笔者想从磁共振成像设备在塞姆斯 - 墨菲诊所，孟菲斯，田纳西州以及神经外科部门田纳西大学健康科学中心在孟菲斯大学，田纳西感谢工作人员。

凝胶幻影1。制备及染料<ol><li>通过以1,000 ml去离子水将2克0.1％的琼脂糖粉制备0.2％琼脂糖凝胶上。搅拌约1分钟，将溶液以保证适当的混合;并立即微波解决方案，在3分钟的间隔为9分钟，或直至清晰，间隔之间搅拌。 </li><li>而琼脂糖凝胶是液体，将溶液倒入5厘米×5厘米×5厘米的容器中。允许空间在容器的顶部，以加水，并允许琼脂糖凝胶冷却并沉降。 </li><li>一旦琼脂糖凝胶凝固后（约1-2小时），添加1厘米的水凝胶与冷藏的顶部。最好是在24-48小时的混合使用的凝胶，但它可以存储多达一周冷藏10。 </li><li>在60毫升注射器由50毫升0.017％溴酚蓝染料（BPB），和钆特醇无线电造影2mM的制备无线电对比染料。 <ol><li>结合8.5 BPB染料毫克50米升去离子水来创建0.017％BPB溶液。 </li><li>加入0.2毫升股票0.5M的钆特醇容量为50毫升0.017％BPB解决方案来创建一个2毫米钆特醇溶液。 </li></ol></li></ol> 2，准备输液系统<ol><li>注射泵的输注系统（优选方法）:用于注射器泵制剂，直接连接输液导管的注射器，通过该压力传感器，减少了输液管线的死体积。注射泵的排出功能可以用于使用大丸剂大于导管以10微升/分钟的速率填充量来清除空气中的行。 </li><li>管泵输注系统（另一种方法）:连接包含无线电造影剂的输液泵注射器。附加的压力传感器，以与连接到第四显示器换能器的泵出口。附加一个地下16输液导管与压力传感器的开口端。注意:在地下16输注导管的前端有一个内直径米为0.2毫米和0.35毫米的外径。尖端是由熔融二氧化硅和小费长度为3毫米。它增加至约0.75毫米，并继续于15 mm，导管那么步骤在一个锥形时尚到1.6毫米或16 G。 </li><li>通过清除系统约15分钟，在16.667微升/分钟，以除去任何气泡制备输注。不要超过16.667μL/ min的流速，因为机器会停止输液，由于高线压力。以下所附的输注导管的线离开的输液泵，空气通过使用输注泵"大丸剂"功能吹扫管线。 </li><li>装上输液导管安装和轨迹帧到凝胶假体容器（5厘米×5厘米×5厘米），发生在MRI检查。 </li></ol> 3，CED凝胶灌注及MR扫描<ol><li>零压力值（毫米汞柱）开始输液前录得的四显示器。 </li><li>插入输液导管进入琼脂糖凝胶瓦特第i个输液泵运行在最低流速可能的，在这种情况下，1.667微升/分钟。 </li><li>开始在MR ​​扫描中，使用表1中列出的参数，并不断注入以1.667微升/分钟的速率。注入的凝胶，以恒定速率输注，直至总体积达到60微升（约38分钟）。 </li><li>连续扫描凝胶在3分钟和50秒的间隔。记录压力读数，每隔60秒。一旦注入量达到60微升，关闭输液泵;和完整的MR扫描，同时继续记录压力读数。 </li></ol> 4，MR数据分析<ol><li>为了分析的MR图像，使用适当的DICOM浏览器与ROI分割功能。 </li><li>选择正确的帧中的每个扫描标记的导管， 如图1所示的横截面。 </li><li>使用"投资回报率 - 矩形"工具，选择凝胶，不包括任何的最大部分输注部位的部分。该软件将输出与标准偏差的平均像素密度。找到对应于三个标准偏差与平均值的值。此值被用作阈值，用于确定当对比度存在99.7％的置信度。 </li><li>使用"投资回报率 - 圈"的工具，环绕输注部位有一个足够大的圆圈，这给一个唯一的名称。 </li><li>选择圆和使用"ROI - 设置像素值"的工具，则在步骤4.3中找到输入到阈值"，如果当前值大于"框中，只要选中这一行。然后在"这个新的价值:"框中，输入一个较大的值（25,000）。重置像素密度来选择前面所定义的阈值所包围的区域。 </li><li>接下来，使用"投资回报率 - 成长区（2D/3D分割）"工具，选择2D的地区，与初始半径参数= 2的信心算法，刷的投资回报率。点击输注部位的软件来计算总面积的内径Ø上本地区。 </li><li>假设一个球形输液云，计算从该区域扩散通过下列公式的体积:V =4/3π（√（面积/π）），3 </li></ol>

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作者宣称，他们有没有竞争的财务权益。

<html>为确保输液成功的关键步骤是:吹扫气的输液管，混合琼脂糖凝胶，分析的MR数据，使用小的内导管的直径，使用阶梯导管设计，以减少回流，并最小化所感受到的压力凝胶或组织到其中的药物被注入。如前所述，主要损害输液的成功是输液管的空气。正确地和彻底吹扫空气的输液管是至关重要的，以确保没有空气进入输液。同样重要的是混合的琼脂糖凝胶电泳图谱。不当合成的凝胶可能导致?...

对流增强输送（CED）已被提议作为用于神经病症包括恶性脑瘤，癫痫，代谢疾病，神经变性疾病（如帕金森病）1，中风和创伤2的广谱治疗选择。 CED采用正压主体流动的药物或其它输注液的分布。 CED提供安全，可靠，且均匀交付分子量化合物，从低到高，在临床相关卷3。传统的药物递送到脑组织受到严重的血-脑屏障4的限制。由内皮细胞间的紧密连接构成的毛细血管在大脑中，血 - 脑屏障块极性和高分子量的分子进入大脑实质内形成。通过海关直接实质内脑灌注可以克服以前的治疗药物传递方式的局限性并允许使用的治疗剂，不会穿过血-脑屏障，因此，此前已作为可行的治疗方案5不可用。 从健康的美国国立卫生研究院（NIH）的研究人员描述土木工程署在上世纪90年代初，作为实现更大的治疗药物浓度的方法比单纯扩散6-8。 CED的第一方法涉及植入一个或多个导管进入大脑，连接输液泵的导管，并直接泵送治疗剂进入目标区域。增加的分布分数和相对稳定的浓度被报道发生如由输液泵产生的正压引起的组织扩张，并允许该药物9的渗透。 最根本的技术CED在很大程度上仍然是它第一次描述的相同。在导管设计10，输液技术进展<su对&gt; 11，管路压力监测2，并实时MRI监测来纠正脑移位12，13，优化多个共线输注14，并监测输液损失15都增加了治疗10的安全性和有效性。额外的重要性已经被放置在导管的设计和输注策略，包括流速。成功的CED，用有限的导管回流和组织损伤，已与相关导管设计和输注速度。使用的导管的具有窄的直径和低输注率，以限制沿脑导管接口倒流以及损坏限制在导管末端16。磁共振成像提供了正确的位置，输液导管置入的视觉确认，因而给药，同时还允许修正输液回流或异常分娩17。 MR图像，也可以用于近似和跟踪分配的体积（VD输注的药物）。在VD是使 ​​用磁共振成像信号强度值大于上述平均值与周围非灌注胶为18分割阈值三个标准差来计算。在Vd为CED为一种有用的测量，因为它代表分布在大脑中的药物的体积。随着输注（六）音量，比率可以生成（VD / VI）量化所涵盖的输注药物的体积。 琼脂糖凝胶体模模仿人类大脑的理解CED等重要几个关键的机械性能:VD，凝胶导管相互作用，多孔弹性性能，并输注云形态10。的0.2％琼脂糖凝胶的混合物已经显示模拟体内由于CED引起凝胶扩张改变当地的孔隙分数。类似的毛孔小部分人脑类似的促进互动和VD 19的精确测量。此外，类似浓度的garose凝胶如0.6％和0.8％也显示类似输液压力分布至脑20。另外，透光性琼脂糖凝胶中提供的导管放置和输注回流的实时可视化的优点。琼脂糖凝胶假体是相对廉价的生产。琼脂糖凝胶假体的费用可能是关键，在整个神经外科未来的广泛培训。由于这些特性，琼脂糖凝胶提供了一个有用的替代，复制了许多人的大脑注入的关键属性，而无需使用脑组织。 如上所述，图像引导到CED琼脂糖凝胶模型提供的体外方法进行测试，研究和培训一个有益的。本文的目的是描述如何重新琼脂糖凝胶假体，勾勒适当CED测试和分析方案，并解决在CED输注用于神经系统疾病的治疗面临的常见错误。

<table><tbody><tr><td>Prohance</td><td>Bracco</td><td></td><td>Gadoteridol radio contrast media</td></tr><tr><td>Bromophenol blue dye</td><td>Biorad</td><td>161-0404</td><td>Dye for infusate visualization</td></tr><tr><td>Agarose gel powder</td><td>Biorad</td><td>161-3101EDU</td><td>Agarose powder for creating gels</td></tr><tr><td>Medrad Veris MR Vital Signs Monitor</td><td>Medrad</td><td></td><td>MR safe infusion pressure monitor</td></tr><tr><td>16 G SmartFlow Catheter</td><td>SurgiVision</td><td></td><td>Infusion catheter</td></tr><tr><td>Medrad Continuum MR Infusion System</td><td>Medrad</td><td></td><td>MR safe infusion pump</td></tr><tr><td>SMART Frame MRI Guided trajectory frame</td><td>ClearPoint</td><td></td><td>Infusion catheter frame</td></tr><tr><td>Osirix imaging software and DICOM Viewer</td><td>Osirix Imaging Software</td><td></td><td>OsiriX 32-bit DICOM Viewer</td></tr></tbody></table>

image-guided convection-enhanced delivery into agarose gel models of the brain

对流增强交付（CED）已被提议作为一个广泛的神经系统疾病的治疗选择。 Neuroinfusion导管海关允许正压大量流量，提供更大量疗法比传统给药方法颅内目标。实时磁共振成像的临床应用指导CED（rCED）在于准确瞄准，监测治疗，并确定并发症的能力。与训练，rCED是有效和并发症可以被最小化。大脑的琼脂糖凝胶模型提供了一种方便的工具CED测试，研究和培训。模拟大脑rCED允许模拟手术的做法，同时还提供可视化的输液反馈。输注的分析允许对分布分数（VD / VI）使学员以验证模型的相似性相比，人脑组织的计算。本文介绍了我们的琼脂糖凝胶假体脑并概述重要的我在同时解决过程中的CED输液面临神经系统疾病的治疗常见的陷阱，一个CED输液和分析协议trics。

解释和分析CED输液涉及的几个重要因素，如分配分数和输液回流。分配比例计算很大程度上取决于Vd的计算。因此，MR图像的准确解释是关键。我们提出了用于可靠地再现这些测量如上面列出的半自动化方法。这些方法客观地确定输液云和横截面面积的大致半径。而可变，在琼脂糖凝胶输液云往往被证明是球形的。假设一个球形输液云，该半径可以被用来确定在VD为CED输注。 VD / VI的琼脂糖凝胶输?...

Watch this Scientific Journal Video about Image-guided Convection-enhanced Delivery into Agarose Gel Models of the Brain at JoVE.com

Image-guided Convection-enhanced Delivery into Agarose Gel Models of the Brain

对流增强交付（CED）已被提议作为一个广泛的神经系统疾病的治疗选择。为了准备专业医护人员采用CED的，需要访问的培训模式。我们描述了使用琼脂糖凝胶作为人脑的这样的试验，研究和训练的模型。

图像引导的对流增强交付到大脑的琼脂糖凝胶模型

Convection-enhanced delivery (CED) has been proposed as a treatment option for a wide range of neurological diseases. Neuroinfusion catheter CED allows ...

image-guided-convection-enhanced-delivery-into-agarose-gel-models

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

11.4K 次观看.  University of Tennessee Health Science Center. 对流增强交付（CED）已被提议作为一个广泛的神经系统疾病的治疗选择。为了准备专业医护人员采用CED的，需要访问的培训模式。我们描述了使用琼脂糖凝胶作为人脑的这样的试验，研究和训练的模型。 

视频: Image-guided Convection-enhanced Delivery into Agarose Gel Models of the Brain

A Rapid Approach to High-Resolution Fluorescence Imaging in Semi-Thick Brain Slices

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are characteristic to neurons in both normal and diseased brain. Towards this, a great deal of effort has been placed on building high-resolution neuroanatomical maps1-3. With the expansion of molecular genetics and advances in light microscopy has come the ability to query not only neuronal morphologies, but also the molecular and cellular makeup of individual neurons and their associated networks4. Major advances in the ability to mark and manipulate neurons through transgenic and gene targeting technologies in the rodent now allow investigators to 'program' neuronal subsets at will5-6. Arguably, one of the most influential contributions to contemporary neuroscience has been the discovery and cloning of genes encoding fluorescent proteins (FPs) in marine invertebrates7-8, alongside their subsequent engineering to yield an ever-expanding toolbox of vital reporters9. Exploiting cell type-specific promoter activity to drive targeted FP expression in discrete neuronal populations now affords neuroanatomical investigation with genetic precision.
Engineering FP expression in neurons has vastly improved our understanding of brain structure and function. However, imaging individual neurons and their associated networks in deep brain tissues, or in three dimensions, has remained a challenge. Due to high lipid content, nervous tissue is rather opaque and exhibits auto fluorescence. These inherent biophysical properties make it difficult to visualize and image fluorescently labelled neurons at high resolution using standard epifluorescent or confocal microscopy beyond depths of tens of microns. To circumvent this challenge investigators often employ serial thin-section imaging and reconstruction methods10, or 2-photon laser scanning microscopy11. Current drawbacks to these approaches are the associated labor-intensive tissue preparation, or cost-prohibitive instrumentation respectively.
Here, we present a relatively rapid and simple method to visualize fluorescently labelled cells in fixed semi-thick mouse brain slices by optical clearing and imaging. In the attached protocol we describe the methods of: 1) fixing brain tissue in situ via intracardial perfusion, 2) dissection and removal of whole brain, 3) stationary brain embedding in agarose, 4) precision semi-thick slice preparation using new vibratome instrumentation, 5) clearing brain tissue through a glycerol gradient, and 6) mounting on glass slides for light microscopy and z-stack reconstruction (Figure 1).
For preparing brain slices we implemented a relatively new piece of instrumentation called the 'Compresstome' VF-200 (http://www.precisionary.com/products_vf200.html). This instrument is a semi-automated microtome equipped with a motorized advance and blade vibration system with features similar in function to other vibratomes. Unlike other vibratomes, the tissue to be sliced is mounted in an agarose plug within a stainless steel cylinder. The tissue is extruded at desired thicknesses from the cylinder, and cut by the forward advancing vibrating blade. The agarose plug/cylinder system allows for reproducible tissue mounting, alignment, and precision cutting. In our hands, the 'Compresstome' yields high quality tissue slices for electrophysiology, immunohistochemistry, and direct fixed-tissue mounting and imaging. Combined with optical clearing, here we demonstrate the preparation of semi-thick fixed brain slices for high-resolution fluorescent imaging.

Here we describe a rapid and simple method to image fluorescently labeled cells in semi-thick brain slices. By fixing, slicing, and optically clearing brain tissue we describe how standard epifluorescent or confocal imaging can be used to visualize individual cells and neuronal networks within intact nervous tissue.

一个快速高分辨率的荧光成像的方法在半厚的脑片

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are ...

科研

神经科学

Modeling Brain Metastases Through Intracranial Injection and Magnetic Resonance Imaging

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are particularly devastating, difficult to treat, and confer a poor prognosis. While the precise incidence of brain metastases in the United States remains hard to estimate, it is likely to increase as extracranial therapies continue to become more efficacious in treating cancer. Thus, it is necessary to identify and develop novel therapeutic approaches to treat metastasis at this site. To this end, intracranial injection of cancer cells has become a well-established method in which to model brain metastasis. Previously, the inability to directly measure tumor growth has been a technical hindrance to this model; however, increasing availability and quality of small animal imaging modalities, such as magnetic resonance imaging (MRI), are vastly improving the ability to monitor tumor growth over time and infer changes within the brain during the experimental period. Herein, intracranial injection of murine mammary tumor cells into immunocompetent mice followed by MRI is demonstrated. The presented injection approach utilizes isoflurane anesthesia and a stereotactic setup with a digitally controlled, automated drill and needle injection to enhance precision, and reduce technical error. MRI is measured over time using a 9.4 Tesla instrument in The Ohio State University James Comprehensive Cancer Center Small Animal Imaging Shared Resource. Tumor volume measurements are demonstrated at each time point through use of ImageJ. Overall, this intracranial injection approach allows for precise injection, day-to-day monitoring, and accurate tumor volume measurements, which combined greatly enhance the utility of this model system to test novel hypotheses on the drivers of brain metastases.

Intracranial brain metastasis modeling is complicated by an inability to monitor tumor size and response to treatment with precise and timely methods. The presented methodology couples intracranial tumor injection with magnetic resonance imaging analysis, which when combined, cultivates precise and consistent injections, enhanced animal monitoring, and accurate tumor volume measurements.

通过颅内注射和磁共振成像对大脑转移进行建模

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are ...

癌症研究

Flexible Organic Electronic Devices for Pulsed Electric Field Therapy of Glioblastoma

Glioblastoma is difficult to eradicate with standard oncology therapies due to its high degree of invasiveness. Bioelectric treatments based on pulsed electric fields (PEFs) are promising for the improvement of treatment efficiency. However, they rely on rigid electrodes that cause acute and chronic damage, especially in soft tissues such as the brain. In this work, flexible electronics were used to deliver PEFs to tumors and the biological response was evaluated with fluorescent microscopy. Interdigitated gold electrodes on a thin, transparent parylene-C substrate were coated with the conducting polymer PEDOT:PSS, resulting in a conformable and biocompatible device. The effects of PEFs on tumors and their microenvironment were examined using various biological models. First, monolayers of glioblastoma cells were cultured on top of the electrodes to investigate phenomena in vitro. As an intermediate step, an in ovo model was developed where engineered tumor spheroids were grafted in the embryonic membrane of a quail. Due to the absence of an immune system, this led to highly vascularized tumors. At this early stage of development, embryos have no immune system, and tumors are not recognized as foreign bodies. Thus, they can develop fast while developing their own vessels from the existing embryo vascular system, which represents a valuable 3D cancer model. Finally, flexible electrode delivery of PEFs was evaluated in a complete organism with a functional immune system, using a syngenic, orthograft (intracranial) mouse model. Tumor spheroids were grafted into the brain of transgenic multi-fluorescent mice prior to the implantation of flexible organic electrode devices. A sealed cranial window enabled multiphoton imaging of the tumor and its microenvironment during treatment with PEFs over a period of several weeks.

This work describes the development of flexible interdigitated electrodes for implementation in 3D brain tumor models, namely, in vitro culture, in ovo model, and in vivo murine model. The proposed method can be used to evaluate the effects of pulsed electric fields on tumors at different levels of complexity.

用于胶质母细胞瘤脉冲电场治疗的柔性有机电子器件

Glioblastoma is difficult to eradicate with standard oncology therapies due to its high degree of invasiveness. Bioelectric treatments based on pulsed ...

Transposon Mediated Integration of Plasmid DNA into the Subventricular Zone of Neonatal Mice to Generate Novel Models of Glioblastoma

An urgent need exists to test the contribution of new genes to the pathogenesis and progression of human glioblastomas (GBM), the most common primary brain tumor in adults with dismal prognosis. New potential therapies are rapidly emerging from the bench and require systematic testing in experimental models which closely reproduce the salient features of the human disease. Herein we describe in detail a method to induce new models of GBM with transposon-mediated integration of plasmid DNA into cells of the subventricular zone of neonatal mice. We present a simple way to clone new transposons amenable for genomic integration using the Sleeping Beauty transposon system and illustrate how to monitor plasmid uptake and disease progression using bioluminescence, histology and immuno-histochemistry. We also describe a method to create new primary GBM cell lines. Ideally, this report will allow further dissemination of the Sleeping Beauty transposon system among brain tumor researchers, leading to an in depth understanding of GBM pathogenesis and progression and to the timely design and testing of effective therapies for patients.

Here we describe an efficient and versatile protocol to induce, monitor and analyze novel glioblastomas (GBM) using transposon DNA injected into the ventricles of neonatal mice. Cells of the subventricular zone, which take up the plasmid, transform, proliferate and generate tumors with histo-pathological characteristics of human GBM.

质粒DNA转座子介导整合到新生小鼠的脑室下区，以生成胶质母细胞瘤的新模型

An urgent need exists to test the contribution of new genes to the pathogenesis and progression of human glioblastomas (GBM), the most common primary ...

医学

A MRI-Based Toolbox for Neurosurgical Planning in Nonhuman Primates

In this paper, we outline a method for surgical preparation that allows for the practical planning of a variety of neurosurgeries in NHPs solely using data extracted from magnetic resonance imaging (MRI). This protocol allows for the generation of 3D printed anatomically accurate physical models of the brain and skull, as well as an agarose gel model of the brain modeling some of the mechanical properties of the brain. These models can be extracted from MRI using brain extraction software for the model of the brain, and custom code for the model of the skull. The preparation protocol takes advantage of state-of-the-art 3D printing technology to make interfacing brains, skulls, and molds for gel brain models. The skull and brain models can be used to visualize brain tissue inside the skull with the addition of a craniotomy in the custom code, allowing for better preparation for surgeries directly involving the brain. The applications of these methods are designed for surgeries involved in neurological stimulation and recording as well as injection, but the versatility of the system allows for future expansion of the protocol, extraction techniques, and models to a wider scope of surgeries.

The method outlined below aims to provide a comprehensive protocol for the preparation of nonhuman primate (NHP) neurosurgery using a novel combination of three-dimensional (3D) printing methods and MRI data extraction.

非人类灵长类动物神经外科规划基于MRI的工具箱

In this paper, we outline a method for surgical preparation that allows for the practical planning of a variety of neurosurgeries in NHPs solely using ...

生物工程

High-resolution Confocal Imaging of the Blood-brain Barrier: Imaging, 3D Reconstruction, and Quantification of Transcytosis

The blood-brain barrier (BBB) is a dynamic multicellular interface that regulates the transport of molecules between the circulation and the brain. Transcytosis across the BBB regulates the delivery of hormones, metabolites, and therapeutic antibodies to the brain parenchyma. Here, we present a protocol that combines immunofluorescence of free-floating sections with laser scanning confocal microscopy and image analysis to visualize subcellular organelles within endothelial cells at the BBB. Combining this data-set with 3D image analysis software allows for the semi-automated segmentation and quantification of capillary volume and surface area, as well as the number and intensity of intracellular organelles at the BBB. The detection of mouse endogenous immunoglobulin (IgG) within intracellular vesicles and their quantification at the BBB is used to illustrate the method. This protocol can potentially be applied to the investigation of the mechanisms controlling BBB transcytosis of different molecules in vivo.

Here we present a microscopy-based protocol for high-resolution imaging and a three-dimensional reconstruction of the mouse neurovascular unit and blood-brain barrier using brain free-floating sections. This method allows for the visualization, analysis, and quantification of intracellular organelles at the BBB.

血脑屏障的高分辨率共聚焦成像: 成像、3D 重建和 Transcytosis 的量化

The blood-brain barrier (BBB) is a dynamic multicellular interface that regulates the transport of molecules between the circulation and the brain. ...

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate tangentially from their place of origin in the ventral telencephalon (including from the medial and caudal ganglionic eminences (MGE, CGE)) to the dorsal cortical plate in response to a variety of intrinsic and extrinsic cues. Different methodologies have been developed over the years to genetically manipulate specific pathways and investigate how they regulate the dynamic cytoskeletal changes required for proper IN migration. In utero electroporation has been extensively used to study the effect of gene repression or overexpression in specific IN subtypes while assessing the impact on morphology and final position. However, while this approach is readily used to modify radially migrating pyramidal cells, it is more technically challenging when targeting INs. In utero electroporation generates a low yield given the decreased survival rates of pups when electroporation is conducted before e14.5, as is customary when studying MGE-derived INs. In an alternative approach, MGE explants provide easy access to the MGE and facilitate the imaging of genetically modified INs. However, in these explants, INs migrate into an artificial matrix, devoid of endogenous guidance cues and thalamic inputs. This prompted us to optimize a method where INs can migrate in a more naturalistic environment, while circumventing the technical challenges of in utero approaches. In this paper, we describe the combination of ex utero electroporation of embryonic mouse brains followed by organotypic slice cultures to readily track, image and reconstruct genetically modified INs migrating along their natural paths in response to endogenous cues. This approach allows for both the quantification of the dynamic aspects of IN migration with time-lapse confocal imaging, as well as the detailed analysis of various morphological parameters using neuronal reconstructions on fixed immunolabeled tissue.

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

Here, we provide a low-cost and reliable method to generate electroporated brain organotypic slice cultures from mouse embryos suitable for confocal microscopy and live-imaging techniques.

前子宫内胚胎小鼠脑电穿孔和脊髓切片培养迁移 GABAergic 中间神经元的活体成像

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate ...

Laser Capture Microdissection of Glioma Subregions for Spatial and Molecular Characterization of Intratumoral Heterogeneity, Oncostreams, and Invasion

Gliomas are primary brain tumors characterized by their invasiveness and heterogeneity. Specific histological patterns such as pseudopalisades, microvascular proliferation, mesenchymal transformation and necrosis characterize the histological heterogeneity of high-grade gliomas. Our laboratory has demonstrated that the presence of high densities of mesenchymal cells, named oncostreams, correlate with tumor malignancy. We have developed a unique approach to understand the mechanisms that underlie glioma&#39;s growth and invasion. Here, we describe a comprehensive protocol that utilizes laser capture microdissection (LMD) and RNA sequencing to analyze differential mRNA expression of intra-tumoral heterogeneous multicellular structures (i.e., mesenchymal areas or areas of tumor invasion). This method maintains good tissue histology and RNA integrity. Perfusion, freezing, embedding, sectioning, and staining were optimized to preserve morphology and obtain high-quality laser microdissection samples. The results indicate that perfusion of glioma bearing mice using 30% sucrose provides good morphology and RNA quality. In addition, staining tumor sections with 4% Cresyl violet and 0.5% eosin results in good nuclear and cellular staining, while preserving RNA integrity. The method described is sensitive and highly reproducible and it can be utilized to study tumor morphology in various tumor models. In summary, we describe a complete method to perform LMD that preserves morphology and RNA quality for sequencing to study the molecular features of heterogeneous multicellular structures within solid tumors.

Laser microdissection (LMD) is a sensitive and highly reproducible technique that can be used to uncover pathways that mediate glioma heterogeneity and invasion. Here, we describe an optimized protocol to isolate discrete areas from glioma tissue using laser LMD followed by transcriptomic analysis.

激光捕获胶质瘤分区域的微切，用于肿瘤内异质性、肿瘤流和入侵的空间和分子表征

Gliomas are primary brain tumors characterized by their invasiveness and heterogeneity. Specific histological patterns such as pseudopalisades, ...

In Situ Visualization of Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures

During neuronal development, axons navigate the cortical environment to reach their final destinations and establish synaptic connections. Growth cones -the sensory structures located at the distal tips of developing axons- execute this process. Studying the structure and dynamics of the growth cone is crucial to understanding axonal development and the interactions with the surrounding central nervous system (CNS) that enable it to form neural circuits. This is essential when devising methods to reintegrate axons into neural circuits following injury in fundamental research and pre-clinical contexts. Thus far, the general understanding of growth cone dynamics is primarily founded on studies of neurons cultured in two dimensions (2D). Although undoubtedly fundamental to the current knowledge of growth cone structural dynamics and response to stimuli, 2D studies misrepresent the physiological three-dimensional (3D) environment encountered by neuronal growth cones in intact CNS tissue. More recently, collagen gels were employed to overcome some of these limitations, enabling the investigation of neuronal development in 3D. However, both synthetic 2D and 3D environments lack signaling cues within CNS tissue, which direct the extension and pathfinding of developing axons. This protocol provides a method for studying axons and growth cones using organotypic brain slices, where developing axons encounter physiologically relevant physical and chemical cues. By combining fine-tuned in utero and ex utero electroporation to sparsely deliver fluorescent reporters along with super-resolution microscopy, this protocol presents a methodological pipeline for the visualization of axon and growth cone dynamics in situ. Furthermore, a detailed toolkit description of the analysis of long-term and live-cell imaging data is included.

In Situ Visualization of Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures

This protocol demonstrates a straightforward and robust method to study in situ axon growth and growth cone dynamics. It describes how to prepare ex vivo physiologically relevant acute brain slices and provides a user-friendly analysis pipeline.

原位 急性 离体 胚胎脑切片培养物中轴突生长和生长锥动力学的可视化

During neuronal development, axons navigate the cortical environment to reach their final destinations and establish synaptic connections. Growth cones ...

A Tissue Clearing Method for Neuronal Imaging from Mesoscopic to Microscopic Scales

A detailed protocol is provided here to visualize neuronal structures from mesoscopic to microscopic levels in brain tissues. Neuronal structures ranging from neural circuits to subcellular neuronal structures are visualized in mouse brain slices optically cleared with ScaleSF. This clearing method is a modified version of ScaleS and is a hydrophilic tissue clearing method for tissue slices that achieves potent clearing capability as well as a high-level of preservation of fluorescence signals and structural integrity. A customizable three dimensional (3D)-printed imaging chamber is designed for reliable mounting of cleared brain tissues. Mouse brains injected with an adeno-associated virus vector carrying enhanced green fluorescent protein gene were fixed with 4% paraformaldehyde and cut into slices of 1-mm thickness with a vibrating tissue slicer. The brain slices were cleared by following the clearing protocol, which include sequential incubations in three solutions, namely, ScaleS0 solution, phosphate buffer saline (&#8211;), and ScaleS4 solution, for a total of 10.5&#8211;14.5 h. The cleared brain slices were mounted on the imaging chamber and embedded in 1.5% agarose gel dissolved in ScaleS4D25(0) solution. The 3D image acquisition of the slices was carried out using a confocal laser scanning microscope equipped with a multi-immersion objective lens of a long working distance. Beginning with mesoscopic neuronal imaging, we succeeded in visualizing fine subcellular neuronal structures, such as dendritic spines and axonal boutons, in the optically cleared brain slices. This protocol would facilitate understanding of neuronal structures from circuit to subcellular component scales.

The protocol provides a detailed method of neuronal imaging in brain slice using a tissue clearing method, ScaleSF. The protocol includes brain tissue preparation, tissue clarification, handling of cleared slices and confocal laser scanning microscopy imaging of neuronal structures from mesoscopic to microscopic levels.

一种从介观尺度到微观尺度的神经元成像组织清除方法

A detailed protocol is provided here to visualize neuronal structures from mesoscopic to microscopic levels in brain tissues. Neuronal structures ranging ...

图像引导的对流增强交付到大脑的琼脂糖凝胶模型

摘要

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一个快速高分辨率的荧光成像的方法在半厚的脑片

通过颅内注射和磁共振成像对大脑转移进行建模

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质粒DNA转座子介导整合到新生小鼠的脑室下区，以生成胶质母细胞瘤的新模型

非人类灵长类动物神经外科规划基于MRI的工具箱

血脑屏障的高分辨率共聚焦成像: 成像、3D 重建和 Transcytosis 的量化

前子宫内胚胎小鼠脑电穿孔和脊髓切片培养迁移 GABAergic 中间神经元的活体成像

激光捕获胶质瘤分区域的微切，用于肿瘤内异质性、肿瘤流和入侵的空间和分子表征

原位急性离体胚胎脑切片培养物中轴突生长和生长锥动力学的可视化

一种从介观尺度到微观尺度的神经元成像组织清除方法

图像引导的对流增强交付到大脑的琼脂糖凝胶模型

摘要

探索更多视频

此视频中的章节

一个快速高分辨率的荧光成像的方法在半厚的脑片

通过颅内注射和磁共振成像对大脑转移进行建模

用于胶质母细胞瘤脉冲电场治疗的柔性有机电子器件

质粒DNA转座子介导整合到新生小鼠的脑室下区，以生成胶质母细胞瘤的新模型

非人类灵长类动物神经外科规划基于MRI的工具箱

血脑屏障的高分辨率共聚焦成像: 成像、3D 重建和 Transcytosis 的量化

前子宫内胚胎小鼠脑电穿孔和脊髓切片培养迁移 GABAergic 中间神经元的活体成像

激光捕获胶质瘤分区域的微切，用于肿瘤内异质性、肿瘤流和入侵的空间和分子表征

原位 急性 离体 胚胎脑切片培养物中轴突生长和生长锥动力学的可视化

一种从介观尺度到微观尺度的神经元成像组织清除方法

原位急性离体胚胎脑切片培养物中轴突生长和生长锥动力学的可视化