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.

모든 동물의 절차는 미에 대학의 윤리위원회의 승인을하고, 국제 가이드 라인에 일본의 동물 복지 규제 &#39;복지와 동물의 관리에 관한 법률&#39;(일본의 환경부)에 따라 수행하고 준수 하였다. 니들 1. 준비 주 : 모든 실험은 마취 하에서 수행하고, 모든 노력이 고통을 최소화 하였다. 안락사, 생선 ≥20 분간 (≤4 ℃에서 5 부 얼음 / 물 1 부)에 빙수 욕에 침지 하였다. <ol><li> 바늘 풀러 (도 1a)과 1.0 mm의 외경 유리 모세관 당기는 유리 마이크로 모세관 바늘을 준비한다. </li><li> 비스듬하게 잘 가위 (그림 1B)를 사용하여 바늘의 끝을 잘라. 200 μm의 (그림 1C) - 이상적인 팁 직경은 약 100이어야합니다. 팁 diamete 경우R은 너무 좁고, 혈액 바늘을 입력하지 않습니다. </li><li> 5 ㎎ / ㎖의 농도로 염수에 헤파린을 녹인다. </li><li> 흡인기 튜브 어셈블리의 코 받침 이요 끝에서 미리 절단 바늘을 놓고 입에 마우스 피스를 보유, 또는 전구 지급기에 바늘을 연결합니다. 헤파린 용액에 바늘 끝을 담그고, 흡입에 의해 솔루션 (그림 1D와 1E)을 통해 불고 바늘을 heparinize. 주 : 흡인기 튜브 조립체는 제브라 피쉬 정자 동결 보존 (16)에 사용되어왔다. 긴 고무 튜브 입으로 비행하는 모든 혈액을 중지 할 수 있습니다. 관의 중간에 필터 설정 위험을 피할 수있다. </li><li> 적어도 1 시간 동안 10 cm 페트리 접시와 공기 건조에 헤파린 주사 바늘을 저장합니다. 바늘의 다수의 미리 제조 될 수있다. </li></ol> 2. 마취 <ol><li> (2- 페녹시 에탄올 100 ㎕ 물고기 200 ml의 물을 혼합하여 2 작은 플라스틱 케이스 마취 용액을 제조-PE). 마취제의 최종 농도는 500 ppm으로합니다. 주의! 2-PE는 빠른 효과가있다. </li><li> 순환 시스템에서 물고기 (AB 변형)의 원하는 번호를 제거합니다. </li><li> 2 분 (그림 1 층) - 1 마취제로 물고기를 전송하기 위해 인터넷을 사용합니다. 1 분 이내에 신속하게 아가미의 움직임을 점차, 수영, 수평 헐떡 거림을 가슴 지느러미를 확산하고,이 물고기를 관찰한다. </li><li> 시간이 지남에 따라, 물고기가 케이스의 바닥에 누워 관찰하고 마지막으로 수영을 중지합니다. 물고기가 말하다을 중지하고 아가미의 움직임이 느린 경우 마취의 수술 비행기에 도달합니다. 이 시점에서, 물고기는 혈액 수집을위한 준비가되어 있습니다. </li><li> 스키머가 부드럽게 2-PE에서 마취 물고기를 들어 올려 사용하면 마취제 (그림 1G)에 적신 종이 타월에 놓습니다. 눈의 건조를 방지하고 부드럽게하는 또 다른 부드러운 마른 티슈 페이퍼를 사용할 수있는 2-PE 용액으로 적신 부드러운 티슈와 물고기의 머리를 커버신체 표면을 건조. </li></ol> 3. 혈액 수집 <ol><li> 흡인기 튜브 어셈블리 (또는 전구 디스펜서)의 코 부분의 끝에서 헤파린 바늘을 놓고 입에 흡인기 튜브 어셈블리의 마우스 피스 끝을 잡고. </li><li> 코 부분의 끝과 함께 바늘을 잡고 조심스럽게 바늘 끝과 간섭 스케일을 제거합니다. 혈액 수집 사이트에 45 ° 각도 - 30 바늘을 삽입합니다. 위장관 (그림 1H)의 구멍을 피하십시오. 상술 한 바와 같이 전구 디스펜서를 사용하는 경우, 엄지 손가락과 가운데 손가락으로 전구를 눌러 먼저 손가락으로 전구의 끝 부분에 구멍을 차단 한 후 바늘을 삽입합니다. 참고 : 혈액 수집이 사이트는 지느러미 대동맥의 영역에서 항문에 몸 축과 후방을 따라입니다. 지느러미 대동맥 (DA) 및 후방 추기경 정맥 (PCV)은 척추에 복부 단지 (그림 2)입니다. </li><li> 빨아 시작바늘이 척추를 만지고 느낄 때 흡입 튜브 어셈블리의 끝을 마우스 피스. 피가 상승하지 않는 경우, 혈액의 흐름을 촉진하기 위해 손으로 미묘 바늘 끝을 이동합니다. 혈액이 바늘로 상승되면, 즉시 흔들어 중지하고 부드럽게 (그림 1I)을 빨아합니다. 전구 디스펜서를 사용하는 경우, 혈액을 흡인하는 전구의 압력을 해제. </li><li> 혈액 천천히 때문에 동맥 혈압 가능성 흡입없이 맥동 방식으로 바늘로 상승 할 것이다 관찰한다. 따라서 바늘이 제대로 동맥을 관통하면 흡인 할 필요가 없다. </li><li> 혈액의 적절한 볼륨이 수집 한 후 흡입을 중지합니다. 물고기에서 바늘을 제거하고 출혈을 멈추게하기 위해 부드러운 티슈를 사용하여 천자 사이트를 누릅니다. 손가락 압력의 20 초 (그림 1J) - 약 10 후 지혈을 준수하십시오. </li><li> 출혈이 정지 된 후, 즉시 CLE로 다시 전송 물고기따뜻한 물 (~ 28 ° C) 탱크. 이 수영을 시작할 때까지 부드럽게 아가미쪽으로 물을 소용돌이로 복구 할 수있는 물고기를 도와주세요. 참고 : 2 L 탱크에 5 postsampling 물고기까지 유지하고 물고기를 산소를하기 위해 순환 시스템에 탱크를 연결합니다. 물고기 물에 항생제를 첨가 할 필요가 없습니다. 일반 주택과 공급에 물고기를 유지한다. </li><li> 파라 필름의 조각 (그림 1K)의 깨끗한 영역에 바늘에서 피를 추방. </li><li> 상업적 휴대용 혈당 측정기 (도 1L)를 사용하여 혈당을 측정한다. 주 : 혈당 측정기는 글루코스 탈수소 효소 플라 빈 아데닌 디 뉴클레오티드 전극을 사용하고, 0.6 μL의 샘플 량을 필요로한다. <ol><li> 미터에 완전히 테스트 스트립을 삽입하고 직접 혈액 방울을 터치합니다. 자동으로 테스트 스트립에 혈액 무승부를 관찰하고 표시 영역에 혈당 결과 5 초를 얻을 수 있습니다. 결과를 기록하고 테스트 스트립을 폐기합니다. 사용각 측정을위한 새로운 테스트 스트립. </li></ol></li><li> (선택 단계) 피펫으로 혈액의 정확한 양을 가지고 더 분석 (헤모글로빈, 중성 지방 (TG), 총 콜레스테롤 등) microcentrifuge 관에 피를 전송합니다. 필요한 경우, 식염수 한 물고기에서 전체 혈액을 희석. RT에서 680 XG에서 3 분 동안 혈액 샘플을 원심 분리하여 플라즈마를 수확. 새로운 튜브에 플라즈마를 전송합니다. 지금부터, 생화학 적 분석에 사용될 준비가되어있다. </li></ol>

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The authors have nothing to disclose.

우리는 직렬 성인 제브라 피쉬에서 혈액을 얻기 위해 여기에 대한 자세한 프로토콜을 제시한다. 이 방법을 수행하는 것이 간단하며 우리는 일상적 실험에 사용. 이러한 혈액 수집 방법에 지브라 피쉬의 등쪽 대동맥 내로 유리 모세관 바늘 삽입에 근거한다. 이 절차를 수행하는 동안, 그것은 지느러미 대동맥를 검색하기위한 기준이 있기 때문에 척추를 브레이션 않도록주의하는 것이 중요합니다. ...

이들 장기 및 인간 유전학 1,2- 마찬가지이기 때문에 지브라 피쉬는 인간 질병의 유용한 모델로 증가 인기를 얻고있다. 발생 생물학의 분야에서 많은 연구는 제브라 피쉬와 인간의 쇼가 조혈 3, 지혈 4,5, 및 myelopoiesis 6 유사성을 표시 것을 증명하고있다. 성인 제브라 피쉬는이 모델 생물을 공유하기 때문에 인간의 질병에 혼란 그와 공통 경로를 면역 학적 7, 신경 퇴행성 8과 비만 관련 질병 (9)을 연구하는 데 사용됩니다. 비만 및 비만 관련 질환 (당뇨병, 간 지방증 및 비 알코올성 지방 간염 및 동맥 경화증), 제브라 피쉬 들어 혈당 및 지질 수준을 충분히 여러 형질 전환 및식이 유도 비만 모델 10-13에서 연구되었다. 개별 동물의 반복 채혈 동물 사용 및 12월을 줄일 수interindividual 차이를 rease. 그러나, 반복 된 시료 채취 때문에 상대적으로 작은 혈액량 쉽게 접근 용기의 부족 등 제브라 작은 동물에서 기술적으로 곤란하다. 이들 방법은 치사 포함 자신 단점을 가지고 있지만 제브라에서 일회성 채혈을위한 여러 방법이, 관련된 조직 손상 및 제한된 혈액량을 개발되어왔다. 예를 들어, 1-5 ㎕의 혈액 등쪽 대동맥 (5)의 영역의 길이는 약 0.3 cm의 횡 절개에서 수확 될 수있다. 팔 이음 뼈를 절단하여 가위로 잘린 5-10 μL 혈액 (10)를 수집 할 수 있습니다. 또 다른 편리한 혈액 샘플링 방법은 꼬리 어블 14이다. 심장 천자 동일한 물고기 반복 채혈을위한 하나의 잠재적 인 대안 방법이지만, 매우 소량이 절차 (약 50 NL)를 수득 할 수있다 perfor 분석의 수를 제한11 클럽 메드. 따라서, 새로운 프로토콜이 유기체는 인간의 질병을위한 표준 모델 생물하기 위해서는 중요한 미리 필요하다 반복적 치명적 혈액 샘플링을 가능하게하는데 필요하다. 이 기술은 약물 반응, 분자 진단용 바이오 마커, 예후 판정 발견과 같은 대사성 질환, 퇴행성 질환 및 악성 종양 등의 다양한 여러 종류의 질환의 모니터링을 테스트 할 수있다. 따라서 우리는 제브라 피쉬 직렬 15에서 혈액을 얻기위한 최소 침습적 방법을 개발했다. 여기에서 우리는 시각적으로 절차를 설명하고이 기​​술에 대한 자세한 프로토콜을 제공합니다. 이 방법을 사용하여, 다양한 파라미터를 포함 헤모글로빈, 공복 혈당 및 건강한 성인 지브라 피쉬의 혈중 지​​질에 기초하여 정상 값을 평가 하였다. 또한, 우리는 또한이 방법은 M에 의해 일련의 샘플을 필요로 연구에 적합한 지 여부를 평가과식 실험시의 혈당 변화를 시간적 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

반복 채혈은 실험 동물에서 수행하는 가장 일반적인 방법 중 하나입니다. 그러나, 제브라 피쉬에서 채혈을위한 비 살상 프로토콜은 확립되어 있지 않다. 제브라 피쉬에서 혈액 수집에 대한 이전의 방법은 횡 절개, 잘린 꼬리 절제로, 치명적입니다. 따라서 우리는 여기에서이 절차를 요약 한 상세한 프로토콜을 소설 &quot;반복&quot;혈액 수집 방법을 개발하고, 존재했다. 이 방법은 침습적이고 따라서 동일인으로부터 반복 채혈이 가능 제브라위한 매우 낮은 사망률 (2.3 %)을 초래한다. 혈액 샘플링의 최대 볼륨은 물고기의 체중에 따라 달라집니다. 간격으로 반복 혈액 샘플링의 볼륨은 혈액의 헤모글로빈의 측정에 의해 평가 하였다 체중 매주 또는 ≤1 % 2 주마다의 ≤0.4 %이어야한다. 또한, 혈색소, 공복 혈당, 혈장 중성 지방 (TG), 총 C남성 및 여성 성인 holesterol 지브라 피쉬의 수준을 측정 하였다. 우리는 또한 다이어트 유발 된 비만에서 포도당 대사의 조절 곤란을 조사하기 위해이 방법을 적용했다. 이러한 혈액 수집 방법은 글루코스 및 지질 대사 및 인간의 질환 모델 생물로서 지브라 피쉬의 사용을 증가시킬 것이다 혈액 학적 연구를 포함하는 많은 애플리케이션을 허용 할 것이다.

(도 1J를 &lt;1mm 천자) 및 2.3 %의 매우 낮은 치사율을 산출 이러한 혈액 수집 방법은 제브라 최소한 부상시킨다. 우리는 하나의 물고기에서 수집 및 체중 (그림 3)의 관계를 평가 될 수있는 혈액의 최대 볼륨을 조사했다. 우리는 수집 된 최대 혈액량이 선형 적으로 체중 (R = 0.813) 상관 관계를 보였다 것으로 나타났습니다. 개별 물고기 (체중 = 1.071 g)으로부터 혈액 채취 큰 부피...

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.

성인 Zebrafish의에서 혈액 검사에 대한 반복 채혈

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 조회수.  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.

비디오: 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.

DNA 복제 타이밍 Zebrafish Vivo에서 모델 시스템으로 사용 하 여 프로 파일링

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

연구

유전학

Screening Sperm for the Rapid Isolation of Germline Edits in Zebrafish

The advent of targeted CRISPR-Cas nuclease technologies has revolutionized the ability to perform precise genome editing in both established and emerging model systems. CRISPR-Cas genome editing systems use a synthetic guide RNA (sgRNA) to target a CRISPR-associated (Cas) endonuclease to specific genomic DNA loci, where the Cas endonuclease generates a double-strand break. The repair of double-strand breaks by intrinsic error-prone mechanisms leads to insertions and/or deletions, disrupting the locus. Alternatively, the inclusion of double-stranded DNA donors or single-stranded DNA oligonucleotides in this process can elicit the inclusion of precise genome edits ranging from single nucleotide polymorphisms to small immunological tags or even large fluorescent protein constructs. However, a major bottleneck in this procedure can be finding and isolating the desired edit in the germline. This protocol outlines a robust method for screening and isolating germline mutations at specific loci in Danio rerio (zebrafish); however, these principles may be adaptable in any model where in vivo sperm collection is possible.

CRISPR-Cas technologies have revolutionized the field of genome editing. However, finding and isolating the desired germline edit remains a major bottleneck. Therefore, this protocol describes a robust method for quickly screening F0 CRISPR-injected zebrafish sperm for germline edits using standard PCR, restriction digest, and gel electrophoresis techniques.

제브라피쉬에서 생식계열 편집의 신속한 분리를 위한 정자 스크리닝

The advent of targeted CRISPR-Cas nuclease technologies has revolutionized the ability to perform precise genome editing in both established and emerging ...

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.

성인 Zebrafish에서 레트로 - 궤도 분사

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

생물학

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.

유동 세포형을 사용하여 개발 중인 제브라피시에서 변경된 혈액 형성을 감지하고 양량화합니다.

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

발생학

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.

근육 질환의 제브라피시 모델에서 근육 재생 검토

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

Intraperitoneal Injection into Adult Zebrafish

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, this method makes it difficult to know how much reagent is absorbed or taken up per fish. Some experimental questions, particularly those related to metabolic studies, may be better addressed by delivering a defined quantity to each fish, based on weight. Here we present a method for intraperitoneal (IP) injection into adult zebrafish. Injection is into the abdominal cavity, posterior to the pelvic girdle. This procedure is adapted from veterinary methods used for larger fish. It is safe, as we have observed zero mortality. Additionally, we have seen bleeding at the injection site in only 5 out of 127 injections, and in each of those cases the bleeding was brief, lasting several seconds, and the quantity of blood lost was small. Success with this procedure requires gentle handling of the fish through several steps including fasting, weighing, anesthetizing, injection, and recovery. Precautions are required to minimize stress throughout the procedure. Our precautions include using a small injection volume and a 35G needle. We use Cortland salt solution as the vehicle, which is osmotically balanced for freshwater fish. Aeration of the gills is maintained during the injection procedure by first bringing the fish into a surgical plane of anesthesia, which allows slow operculum movements, and second, by holding the fish in a trough within a water-saturated sponge during the injection itself. We demonstrate the utility of IP injection by injecting glucose and monitoring the rise in blood glucose level and its subsequent return to normal. As stress is known to increase blood glucose in teleost fish, we compare blood glucose levels in vehicle-injected and non-injected adults and show that the procedure does not cause a significant rise in blood glucose.

We demonstrate intraperitoneal injection into adult zebrafish. We use a 10 &mu;l NanoFil microsyringe controlled by a Micro4 controller and UltraMicroPump III. This demonstration includes the use of cold water as an anesthetic.

성인 Zebrafish에 Intraperitoneal 사출

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, ...

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.

성체 제브라피쉬에 최적화된 정맥 주사

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

암 연구학

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.

성인 Zebrafish의 생물 화학 분석을위한 혈액 수집

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 ...

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.

성인 Zebrafish의를 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.

높은 처리량 응용 프로그램에 대한 성인 Zebrafish의 마음의 복구

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