Name: a doxorubicin-induced cardiomyopathy model in adult zebrafish
Uploaded: 2018-06-07T13:30:00
Description: Watch this Scientific Journal Video about A Doxorubicin-induced Cardiomyopathy Model in Adult Zebrafish at JoVE.com

이 작품에서 미국 심장 협회 과학자 개발 교부 금에 의해 부분적으로 지원 되었다 (14SDG18160021 야드), 미국 NIH R01 보조금 HL 81753와 HL 107304 xx, xx 메이 요 재단.

여기에서 설명한 모든 절차 관리 및 실험 동물 사용 (국가 아카데미 압박. 2011), 대 한 가이드에 따라 수행 했다 그리고 그들은 메이 요 클리닉 기관 동물 관리 및 사용 위원회에 의해 승인 했다.
1. 성인 Zebrafish 준비
<ol><li>얻으려면 적어도 두 배나 많은 DOX 주입에 필요한 총 물고기 탱크를 건너에 충분 한 번 식 쌍을 설정 합니다. 서로 다른 유전적 배경 가진 물고기를 비교 하?...

<ol>
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진보적인 DIC 모델, DOX 20 mg/kg의 복용량은 물고기 죽음에 1 wpi 하지만 여전히 결과 중 중요 한 물고기 죽음 및 4 wpi (그림 3 및 후 심장 기능의 감소를 발생 하지 않는 가장 높은 복용량으로 실험적으로 결정 그림 4C). 이 복용량은 설치류 DIC 모델 (15-25 mg/kg)에서 자주 사용 되 고 인간 (550 m g/m2는 15 mg/kg에 해당)에 누적 복용량을4,...

독 소 루비 (DOX), Adriamycin, 라는 1960 년대1,2이후는 항 종양 약 약물으로 개발 되었습니다. 그것은 지금 아직도 적극적으로 광범위 한 종양에 대 한 중요 한 화학요법 약물으로 사용. 그러나, DOX의 임상 응용의 복용량 의존 독성에 의해 방해 되었다, 특히 cardiotoxicity asymptomatic electrocardiographic 변경에서 pericarditis에 배열 하는 가변 증상이 특징 및 decompensated 심장 근육 병 증 1 , 2. 현재까지, 적어도 3 개의 주요 가설 일어났습니다 DIC, 활성화 된 반응성 산소 종 (선생님)1,3,,45, topoisomerase II-β (의 억제를 포함 하 여 설명 하 TOP2β)6,7, 그리고 세포내 칼슘의 릴리스1,,89. 또한 DIC10,11,,1213대 중요 위험 요인으로 유전적 제안 증거를 축적. 그러나 이러한 DIC 선천적인 유전자 정체성 관련, 크게 알려지지 않은 남아 있다. Dexrazoxane 유일한 보조 에이전트는 하지만 제한 된 구현14,,1516, DIC, 치료 하는 미국 식품 및 의약품 안전 청 (FDA)에 의해 승인 추가 확인 하는 필요를 강조 치료 전략입니다. DIC의 동물 모델 따라서 이러한 목적에 대 한 탐구 된다. 때문에 그들의 접근 및 단순, DIC 모델에 기계 연구 수 잠재적으로 영향을 미칠 광범위 한 cardiomyopathies의 다른 종류에: 일반적인 병 인 수 들 간에 공유 될 다른 etiologies의 cardiomyopathies에서 특히 나중 병 적인 단계17,18,,1920.
DIC의 설치류 모델, 뿐만 아니라 더 높은 처리량을 가진 zebrafish DIC 모델 새로운 유전적 요인 및 치료의 발견을 촉진 하기 위하여 개발 되었습니다. 배아 DIC 모델 치료 화합물21심사 투명 zebrafish 태아에 설립 되었습니다. 감안할 때 그는 cardiomyopathies는 진보적인 병을 가진 성인 개시 질병, 성인 zebrafish 심근 모델 되었습니다22,,2324,,2526개발. 만성 빈 혈24, DIC 성인 zebrafish23의 두 번째 인수 심근 모델 뒤에서 발생 하는 심근에 대 한 첫 번째 인수 모델을 생성 되었습니다. 우리는 성인 zebrafish에 DOX의 단일 bolus 주사 뒤 심장 근육 병 증의 만성 단계 6 개월 후 주입 약 1 주 후 주입 (wpi), 이내 급성 단계 구성 된 cardiotoxicity 유도 발견. Rapamycin의 기계적 대상의 haploinsufficiency 동안(mtor) 심근을 살펴볼 만성 단계에서 그것은 과장 물고기 사망률 급성 단계에 단계-종속 분별을 성인 DIC 모델의 가치를 강조 메커니즘23. 우리 성인 DIC 모델 스트레스 zebrafish insertional 심장 (ZIC) 돌연변이의27insertional mutagenesis transposon 기반 접근 통해 생성 되는 컬렉션을 사용할 수 있습니다 입증 되었습니다. 3 알려진된 심근 유전자로 DnaJ (Hsp40) 체, 인도의 B, 및 회원 6b 파일럿 화면 확인 (dnajb6b) 새로운 DIC 민감성 유전자28. 따라서, zebrafish에 성인 DIC 모델의 세대 주도 체계적으로 보완 기존 게놈 넓은 협회 연구 결과 (GWAS) 및 양이 많은 특색 소재 시 (QTL DIC에 대 한 유전 한정자의 식별을 가능 하 게 하는 새로운 방법론 ) 분석.
생성 및 성인 zebrafish DIC 모델의 구현 동안 우리는 다른 연구자 들 사이 또는 다른 주사 같은 조사에 의해 수행 중 에서도 상당한 변화 것으로 나타났습니다. 모델의 경도 특성 도전 다른 조사에서 결과 등록 하 고 순차 문제 해결 과정을 부과 한다. 이 간단한 심근 유도 스트레스 방법의 연구 공동체에 의해 사용을 촉진, 하 우리 자세히, 현재 두 가지 유형의 IP 주입, 우리의 프로토콜을 설명 하 고 다른 연구자 들 사이 유사를 줄이기 위해 고려 사항에 대해.

<table>
	<tbody>
		<tr>
			<td>Crossing tank</td>
			<td>Aquaneering</td>
			<td>ZHCT100</td>
			<td>Fish breeding</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>ThermoFisher</td>
			<td></td>
			<td>Maintaining embryo</td>
		</tr>
		<tr>
			<td>3 L medium tank</td>
			<td>Aquaneering</td>
			<td>ZT280</td>
			<td>Maintaining fish</td>
		</tr>
		<tr>
			<td>Paramecia</td>
			<td>Carolina</td>
			<td>131560</td>
			<td>Food for juvenile fish</td>
		</tr>
		<tr>
			<td>Live hatched brine shrimp</td>
			<td>in house</td>
			<td></td>
			<td>Food for adult fish</td>
		</tr>
		<tr>
			<td>Doxorubicin hydrochloride</td>
			<td>Sigma</td>
			<td>D1515-10MG</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 ml safe-lock tube</td>
			<td>Eppendorf</td>
			<td>No. 022363204</td>
			<td>For drug storage</td>
		</tr>
		<tr>
			<td>Aluminum foil paper</td>
			<td>Fisher</td>
			<td>1213104</td>
			<td>For preventing light exposure</td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Roche</td>
			<td>No. 03115887001</td>
			<td>For dechorionating embryo</td>
		</tr>
		<tr>
			<td>Hank&#39;s balanced salt solution (HBBS)</td>
			<td>ThermoFisher</td>
			<td>14025076</td>
			<td>Vehicle for DOX</td>
		</tr>
		<tr>
			<td>100 mm petri dish</td>
			<td>Falcon</td>
			<td>431741</td>
			<td></td>
		</tr>
		<tr>
			<td>10 &#956;L NanoFil micro-syringe</td>
			<td>WPI</td>
			<td>NANOFIL</td>
			<td>For injection</td>
		</tr>
		<tr>
			<td>34 gauge needle&#160;</td>
			<td>WPI</td>
			<td>NF34BV-2</td>
			<td>For injection</td>
		</tr>
		<tr>
			<td>Tricaine</td>
			<td>Argent</td>
			<td>MS-222</td>
			<td>Anesthetizing fish</td>
		</tr>
		<tr>
			<td>96 well plate</td>
			<td>Costar</td>
			<td>3539</td>
			<td>For embryo drug treatment</td>
		</tr>
		<tr>
			<td>Transfer pipette</td>
			<td>Bel-art product</td>
			<td>F37898</td>
			<td>For transfering embryo</td>
		</tr>
	</tbody>
</table>

a doxorubicin-induced cardiomyopathy model in adult zebrafish

유전자 액세스할 수 성인 제 브라 (Danio rerio) 점점 척추 모델 심근과 같은 인간의 질병을 이해 하기 위한를 사용 했다. 편의 및 높은 처리량 유전자 조작에 순종 획득된 심근 모델의 세대 성인 zebrafish, 독 소 루비 유도 심장 근육 병 증 (DIC) 모델 등은 문을 열고 새로운 연구도로에 앞으로 유전자 검사를 통해 심근 한정자를 발견 포함 하 여. 다른 배아 zebrafish DIC 모델, 초기 급성 및 만성 단계 후 심근의 무대-종속 신호 메커니즘 및 치료 전략의 연구를 활성화 성인 zebrafish DIC 모델에서 확인할 수 있습니다. 그러나, 경험 있는 수 사관의 손 에서도 현재 모델 변수 결과 얻을 수 있습니다. DIC 모델의 미래 구현을 촉진 하기 위하여 성인 zebrafish이 DIC 모델을 생성 하 고 복 (IP) 주입의 두 가지 대체 방법을 설명 하는 방법에 자세한 프로토콜을 선물이. 우리는 더 신뢰할 수 있는 결과 얻을 결과 적절 하 게 해석 하는 방법에 제안을 제공 하는 유사에 옵션을 논의.

여기, 성인 zebrafish 모델 DIC에 IP 삽입을 수행 하는 두 가지 방법이 제공 됩니다. 고전, 설립 IP 주입 방법29를 사용 하 여, 하는 동안 그것은 주사 된 DOX 솔루션 (빨간색) 바늘 관통 하는 위치에서 밖으로 가끔 진흙 수 지적 했다. 대체 IP 주입은 복 막에서 3-4 m m는 DOX 출시 하는 바늘 침투에 대 한 다른 위치를 사용 하 여 (그림 1A)는 효과?...

Watch this Scientific Journal Video about A Doxorubicin-induced Cardiomyopathy Model in Adult Zebrafish at JoVE.com

A Doxorubicin-induced Cardiomyopathy Model in Adult Zebrafish

성인 제 브라에서 독 소 루비 유발 심근 모델

성인 zebrafish (Danio rerio)에서 독 소 루비 유발 심근 모델을 생성 하는 메서드는 여기에 설명 되어 있습니다. 복 주입의 2 개의 다른 방법을 제시 하 고 다른 실험적인 그룹 사이 변이 줄이기 위해 조건을 설명.

The genetically accessible adult zebrafish (Danio rerio) has been increasingly used as a vertebrate model for understanding human diseases such as ...

This method can help determine key factors affecting viral agents during the development of an adaptive model for ensocycline induced catatoxicity. The main advantage of this technique is that it is a simple ensocycline induced catatoxicity model which can be used for high-throughput genetic and drug-based screenings. When making DOX stock solution, thoroughly dissolve the DOX powder in deionized water until absolutely no clumps are visible.For quality control testing, collect a wild-type Zebra fish embryos from at least two pair of fish in two separate collections. Manually dechorionate the embryos 24 hours post-fertilization using fine needles. After the dechorionation, refresh the embryo water and remove any dead embryos.Each collection of embryos must have at least 36 embryos for a quality control test. Now, dilute the stock DOX solution in fresh embryo water to a final concentration of 100 micromolar. Prepare at least 100 microliters per three embryos.Mix the dilution using a vortex. The final dilution should have a light red color. Next, load the wells of a 96 well plate with 100 microliters of diluted DOX solution.Prepare one well per three embryos. Then, transfer the embryos to the wells using a plastic transfer pipette. While transferring embryos, keep the embryos close to the end of the pipette tip.Then, put the pipette tip into solution and allow the embryos to swim naturally into the well. Do not eject the embryos with solution as this will alter the DOX concentration. 48 hours post-fertilization, refresh the DOX solution.At this time, look under 10X magnification to identify dead embryos, which have no heartbeat and identify embryos with edema. Be quick to count and remove any dead embryos, otherwise the remaining embryos in the well will also die. Repeat the dead count at 72 hours post-fertilization.If there is at least 25%death then the DOX solution has a good drug efficacy and can be used for the experiment. Prior to injecting the fish with DOX, fast the fish for 24 hours. Next, group the fish.While anesthetized, use a clean filter paper to dry each fish and measure its body weight. Make groups of fish that are all within 10%of the same body weight, so each group can be injected with the same dose of DOX. Next, calculate the working concentration of DOX for each group so that each group is injected with five microliters of solution and receives the same dose of DOX by body weight.Then, dilute the stock of DOX in 1X HBSS. Mix the dilution with a vortex and then pulse spin the dilution to keep it cooled together in the tube. For the injection, prepare an injecting platform.In a clean, 100 milliliter Petri dish, place a sponge. With the aid of a dissection microscope, cut a cavity into the sponge to hold one fish. Four centimeters usually works.Next, unto a 34-gage needle, attach a 10 microliter microsyringe. Then, rinse the needle with one 1X HBSS to remove any bubbles or blocks from the syringe and tubing. Now, briefly anesthetize the adult fish with tricaine.Then, soak the sponge in the embryo water with tricaine and transfer a fish onto the sponge with its abdomen up for the injection. Now, intraperitoneally inject DOX solution by quickly inserting the needle at a 45 degree angle into the midline between the pelvic fins about one to two millimeters deep. Then, slowly release all of the DOX solution.Before retracting the needle, wait five seconds. Alternatively, the fish can be positioned laterally, with the anterior to the right. Then, inject at the lateral line above the pelvic fin, with the bevel up pointing towards seven o'clock at a 45 degree angle and three to four millimeters deep.If either method for injection was successful, there will be a red tint in the belly. After the injection, quickly transfer the fish to a clean crossing tank filled with fresh system water where it can recover. Then, rinse the needle with HBSS and proceed to the inject the next fish.Later, return the injected fish to a system with running circulation, but separate from the main system to avoid cross-contamination. For the next 24 hours, continue to fast the fish. Over the first week post-injection, be certain to check the fish daily and remove any dead fish.When using the classic belly-up IP injection method, it was noted that the injected DOX solution could sometimes ooze out from the location where the needle penetrated. The alternative lateral IP injection effectively prevented the leakage. Injection of DOX dosed at 50 milligrams per kilogram led to severe toxicity where the majority of fish died within one week.However, at a lower dose of 20 milligrams per kilogram, the injected Zebra fish could be maintained for chronic observation. With the alternative injection, there was no fish death during the first two weeks, and only about 10%death at four weeks. The classic method for injection at the same dose led to about 30%dead by four weeks.Next, transgenic fish with a casper background expressing DsRed and cardiomyocytes were used to assess the progression of cardiac dysfunction in the DIC model. Their red heart could be viewed at both the systolic and diastolic stages under a fluorescent microscope. After injection of 20 milligrams per kilogram of DOX using the alternative lateral injection method, ventricular function decline was detected beginning after four weeks.Once mastered, delivery of Doxorubicin with either technique can be performed on about 30 to 50 fish an hour. Following this procedure, other methods like echocardiography, electrocardiography or ex vivo heart function essay can be performed in order to profile heart function of this DIC model at different stages. While attempting this procedure, it is important to remember to use freshly prepared Doxorubicin and to test its drug efficacy after long-term storage, before the injection.Also, always handle Doxorubicin taking the appropriate safety precautions.

a-doxorubicin-induced-cardiomyopathy-model-in-adult-zebrafish

Research

JoVE Journal

Medicine

Induction of Myocardial Infarction in Adult Zebrafish Using Cryoinjury

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

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

성인 Zebrafish에서 심근 경색의 유도는 Cryoinjury 사용

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

연구

의학

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

태아 알코올 노출의 Zebrafish 모델에서 Teratogenic 변경 사항 평가

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

Genomic studies of human cancers have yielded a wealth of information about genes that are altered in tumors1,2,3. A challenge arising from these studies is that many genes are altered, and it can be difficult to distinguish genetic alterations that drove tumorigenesis from that those arose incidentally during transformation. To draw this distinction it is beneficial to have an assay that can quantitatively measure the effect of an altered gene on tumor initiation and other processes that enable tumors to persist and disseminate. Here we present a rapid means to screen large numbers of candidate melanoma modifiers in zebrafish using an autochthonous tumor model4 that encompasses steps required for melanoma initiation and maintenance. A key reagent in this assay is the miniCoopR vector, which couples a wild-type copy of the mitfa melanocyte specification factor to a Gateway recombination cassette into which candidate melanoma genes can be recombined5. The miniCoopR vector has a mitfa rescuing minigene which contains the promoter, open reading frame and 3'-untranslated region of the wild-type mitfa gene. It allows us to make constructs using full-length open reading frames of candidate melanoma modifiers. These individual clones can then be injected into single cell Tg(mitfa:BRAFV600E);p53(lf);mitfa(lf)zebrafish embryos. The miniCoopR vector gets integrated by Tol2-mediated transgenesis6 and rescues melanocytes. Because they are physically coupled to the mitfa rescuing minigene, candidate genes are expressed in rescued melanocytes, some of which will transform and develop into tumors. The effect of a candidate gene on melanoma initiation and melanoma cell properties can be measured using melanoma-free survival curves, invasion assays, antibody staining and transplantation assays.

A rapid way to screen for melanoma modifiers using a zebrafish autochthonous tumor model is presented. It takes advantage of the miniCoopR vector which allows for expression of candidate melanoma genes in melanocytes. A method to obtain melanoma-free survival curves, an invasion assay, a protocol for antibody staining of scale melanocytes and a melanoma transplantation assay are described.

Zebrafish Autochthonous 종양 모델을 사용하여 흑종 조절을위한 검사

Genomic studies of  human cancers have yielded a wealth of information about genes that are altered  in tumors1,2,3. A challenge arising from these ...

A Zebrafish Model of Diabetes Mellitus and Metabolic Memory

Diabetes mellitus currently affects 346 million individuals and this is projected to increase to 400 million by 2030. Evidence from both the laboratory and large scale clinical trials has revealed that diabetic complications progress unimpeded via the phenomenon of metabolic memory even when glycemic control is pharmaceutically achieved. Gene expression can be stably altered through epigenetic changes which not only allow cells and organisms to quickly respond to changing environmental stimuli but also confer the ability of the cell to "memorize" these encounters once the stimulus is removed. As such, the roles that these mechanisms play in the metabolic memory phenomenon are currently being examined.
We have recently reported the development of a zebrafish model of type I diabetes mellitus and characterized this model to show that diabetic zebrafish not only display the known secondary complications including the changes associated with diabetic retinopathy, diabetic nephropathy and impaired wound healing but also exhibit impaired caudal fin regeneration. This model is unique in that the zebrafish is capable to regenerate its damaged pancreas and restore a euglycemic state similar to what would be expected in post-transplant human patients. Moreover, multiple rounds of caudal fin amputation allow for the separation and study of pure epigenetic effects in an in vivo system without potential complicating factors from the previous diabetic state. Although euglycemia is achieved following pancreatic regeneration, the diabetic secondary complication of fin regeneration and skin wound healing persists indefinitely. In the case of impaired fin regeneration, this pathology is retained even after multiple rounds of fin regeneration in the daughter fin tissues. These observations point to an underlying epigenetic process existing in the metabolic memory state. Here we present the methods needed to successfully generate the diabetic and metabolic memory groups of fish and discuss the advantages of this model.

Metabolic memory is the phenomenon by which diabetic complications persist and progress unimpeded even after euglycemia is achieved pharmaceutically. Here we describe a diabetes mellitus zebrafish model which is unique in that it allows for the examination of the mitotically transmissible epigenetic components of metabolic memory in vivo.

당뇨병과 신진 대사 메모리의 Zebrafish 모델

Diabetes mellitus currently affects 346 million individuals and this is projected to increase to 400 million by 2030. Evidence from both the laboratory ...

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The molecular mechanisms leading to cyst formation in PKD remain somewhat unclear, but many genes are thought to be involved. Wnt5a is a non-canonical glycoprotein that regulates a wide range of developmental processes. Wnt5a works through the planar cell polarity (PCP) pathway that regulates oriented cell division during renal tubular cell elongation. Defects of the PCP pathway have been found to cause kidney cyst formation. Our paper describes a method for developing a zebrafish cystic kidney disease model by knockdown of the wnt5a gene with wnt5a antisense morpholino (MO) oligonucleotides. Tg(wt1b:GFP) transgenic zebrafish were used to visualize kidney structure and kidney cysts following wnt5a knockdown. Two distinct antisense MOs (AUG - and splice-site) were used and both resulted in curly tail down phenotype and cyst formation after wnt5a knockdown. Injection of mouse Wnt5a mRNA, resistant to the MOs due to a difference in primary base pair structure, rescued the abnormal phenotype, demonstrating that the phenotype was not due to &#8220;off-target&#8221; effects of the morpholino. This work supports the validity of using a zebrafish model to study wnt5a function in the kidney.

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

We describe a method of generating a possible zebrafish model of polycystic kidney disease. We used Tg(wt1b:GFP) fish to visualize kidney structure. Knockdown of wnt5a was by morpholino injection. Pronephric cyst formation after wnt5a knockdown was observed in this GFP transgenic zebrafish.

다낭성 신장 질환의 가능한 제브라 피쉬 모델 :의 넉다운<em&gt; wnt5a</em&gt; Zebrafish의 신장의 낭종 원인

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The ...

Tachycardia-Induced Cardiomyopathy As a Chronic Heart Failure Model in Swine

A stable and reliable model of chronic heart failure is required for many experiments to understand hemodynamics or to test effects of new treatment methods. Here, we present such a model by tachycardia-induced cardiomyopathy, which can be produced by rapid cardiac pacing in swine.
A single pacing lead is introduced transvenously into fully anaesthetized healthy swine, to the apex of the right ventricle, and fixated. Its other end is then tunneled dorsally to the paravertebral region. There, it is connected to an in-house modified heart pacemaker unit that is then implanted in a subcutaneous pocket. 
After 4 - 8 weeks of rapid ventricular pacing at rates of 200 - 240 beats/min, physical examination revealed signs of severe heart failure - tachypnea, spontaneous sinus tachycardia, and fatigue. Echocardiography and X-ray showed dilation of all heart chambers, effusions, and severe systolic dysfunction. These findings correspond well to decompensated dilated cardiomyopathy and are also preserved after the cessation of pacing.
This model of tachycardia-induced cardiomyopathy can be used for studying the pathophysiology of progressive chronic heart failure, especially hemodynamic changes caused by new treatment modalities like mechanical circulatory supports. This methodology is easy to perform and the results are robust and reproducible.

Here, we present a protocol to produce tachycardia-induced cardiomyopathy in swine. This model represents a potent way to study the hemodynamics of progressive chronic heart failure and the effects of applied treatment.

돼지에서 만성 심장 마비 모델로 tachycardia 유발 심근

A stable and reliable model of chronic heart failure is required for many experiments to understand hemodynamics or to test effects of new treatment ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

Myography 와이어를 사용 하 여 마우스 Mesenteric 동맥의 isometric 수축 측정

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Murine Myocardial Infarction Model using Permanent Ligation of Left Anterior Descending Coronary Artery

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine models of MI with permanent ligation of left-anterior descending (LAD) coronary artery closely mimics MI in humans. Murine models benefit from the extensive genetic engineering available nowadays. Here we propose a reproducible murine surgical model of myocardial infarction by permanent LAD coronary ligation. Our technique comprises anesthesia with ketamine/xylazine that can be rapidly reversed by administration of an antagonist, intubation without tracheotomy for mechanical-assisted ventilation, ventilation with application of extrinsic positive end-expiratory pressure (PEEP) to avoid alveolar collapse, a thoracotomy method limiting to the minimum surgical lesions made to skeletal muscles, and lung inflation without thoracentesis. This method is sparsely invasive, reproducible and reduces post-surgery mortality and complications.

Herein we describe a surgical procedure showing how to achieve permanent ligation of the left-anterior descending coronary artery in mice. This model is of high relevance to investigate the pathophysiology of myocardial infarction and the concomitant biological processes.

왼쪽 전방 내림차순 관상 동맥의 영구 결찰을 사용하는 뮤린 심근 경색 모델

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine ...

A Doxorubicin-Induced Murine Model of Dilated Cardiomyopathy In Vivo

Dilated cardiomyopathy (DCM) refers to a spectrum of heterogeneous myocardial disorders characterized by ventricular dilation and depressed cardiac performance in the absence of hypertension, valvular, congenital, or ischemic heart diseases, and which may be related to infection, autoimmune or metabolic abnormalities, or family inheritance. It can progress into congestive heart failure with a poor prognosis. Doxorubicin (Dox) is widely employed as a chemotherapeutic drug, but its use is limited because it causes DCM-like changes of the myocardium. Its myocardial toxicity is attributed to oxidative stress, chronic inflammation, and cardiomyocyte apoptosis. A model of DCM exploiting these Dox-induced DCM symptoms has not been established.

Described is a protocol to establish a Doxorubicin-induced dilated cardiomyopathy (DCM) model in mice via long-term intraperitoneal injection of Doxorubicin.

비보에서 확장 된 심근병증의 독소루비신 유도 뮤린 모델

Dilated cardiomyopathy (DCM) refers to a spectrum of heterogeneous myocardial disorders characterized by ventricular dilation and depressed cardiac ...

Acute Kidney Injury Model Induced by Cisplatin in Adult Zebrafish

Cisplatin is commonly used as chemotherapy. Although it has positive effects in cancer-treated individuals, cisplatin can easily accumulate in the kidney due to its low molecular weight. Such accumulation causes the death of tubular cells and can induce the development of Acute Kidney Injury (AKI), which is characterized by a quick decrease in kidney function, tissue damage, and immune cells infiltration. If administered in specific doses cisplatin can be a useful tool as an AKI inducer in animal models. The zebrafish has appeared as an interesting model to study renal function, kidney regeneration, and injury, as renal structures conserve functional similarities with mammals. Adult zebrafish injected with cisplatin shows decreased survival, kidney cell death, and increased inflammation markers after 24 h post-injection (hpi). In this model, immune cells infiltration and cell death can be assessed by flow cytometry and TUNEL assay. This protocol describes the procedures to induce AKI in adult zebrafish by intraperitoneal cisplatin injection and subsequently demonstrates how to collect the renal tissue for flow cytometry processing and cell death TUNEL assay. These techniques will be useful to understand the effects of cisplatin as a nephrotoxic agent and will contribute to the expansion of AKI models in adult zebrafish. This model can also be used to study kidney regeneration, in the search for compounds that treat or prevent kidney damage and to study inflammation in AKI. Moreover, the methods used in this protocol will improve the characterization of tissue damage and inflammation, which are therapeutic targets in kidney-associated comorbidities.

This protocol describes the procedures to induce Acute Kidney Injury (AKI) in adult zebrafish using cisplatin as a nephrotoxic agent. We detailed the steps to evaluate the reproducibility of the technique and two techniques to analyze inflammation and cell death in the renal tissue, flow cytometry and TUNEL, respectively.

성인 얼룩말 피시에 있는 Cisplatin에 의해 유도된 급성 신장 상해 모형

Cisplatin is commonly used as chemotherapy. Although it has positive effects in cancer-treated individuals, cisplatin can easily accumulate in the kidney ...