我们希望感谢质粒的礼物蒙特卡罗韦斯特小林诚和亚切克Topczewski的的原位杂交和基因微量注射。我们是感谢Steph艾哈德在视频中出现的。我们也愿意承认，在视频中显示的共焦显微镜威廉古森斯援助。泰勒Schwend提供视频图像，并协助罗德尼·戴尔准备视频。我们也承认他们与斑马鱼的饲养工作，的CMRC动物保健人员。这项工作是由美国国立卫生研究院授予R21的AA13596政制事务局局长。

1。乙醇处理的胚胎<ol><li>提出了从野生型交配而得的斑马鱼胚胎在28°C在胚胎水5毫升。 （60 mg / L的即时海洋的Milli-Q水）。野生型胚胎的不同菌株具有略微不同的公差乙醇暴露9。 </li><li>暴露于乙醇所需的时间长度，在圆顶阶段（〜4.3小时后受精，HPF）发起长达24小时的脉冲。这起步阶段，大致相当于哺乳动物胚胎的植入，只是前原肠阶段。酒精浓度使用范围从0.2％-2.5％，体积/体积。试验表明，在水溶液中之间的25-50％结束了在胚胎发育10，11。胚胎保持在28°C所需的时​​间长度。乙醇暴露后所需要的长度，被删除，取而代之的乙醇水溶液三个定期斑马鱼水清洗，并保持FOR的所需的时间长度。发病时间，剂量，对乙醇的脉冲长度取决于缺陷的特异性。 </li></ol> 2。收集胚胎的RNA 原位杂交，抗体，和软骨染色<ol><li>收集的定量PCR基因，年龄相匹配的胚胎收集Eppendorf管中。去除斑马鱼水后，添加与TCEP的裂解缓冲。机动粉碎机用于裂解液中分离胚胎。这随后被传递通过预清除列，其中删除任何未解离大块。最终的解决方案包含了所有的裂解过程中释放的大分子。然后提取RNA是由一列运行这一解决方案，其次是清洗和DNase处理。然后使用水或由制造商（5&#39;总理）提供的洗脱液洗脱。的RNA可以转化为cDNA，使用标准的qPCR技术或微阵列分析。 </li><li> 在原位杂交和抗体染色，胚胎从6日至24 HPF被固定在4％多聚甲醛在一夜之间在4°C其次是3清洗在PBS + 0.1％吐温（PBT）的粘在一起，以保持胚胎。然后转移胚胎通过一系列3甲醇:PBT的洗到100％的甲醇，在这一点，他们可以在-20°C存储这些胚胎可用于原位杂交或抗体染色。甲醇处理后，所以这个警告必须考虑和您的特异性抗体测试，以确定它是否将甲醇处理后，一些抗体不起作用。 </li><li>软骨染色，胚胎需要提高到5或6天受精后（DPF）。然后，他们被固定在4％多聚甲醛过夜4°C间，在PBT洗。 </li></ol> 3。评估乙醇诱导基因表达的变化和发展后果<ol><li要确定哪些基因表达水平改变，双方的qPCR 和原位杂交检测靶基因。 qPCR可以实现使用与集成DNA技术（IDT）的软件，然后用3个独立的生物样品的验证，设计引物。一旦引物，已验证，定量PCR进行基因从收集RNA样品。每个样品运行在一式三份。 PCR反应色度-4的PCR仪（BioRad公司）或类似的PCR仪与多色检测系统上运行白金的SYBR Green qPCR的超级组合（Invitrogen公司）。除了正在审讯的基因，这是关键，引物控制用于平衡cDNA或RNA制备的小差异。对于我们的斑马鱼胚胎中，我们使用GAPDH:5&#39;GAAGGTGGGAAACTGGTCAT3和5&#39;TTGCACCACCCTTAATGTGA3"。被分析的基因GAPDH水平正常化，用2 Pfaffl法变异的基因表达相对定量计算<sup- CT，显示的数据作为实验相对于野生型12倍的差异。正常的计算，假定所有基因每个周期重复两次的DNA，从而表现为比例超过参照基因变化的实验基因的变化数据，为功率计算在整数"2"表示，这是一个完美的PCR反应中看到每个周期的DNA加倍效率。 Pfaffl法取代通用的"2"实验者选择的PCR引物产生真正的效率。它反映了在PCR反应的差异，更准确地反映。完整的计算公式为: <table border="0"><tbody><tr><td> （实验基因效率）（CT控制样品处理样品的CT） </td></tr><tr><td> -------------------------------------------------- ----------------------------------- </td></tr><tr><td> （参照基因的效率）（CT对控制样本- CT处理样品） </td></tr></tbody></table></li><li> 在原位杂交，地高辛（DIG）标记riboprobes的构造含有部分感兴趣的基因质粒。年龄相匹配的胚胎暴露riboprobes使用13种标准条件和检测碱性磷酸酶允许使用显色反应（NBT / BCIP）的标记riboprobes的位置加上反掏抗体。胚胎，然后使用徕卡解剖显微镜（ 图1B-G）的可视化。 </li><li>评估形态发生发展，胚胎收集所需的时间，并在解剖显微镜下观察。活胚胎体节形状，成像14。眼间距离和身体长度，胚胎固定在煤灰。在所有情况下，所需区域的图像被捕获在一个固定的放大倍率和测量，解剖显微镜使用Adobe Photoshop softwar北京时间10,14。 </li><li>检查治疗发展斑马鱼幼虫的软骨结构的影响，我们用阿尔新蓝染色。固定斑马鱼幼虫被视为与阿尔新蓝溶于80％乙醇溶液:20％冰醋酸几个小时或过夜（酸酒精）。幼虫脱色酸酒精清洗，然后被转移到1％KOH进一步清除色素细胞的过氧化氢溶液:3％。斑马鱼需要至少4天显示软骨结构，这些都更好地定义在5或6天的鱼。 </li><li>用吖啶橙（AO）的评估在活胚胎细胞死亡。 AO是不是渗透到活细胞的细胞，受损的细胞膜，包括那些发生凋亡和坏死细胞的DNA结合。活胚胎与5毫克/毫升敖在PBS孵育1小时，3 PBS洗涤。胚胎，然后利用共聚焦显微镜成像。数字的Z-SER而IES图像结合创建复合材料。 </li></ol> 4。操纵斑马鱼胚胎<ol><li>减少乙醇暴露的基因鉴定后，有可能尝试使用注射的设计，以取代丢失的基因表达，以取代他们。皑皑的mRNA转录从线性质粒DNA使用RNA聚合酶体外转录试剂盒，根据制造商的指示（mMessage机，应用生物系统公司）。皮克/ NL的RNA 25-200之间被注入到1-2细胞阶段，斑马鱼的卵，在解决了0.1米氯化钾。胚胎，然后恢复之前，用乙醇视为以上（ 图5）。 </li><li>对于任何基因转录增加乙醇暴露，他们可以减少，或"敲出"用反义吗啉技术。反义吗啉代（AMOS）是由该公司设计的基因工具，阻止RNA翻译成蛋白质，或BLOCK的成熟和RNA剪接，根据实验需要。这种行动吗啉代活性蛋白的数量限制为目标基因，可以测量使用anitibodies如果他们亏。 RNA剪接吗啉代的效率可以测量采用RT-PCR检测剪接和未拼接的成绩单的相对含量。吗啉的剂量可以导致剂量依赖性的活动15。阿莫斯稀释工作浓度在Danieau解决方案（纳克的浓度滴定PG）（58毫米氯化钠，0.7毫米氯化钾， 硫酸镁 0.4毫米，0.6毫米的Ca（NO 3）2，5毫米肝素钠，pH值7.6）。注射,1-2细胞阶段的地方，然后胚胎被允许之前受到乙醇处理上述发展。 </li></ol> 5。代表结果斑马鱼胚胎暴露于乙醇结果在一些发育和遗传缺陷有关表型的发现在其他脊椎动物。我们已经证明了轴组织发育异常，包括脊索（数据未显示14）。乙醇生产的发育迟缓，显然是导致适当的脊索伸长受阻，导致缩短偶尔打乱的脊索（数据未显示14）。这种早期的延迟和脊索缺陷可能导致在体节的后异常（ 图2），这已经失去了他们强烈的雪佛龙的形状，在未经处理的控制部分，是接近U形体节时看到的表型特征声波信号减少14。体节的角度，可以采用标准的软件，从92.1±4.6°，在未经处理的控制为122.6±6.6°的乙醇处理的胚胎（P &lt;0.001）的角度增加。 另一个乙醇暴露后果可能downstrea的m的早期发育迟缓，后来脊索缺陷的胚胎长度缩短（ 图3）。即使测量胚胎考虑到乙醇暴露产生的曲率，乙醇暴露导致缩短主干，是依赖于剂量的乙醇胚胎暴露（ 图3E 14）。这一发现是持续暴露在发展16，17乙醇的儿童在青春期前短身材发现的类似，这表明斑马鱼模型是了解人类出生缺陷有关。 FAS的典型特征之一，是一个典型的相，其中包括减少下颌，小眼开，并顺利人中。这些功能大部分都在减少中线组织有关。它已被证明在动物模型中，重度乙醇暴露导致更显着的表型，包括synopthalmia和独眼。当世博会增加剂量乙醇唱斑马鱼胚胎，人工距离（IOD）以剂量依赖的方式降低（ 图4）与人类出生缺陷的性质是一致的。独眼只发现具有非常高的剂量，但低剂量产生的IOD（ 图4）在显着减少，表明这种动物模型有类似的缺陷面部中线发展10。 为了了解机制在身体和面对生产乙醇暴露的缺陷，我们试图建立基因表达的变化发生在发展初期，可合理预期，有助于以后的发育缺陷。我们做这两种方式，表达从胚胎提取可以定量评价了乙醇处理的胚胎中的基因表达水平较对照组（ 图1A）。此外，我们可以执行原位杂交基因的格局看，不管与否的信号下降（ 图1B-G的 18）。在这个例子中，两个基因的表达，减少乙醇暴露后，GLI1和six3b。当我们在原位为six3b模式研究，我们发现在该基因的表达的空间范围，在暴露于乙醇胚胎（ 图1，EG）的减少。类似的减少被发现的基因GSC（ 图BD）。都six3b和GSC表示在注定有助于颅面中线组织的组织。所以减少8 HPF这些基因的发现后， 如图4所示，在IOD的减少是一致的。 一旦确定乙醇影响的基因，也有机制，通过他们的表现可以增加或减少。在这个特别的EXAmple，我们已经表明下降的qPCR 2基因（GLI1及six3b）。我们选择注入基因，以确定如果扭转这些基因的变化，可以改善预后。对于这个实验，而不是注射GLI1，我们选择了注入SHH，配体，增加GLI1水平。为six3b，我们能够注入six3b本身。我们没有发现任何变化，当我们注入six3b（数据未显示），但能够从根本上挽救的补充，SHH注射（ 图5）在斑马鱼胚胎的总缺陷。 <img alt="图1" src="/files/ftp_upload/3704/3704fig1.jpg" /> 图1。乙醇暴露改变了早期的模式和选定的发育基因的基因表达水平。一）胚胎在8hpf qPCR的结果。这两个基因的结果显示:six3b和GLI1。平均为三个独立的实验结果表明，乙醇剂量为2.5％。这两个基因的方式，是从控制（t检验，P &lt;0.05）显著减少。 BG） 在 8个HPF斑马鱼胚胎的原位杂交。改变GSC和six3b模式都在这个时间点与对照组相比。 （劳克斯等2007年的许可改性）。 <img alt="图2" src="/files/ftp_upload/3704/3704fig2.jpg" /> 图2。体节的发展改变了处理的胚胎。体节的结构和角度研究在48 HPF。 （一），在控制胚胎体节有雪佛龙的形状和尖尖角。乙醇暴露斑马鱼有更多的I或U形的体节，少锐利的角度。 （改性劳克斯和Ahlgren 2009的权限）。 <img alt="图3" src="/files/ftp_upload/3704/3704fig3.jpg" /> 图3。乙醇处理显着降低在斑马鱼的体长。在受精后5天未处理和乙醇暴露胚胎的长度测量。在所有处理的胚胎被视为一个长度显着减少（ANOVA，P &lt;0.001）。屋宇署。在总长度的1.0％和1.5％的乙醇处理的胚胎有一个轻微但显着减少，而更大的减少了2.0％和2.5％的乙醇剂量的胚胎。 E.要控制在每个离合器的大小差异，每个实验的控制胚胎正常化至100和100的百分比表示的兄弟处理的胚胎。 （改性劳克斯和Ahlgren 2009的权限）。 <img alt="图4" src="/files/ftp_upload/3704/3704fig4.jpg" /> 图4。减少在眼内的距离（IOD）在斑马鱼胚胎中的乙醇暴露的结果。一）正面看法的胚胎表明正常和融合的眼表型。乙）乙醇的不同的给药剂量为3个小时在原肠水库在ULTS下降IOD值时，鱼类24小时后检查。熔融的眼睛和独眼只看到在最高剂量测试（2.4％）。 *表示在IOD显着减少，与对照组相比。 （从2004年Ahlgren，许可修改）。 <img alt="图5" src="/files/ftp_upload/3704/3704fig5.jpg" /> 图5。SHH-N 的基因注射抢救斑马鱼暴露乙醇生产总值的缺陷。一个个细胞的胚胎注入100皮克/ NL SHH-N的基因注入胚胎的一半暴露乙醇剂量的标准，从4.3至24 HPF。胚胎进行了分析5 DPF。 2.0％的乙醇处理的胚胎表现出背侧弯曲，短尾巴，我形体节和眼的缺陷，包括独眼。这些表型的救援被认为是在注射胚胎（93％）71/76。在这些胚胎，身体直，体节是雪佛龙形，眼睛是完全分开的。

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fetal+alcohol+spectrum+disorders+in+young+adulthood.">Fetal alcohol spectrum disorders in young adulthood.</a> J. Pediatr. 150 (2), 175-179 (2007).</li><li>Strauss, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+the+intrauterine+environment+on+childhood+growth.">Effects of the intrauterine environment on childhood growth.</a> Br. Med. Bull. 53 (1), 81-95 (1997).</li><li>Loucks, E. J., Schwend, T., Ahlgren, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+changes+associated+with+teratogen-induced+cyclopia.">Molecular changes associated with teratogen-induced cyclopia.</a> Birth Defects Res. A. Clin. Mol. Teratol. 79 (9), 642-651 (2007).</li><li>Schwend, T., Loucks, E. J., Ahlgren, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+Gli+activity+in+craniofacial+tissues+of+hedgehog-pathway+reporter+transgenic+zebrafish.">Visualization of Gli activity in craniofacial tissues of hedgehog-pathway reporter transgenic zebrafish.</a> PLoS One. 5 (9), 14396-14396 (2011).</li><li>Chrisman, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gestational+ethanol+exposure+disrupts+the+expression+of+FGF8+and+Sonic+hedgehog+during+limb+patterning.">Gestational ethanol exposure disrupts the expression of FGF8 and Sonic hedgehog during limb patterning.</a> Birth Defects Res. A. Clin. Mol. Teratol. 70 (4), 163-171 (2004).</li></ol>

我们什么都没有透露。

这里介绍的方法，结果表明只是如何斑马鱼可以用来审问发育缺陷的一角。由于胚胎的辅助，每个离合器，高重复性的结果产卵数量高，这些脊椎动物是致畸研究的理想。鱼也可以操纵含有荧光分子，利用视觉作为一个特定的基因或通路利息19记者。在乙醇的研究，使用剂量出现相比，哺乳动物的血液酒精含量相当高。然而，这些级别反映了什么是水，不是所有的乙醇被传递到胚胎。 两者合计，描述和上面详细介绍的方法表明，斑马鱼的胚胎是乙醇暴露相关的模拟人类出生缺陷非常有用。此外，发现在斑马鱼的基因表达的变化已被证实由他人在哺乳动物模型系统20，表明乙醇的发展影响整个脊椎动物保留。上面显示的技术可用于许多其他环境或制药侮辱，快速，方便地检测潜在的致畸和判断，有助于特定物质的致畸的潜在机制。

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assessing teratogenic changes in a zebrafish model of fetal alcohol exposure

胎儿酒精综合征（FAS）是一个严重的表现胚胎暴露于乙醇。它提出了缺陷的脸和器官，包括精神发育迟滞，由于紊乱和脑损伤发展的特点。胎儿酒精谱系障碍（FASD）是用于支付出生缺陷的发生是由于产妇饮酒连续任期，并在发生在美国出生的儿童约4％。与育龄妇女的50％食用酒精，一半的怀孕是无计划的，无意的暴露是一个持续的问题2。 ，以最好地理解乙醇产生的破坏，加上产生的模型来测试潜在的干预，我们开发了一个使用斑马鱼胚胎发育乙醇曝光模式。斑马鱼是理想的这种致畸研究3-8。每对奠定了数百个鸡蛋，然后可以不损害成年人的情况下收集鱼。斑马鱼的胚胎是透明的，可以很容易地与任何数量的污渍成像。分析这些胚胎后，暴露在不同剂量和时间期限和应用表明乙醇，乙醇生产总值的发育缺陷与人类的出生缺陷是一致的。这里描述的是用于研究和操纵斑马鱼FAS模型的基本技巧。

Watch this Scientific Journal Video about Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure at JoVE.com

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

为了了解乙醇诱导发育损伤的分子机制，我们已经开发的乙醇暴露斑马鱼模型，并正在探索乙醇暴露后发生的物理，细胞和基因改变<sup&gt; 1</sup&gt;。然后，我们来寻求潜在的干预和快速测试他们在这个动物模型。

在斑马鱼模型的胎儿酒精暴露评估致畸的变化

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

assessing-teratogenic-changes-zebrafish-model-fetal-alcohol

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

26.2K 次观看.  Children's Memorial Research Center. 为了了解乙醇诱导发育损伤的分子机制，我们已经开发的乙醇暴露斑马鱼模型，并正在探索乙醇暴露后发生的物理，细胞和基因改变 1。然后，我们来寻求潜在的干预和快速测试他们在这个动物模型。

视频: Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Moderate Prenatal Alcohol Exposure and Quantification of Social Behavior in Adult Rats

Alterations in social behavior are among the major negative consequences observed in children with Fetal Alcohol Spectrum Disorders (FASDs). Several independent laboratories have demonstrated robust alterations in the social behavior of rodents exposed to alcohol during brain development across a wide range of exposure durations, timing, doses, and ages at the time of behavioral quantification. Prior work from this laboratory has identified reliable alterations in specific forms of social interaction following moderate prenatal alcohol exposure (PAE) in the rat that persist well into adulthood, including increased wrestling and decreased investigation. These behavioral alterations have been useful in identifying neural circuits altered by moderate PAE1, and may hold importance for progressing toward a more complete understanding of the neural bases of PAE-related alterations in social behavior. This paper describes procedures for performing moderate PAE in which rat dams voluntarily consume ethanol or saccharin (control) throughout gestation, and measurement of social behaviors in adult offspring.

The goal of the protocol presented here is to describe procedures to expose rats to moderate levels of alcohol during prenatal brain development and to quantify resulting alterations in social behavior during adulthood.

适度的产前酒精暴露在成年大鼠社会行为的量化

Alterations in social behavior are among the major negative consequences observed in children with Fetal Alcohol Spectrum Disorders (FASDs). Several ...

科研

行为学

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.

斑马鱼急性酒精性肝损伤的组织学分析

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 ...

发育生物学

Automated High-throughput Behavioral Analyses in Zebrafish Larvae

We have created a novel high-throughput imaging system for the analysis of behavior in 7-day-old zebrafish larvae in multi-lane plates. This system measures spontaneous behaviors and the response to an aversive stimulus, which is shown to the larvae via a PowerPoint presentation. The recorded images are analyzed with an ImageJ macro, which automatically splits the color channels, subtracts the background, and applies a threshold to identify individual larvae placement in the lanes. We can then import the coordinates into an Excel sheet to quantify swim speed, preference for edge or side of the lane, resting behavior, thigmotaxis, distance between larvae, and avoidance behavior. Subtle changes in behavior are easily detected using our system, making it useful for behavioral analyses after exposure to environmental toxicants or pharmaceuticals.

Our laboratory developed a novel high-throughput automated imaging system that is useful for the detection of several different behaviors in 7-day-old zebrafish larvae. The system can be used for detecting subtle changes in behavior after the larvae have been exposed to environmental toxicants or pharmaceuticals.

在斑马鱼幼虫的高通量自动化的行为分析

We have created a novel high-throughput imaging system for the analysis of behavior in 7-day-old zebrafish larvae in multi-lane plates. This system ...

神经科学

Zebrafish Larvae as a Model to Evaluate Potential Radiosensitizers or Protectors

Zebrafish are extensively used in several kinds of research because they are one of the easily maintained vertebrate models and exhibit several features of a unique and convenient model system. As highly proliferative cells are more susceptible to radiation-induced DNA damage, zebrafish embryos are a front-line in vivo model in radiation research. In addition, this model projects the effect of radiation and different drugs within a short time, along with major biological events and associated responses. Several cancer studies have used zebrafish, and this protocol is based on the use of radiation modifiers in the context of radiotherapy and cancer. This method can be readily used to validate the effects of different drugs on irradiated and control (non-irradiated) embryos, thus identifying drugs as radio sensitizing or protective drugs. Although this methodology is used in most drug screening experiments, the details of the experiment and the toxicity assessment with the background of X-ray radiation exposure are limited or only briefly addressed, making it difficult to perform. This protocol addresses this issue and discusses the procedure and toxicity evaluation with a detailed illustration. The procedure describes a simple approach for using zebrafish embryos for radiation studies and radiation-based drug screening with much reliability and reproducibility.

The zebrafish has recently been exploited as a model to validate potential radiation modifiers. The present protocol describes the detailed steps to use zebrafish embryos for radiation-based screening experiments and some observational approaches to evaluate the effect of different treatments and radiation.

斑马鱼幼虫作为评估潜在放射增敏剂或保护剂的模型

Zebrafish are extensively used in several kinds of research because they are one of the easily maintained vertebrate models and exhibit several features ...

癌症研究

Construction of Vapor Chambers Used to Expose Mice to Alcohol During the Equivalent of all Three Trimesters of Human Development

Exposure to alcohol during development can result in a constellation of morphological and behavioral abnormalities that are collectively known as Fetal Alcohol Spectrum Disorders (FASDs). At the most severe end of the spectrum is Fetal Alcohol Syndrome (FAS), characterized by growth retardation, craniofacial dysmorphology, and neurobehavioral deficits. Studies with animal models, including rodents, have elucidated many molecular and cellular mechanisms involved in the pathophysiology of FASDs. Ethanol administration to pregnant rodents has been used to model human exposure during the first and second trimesters of pregnancy. Third trimester ethanol consumption in humans has been modeled using neonatal rodents. However, few rodent studies have characterized the effect of ethanol exposure during the equivalent to all three trimesters of human pregnancy, a pattern of exposure that is common in pregnant women. Here, we show how to build vapor chambers from readily obtainable materials that can each accommodate up to six standard mouse cages. We describe a vapor chamber paradigm that can be used to model exposure to ethanol, with minimal handling, during all three trimesters. Our studies demonstrate that pregnant dams developed significant metabolic tolerance to ethanol. However, neonatal mice did not develop metabolic tolerance and the number of fetuses, fetus weight, placenta weight, number of pups/litter, number of dead pups/litter, and pup weight were not significantly affected by ethanol exposure. An important advantage of this paradigm is its applicability to studies with genetically-modified mice. Additionally, this paradigm minimizes handling of animals, a major confound in fetal alcohol research.

We demonstrate the construction of alcohol vapor chambers using readily available materials that simultaneously house 6 mouse cages. We further describe their use in a mouse model of fetal alcohol exposure equivalent to all 3&#160;trimesters of human pregnancy. This paradigm exposes animals during gestation and postnatal days 1-12.

蒸汽腔体建设用于公开小鼠酒精在所有三个学期人类发展的等效

Exposure to alcohol during development can result in a constellation of morphological and behavioral abnormalities that are collectively known as Fetal ...

医学

Quantification of Ethanol Levels in Zebrafish Embryos Using Head Space Gas Chromatography

Fetal Alcohol Spectrum Disorders (FASD) describe a highly variable continuum of ethanol-induced developmental defects, including facial dysmorphologies and neurological impairments. With a complex pathology, FASD affects approximately 1 in 100 children born in the United States each year. Due to the highly variable nature of FASD, animal models have proven critical in our current mechanistic understanding of ethanol-induced development defects. An increasing number of laboratories has focused on using zebrafish to examine ethanol-induced developmental defects. Zebrafish produce large numbers of externally fertilized, genetically tractable, translucent embryos. This allows researchers to precisely control timing and dosage of ethanol exposure in multiple genetic contexts and quantify the impact of embryonic ethanol exposure through live imaging techniques. This, combined with the high degree of conservation of both genetics and development with humans, has proven zebrafish to be a powerful model in which to study the mechanistic basis of ethanol teratogenicity. However, ethanol exposure regimens have varied between different zebrafish studies, which has confounded the interpretation of zebrafish data across these studies. Here is a protocol to quantify ethanol concentrations in zebrafish embryos using head space gas chromatography.

This work describes a protocol to quantify ethanol levels in a zebrafish embryo using head space gas chromatography from proper exposure methods to embryo processing and ethanol analysis.

使用头太空气色谱对斑马鱼胚胎中的乙醇水平进行定量

Fetal Alcohol Spectrum Disorders (FASD) describe a highly variable continuum of ethanol-induced developmental defects, including facial dysmorphologies ...

Zebrafish as a Model to Assess the Teratogenic Potential of Nitrite

High nitrate levels in the environment may&#160;result in congenital defects or miscarriages in humans. Presumably, this is due to the conversion of nitrate to nitrite by gut and salivary bacteria. However, in other mammalian studies, high nitrite levels do not cause birth defects, although they can lead to poor reproductive outcomes. Thus, the teratogenic potential of nitrite is not clear. It would be useful to have a vertebrate model system to easily assess teratogenic effects of nitrite or any other chemical of interest. Here, we demonstrate the utility of zebrafish (Danio rerio) to screen compounds for toxicity and embryonic defects. Zebrafish embryos are fertilized externally and have rapid development, making them a good model for teratogenic studies. We show that increasing the time of exposure to nitrite negatively affects survival. Increasing the concentration of nitrite also adversely affects survival, whereas nitrate does not. For embryos that survive nitrite exposure, various defects can occur, including pericardial and yolk sac&#160;edema, swim bladder noninflation, and craniofacial malformation. Our results indicate that the zebrafish is a convenient system for studying the teratogenic potential of nitrite. This approach can easily be adapted to test other chemicals for their effects on early vertebrate development.

Exposure to teratogens may cause birth defects. Zebrafish are useful for determining the teratogenic potential of chemicals. We demonstrate the utility of zebrafish by exposing embryos to various levels of nitrite and also at different times of exposure. We show that nitrite can be toxic and cause severe developmental defects.

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 ...

免疫与感染

Modeling Fetal Alcohol Spectrum Disorders in Zebrafish to Characterize the Impact of an Adverse Embryonic Environment on Adult Social Behavior

Fetal alcohol spectrum disorders (FASD) describe all alcohol-induced birth defects. Birth defects such as growth deficiencies, craniofacial, behavioral, and cognitive abnormalities are associated with FASD. Social difficulties are common behavioral abnormalities associated with FASD and often result in serious health issues. Animal models are critical to understanding the mechanisms responsible for ethanol-induced social defects. Zebrafish are social vertebrates that produce externally fertilized transparent eggs; these characteristics provide researchers with a precise yet simple procedure for creating the FASD phenotype and an innate behavior that can be leveraged to model the social deficits associated with FASD. Thus, zebrafish are ideal for characterizing the social deficits of FASD. The goal of the current protocol is to provide the user with a simple behavioral assay that can be used to characterize the consequences of a negative environment early during development and the effects it can have on social behavior in adulthood. The protocol can be used to characterize the effect mutations or teratogens have on adult social behavior. The protocol outlined here demonstrates how to characterize the social behavior of individual fish during a 20-min social assay. Furthermore, the data obtained using the current protocol provides evidence that the protocol can be used to characterize the effects of embryonic ethanol-induced social defects in adult zebrafish.

The goal of the current protocol is to outline the steps necessary to establish and use a social preference assay for adult zebrafish and demonstrate that it can be used to characterize ethanol-induced social defects.

Fetal alcohol spectrum disorders (FASD) describe all alcohol-induced birth defects. Birth defects such as growth deficiencies, craniofacial, behavioral, ...

The Zebrafish Tol2 System: A Modular and Flexible Gateway-Based Transgenesis Approach

Fetal alcohol spectrum disorders (FASD) are characterized by a highly variable set of structural defects and cognitive impairments that arise due to prenatal ethanol exposure. Due to the complex pathology of FASD, animal models have proven critical to our current understanding of ethanol-induced developmental defects. Zebrafish have proven to be a powerful model to examine ethanol-induced developmental defects due to the high degree of conservation of both genetics and development between zebrafish and humans. As a model system, zebrafish possess many attributes that make them ideal for developmental studies, including large numbers of externally fertilized embryos that are genetically tractable and translucent. This allows researchers to precisely control the timing and dosage of ethanol exposure in multiple genetic contexts. One important genetic tool available in zebrafish is transgenesis. However, generating transgenic constructs and establishing transgenic lines can be complex and difficult. To address this issue, zebrafish researchers have established the transposon-based Tol2 transgenesis system. This modular system uses a multisite Gateway cloning approach for the quick assembly of complete Tol2 transposon-based transgenic constructs. Here, we describe the flexible Tol2 system toolbox and a protocol for generating transgenic constructs ready for zebrafish transgenesis and their use in ethanol studies.

This work describes a protocol for the modular Tol2 transgenesis system, a gateway-based cloning method to create and inject transgenic constructs into zebrafish embryos.

斑马鱼 Tol2 系统:一种模块化且灵活的基于网关的转基因方法

Fetal alcohol spectrum disorders (FASD) are characterized by a highly variable set of structural defects and cognitive impairments that arise due to ...

Small Molecule Screening and Toxicity Testing in Early-stage Zebrafish Larvae

Zebrafish have become a prominent model organism for human translational research, toxicity testing of small molecules and environmental pollutants, therapeutic drug discovery, and other biomedical research applications. The presented protocol aims to provide a clear guide for drug screening and toxicity testing in early-stage zebrafish larvae (aged 3-7 days post fertilization, dpf). Several methodologies describing toxicity testing in zebrafish larvae have been published, albeit lacking a standardized protocol for exposure durations, rate of media changes, and morphology scoring, amongst others. We experimentally determined the ideal larvae ages for conducting the experiments, the plate sizes that work best for those ages, the rate of media changes that provide reliable results while limiting wastage&#160;of test compound, and a morphology scoring system that is easily quantifiable in a statistical manner. The manuscript describes in detail a protocol for toxicity testing in zebrafish larvae aged between&#160;3 to 7 dpf, aiming to target the gap in drug toxicity testing and taking the first step towards a standardized method similar to the Fish Embryo Acute Toxicity test.

This protocol describes a drug screening procedure in zebrafish larvae 3-7 days post fertilization followed by morphological assessment.

早期斑马鱼幼虫的小分子筛选和毒性试验

Zebrafish have become a prominent model organism for human translational research, toxicity testing of small molecules and environmental pollutants, ...