Name: imaging serotonergic fibers in the mouse spinal cord using the clarity/cubic technique
Uploaded: 2016-02-26T14:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique at JoVE.com

This work was supported by the Australian Research Council Centre of Excellence for Integrative Brain Function (ARC Centre Grant CE140100007), an NHMRC project grant (#1086643). Prof. George Paxinos is supported by a Senior Principal Research Fellow NHMRC grant (#1043626).

伦理学声明:所有涉及动物主题程序遵循的动物护理和伦理委员会（ACEC）在新南威尔士大学的准则（经批准的ACEC号为14 / 94A）。 1.透明小鼠脊髓的制备<ol><li>冰冷水凝胶液的制备<ol><li>的16％多聚甲醛溶液的制备（PFA） <ol><li>将16多聚甲醛粉末成70毫升预热蒸馏水（50-55℃）和直至多聚甲醛溶解在加热的磁力搅拌器搅拌。注意:不要让溶液加热超过55℃，要知道，多聚甲醛是有毒的。 </li><li>的多聚甲醛溶液转移到一个圆柱体，并添加蒸馏水至100ml。冷却用4℃冰箱内的多聚甲醛溶液。 </li></ol></li><li>溶解125毫克的VA-044引发剂在26.25毫升蒸馏水，并在4冷却溶液°Ç冰箱。 </li><li>凉爽5毫升丙烯酰胺溶液（40％），1.25毫升二溶液（2％）（小心:双丙烯酰胺溶液是有毒的，可能会导致遗传性缺陷或癌症）中，加入5ml 10×PBS，12.5毫升16％PFA溶液中，将在冰上的VA-044引发剂溶液。 </li><li>混合冰上述解决方案。 注意:总体积为50毫升</li></ol></li><li>灌注和收集的小鼠脊髓的<ol><li>麻醉小鼠，在0.9％的生理盐水稀释氯胺酮（80毫克/千克）和xyzaline（5毫克/千克）的腹膜内注射。对小鼠的剂量可以比老鼠低。 </li><li>在通风橱合适的塑料表面，固定鼠标的四肢远离身体（胶带是有效的），一旦鼠标不能捏响应其足部皮肤。 </li><li>切过胸部小鼠皮肤，然后胸口骨头用剪刀暴露心脏。 </li><li>使用蠕动泵，以地下25针连接到第管电子端，针头插入心脏的左心室，剪断右心房以创建血液出口点，然后用40毫升的0.9％的生理盐水开始洗涤的约10毫升/分钟的速率。 </li><li>当完成时，灌注用35ml冰冷的水凝胶溶液中的鼠标。 注意:灌注以10ml / min的速度，以避免对神经组织的肿胀。 </li><li>切开皮肤上用刀片的背面，然后除去旁边的脊椎动物的肌肉。切割一侧椎弓，并将其翻转到另一边后，将脊髓出脊柱用细剪刀。 </li></ol></li><li>清除小鼠脊髓<ol><li>放置在15毫升的水凝胶溶液中的脊髓在4℃下过夜。 </li><li>除去将10毫升的水凝胶溶液和倾与脊髓节段的溶液的其余部分5ml的管中。填5毫升管与水凝胶溶液，直到它完全满。 </li><li>伸一小块封口膜超过5毫升管的顶部，然后包裹封口膜围绕管的颈部。注:确保封口膜接触水凝胶解决方案，并有水凝胶液和封口膜之间没有气泡。 </li><li>把5毫升管在37 &#160; ℃的烘箱过夜。观察水凝胶溶液，直到它变成了凝胶。 </li><li>取5毫升管出炉并从用扳手管取出凝胶（如铲1毫升枪头）。通过坚持粗组织凝胶取下脊髓组织的凝胶，然后把粗的组织远（凝胶将坚持组织，因此将通过组织被删除）。 </li><li>从脊髓组织除去凝胶后，洗脊髓4次用1×PBS（pH7.4）中为在振荡器上24小时。 </li><li> 3.85克尿素3.85克N-，N，N&#39;，N&#39;-四（2-羟丙基）ethylenediam溶解制备立方结算溶液INE在5.38毫升蒸馏水。 注:加热搅拌器使用。添加2.31克聚乙二醇单对异辛基苯基醚/的Triton X-100的溶液，一旦它是明确的，冷却到室温。 注为10g这三种化学品是一个鼠标。 </li><li>冠状切开小鼠脊髓与刀片2-3毫米长的段，放入5ml上述CUBIC结算解决方案。上于37在烤箱摇动器放置&#160; °℃下3天。 </li><li>三天后，改变它的结算溶液至新鲜的。注意:将上述溶液在使用前制备。 </li><li>两三天后刷新CUBIC结算解决方案后，核对与字体8个字符的文件组织的透明度。如果它是透明的，字母可通过清除组织中看到。除去信息交换溶液和添加的PBST 4毫升（0.1％的Triton-X100）洗组织每天（每6小时）的4倍。 </li></ol></li></ol><ol><li>在37℃的烘箱中3天振荡器上:孵育在一次抗体溶液中的脊髓节段（在PBST 100抗血清素，兔凸起，稀释1）。 </li><li>除去抗体溶液，并添加的PBST 4毫升（0.1％的Triton-X100）洗脊髓节段，每天4次（每6小时）在37℃的烘箱。 </li><li>除去PBST溶液，并添加二次抗体溶液（594缀合的山羊抗兔IgG，1:100稀释在PBST）温育3天的组织上的摇动器在37℃的烘箱。 </li><li>去除二级抗体溶液并在振荡器上在37℃的烘箱加4毫升的PBST洗脊髓节段每日4次（每6小时）。次日将组织准备成像。 </li></ol> 3.成像<ol><li>除去PBST溶液，并添加4毫升85％甘油，以使所述组织的折射率均匀。 </li><l我&gt;检查组织的清晰度。一旦它在大约1小时清晰，把组织在其上适合多张照片荧光显微镜的保持框大，薄玻璃幻灯片。 </li><li>把85％的甘油几滴旁边的脊髓组织，并用22×50毫米2盖玻片玻璃盖玻片它。 注意:脊髓的取向决定了图象的样子。 </li><li>把玻璃载片上的多光子荧光显微镜的保持框和移动组织进入光路。 </li><li>选择氦氖激光线594 nm和通过检查阳性信号的亮度的实时图像中调整激光的最佳水平的强度。 </li><li>选择使用20X物镜（在水中，NA 0.7）的脊髓组织的扫描区域和准备创建的z栈，并设置为3微米的每一个步骤的深度。 </li><li>扫描从顶部组织于z叠层的20X objec下底略去然后63X物镜（在油中，NA 1.4）（步长为1微米），分别。重建使用3D软件28 3D视频。 </li></ol>

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The authors have nothing to disclose.

协议中所述显示了如何像血清素纤维在小鼠脊髓与组合的清晰度和CUBIC技术。它与由Cheung等 14和托默等人 15开发的被动资料交换协议引入了更快的结算处理，并允许脊髓组织要由水凝胶清除期间很好的支持。 鼠标脊髓定影期间的一个重要步骤，所报告的Cheung等 14和托默等人 15，是保持所有的解决方案在冰上，其防止?...

Supraspinal projections are responsible for the modulation of diverse behaviors such as pain perception. One of the projections carrying nociceptive information contains serotoninergic fibers, which originate from the hindbrain raphe and adjacent reticular nuclei1,2. Physiological and pharmacological studies have demonstrated an increased release of serotonin in the dorsal horn of the spinal cord after electrical stimulation of the raphe nuclei in the hindbrain3-5. In the rat and opossum, serotonergic raphespinal fibers have dense terminals, not only in the dorsal horn6-8, but also in the intermediate zone7,9,10, the ventral horn7,11, and even lamina 1012,13. There are no similar studies in the mouse. The present study aimed to map the termination pattern of serotonergic fibers arising from the hindbrain raphe nuclei and their adjacent reticular nuclei in the mouse spinal cord using the recently published CLARITY14 method and its modification - CUBIC15. 
Conventional fluorescence or peroxidase immunohistochemistry of the spinal cord clearly shows the distribution of serotonergic fibers in the gray matter of the spinal cord in 30-40 &#181;m thick cross-sections. However, this approach does not show the continuity of the serotonergic fiber tracts in the white matter and their collaterals in the gray matter. Although the 3D reconstruction of histological sections has advanced our knowledge of fiber tracts, it remains a challenge for histologists and anatomists to follow a single tract due to small distortions in the tissue caused by cutting. To circumvent this obstacle a number of researchers have developed various protocols for making the whole tissue structure transparent, and collecting an image of unaltered tissue in a single video file17-21. So far, the clear, lipid-exchanged, acrylamide-hybridized rigid, imaging/ immunostaining compatible, tissue hydrogel (CLARITY) technique, developed by Deisseroth's group14,15, as well as CUBIC, developed by Susaki et al16 are the most successful. Since the publication of the protocols, many researchers have started using these techniques to investigate various aspects of biological tissues, including, not only the brain22-25, but also the heart, kidneys, intestine, and the lungs26,27. 
By fixing the mouse spinal cord with the hydrogel solution (CLARITY) and clearing with the CUBIC reagents (which is a much faster method than that described by the original CLARITY protocol14,15), a spinal cord tissue block of 2-3 mm long was cleared within two weeks and immunofluorescence staining for serotonin completed in eight days. With just a combination of chemical agents, conventional immunohistochemistry can be used to create an image of individual fiber tracts in a 3D video file in approximately one month.

<table><tbody><tr><td>Photoinitiator VA044</td><td>Wako</td><td>va-044/225-02111</td><td>http://www.wako-chem.co.jp/specialty/waterazo/VA-044.htm</td></tr><tr><td>40% acrylamide solution</td><td>Bio Rad</td><td>161-0140</td><td>http://www.bio-rad.com/en-au/sku/161-0140-40-acrylamide-solution</td></tr><tr><td>2% Bis Solution</td><td>Bio Rad</td><td>161-0142</td><td>http://www.bio-rad.com/en-au/sku/161-0142-2-bis-solution?parentCategoryGUID=5e7a4f31-&lt;br/&gt;879c-4d63-ba0b-82556a0ccf1d</td></tr><tr><td>paraformaldehyde</td><td>Sigma</td><td>158127</td><td>http://www.sigmaaldrich.com/catalog/product/sial/158127?lang=en&amp;amp;region=AU</td></tr><tr><td>urea</td><td>Merck Millipore</td><td>66612</td><td>http://www.merckmillipore.com/AU/en/product/Urea---CAS-57-13-6---Calbiochem,EMD_BIO-66612</td></tr><tr><td>N,N,N&amp;rsquo;,N&amp;rsquo;-tetrakis (2-hydroxypropyl) ethylenediamine</td><td>Merck Millipore</td><td>821940</td><td>http://www.merckmillipore.com/AU/en/product/Ethylenediamine-N,N,N&#039;,N&#039;-tetra-2-propanol,MDA_CHEM-821940</td></tr><tr><td>Triton-X 100</td><td>Merck Millipore</td><td>648462</td><td>http://www.merckmillipore.com/AU/en/product/TRITON&amp;reg;-X-100-Detergent---CAS-9002-93-1---Calbiochem,EMD_BIO-648462</td></tr><tr><td>sucrose</td><td>Sigma</td><td>S0389</td><td>http://www.sigmaaldrich.com/catalog/product/sigma/s0389?lang=en&amp;amp;region=AU</td></tr><tr><td>serotonin antibody</td><td>Merck Millipore</td><td>AB938</td><td>http://www.merckmillipore.com/AU/en/product/Anti-Serotonin-Antibody,MM_NF-AB938</td></tr><tr><td>goat anti rabbit IgG (H+L) Secondary Antibody, Alexa Fluor&amp;reg; 594 conjugate</td><td>Life Technologies</td><td>&amp;nbsp;A-11012</td><td>https://www.lifetechnologies.com/order/genome-database/antibody/Rabbit-IgG-H-L-Secondary-Antibody-Polyclonal/A-11012</td></tr><tr><td>multi-photon microscope</td><td>Leica</td><td>Leica TCS SP5 MP STED</td><td>http://www.leica-microsystems.com/products/confocal-microscopes/details/product/leica-tcs-sp5-mp/</td></tr></tbody></table>

imaging serotonergic fibers in the mouse spinal cord using the clarity/cubic technique

长递减纤维脊髓是运动，疼痛的感知，和其他行为的关键。在大多数这些光纤系统的脊髓纤维端接图案没有在任何物种被彻底调查。血清素纤维，其投射到脊髓，已经研究了在大鼠和负鼠上组织切片和它们的功能意义已经基于在脊髓其光纤端接图案被推断出来。与净度和立方技术的发展，有可能研究这种纤维系统及其在脊髓分布，这是可能揭示血清素脊髓上通路的先前未知的功能。在这里，我们使用相结合的清晰度和CUBIC成像技术在小鼠脊髓血清素纤维提供了详细的方案。该方法涉及的小鼠的灌注的水凝胶溶液中，澄清的组织与COMBIN清除试剂的通货膨胀。脊髓组织在不到2周清除，5-羟色胺随后的免疫荧光染色在不到十天完成。用多光子荧光显微镜，所述组织被扫描，并使用Osirix软件的3D图像被重建。

本节将展示使用的清晰度和CUBIC协议的组合在透明小鼠脊髓血清素从抗体染色的结果。我们表明，血清素纤维的存在与腹角的腹侧部的优势脊髓所有薄片（ 图1中 ，也看到视频1）。控制的组织没有阳性纤维（结果未示出）。在腹角，密密麻麻的血清素纤维在腹角的腹内侧部分，他们对腹角的横向部分延伸（ 图2，见视频2）。</str...

Watch this Scientific Journal Video about Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique at JoVE.com

Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique

Supraspinal projections are important for pain perception and other behaviors, and serotonergic fibers are one of these fiber systems. The present study focused on the application of the combined CLARITY/CUBIC protocol to the mouse spinal cord in order to investigate the termination of these serotonergic fibers.

影像学检查HT能纤维在小鼠脊髓使用CLARITY / CUBIC技术

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal ...

imaging-serotonergic-fibers-mouse-spinal-cord-using-claritycubic

Research

JoVE Journal

Neuroscience

10.4K 次观看.  Neuroscience Research Australia. Supraspinal projections are important for pain perception and other behaviors, and serotonergic fibers are one of these fiber systems. The present study focused on the application of the combined CLARITY/CUBIC protocol to the mouse spinal cord in order to investigate the termination of these serotonergic fibers.

视频: Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has significantly broadened our knowledge of physiological and pathological processes in numerous tissues in vivo 4-7. In studies of the central nervous system (CNS), there has been a broad application of in vivo imaging in the brain, which has produced a plethora of novel and often unexpected findings about the behavior of cells such as neurons, astrocytes, microglia, under physiological or pathological conditions 8-17. However, mostly technical complications have limited the implementation of in vivo imaging in studies of the living mouse spinal cord. In particular, the anatomical proximity of the spinal cord to the lungs and heart generates significant movement artifact that makes imaging the living spinal cord a challenging task.
We developed a novel method that overcomes the inherent limitations of spinal cord imaging by stabilizing the spinal column, reducing respiratory-induced movements and thereby facilitating the use of two-photon microscopy to image the mouse spinal cord in vivo. This is achieved by combining a customized spinal stabilization device with a method of deep anesthesia, resulting in a significant reduction of respiratory-induced movements. This video protocol shows how to expose a small area of the living spinal cord that can be maintained under stable physiological conditions over extended periods of time by keeping tissue injury and bleeding to a minimum. Representative raw images acquired in vivo detail in high resolution the close relationship between microglia and the vasculature. A timelapse sequence shows the dynamic behavior of microglial processes in the living mouse spinal cord. Moreover, a continuous scan of the same z-frame demonstrates the outstanding stability that this method can achieve to generate stacks of images and/or timelapse movies that do not require image alignment post-acquisition. Finally, we show how this method can be used to revisit and reimage the same area of the spinal cord at later timepoints, allowing for longitudinal studies of ongoing physiological or pathological processes in vivo.

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

A minimally invasive protocol to stabilize the mouse spinal column and perform repetitive in vivo spinal cord imaging using two-photon microscopy is described. This method combines a spinal stabilization device and an anesthetic regimen to minimize respiratory-induced movements and produce raw imaging data that require no alignment or other post-processing.

在体内脊髓的鼠标线使用双光子显微镜成像

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has ...

科研

神经科学

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by repeated, time-lapse in vivo imaging in live rodents. These studies were made possible by straightforward surgical procedures, which enabled optical access for a prolonged period of time without repeat surgical procedures. In contrast, analogous studies of the spinal cord have been previously limited to only a few imaging sessions, each of which required an invasive surgery. As previously described, we have developed a spinal chamber that enables continuous optical access for upwards of 8 weeks, preserves mechanical stability of the spinal column, is easily stabilized externally during imaging, and requires only a single surgery. Here, the design of the spinal chamber with its associated surgical implements is reviewed and the surgical procedure is demonstrated in detail. Briefly, this video will demonstrate the preparation of the surgical area and mouse for surgery, exposure of the spinal vertebra and appropriate tissue debridement, the delivery of the implant and vertebral clamping, the completion of the chamber, the removal of the delivery system, sealing of the skin, and finally, post-operative care. The procedure for chronic in vivo imaging using nonlinear microscopy will also be demonstrated. Finally, outcomes, limitations, typical variability, and a guide for troubleshooting are discussed.

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

In this video, we describe a procedure for implanting a chronic optical imaging chamber over the dorsal spinal cord of a live mouse. The chamber, surgical procedure, and chronic imaging are reviewed and demonstrated.

一个程序，植入脊柱商会纵<em&gt;在体内</em&gt;鼠标脊髓成像

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by ...

Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for live-cell imaging in the murine spinal cord. This technique is uniquely suited to analyze neural precursor cell (NPC) dynamics following transplantation into spinal cords undergoing neuroinflammatory demyelinating disorders. NPCs migrate to sites of axonal damage, proliferate, differentiate into oligodendrocytes, and participate in direct remyelination. NPCs are thereby a promising therapeutic treatment to ameliorate chronic demyelinating diseases. Because transplanted NPCs migrate to the damaged areas on the ventral side of the spinal cord, traditional intravital 2P imaging is impossible, and only information on static interactions was previously available using histochemical staining approaches. Although this method was generated to image transplanted NPCs in the ventral spinal cord, it can be applied to numerous studies of transplanted and endogenous cells throughout the entire spinal cord. In this article, we demonstrate the preparation and imaging of a spinal cord with enhanced yellow fluorescent protein-expressing axons and enhanced green fluorescent protein-expressing transplanted NPCs.

A new ex vivo preparation for imaging the mouse spinal cord. This protocol allows for two-photon imaging of live cellular interactions throughout the spinal cord.

细胞动力学在小鼠脊髓双光子成像

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for ...

Optical Clearing of the Mouse Central Nervous System Using Passive CLARITY

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique non-linear deformations, dramatically hampering one's ability to evaluate cellular morphology, distribution and connectivity in the central nervous system (CNS). However, optical clearing techniques are changing the way tissues are visualized. These approaches permit one to probe deeply into intact organ preparations, providing tremendous insight into the structural organization of tissues in health and disease. Techniques such as Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) achieve this goal by providing a matrix that binds important biomolecules while permitting light-scattering lipids to freely diffuse out. Lipid removal, followed by refractive index matching, renders the tissue transparent and readily imaged in 3 dimensions (3D). Nevertheless, the electrophoretic tissue clearing (ETC) used in the original CLARITY protocol can be challenging to implement successfully and the use of a proprietary refraction index matching solution makes it expensive to use the technique routinely. This report demonstrates the implementation of a simple and inexpensive optical clearing protocol that combines passive CLARITY for improved tissue integrity and 2,2&#8242;-thiodiethanol (TDE), a previously described refractive index matching solution.

Optical clearing techniques are revolutionizing the way tissues are visualized. In this report we describe modifications of the original Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) protocol that yields more consistent and less expensive results.

鼠标中枢神经系统的光透明使用被动CLARITY

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique ...

Immunostaining of Whole-Mount Retinas with the CLARITY Tissue Clearing Method

The tissue hydrogel delipidation method (CLARITY), originally developed by the Deisseroth laboratory, has been modified and widely used for immunostaining and imaging of thick brain samples. However, this advanced technology has not yet been used for whole-mount retinas. Although the retina is partially transparent, its thickness of approximately 200 &#181;m (in mice) still limits the penetration of antibodies into the deep tissue as well as reducing light penetration for high-resolution imaging. Here, we adapted the CLARITY method for whole-mount mouse retinas by polymerizing them with an acrylamide monomer to form a nanoporous hydrogel and then clearing them in sodium dodecyl sulfate to minimize protein loss and avoid tissue damage. CLARITY-processed retinas were immunostained with antibodies for retinal neurons, glial cells, and synaptic proteins, mounted in a refractive index matching solution, and imaged. Our data demonstrate that CLARITY&#160;can improve the quality of standard immunohistochemical staining and imaging for retinal neurons and glial cells in whole-mount preparation. For instance, 3D resolution of fine axon-like and dendritic structures of dopaminergic amacrine cells were much improved by CLARITY. Compared to non-processed whole-mount retinas, CLARITY can reveal immunostaining for synaptic proteins such as postsynaptic density protein 95. Our results show that CLARITY renders the retina more optically transparent after the removal of lipids and preserves fine structures of retinal neurons and their proteins, which can be routinely used for obtaining high-resolution imaging of retinal neurons and their subcellular structures in whole-mount preparation.

Here we present a protocol to adapt the CLARITY method of the brain tissues for whole-mount retinas to improve the quality of standard immunohistochemical staining and high-resolution imaging of retinal neurons and their subcellular structures.

使用清晰组织清除方法对全山视网膜进行免疫染色

The tissue hydrogel delipidation method (CLARITY), originally developed by the Deisseroth laboratory, has been modified and widely used for immunostaining ...

Imaging and Quantification of Intact Neuronal Dendrites via CLARITY Tissue Clearing

Brain activity, the electrochemical signals passed between neurons, is determined by the connectivity patterns of neuronal networks, and from the morphology of processes and substructures within these neurons. As such, much of what is known about brain function has arisen alongside developments in imaging technologies that allow further insight into how neurons are organized and connected in the brain. Improvements in tissue clearing have allowed for high-resolution imaging of thick brain slices, facilitating morphological reconstruction and analyses of neuronal substructures, such as dendritic arbors and spines. In tandem, advances in image processing software provide methods of quickly analyzing large imaging datasets. This work presents a relatively rapid method of processing, visualizing, and analyzing thick slices of labeled neural tissue at high-resolution using CLARITY tissue clearing, confocal microscopy, and image analysis. This protocol will facilitate efforts toward understanding the connectivity patterns and neuronal morphologies that characterize healthy brains, and the changes in these characteristics that arise in diseased brain states.

Neuronal dendritic morphology often underlies function. Indeed, many disease processes that affect the development of neurons manifest with a morphological phenotype. This protocol describes a simple and powerful method for analyzing intact dendritic arbors and their associated spines.

通过 CLARITY 组织清除对完整神经元树突进行成像和定量

Brain activity, the electrochemical signals passed between neurons, is determined by the connectivity patterns of neuronal networks, and from the ...

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 ...

Establishing a Mouse Contusion Spinal Cord Injury Model Based on a Minimally Invasive Technique

Using minimally invasive methods to model spinal cord injury (SCI) can minimize behavioral and histological differences between experimental animals, thereby improving the reproducibility of the experiments.
These methods need two requirements to be fulfilled: clarity of the surgical anatomical pathway and simplicity and convenience of the laboratory device. Crucially for the operator, a clear anatomical pathway provides minimally invasive exposure, which avoids additional damage to the experimental animal during the surgical procedures and allows the animal to maintain a consistent and stable anatomical morphology during the experiment.
In this study, the use of a novel integrated platform called the SCI coaxial platform for spinal cord injury in small animals to expose the T9 level spinal cord in a minimally invasive way and stabilize and immobilize the vertebra of mice using a vertebral stabilizer is researched, and, finally, a coaxial gravity impactor is used to contuse the spinal cord of mice to approach different degrees of T9 spinal cord injury. Finally, histological results are provided as a reference for the readers.

Minimally invasive techniques and a simple laboratory device improve the reproducibility of the spinal cord injury model by reducing operative damage to the experimental animals and allowing anatomical morphology maintenance. The method is worthwhile because the reliable results and reproducible procedure facilitate investigations of the mechanisms of disease reparation.

基于微创技术的小鼠挫伤脊髓损伤模型的建立

Using minimally invasive methods to model spinal cord injury (SCI) can minimize behavioral and histological differences between experimental animals, ...

Establishment of Central Cord Syndrome Model in C57BL/6J Mouse

Animal models of central cord syndrome (CCS) could substantially benefit preclinical research. Identifiable anatomical pathways can give minimally invasive exposure approaches and reduce extra injury to experimental animals during operation, enabling the maintenance of consistent and stable anatomical morphology during experiments to minimize behavioral and histological differences between individuals to improve the reproducibility of experiments. In this study, the C6 level spinal cord was exposed using a spinal cord injury coaxial platform (SCICP) and combination with a minimally invasive technique. With the assistance of a vertebral stabilizator, we fixed the vertebrae and compressed the spinal cords of C57BL/6J mice with 5 g/mm2 and 10 g/mm2 weights with SCICP to induce different degrees of C6 spinal cord injury. In line with the previous description of CCS, the results reveal that the lesion in this model is concentrated in the gray matter around the central cord, enabling further research into CCS. Finally, histological results are provided as a reference for the readers.

The present protocol simulating central cord syndrome (CCS) in mice has improved repeatability and minimized operation damage to the experimental animals, avoiding disrupting the anatomical structure excessively. The strategy in this study is advantageous because it allows for research into injury mechanisms by producing consistent results.

C57BL/6J小鼠中索综合征模型的建立

Animal models of central cord syndrome (CCS) could substantially benefit preclinical research. Identifiable anatomical pathways can give minimally ...

Imaging the Gut with "CLARITY"

CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging compatible Tissue hYdrogel) has recently evolved as a valuable technique involving acrylamide embedding to delipidate tissue (without sectioning) and to preserve the 3-D tissue structure for immunostaining. The technique is highly relevant in imaging the dynamic gut environment where different cell types interact during homeostasis and disease states. This method optimized for the mouse gut is described here, which helps to trace cell types like epithelia, enteroendocrine, neurons, glia, and the neuronal projections into the epithelia or enteroendocrine cells that mediate microbial sensing or nutrient chemo sensing respectively. The gut tissue (1-1.5 cm) is fixed in 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS) at 4 &#176;C overnight on day 1. On day 2, PFA is discarded, and the tissue is washed thrice with PBS. The tissue is hydrogel embedded to preserve its integrity by incubation in 4% hydrogel (acrylamide) solution in PBS (diluted from 30% ProtoGel) overnight at 4 &#176;C. On day 3, the tissue-hydrogel solution is incubated at 37&#160;&#176;C for 1 h to allow hydrogel polymerization. Tissue is then washed thrice gently with PBS to remove excess hydrogel. The subsequent step of delipidation (clearing) involves tissue incubation in sodium dodecyl sulfate (8% SDS in PBS) at 37 &#176;C for 2 days (days 4 &#38; 5) on a shaker at room temperature (RT). On day 6, the cleared tissue is thoroughly washed with PBS to remove SDS. Tissue can be immunostained by incubation in primary antibodies (diluted in 0.5% normal donkey serum in PBS containing 0.3% Triton X-100), overnight at 4&#176;C, and subsequent incubation in appropriate secondary Alexa Fluor antibodies for 1.5 h at RT, and nuclear staining with DAPI (1: 10000). The tissue is transferred onto a clean glass slide and mounted using VectaShield for confocal imaging.

We describe the protocol for passive CLARITY (PACT) staining of mouse intestine to enable visualization of subepithelial tissues, including neurons, glia and enteroendocrine cells (EEC) without tissue sectioning. The protocol involves hydrogel embedding of formaldehyde-fixed tissue, and subsequent delipidation using an anionic detergent to "clear" the tissue.

CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging compatible Tissue hYdrogel) has recently evolved as a valuable technique involving ...