这项工作是从艾科卡家庭基金会，罗莎琳德富兰克林大学启动资金，和美国国立卫生研究院格兰特DK092721（RVI）的研究经费支持的。作者希望感谢尼克内壁的援助在准备论文。

所有程序中的“实验动物护理原则的指引”（美国国立卫生研究院出版。85-23号，1985年修订）和罗莎琳德·富兰克林大学实验动物护理和使用委员会的动物协议08-19。 有2个重要的这个手稿中所使用的缩写。 1）DM：是指鱼（300毫克/分升）在急性高血糖状态，并已在至少3个星期。 2）MM：是指鱼，分别为21天（见协议）DM鱼和胰腺再生，恢复血糖控制。这是实现内的去除药物（14）天。从这一点鱼被认为是MM鱼。同样重要的是要注意，鱼，被统称为控制方面的注射次数DM或MM鱼（盐水），在各种温度下的孵育时间和数量和定时的处理是完全相同的尾鳍截肢。 1。糖尿病，DM鱼斑马鱼产生的<ol><li>准备恢复和麻醉水箱。恢复水是正常的鱼水。补充足够的麻醉水2 - 苯氧乙醇，1:1,000稀释在正常鱼水的。 </li><li>准备的链脲佐菌素（STZ）的0.3％的溶液（在通风橱中）通过添加6毫克STZ 2毫升0.09％的氯化钠，并立即在冰上放置该溶液。这将提供足够的注射液，注射20分钟约20鱼。如果你超过20分钟的标记，停，并作出新的STZ溶液，然后再继续。在一个单独的管等分足够的生理盐水溶液用于控制鱼。一旦STZ溶解所有的后续步骤不需要通风橱中使用。 </li><li>填写一个半毫升注射器配有1/2的27号针STZ或控制解决方案，确保没有气泡被困。 </li><li>麻醉医师个别tize每条鱼将鱼放置在麻醉水，并等待，直到他们停止游泳运动（1-2分钟）。 </li><li>一旦麻醉简要的鱼放在纸巾上，以吸收任何多余的水，将鱼在权衡船和测量质量的鱼。 </li><li>将鱼放在一个坚固的表面上（培养皿盖子）注射。 </li><li>过去的斜角到腹侧腹膜后方面通过插入针进入腹膜腔的鱼注射链脲佐菌素或对照溶液。 </li><li> 0.35毫克/克（350毫克/千克）的链脲佐菌素应交付每个鱼和需要，可以以下面的方式计算的0.3％的溶液的体积。 <ol class="lalpha"><li>鱼的质量（克）乘以0.35以产生所需要的毫克量的STZ。 </li><li>划分产品上面生成3，得到所需的注射在微升0.3％的溶液的体积。 示例：对于一个0.5克的鱼：A）0.5×0.35 = 0.175）0.0175 / 3 =0.058毫升= 58微升。 同样的体积，注射STZ没有控制鱼。一张卷注入每条鱼的质量应该产生和快速参考使用。 </li></ol></li><li>注射后，将恢复水箱中的鱼和他们正常的游泳活动进行监视。一旦这个已经实现了鱼被转移到一个正常的生活罐维持在降低的温度范围为22℃ - 24℃。这减少的温度为高效诱导的高血糖症的糖尿病（DM）的关键。 </li><li>虽然高血糖在检测到第一次注射的24小时的范围内，以非常高的高血糖状态的诱导长期斑马鱼需要频繁注射诱导阶段，然后由注射每周检修，如下所示。 </li></ol>第1周：3次注射（第1天，3，5），第2周（12天）：注射1次，3周注射1次（19天）， 第4周（21天）执行分析的兴趣。 在这一点上，斑马鱼被认为是已经在长期的高血糖状态，并表现出糖尿病视网膜病变，肾病和也鳍的再生受损的并发症。这些被称为作为DM鱼。此外，如果需要的鱼可以保持在高血糖状态下与注射每周检修。在此过程中，大约有5％的死亡可以预期的。 2。采血车和空腹血糖的水平（FBGL）测定<ol><li>各组DM和控制鱼类必须有足够的鱼，此法需要这些鱼被牺牲准确地确定本集团的平均FBGL。 </li><li>准备标记的PCR管，每一个血液样本含5μl的无菌生理盐水。 </li><li>血液采集，麻醉鱼上面，删除所有的水，将鱼放在显微镜载片上，用手术刀，去除头的鱼在基地的厣。 </li><li>收集血液（最多2微升），从鱼到幻灯片上被释放，并迅速将其添加到5微升无菌生理盐水上下吹打确保血液不会堵塞。立即将样品放置在冰上。 </li><li>确定的血液样品体积通过测量该液体的总体积（生理盐水+血），再减去5微升生理盐水。 </li><li>从每个PCR管5微升稀释血液转移到1.5毫升微量离心管中，并确定使用QuantiChrome葡萄糖分析试剂盒的血液中的葡萄糖浓度。这是通过按照制造商的协议无一例外。预期的结果：正常/控制鱼60 mg / dl和DM鱼310毫克/分升。 </li></ol> 3。尾鳍再生研究<ol><li>麻醉鱼在1.0-1.4。 </li><li>地点在培养皿盖和截肢鱼的尾鳍在一条直线上使用无菌大小的10手术刀近端的第一lepidotrichia分支点，同时通过在解剖显微镜观看翅片。 </li><li>让鱼恢复，但在1.10的鱼在33°C的再生生长阶段的检测。这是一个既定的温度加速鳍的再生分析。 </li><li>的散热片可以在任何时间后截肢成像，但是我们例行检查在24，28和72小时后，反式截面的再生生长。 </li><li>麻醉鱼像以前那样（见1.0-1.4），在解剖显微镜下配备了一个摄像头（使用尼康SMZ-1500配备了一个Q-成像相机）的鱼，并收集所有的鳍在1倍放大率的图像与NIS Elements软件。应传播的翅片用于成像，以便它完全伸展不用拉伸或损坏的组织，并应该是免费的，任何水滴。为了一致性，始终把背侧到右侧。 </li><li>打印的图像和测量的再生GRO一族的区域使用Image J软件和使用图垫。跟踪围绕新的增长的整个区域，并确定该区域。每次测量应进行5次，取平均值，以确保准确的跟踪。重要的是，所使用的图像没有阴影或水滴的，因为这些有助于扩展面积测量中的错误。 </li><li>测量沿背腹轴的截肢站点的长度划分确定在3.6的区域，通过该测定。这允许直接进行比较，不同大小的鱼。 </li></ol> 4。产生的代谢记忆（MM）斑马鱼<ol><li>启动DM鱼和一组适当的控制，如第1节所述。 </li><li>在21天中，确定FBGL本集团的一个子集，分2组，DM成鱼。继续每周STZ注射作为实验的持续时间的DM控制的基团之一。停止注射STZ为第二组和incubat的e的鱼在正常的温度。这些斑马鱼在14天内将恢复正常的血液中的胰岛素和血糖控制通过胰腺再生。这些鱼被称为MM（代谢记忆的鱼）。 </li><li>在30天的药物清除后截肢的尾鳍控制，DM和MM鱼类的通过3.2中所描述的方法。 </li><li>返回鱼的鳍的再生为期30天的正常供水条件。这使得我们称之为代谢记忆组织的生长。 </li><li>在第60天，进行第二次截肢（如3.2中所述）被再生期间，在30-60天的组织内，并执行图1的尾鳍再生吸附试验（3.1-3.7）。 </li><li>隔离组织的利益进行分析。协议摘要，请参阅表1。 </li></ol>

没有利益冲突的声明。

糖尿病是一种疾病，代谢失调，高血糖，最终导致血管损伤，导致许多并发症，坚持，即使血糖正常实现，但药物干预后，初步诊断为。这种持久性的并发症，被称为“代谢记忆和最近一些研究研究，表观遗传机制的作用发挥这种现象。在这里，我们已经详细介绍了协议，允许糖尿病急性代谢记忆（恢复血糖控制）斑马鱼产生的。我们进一步描述的方法，可以采用从以前的糖尿病状态的组件的潜?...

糖尿病（DM）是一个日益严重的健康问题，缩短预期寿命，由于特定的微血管疾病（视网膜病变，肾病，神经病变，影响伤口愈合）和大血管并发症（心脏疾病和中风）1。一旦启动，糖尿病并发症的不断进步，即使血糖控制是实现不间断2,3，这种现象被称为代谢记忆或旧的效果。这种现象的存在，临床上在20世纪90年代初的“糖尿病控制和并发症试验（DCCT）”的进展，因为已经得到了多个额外的临床试验4,5,6,7,8,9,10， 11,12,13,14。 DM动物模型，发现相关的病理生理学，糖尿病并发症和代谢记忆的关键。事实上，糖尿病并发症的最早记载于持久性糖尿病犬模型视网膜病变这已被使用各种在体外培养系统和动物模型15,16,17,18,19,20,21几行的实验证据支持的。这些研究清楚地表明，一个的初始高血糖期间的查询结果的永久异常（包括异常的基因表达）的靶器官/细胞和机械地表明表观基因参与。 表观基因组包括所有的染色质修饰为一个特定的细胞类型，并负责细胞的独特的基因表达谱。染色体修改是动态的，在开发过程中，支持细胞分化，对外界刺激反应的，是有丝分裂稳定遗传22,23和可以改变疾病24,25,26。这些表观遗传机制，包括：发布翻译组蛋白修饰，非规范的组蛋白变体包含在octomers通过DNA甲基化，染色质的访问变化，基因通过微RNA的非编码27,28,29,30的表达调控。总之，后生过程使细胞/生物能够快速响应不断变化的环境刺激31,32,33，他们还赋予细胞的能力“记住”这些一旦遇到刺激被删除23,22。因此，改变基因的表达谱是稳定的表观遗传过程产生启动信号（S）的情况下，是可遗传的，通过细胞分裂，他们都获得了极大的兴趣相关的人类疾病包括代谢记忆的分子机制。在DM和表观遗传学在其他疾病中过多的表观遗传变化引起的高血糖会导致细胞的基因调控网络的不断变化显着（审查34,35,36,37,38）平行进步的背景下，新兴的结果。 斑马鱼一直是最大的模式生物，以学生DY脊椎动物的发展，但过去15年来一直呈指数级增长，利用这种微生物为研究人类疾病的39。人类疾病的斑马鱼模型已经建立了覆盖广泛的人类疾病，包括遗传性疾病和后天疾病40,41,42。比其他脊椎动物模式生物斑马鱼的诸多优点，包括高繁殖力，世代时间短，通过成年早期的透明度，降低住房成本和阵列的基因操纵的工具。此外，由于遗传途径和细胞之间的脊椎动物的生理和能力进行高通量药物筛选的广泛保护，斑马鱼已被成功地用于药物发现。 我们已经开发了一个成年斑马鱼模型的I型糖尿病，链脲佐菌素致糖尿病药物。我们这一模型显示，糖尿病斑马鱼T仅显示已知的人类继发性并发症，但除此之外，表现出肢体再生障碍（尾鳍再生）的高血糖环境的后果。此外，我们曾报道过，高血糖斑马鱼恢复到正常血糖水平在2周内由于在生理上正常的血糖状态的内源性胰岛β细胞再生的药物清除。然而，与此相反，这些鱼的肢体再生的仍然减值到相同的程度，在这种并发症的糖尿病急性状态仍然存在，并容易代谢记忆。产生这种模式的主要动力是提供一个系统研究的有丝分裂稳定的表观遗传代谢记忆现象的组件，支持在以前的高血糖环境的背景噪音的情况下。在该协议的结论这里提供的斑马鱼和或选择性组织可以处理任何适合的resea测定rchers需要。我们已经成功地使用了此程序，以确定在DNA甲基化的基因组范围内的持久改变代谢内存状态21被保持在由高血糖引起的。 我们认为，这种斑马鱼模型的I型糖尿病有几个创新的优势，其他的模型系统研究代谢记忆。 1）所有我们的研究可以在体内进行的和以前的高血糖鱼通过返回到正常血糖的内源性胰岛素生产的再生，它们不需要外源性胰岛素注射。因此，这样就避免了复杂的血糖控制可能发生的需要外源性胰岛素在动物的峰值和低谷。 2）如上所述，从以前的糖尿病状态（ 即晚期糖基化终端产品和活性氧标志物的继续存在）的背景刺激被淘汰，因此可以检查纯粹epig代谢记忆的enetic因素。 3）的实验，可以迅速地进行，因为它需要约80天诱导糖尿病直到代谢记忆检查。 4）尾鳍再生实验非常平易近人，和允许容易的遗传和实验操作，有大量的工具。 5）尾鳍再生提供了一个非常简单的，可量化的方法来评估代谢记忆，因此将允许未来的药物发现。

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td>链脲佐菌素</td><td> Sigma Aldrich公司</td><td> S0130 </td><td></td></tr><tr><td> 2苯氧乙醇</td><td> Sigma Aldrich公司</td><td> P1126 </td><td></td></tr><tr><td>手术刀（规格10） </td><td> Fisher Scientific则</td><td> 089275A </td><td></td></tr><tr><td>培养皿</td><td> Fisher Scientific则</td><td> 08-757-13 </td><td></td></tr><tr><td> ½毫升的注射器，针头27 1/2 </td><td> Fisher Scientific则</td><td> 305620 </td><td></td></tr><tr><td> QuantiChrome GLUCOSE检测试剂盒。 </td><td>生物测定系统</td><td> DIGL-100 </td><td></td></tr><tr><td>氯化钠</td><td> Sigma Aldrich公司</td><td> S3014 </td><td></td></tr><tr><td>解剖显微镜</td><td>尼康</td><td> TMZ-1500 </td><td>任何解剖显微镜的罚款。 </td></tr><tr><td>相机的成像</td><td>尼康</td><td> Q成像</td><td>相机的任何适合的。 </td></tr><tr><td> Image J软件</td><td>美国国立卫生研究院</td><td>美国国立卫生研究院图片</td><td></td></tr><tr><td> NIS元素</td><td>尼康</td><td></td><td>任何成像软件是合适的。 </td></tr></tbody></table>

a zebrafish model of diabetes mellitus and metabolic memory

糖尿病目前影响3.46亿人，这是到2030年预计将增加至400万。从实验室和大规模临床试验的证据已显示，糖尿病并发症的进展不受阻碍地通过即使当血糖控制药学实现代谢记忆现象。基因的表达，可以稳定地改变表观遗传变化，不仅让细胞和生物体能够快速响应不断变化的环境刺激，但也赋予能力的细胞的“记忆”一旦遇到刺激被删除。因此，角色，这些机制在代谢记忆的现象，目前正在研究之中。 我们最近报道的斑马鱼模型的Ⅰ型糖尿病的发展，这种模式的特点表明，糖尿病斑马鱼不仅显示已知的继发性并发症，包括有关的变化糖尿病视网膜病变，糖尿病肾病，影响伤口愈合，但也表现出减值尾鳍再生。这种模式是独特的斑马鱼能够再生受损的胰腺和类似什么，将可望在人类患者移植后恢复正常血糖状态。此外，多个轮尾鳍截肢允许在体内系统中的分离和纯后生效应研究没有潜在的复杂因素，从以前的糖尿病状态。虽然血糖正常胰腺再生，鳍的再生和皮肤伤口愈合的糖尿病继发的持续存在下去。在鳍的再生受损的情况下，这种病理鳍的再生女儿鳍组织的多轮之后仍然保留。这些观察指出，一个基本的表观遗传过程中存在的代谢记忆状态。在这里，我们目前的需要的方法，成功地根全方位的糖尿病和代谢记忆的鱼，并讨论了该模型的优势。

Ⅰ型糖尿病的斑马鱼不仅显示已知的继发性并发症视网膜病变和肾病患者，但也表现出额外的复杂性：尾鳍再生受损。这以后的并发症仍然存在，由于代谢记忆中的鱼，已经恢复了正常的血糖控制高血糖期。在图2A中（对照）和图2B（代谢记忆）代表图像再生的散热片，在72小时后截肢捕获。可以量化和图2C DM和MM斑马鱼表现出赤字约为40％，在72小时时相比，?...

Watch this Scientific Journal Video about A Zebrafish Model of Diabetes Mellitus and Metabolic Memory at JoVE.com

A Zebrafish Model of Diabetes Mellitus and Metabolic Memory

代谢记忆的现象，其中糖尿病并发症的坚持和畅通，即使血糖正常，取得药学进展。在这里，我们描述了一个糖尿病斑马鱼模型的独特之处在于它允许参加考试的有丝分裂传播后生成分代谢记忆<em在体内</em&gt;。

斑马鱼模型的糖尿病和代谢记忆

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

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25.5K 次观看.  Rosalind Franklin University of Medicine and Science. 代谢记忆的现象，其中糖尿病并发症的坚持和畅通，即使血糖正常，取得药学进展。在这里，我们描述了一个糖尿病斑马鱼模型的独特之处在于它允许参加考试的有丝分裂传播后生成分代谢记忆

视频: A Zebrafish Model of Diabetes Mellitus and Metabolic Memory

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.

对黑色素瘤修饰符一个斑马鱼原地肿瘤模型的筛选

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 ...

科研

医学

A Model of Chronic Nutrient Infusion in the Rat

Chronic exposure to excessive levels of nutrients is postulated to affect the function of several organs and tissues and to contribute to the development of the many complications associated with obesity and the metabolic syndrome, including type 2 diabetes. To study the mechanisms by which excessive levels of glucose and fatty acids affect the pancreatic beta-cell and the secretion of insulin, we have established a chronic nutrient infusion model in the rat. The procedure consists of catheterizing the right jugular vein and left carotid artery under general anesthesia; allowing a 7-day recuperation period; connecting the catheters to the pumps using a swivel and counterweight system that enables the animal to move freely in the cage; and infusing glucose and/or Intralipid (a soybean oil emulsion which generates a mixture of approximately 80% unsaturated/20% saturated fatty acids when infused with heparin) for 72 hr. This model offers several advantages, including the possibility to finely modulate the target levels of circulating glucose and fatty acids; the option to co-infuse pharmacological compounds; and the relatively short time frame as opposed to dietary models. It can be used to examine the mechanisms of nutrient-induced dysfunction in a variety of organs and to test the effectiveness of drugs in this context.

A protocol for chronic infusions of glucose and Intralipid in rats is described. This model can be used to study the impact of nutrient excess on organ function and physiological parameters.

在大鼠模型慢性营养输液

Chronic exposure to excessive levels of nutrients is postulated to  affect the function of several organs and tissues and to contribute to the  ...

A Modified Precipitation Method to Isolate Urinary Exosomes

Identification of biomarkers that allow early detection of kidney diseases in urine and plasma has been an area of active interest for several years. Urinary exosome vesicles, 40-100 nm in size, are released into the urine under normal conditions by cells from all nephron segments and may contain protein, mRNA and microRNA representative of their cell type of origin. Under conditions of renal dysfunction or injury, exosomes may contain altered proportions of these components, which may serve as biomarkers for disease. There are currently several methods available for isolation of urinary exosomes, and we have previously conducted an experimental comparison of each of these approaches, including three based on ultracentrifugation, one using a nanomembrane ultrafiltration concentrator, one using a commercial precipitation reagent and one using a modification of the precipitation technique using ExoQuick reagent that we developed in our laboratory. We found the modified precipitation method produced the highest yield of exosome particles, miRNA, and mRNA, making this approach suitable for the isolation of exosomes for subsequent RNA profiling. We conclude that the modified exosome precipitation method offers a quick, scalable, and effective alternative for the isolation of exosomes from urine. In this report, we describe our modified precipitation technique using ExoQuick reagent for isolating exosomes from human urine.

This manuscript describes a protocol for isolating exosomes from human urine using a modified precipitation technique.

一种改进沉淀法来隔离尿外来体

Identification of biomarkers that allow early detection of kidney diseases in urine and plasma has been an area of active interest for several years. ...

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.

多囊肾一种可能的斑马鱼型号：击倒<em&gt; Wnt5a的</em&gt;原因在斑马鱼肾囊肿

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 ...

Surgical Injury to the Mouse Pancreas through Ligation of the Pancreatic Duct as a Model for Endocrine and Exocrine Reprogramming and Proliferation

Expansion of pancreatic beta cells in vivo or ex vivo, or generation of beta cells by differentiation from an embryonic or adult stem cell, can provide new expandable sources of beta cells to alleviate the donor scarcity in human islet transplantation as therapy for diabetes. Although recent advances have been made towards this aim, mechanisms that regulate beta cell expansion and differentiation from a stem/progenitor cell remain to be characterized. Here, we describe a protocol for an injury model in the adult mouse pancreas that can function as a tool to study mechanisms of tissue remodeling and beta cell proliferation and differentiation. Partial duct ligation (PDL) is an experimentally induced injury of the rodent pancreas involving surgical ligation of the main pancreatic duct resulting in an obstruction of drainage of exocrine products out of the tail region of the pancreas. The inflicted damage induces acinar atrophy, immune cell infiltration and severe tissue remodeling. We have previously reported the activation of Neurogenin (Ngn) 3 expressing endogenous progenitor-like cells and an increase in beta cell proliferation after PDL. Therefore, PDL provides a basis to study signals involved in beta cell dynamics and the properties of an endocrine progenitor in adult pancreas. Since, it still remains largely unclear, which factors and pathways contribute to beta cell neogenesis and proliferation in PDL, a standardized protocol for PDL will allow for comparison across laboratories.

This protocol describes an injury model involving the surgical ligation of the pancreatic duct in the adult mouse pancreas, resulting in severe injury that establishes an environment that allows beta cell neogenesis and proliferation. This model can be used as a tool to study mechanisms involved in beta cell formation.

通过胰管结扎手术损伤的小鼠胰腺作为模型内分泌和外重新规划和扩散

Expansion of pancreatic beta cells in vivo or ex vivo, or generation of beta cells by differentiation from an embryonic or adult stem cell, can provide ...

Ferric Chloride-induced Thrombosis Mouse Model on Carotid Artery and Mesentery Vessel

Severe thrombosis and its ischemic consequences such as myocardial infarction, pulmonary embolism and stroke are major worldwide health issues. The ferric chloride injury is now a well-established technique to rapidly and accurately induce the formation of thrombi in exposed veins or artery of small and large diameter. This model has played a key role in the study of the pathophysiology of thrombosis, in the discovery and validation of novel antithrombotic drugs and in the understanding of the mechanism of action of these new agents. Here, the implementation of this technique on a mesenteric vessel and carotid artery in mice is presented. The method describes how to label circulating leukocytes and platelets with a fluorescent dye and to observe, by intravital microscopy on the exposed mesentery, their accumulation at the injured vessel wall which leads to the formation of a thrombus. On the carotid artery, the occlusion caused by the clot formation is measured by monitoring the blood flow with a Doppler probe.

The FeCl3 induced thrombosis model in mice is described herein. A method to monitor thrombus growth by intravital microscopy observation on a mesenteric vessel and by blood flow measurement in the carotid artery is presented.

三氯化铁诱发血栓形成小鼠模型上的颈动脉和肠系膜血管

Severe thrombosis and its ischemic consequences such as myocardial infarction, pulmonary embolism and stroke are major worldwide health issues. The ferric ...

Dissection of the Mouse Pancreas for Histological Analysis and Metabolic Profiling

We have been investigating the pancreas specific transcription factor, 1a cre-recombinase; lox-stop-lox- Kristen rat sarcoma, glycine to aspartic acid at the 12 codon (Ptf1acre/+;LSL-KrasG12D/+) mouse strain as a model of human pancreatic cancer. The goal of our current studies is to identify novel metabolic biomarkers of pancreatic cancer progression. We have performed metabolic profiling of urine, feces, blood, and pancreas tissue extracts, as well as histological analyses of the pancreas to stage the cancer progression. The mouse pancreas is not a well-defined solid organ like in humans, but rather is a diffusely distributed soft tissue that is not easily identified by individuals unfamiliar with mouse internal anatomy or by individuals that have little or no experience performing mouse organ dissections. The purpose of this article is to provide a detailed step-wise visual demonstration to guide novices in the removal of the mouse pancreas by dissection. This article should be especially valuable to students and investigators new to research that requires harvesting of the mouse pancreas by dissection for metabolic profiling or histological analyses.

This video article provides a detailed demonstration of the procedures required to successfully remove the pancreas from a mouse by dissection for histological analysis and metabolic profiling.

解剖的小鼠胰腺的组织学分析与代谢谱

We have been investigating the pancreas specific transcription factor, 1a cre-recombinase; lox-stop-lox- Kristen rat sarcoma, glycine to aspartic acid at ...

Studying Diabetes Through the Eyes of a Fish: Microdissection, Visualization, and Analysis of the Adult tg(fli:EGFP) Zebrafish Retinal Vasculature

Diabetic retinopathy is the leading cause of blindness among middle-aged adults. The rising prevalence of diabetes worldwide will make the prevention of diabetic microvascular complications one of the key research fields of the next decades. Specialized, targeted therapy and novel therapeutic drugs are needed to manage the increasing number of patients at risk of vision-loss. The zebrafish is an established animal model for developmental research questions with increasing relevance for modeling metabolic multifactorial disease processes. The advantages of the species allow for optimal visualization and high throughput drug screening approaches, combined with the strong ability to knock out genes of interest. Here, we describe a protocol which will allow easy analysis of the adult tg(fli:EGFP) zebrafish retinal vasculature as a fast read-out in settings of long-term vascular pathologies linked to neoangiogenesis or vessel damage. This is achieved via dissection of the zebrafish retina and whole-mounting of the tissue. Visualization of the exposed vessels is then achieved via confocal microscopy of the green EGFP reporter expressed in the adult retinal vasculature. Correct handling of the tissue will lead to better outcomes and less internal vessel breakage to assure the visualization of the unaltered vascular structure. The method can be utilized in zebrafish models of retinal vasculopathy linked to changes in the vessel architecture as well as neoangiogenesis.

Studying Diabetes Through the Eyes of a Fish: Microdissection, Visualization, and Analysis of the Adult tgfli:EGFP Zebrafish Retinal Vasculature

Here, we discuss a method protocol which will allow an easy analysis of the adult tg(fli:EGFP) zebrafish retinal vasculature as a fast read-out in settings of long-term vascular pathologies linked to neoangiogenesis and structural changes.

通过鱼眼研究糖尿病: 成人 tg (fli: EGFP) 斑马鱼视网膜血管的显微解剖、可视化和分析

Diabetic retinopathy is the leading cause of blindness among middle-aged adults. The rising prevalence of diabetes worldwide will make the prevention of ...

Body Composition and Metabolic Caging Analysis in High Fat Fed Mice

Alterations to body composition (fat or lean mass), metabolic parameters such as whole-body oxygen consumption, energy expenditure, and substrate utilization, and behaviors such as food intake and physical activity can provide important information regarding the underlying mechanisms of disease. Given the importance of body composition and metabolism to the development of obesity and its subsequent sequelae, it is necessary to make accurate measures of these parameters in the pre-clinical research setting. Advances in technology over the past few decades have made it possible to derive these measures in rodent models in a non-invasive and longitudinal fashion. Consequently, these metabolic measures have proven useful when assessing the response of genetic manipulations (for example knockout or transgenic mice, viral knock-down or overexpression of genes), experimental drug/compound screening and dietary, behavioral or physical activity interventions. Herein, we describe the protocols used to measure body composition and metabolic parameters using an animal monitoring system in chow-fed and high fat diet-fed mice.

This protocol describes the use of a body composition analyzer and metabolic animal monitoring system to characterize body composition and metabolic parameters in mice. An obesity model induced by high-fat feeding is used as an example for the application of these techniques.

高脂喂养小鼠体内成分及代谢锁定分析

Alterations to body composition (fat or lean mass), metabolic parameters such as whole-body oxygen consumption, energy expenditure, and substrate ...

A Doxorubicin-induced Cardiomyopathy Model in Adult Zebrafish

The genetically accessible adult zebrafish (Danio rerio) has been increasingly used as a vertebrate model for understanding human diseases such as cardiomyopathy. Because of its convenience and amenability to high throughput genetic manipulations, the generation of acquired cardiomyopathy models, such as the doxorubicin-induced cardiomyopathy (DIC) model in adult zebrafish, is opening the doors to new research avenues, including discovering cardiomyopathy modifiers via forward genetic screening. Different from the embryonic zebrafish DIC model, both initial acute and later chronic phases of cardiomyopathy can be determined in the adult zebrafish DIC model, enabling the study of stage-dependent signaling mechanisms and therapeutic strategies. However, variable results can be obtained with the current model, even in the hands of experienced investigators. To facilitate future implementation of the DIC model, we present a detailed protocol on how to generate this DIC model in adult zebrafish and describe two alternative ways of intraperitoneal (IP) injection. We further discuss options on how to reduce variations to obtain reliable results and provide suggestions on how to appropriately interpret the results.

A method to generate a doxorubicin-induced cardiomyopathy model in adult zebrafish (Danio rerio) is described here. Two alternative ways of intraperitoneal injection are presented and conditions to reduce variations among different experimental groups are discussed.