Name: zebrafish as a model to assess the teratogenic potential of nitrite
Uploaded: 2016-02-16T14:00:00
Description: Watch this Scientific Journal Video about Zebrafish as a Model to Assess the Teratogenic Potential of Nitrite at JoVE.com

VK was funded by grants from the IUP Department of Biology and School of Graduate Studies and Research (Graduate Student Professional Development). CQD and TWS were supported by the IUP School of Graduate Studies and Research (Faculty Publication Costs/Incidental Research Expenses). We also thank members of the Diep laboratory for maintaining the zebrafish facility.

在本协议中所描述的程序进行的机构动物护理和使用委员会在宾夕法尼亚州印第安纳大学的批准。 1.收获胚胎<ol><li>在28.5℃，pH为7，500-1,500μS之间电导率维持斑马鱼，和14小时光照和10小时黑暗的24的光/暗周期。使用野生型菌株，如涂，AB或TU / AB混合。不同的菌株可能有不同的反应化学处理25。 </li><li>建立了鱼通过鱼加水系统到配合坦克交配收获鸡蛋的前一?...

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这里介绍的方法证明了评估硝酸盐和亚硝酸盐的潜在致畸斑马鱼的效用。相比其他脊椎动物斑马鱼有优势，包括高繁殖力，体外受精，光学透明，快速发展。缺乏色素沉着（如斑马鱼的Casper 36）可用的突变体也有助于增强内脏器官的知名度。它也很容易产生与报告基因的转基因斑马鱼便于分析在活鱼37。因为斑马鱼基因组与人类保守的，从他们的研究中获得的信息可以导致在人中<...

畸变是由造成永久性的结构和功能异常，生长发育迟缓，或流产情况严重扰乱了1胚胎或胎儿的正常发育过程。它可以通过某些天然剂（致畸），其与胚胎发育以多种方式2干扰引起的。在人的胎儿发育，共同致畸如辐射，感染剂，有毒金属，和有机化工已报道引起epicanthic褶皱（弯曲手指或脚趾）通过形态发生错误的缺陷（皮肤褶皱中的上眼睑）和五指1。 理解致畸的分子机制是朝着显影治疗和预防的第一步。一些脊椎动物模型如非洲爪蛙（ 爪蟾 ）和斑马鱼（ 斑马鱼 ）已被用来确定受terat的分子途径ogens。以前的研究已经使用斑马鱼作为流行病学，毒理学和致畸3-7的典范。 Scholz等人 。考虑斑马鱼作为“金标准”环境毒性评估。这是由于在某种程度上，对斑马鱼胚胎，这允许它发生8研究者形象化发育缺陷的透明度。人类基因的大约70％的具有在斑马鱼直系同源物，使得斑马鱼的理想脊椎动物模型研究人类的缺陷9。 一些流行病学研究表明，硝酸盐和亚硝酸盐，通常存在于农场的食物和水，与出生缺陷或自然流产10,11相关联，而其它研究不支持此关联12。硝酸盐（NO 3 - ）和亚硝酸根（NO 2 - ）是在土壤和水自然存在。它们是氮植物的来源，并且是n的一部分itrogen周期13。食物，如绿豆，胡萝卜，南瓜，菠菜，甜菜，并从使用化肥高硝酸盐农场显著增强硝酸盐和亚硝酸盐7的水平。从高硝酸盐的食物和鱼高硝酸盐水（主要是从土壤中流失30）喂牛奶会导致人体消耗大量的硝酸盐和亚硝酸盐14。硝酸盐和亚硝酸盐也常用于食品保鲜，这大大增加了由人类12摄取的量使用。 硝酸盐和亚硝酸盐的最佳水平像血管稳态和功能，神经传递及免疫宿主防御机制13-15生理过程中发挥根本性的作用。然而，暴露于高水平的硝酸盐和亚硝酸盐的可能导致不利的影响，特别是在婴儿和儿童16。摄入硝酸盐是由微生物，并在第进一步转化为亚硝酸盐在口腔中由肠道菌群16 E胃肠道。 硝酸盐使婴儿在蓝色婴儿综合症高风险的血红蛋白氧化成高铁血红蛋白，其携氧能力18损害血红蛋白。这导致皮肤的蓝色延伸到在更严重的情况下外周组织。在其他症状组织结果抑制氧化，最严重导致昏迷和死亡19,20。类似症状中的婴儿和成人在较高浓度的硝酸21的观察。由于紫绀，头痛亚硝酸盐中毒导致高铁血红蛋白成人水平升高，呼吸障碍31，死如不及时治疗，由于涉及重要组织缺氧32,33并发症。 硝酸盐在较高水平的摄入也可以导致各种健康并发症。儿童糖尿病，反复腹泻和反复呼吸道感染患儿已与硝酸盐摄入11,17,22相连。长期暴露于高水平硝酸盐与排尿和脾出血有关。急性高剂量照射硝酸盐可导致像腹痛，肌肉无力，便血和尿，昏厥，甚至死亡11医疗条件范围广泛。在较高水平产前暴露于硝酸一直与神经管缺陷的肌肉骨骼相关的11。 最近的一份报告显示，治疗斑马鱼胚胎与亚导致卵黄囊水肿，颅面和轴向畸形和鱼鳔noninflation 5。在这项研究中，我们证明了治疗斑马鱼的胚胎与硝酸盐和亚硝酸盐，以确定他们的致畸潜力的方法。胚胎在不同浓度和不同的时间长度暴露于亚硝酸盐。乙醇用作阳性对照，因为它是一个既定的致畸23。 Ø乌尔法表明，两种高浓度，长的曝光时间为亚硝酸盐不利于生存和导致各种表型，从轻度（水肿），重度（毛发育缺陷）。因此，斑马鱼是直接在探索胚胎硝酸盐和亚硝酸盐的潜在致畸作用，以补充流行病学研究的有用模型。

<table border="0" cellpadding="0" cellspacing="0" width="647">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:191px;">DREL/2010 instrument</td>
			<td style="width:185px;">Hach</td>
			<td style="width:104px;">26700-03</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:191px;">Ethanol</td>
			<td style="width:185px;">Sigma-Aldrich</td>
			<td style="width:104px;">E7023</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:191px;">KIMAX glass Petri Dish</td>
			<td style="width:185px;">VWR</td>
			<td style="width:104px;">89001-244</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:191px;">MS-222</td>
			<td style="width:185px;">Sigma-Aldrich</td>
			<td style="width:104px;">E10521</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:191px;">NitraVer 5 Nitrate Reagent</td>
			<td style="width:185px;">Hach</td>
			<td style="width:104px;">14034-46</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:191px;">NitriVer 3 Nitrite Reagent</td>
			<td style="width:185px;">Hach</td>
			<td style="width:104px;">14065-99</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:191px;">Parafilm</td>
			<td style="width:185px;">Fisher Scientific</td>
			<td style="width:104px;">3-374-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:191px;">Paraformaldehyde</td>
			<td style="width:185px;">Sigma-Aldrich</td>
			<td style="width:104px;">158127</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:191px;">S6E stereomicroscope</td>
			<td style="width:185px;">Leica</td>
			<td style="width:104px;">10446294</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:191px;">Sodium nitrate</td>
			<td style="width:185px;">Fisher Scientific</td>
			<td style="width:104px;">S343</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:191px;">Sodium nitrite</td>
			<td style="width:185px;">Fisher Scientific</td>
			<td style="width:104px;">S347</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:191px;">Transfer pipets</td>
			<td style="width:185px;">Laboratory Products Sales</td>
			<td style="width:104px;">L320072</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:191px;">Glass vials</td>
			<td style="width:185px;">Fisher Scientific</td>
			<td style="width:104px;">03-338B</td>
			<td></td>
		</tr>
	</tbody>
</table>

zebrafish as a model to assess the teratogenic potential of nitrite

在环境中的高硝酸盐水平可能导致在人体中的先天性缺陷或流产。据推测，这是由于硝酸根的由肠道和唾液细菌转化为亚硝酸盐。然而，在其他哺乳动物的研究中，高亚硝酸盐的含量不导致出生缺陷，尽管它们可能会导致差的生殖结局。因此，亚硝酸盐的致畸潜在不明确。这将是有脊椎动物模型系统轻松评估亚硝酸盐或任何其他感兴趣的化学物质的致畸作用非常有用。这里，我们证明斑马鱼（ 斑马鱼 ）的工具来筛选毒性和胚胎缺陷化合物。斑马鱼胚胎受精外，并有快速发展，使他们致畸研究的一个很好的模式。我们表明，增加曝光时间以负亚硝酸盐影响生存。增加亚硝酸盐的浓度也有不利影响的生存，而硝酸盐没有。对于胚胎塔ŧ生存亚硝酸盐暴露，可能会出现各种缺陷，包括心包和卵黄囊水肿，鱼鳔noninflation和颅面畸形。我们的结果表明，该斑马鱼是研究亚硝酸盐的致畸电位的方便的系统。这种方法可以容易地适用于测试其他化学品的早期脊椎动物发育的影响。

曝光至300mM乙醇22小时对存活率（数据未示出），与以前的报告5,23,26一致没有影响。这是意料之中的，因为乙醇是一种已知的致畸，并担任阳性对照。观测到的表型包括心包水肿，鱼鳔noninflation（图1），颅面畸形和发育迟缓（数据未示出）。 用亚硝酸盐处理导致轻度至对生存造成严重影响，这取决于曝光?...

Watch this Scientific Journal Video about Zebrafish as a Model to Assess the Teratogenic Potential of Nitrite at JoVE.com

Zebrafish as a Model to Assess the Teratogenic Potential of Nitrite

接触致畸可能导致出生缺陷。斑马鱼是确定的化学物质的致畸潜在有用的。我们通过暴露胚胎各级亚硝酸盐，并且也在曝光不同的时间证明斑马鱼的效用。我们表明，亚硝酸盐可能是有毒的，并导致严重的发育缺陷。

斑马鱼作为一种模型来评估亚硝酸盐的潜在致畸

High nitrate levels in the environment may&#160;result in congenital defects or miscarriages in humans. Presumably, this is due to the conversion of ...

Research

Education

JoVE Core

JoVE Journal

Pharmacology

Immunology and Infection

Unit 1

Adverse Drug Effects and Chemical Toxicity

SCIENTISTS IN ACTION

Introducing Shear Stress in the Study of Bacterial Adhesion

在细菌粘附的研究剪应力

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

科研

免疫与感染

Using the BLT Humanized Mouse as a Stem Cell based Gene Therapy Tumor Model

BLT人性化的鼠标使用一个基于干细胞的基因治疗肿瘤模型的建立

Small animal models such as mice have been extensively used to study human  disease and to develop new therapeutic interventions. Despite the wealth of  ...

The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis

The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis

昆虫<em&gt;大蜡螟</em作为一个强大的的感染模型探讨细菌致病

The study of bacterial virulence often requires a suitable animal model. Mammalian models of infection are costly and may raise ethical issues. The use of ...

Quantification of the Respiratory Burst Response as an Indicator of Innate Immune Health in Zebrafish

呼吸爆发的响应为先天免疫健康的斑马鱼中的指标量化

The phagocyte respiratory burst is part of the innate immune response to  pathogen infection and involves the production of reactive oxygen species (ROS). ...

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

使用<em&gt;大蜡螟</em&gt;作为模式生物来研究<em&gt;嗜肺军团菌</em&gt;感染

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and ...

Cecal Ligation and Puncture-induced Sepsis as a Model To Study Autophagy in Mice

盲肠结扎穿孔致脓毒症作为模型小鼠中自噬研究

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method, which causes polymicrobial sepsis. Here, a protocol is ...

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

先天免疫反应中的斑马鱼幼体型号无创成像<em&gt;链球菌iniae</em&gt;感染

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

Retroviral Transduction of Helper T Cells as a Genetic Approach to Study Mechanisms Controlling their Differentiation and Function

辅助性T细胞作为一种遗传方法来研究逆转录病毒转导机制控制其分化和功能

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents ...

The Madagascar Hissing Cockroach as an Alternative Non-mammalian Animal Model to Investigate Virulence, Pathogenesis, and Drug Efficacy

马达加斯加嘘声蟑螂作为非哺乳动物的替代动物模型来研究毒力、致病机制和药效

Many aspects of innate immunity are conserved between mammals and insects. An insect, the Madagascar hissing cockroach from the genus Gromphadorhina, can ...

Using the Protozoan Paramecium caudatum as a Vehicle for Food-borne Infections in Zebrafish Larvae

Using the Protozoan Paramecium caudatum as a Vehicle for Food-borne Infections in Zebrafish Larvae

用原生动物给鱼作动物感染的载体

Due to their transparency, genetic tractability, and ease of maintenance, zebrafish (Danio rerio) have become a widely-used vertebrate model for ...