Edith Suss-Toby is thanked for her assistance in the time-lapse microscopy and image analysis. Nofar Rada is thanked for the artistic work.

四到六周大的雌性C57BL / CJ小鼠（Harlan，耶路撒冷，以色列）在实验中根据协会评估和实验动物管理规范评审使用。所有实验程序均按照机构动物护理和使用委员会和农业法规部完成。 1.收获脊髓<ol><li>安乐死使用CO 2室中的小鼠。避免颈椎脱位，因为它可能会导致因剪切力根神经节破坏。从这里开始，执行无菌条件下的所有步骤。 </li><li>浸泡动物向下，用70％的乙醇。这个步骤是用于杀菌和防止头发从通过内器官拖动重要。 </li><li>离开鼠标乙醇干燥。防止乙醇与因神经毒性的内脏有任何接触。 </li><li>干燥后鼠标，在俯卧位使用引脚放置它（面朝下）。将每个后肢和前肢的针脚。 </li><li>使用镊子，使从后颈部到腰椎craniocaudally延伸的中线切口。其次，提高真皮皮瓣两侧使用镊子，揭露皮下组织。 </li><li>触诊鼠标头骨直至到达颅颈交界区。使用垂直于动物通过颈椎部肌肉和脊椎在颅颈交界区（ 第 7颈椎）切剪刀，采用重钳。尾部解剖脊椎和消费重钳脊柱直到达到腰椎水平（后肢级）。 </li><li>在使用重型钳腰椎水平（ 第 5腰椎）执行一个完整的尾部横切。 </li><li>将鼠标移到（面朝上）。没有必要覆盖切口自DRG选自低级椎骨水平抽取，而不是颈椎。 </li><li>从颈部到腹部正中线砍掉，收回皮肤侧向然后切打开腹膜。 </li><li> 整块除去腹膜内的内部器官（肝，脾，胰，胃和肠）。接下来，取出腹膜后脏器（ 如肾脏和胰腺）。颅，打开胸壁。 </li><li>使用镊子和手术刀片，切开肋，留下肋5毫米从脊柱双边远。在这一点上，脊柱是从身体的其余部分隔开。 </li><li>用冷的（4℃）新鲜磷酸盐缓冲盐水（PBS）洗两次，从血液中的脊柱。 </li></ol> 2.隔离背根神经节（DRG） <ol><li>使用具有放大4倍立体显微镜。 </li><li>上的非贴壁非吸收性平台（Telfa /尼龙）脊椎的位置。脊柱面朝上在同一头尾定向位置。 </li><li>卸下所有脊柱的肌肉和结缔组织。使用肋骨作为神经，因为他们具有里程碑意义离开脊椎。排骨也保护神经从外科手术工具。背根神经节位于颈椎，胸椎，腰椎的椎骨的位点。 </li><li>接着，使用剪刀切割椎体在中线和椎体的温和横向缩回暴露脊髓和背根神经节的根。 </li><li>在肋间，按照周围神经内侧沿肋部横向到DRG。 </li><li>识别DRG。它看起来像&#39;荷包蛋&#39;躺在神经的蛋黄。 </li><li>切肋间神经远离DRG离去2-3毫米神经远端的向神经节，以用于回缩。这个"尾巴"，将收获之后去除。 </li><li>使用镊子抓住神经。捏DRG体本身会引起神经细胞损伤和应当避免。 </li><li>接近DRG沿其附着到脊髓近侧，通过其前部和后部的根。 </li><li>通过拉动EF适用于温和回缩到DRG同的神经（肋间神经断端）横向，并切断前部和后部根部靠近DRG。 </li><li>保持DRG在35毫米的培养皿中装有补充有10％胎牛血清（FCS）的新鲜，冰冷的DMEM，1％青霉素 - 链霉素，1％非必需氨基酸和1％丙酮酸钠。 </li></ol> 3.植入培养皿<ol><li>使用预冷枪头，以防止ECM凝固执行冰下一个步骤。 </li><li>根据在2-4X放大立体显微镜，放在一个纸格35毫米的玻璃底培养皿。 重要提示:为了保持ECM液态冰的工作。在网格的中心创建生长因子耗尽的ECM的1mm直径的点（约20微升）。 </li><li>在直视下，将DRG在ECM点靠近盘底部的中心。 注:本协议所使用的癌细胞的小鼠胰腺癌细胞（KPC）establis如其他地方19说明从小鼠KPC新鲜分离的肿瘤标本建置。 </li><li>收获40000癌细胞融合的培养，用PBS洗他们一次，并在冰40微升ECM重悬。 </li><li>下，使用立体显微镜量度500微米在从DRG每个方向上的网格的直接可视化。此时慢慢种子10,000个细胞/ 10微升的ECM。使用2-10微升预冷尖端注入靠近培养皿底部的细胞，从而避免在基质的基质或脱离其蔓延。 （ 图1）。 </li><li>发表层流罩内的菜10-15分钟固化。避免ECM干燥。 </li><li>慢慢加入DMEM，如前所述制备，对板的侧壁。添加足够的介质，以覆盖的ECM（约2毫升）中。 </li><li>把盘返回到培养箱中于37℃。 </li><li>替换于次日新鲜培养基中。 </li><li>更换培养基，每2天。&lt;/ LI&gt; </ol> 4.数据采集，定时电视显微镜<ol><li>在植入后第7天，采取时间的推移显微镜菜。请注意，某些细胞可能会更早需要获取由于更快的迁移。 </li><li>在实时成像，放置在一个封闭环境中的培养皿，在37±0.1℃和5％的CO 2的平均温度。 </li><li>通过使用放置在显微镜电荷耦合器件照相机记录的细胞。 </li><li>取数字化图象，每10分钟达72小时，从三个到六个不同的细胞遵循细胞运动。 </li><li> 24小时后更换培养基。 </li><li>进行数据采集和使用显微镜软件简单的分析。 </li></ol> 5.数据分析<ol><li>对于更高级的应用程序（ 即 ，在二维或三维细胞跟踪）使用专门的软件（如了Imaris 4D）。注意:该软件在决定了小区的XY坐标任何给定的时间，用自原点运动的距离和速度沿。 </li><li>通过测量在10分钟的间隔后续位置之间的行驶距离计算平均流速。计算前向迁移指数（FMI），它是细胞前从突起的距离，并描述朝向DRG细胞的单向运动。 </li></ol>

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This research was supported by the ICRF Barbara S. Goodman endowed research career development award (2011-601-BGPC) and a grant from the US-Israel Binational Science Foundation. No other financial disclosures from the authors are to be made.

本文给出了一个概括的神经利基癌微环境中，DRG模型的体外模型。视频演示从识别解剖标志诸如在小鼠的背根神经节，其提取，最后，它在细胞外基质培养开始的所有步骤。共培养与癌细胞的DRG旁边还提出。有制作用于体外研究神经周围生态位微此模型必需在文献中描述没有其他模型体外神经浸润研究。 在视频中介绍的协议有两个关键步骤。首先，抓邻?...

直接浸润，淋巴扩散和传播血源性:固体肿瘤的三种主要方式传播。然而，有癌症扩散的第四手段是经常忽视，传播沿着神经。癌神经侵袭（CNI）是癌症扩散的一个公知的途径，尤其是在头部和颈部，1前列腺2，第3和胰腺的癌症。4-8 CNI与胰腺癌的个体的80％以上发生，导致到腹膜后肿瘤，通过腹腔神经节的神经传播。这些神经营养癌细胞具有沿着朝向中枢神经系统（CNS）的神经单向迁移的独特能力。9这一发现表明，在神经周围的微环境可以由癌细胞被利用，提供支持恶性生长因子。 为数不多的体外模型CNI研究的是背根神经节（DRG）/肿瘤细胞模型。这个mod埃尔经常用于研究神经基质和癌细胞10-18之间。旁分泌相互作用在该模型中，小鼠或人肿瘤细胞株中相邻新鲜分离培养的DRG的制剂外基质（ECM）中生长。 此视频介绍了在体外CNI在胰腺导管腺癌中的应用。

<table><tbody><tr><td>&lt;strong&gt;Equipment:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Operating microscope</td><td>Leica</td><td>M205</td><td></td></tr><tr><td>Time Lapse System</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Forceps</td><td>Sigma-Aldrich</td><td>F4142&amp;nbsp;</td><td></td></tr><tr><td>Surgical blade</td><td>Sigma-Aldrich</td><td>Z309036</td><td></td></tr><tr><td>Scissors</td><td>Sigma-Aldrich</td><td>S3271</td><td></td></tr><tr><td>35 mm Petri dishes, glass bottom</td><td>de groot</td><td>60-627860</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Materials:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>70% ethanol</td><td>Sigma</td><td></td><td></td></tr><tr><td>Cold PBS</td><td>Biological industries</td><td>02-023-1A</td><td></td></tr><tr><td>DMEM</td><td>Biological industries</td><td>01-055-1A</td><td></td></tr><tr><td>FCS</td><td>Rhenium</td><td>10108165</td><td></td></tr><tr><td>Penicillin and streptomycin</td><td>Biological industries</td><td>01-031-1B</td><td></td></tr><tr><td>Sodium Pyruvate</td><td>Biological industries</td><td>03-042-1B</td><td></td></tr><tr><td>L-Glutamine</td><td>Biological industries</td><td>03-020-1B</td><td></td></tr><tr><td>Growth factor-depleted matrigel</td><td>Trevigen</td><td>3433-005-01</td><td></td></tr></tbody></table>

in vitro modeling of cancerous neural invasion: the dorsal root ganglion model

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These neurotropic cancer cells have a unique ability to migrate unidirectionally along nerves towards the central nervous system (CNS). The dorsal root ganglia (DRG)/cancer cell model is a three dimensional (3D) in vitro model frequently used for studying the interaction between neural stroma and cancer cells. In this model, mouse or human cancer cell lines are grown in ECM adjacent to preparations of freshly dissociated cultured DRG. In this article, the DRG isolation protocol from mice, and implantation in petri dishes for co-culturing with pancreatic cancer cells are demonstrated. Five days after implantation, the cancer cells made contact with the DRG neurites. Later, these cells formed bridgeheads to facilitate more extensive polarized, neurotropic migration of cancer cells.

<html>使用视频显微成像，在DRG可以看出植入后轴突发芽5-7天，而癌细胞他们对DRG殖民地迁移了。通过在植入后第 7天，肿瘤细胞来与所述突起（ 图2）接触。 在协议中使用的胰腺癌细胞的前向迁移指数为3-4倍比其它细胞系（QLL2，B16F）（ 图3a）更高。 图3b给出一个代表X和Y坐标图，?...

Watch this Scientific Journal Video about In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model at JoVE.com

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

This video article shows the use of the dorsal root ganglia (DRG)/cancer cell model in pancreatic ductal adenocarcinoma.

在癌神经侵袭的体外模型:背根神经节型号

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These ...

in-vitro-modeling-cancerous-neural-invasion-dorsal-root-ganglion

Research

JoVE Journal

Neuroscience

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

9.0K 次观看.  Rambam Medical Healthcare Campus. This video article shows the use of the dorsal root ganglia (DRG)/cancer cell model in pancreatic ductal adenocarcinoma.

视频: In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

A Model for Perineural Invasion in Head and Neck Squamous Cell Carcinoma

Perineural invasion (PNI) is found in approximately 40% of head and neck squamous cell carcinomas (HNSCC). Despite multimodal treatment with surgery, radiation, and chemotherapy, locoregional recurrences and distant metastases occur at higher rates, and overall survival is decreased by 40% compared to HNSCC without PNI. In vitro studies of the pathways involved in HNSCC PNI have historically been challenging given the lack of a consistent, reproducible assay. Described here is the adaptation of the dorsal root ganglion (DRG) assay for the examination of PNI in HNSCC. In this model, DRG are harvested from the spinal column of a sacrificed nude mouse and placed within a semisolid matrix. Over the subsequent days, neurites are generated and grow in a radial pattern from the cell bodies of the DRG. HNSCC cell lines are then placed peripherally around the matrix and invade preferentially along the neurites toward the DRG. This method allows for rapid evaluation of multiple treatment conditions, with very high assay success rates and reproducibility.

Perineural invasion (PNI) is a common feature of head and neck squamous cell carcinoma (HNSCC), conferring lower survival rates. Its mechanisms are poorly understood. Utilizing neurites generated from murine dorsal root ganglia confined to a semisolid matrix, the pathways involved in the PNI of HNSCC cell lines can be investigated.

在头颈部鳞状细胞癌的神经浸润模型

Perineural invasion (PNI) is found in approximately 40% of head and neck squamous cell carcinomas (HNSCC). Despite multimodal treatment with surgery, ...

科研

癌症研究

An In Vivo Murine Sciatic Nerve Model of Perineural Invasion

Cancer cells invade nerves through a process termed perineural invasion (PNI), in which cancer cells proliferate and migrate in the nerve microenvironment. This type of invasion is exhibited by a variety of cancer types, and very frequently is found in pancreatic cancer. The microscopic size of nerve fibers within mouse pancreas renders the study of PNI difficult in orthotopic murine models. Here, we describe a heterotopic in vivo model of PNI, where we inject syngeneic pancreatic cancer cell line Panc02-H7 into the murine sciatic nerve. In this model, sciatic nerves of anesthetized mice are exposed and injected with cancer cells. The cancer cells invade in the nerves proximally toward the spinal cord from the point of injection. The invaded sciatic nerves are then extracted and processed with OCT for frozen sectioning. H&amp;E and immunofluorescence staining of these sections allow quantification of both the degree of invasion and changes in protein expression. This model can be applied to a variety of studies on PNI given its versatility. Using mice with different genetic modifications and/or different types of cancer cells allows for investigation of the cellular and molecular mechanisms of PNI and for different cancer types. Furthermore, the effects of therapeutic agents on nerve invasion can be studied by applying treatment to these mice.

An In Vivo Murine Sciatic Nerve Model of Perineural Invasion

We describe an in vivo murine model of perineural invasion by injecting syngeneic pancreatic cancer cells into the sciatic nerve. The model allows for quantification of the extent of nerve invasion, and supports investigation of the cellular and molecular mechanisms of perineural invasion.

神经入侵的体内小鼠坐骨神经模型

Cancer cells invade nerves through a process termed perineural invasion (PNI), in which cancer cells proliferate and migrate in the nerve ...

Dorsal Root Ganglion Injection and Dorsal Root Crush Injury as a Model for Sensory Axon Regeneration

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each other anatomically, it is important to identify an appropriate model to use for the study of axon regeneration. By using a suitable model, we can formulate a specific treatment based on the severity of injury, the neuronal cell type of interest, and the desired spinal tract for assessing regeneration. Within the sensory pathway, DRG neurons are responsible for relaying sensory information from the periphery to the CNS. We present here a protocol that uses a DRG injection with a viral vector and a concurrent dorsal root crush injury in the lower cervical spinal cord of an adult rat as a model to study sensory axon regeneration. As demonstrated using a control virus, AAV5-GFP, we show the effectiveness of a direct DRG injection in transducing DRG neurons and tracing sensory axons into the spinal cord. We also show the effectiveness of the dorsal root crush injury in denervating the forepaw as an injury model for evaluating axon regeneration. Despite the requirement for specialized training to perform this invasive surgical procedure, the protocol is flexible, and potential users can modify many parts to accommodate their experimental requirements. Importantly, it can serve as a foundation for those in search of a suitable animal model for their studies. We believe that this article will help new users to learn the procedure in a very efficient and effective manner.

This protocol presents the use of a dorsal root ganglion (DRG) injection with a viral vector and a concurrent dorsal root crush injury in an adult rat as a model to study sensory axon regeneration. This model is suitable for investigating the use of gene therapy to promote sensory axon regeneration.

背根神经节内注射和背根挤压伤为感官轴突再生模型

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each ...

神经科学

The Chick Chorioallantoic Membrane In Vivo Model to Assess Perineural Invasion in Head and Neck Cancer

Perineural invasion is a phenotype in which cancer surrounds or invades the nerves. It is associated with poor clinical outcome for head and neck squamous cell carcinoma and other cancers. Mechanistic studies have shown that the molecular crosstalk between nerves and tumor cells occurs prior to physical interaction. There are only a few in vivo models to study perineural invasion, especially to investigate early progression, before physical nerve-tumor interactions occur. The chick chorioallantoic membrane model has been used to study cancer invasion, because the basement membrane of the chorionic epithelium mimics that of human epithelial tissue. Here we repurposed the chick chorioallantoic membrane model to investigate perineural invasion, grafting rat dorsal root ganglia and human head and neck squamous cell carcinoma cells onto the chorionic epithelium. We have demonstrated how this model can be useful to evaluate the ability of cancer cells to invade neural tissue in vivo.&#160;

The Chick Chorioallantoic Membrane In Vivo Model to Assess Perineural Invasion in Head and Neck Cancer

Perineural invasion is an aggressive phenotype for head and neck squamous cell carcinomas and other tumors. The chick chorioallantoic membrane model has been used for studying angiogenesis, cancer invasion, and metastasis. Here we demonstrate how this model can be utilized to assess perineural invasion in vivo.

Vivo模型中的小鸡胆膜模型,用于评估头颈部癌症的围神经入侵

Perineural invasion is a phenotype in which cancer surrounds or invades the nerves. It is associated with poor clinical outcome for head and neck squamous ...

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 ...

生物工程

Establishing a Three-dimensional Co-culture Model of Murine Dorsal Root Ganglia and Cancer Cells: An In Vitro Model to Study Perineural Cancer Cell Invasion

Source: Huyett, P. et al. <a href="https://www.jove.com/video/55043" target="_blank">A Model for Perineural Invasion in Head and Neck Squamous Cell Carcinoma.</a> J. Vis. Exp. (2017)
In this video, we demonstrate the procedure for developing an in vitro perineural cancer cell invasion model by co-culturing mouse dorsal root ganglion and neurotropic cancer cells. The model can be utilized to conduct cancer assays, producing rapid and reproducible results.

建立小鼠背根神经节和癌细胞的三维共培养模型:研究神经周围癌细胞侵袭的体外模型

Source: Huyett, P. et al. A Model for Perineural Invasion in Head and Neck Squamous Cell Carcinoma. J. Vis. Exp. (2017)
In this video, we demonstrate the ...

JoVE Encyclopedia of Experiments

Head and Neck Cancer

Assays & techniques

Murine Perineural Invasion Model: Generating an In Vivo Model to Simulate Perineural Invasion in Mouse Sciatic Nerve

Source: Deborde, S. et al. <a href="https://www.jove.com/video/56857" target="_blank">An In Vivo Murine Sciatic Nerve Model of Perineural Invasion.</a> J. Vis. Exp. (2018)
In this video, we develop an in vivo mouse model for perineural invasion by injecting pancreatic cancer cells into the sciatic nerve. This model helps quantify the extent of invasion and study the interactions between cancer cells and nerves.

小鼠神经周围浸润模型:生成体内模型以模拟小鼠坐骨神经中的神经周围浸润

Source: Deborde, S. et al. An In Vivo Murine Sciatic Nerve Model of Perineural Invasion. J. Vis. Exp. (2018)
In this video, we develop an in vivo mouse ...

Cancers of the Nervous System

In vivo studies

Modeling Cancerous Neural Invasion In Vitro: A Method to Co-culture Dorsal Root Ganglia and Cancer Cells

Source: Na&#39;ara, S. et al. <a href="https://www.jove.com/video/52990" target="_blank">In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model.</a> J. Vis. Exp. (2016)
In this video, we demonstrate an in vitro method for co-culturing DRG and cancer cells to study neurotropism and neural invasion.

体外癌性神经侵袭建模:一种共培养背根神经节和癌细胞的方法

Source: Na&#39;ara, S. et al. In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model. J. Vis. Exp. (2016)
In this video, we ...

在癌神经侵袭的体外模型:背根神经节型号

摘要

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此视频中的章节

在头颈部鳞状细胞癌的神经浸润模型

神经入侵的体内小鼠坐骨神经模型

背根神经节内注射和背根挤压伤为感官轴突再生模型

Vivo模型中的小鸡胆膜模型,用于评估头颈部癌症的围神经入侵

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

建立小鼠背根神经节和癌细胞的三维共培养模型:研究神经周围癌细胞侵袭的体外模型

小鼠神经周围浸润模型:生成体内模型以模拟小鼠坐骨神经中的神经周围浸润

体外癌性神经侵袭建模:一种共培养背根神经节和癌细胞的方法