The authors would like to thank Drs. Stephen Wilson and Lisa Wilsbacher for helpful discussions. This work was supported in part by NIH HL054737.

NOTA: Os procedimentos que utilizam peixe-zebra foram aprovados pelo Institutional Animal Care da UCSF e Comitê de Uso. 1. Preparação de Ferramentas <ol><li> Insira pin minúcia em um suporte de pino e prenda o pino. </li><li> Utilizando uma pinça de ponta fina, dobre com cuidado a ponta do pino para criar um ligeiro gancho. </li><li> Para manipulação e estabilização do embrião durante a lesão, dobre a extremidade de um calibre 28 ½ polegadas agulha montada em uma seringa de insulina usando alicates. </li></ol> 2. Preparação de embriões Zebrafish por lesão <ol><li> Configure casais reprodutores de peixes-zebra e recolher os ovos em água ovo (60ug / sais aquário ml) como mostrado anteriormente 22. </li><li> Adicionar 0,003% de N-feniltioureia (PTU) para a água de ovos quando os embriões são aproximadamente 8 horas pós-fertilização (hpf) para impedir que a melanização. </li><li> Dechorionate dois dia após a fertilização (DPF) embriões antes da experiência com a pinça de ponta fina. <br/&gt; NOTA: Os embriões podem ser ferido a qualquer momento após a circulação começa. Os dados aqui apresentados são para 2 dpf embriões, mas a técnica foi aplicada com sucesso em embriões de até 5 dpf. </li><li> Anestesiar os embriões com 0,02% tamponado com ácido 3-aminobenzóico (tricaina) aproximadamente 10 min antes de manipulações. </li></ol> 3. Vessel Mecânica Lesão de Embriões <ol><li> Transferência de embriões anestesiados para uma lâmina de depressão em um microscópio estereoscópico de dissecação usando uma pipeta de transferência. </li><li> Usando o lado curto plana da agulha da seringa para manipular o embrião com a mão dominante, posicionar o embrião de lado, com a superfície ventral virada para fora da agulha. </li><li> Posicionar o pino minúcia com a ponta virada directamente contra a superfície ventral do peixe para a posterior abertura urogenital. Posicionar o pino minúcia com um ligeiro ângulo de tal modo que a ponta curvada é capaz de perfurar o periderme directamente na veia caudal (Figura 1 &lt;/ Strong&gt;). </li><li> Usando a agulha da seringa para manipular o embrião, perfurar a veia caudal com o pino de minúcia tocando no embrião para o pino de gancho ligeiramente o pino para dentro da veia. </li><li> Usando a agulha da seringa, puxe o embrião para longe do pino de minúcia para criar um pequeno rasgo no recipiente. NOTA: A lesão de sucesso vai resultar em sangramento imediato a partir da veia. </li></ol> 4. Análise da hemostasia <ol><li> Escolha apenas os embriões com visivelmente células circulantes no sangue para este procedimento. </li><li> Prepare-se para iniciar o temporizador assim que o pino de minúcia é puxado a partir do vaso. </li><li> Inicie o cronometro logo que a perda de sangue pode ser visualizado a partir da ferida. Quando a perda de sangue da ferida cessa, parar o temporizador e registrar o tempo total, como o tempo de sangramento. Se a coagulação é inibida, o tempo para gravar quando não há mais visivelmente células circulantes no sangue. </li></ol> 5. Análise da cicatrização de feridas <ol><li> Transferência posanimais t-lesão em placas de imagem com fundo de vidro para microscopia. </li><li> Remover a maior parte da água do ovo. </li><li> Cubra os embriões em 0,3-1,2% baixo ponto de fusão agarose dissolvidos na água do ovo, aquecido a entre 42 e 45 ºC e suplementadas com 0,02% tricaina. </li><li> Embriões posição sobre os seus lados, usando uma pinça. </li><li> Após a agarose esfria, encher o prato com 0,02% tricaina em água ovo. </li><li> Adquirir imagens usando brightfield, epifluorescência, ou microscopia confocal. </li><li> Remover embrião do agarose usando uma pinça e transferência de volta para a água ovo. </li></ol>

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The authors declare that they have no competing financial interests.

Zebrafish têm sido utilizados com sucesso como um modelo para vários tipos de feridas, incluindo lesão de laser 13-15, trombose induzida por laser 16, e epitelial ferindo 10. Relata-se um método de ferimento mecânico que é simples de executar e produz uma lesão controlada num modelo in vivo, que é altamente favorável à microscopia em tempo real. Resultados da lesão em um resposta hemostática rápida e mensurável e um programa de reparação ferida reprodutível que pode ser controlada por meio de microscopia de vídeo e timelapse. A sua anatomia simples e estereotipado vascular, o que permite lesão reprodutível num local definido microscopicamente e acessível, e a presença de mais vascular e tipos de células hematopoiéticas fazer embriões de peixe-zebra particularmente úteis para estudar as respostas à lesão. No entanto, os embriões de peixes-zebra não tem linfócitos funcionais durante as primeiras semanas de desenvolvimento 5,6, tornando este sistema mais appropriate para estudar a contribuição da imunidade inata na inflamação e reparo. Actualmente, uma ampla variedade de peixes-zebra transgénicos existe com os marcadores para as células e proteínas que participam na formação de trombos, coagulação, inflamação e reparação de feridas, incluindo as linhas que rotulam trombócitos, fibrinogénio, eritrócitos, leucócitos e ao endotélio vascular 17,21,25- 31. Estas e outras ferramentas devem torná-lo possível acompanhar os processos envolvidos na hemostasia e reparo em detalhe significativo. A lesão mecânica complementa lesão laser para o estudo da hemostasia em peixes-zebra. Enquanto lesão induzida por laser tem sido usado há anos para desencadear a formação de trombos em embriões de peixes-zebra e modelos de mouse, os mecanismos pelos quais os danos de laser dispara coagulação e ativação thrombocyte / plaquetas não são totalmente conhecidos 16,32. A lesão mecânica proporciona um método fisiologicamente relevante para a indução de coagulação por ruptura vascular e, presumivelmente,-tecido dependente do factor de iniciação da cascata de coagulação. A constatação de que o tratamento com hirudina aumentou significativamente o tempo de sangramento após lesão sugere que este modelo é dependente de trombina. A lesão mecânica, adicionalmente, complementa lesão de laser, fornecendo perturbação suficiente de um vaso sanguíneo para fornecer uma oportunidade de seguir reparação navio. Estudos anteriores têm usado com sucesso lesão mecânica por incisão bisturi e punção com agulha para mostrar diferenças nos tempos de sangramento em condições de manipulação farmacológica e genética 19,33. A lesão pin minúcia usado no modelo atual pode complementar outros modelos de lesão, fornecendo uma lesão mais reprodutível devido ao tamanho pequeno e definido da ferida que produz e proporcionando uma oportunidade de estudar melhor recanalização do vaso e reparação. Epithelial ferindo no peixe-zebra tem provado ser um poderoso modelo para o estudo de inflamação e reparo de feridas 10. A capacidade deintroduzir uma lesão vascular fornece uma oportunidade para avaliar a reparação de mais feridas complexas em ambientes onde fibrina fornece uma matriz provisória, trombos e detritos são apagadas, e os vasos se regenerar. Como estes processos de participar na reparação de tecidos normal e na inflamação aguda e crónica e patologia vascular, este método deve contribuir para modelar aspectos da doença humana num sistema onde os comportamentos celulares pode ser monitorado em tempo real em um modelo animal inteiro.

Zebrafish have been used extensively to study a variety of topics in vascular biology, including vascular development, angiogenesis, and hematopoietic development and pathology1-3. Embryos develop a functional circulation as well as leukocytes and other components of the innate immune system by 1 day post fertilization (dpf) 1,4,5. The conservation of the inflammatory and leukocyte response to injury has made the zebrafish embryo an informative model for such diverse inflammatory processes as tuberculous infection, enterocolitis, and tissue regeneration6-9. Zebrafish embryos have been used to study injury-related inflammation particularly in the context of epithelial wounding and the neutrophil response10,11. Injury to the embryo results in a highly conserved cellular response from cells at the injury site and the innate immune cells recruited to respond to the injury and regulate its resolution11,12. Other injury models have used focused laser pulses to spatially localize injury to specific cell types including neurons, muscle cells, and cardiomyocytes13-15. 
Zebrafish embryos have been used as a model to study hemostasis and thrombosis in conditions of pharmacological and genetic manipulation, using both mechanical and laser-induced thrombus formation16-19. Components of the coagulation cascade appear to be well-conserved and transgenics have allowed for detailed studies of thrombocyte and fibrin deposition at the site of coagulation17,20,21. The procedure presented in this paper complements these methods by providing a system for studying mechanical vessel injury resulting in vessel breach, thrombus formation and resolution, and vessel repair.

<table border="0" cellpadding="0" cellspacing="0" width="867">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Name of Material/ Equipment</td>
			<td style="width:159px;">Company</td>
			<td style="width:159px;">Catalog Number</td>
			<td style="width:303px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Minutia Pins</td>
			<td style="width:159px;">Fine Science Tools</td>
			<td>26002-10</td>
			<td>Tip diameter 0.0125 mm, rod diameter 0.1 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Pin Holder</td>
			<td style="width:159px;">Fine Science Tools</td>
			<td>26016-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dumont #5 Fine Tip Forceps</td>
			<td style="width:159px;">Fine Science Tools</td>
			<td>11254-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Glass Depression Slide</td>
			<td>Aquatic Eco-Systems</td>
			<td style="width:159px;">M30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Low Melting Agarose</td>
			<td>Lonza&#160;</td>
			<td style="width:159px;">50081</td>
			<td>Preheated to 42 &#186; C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N-Phenylthiourea (PTU)</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:159px;">P7629</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">3-aminobenzoic acid (Tricaine)</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:159px;">E10521</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Hirudin</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:159px;">H7016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass bottom imaging dishes</td>
			<td style="width:159px;">Mattek</td>
			<td>P35G-1.5-14-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissecting microscope</td>
			<td style="width:159px;">Olympus</td>
			<td style="width:159px;">SZH10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Fluorescence microscope</td>
			<td style="width:159px;">Zeiss</td>
			<td>Axio Observer</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Aquarium salts</td>
			<td style="width:159px;">Instant Ocean</td>
			<td style="width:159px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Insulin syringe with 28G1/2 needle</td>
			<td style="width:159px;">Becton Dickinson&#160;</td>
			<td style="width:159px;">329461</td>
			<td></td>
		</tr>
	</tbody>
</table>

mechanical vessel injury in zebrafish embryos

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes. External development and optical transparency make these embryos especially amenable to microscopy, and numerous transgenic lines that label specific cell types with fluorescent proteins are available, making the zebrafish embryo an ideal system for visualizing the interaction of vascular, hematopoietic, and other cell types during injury and repair in vivo. Forward and reverse genetics in zebrafish are well developed, and pharmacological manipulation is possible. We describe a mechanical vascular injury model using micromanipulation techniques that exploits several of these features to study responses to vascular injury including hemostasis and blood vessel repair. Using a combination of video and timelapse microscopy, we demonstrate that this method of vascular injury results in measurable and reproducible responses during hemostasis and wound repair. This method provides a system for studying vascular injury and repair in detail in a whole animal model.

A lesão mecânica vaso foi realizada em 2 dpf embriões (Figura 2A - C). Resultados da lesão em um resposta rápida e confiável de coagulação medida pelo tempo de interrupção do sangramento (Figura 2D). Para determinar se ou não diferenças na resposta de coagulação pode ser medido, o anticoagulante hirudina foi administrada aos embriões por injecção para dentro da conduta de Cuvier imediatamente antes do ferimento (5-10 nl de 1 unidade por ul de hirudina dissolvidos em água) (por demonstração de injeções no duto de Cuvier, consulte o artigo anterior JoVE 23) 24. A administração de hirudina a uma lesão anterior resultou em aumento significativo vezes versus controlo veículo (Figura 2D) o sangramento. A evidência de danos nos vasos e coagulação podem ser vistos imediatamente após a lesão usando linhagens transgênicas para endotelial (kdrl: EGFP) e glóbulos vermelhos (gata1a: dsRed </em&gt;) Os marcadores 25,26. As imagens foram adquiridas sequencialmente a cada 5 min durante um período de 12 horas utilizando epifluorescência. Representante ainda imagens são mostradas ao longo de diferentes fases da reparação da ferida (Figura 3). Usando uma combinação de contraste de interferência diferencial (DIC) e microscopia de fluorescência, é possível medir os parâmetros distintos de reparação de feridas. A fim de determinar se ou não a reparação de feridas seguiu um padrão reprodutível através de experiências, o tempo de restabelecimento do fluxo sanguíneo foi medida em 4 grupos de peixe. Lesão do vaso resultou em uma resposta estereotipada confiável de 253 ± 16 min para o restabelecimento do fluxo sanguíneo através do vaso ferido (n = 4-5 peixe por experimento, média ± SEM). <img alt="Figura 1" src="/files/ftp_upload/52460/52460fig1highres.jpg" /> Figura 1: Diagrama de 2 dpf embrião mostrando a colocação de pino minúcia para perf<html> orming lesão mecânica da veia caudal (CV). compartimento Vascular é sombreado em cinza. <img alt="Figura 2" src="/files/ftp_upload/52460/52460fig2highres.jpg" /> Figura 2: o tempo de sangramento pode ser medida visualmente após a lesão mecânica Stills de vídeo em tempo real da lesão do vaso no peixe-zebra 2 dpf embriões.. As imagens são apresentadas na altura da lesão (A), durante a perda de sangue do ferimento activa (B), e, após cessação da perda de sangue (C). Todos os horários são indicados em segundos. Os embriões são orientados lateralmente com a superfície de topo anterior ventral e voltado para a esquerda. A barra de escala 100? M. A administração do anticoagulante hirudina levou a um aumento significativamente os tempos de hemorragia versus controlo veículo (D) (p &lt;0,0001, teste t de Student).pg &quot;target =&quot; _ blank &quot;&gt; Clique aqui para ver uma versão maior desta figura. <img alt="Figura 3" src="/files/ftp_upload/52460/52460fig3highres.jpg" /> Figura 3:. Visualizando lesão mecânica e reparação utilizando marcadores transgênicos Stills de timelapse DIC e microscopia de fluorescência após a lesão do vaso utilizando marcadores para endotélio vascular (kdrl: egfp) e células vermelhas do sangue (gata1a: dsRed) em 2 dpf embriões. Imagens demonstraram que a distância em vasos e a acumulação local de células vermelhas do sangue (t = 25), reparação parcial com fluxo de re-estabelecida de sangue (t = 210), e aparentemente a restauração completa da estrutura do vaso normal (t = 615). O tempo é indicado em minutos. Os embriões são orientados lateralmente com a superfície de topo anterior ventral e voltado para a esquerda. * Indica a posição da aorta dorsal. A lesão (seta) interrompeu a veia caudal e parte do plexo caudal. Escalabarra 25 um.

Watch this Scientific Journal Video about Mechanical Vessel Injury in Zebrafish Embryos at JoVE.com

Mechanical Vessel Injury in Zebrafish Embryos

This article describes a method for creating a mechanical vessel injury in zebrafish embryos. This injury model provides a platform for studying hemostasis, injury-related inflammation, and wound healing in an organism ideally suited for real-time microscopy.

Lesão Vessel Mecânica em embriões Zebrafish

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes.  External development ...

mechanical-vessel-injury-in-zebrafish-embryos

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

7.2K visualizações.  University of California. This article describes a method for creating a mechanical vessel injury in zebrafish embryos. This injury model provides a platform for studying hemostasis, injury-related inflammation, and wound healing in an organism ideally suited for real-time microscopy. 

Vídeo: Mechanical Vessel Injury in Zebrafish Embryos

Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is the nephron. Mammals are able to repair existing nephrons after injury, but lose the ability to form new nephrons soon after birth. In contrast to mammals, adult fish produce new nephrons (neonephrogenesis) throughout their lives in response to growth requirements or injury. Recently, lhx1a has been shown to mark nephron progenitor cells in the adult zebrafish kidney, however mechanisms controlling the formation of new nephrons after injury remain unknown. Here we show our method for robust and reproducible injury in the adult zebrafish kidney by intraperitoneal (i.p.) injection of gentamicin, which uses a noninvasive visual screening process to select for fish with strong but nonlethal injury. Using this method, we can determine optimal gentamicin dosages for injury and go on to demonstrate the effect of higher temperatures on kidney regeneration in zebrafish.

Here we present a reliable method to study adult kidney regeneration by inducing acute kidney injury by gentamicin injection. We show that injury is dependent on gentamicin dosage and environmental temperature using in situ hybridization to label lhx1a+ developing new nephrons.

A regeneração renal em Zebrafish Adulto por lesão induzida pela gentamicina

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is ...

Biologia do Desenvolvimento

Isolation and Characterization of Single Cells from Zebrafish Embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

Isolamento e caracterização de células individuais de embriões Zebrafish

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

Affinity Labeling Detection of Endogenous Receptors from Zebrafish Embryos

By combining the powers of Affinity Labeling and Immunoprecipitation (AFLIP), a technique for the detection of low abundance receptors in zebrafish embryos has been implemented. This technique takes advantage of the selectivity and sensitivity conferred by affinity labeling of a given receptor by its ligand with the specificity of the immunoprecipitation. We have used AFLIP to detect the type III TGF-&#946; receptor (TGFBR3), also know as betaglycan, during early zebrafish development. AFLIP was instrumental in validating the efficacy of a TGFBR3 morphant zebrafish phenotype. In the first step, embryo protein extracts are prepared and used to generate 125I-TGF-&#946;2-TGFBR3 complexes that are purified by immunoprecipitation. Later, these complexes are covalently cross-linked and revealed using SDS-PAGE separation and autoradiography detection. This technique requires the availability of a labeled ligand for, and a specific antibody against, the receptor to be detected, and shall be easily adapted to identify any growth factor or cytokine receptor that meets these requirements.

A novel technique for the detection of low abundance endogenous receptors present in zebrafish embryos is described. We have named it AFLIP because it consists of affinity labeling of the receptor by its ligand linked to immunoprecipitation.

Afinidade Detecção de Etiquetagem endógena Receptores de embriões Zebrafish

By combining the powers of Affinity Labeling and Immunoprecipitation (AFLIP), a technique for the detection of low abundance receptors in zebrafish ...

Ploidy Manipulation of Zebrafish Embryos with Heat Shock 2 Treatment

Manipulation of ploidy allows for useful transformations, such as diploids to tetraploids, or haploids to diploids. In the zebrafish Danio rerio, specifically the generation of homozygous gynogenetic diploids is useful in genetic analysis because it allows the direct production of homozygotes from a single heterozygous mother. This article describes a modified protocol for ploidy duplication based on a heat pulse during the first cell cycle, Heat Shock 2 (HS2). Through inhibition of centriole duplication, this method results in a precise cell division stall during the second cell cycle. The precise one-cycle division stall, coupled to unaffected DNA duplication, results in whole genome duplication. Protocols associated with this method include egg and sperm collection, UV treatment of sperm, in vitro fertilization and heat pulse to cause a one-cell cycle division delay and ploidy duplication. A modified version of this protocol could be applied to induce ploidy changes in other animal species.

A modified protocol for ploidy manipulation uses a heat shock to induce a one-cycle stall in cytokinesis in the early embryo. This protocol is demonstrated in the zebrafish but may be applicable to other species.

Manipulação de ploidia de embriões Zebrafish com choque térmico 2 Tratamento

Manipulation of ploidy allows for useful transformations, such as diploids to tetraploids, or haploids to diploids. In the zebrafish Danio rerio, ...

Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the cells are located. Specifically, they have been developed to analyze the electrical activity of excitable cells, such as spinal neurons. In such a scenario, it is crucial to preserve the integrity of the spinal cell, but it is also important to preserve the anatomy and physiological shape of the systems involved. Indeed, the comprehension of the manner in which the nervous system-and other complex systems-works must be based on a systemic approach. For this reason, the live zebrafish embryo was chosen as a model system, and the spinal neuron membrane voltage changes were evaluated without interfering with the physiological conditions of the embryos. 
Here, an approach combining the employment of zebrafish embryos with a FRET-based biosensor is described. Zebrafish embryos are characterized by a very simplified nervous system and are particularly suited for imaging applications thanks to their transparency, allowing for the employment of fluorescence-based voltage indicators at the plasma membrane during zebrafish development. The synergy between these two components makes it possible to analyze the electrical activity of the cells in intact living organisms, without perturbing the physiological state. Finally, this non-invasive approach can co-exist with other analyses (e.g., spontaneous movement recordings, as shown here).

Protocols described here allow for the study of the electrical properties of excitable cells in the most non-invasive physiological conditions by employing zebrafish embryos in an in vivo system together with a fluorescence resonance energy transfer (FRET)-based genetically encoded voltage indicator (GEVI) selectively expressed in the cell type of interest.

Potencial de membrana de neurônio motor biossensível em embriões vivos de peixe-zebra

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the ...

Precise Cellular Ablation Approach for Modeling Acute Kidney Injury in Developing Zebrafish

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore adequate kidney function after supportive treatment. However, a better understanding of how nephron cell death and repair occur on the cellular level is required to minimize cell death and to enhance the regenerative process. The zebrafish pronephros is a good model system to accomplish this goal because it contains anatomical segments that are similar to the mammalian nephron. Previously, the most common model used to study kidney injury in fish was the pharmacological gentamicin model. However, this model does not allow for precise spatiotemporal control of injury, and hence it is difficult to study cellular and molecular processes involved in kidney repair. To overcome this limitation, this work presents a method through which, in contrast to the gentamicin approach, a specific Green Fuorescent Protein (GFP)-expressing nephron segment can be photoablated using a violet laser light (405 nm). This novel model of AKI provides many advantages that other methods of epithelial injury lack. Its main advantages are the ability to &#34;dial&#34; the level of injury and the precise spatiotemporal control in the robust in vivo animal model. This new method has the potential to significantly advance the level of understanding of kidney injury and repair mechanisms.

This work presents a new in vivo model of segmental kidney injury using kidney GFP transgenic zebrafish. The model allows for the induction of the targeted ablation of kidney epithelial cells to show the cellular mechanisms of nephron injury and repair.

Abordagem precisa de ablação celular para modelagem de lesão renal aguda no desenvolvimento de peixe-zebra

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore ...

Histological Analyses of Acute Alcoholic Liver Injury in Zebrafish

Alcoholic Liver Disease (ALD) refers to damage to the liver due to acute or chronic alcohol abuse. It is among the leading causes of alcohol-related morbidity and mortality and affects more than 2 million people in the United States. A better understanding of the cellular and molecular mechanisms underlying alcohol-induced liver injury is crucial for developing effective treatment for ALD. Zebrafish larvae exhibit hepatic steatosis and fibrogenesis after just 24 h of exposure to 2% ethanol, making them useful for the study of acute alcoholic liver injury. This work describes the procedure for acute ethanol treatment in zebrafish larvae and shows that it causes steatosis and swelling of the hepatic blood vessels. A detailed protocol for Hematoxylin and Eosin (H&#38;E) staining that is optimized for the histological analysis of the zebrafish larval liver, is also described. H&#38;E staining has several unique advantages over immunofluorescence, as it marks all liver cells and extracellular components simultaneously and can readily detect hepatic injury, such as steatosis and fibrosis. Given the increasing usage of zebrafish in modeling toxin and virus-induced liver injury, as well as inherited liver diseases, this protocol serves as a reference for the histological analyses performed in all these studies.

This protocol describes histological analyses of the livers from zebrafish larvae that have been treated with 2% ethanol for 24 h. Such acute ethanol treatment results in hepatic steatosis and swelling of the hepatic vasculature.

Análises histológicas de lesão hepática alcoólica aguda em peixe-zebra

Alcoholic Liver Disease (ALD) refers to damage to the liver due to acute or chronic alcohol abuse. It is among the leading causes of alcohol-related ...

Induction of Hypoxia in Living Frog and Zebrafish Embryos

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and zebrafish embryos. Our system comprises a chamber featuring a simple setup that is nevertheless robust to induce and maintain a specific oxygen concentration and temperature in any experimental solution of choice. The presented system is very cost-effective but highly functional, it allows induction and sustainment of hypoxia for direct experiments in vivo and for various time periods up to 48 h.
To monitor and study the effects of hypoxia, we have employed two methods - measurement of levels of hypoxia-inducible factor 1alpha (HIF-1&#945;) in whole embryos or specific tissues and determination of retinal stem cell proliferation by 5-ethynyl-2&#8242;-deoxyuridine (EdU) incorporation into the DNA. HIF-1&#945; levels can serve as a general hypoxia marker in the whole embryo or tissue of choice, here embryonic retina. EdU incorporation into the proliferating cells of embryonic retina is a specific output of hypoxia induction. Thus, we have shown that hypoxic embryonic retinal progenitors decrease proliferation within 1 h of incubation under 5% oxygen of both frog and zebrafish embryos.
Once mastered, our setup can be employed for use with small aquatic model organisms, for direct in vivo experiments, any given time period and under normal, hypoxic or hyperoxic oxygen concentration or under any other given gas mixture.

We introduce a novel hypoxic chamber system for use with aquatic organisms such as frog and zebrafish embryos. Our system is simple, robust, cost-effective and allows the induction and sustainment of hypoxia in vivo and for up to 48 h. We present 2 reproducible methods to monitor the effectiveness of hypoxia.

Indução de hipóxia em sapos vivos e embriões de peixe-zebra

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and ...

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling processes of excitable neuronal and muscular cells and many other biological processes, such as embryonic developmental patterning. However, there is a need for in vivo electrical activity monitoring in vertebrate embryogenesis. The advances of genetically encoded fluorescent voltage indicators (GEVIs) have made it possible to provide a solution for this challenge. Here, we describe how to create a transgenic voltage indicator zebrafish using the established voltage indicator, ASAP1 (Accelerated Sensor of Action Potentials 1), as an example. The Tol2 kit and a ubiquitous zebrafish promoter, ubi, were chosen in this study. We also explain&#160;the processes of Gateway site-specific cloning, Tol2 transposon-based zebrafish transgenesis, and the imaging process for early-stage fish embryos and fish tumors using regular epifluorescent microscopes. Using this fish line, we found that there are cellular electric voltage changes during zebrafish embryogenesis, and fish larval movement. Furthermore, it was observed that in a few zebrafish malignant peripheral nerve sheath tumors, the tumor cells were generally polarized compared to the surrounding normal tissues.

Here, we show the process of creating a cellular electric voltage reporter zebrafish line to visualize embryonic development, movement, and fish tumor cells in vivo.

Visualização da atividade elétrica celular no Zebrafish embriões adiantados e tumores

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling ...

Whole Mount Immunohistochemistry in Zebrafish Embryos and Larvae

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. Although many immunohistochemistry protocols have been optimized for mammalian tissue and tissue sections, these protocols often require modification and optimization for non-mammalian model organisms. Zebrafish are increasingly used as a model system in basic, biomedical, and translational research to investigate the molecular, genetic, and cell biological mechanisms of developmental processes. Zebrafish offer many advantages as a model system but also require modified techniques for optimal protein detection. Here, we provide our protocol for whole-mount fluorescence immunohistochemistry in zebrafish embryos and larvae. This protocol additionally describes several different mounting strategies that can be employed and an overview of the advantages and disadvantages each strategy provides. We also describe modifications to this protocol to allow detection of chromogenic substrates in whole mount tissue and fluorescence detection in sectioned larval tissue. This protocol is broadly applicable to the study of many developmental stages and embryonic structures.

Here, we present a protocol for fluorescent antibody-mediated detection of proteins in whole preparations of zebrafish embryos and larvae.

Imunohistoquímica do Monte Inteiro em Embriões e Larvas de Zebrafish

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. ...