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.

Bütün hayvan prosedürleri Mie Üniversitesi Etik Kurulu tarafından onaylanmış ve uluslararası kurallara Japon hayvan refahı yönetmelik &#39;Refah ve Hayvanların Yönetimi Yasası&#39; (Japonya Çevre Bakanlığı) göre gerçekleştirilmiştir ve uyulması bulundu. Needle 1. Hazırlık NOT: Bütün deneyler anestezi altında gerçekleştirildi ve tüm çabaları acıyı en aza indirmek için yapılmıştır. Ötenazi için, balık ≥20 dakika boyunca (≤4 ° C&#39;de 5 parça buz / 1 kısım su), bir buz-su banyosu içine daldırıldı. <ol><li> Bir iğne çektirmesi (Şekil 1A) ile 1,0 mm dış çaplı cam kapiller çekerek cam iğneler microcapillary hazırlayın. </li><li> Oblik ince makas (Şekil 1B) kullanarak iğne uçlarını kesin. 200 mikron (Şekil 1C) - İdeal uç çapı yaklaşık 100 olmalıdır. İpucu Diamete Eğerr çok dar, kan iğne girmeyecektir. </li><li> 5 mg / ml &#39;lik bir konsantrasyona kadar tuzla içinde heparin çözülür. </li><li> Bir aspiratör borusu düzeneğinin burunluk sonunda bir precut iğne yerleştirin ve ağız ağızlık tutun, ya da bir ampul dağıtıcı iğne bağlayın. Heparin çözeltisi içine iğne ucu daldırın ve emme ve çözelti (Şekil 1D ve 1E) ile üfleme iğnesi heparinize. Not: aspiratör boru montaj Zebra balığı sperm dondurulması 16 kullanılmıştır. Uzun kauçuk tüp ağzına uçan herhangi bir kan durdurabilirsiniz. Tüpün yarıda bir filtre ayarlama tehlikeleri önleyebilirsiniz. </li><li> En az 1 saat süre ile 10 cm&#39;lik bir Petri kabı ve hava-kuru heparinize iğneler saklayın. Iğneler çok sayıda önceden hazırlanabilir. </li></ol> 2. Anestezi <ol><li> (2-fenoksietanol, 100 ul balık 200 ml su karıştırılarak 2 küçük bir plastik durumda anestezi çözeltisi hazırlayın-PE). Anestezik nihai konsantrasyonu 500 ppm dir. DİKKAT! 2-PE hızlı bir etkiye sahiptir. </li><li> Dolaşım sisteminden balık (AB zorlanma) istenen sayıda kaldırın. </li><li> 2 dakika (Şekil 1F) - 1 Anesteziyoloji içine balık aktarmak için bir net kullanın. 1 dakika içinde hızlı operkulum hareketlerini yavaş yavaş, yüzmek, yatay gasp göğüs yüzgeçleri yayıldı ve sahip balık gözlemleyin. </li><li> Zaman geçtikçe, balık kasanın altında yatıyordu gözlemlemek ve nihayet yüzme durdurun. Balık gasp durur ve operkulum hareketleri yavaş olduğunda anestezi cerrahi uçağı ulaşacak. Bu noktada, balık kan toplama için hazırdır. </li><li> Bir skimmer hafifçe 2-PE anestezi balık kaldırın ve kullanma anestezik (Şekil 1G) ile ıslatılmış kağıt havlu üzerine yerleştirin. Göz kuruluğunu önlemek ve yavaşça başka bir kuru ve yumuşak doku kağıt kullanmak da 2-PE çözeltisi ile ıslatılmış yumuşak doku kağıt ile balığın baş örtüsüvücut yüzeyinden kurulayın. </li></ol> 3. Kan Toplama <ol><li> Aspiratör tüp montajı (ya da ampul dağıtıcı) burunluğu sonunda bir heparinize iğne yerleştirin ve ağızda aspiratör borusu düzeneğinin ağızlık ucunu tutun. </li><li> Burunluk ucu bir arada iğne tutun ve dikkatlice iğne ucu ile müdahale ölçekleri kaldırın. Kan toplama sitesine 45 ° açı - 30 de iğne takın. Gastrointestinal sistem (Şekil 1H) bir patlağı kaçının. Yukarıda açıklandığı gibi ampul dağıtıcı kullanılması durumunda, başparmak ve orta parmak ile ampul basın ve ilk parmak ile ampulün ucundaki delik blok, sonra iğneyi. Not: Kan toplama sitesi dorsal aorta bölgesinde anüs vücut ekseni ve posterior boyunca olduğunu. Dorsal aorta (DA) ve posterior kardinal ven (PCV) omurga ventral sadece (Şekil 2). </li><li> Emmek başlayıniğne omurgayı dokunmadan hissettim zaman aspirasyon borusu düzeneğinin sonuna ağızlık. Kan yükselecek etmezse, kan akışını teşvik etmek için elle kurnazca iğne ucu hareket ettirin. Kan iğne içine yükseliyor kez hemen sallayarak durdurun ve yavaşça (Şekil 1i) emmek unutmayın. Ampul dağıtıcı kullanılması durumunda, kan aspire ampul basıncı boşaltın. </li><li> Kan yavaş nedeniyle arteriyel kan basıncının muhtemelen emme olmadan pulsatil bir tarzda iğne içine artacak gözlemleyin. Bu nedenle, iğne doğru arteri nüfuz halinde emmek için gerekli değildir. </li><li> Kanın uygun hacmi toplanır sonra emme durdurun. Balık iğneyi çıkarın ve herhangi bir kanamayı durdurmak için yumuşak doku kağıdı kullanarak ponksiyon sitesine basın. Parmak basıncı 20 sn (Şekil 1J) - yaklaşık 10 sonrası kanama durdurma gözlemleyin. </li><li> Kanama durdurulduktan sonra, derhal bir cle geri balık transferiBir sıcak su (~ 28 ° C) tankı. Yüzmek başlayana kadar yavaşça solungaçları doğru su dönen kurtarmak için balık yardımcı olur. Not: 2 L tankında 5 postsampling balık tutun ve balık oksijen sirkülasyon sistemine tankı bağlayın. Balık su antibiyotik ilave edilmesi gerekli değildir. Normal konut ve beslenme ile balık koruyun. </li><li> Parafilm bir parça (Şekil 1K) temiz bir alan üzerine iğne kan kovmak. </li><li> Herhangi bir ticari el glukometre (Şekil 1 L) kullanılarak kan şekeri ölçün. Not: Glucometer bir glikoz dehidrojenaz-flavin adenin dinükleotid elektrot kullanır ve 0,6 ul örnek bir hacimde kullanılmasını gerektirir. <ol><li> Metre içine tamamen bir test bandı takın ve doğrudan kan damlası dokunun. Otomatik olarak test şeridi içine kan beraberlik gözlemleyin ve görüntüleme alanında kan şekeri sonucu 5 sn edinin. Sonucu kaydedin ve test bandını atın. KullanınHer ölçüm için yeni bir test şeridi. </li></ol></li><li> (isteğe bağlı adım) bir pipetleme kanın doğru miktarda alın ve daha analizler (hemoglobin, triaçilgliserol (TG), total kolesterol, vs.) bir mikrosantrifüj tüp kan transferi. Gerekirse, tuzlu su ile bir balık tam kan seyreltin. RT&#39;de 680 x g&#39;de 3 dakika boyunca kan örnekleri santrifüj ve plazma hasat. Yeni bir tüp içine plazma aktarın. Bu noktada, bu biyokimyasal analizlerde kullanılmak üzere hazırdır. </li></ol>

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The authors have nothing to disclose.

Biz seri erişkin zebrabalıkları kan elde etmek için burada ayrıntılı bir protokol mevcut. Bu yöntem gerçekleştirmek için basittir ve günlük olarak laboratuvarda kullanın. Bu kan toplama yöntemi Zebra balığı en dorsal aort içine bir cam kılcal iğne takmadan dayanmaktadır. Bu işlem sırasında, o dorsal aorta arıyor için kriter olduğu için omurgayı baskılamak için dikkatli olmak önemlidir. Omurga yaralanması azaltılması hayatta kalma oranını artıracaktır. Bu teknik, basit ve usta kola...

Organları ve genetik insanlarda 1,2 benzemektedir, çünkü balığı insan hastalıklarında değerli bir model olarak artan bir popülerlik kazanmaktadır. Gelişimsel biyoloji alanında birçok çalışma zebra balığı ve insan gösteri hematopoiesis 3, hemostaz 4,5 ve myelopoiesis 6 benzerliği işaretli olduğunu göstermiştir. Yetişkin Zebra balığı da bu model organizma hisseleri nedeniyle insan hastalıklarında bozulduğu olanlarla ortak yollar immünolojik 7, nörodejeneratif 8 ve obezite ile ilişkili hastalıkların 9 çalışmak için kullanılır. Obezite ve obezite ile ilişkili hastalıkların (diyabet, karaciğer yağlanması ve alkolsüz steatohepatit ve ateroskleroz) için zebrabalıkları kan şekeri ve lipidler düzeyleri iyice birçok transgenik ve diyete bağlı obezite modellerinde 10-13 araştırılmıştır. Bireysel hayvanlardan Tekrarlanan kan örneklemesi hayvan kullanımı ve dec azaltacaktırbireyler farklılıkları artma. Ancak, tekrarlanan numune toplama nedeniyle nispeten küçük kan hacmi ve kolay erişilebilir damarların olmaması gibi zebrabalıkları gibi küçük hayvanlarda teknik olarak zordur. Bu yöntemler de dahil olmak üzere öldürücülüğü kendi dezavantajları, olmasına rağmen zebrafish bir defalık kan toplama için çeşitli yöntemler, ilişkili doku hasarı ve sınırlı kan hacmi geliştirilmiştir. Örneğin, 1-5 ul kan dorsal aorta 5 bölgesinde yaklaşık olarak boyuna 0.3 cm bir yanal kesi hasat edilebilir. Göğüs kuşak boyunca keserek makas ile Decapitation 5-10 ul kan 10 toplayabilir. Başka uygun kan örnekleme metodu kuyruk ablasyon 14&#39;tür. Kardiyak delinme aynı balık tekrarlanan kan toplama için tek potansiyel alternatif bir yöntemdir, ancak çok küçük bir miktar bu prosedüre (yaklaşık 50 nl) elde perfor olabilir analizlerin sayısını sınırlar11 med. Bu duruma göre, yeni bir protokol bu organizma insan hastalıkları için standart model organizma için önemli bir ilerleme gerekli olacaktır tekrarlanan öldürücü olmayan kan örnekleme sağlamak için gereklidir. Bu teknik farmakolojik cevabı, moleküler tanı için biyobelirteçlerin, prognoz tayini keşif ve metabolik hastalıklar, dejeneratif hastalıklar ve maligniteler çeşitli türleri gibi çeşitli hastalıkların izlenmesi test etmek için izin verecek. Bu nedenle Zebra balığı seri 15 kan elde etmek için minimal invaziv bir yöntem geliştirdi. Burada görsel prosedür göstermek ve bu teknik için ayrıntılı bir protokol sağlar. Bu yöntemi kullanarak, çeşitli hemoglobin içeren parametreler, açlık kan şekeri, sağlıklı erişkin zebrabalıkları kanında lipidler dayalı normal değer değerlendirildi. Ayrıca, aynı zamanda, bu yöntem, m seri örnekleri gerektiren çalışmalar için uygun olup olmadığını değerlendiriroverfeeding deneyler sırasında kan glukoz düzeylerinde geçici değişiklikler onitoring.

<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

Tekrarlanan kan toplama laboratuvar hayvanları üzerinde yapılan en yaygın tekniklerden biridir. Ancak, Zebra balığı kan toplama için bir öldürücü olmayan protokol kurulmamıştır. Zebra balığı kan toplama için önceki metotlar, lateral insizyon, dekapitasyon ve kuyruk ablasyon gibi öldürücü bulunmaktadır. Böylece biz burada bu yordamı özetleyen ayrıntılı bir protokol, bir roman &quot;tekrarlanan&quot; kan toplama yöntemi geliştirdi ve mevcut var. Bu yöntem, minimal invaziv ve bu nedenle, aynı bireyden elde tekrarlanan kan örnekleme sağlayan zebrabalıkları için çok düşük bir ölüm oranına (% 2.3) elde edilir. Kan örneği maksimum hacim balık vücut ağırlığına bağlıdır. Aralıklarla tekrarlanan kan örneklemesi için hacim kan hemoglobin ölçümleri ile değerlendirildi vücut ağırlığının her hafta ya da ≤1% 2 haftada, bir ≤0.4% olmalıdır. Ayrıca, hemoglobin, açlık kan şekeri, plazma triaçilgliserol (TG) ve toplam cerkek ve dişi yetişkin zebrafish holesterol düzeyleri ölçüldü. Ayrıca diyet indükte obeziteye glikoz metabolizmasının bozulmasına araştırmak için, bu yöntemi uygulanır. Bu kan toplama yöntemi glukoz ve lipit metabolizması ve bir insan hastalık model organizma olarak zebrafish kullanımını artıracak hematolojik çalışmalar da dahil olmak üzere birçok uygulama, sağlayacaktır.

(Şekil 1J &lt;1 mm delinmesi) ve% 2.3 gibi çok düşük bir mortalite oranı veren bu kan toplama yöntemi zebrabalıkları minimal hasara neden olmaktadır. Tek bir balık toplanan ve vücut ağırlığı (Şekil 3) bir ilişki değerlendirilebilir kan maksimum ses incelenmiştir. Biz toplanan maksimum kan hacmi doğrusal vücut ağırlığının (R = 0,813) ile ilişkili olduğunu bulduk. Tek bir balık (vücut ağırlığı = 1,071 g) toplanan kanın en büyük hacmi 25 ul oldu ve ...

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.

Yetişkin Zebra balığı Kan Testleri için Tekrarlanan Kan Alma

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

repeated-blood-collection-for-blood-tests-in-adult-zebrafish

Research

JoVE Journal

Biology

34.9K Görüntüleme.  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.

Zebra balığı bir Vivo içinde Model sistemi olarak kullanarak DNA çoğaltma zamanlaması profil oluşturma

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

Genetik

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.

Yetişkin Zebra balığı retro-orbital Enjeksiyon

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

Biyoloji

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.

Gelişmekte Olan Zebra Balıklarında Değiştirilmiş Kan Oluşumunu Tespit Etmek ve Nicel hale Getirmek için Akış Sitometrisini Kullanma

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

Gelişim Biyolojisi

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.

Kas Hastalığının Zebra Balığı Modellerinde Kas Yenilenmesinin İncelenmesi

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.

Yetişkin Zebra Balıklarında Optimize Edilmiş İntravenöz Enjeksiyon

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

Kanser Araştırmaları

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.

Yetişkin Zebra balığı Biyokimyasal Analiz için Kan Alma

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.

Diyabet ile gözleri bir balık eğitim: mikrodiseksiyon, görselleştirme ve yetişkin tg(fli:EGFP) Zebra balığı retina damarlara analizi

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

Tıp

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.

Yetişkin Zebra balığı Gavaging

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.

Yüksek verim Uygulamaları Yetişkin Zebra Hearts Kurtarma

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.

Yetişkin zebrafish için In vivo yüzey elektrokardiyografi

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