在实验室的工作是由NSF（MCB0919933）和国立卫生研究院（GM088790）的支持。

1。中继片段再生实验<ol><li>再生实验前至少5天停喂的蠕虫病毒的队列，以确保动物废物和摄取食物（30〜完整的蠕虫，每个蠕虫〜8-10毫米长的饥饿后）的。 </li><li>在检测的一天，冲洗与水预先冷冻的平整的冰盘，并用保鲜膜覆盖平的冰面。使用镊子，一个滤纸上的保鲜膜。 </li><li>几滴泉水浸湿的滤纸。转移到过滤器使用的移液管（&lt;20％过滤蠕虫）涡虫。蠕虫过滤器，如果有必要可以重新定位，通过与更多的泉水repipetting。删除多余的液体。 </li><li>使用手术刀，截肢动物的前尖，咽（ 图1A）前结束之间约中途一个单切头。 </li><li>使用手术刀，截去尾部区域，由一个单一的削减约中间动物的尾部和后咽（ 图 1A）结束。删除多余的粘液，用手术刀切割后蘸用70％乙醇的纸巾。用手术刀多余的乙醇。 </li><li>转移到培养皿中（100 x 25毫米深）通过含有泉水钳过滤器。等待〜3分钟（伤口缝合，观察一个捏和伤口变黑）。 </li><li>冲洗干净，滤纸的Petri菜含有所需再生法和转移到恒温培养箱（24℃）介质的主干片段。 </li><li>分数再生表型〜1周后，当再生结构识别（ 图 1A） 。 </li></ol> 2。吡喹酮诱导再生:两极药理操纵<ol><li>准备溶药物在DMSO（62.4mg 1ML PZQ 200MM股票）吡喹酮（PZQ）的股票。当天使用。 </li><li>设为50毫升含药溶液中缓冲Montjuch盐（PZQ 90微米）。涡〜1分钟，以确保分散。 </li><li>执行主干片段[＃1]，露出的树干碎片队列的最后一步[1.7] PZQ协议的详细以上的再生法。 </li><li> 24-48小时后，交换PZQ含有温泉水的媒体，洗衣机蠕虫（&gt; 3）次。 </li><li>返回蠕虫孵化器。分数两极（两个头， 图1B）切割后的一周。 </li></ol> 3。再生的基因操作:在体内RNAi的喂养协议<ol><li>检测前，准备鸡肝匀浆。丢弃从foodstock脂肪，黄颜色的部分，菜泥其余肝。过滤菜泥通过滤网和存储等分悬浮液（1.5ml离心管，-20℃）。 </li><li>检测之前，准备aliquoting到1ML样品供应，储存于-20 ° C牛的红血细胞（红细胞） </li><li>选择用于RNAi的蠕虫病毒。三个同伙:感兴趣的基因，阳性对照组（基因与已知的RNA干扰表型;图1C，D和 E）和负控制（无RNAi的表型的基因，也有用的评估击倒水平相比，实验队列）。对于每个队列，使用〜250的蠕虫（〜8-10毫米长至少5天的饥饿后）。存放在塑料桶泉水。 </li><li>对于每个RNAi的队列中，大肠杆菌表达dsRNA的目标感兴趣的基因大肠杆菌解冻股票[8]，随着鸡肝匀浆管[3.1]和红细胞管[3.2]。 </li><li>佩莱细菌（13000克，1分钟）。移除上清液2xYT媒体（〜700μl）和重新悬浮颗粒。 Recentrifuge，在冰上，丢弃上清液，沉淀的地方。 </li><li>创建大缸径P200的枪头，切断提示结束。在鸡肝匀浆（150μL）和红细胞（50μL）的混合物创建RNAi的喂养混合彻底重悬细菌沉淀。取出离心中的气泡。 </li><li>觅食，删除包含蠕虫的塑料浴盆的水多数（离开〜1英寸深入）。涡流浴缸集中在这个容器中的中心的蠕虫，和吸管RNAi在一个网罗蠕虫圈饲养的组合。请使用移液管，仔细同轴电缆上的RNAi喂养混合逃出，没有其他食客的干扰！ </li><li>喂奶后（约1小时），确定，美联储以及涡虫。这些蠕虫从摄入的红细胞深红色着色。丢弃他人。 </li><li>小心地更换新鲜的泉水喂养的解决方案，最大限度地减少干扰，防止食物的排泄。要做到这一点，轻轻地本地化蠕虫使用移液管的管的一侧，倒入水混浊，并仔细地用清水取代倾盆而下的浴缸对面的墙上。长时间暴露在空气Resubmerge涡虫很快也会导致食物排泄。 </li><li>松散SE铝容器的盖子，并返回到培养箱（24℃） </li><li>重复RNAi在多个周期中喂养协议（〜2-3天），穿插协议＃1]屏幕RNAi的表型再生周期。许多基因的有效供料和再生的一个标准协议显示在图2〜1个月，总工期。 </li></ol>修改这个计划，针对不同的基因作为最佳的协议将取决于mRNA的稳定性，组织定位，蛋白质perdurance，或排除多个喂养周期后再生一个表型的发展等因素。评估只是表型的显性筛选（明显），或定量PCR方法，有针对性的mRNA水平与阴性对照队列击倒水平。 4。代表性的成果: 主干片段再生实验是健壮的所有蠕虫应重新正常前后（"头到尾"）极性。这可以切割后的5天，前眼点（asterixed图1A）的出现，促成尽快拿下。然而，一周后完成恢复失去结构的形态发生。药理操纵主干片段再生PZQ产量双头蠕虫（ 图1B）也很强大（欧共体50 = 87（+ - ）11％的双极片段，70μM为48 小时 20 PZQ） 。两极发生在一个单一的再生周期。下在这些实验的疗效可能与药物的溶解度和/或存储，或使用不同的扁虫种PZQ是无效的问题。对于外显率较 ​​低的药物，通常用于anteriorization指数得分从 22完整的两极的中间表型搁置。表型从积极的控制结构的RNAi， 如图1所示:RNAi 的日本三角涡虫6 - 1（D J六个- 1）的结果，在无眼表型23（ 图 1C），日本三角涡虫的PC2的RNAi（DJ - PC2）在损失厌恶的光响应24（ 图1D）和RNAi的结果， 日本三角涡虫βcatenin- 1（βcateninDJ - 1）的结果在两个为首的表型16-19日，从主干片段（ 图1E）21。这些表型，可以实现用下面这个简单的RNA干扰协议:DJ -六（FFxFx），DJ - PC2（FFFx），DJ -βcatenin- 1（FFxFx），其中F =饲养周期和x =分别再生周期。 <img src="/files/ftp_upload/3058/3058fig1.jpg" alt="图1"/> 图1:主干片段再生检测涡虫（上）和原理图（中） 左 ，形象地展现切除主干片段的位置。右键，躯干片段再生timecourse显示外观再生片段在指定的时间（天）乙PZQ曝光产生。双头涡虫的图像C"无眼"DJ -六- 1 RNA干扰，D动的DJ - PC2的RNAi蠕虫（染成红色）轻厌恶反应，亏损相比，移动控制蠕虫（染色产生的蠕虫绿色），E双极型涡虫的DJ -βcatenin1的RNAi 。在Be，原来的RNAi蠕虫前到底是面向左侧。 <img src="/files/ftp_upload/3058/3058fig2.jpg" alt="图2"/> 图2:典型RNAi的协议包括多个喂养（F）和再生周期（X）是有效的 D.许多基因敲除喂养和切割周期序列粳稻 。本议定书的个体基因可能需要修改，​​以产生最佳的结果，使用例如一个较少（括号）或更大数量的喂养和再生周期。

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没有利益冲突的声明。

这里描述协议详细分析，研究和操纵涡虫三角涡虫再生的粳稻。他们很简单，不需要专用设备等，他们可以很容易地在实验室或教室进行。分析可以单独进行，或合并化学药物药效在体内的基因筛查，可以在候选基因水平上进行，或适应无偏见的，高通量筛选 8 。无论是耐人寻味的涡虫的生物学研究自己的权利，或在体内功能在哺乳动物的同源性研究组织再生的替代模型的评估，这些方法应推动多元化的研究人员的兴趣。

<table border="1"><tbody><tr><th>试剂</th><th>供应商</th><th>目录编号</th><th>评论</th></tr><tr><td>泉水</td><td> Kandiyohi。高级沃特公司明尼苏达州明尼阿波利斯</td><td> N / A </td><td>其他形式的泉水也很好。审判第一的可行性分析。 </td></tr><tr><td> 1 ×缓冲蒙特伊克（Montjuich）盐:氯化钠（1.6毫米），氯化钙（1MM），硫酸镁（1MM），氯化镁（0.1MM），氯化钾（0.1毫米），碳酸氢钠（1.2MM），HEPES（1.5）。 pH值7.4在24 ° C。 </td><td>多个供应商</td><td> N / A </td><td>人工水再生实验期间的药物治疗，以确保pH缓冲。 5 / 8 Holtfreter的解决方案是一个另类。 </td></tr><tr><td> 2xYT肉汤</td><td> Fisher Scientific则</td><td> BP2467 - 500 </td><td>媒体= 31 g / L的。高压灭菌。 </td></tr><tr><td>培养皿（100x25mm） </td><td> Fisher Scientific则</td><td> 08-757-11 </td><td>在再生周期的房屋蠕虫</td></tr><tr><td>广场菜（100x100x15mm） </td><td> Fisher Scientific则</td><td> 08 - 757 - 11A </td><td>与水，冻结填补蠕虫切割面的冰盘</td></tr><tr><td>塑料桶:密保诺拉链式扭&#39;N LOC（16盎司）。 </td><td>各种零售商</td><td> N / A </td><td>便民水密封容器内的RNAi同伙</td></tr><tr><td>鸡肝</td><td>商业杂货店</td><td> N / A </td><td>偏见，对有机用品，由于避免抗生素。 </td></tr><tr><td>手搅拌机</td><td>任何厨房供应商</td><td> N / A </td><td>使用鸡肝脏原浆</td></tr><tr><td>电线1毫米滤网</td><td>任何厨房供应商</td><td> N / A </td><td>使用使劲鸡肝脏原浆</td></tr><tr><td>牛红细胞</td><td> Lampire生物实验室</td><td> 7240807 </td><td>冲到100％，并汇集了红细胞悬浮液</td></tr><tr><td>圆形滤纸</td><td>的Whatman＃3 </td><td> 1003 055 </td><td></td></tr><tr><td>移液管</td><td> Fisher Scientific则</td><td> 13-711-41 </td><td></td></tr><tr><td>无菌，手术刀</td><td>多个供应商</td><td> N / A </td><td></td></tr><tr><td> ±吡喹酮</td><td>西格玛爱秩序</td><td> P4668 </td><td>在黑暗中商店粉末分装在4 ° C变干。 </td></tr><tr><td> DJ -六1 </td><td></td><td> GenBank中AJ557022.1 </td><td> RNAi的"眼"23表型阳性对照</td></tr><tr><td> DJ -βcatenin- 1 </td><td></td><td> GenBank中AB181913.1 </td><td> RNAi的阳性对照两个为首的表型 17 </td></tr><tr><td> DJ - PC2 </td><td></td><td></td><td> RNAi的阳性对照光厌恶表型24损失</td></tr></tbody></table>

pharmacological and functional genetic assays to manipulate regeneration of the planarian dugesia japonica

自由生活涡虫扁虫有一个历史悠久的实验使用，因为他们非凡的再生能力 1 。从这些动物切除的小片断，改革原体计划，缺少的车身结构的再生。例如，如果一个"主干"的片段是从一个完整的蠕虫切割，一个新的"头"将再生前方一个"尾巴"后方将重新恢复了原有的"头部到尾部的车身结构的极性截肢前（图1A）。 涡虫被称为"neoblasts&#39;〜30种不同类型的细胞分化，在正常的身体动态平衡和执行组织再生的干细胞，是由再生。这种再生过程是可靠和容易证明。由于几个创业实验室，许多工具和功能基因的方法的奉献精神，现在已经为这个模型系统进行了优化。因此，相当最近已经取得了进展，在理解和操作基础 2-9涡虫的发育可塑性的分子事件。 涡模型系统将会以一个科学家的广泛利益。神经学家，模型给予机会研究整个神经系统的再生，而不是简单的再生/修复单个神经细胞的过程中，通常是在许多已建立的模型研究的重点。涡虫表达的神经递质10过多，代表的一项重要制度，为研究中枢神经系统的11，12的演变，行为甄别潜在的13，14 。 再生的结果是经得起药理和遗传apparoaches操纵。例如，可以进行筛选药物对再生的影响，只需放置在身体片段的药物含截肢后在不同时间点的解决方案。使用击倒方法（ 在体内的RNAi），这可以实现，通过显微注射周期或喂食细菌表达双链RNA的结构8，9，15，可以研究单个基因的作用。这两种方法都可以产生高外显率，例如，双极动物16-21再生的视觉冲击的表型。为了便于采用这种模式和实施这种方法，我们将展示使用涡虫日本三角涡虫的药理学和基因检测（在体内RNAi技术通过喂食）的视频在这条协议。

Watch this Scientific Journal Video about Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica at JoVE.com

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

在一个活的动物研究干细胞分化的一个有吸引力的模式是涡虫扁虫。再生是由简单的截肢手术，很容易在一个基本的实验室进行的实验研究和药理学和基因（适合<em&gt;在体内</em&gt; RNAi）的操纵，在这篇文章中的协议详细。

药理和功能的遗传分析处理涡虫的再生日本三角涡虫</em

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from ...

pharmacological-functional-genetic-assays-to-manipulate-regeneration

Nanyang Technological University

Research

JoVE Journal

Biology

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

36.8K 次观看.  University of Minnesota Medical School. 在一个活的动物研究干细胞分化的一个有吸引力的模式是涡虫扁虫。再生是由简单的截肢手术，很容易在一个基本的实验室进行的实验研究和药理学和基因（适合在体内 RNAi）的操纵，在这篇文章中的协议详细。

视频: Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson's Disease Genes

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations at risk. In the process of attempting to identify novel genetic factors associated with PD, scientists have generated many lists of candidate genes, polymorphisms, and proteins that represent important advances, but these leads remain mechanistically undefined. Our work is aimed toward significantly narrowing such lists by exploiting the advantages of a simple animal model system. While humans have billions of neurons, the microscopic roundworm Caenorhabditis elegans has precisely 302, of which only eight produce dopamine (DA) in hemaphrodites. Expression of a human gene encoding the PD-associated protein, alpha-synuclein, in C. elegans DA neurons results in dosage and age-dependent neurodegeneration.
Worms expressing human alpha-synuclein in DA neurons are isogenic and express both GFP and human alpha-synuclein under the DA transporter promoter (Pdat-1). The presence of GFP serves as a readily visualized marker for following DA neurodegeneration in these animals. We initially demonstrated that alpha-synuclein-induced DA neurodegeneration could be rescued in these animals by torsinA, a protein with molecular chaperone activity 1. Further, candidate PD-related genes identified in our lab via large-scale RNAi screening efforts using an alpha-synuclein misfolding assay were then over-expressed in C. elegans DA neurons. We determined that five of seven genes tested represented significant candidate modulators of PD as they rescued alpha-synuclein-induced DA neurodegeneration 2. Additionally, the Lindquist Lab (this issue of JoVE) has performed yeast screens whereby alpha-synuclein-dependent toxicity is used as a readout for genes that can enhance or suppress cytotoxicity. We subsequently examined the yeast candidate genes in our C. elegans alpha-synuclein-induced neurodegeneration assay and successfully validated many of these targets 3, 4.
Our methodology involves generation of a C. elegans DA neuron-specific expression vector using recombinational cloning of candidate gene cDNAs under control of the Pdat-1 promoter. These plasmids are then microinjected in wild-type (N2) worms, along with a selectable marker for successful transformation. Multiple stable transgenic lines producing the candidate protein in DA neurons are obtained and then independently crossed into the alpha-synuclein degenerative strain and assessed for neurodegeneration, at both the animal and individual neuron level, over the course of aging.

This video demonstrates how to use C. elegans to assess dopaminergic neuron neurodegeneration as a model for Parkinson's disease. Furthermore, genetic screens are used to identify factors that either enhance degeneration or are neuroprotective.

一个线虫多巴胺神经元退化含量的应用验证潜在的帕金森氏症基因

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations ...

科研

生物学

Surgical Ablation Assay for Studying Eye Regeneration in Planarians

In the study of adult stem cells and regenerative mechanisms, planarian flatworms are a staple in vivo model system. This is due in large part to their abundant pluripotent stem cell population and ability to regenerate all cell and tissue types after injuries that would be catastrophic for most animals. Recently, planarians have gained popularity as a model for eye regeneration. Their ability to regenerate the entire eye (comprised of two tissue types: pigment cells and photoreceptors) allows for the dissection of the mechanisms regulating visual system regeneration. Eye ablation has several advantages over other techniques (such as decapitation or hole punch) for examining eye-specific pathways and mechanisms, the most important of which is that regeneration is largely restricted to eye tissues alone. The purpose of this video article is to demonstrate how to reliably remove the planarian optic cup without disturbing the brain or surrounding tissues. The handling of worms and maintenance of an established colony is also described. This technique uses a 31 G, 5/16-inch insulin needle to surgically scoop out the optic cup of planarians immobilized on a cold plate. This method encompasses both single and double eye ablation, with eyes regenerating within 1-2 weeks, allowing for a wide range of applications. In particular, this ablation technique can be easily combined with pharmacological and genetic (RNA interference) screens for a better understanding of regenerative mechanisms and their evolution, eye stem cells and their maintenance, and phototaxic behavioral responses and their neurological basis.

This protocol shows how to consistently excise planarian eyes (optic cups) without disturbing surrounding tissues. Using an insulin needle and syringe, either one or both eyes can be ablated to facilitate investigations into the mechanisms regulating eye regeneration, the evolution of visual regeneration, and the neural basis of light-induced behavior.

手术消融分析在研究涡虫再生眼

In the study of adult stem cells and regenerative mechanisms, planarian flatworms are a staple in vivo model system. This is due in large part to their ...

发育生物学

Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability to support axon growth and a hostile environment in the mature CNS1,2. In contrast, mature neurons in the peripheral nervous system (PNS) regenerate readily after injuries3. Adult dorsal root ganglion (DRG) neurons are well known to regenerate robustly after peripheral nerve injuries. Each DRG neuron grows one axon from the cell soma, which branches into two axonal branches: a peripheral branch innervating peripheral targets and a central branch extending into the spinal cord. Injury of the DRG peripheral axons results in substantial axon regeneration, whereas central axons in the spinal cord regenerate poorly after the injury. However, if the peripheral axonal injury occurs prior to the spinal cord injury (a process called the conditioning lesion), regeneration of central axons is greatly improved4. Moreover, the central axons of DRG neurons share the same hostile environment as descending corticospinal axons in the spinal cord. Together, it is hypothesized that the molecular mechanisms controlling axon regeneration of adult DRG neurons can be harnessed to enhance CNS axon regeneration. As a result, adult DRG neurons are now widely used as a model system to study regenerative axon growth5-7.
Here we describe a method of adult DRG neuron culture that can be used for genetic study of axon regeneration in vitro. In this model adult DRG neurons are genetically manipulated via electroporation-mediated gene transfection6,8. By transfecting neurons with DNA plasmid or si/shRNA, this approach enables both gain- and loss-of-function experiments to investigate the role of any gene-of-interest in axon growth from adult DRG neurons. When neurons are transfected with si/shRNA, the targeted endogenous protein is usually depleted after 3-4 days in culture, during which time robust axon growth has already occurred, making the loss-of-function studies less effective. To solve this problem, the method described here includes a re-suspension and re-plating step after transfection, which allows axons to re-grow from neurons in the absence of the targeted protein. Finally, we provide an example of using this in vitro model to study the role of an axon regeneration-associated gene, c-Jun, in mediating axon growth from adult DRG neurons9.

An in vitro model for genetic study of axon regeneration using cultured adult mouse dorsal root ganglion neurons is described. The method includes a re-suspension/re-plating step to allow axon re-growth from neurons undergoing genetic manipulation. This approach is especially useful for loss-of-function studies of axon regeneration using RNAi-based protein knockdown.

培养成年的背根神经节神经元轴突再生的遗传研究

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability ...

神经科学

Chemical Amputation and Regeneration of the Pharynx in the Planarian Schmidtea mediterranea

Planarians are flatworms that are extremely efficient at regeneration. They owe this ability to a large number of stem cells that can rapidly respond to any type of injury. Common injury models in these animals remove large amounts of tissue, which damages multiple organs. To overcome this broad tissue damage, we describe here a method to selectively remove a single organ, the pharynx, in the planarian Schmidtea mediterranea. We achieve this by soaking animals in a solution containing the cytochrome oxidase inhibitor sodium azide. Brief exposure to sodium azide causes extrusion of the pharynx from the animal, which we call &#34;chemical amputation.&#34;&#160;Chemical amputation removes the entire pharynx, and generates a small wound where the pharynx attaches to the intestine. After extensive rinsing, all amputated animals regenerate a fully functional pharynx in approximately one week. Stem cells in the rest of the body drive regeneration of the new pharynx. Here, we provide a detailed protocol for chemical amputation, and describe both histological and behavioral methods to assess successful amputation and regeneration.

Chemical Amputation and Regeneration of the Pharynx in the Planarian Schmidtea mediterranea

The planarian Schmidtea mediterranea is an excellent model for studying stem cells and tissue regeneration. This publication describes a method to selectively remove one organ, the pharynx, by exposing animals to the chemical sodium azide. This protocol also outlines methods for monitoring pharynx regeneration.

真涡虫的化学截肢和咽部再生Schmidtea mediterranea

Planarians are flatworms that are extremely efficient at regeneration. They owe this ability to a large number of stem cells that can rapidly respond to ...

A Planarian Motility Assay to Gauge the Biomodulating Properties of Natural Products

A straightforward, controllable means of using the non-parasitic planarian, Dugesia tigrina, a free-living aquatic flatworm, to study the stimulant and withdrawal properties of natural products is described. Experimental assays benefitting from unique aspects of planarian physiology have been applied to studies on wound healing, regeneration, and tumorigenesis. In addition, because planarians exhibit sensitivity to a variety of environmental stimuli and are capable of learning and developing conditioned responses, they can be used in behavioral studies examining learning and memory. Planarians possess a basic bilateral symmetry and a central nervous system that uses neurotransmitter systems amenable to studies examining the effects of neuromuscular biomodulators. Consequently, experimental systems monitoring planarian movement and motility have been developed to examine substance addiction and withdrawal. Because planarian motility offers the potential for a sensitive, easily standardized motility assay system to monitor the effect of stimuli, the planarian locomotor velocity (pLmV) test was adapted to monitor both stimulation and withdrawal behaviors by planarians through the determination of the number of grid lines crossed by the animals with time. Here, the technique and its application are demonstrated and explained.

Planarian motility is used to gauge the stimulant and withdrawal properties of natural products when compared to the movement of the animals in spring water alone.

测量天然产品生物调节特性的平面运动分析

A straightforward, controllable means of using the non-parasitic planarian, Dugesia tigrina, a free-living aquatic flatworm, to study the stimulant and ...

生物化学

Planarian Immobilization, Partial Irradiation, and Tissue Transplantation

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian regeneration of any missing or damaged tissues is made possible by adult stem cells termed neoblasts3. Although these stem cells have been definitively shown to be pluripotent and singularly capable of reconstituting an entire animal4, the heterogeneity within the stem cell population and the dynamics of their cellular behaviors remain largely unresolved. Due to the large number and wide distribution of stem cells throughout the planarian body plan, advanced methods for manipulating subpopulations of stem cells for molecular and functional study in vivo are needed.
Tissue transplantation and partial irradiation are two methods by which a subpopulation of planarian stem cells can be isolated for further study. Each technique has distinct advantages. Tissue transplantation allows for the introduction of stem cells, into a na&iuml;ve host, that are either inherently genetically distinct or have been previously treated pharmacologically. Alternatively, partial irradiation allows for the isolation of stem cells within a host, juxtaposed to tissue devoid of stem cells, without the introduction of a wound or any breech in tissue integrity. Using these two methods, one can investigate the cell autonomous and non-autonomous factors that control stem cell functions, such as proliferation, differentiation, and migration.
Both tissue transplantation5,6 and partial irradiation7 have been used historically in defining many of the questions about planarian regeneration that remain under study today. However, these techniques have remained underused due to the laborious and inconsistent nature of previous methods. The protocols presented here represent a large step forward in decreasing the time and effort necessary to reproducibly generate large numbers of grafted or partially irradiated animals with efficacies approaching 100 percent. We cover the culture of large animals, immobilization, preparation for partial irradiation, tissue transplantation, and the optimization of animal recovery. Furthermore, the work described here demonstrates the first application of the partial irradiation method for use with the most widely studied planarian, Schmidtea mediterranea. Additionally, efficient tissue grafting in planaria opens the door for the functional testing of subpopulations of na&iuml;ve or treated stem cells in repopulation assays, which has long been the gold-standard method of assaying adult stem cell potential in mammals8. Broad adoption of these techniques will no doubt lead to a better understanding of the cellular behaviors of adult stem cells during tissue homeostasis and regeneration.

An effective method for grafting tissue of defined and consistent size between planaria is described. Also included is a description of how the immobilization technique used for transplantation can be adapted, in conjunction with lead shields, for the partial irradiation of live animals.

涡固定，局部照射，组织移植

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian ...

A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System

The regrowth capacity of damaged neurons governs neuroregeneration and functional recovery after nervous system trauma. Over the past few decades, various intrinsic and extrinsic inhibitory factors involved in the restriction of axon regeneration have been identified. However, simply removing these inhibitory cues is insufficient for successful regeneration, indicating the existence of additional regulatory machinery. Drosophila melanogaster, the fruit fly, shares evolutionarily conserved genes and signaling pathways with vertebrates, including humans. Combining the powerful genetic toolbox of flies with two-photon laser axotomy/dendriotomy, we describe here the Drosophila sensory neuron &#8211; dendritic arborization (da) neuron injury model as a platform for systematically screening for novel regeneration regulators. Briefly, this paradigm includes a) the preparation of larvae, b) lesion induction to dendrite(s) or axon(s) using a two-photon laser, c) live confocal imaging post-injury and d) data analysis. Our model enables highly reproducible injury of single labeled neurons, axons, and dendrites of well-defined neuronal subtypes, in both the peripheral and central nervous system.

A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System

Here, we present a protocol using the Drosophila sensory neuron - dendritic arborization (da) neuron injury model, which combines in vivo live imaging, two-photon laser axotomy/dendriotomy, and the powerful fly genetic toolbox, as a platform for screening potential promoters and inhibitors of neuroregeneration.

一种研究外周和中枢神经系统 Neuroregeneration 的果蝇体内损伤模型

The regrowth capacity of damaged neurons governs neuroregeneration and functional recovery after nervous system trauma. Over the past few decades, various ...

医学

用于研究 DRG 培养中神经元超敏反应的高级体外模型

Harvesting, Embedding, and Culturing Dorsal Root Ganglia in Multi-compartment Devices to Study Peripheral Neuronal Features

The most common peripheral neuronal feature of pain is a lowered stimulation threshold or hypersensitivity of terminal nerves from the dorsal root ganglia (DRG). One proposed cause of this hypersensitivity is associated with the interaction between immune cells in the peripheral tissue and neurons. In vitro models have provided foundational knowledge in understanding how these mechanisms result in nociceptor hypersensitivity. However, in vitro models face the challenge of translating efficacy to humans. To address this challenge, a physiologically and anatomically relevant in vitro model has been developed for the culture of intact dorsal root ganglia (DRGs) in three isolated compartments in a 48-well plate. Primary DRGs are harvested from adult Sprague Dawley rats after humane euthanasia. Excess nerve roots are trimmed, and the DRG is cut into appropriate sizes for culture. DRGs are then grown in natural hydrogels, enabling robust growth in all compartments. This multi-compartment system offers anatomically relevant isolation of the DRG cell bodies from neurites, physiologically relevant cell types, and mechanical properties to study the interactions between neural and immune cells. Thus, this culture platform provides a valuable tool for investigating treatment isolation strategies, ultimately leading to an improved screening approach for predicting pain.

作者聚焦：提高慢性疼痛治疗效果的体外和体内模型

This protocol provides a technique to harvest and culture explanted dorsal root ganglion (DRG) from adult Sprague Dawley rats in a multi-compartment (MC) device.

在多室装置中收获、包埋和培养背根神经节以研究外周神经元特征

The most common peripheral neuronal feature of pain is a lowered stimulation threshold or hypersensitivity of terminal nerves from the dorsal root ganglia ...

药理和功能的遗传分析处理涡虫的再生日本三角涡虫

摘要

探索更多视频

此视频中的章节

一个线虫多巴胺神经元退化含量的应用验证潜在的帕金森氏症基因

手术消融分析在研究涡虫再生眼

培养成年的背根神经节神经元轴突再生的遗传研究

真涡虫的化学截肢和咽部再生Schmidtea mediterranea

测量天然产品生物调节特性的平面运动分析

涡固定，局部照射，组织移植

一种研究外周和中枢神经系统 Neuroregeneration 的果蝇体内损伤模型

在多室装置中收获、包埋和培养背根神经节以研究外周神经元特征

在多室装置中收获、包埋和培养背根神经节以研究外周神经元特征

在多室装置中收获、包埋和培养背根神经节以研究外周神经元特征