Name: a hydrophobic tissue clearing method for rat brain tissue
Uploaded: 2020-12-23T15:00:00.000+00:00
Duration: 8 min 24 s
Description: Watch this Scientific Journal Video about A Hydrophobic Tissue Clearing Method for Rat Brain Tissue at JoVE.com

这项工作由国家卫生研究院赠款资助:NS100624、DA013137、HD043680、MH106392 – T32 培训补助金 5T32GM081740

所有动物协议都经过南卡罗来纳大学动物护理和使用委员会的审查和批准。
1. 库存解决方案准备
<ol><li>解决方案 1 （1 L）: 到 900 mL 的去离子 H2O 添加 100 mL 的 10 倍磷酸盐缓冲盐水 （PBS） 和 2 mL 的 Triton X-100。</li>
	<li>解决方案 2 （1 L）: 至 900 mL 的去离子 H2O， 添加 100 mL 的 10 倍 PBS， 2mL 的 Tween-20 和 1 mL 的 10 毫克/mL 肝素库存解决方案。</li>
	<li>解决方案 3 （500 mL）: 至 400 mL 的解决方案 1，添加 11.5 克甘氨酸和 100 mL 二甲基硫化物 （DMSO）。</li>
	<li>解决方案 4 （50 mL）:要达到 42 mL 的解决方案 1，添加 3 mL 的相应血清和 5 mL 的 DMSO。</li>
	<li>初级抗体溶液 （50 mL）:要达到 46 mL 的解决方案 2，添加 2.5 mL 的 DMSO 和 1.5 mL 的相应血清。</li>
	<li>二级抗体溶液 （50 mL）:要达到 48.5 mL 的解决方案 2，添加 1.5 mL 的相应血清。 注:对于解决方案 4、初级溶液和二级溶液，请使用与二级抗体（例如山羊、马、牛）对应的血清。</li>
</ol>2. 样品制备
注:由于限制对 PFA 的暴露，请在烟雾罩中执行步骤 2.1 至 2.7。
<ol><li>使用 sevoflurane 对幼鼠（3-6 周）进行深度麻醉;当大鼠没有反应，对脚趾和尾巴捏没有反应时，继续到2.2。</li>
	<li>将老鼠放在一个超音位，用一把虹膜剪刀切开腹部壁。</li>
	<li>用梅奥剪刀切开肋骨笼底部，切到肋骨笼两侧的锁骨上。</li>
	<li>用下皮将胸骨和肋骨与心脏和肺部擦开。</li>
	<li>用23G针刺穿左心室，连接到灌注泵上，用虹膜剪刀切开右心房。</li>
	<li>用约 75 mL 的 100 mM （1x） PBS 进行跨心灌注。</li>
	<li>继续转心灌注，在 1x PBS 中缓冲约 100 mL 的 4% 半甲醛 （PFA）。</li>
	<li>用钳子把大脑从头骨上取下。</li>
	<li>将大脑置于下垂位置，然后使用剃须刀刀片和鼠脑矩阵切成4个相等部分（宽度约3毫米）。</li>
	<li>在密封的 5mL 塑料管中将每个部分在 1x PBS 中将每个部分在 4% PFA 中固定在摇床上，在 4 °C 处过夜。</li>
	<li>在室温下（RT）在摇床上将 4% PFA 固定在 1x PBS 中，持续固定 1 小时。</li>
	<li>在 RT 中用 1x PBS 清洗每个部分 30 分钟，3 次。</li>
</ol>3. 脱水和脱皮
<ol><li>在 RT 的 20% 甲醇/80% 去离子水溶液中孵育样品 1 小时。</li>
	<li>在 RT 的 40% 甲醇/60% 去离子水溶液中将样品孵育 1 小时。</li>
	<li>在 RT 的 60% 甲醇/40% 去离子水溶液中孵育样品 1 小时。</li>
	<li>在 RT 的 80% 甲醇/20% 去离子水溶液中将样品孵育 1 小时。</li>
	<li>在 Rt. 用超过 100% 甲醇重复 1 小时的 100% 甲醇孵化样品， 在 Rt 孵育 1 小时。</li>
	<li>将样品在 4° C 下 100% 甲醇中冷却约 10 分钟。</li>
	<li>准备 66% 二氯甲烷 （DCM）/33% 甲醇溶液。</li>
	<li>在 RT 的 DCM/甲醇溶液中将样品在一夜之间孵化，并进行摇晃。</li>
	<li>在 Rt 用甲醇清洗 30 分钟， 2 次。</li>
	<li>将样品在 4° C 下 100% 甲醇中冷却约 10 分钟。</li>
	<li>在 4° C 下，在甲醇中冷冻、新鲜制备的 5% 过氧化氢中漂白过夜。</li>
	<li>在 RT 的 80% 甲醇/20% 去离子水溶液中将样品孵育 1 小时。</li>
	<li>在 RT 的 60% 甲醇/40% 去离子水溶液中孵育样品 1 小时。</li>
	<li>在 RT 的 40% 甲醇/60% 去离子水溶液中将样品孵育 1 小时。</li>
	<li>在 RT 的 20% 甲醇/80% 去离子水溶液中孵育样品 1 小时。</li>
	<li>在 RT 将样品在 1x PBS 中孵育 1 小时。</li>
	<li>在 RT 中将溶液 1 洗涤 1 小时，2 次。</li>
</ol>4. 霍乱毒素子字组 B 标签
<ol><li>将 1 mL 注射器的针尖插入原子核累积位点。</li>
	<li>慢慢注射 2.5 μL 的 1% 生物素 CTB 超过 20 秒。</li>
	<li>将针尖留在原位 1 分钟，然后缓慢取出超过 10 秒，以防止泄漏。</li>
</ol>5. 抗体应用
<ol><li>在水浴中将样品在 37 °C 下在解决方案 3 中孵育 2 天。</li>
	<li>在水浴中将样品在溶液 4 中孵育 2 天，在 37 °C 下。</li>
	<li>在水浴中用原性抗体在 37 °C 下孵育 7 天。</li>
	<li>在解决方案 2 中清洗 5 次，每次洗涤孵育 1 小时。在 Rt 过夜存储解决方案 2 。</li>
	<li>在水浴中37°C下用二次抗体孵育7天。</li>
	<li>在解决方案 2 中清洗 5 次，每次洗涤孵化 1 小时;在 Rt 过夜的解决方案 2 中存储。 注:从 5.6 开始的所有步骤，包括最终存储，应在低光下进行。样品管可以用铝箔屏蔽。本协议中用于抗体的浓度为:TH在1:100，Iba1在1:200，二级抗体在1:100。在步骤 5.1-5.6 中添加额外的解决方案 2，以确保管子完全充满。</li>
</ol>6. 组织清除
<ol><li>在 RT 的 20% 甲醇/80% 去离子水溶液中孵育样品 1 小时。</li>
	<li>在 RT 的 40% 甲醇/60% 去离子水溶液中将样品孵育 1 小时。</li>
	<li>在 RT 的 60% 甲醇/40% 去离子水溶液中孵育样品 1 小时。</li>
	<li>在 RT 的 80% 甲醇/20% 去离子水溶液中将样品孵育 1 小时。</li>
	<li>在 RT 将样品在 100% 甲醇中孵育 1 小时。</li>
	<li>在 Rt 过夜孵育新鲜 100% 甲醇。</li>
	<li>在室温下以 66% DCM/33% 甲醇孵育 3 小时，并具有抖动。</li>
	<li>在二苯醚 （DBE） 中孵育，无需摇晃。将样品留在 DBE 中直到清除，并存储在 DBE 中直到成像。</li>
</ol>7. 安装
<ol><li>获取由 Visijet M3 水晶树脂制成的 3D 打印室（idisco.info 网站上提供模板）。</li>
	<li>使用环氧树脂将腔室固定在显微镜滑动器上。</li>
	<li>将样品放在房间中间的方形空间中。</li>
	<li>将房间完全填充 DBE。</li>
	<li>将 0.17 毫米厚的盖唇放在腔室上并施加压力，直到盖唇与腔室墙壁完全接触。</li>
	<li>使用环氧树脂将盖唇边缘密封到腔室。</li>
	<li>旋转腔室，让气泡从填充入口中逸出。</li>
	<li>给会议厅加满额外的 DBE。</li>
	<li>用环氧树脂密封灌装入口，并允许治愈。 注意:在安装过程中，多余的 DBE 会溢出。</li>
</ol>8. 成像
<ol><li>使用共聚焦显微镜系统进行 4 倍放大的 z 扫描（参见所附 的 3D 视频）。</li>
	<li>使用激光设置以类似于二级抗体指定的波长的波长发出样品的荧光激发。利用 HeNe 激光集以 632 nm 的速度发射，实现了 TH 激发。</li>
	<li>将探测器设置为接近激光发射的波长。对于当前协议，TH 探测器设置为 650 nm。</li>
	<li>提高探测器的增益，直到有可见信号。图像的 650LP 增益设置为 7.50 B。</li>
	<li>使用共聚焦成像软件中的 扫描 选项，移动显微镜阶段，直到样品成为焦点。</li>
	<li>在所有平原的组织周围导航，并使用共焦成像软件获取单个图像或 z 堆栈图像。</li>
	<li>成像完成后，将样品从腔室中取出。将其放回完全充满 DBE 的管子中，并存放在 RT 的黑暗地方，最好用铝箔覆盖。 注:只要荧光信号仍然强烈，样品就可以重新安装并成像。</li>
</ol>

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作者没有利益冲突要申报。

组织清除为传统 IHC 协议的局限性提供了解决方案。透明的样品可最大限度地减少光的散射和吸收，从而提供细胞水平的光学访问到完整的组织18，19。组织清除技术通过脱毛、去装饰和贴花与溶剂的管理使组织透明化。一旦组织样本的折射指数与所选成像介质的折射索引匹配，样本将呈现完全透明，并可以正确成像3。目前的疏水方案?...

一些组织清除技术已被验证用于大脑:水凝胶，亲水性和疏水性。这些技术旨在通过溶剂的管理，通过脱毛、去装饰和贴花来使组织透明化。一旦组织样本的折射指数与所选成像介质的折射指数匹配，就可以获得样本的清晰图像。水凝胶技术，如 CLARITY，通过将生物分子与丙烯酸酯水凝胶连接起来，确保组织中的生物分子的安全，从而防止结构损伤和蛋白质损失1.然而，水凝胶技术使用的苛刻化学物质比有可能损害更脆弱的组织，更密集的组织样本可能不符合水凝胶协议。某些水凝胶技术可能需要昂贵的设备或导致组织扩张。亲水技术，如立方体，通过在组织2内形成氢键来保存3D结构。组织扩张也可能发生在某些亲水协议中。亲水技术的清除能力往往与疏水技术的能力不相称，这对更稠密、更厚的组织非常重要。
疏水技术通常速度快，不需要特殊设备，并产生易于处理和存储的样品。iDISCO 是一种疏水技术，可消除其他疏水协议中可能发生的收缩。最初，DISCO组织清除技术是由Renier等人描述的 ，用于小鼠的胚胎和密集的成人器官。该技术在初始脱水步骤中去除组织中的水，从而减少光散射。组织在预处理过程中渗透，允许深层抗体渗透。远红光谱中的亚历克萨氟染料用于免疫标签，以避免组织在低波长5的自身发光。经过疏水清除协议的组织能够轻松处理，并且由于 Alexa 氟染料的寿命长，可以成像几次。如果储存得当，组织可以在初始处理后提供数月到一年的图像。
在本协议中，酪氨酸羟基酶（TH）抗体、Iba1抗体和霍乱毒素亚细胞B（CTB）用于男性和女性控制以及HIV-1转基因（Tg）大鼠大脑。HIV-1 Tg大鼠拥有构成HIV-1病毒基因组的9个基因中的7个，这导致了长期HIV-1蛋白暴露的非传染性模型6，7，8。多巴胺变种以前在HIV-1 Tg大鼠身上已经证明，HIV-1本身是一种炎症性疾病，所以抗体选择与实验设计9，10，11，12有关。CTB是一种通过结核结合附着在神经元的示踪器，可用于追踪大脑中令人不关心的投影。CTB以前曾被用来研究从细胞核到亚斯坦蒂娅黑鬼区域的投影，大脑的两个区域严重涉及多巴胺性通路13、14、15。
在此协议中，TH 将作为多巴胺生产的标志，Iba1 将标记激活的微胶质。TH 抗体使用选择性地标记单个带在大约 62 kDa，对应酪氨酸羟基酶。Iba1 抗体对应于 Iba1 卡盒终端序列。这两种抗体都是在兔子身上培养的，是多克隆的。CTB 将用于逆行追踪神经元从细胞核区到亚斯坦蒂娅黑鬼区域。当前协议还以两种不同的方式为原 iDISCO 协议的调整提供了指导:1） 通过更改阻塞步骤中的血清试剂来全面减少背景染色，2） 扩大孵化时间以适合较大的组织样本。总体而言，本协议提供了持续的证据，证明疏水清除技术在大鼠的脑组织中是可行的，与HIV-1病毒蛋白没有明显的有害相互作用，并且与TH抗体、Iba1抗体和CTB相容。

<table><tbody><tr><td>Cholera Toxin Subunit B (Recombinant), Alexa Fluor 488</td><td>Invitrogen</td><td>C34775</td><td></td></tr><tr><td>DBE</td><td>Sigma-Aldrich</td><td>108014-1KG</td><td></td></tr><tr><td>DCM</td><td>Sigma-Aldrich</td><td>270997-100mL</td><td></td></tr><tr><td>DMSO</td><td>Sigma-Aldrich</td><td>472301-1L</td><td></td></tr><tr><td>Glycine</td><td>Fisher Chemical</td><td>G46-500</td><td></td></tr><tr><td>Goat anti-rabbit Alexa Fluor Plus 647</td><td>Invitrogen</td><td>A32733</td><td></td></tr><tr><td>Goat serum</td><td>Sigma Life Science</td><td>G9023-10mL</td><td></td></tr><tr><td>Heparin</td><td>Acros Organics</td><td>41121-0010</td><td></td></tr><tr><td>Iba1 primary antibody</td><td>FUJIFILM Wako</td><td>019-19741</td><td></td></tr><tr><td>Kwik-sil epoxy</td><td>VWR</td><td>70730-062</td><td></td></tr><tr><td>Methanol</td><td>Sigma-Aldrich</td><td>34860-1l-R</td><td></td></tr><tr><td>PBS</td><td>Fisher Bioreagents</td><td>BP2944-100</td><td></td></tr><tr><td>Perfusion machine</td><td>VWR</td><td>70730-062</td><td>mini pump variable flow</td></tr><tr><td>PFA</td><td>Sigma-Aldrich</td><td>158127-3KG</td><td></td></tr><tr><td>TH primary antibody</td><td>Millipore Sigma</td><td>AB152</td><td></td></tr><tr><td>TritonX-100</td><td>Fisher Bioreagents</td><td>BP151-500</td><td></td></tr><tr><td>Tween-20</td><td>Fisher Bioreagents</td><td>BP337-500</td><td></td></tr></tbody></table>

a hydrophobic tissue clearing method for rat brain tissue

疏水组织清除方法易于调节、快速和低成本的程序，允许研究未受变组织样本中感兴趣的分子。传统的免疫标签程序要求将样品切成薄部分，这限制了标记和检查完整结构的能力。但是，如果脑组织在处理过程中能够保持完整，则结构和回路可以保持完好无损进行分析。先前建立的清除方法需要大量时间才能完全清除组织，而苛刻的化学物质通常会损害敏感的抗体。iDISCO 方法快速、完全地清除组织，与许多抗体兼容，无需特殊的实验室设备。这项技术最初被验证用于小鼠组织，但目前的协议使这种方法适应了控制半球和转基因大鼠大脑的图像。除此之外，本协议还对预先存在的协议进行了若干调整，以提供更清晰的图像，减少背景污渍。使用目前的疏水组织清除方法，在HIV-1转基因大鼠和F344/N控制大鼠中验证了Iba-1和酪氨酸羟基酶的抗体。大脑是一个交织在一起的网络，结构相互作用的频率高于彼此的相互作用。分析整个系统的大脑，而不是单个片段的组合，是整个大脑清理方法的最大好处。

利用这种经过修改的疏水组织清除方案，实现了F344/N和HIV-1大鼠脑组织的大面积完全清除。 图1 显示了在亚斯坦蒂娅尼格拉区域TH的典型共聚焦图像。 图 1A 表示密集、正色。通过"Z"平面聚焦以确认正染色和适当的细胞形态，可以分析出像这样的密集区域。 图1B 表示稀疏的正染色，可以通过TH神经元独特的形态来识别。 <strong class="...

Watch this Scientific Journal Video about A Hydrophobic Tissue Clearing Method for Rat Brain Tissue at JoVE.com

A Hydrophobic Tissue Clearing Method for Rat Brain Tissue

在这里，我们提出了一个疏水组织清除方法，允许查看目标分子作为完整的大脑结构的一部分。这项技术现已得到F344/N控制和艾滋病毒-1转基因两性大鼠的验证。

鼠脑组织疏水组织清除方法

Hydrophobic tissue clearing methods are easily adjustable, fast, and low-cost procedures that allows for the study of a molecule of interest in unaltered ...

a-hydrophobic-tissue-clearing-method-for-rat-brain-tissue

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

3.9K 次观看.  University of South Carolina. 在这里，我们提出了一个疏水组织清除方法，允许查看目标分子作为完整的大脑结构的一部分。这项技术现已得到F344/N控制和艾滋病毒-1转基因两性大鼠的验证。

视频: A Hydrophobic Tissue Clearing Method for Rat Brain Tissue

2-Vessel Occlusion/Hypotension: A Rat Model of Global Brain Ischemia

Cardiac arrest followed by resuscitation often results in dramatic brain damage caused by ischemia and subsequent reperfusion of the brain. Global brain ischemia produces damage to specific brain regions shown to be highly sensitive to ischemia 1. Hippocampal neurons have higher sensitivity to ischemic insults compared to other cell populations, and specifically, the CA1 region of the hippocampus is particularly vulnerable to ischemia/reperfusion 2.
The design of therapeutic interventions, or study of mechanisms involved in cerebral damage, requires a model that produces damage similar to the clinical condition and in a reproducible manner. Bilateral carotid vessel occlusion with hypotension (2VOH) is a model that produces reversible forebrain ischemia, emulating the cerebral events that can occur during cardiac arrest and resuscitation. We describe a model modified from Smith et al. (1984) 2, as first presented in its current form in Sanderson, et al. (2008) 3, which produces reproducible injury to selectively vulnerable brain regions 3-6. The reliability of this model is dictated by precise control of systemic blood pressure during applied hypotension, the duration of ischemia, close temperature control, a specific anesthesia regimen, and diligent post-operative care. An 8-minute ischemic insult produces cell death of CA1 hippocampal neurons that progresses over the course of 6 to 24 hr of reperfusion, while less vulnerable brain regions are spared. This progressive cell death is easily quantified after 7-14 days of reperfusion, as a near complete loss of CA1 neurons is evident at this time.
In addition to this brain injury model, we present a method for CA1 damage quantification using a simple, yet thorough, methodology. Importantly, quantification can be accomplished using a simple camera-mounted microscope, and a free ImageJ (NIH) software plugin, obviating the need for cost-prohibitive stereology software programs and a motorized microscopic stage for damage assessment.

Bilateral carotid occlusion coupled with systemic hypotension produces global brain ischemia in the rat, resulting in damage to the hippocampus with reproducible severity. Animal subjects are impaired with predictable patterns of brain damage, they recover expediently, and mortality rates are comparatively low.

2血管闭塞/低血压：全脑缺血模型大鼠

Cardiac arrest followed by resuscitation often results in dramatic brain damage caused by ischemia and subsequent reperfusion of the brain. Global brain ...

科研

医学

Metabolic Support of Excised, Living Brain Tissues During Magnetic Resonance Microscopy Acquisition

This protocol describes the procedures necessary to support normal metabolic functions of acute brain slice preparations during the collection of magnetic resonance (MR) microscopy data. While it is possible to perform MR collections on living, excised mammalian tissue, such experiments have traditionally been constrained by resolution limits and are thus incapable of visualizing tissue microstructure. Conversely, MR protocols that did achieve microscopic image resolution required the use of fixed samples to accommodate the need for static, unchanging conditions over lengthy scan times. The current protocol describes the first available MR technique that enables imaging of living, mammalian tissue samples at microscopic resolutions. Such data is of great importance to the understanding of how pathology-based contrast changes occurring at the microscopic level influence the content of macroscopic MR scans such as those used in the clinic. Once such an understanding is realized, diagnostic methods with greater sensitivity and accuracy can be developed, which will translate directly to earlier disease treatment, more accurate therapy monitoring and improved patient outcomes.
While the described methodology focuses on brain slice preparations, the protocol is adaptable to any excised tissue slice given that changes are made to the gas and perfusate preparations to accommodate the tissue&#39;s specific metabolic needs. Successful execution of the protocol should result in living, acute slice preparations that exhibit MR diffusion signal stability for periods up to 15.5 h. The primary advantages of the current system over other MR compatible perfusion apparatuses are its compatibility with the MR microscopy hardware required to attain higher resolution images and ability to provide constant, uninterrupted flow with carefully regulated perfusate conditions. Reduced sample throughput is a consideration with this design as only one tissue slice may be imaged at a time.

The current protocol describes a method by which users can maintain viability of acute hippocampal and cortical slice preparations during the collection of magnetic resonance microscopy data.

磁共振显微镜下切除活体脑组织的代谢支持

This protocol describes the procedures necessary to support normal metabolic functions of acute brain slice preparations during the collection of magnetic ...

生物工程

Sublimation of DAN Matrix for the Detection and Visualization of Gangliosides in Rat Brain Tissue for MALDI Imaging Mass Spectrometry

Sample preparation is key for optimal detection and visualization of analytes in Matrix-assisted Laser Desorption/Ionization (MALDI) Imaging Mass Spectrometry (IMS) experiments. Determining the appropriate protocol to follow throughout the sample preparation process can be difficult as each step must be optimized to comply with the unique characteristics of the analytes of interest. This process involves not only finding a compatible matrix that can desorb and ionize the molecules of interest efficiently, but also selecting the appropriate matrix deposition technique. For example, a wet matrix deposition technique, which entails dissolving a matrix in solvent, is superior for desorption of most proteins and peptides, whereas dry matrix deposition techniques are particularly effective for ionization of lipids. Sublimation has been reported as a highly efficient method of dry matrix deposition for the detection of lipids in tissue by MALDI IMS due to the homogeneity of matrix crystal deposition and minimal analyte delocalization as compared to many wet deposition methods 1,2. Broadly, it involves placing a sample and powdered matrix in a vacuum-sealed chamber with the samples pressed against a cold surface. The apparatus is then lowered into a heated bath (sand or oil), resulting in sublimation of the powdered matrix onto the cooled tissue sample surface. Here we describe a sublimation protocol using 1,5-diaminonaphthalene (DAN) matrix for the detection and visualization of gangliosides in the rat brain using MALDI IMS.

A protocol for the sublimation of DAN matrix onto rat brain tissue for the detection of gangliosides using MALDI Imaging Mass Spectrometry is presented.

DAN矩阵升华为神经节苷脂对大鼠脑组织的检测和可视化MALDI成像质谱

Sample preparation is key for optimal detection and visualization of analytes in Matrix-assisted Laser Desorption/Ionization (MALDI) Imaging Mass ...

神经科学

A Micro-CT-based Method for Characterizing Lesions and Locating Electrodes in Small Animal Brains

Lesion and electrode location verification are traditionally done via histological examination of stained brain slices, a time-consuming procedure that requires manual estimation. Here, we describe a simple, straightforward method for quantifying lesions and locating electrodes in the brain that is less laborious and yields more detailed results. Whole brains are stained with osmium tetroxide, embedded in resin, and imaged with a micro-CT scanner. The scans result in 3D digital volumes of the brains with resolutions and virtual section thicknesses dependent on the sample size (12&#8211;15 and 5&#8211;6 &#181;m&#160;per voxel for rat and zebra finch brains, respectively). Surface and deep lesions can be characterized, and single tetrodes, tetrode arrays, electrolytic lesions, and silicon probes can also be localized. Free and proprietary software allows experimenters to examine the sample volume from any plane and segment the volume manually or automatically. Because this method generates whole brain volume, lesions and electrodes can be quantified to a much higher degree than in current methods, which will help standardize comparisons within and across studies.

This article describes a straightforward method to prepare small animal brains for micro-CT imaging, in which lesions can be quantified and electrodes located with high precision in the context of the whole brain.

一种基于微 ct 的小动物脑损伤和定位电极表征方法

Lesion and electrode location verification are traditionally done via histological examination of stained brain slices, a time-consuming procedure that ...

Massive Pontine Hemorrhage by Dual Injection of Autologous Blood

We provide a protocol to establish a massive pontine hemorrhage model in a rat. Rats weighing about 250 grams were used in this study. One hundred microliters of autologous blood was taken from the tail vein and stereotaxically injected into the pons. The injection process was divided into 2 steps: First, 10 &#181;L of blood was injected into a specific location, anteroposterior position (AP) -9.0 mm; lateral (Lat) 0 mm; vertical (Vert) -9.2 mm, followed by a second injection of the residual blood located at AP -9.0 mm; Lat 0 mm; Vert -9.0 mm with a 20-minute interval. The balance beam test, limb placement test, and the modified Voestch neuroscore were used to evaluate neurological function. Magnetic Resonance Imaging (MRI) was used to assess the volume of hemorrhage in vivo. The symptoms of this model were in line with patients with massive pontine hemorrhage.

We present a protocol to establish a massive pontine hemorrhage model in a rat via dual injection of autologous blood.

大量庞廷出血通过双注射自体血

We provide a protocol to establish a massive pontine hemorrhage model in a rat. Rats weighing about 250 grams were used in this study. One hundred ...

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 ...

Investigation of Spatial Interaction Between Astrocytes and Neurons in Cleared Brains

Combining viral vector transduction and tissue clearing using the CLARITY method makes it possible to simultaneously investigate several types of brain cells and their interactions. Viral vector transduction enables the marking of diverse cell types in different fluorescence colors within the same tissue. Cells can be identified genetically by activity or projection. Using a modified CLARITY protocol, the potential sample size of astrocytes and neurons has grown by 2-3 orders of magnitude. The use of CLARITY allows the imaging of complete astrocytes, which are too large to fit in their entirety in slices, and the examination of the somata with all their processes. In addition, it provides the opportunity to investigate the spatial interaction between astrocytes and different neuronal cell types, namely, the number of pyramidal neurons in each astrocytic domain or the proximity between astrocytes and specific inhibitory neuron populations. This paper describes, in detail, how these methods are to be applied.

Combining viral vector transduction and brain clearing using the CLARITY method allows the investigation of a large number of neurons and astrocytes simultaneously.

研究清除大脑中星形胶质细胞和神经元之间的空间相互作用

Combining viral vector transduction and tissue clearing using the CLARITY method makes it possible to simultaneously investigate several types of brain ...

Extraction and Tissue Clearing Preparation of Mouse Brain-Spinal Cord Samples

Neuroscience research focused on the central nervous system necessitates a thorough understanding of various factors, including cell distribution, neuronal connectivity, and molecular dynamics. Traditional methodologies for investigating the entire brain and spinal cord often involve isolating, sectioning, and scanning tissue slices, followed by the labor-intensive process of three-dimensional image reconstruction. This conventional approach can be both time-consuming and cumbersome. Advancements in tissue clearing and whole-organ imaging techniques have revolutionized the analysis of the entire brain and spinal cord. To maximize the potential of these innovative methods, it is essential to extract and clear brain samples while maintaining their connection to the spinal cord. This protocol provides a detailed and systematic guide for preparing samples of the brain connected to the spinal cord, outlining the extraction and clearing procedures. By streamlining these processes, this approach significantly enhances experimental efficiency and data integrity, thereby fostering advancements in neuroscience research and enabling more comprehensive investigations into the complexities of the central nervous system.

This study presents a protocol for extracting and preparing cleared whole-brain and spinal cord samples with preserved fluorescent signals, enhancing experimental efficiency and data integrity to advance neuroscience research.

小鼠脑脊髓样品的提取和组织透明化制备

Neuroscience research focused on the central nervous system necessitates a thorough understanding of various factors, including cell distribution, ...

A Hydrophobic Tissue Clearing Method for a Rat Brain Tissue Sample

<a href='https://appjove.unicartagenaproxy.elogim.com/v/61821/a-hydrophobic-tissue-clearing-method-for-rat-brain-tissue'>Source: Kirchner, K.N., et al.,&#160;A Hydrophobic Tissue Clearing Method for Rat Brain Tissue. J. Vis. Exp.&#160;(2020).</a>This video demonstrates the method of hydrophobic tissue clearing of a rat brain tissue sample. The sample is sequentially dehydrated in methanol, treated with a dichloromethane and methanol mixture to dissolve lipids and pigments, and transferred to dibenzyl ether for complete tissue clearing, enabling detailed imaging without thin sectioning.

Source: Kirchner, K.N., et al.,&#160;A Hydrophobic Tissue Clearing Method for Rat Brain Tissue. J. Vis. Exp.&#160;(2020).This video demonstrates the ...

JoVE Encyclopedia of Experiments

Neuropathology

Neurodegenerative Diseases

A Method for the Isolation of Neurovascular Units from Rat Brain

Source: Brzica, H., et al,. <a href="https://appjove.unicartagenaproxy.elogim.com/v/57698">A simple and reproducible method to prepare membrane samples from freshly isolated rat brain microvessels.</a> J. Vis. Exp. (2018).
This video demonstrates the process of isolating brain microvessels from euthanized rats. It includes the dissection of the brain, cleaning of the tissue, and mechanical digestion, followed by repeated centrifugation to isolate the pure fraction of microvessels.

一种从大鼠脑中分离神经血管单位的方法

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines. The procedural flow for the protocol ...