Name: toluidine blue staining of resin-embedded sections for evaluation of peripheral nerve morphology
Uploaded: 2018-07-03T15:00:00.000+00:00
Duration: 8 min 17 s
Description: Watch this Scientific Journal Video about Toluidine Blue Staining of Resin-Embedded Sections for Evaluation of Peripheral Nerve Morphology at JoVE.com

作者希望 thankthe 詹金斯显微镜设施在怀俄明大学为他们的帮助, 并且布什曼实验室的成员, 凯利 Roballo, 海登真实, Wupu Osimanjiang 并且 Subash Dhunghana, 为动物关心的协助。这份出版物是由国立卫生研究院国立医学研究院的一个机构发展奖 (2P20GM103432) 授予的。

本项目使用成人大大鼠, 所有程序均经怀俄明州大学动物保育和使用委员会批准。
1. 手术和体内神经固定
注: 本协议中使用的所有材料和设备的供应商信息列在材料表中。
注:体内神经固定用于保存组织并减少在死亡时间和神经收集之间可能发生的结构退化。在体内组织固定是一种标准的做法, 以制备神经系统组织的组织学, 其中灌注往往是这一前兆。周围神经的位置和大小允许原位固定。我们不建议在安乐死后, 由于组织退化的可能性, 收集和固定组织。
<ol><li>准备大鼠全身麻醉, 将其放置在2-3% 异氟醚的感应腔内, 然后将麻醉大鼠置于解剖垫顶端的俯卧位, 并通过麻醉鼻锥维持1-2% 的异氟醚。为了确保足够深度的麻醉, 测试动物的踏板撤退的后腿, 并检查是否没有眼睑反射在眼睛通过触摸的眼睛内侧眼角。 注: 腹腔注射氯胺酮是一种常用的替代吸入异氟醚。</li>
	<li>为了暴露坐骨神经, 剃掉后肢的毛发, 用70% 乙醇清洁剃须的区域。用手术刀或剪刀, 在皮肤沿后腿的2-3 厘米切口从膝到更大的粗隆, 在那里触诊股骨使用无菌手术刀, 以确保切口的正确位置。股骨位于坐骨神经最接近的部分。虽然这是一个非生存手术, 保持无菌技术。</li>
	<li>在做皮肤切口后, 定位在两头肌肌肉和臀阔肌肌肉之间的平面, 并使用显微切割剪刀分开的基础筋膜和暴露2-3 厘米的下划线坐骨神经。为了暴露坐骨神经的明显部分, 使用牵引器扩大两个肌肉之间的间隙 (图 1A)。用细镊子和虹膜剪刀, 小心地将神经从周围的结缔组织中分离出来, 注意不要压缩或切断神经。</li>
	<li>添加足够的王牌的固定剂 (4% 甲醛, 1% 戊二醛在 1x PBS (磷酸盐缓冲盐水) 与1.16 克 NaH2PO4 ·H2O 每100毫升) 进入含有暴露神经的腔内以遮盖神经, 让其坐10分钟。如有必要, 在后腿下放置纱布以改善角度, 使更多的固定剂可以加入腔内。10分钟后取出固定件, 再重复此步骤两次。</li>
	<li>使用解剖显微镜, 切断双方的神经使用细解剖剪刀, 确保不伸展或捏坐骨神经, 并立即把神经节在15毫升管包含王牌的固定在4°c 一个星期, 改变固定每48小时。</li>
	<li>在手术过程中, 不要把动物留在无人看管的地方。神经切除后, 弄死在麻醉下颈椎脱位。</li>
</ol>2. 锇毒气处理和树脂嵌入
<ol><li>后固定, 小心地清除任何剩余的脂肪和结缔组织从神经和使用锋利的手术刀把神经切成段约5毫米长度 (神经段可以分离和标记在不同的管)。准备新鲜的2% 锇毒气稀释在王牌的固定液, 并保持在玻璃管。将2% 锇毒气中的神经段浸入2小时, 这在短时间内可以在塑料管上。 注: 一些塑料管与锇毒气反应, 导致溶液变暗, 因此不推荐在塑料管中长时间储存锇毒气。</li>
	<li>使用环氧树脂嵌入介质套件, 准备最后的嵌入公式:<ol>
<li>将环氧树脂嵌入介质与 DDSA 溶液 (dodecenylsuccinic 酸酐; 混合物 A) 混合, 并将环氧树脂嵌入介质与 NMA 溶液 (methylnadic 酸酐; 混合物 B) 混合。让混合物 a 和 B 混合至少20分钟通过搅拌与磁性搅拌器。</li>
		<li>在使用前, 混合混合物 A 和 B 并加入加速器 DPM-30 (24, 6-三 (dimethylaminomethyl) 苯酚) 的比例为1.5 到2.0% 的混合物的总体积。请注意, DPM-30 的解决方案应该精确地测量, 以防止块变暗的颜色和脆性。所有树脂化学物质都是有毒的;采取额外的照顾, 以防止皮肤接触或吸入。</li>
	</ol>
</li>
	<li>使用1.5 毫升管, 洗涤神经段与 PBS 和开始脱水过程通过安置神经段在不同的丙酮或蒸馏水段落: 30%, 60%, 90%, 浓度为10分钟每, 然后在100% 丙酮3次为10分钟每。 请注意, 乙醇可用于丙酮的地方。</li>
	<li>为树脂渗透, 安置神经片断在最后的嵌入的混合物的1个部分的混合物和1部分100% 丙酮为30分钟, 然后在最后的嵌入的混合物2个部分的混合物和1个部分100% 丙酮为30分钟, 最后将神经段放在最后的嵌入混合物中30分钟。</li>
	<li>将神经段放入硅橡胶嵌入模具 (我们使用了106毫米长 x 71 毫米宽度 x 7 毫米深度; 块尺寸 11 mm 长度 x 6 毫米宽度 x 3 毫米深度)。轻轻地将树脂放在神经的顶端, 确保覆盖整个神经段, 避免制造气泡。将含有神经段的模具留于树脂中, 聚合60摄氏度。</li>
</ol>3. Ultramicrotome 切片
<ol><li>用玻璃刀制造者 (图 1B) 制作玻璃刀, 用小船制造玻璃刀 (图 1C), 用来收集在蒸馏水上漂浮的神经部分。</li>
	<li>将树脂嵌入的神经块放置到 ultramicrotome 支架上, 梯形侧朝上。使用 ultramicrotome 的范围, 修剪任何多余的树脂周围的神经组织, 并使该块面对一个梯形形状和接近的纵向表面的神经尽可能使用单刃刀片, 如剃刀刀片。不要穿透神经段的纵面, 由锇毒气的深色染色可辨认。 注: 多余树脂的修整将减少树脂的面积, 提高刀片的性能, 并在随后的步骤中进行切割。</li>
	<li>使用 ultramicrotome, 使多个横断面足以暴露一个统一的断面表面的神经组织使用普通的玻璃刀。一旦完成, 切换到一个玻璃刀, 其船在室温下充满蒸馏水, 并调整 ultramicrotome 到1-2 微米, 以削减薄部分。</li>
	<li>将浮动薄剖面从玻璃刀船转移到玻璃滑梯上的去离子水上, 使用金属环 (图 1D)。干燥的幻灯片包含部分, 通过多次在火焰上传递几秒钟, 确保不过热的部分。染色后, 立即用甲苯胺蓝染干切片或在室温下贮存几天。 注意: 切片也可以用60摄氏度的热板干燥15分钟。缓慢的干燥过程使剖面平滑。</li>
</ol>4. 甲苯胺蓝染色
<ol><li>在100毫升去离子水中溶解2克硼酸钠, 然后加入1克甲苯胺蓝, 搅拌直至溶解, 制备甲苯胺蓝1% 溶液。用滤纸 (孔径:11 µm) 过滤溶液, 在室温下将溶液保持在不透明的瓶子中长达两周。</li>
	<li>使用塑料吸管或微 pipettor, 增加一滴甲苯胺蓝溶液的顶部的神经部分, 并离开 20-30 s。</li>
	<li>冲洗所有甲苯胺蓝溶液过剩通过轻轻地把幻灯片浸入去离子水罐子, 重复3-4 次, 直到部分是明确的。干燥幻灯片至少15分钟, 在60摄氏度或隔夜在室温下, 然后覆盖的部分与盖玻片使用定期安装介质。立即检查已安装的幻灯片或在室温下存储。</li>
	<li>在光显微镜下检查。对于用于计算 g 比值的详细图像, 建议使用100X 油浸出透镜。</li>
</ol>

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作者声明他们没有利益冲突。

对周围神经损伤和再生的形态学结构的检查是研究的13个经常科目。在本协议中, 我们描述了获取高品质图像的步骤, quantifications 利用大鼠坐骨神经组织嵌入树脂块和染色甲苯胺蓝的组织学数据。该技术提供了一种神经形态学的图像, 可以通过测量轴突数、髓鞘形成程度、浸润性纤维化组织的存在和健康神经来量化神经再生。虽然图像显示切片和处理未受伤的神经作为样本, 同样的步骤和材料适用于受伤的神经和神经再生的导管或组织移植, 只要组织的固定是足够的20, 21.
虽然该协议的所有步骤都是必不可少的, 但其中一些步骤更可能导致质量较差的部分或图像。组织脱水不足可能导致神经部分的空洞 (图 5B)。一个可能的解释是树脂与水的不混溶, 组织中多余的水分防止树脂渗入组织。因此, 允许充分的时间和使用绝对丙酮是必不可少的, 以确保适当的脱水步骤。虽然我们使用丙酮在这个协议, 乙醇也可以使用。然而, 许多树脂对乙醇没有反应, 因此必须用环氧丙烷处理组织, 作为乙醇脱水和树脂嵌入之间的过渡。
由于切片的薄, 将神经部分从玻璃刀转移到玻璃滑梯应该非常小心 (图 5C)。1-2 微米的断面非常脆弱, 不适当的转移会导致剖面断裂。如果该节是用针在部分的中心部分拾取的, 则该部分容易折叠起来, 并且在转移到幻灯片时往往很难展开。不同的工具可用于将截面转移到玻璃滑轨上, 包括针和环, 每一个都应测试特定用户, 以确定哪些将产生最佳的传输结果。对于我们的目的, 使用一个可以包含整个部分的循环大大减少了部分的折叠, 当他们从玻璃刀转移到显微镜幻灯片 (图 1D)。
一般来说, 正确的处理神经是必不可少的, 因为压迫神经可能导致裂缝发生在神经部分 (图 5A)。神经组织压缩可能发生在神经节转移过程中的动物暴露, 固定, 脱水和树脂嵌入。为了避免神经组织压迫, 神经段应通过细钳转移, 并在段的一端通过外膜层来选择, 所以整个神经不会受到影响。在神经部分出现裂纹和线 (刀痕) 也可能是由于钝或过度用过的玻璃刀造成的。为了保证最佳切片, 我们建议制作新鲜的玻璃刀和改变玻璃刀每25-30 削减, 更少, 如果通过神经包裹在管道内。
树脂可以是相对昂贵的, 是不够的, 当纵向神经部分是必需的, 并可能需要昂贵的工具, 如 ultramicrotome 和玻璃刀制造商。尽管有这些局限性, 甲苯胺蓝染色的树脂嵌入周围神经部分仍然被认为是金标准的可视化的外围神经形态学4,5。纵断面也可以揭示周围神经的重要特征, 如轴突的连续性和 Ranvier 的节点, 但最有用的免疫组化。在这种情况下, 一般的做法是差异处理两个部分的同一神经, 一个为甲苯胺蓝横断面, 另一个用于纵向免疫组化。由于周围神经损伤和损伤的发生率增加以及神经形态学结构评估方法的需要, 该方法可作为获取神经损伤和再生组织学数据的重要工具。

周围神经延伸整个身体, 支配靶组织与马达或感觉轴突1。由医疗失调和外伤引起的周围神经缺损是一个重大的公共卫生问题, 对2、3的经济影响很大。尽管评估周围神经损伤的结果和了解神经再生的进展, 传统的方法, 如神经组织学和染色技术是定性和定量评估神经健康的基本工具作为最终的结果测量动物模型或切除人体组织。这通常与周围神经功能的电生理测量成配对, 在那里, 形态计量学可以揭示功能性神经再生的原因或没有发生。
甲苯胺蓝染色的树脂嵌入半薄外围神经切片是一种专门的方法, 以成像髓神经纤维, 提供高质量和清晰的详细图像的神经结构4,5,6.甲苯胺蓝是一个耐热 metachromatic 染色, 发现的威廉亨利珀在 1856年7, 并已用于几个医疗应用8。甲苯胺蓝染色的周围神经部分获得的树脂嵌入神经段允许清晰的可视化神经结构。使用锇毒气后固定4,9可增强髓鞘结构的可视化。锇毒气是一种有毒的氧化剂和脂质固定剂, 与脂质中的双键相互作用, 导致了明显的富含脂质的髓鞘10。然而, 锇毒气是有毒的, 昂贵的, 需要更长的神经段的孵化, 并不总是使用。
对周围神经形态学的可视化处理和染色方法进行了研究;石蜡, 低温切片, 环氧树脂嵌入神经切片后, 以甲苯胺蓝或苯二胺溶液染色, 用于定量测定周围神经再生11,12 的形态学变化..这些方法各自有各自的优势和产生的关键数据的轴突, 髓鞘厚度, 轴突直径, 轴突直径髓纤维直径 (g 比) 11,13,14,15.
该协议中的树脂嵌入的主要区别是它促进获得1-2 微米厚度剖面由于树脂的硬度, 同时保持神经的组织学质量。这些稀薄的部分, 相对于从石蜡嵌入获得的4-5 微米厚度部分, 提供了更高的分辨率的外围神经部分, 允许更准确的量化轴突髓鞘形成, 如 g 比, 这不能从较厚的部分16获得。虽然低温切片可用于获得1-2 µm 剖面, 但我们的经验是, 很难获得部分没有大量的大裂缝。这些破裂的部分可能导致不准确计数的轴突的数量和方面的髓鞘形成。
除了甲苯胺蓝染色17, 银染色法18和马尾松的三色染色4也可以用来显示神经轴突。然而, 使用树脂嵌入的大鼠中位神经切片染色的苏木精和三色或马尾的, 显示微弱的髓鞘和无法识别的结构, 而甲苯胺蓝染色显示明确的髓鞘图像, 容易能够量化4。尽管有一定的局限性, 甲苯胺蓝染色的树脂嵌入周围神经是一个宝贵的技术, 可用于当高分辨率图像的神经形态学需要。
树脂嵌入的主要缺点是它是费时的, 并且不允许同一个组织的染色由于抗原检索的困难与石蜡和被冻结的嵌入的部分技术相比。因此, 一般不可能利用相同的组织为染色, 通过树脂嵌入处理甲苯胺蓝染色。虽然在这里不使用, 如果在树脂嵌入部分需要免疫组化, 使用乙二醇甲基丙烯酸酯嵌入树脂允许免疫组化在组织切片, 但它是相对昂贵的19。这可以通过将周围神经分割成分开的部分来减轻, 有些用于植入树脂, 或者在固定后直接染色。
甲苯胺蓝染色的树脂嵌入周围神经的过程, 如大多数病理学分析, 可分为五个阶段, 包括固定, 脱水, 植入, 切片, 染色20。本实验旨在为应用树脂嵌入甲苯胺蓝染色的大鼠坐骨神经切片提供一种实用的方案和实践指导, 以获得高质量的图像。

<table><tbody><tr><td>Dulbecco&#039;s Phosphate Buffer Saline</td><td>Gibco</td><td>14200-075</td><td></td></tr><tr><td>Glutaraldehyde Solution</td><td>Sigma-Aldrich</td><td>G6257</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>P6148</td><td></td></tr><tr><td>Sodium Phosphate Monobasic Monohydrate</td><td>Sigma-Aldrich</td><td>S9638</td><td></td></tr><tr><td>Toluidine Blue O</td><td>Sigma-Aldrich</td><td>T3260</td><td></td></tr><tr><td>Sodium Tetraborate Decahydrate</td><td>Acros Organics</td><td>205950010</td><td></td></tr><tr><td>Isoflurane</td><td>Piramal</td><td>NDC 66794-013-25</td><td></td></tr><tr><td>Epoxy Embedding Medium Kit</td><td>Sigma-Aldrich</td><td>45359</td><td></td></tr><tr><td>Sodium Hydroxide Solution</td><td>Sigma-Aldrich</td><td>72068</td><td>To adjust Trump&#039;s fixative pH</td></tr><tr><td>Acetone</td><td>Fisher Chemical</td><td>170942</td><td></td></tr><tr><td>Osmium Tetroxide Solution</td><td>Sigma-Aldrich</td><td>75632</td><td></td></tr><tr><td>VWR Micro Slides, Superfrost Plus</td><td>VWR</td><td>48311-703</td><td></td></tr><tr><td>Microscope Cover Glass</td><td>Fisher Scientific</td><td>12545102</td><td></td></tr><tr><td>Pelco Embedding Cast</td><td>Fisher Scientific</td><td>NC9671811</td><td></td></tr><tr><td>Glass Knife Maker</td><td>RMC Products</td><td>GKM-2</td><td></td></tr><tr><td>Ultramicrotome</td><td>RMC Products</td><td>MT-XL</td><td></td></tr><tr><td>15 mL Conical Tube</td><td>Falcon</td><td>ISO 9001</td><td></td></tr><tr><td>Eppendorf 1.5 mL microcentrifuge tubes</td><td>Sigma-Aldrich</td><td>T9661</td><td></td></tr><tr><td>4 mL Glass Vial</td><td>Sigma-Aldrich</td><td>854190</td><td></td></tr><tr><td>Razor Blades</td><td>VWR</td><td>55411-050</td><td>For trimming resin block</td></tr><tr><td>Perfect Loop</td><td>Electron Microscopy Sciences</td><td>70944</td><td>For picking up thin resin sections</td></tr><tr><td>Ultra Glass Knife Strips 6.4 mm x 25 mm x 400 mm</td><td>Electron Microscopy Sciences</td><td>71012</td><td></td></tr><tr><td>100 Watt Oven</td><td>Millipore&amp;nbsp;</td><td>6350115</td><td></td></tr><tr><td>Whatman Filter Paper</td><td>Sigma-Aldrich</td><td>WHA10010155</td><td></td></tr><tr><td>3 mL plastic pipette</td><td>Sigma-Aldrich</td><td>Z331740</td><td></td></tr><tr><td>Micro-surgical Kit</td><td>World precision instruments</td><td></td><td></td></tr><tr><td>Olympus fluorescence microscope</td><td></td><td></td><td>Dual CCD Color and Monochrome Camera, DP80</td></tr></tbody></table>

toluidine blue staining of resin-embedded sections for evaluation of peripheral nerve morphology

周围神经延伸整个身体, 支配靶组织与马达或感觉轴突。由于广泛的分布, 周围神经经常因外伤或疾病而受损。由于对周围神经损伤的动物模型、功能和再生方法和策略进行了评估, 分析周围神经的形态计量已成为一个重要的终端结果测量。甲苯胺蓝染色神经横断面从树脂嵌入神经切片是一种重现性的方法, 定性和定量评估周围神经, 使可视化的形态学数字的轴突和程度髓鞘形成.这项技术, 和其他许多组织学方法一样, 很难用标准的书面协议来学习和掌握。因此, 本出版物的目的是强调书面协议, 以甲苯胺蓝染色的周围神经与录像的方法, 利用从大鼠的坐骨神经。在本协议中, 我们描述了在体外周神经固定和组织收集, 并后固定与2% 锇毒气, 嵌入神经在环氧树脂, ultramicrotome 切片的神经1-2μm 厚度。神经部分然后转移到玻璃滑动和染甲苯胺蓝色, 在之后他们定量地和定性地被评估。显示了最常见问题的例子, 以及缓解这些问题的步骤。

树脂嵌入周围神经部分染色甲苯胺蓝允许准确的组织学数据 quantifications。过程概述 (图 2)。在树脂培养基中嵌入的坐骨神经部分和甲苯胺蓝染色显示清晰的图像, 最佳分辨率 (图 3)。神经损伤可引起神经形态学结构的许多变化, 如神经纤维、轴突直径和髓鞘厚度的变化。该方法能保持神经结构的自然形态, 有利于测量轴突直径与总纤维直径的比值 (g 比;图 4)。由于神经处理不当, 各种因素可能导致不到最佳的外围神经部分, 包括出现裂纹 (图 5A)。由于脱水不足, 部分也可能出现孔 (图 5B)。该过程中的另一个可能的错误是折叠的部分 (图 5C), 这可以通过使用接种循环的部分转移到玻璃滑梯上补救。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58031/58031fig1.jpg"> 图 1: 手术和切片.(A)大鼠坐骨神经在体内固定期间。(B)玻璃刀制造者(C)带船的玻璃刀。(D)金属循环用于从玻璃刀上转移漂薄的部分到玻璃滑梯上的去离子水滴。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58031/58031fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58031/58031fig2.jpg"> 图 2: 本协议中使用的方法示意图.(A)成人大大鼠麻醉, 其次为体内神经固定(B)和神经收集(C)。下一步是固定后的2% 锇毒气(D)和树脂嵌入(E)。树脂嵌入神经部分然后修剪(F)和切片(G)使用 ultramicrotome。最后, 用甲苯胺蓝(H)染色玻璃滑块上的神经切片。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58031/58031fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58031/58031fig3.jpg"> 图 3: 用甲苯胺蓝染色的大鼠坐骨神经横断面.以最佳分辨率显示清晰图像的不同放大 (10X、20X、40X 和 60X) 下的节。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58031/58031fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58031/58031fig4.jpg"> 图 4: 用甲苯胺蓝染色的大鼠坐骨神经横断面.该高分辨率图像 (100X) 可用于测量轴突直径与总纤维直径 (g 比值) 的比值。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58031/58031fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58031/58031fig5.jpg"> 图 5: 在不同放大下, 大鼠坐骨神经的横断面染色甲苯胺蓝.显示了一些在执行本协议时可能遇到的问题。(a)和(B)。在神经节内存在裂纹和孔洞, 原因是该段处理不当以及组织的脱水不足。(C)折叠的部分, 这可能发生在把部分从玻璃刀转移到幻灯片。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58031/58031fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Toluidine Blue Staining of Resin-Embedded Sections for Evaluation of Peripheral Nerve Morphology at JoVE.com

Toluidine Blue Staining of Resin-Embedded Sections for Evaluation of Peripheral Nerve Morphology

在这里, 我们提出了一个通过获取和染色1-2 µm 段与甲苯胺蓝可视化周围神经精细结构的协议

甲苯胺蓝染色树脂嵌段评价周围神经形态学

Peripheral nerves extend throughout the body, innervating target tissues with motor or sensory axons. Due to widespread distribution, peripheral nerves ...

toluidine-blue-staining-resin-embedded-sections-for-evaluation

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

18.8K 次观看.  University of Wyoming. 在这里, 我们提出了一个通过获取和染色1-2 µm 段与甲苯胺蓝可视化周围神经精细结构的协议

视频: Toluidine Blue Staining of Resin-Embedded Sections for Evaluation of Peripheral Nerve Morphology

Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

Going beyond single gene function to cut deeper into gene regulatory networks requires multiple mutations combined in a single animal. Such analysis of two or more genes needs to be complemented with in situ hybridization of other genes, or immunohistochemistry of their proteins, both in whole mounted developing organs or sections for detailed resolution of the cellular and tissue expression alterations. Combining multiple gene alterations requires the use of cre or flipase to conditionally delete genes and avoid embryonic lethality. Required breeding schemes dramatically enhance effort and cost proportional to the number of genes mutated, with an outcome of very few animals with the full repertoire of genetic modifications desired. Amortizing the vast amount of effort and time to obtain these few precious specimens that are carrying multiple mutations necessitates tissue optimization. Moreover, investigating a single animal with multiple techniques makes it easier to correlate gene deletion defects with expression profiles. We have developed a technique to obtain a more thorough analysis of a given animal; with the ability to analyze several different histologically recognizable structures as well as gene and protein expression all from the same specimen in both whole mounted organs and sections. Although mice have been utilized to demonstrate the effectiveness of this technique it can be applied to a wide array of animals. To do this we combine lipophilic dye tracing, whole mount in situ hybridization, immunohistochemistry, and histology to extract the maximal possible amount of data.

Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

A combination of different techniques to maximize data collection from mouse tissue is presented.

结合亲脂性染料，原位杂交，免疫组织化学和组织学

Going beyond single gene function to cut deeper into gene regulatory networks requires multiple mutations combined in a single animal.  Such analysis of ...

科研

神经科学

Tissue Preparation and Immunostaining of Mouse Sensory Nerve Fibers Innervating Skin and Limb Bones

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in animals and humans. These peripheral sensory nerve fibers express a plethora of receptors and ion channel proteins which detect and initiate specific sensory stimuli. Methods are available to characterize the electrical properties of peripheral sensory nerve fibers innervating the skin, which can also be utilized to identify the functional expression of specific ion channel proteins in these fibers. However, similar electrophysiological methods are not available (and are also difficult to develop) for the detection of the functional expression of receptors and ion channel proteins in peripheral sensory nerve fibers innervating other visceral organs, including the most challenging tissues such as bone. Moreover, such electrophysiological methods cannot be utilized to determine the expression of non-excitable proteins in peripheral sensory nerve fibers. Therefore, immunostaining of peripheral/visceral tissue samples for sensory nerve fivers provides the best possible way to determine the expression of specific proteins of interest in these nerve fibers. So far, most of the protein expression studies in sensory neurons have utilized immunostaining procedures in sensory ganglia, where the information is limited to the expression of specific proteins in the cell body of specific types or subsets of sensory neurons. Here we report detailed methods/protocols for the preparation of peripheral/visceral tissue samples for immunostaining of peripheral sensory nerve fibers. We specifically detail methods for the preparation of skin or plantar punch biopsy and bone (femur) sections from mice for immunostaining of peripheral sensory nerve fibers. These methods are not only key to the qualitative determination of protein expression in peripheral sensory neurons, but also provide a quantitative assay method for determining changes in protein expression levels in specific types or subsets of sensory fibers, as well as for determining the morphological and/or anatomical changes in the number and density of sensory fibers during various pathological states. Further, these methods are not confined to the staining of only sensory nerve fibers, but can also be used for staining any types of nerve fibers in the skin, bones and other visceral tissue.

Immunocytochemical identification of peripheral sensory nerve fiber subtypes (and detection of protein expression therein) are key to the understanding of molecular mechanisms underlying peripheral sensation. Here we describe methods for preparation of peripheral/visceral tissue samples, such as skin and limb bones, for specific immunostaining of peripheral sensory nerve fibers.

鼠标感觉神经支配的皮肤和四肢骨骼纤维组织的制备及免疫染色

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in ...

Techniques for Processing Eyes Implanted With a Retinal Prosthesis for Localized Histopathological Analysis

With the recent development of retinal prostheses, it is important to develop reliable techniques for assessing the safety of these devices in preclinical studies. However, the standard fixation, preparation, and automated histology procedures are not ideal. Here we describe new procedures for evaluating the health of the retina directly adjacent to an implant. Retinal prostheses feature electrode arrays in contact with eye tissue. Previous methods have not been able to spatially localize the ocular tissue adjacent to individual electrodes within the array. In addition, standard histological processing often results in gross artifactual detachment of the retinal layers when assessing implanted eyes. Consequently, it has been difficult to assess localized damage, if present, caused by implantation and stimulation of an implanted electrode array. Therefore, we developed a method for identifying and localizing the ocular tissue adjacent to implanted electrodes using a (color-coded) dye marking scheme, and we modified an eye fixation technique to minimize artifactual retinal detachment. This method also rendered the sclera translucent, enabling localization of individual electrodes and specific parts of an implant. Finally, we used a matched control to increase the power of the histopathological assessments. In summary, this method enables reliable and efficient discrimination and assessment of the retinal cytoarchitecture in an implanted eye.

Techniques for visualizing retinal cytoarchitecture directly adjacent to individual electrodes within a retinal stimulator.

技术处理眼睛的视网膜假体植入局部组织病理学分析

With  the recent development of retinal prostheses, it is important to develop  reliable techniques for assessing the safety of these devices in ...

医学

Transplantation of Olfactory Ensheathing Cells to Evaluate Functional Recovery after Peripheral Nerve Injury

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory system is characterized by its ability to give rise to new neurons even in adult animals. This particular ability is partly due to the presence of OECs which create a favorable microenvironment for neurogenesis. This property of OECs has been used for cellular transplantation such as in spinal cord injury models. Although the peripheral nervous system has a greater capacity to regenerate after nerve injury than the central nervous system, complete sections induce misrouting during axonal regrowth in particular after facial of laryngeal nerve transection. Specifically, full sectioning of the recurrent laryngeal nerve (RLN) induces aberrant axonal regrowth resulting in synkinesis of the vocal cords. In this specific model, we showed that OECs transplantation efficiently increases axonal regrowth.
OECs are constituted of several subpopulations present in both the olfactory mucosa (OM-OECs) and the olfactory bulbs (OB-OECs). We present here a model of cellular transplantation based on the use of these different subpopulations of OECs in a RLN injury model. Using this paradigm, primary cultures of OB-OECs and OM-OECs were transplanted in Matrigel after section and anastomosis of the RLN. Two months after surgery, we evaluated transplanted animals by complementary analyses based on videolaryngoscopy, electromyography (EMG), and histological studies. First, videolaryngoscopy allowed us to evaluate laryngeal functions, in particular muscular cocontractions phenomena. Then, EMG analyses demonstrated richness and synchronization of muscular activities. Finally, histological studies based on toluidine blue staining allowed the quantification of the number and profile of myelinated fibers.
All together, we describe here how to isolate, culture, identify and transplant OECs from OM and OB after RLN section-anastomosis and how to evaluate and analyze the efficiency of these transplanted cells on axonal regrowth and laryngeal functions.

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth of the primary olfactory neurons. This specific property can be used for cellular transplantation. We present here a model of cellular transplantation based on the use of OECs in a laryngeal nerve injury model.

嗅鞘细胞移植周围神经损伤后，以评估功能恢复

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory ...

Assessment of Ultrastructural Neuroplasticity Parameters After In Utero Transduction of the Developing Mouse Brain and Spinal Cord

The present study combines in utero transduction with transmission electron microscopy (TEM) aiming at a precise morphometrical analysis of ultrastructural parameters in unambiguously identified topographical structures, affected by a protein of interest that is introduced into the organism via viral transfer. This combined approach allows for a smooth transition from macrostructural to ultrastructural identification by following topographical navigation maps in a tissue atlas. High-resolution electron microscopy of the in-utero-transduced tissue reveals the fine ultrastructure of the neuropil and its plasticity parameters, such as cross-sectioned synaptic bouton areas, the number of synaptic vesicles and mitochondria within a bouton profile, the length of synaptic contacts, cross-sectioned axonal areas, the thickness of myelin sheaths, the number of myelin lamellae, and cross-sectioned areas of mitochondria profiles. The analysis of these parameters reveals essential insights into changes of ultrastructural plasticity in the areas of the nervous system that are affected by the viral transfer of the genetic construct. This combined method can not only be used for studying the direct effect of genetically engineered biomolecules and/or drugs on neuronal plasticity but also opens the possibility to study the in utero rescue of neuronal plasticity (e.g., in the context of neurodegenerative diseases).

The combination of transmission electron microscopy and in utero transduction is a powerful approach for studying morphological changes in the fine ultrastructure of the nervous system during development. This combined method allows deep insights into changes in structural details underlying neuroplasticity with respect to their topographical representation.

小鼠脑脊髓子宫转导后超微结构神经可塑性参数的评价

The present study combines in utero transduction with transmission electron microscopy (TEM) aiming at a precise morphometrical analysis of ...

发育生物学

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

Nerve endings in skin are involved in physiological processes such as sensing1 as well as in pathological processes such as neuropathic pain2. Their close-to-surface positioning facilitates microscopic imaging of skin nerve endings in living intact animal. Using multiphoton microscopy, it is possible to obtain fine images overcoming the problem of strong light scattering of the skin tissue. Reporter transgenic mice that express EYFP under the control of Thy-1 promoter in neurons (including periphery sensory neurons) are well suited for the longitudinal studies of individual nerve endings over extended periods of time up to several months or even life-long. Furthermore, using the same femtosecond laser as for the imaging, it is possible to produce highly selective lesions of nerve fibers for the studies of the nerve fiber restructuring. Here, we present a simple and reliable protocol for longitudinal multiphoton in vivo imaging and laser-based microsurgery on mouse skin nerve endings.

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

The protocol for dynamic longitudinal imaging and selective laser lesion of nerve endings in reporter transgenic mice is presented.&#160;

在皮肤中的单神经末梢体内双光子显微镜

Nerve endings in skin are involved in physiological processes such as sensing1 as well as in pathological processes such as neuropathic pain2. Their ...

Experimental Demyelination and Remyelination of Murine Spinal Cord by Focal Injection of Lysolecithin

Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system characterized by plaque formation containing lost oligodendrocytes, myelin, axons, and neurons. Remyelination is an endogenous repair mechanism whereby new myelin is produced subsequent to proliferation, recruitment, and differentiation of oligodendrocyte precursor cells into myelin-forming oligodendrocytes, and is necessary to protect axons from further damage. Currently, all therapeutics for the treatment of multiple sclerosis target the aberrant immune component of the disease, which reduce inflammatory relapses but do not prevent progression to irreversible neurological decline. It is therefore imperative that remyelination-promoting strategies be developed which may delay disease progression and perhaps reverse neurological symptoms. Several animal models of demyelination exist, including experimental autoimmune encephalomyelitis and curprizone; however, there are limitations in their use for studying remyelination. A more robust approach is the focal injection of toxins into the central nervous system, including the detergent lysolecithin into the spinal cord white matter of rodents. In this protocol, we demonstrate that the surgical procedure involved in injecting lysolecithin into the ventral white matter of mice is fast, cost-effective, and requires no additional materials than those commercially available. This procedure is important not only for studying the normal events involved in the remyelination process, but also as a pre-clinical tool for screening candidate remyelination-promoting therapeutics.

Demyelinating diseases can be modeled in animals by focal application of lysolecithin into the CNS. A single injection of lysolecithin into mouse spinal cord produces a lesion that spontaneously repairs over time. The goal is to study factors involved in de- and remyelination, and to test agents for enhancing repair.

实验脱髓鞘和小鼠脊髓髓鞘再生由溶血卵磷脂的焦点注射

Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system characterized by plaque formation containing lost ...

Optical Monitoring of Living Nerve Terminal Labeling in Hair Follicle Lanceolate Endings of the Ex Vivo Mouse Ear Skin

A novel dissection and recording technique is described for optical monitoring staining and de-staining of lanceolate terminals surrounding hair follicles in the skin of the mouse pinna. The preparation is simple and relatively fast, reliably yielding extensive regions of multiple labeled units of living nerve terminals to study uptake and release of styryl pyridinium dyes extensively used in studies of vesicle recycling. Subdividing the preparations before labeling allows test vs. control comparisons in the same ear from a single individual. Helpful tips are given for improving the quality of the preparation, the labeling and the imaging parameters. This new system is suitable for assaying pharmacologically and mechanically-induced uptake and release of these vital dyes in lanceolate terminals in both wild-type and genetically modified animals. Examples of modulatory influences on labeling intensity are given.

Optical Monitoring of Living Nerve Terminal Labeling in Hair Follicle Lanceolate Endings of the Ex Vivo Mouse Ear Skin

Here we describe a novel preparation for imaging live lanceolate sensory terminals of palisade endings that innervate mouse ear skin hair follicles during staining and destaining with styryl pyridinium dyes.

在毛囊披针形结局生活神经末端标记的光学监测<em&gt;离体</em&gt;鼠耳皮肤

A novel dissection and recording technique is described for optical monitoring staining and de-staining of lanceolate terminals surrounding hair follicles ...

Establishing a Mouse Model of a Pure Small Fiber Neuropathy with the Ultrapotent Agonist of Transient Receptor Potential Vanilloid Type 1

Patients with diabetes mellitus (DM) or those experiencing the neurotoxic effects of chemotherapeutic agents may develop sensation disorders due to degeneration and injury of small-diameter sensory neurons, referred to as small fiber neuropathy. Present animal models of small fiber neuropathy affect both large- and small-diameter sensory fibers and thus create a neuropathology too complex to properly assess the effects of injured small-diameter sensory fibers. Therefore, it is necessary to develop an experimental model of pure small fiber neuropathy to adequately examine these issues. This protocol describes an experimental model of small fiber neuropathy specifically affecting small-diameter sensory nerves with resiniferatoxin (RTX), an ultrapotent agonist of transient receptor potential vanilloid type 1 (TRPV1), through a single dose of intraperitoneal injection, referred to as RTX neuropathy. This RTX neuropathy showed pathological manifestations and behavioral abnormalities that mimic the clinical characteristics of patients with small fiber neuropathy, including intraepidermal nerve fiber (IENF) degeneration, specifically injury in small-diameter neurons, and induction of thermal hypoalgesia and mechanical allodynia. This protocol tested three doses of RTX (200, 50, and 10 &#181;g/kg, respectively) and concluded that a critical dose of RTX (50 &#181;g/kg) is required for the development of typical small fiber neuropathy manifestations, and prepared a modified immunostaining procedure to investigate IENF degeneration and neuronal soma injury. The modified procedure is fast, systematic, and economic. Behavioral evaluation of neuropathic pain is critical to reveal the function of small-diameter sensory nerves. The evaluation of mechanical thresholds in experimental rodents is particularly challenging and this protocol describes a customized metal mesh that is suitable for this type of assessment in rodents. In summary, RTX neuropathy is a new and easily established experimental model to evaluate the molecular significance and intervention underlying neuropathic pain for the development of therapeutic agents.

This study establishes an experimental model of pure small fiber neuropathy with resiniferatoxin (RTX). A unique dose of RTX (50 &#181;g/kg) is optimal for developing a small fiber neuropathy model that mimics patient characteristics and could help investigate the nociceptive molecular significance underlying neuropathic pain.

利用瞬态受体电位辣椒1型 Ultrapotent 激动剂建立单纯小纤维神经病变的小鼠模型

Patients with diabetes mellitus (DM) or those experiencing the neurotoxic effects of chemotherapeutic agents may develop sensation disorders due to ...

Imaging and Analysis of Neurofilament Transport in Excised Mouse Tibial Nerve

Neurofilament protein polymers move along axons in the slow component of axonal transport at average speeds of ~0.35-3.5 mm/day. Until recently the study of this movement in situ was only possible using radioisotopic pulse-labeling, which permits analysis of axonal transport in whole nerves with a temporal resolution of days and a spatial resolution of millimeters. To study neurofilament transport in situ with higher temporal and spatial resolution, we developed a hThy1-paGFP-NFM transgenic mouse that expresses neurofilament protein M tagged with photoactivatable GFP in neurons. Here we describe fluorescence photoactivation pulse-escape and pulse-spread methods to analyze neurofilament transport in single myelinated axons of tibial nerves from these mice ex vivo. Isolated nerve segments are maintained on the microscope stage by perfusion with oxygenated saline and imaged by spinning disk confocal fluorescence microscopy. Violet light is used to activate the fluorescence in a short axonal window. The fluorescence in the activated and flanking regions is analyzed over time, permitting the study of neurofilament transport with temporal and spatial resolution on the order of minutes&#160;and microns, respectively. Mathematical modeling can be used to extract kinetic parameters of neurofilament transport including the velocity, directional bias and pausing behavior from the resulting data. The pulse-escape and pulse-spread methods can also be adapted to visualize neurofilament transport in other nerves. With the development of additional transgenic mice, these methods could also be used to image and analyze the axonal transport of other cytoskeletal and cytosolic proteins in axons.

We describe fluorescence photoactivation methods to analyze the axonal transport of neurofilaments in single myelinated axons of peripheral nerves from transgenic mice that express a photoactivatable neurofilament protein.

切除小鼠神经神经纤维神经丝的成像与分析

Neurofilament protein polymers move along axons in the slow component of axonal transport at average speeds of ~0.35-3.5 mm/day. Until recently the study ...