" من الأسماك: ميكروديسيكتيون، والتصور، وتحليل tg(fli:EGFP) الكبار "الزرد الشبكية المفرج"

Diabetic retinopathy is the leading cause of blindness among middle-aged adults. The rising prevalence of diabetes worldwide will make the prevention of ...

Medicine

Gavaging Adult Zebrafish

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or agents in solutions for scientific research. Current methods for administering compounds orally to adult zebrafish are inaccurate due to variability in voluntary consumption by the fish. A gavage procedure was developed to deliver precise quantities of infectious agents to zebrafish for study in biomedical research. Adult zebrafish over 6 months of age were anesthetized with 150 mg/L of buffered MS-222 and gavaged with 5 &mu;l of solution using flexible catheter implantation tubing attached to a cut 22-G needle tip. The flexible tubing was lowered into the oral cavity of the zebrafish until the tip of the tubing extended past the gills (approximately 1 cm). The solution was then injected slowly into the intestinal tract. This method was effective 88% of the time, with fish recovering uneventfully. This procedure is also efficient as one person can gavage 20-30 fish in one hour. This method can be used to precisely administer agents for infectious diseases studies, or studies of other compounds in adult zebrafish.

The increasing use of zebrafish as an animal model requires the development of effective methods for the delivery of known quantities of compounds and solutions. The gavage procedure described below allows for the oral delivery of precise volumes of solution reliably, safely and efficiently to adult zebrafish.

Gavaging اسماك الزرد الكبار

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

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 ...

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 ...