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>

<ol>
	<li>Zacchigna, S., Ruiz de Almodovar, C., Carmeliet, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Similarities+between+angiogenesis+and+neural+development:+What+small+animal+models+can+tell+us.">Similarities between angiogenesis and neural development: What small animal models can tell us.</a> Current Topics in Developmental Biology. 80, 1-55 (2008).</li><li>Grinsell, D., Keating, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+nerve+reconstruction+after+injury:+A+review+of+clinical+and+experimental+therapies.">Peripheral nerve reconstruction after injury: A review of clinical and experimental therapies.</a> BioMed Research International. 2014, 1-13 (2014).</li><li>Chen, M. B., Zhang, F., Lineaweaver, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Luminal+fillers+in+nerve+conduits+for+peripheral+nerve+repair.">Luminal fillers in nerve conduits for peripheral nerve repair.</a> Annals of Plastic Surgery. 57 (4), 462-471 (2006).</li><li>Scipio, F., Raimondo, S., Tos, P., Geuna, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+protocol+for+paraffin-embedded+myelin+sheath+staining+with+osmium+tetroxide+for+light+microscope+observation.">A simple protocol for paraffin-embedded myelin sheath staining with osmium tetroxide for light microscope observation.</a> Microscopy Research and Technique. 71, 497-502 (2008).</li><li>Raimondo, S., Fornaro, M., Di Scipio, F., Ronchi, G., Giacobini-Robecchi, M. G., Geuna, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+5:+Methods+and+protocols+in+peripheral+nerve+regeneration+experimental+research:+part+II-morphological+techniques.">Chapter 5: Methods and protocols in peripheral nerve regeneration experimental research: part II-morphological techniques.</a> International Review of Neurobioly. 87, 81-103 (2009).</li><li>Carriel, V., Garzon, I., Alaminos, M., Campos, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+myelin+sheath+and+collagen+reorganization+pattern+in+a+model+of+peripheral+nerve+regeneration+using+an+integrated+histochemical+approach.">Evaluation of myelin sheath and collagen reorganization pattern in a model of peripheral nerve regeneration using an integrated histochemical approach.</a> Histochemistry and Cell Biology. , 709-717 (2011).</li><li>Sridharan, G., Shankar, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toluidine+blue:+A+review+of+its+chemistry+and+clinical+utility.">Toluidine blue: A review of its chemistry and clinical utility.</a> Journal of Oral and Maxillofacial Pathology. 16, 251-255 (2012).</li><li>Epstein, J. B., Scully, C., Spinelli, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toluidine+blue+and+Lugol's+iodine+application+in+the+assessment+of+oral+malignant+disease+and+lesions+at+risk+of+malignancy.">Toluidine blue and Lugol's iodine application in the assessment of oral malignant disease and lesions at risk of malignancy.</a> Journal of Oral Pathology &amp; Medicine. 21, 160-163 (1992).</li><li>Carriel, V., Garzon, I., Alaminos, M., Cornelissen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histological+assessment+in+peripheral+nerve+tissue+engineering.">Histological assessment in peripheral nerve tissue engineering.</a> Neural Regeneration Research. 9, 1657-1660 (2014).</li><li>Dykstra, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> A manual of applied techniques for biological electron microscopy. , 257 (1993).</li><li>Vleggeert-Lankamp, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+evaluation+methods+in+the+assessment+of+peripheral+nerve+regeneration+through+synthetic+conduits:+A+systematic+review.">The role of evaluation methods in the assessment of peripheral nerve regeneration through synthetic conduits: A systematic review.</a> Journal of Neurosurgery. 107 (6), 1168-1189 (2007).</li><li>Williams, P., Wendell-Smith, C. P., Finch, A., Stevens, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Further+uses+and+methods+of+processing+of+fresh+frozen+sections+of+peripheral+nerve.">Further uses and methods of processing of fresh frozen sections of peripheral nerve.</a> Journal of Cell Science. 3 (69), 99-105 (1964).</li><li>Castro, J., Negredo, P., Avenda&#241;o, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiber+composition+of+the+rat+sciatic+nerve+and+its+modification+during+regeneration+through+a+sieve+electrode.">Fiber composition of the rat sciatic nerve and its modification during regeneration through a sieve electrode.</a> Brain research. , 65-77 (2008).</li><li>Raimondo, S., Fornaro, M., Di Scipio, F., Ronchi, G., Giacobini-Robecchi, M. G., Geuna, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+and+protocols+in+peripheral+nerve+regeneration+experimental+research:+Part+II-morphological+techniques.">Methods and protocols in peripheral nerve regeneration experimental research: Part II-morphological techniques.</a> International review of neurobiology. 87, 81-103 (2009).</li><li>Bozkurt, A., Lassner, F., O'Dey, D., Deumens, R., B&#246;cker, A., Schwendt, T., Janzen, C., Suschek, C. V., Tolba, R., Kobayashi, E., Sellhaus, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+microstructured+and+interconnected+pore+channels+in+a+collagen-based+nerve+guide+on+axonal+regeneration+in+peripheral+nerves.">The role of microstructured and interconnected pore channels in a collagen-based nerve guide on axonal regeneration in peripheral nerves.</a> Biomaterials. 33 (5), 1363-1375 (2012).</li><li>Weis, J., Brandner, S., Lammens, M., Sommer, C., Vallat, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Processing+of+nerve+biopsies:+A+practical+guide+for+neuropathologists.">Processing of nerve biopsies: A practical guide for neuropathologists.</a> Clinical Neuropathology. 31 (1), 7-23 (2012).</li><li>Battiston, B., Tos, P., Geuna, S., Giacobini-Robecchi, M. G., Guglielmone, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nerve+repair+by+means+of+vein+filled+with+muscle+grafts.+II.+Morphological+analysis+of+regeneration.">Nerve repair by means of vein filled with muscle grafts. II. Morphological analysis of regeneration.</a> Microsurgery. 20 (1), 37-41 (2000).</li><li>Bhattacharyya, T. K., Thomas, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+staining+methods+for+resin-embedded+peripheral+nerve.">Comparison of staining methods for resin-embedded peripheral nerve.</a> Journal of Histotechnology. 27, 161-164 (2004).</li><li>Zbaeren, J., Zbaeren-Colbourn, D., Haeberli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+immunohistochemistry+on+improved+glycol+methacrylate-resin+sections.">High-resolution immunohistochemistry on improved glycol methacrylate-resin sections.</a> Journal of Histotechnology. 30 (1), 27-33 (2007).</li><li>Alturkistani, H. A., Tashkandi, F. M., Mohammedsaleh, Z. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histological+stains:+A+literature+review+and+case+study.">Histological stains: A literature review and case study.</a> Global Journal of Health Science. 8 (3), 72-79 (2016).</li><li>Ezra, M., Bushman, J., Shreiber, D., Schachner, M., Kohn, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Porous+and+nonporous+nerve+conduits:+the+effects+of+a+hydrogel+luminal+filler+with+and+without+a+neurite-promoting+moiety.">Porous and nonporous nerve conduits: the effects of a hydrogel luminal filler with and without a neurite-promoting moiety.</a> Tissue Engineering Part A. (9-10), 818-826 (2016).</li><li>Bhatnagar, D., Bushman, J. S., Murthy, N. S., Merolli, A., Kaplan, H. M., Kohn, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrin+glue+as+a+stabilization+strategy+in+peripheral+nerve+repair+when+using+porous+nerve+guidance+conduits.">Fibrin glue as a stabilization strategy in peripheral nerve repair when using porous nerve guidance conduits.</a> Journal of Materials Science: Materials in Medicine. 28 (5), 79 (2017).</li></ol>

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?...

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 border="0" cellpadding="0" cellspacing="0" width="1092">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Dulbecco&#39;s Phosphate Buffer Saline</td>
			<td style="width:216px;">Gibco</td>
			<td style="width:137px;">14200-075</td>
			<td style="width:384px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Glutaraldehyde Solution</td>
			<td style="width:216px;">Sigma-Aldrich</td>
			<td style="width:137px;">G6257</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Paraformaldehyde</td>
			<td style="width:216px;">Sigma-Aldrich</td>
			<td style="width:137px;">P6148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Sodium Phosphate Monobasic Monohydrate</td>
			<td style="width:216px;">Sigma-Aldrich</td>
			<td style="width:137px;">S9638</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Toluidine Blue O</td>
			<td style="width:216px;">Sigma-Aldrich</td>
			<td style="width:137px;">T3260</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Sodium Tetraborate Decahydrate</td>
			<td style="width:216px;">Acros Organics</td>
			<td style="width:137px;">205950010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Isoflurane</td>
			<td style="width:216px;">Piramal</td>
			<td style="width:137px;">NDC 66794-013-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Epoxy Embedding Medium Kit</td>
			<td style="width:216px;">Sigma-Aldrich</td>
			<td style="width:137px;">45359</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Sodium Hydroxide Solution</td>
			<td style="width:216px;">Sigma-Aldrich</td>
			<td style="width:137px;">72068</td>
			<td>To adjust Trump&#39;s fixative pH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Acetone</td>
			<td style="width:216px;">Fisher Chemical</td>
			<td style="width:137px;">170942</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Osmium Tetroxide Solution</td>
			<td style="width:216px;">Sigma-Aldrich</td>
			<td style="width:137px;">75632</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">VWR Micro Slides, Superfrost Plus</td>
			<td style="width:216px;">VWR</td>
			<td style="width:137px;">48311-703</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Microscope Cover Glass</td>
			<td style="width:216px;">Fisher Scientific</td>
			<td>12545102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Pelco Embedding Cast</td>
			<td style="width:216px;">Fisher Scientific</td>
			<td>NC9671811</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Glass Knife Maker</td>
			<td style="width:216px;">RMC Products</td>
			<td style="width:137px;">GKM-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Ultramicrotome</td>
			<td style="width:216px;">RMC Products</td>
			<td style="width:137px;">MT-XL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">15 mL Conical Tube</td>
			<td style="width:216px;">Falcon</td>
			<td>ISO 9001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Eppendorf 1.5 mL microcentrifuge tubes</td>
			<td style="width:216px;">Sigma-Aldrich</td>
			<td>T9661</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">4 mL Glass Vial</td>
			<td style="width:216px;">Sigma-Aldrich</td>
			<td>854190</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:355px;">Razor Blades</td>
			<td style="width:216px;">VWR</td>
			<td style="width:137px;">55411-050</td>
			<td>For trimming resin block</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:355px;">Perfect Loop</td>
			<td style="width:216px;">Electron Microscopy Sciences</td>
			<td style="width:137px;">70944</td>
			<td>For picking up thin resin sections</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Ultra Glass Knife Strips 6.4 mm x 25 mm x 400 mm</td>
			<td style="width:216px;">Electron Microscopy Sciences</td>
			<td style="width:137px;">71012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">100 Watt Oven</td>
			<td style="width:216px;">Millipore&#160;</td>
			<td style="width:137px;">6350115</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Whatman Filter Paper</td>
			<td style="width:216px;">Sigma-Aldrich</td>
			<td>WHA10010155</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">3 mL plastic pipette</td>
			<td style="width:216px;">Sigma-Aldrich</td>
			<td style="width:137px;">Z331740</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Micro-surgical Kit</td>
			<td style="width:216px;">World precision instruments</td>
			<td style="width:137px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Olympus fluorescence microscope</td>
			<td style="width:216px;"></td>
			<td style="width:137px;"></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...

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 de Cartagena UDEC

Research

JoVE Journal

Neuroscience

18.7K 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

Laser Capture Microdissection of Drosophila Peripheral Neurons

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the molecular mechanisms underlying class-specific dendrite morphogenesis1,2. To facilitate molecular analyses of class-specific da neuron development, it is vital to obtain these cells in a pure population. Although a range of different cell, and tissue-specific RNA isolation techniques exist for Drosophila cells, including magnetic bead based cell purification3,4, Fluorescent Activated Cell Sorting (FACS)5-8, and RNA binding protein based strategies9, none of these methods can be readily utilized for isolating single or multiple class-specific Drosophila da neurons with a high degree of spatial precision. Laser Capture Microdissection (LCM) has emerged as an extremely powerful tool that can be used to isolate specific cell types from tissue sections with a high degree of spatial resolution and accuracy. RNA obtained from isolated cells can then be used for analyses including qRT-PCR and microarray expression profiling within a given cell type10-16. To date, LCM has not been widely applied in the analysis of Drosophila tissues and cells17,18, including da neurons at the third instar larval stage of development. 
Here we present our optimized protocol for isolation of Drosophila da neurons using the infrared (IR) class of LCM. This method allows for the capture of single, class-specific or multiple da neurons with high specificity and spatial resolution. Age-matched third instar larvae expressing a UAS-mCD8::GFP19 transgene under the control of either the class IV da neuron specific ppk-GAL420 driver or the pan-da neuron specific 21-7-GAL421 driver were used for these experiments. RNA obtained from the isolated da neurons is of very high quality and can be directly used for downstream applications, including qRT-PCR or microarray analyses. Furthermore, this LCM protocol can be readily adapted to capture other Drosophila cell types a various stages of development dependent upon the cell type specific, GAL4-driven expression pattern of GFP.

Laser Capture Microdissection of Drosophila Peripheral Neurons

In this video-article we present a method for isolating single or multiple Drosophila da neurons from third instar larvae using the infrared capture (IR) class of Laser Capture Microdissection (LCM). RNA obtained from the isolated neurons can be readily used for downstream applications including qRT-PCR or microarray analyses.

激光捕获显微切割技术的果蝇外周神经元

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the ...

科研

神经科学

Organotypic Slice Culture of GFP-expressing Mouse Embryos for Real-time Imaging of Peripheral Nerve Outgrowth

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in which the enhanced version of the green fluorescent protein (EGFP) is exclusively expressed in all neurons of the developing central and peripheral nervous system1, allowing the possibility to both film the innervation of the forelimb and to manipulate this process with pharmacological and genetic techniques2. The most critical parameter in the successful cultivation of such slice cultures is the method by which the slices are prepared. After extensive testing of a variety of methods, we have found that a vibratome is the best possible device to slice the embryos such that they routinely result in a culture that demonstrates viability over a period of several days, and most importantly, develops in an age-specific manner. For mid-gestation embryos, this includes the normal outgrowth of spinal nerves from the spinal cord and the dorsal root ganglia to their targets in the periphery and the proper determination of skeletal and muscle tissue. 
In this work, we present a method for processing whole embryos of embryonic day (E) E10 to E12 into 300 - 400 micrometer slices for cultivation in a standard tissue culture incubator, which can be studied for up to two days after slice preparation. Critical for the success of this approach is the use of a vibratome to slice each agarose-embedded embryo. This is followed by the cultivation of the slices upon Millicell culture membrane inserts placed upon a small volume of medium, resulting in an interface culture technique. One litter with an average of 7 embryos routinely produces at least 14 slices (2-3 slices of the forelimb region per embryo), which varies slightly due to the age of the embryos as well as to the thickness of the slices. About 80% of the cultured slices show nerve outgrowth, which can be measured througout the culturing period2. Representative results using the tauGFP mouse line are demonstrated.

We present a method to prepare organotypic slices of mid-gestation mouse embryos for the cultivation and time-lapse imaging of peripheral nerve outgrowth.

器官型切片文化绿色荧光蛋白表达的实时成像周围神经生长的小鼠胚胎

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in ...

Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based multicolor labeling is challenging, especially when utilized for assessing interrelations between target proteins in the tissue with a high fat content such as the central nervous system (CNS).
Our protocol represents a refinement of the standard immunolabeling technique particularly adjusted for detection of both structural and soluble proteins in the rat CNS and peripheral lymph nodes (LN) affected by neuroinflammation. Nonetheless, with or without further modifications, our protocol could likely be used for detection of other related protein targets, even in other organs and species than here presented.

We here present an optimized, detailed protocol for double immunostaining in formalin-fixed, paraffin-embedded rat central nervous system (CNS) and peripheral lymph node (LN) tissue sections.

免疫组化分析大鼠中枢神经系统和周围淋巴结组织切片

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based ...

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 ...

Visualizing the Effects of a Positive Early Experience, Tactile Stimulation, on Dendritic Morphology and Synaptic Connectivity with Golgi-Cox Staining

To generate longer-term changes in behavior, experiences must be producing stable changes in neuronal morphology and synaptic connectivity. Tactile stimulation is a positive early experience that mimics maternal licking and grooming in the rat. Exposing rat pups to this positive experience can be completed easily and cost-effectively by using highly accessible materials such as a household duster. Using a cross-litter design, pups are either stroked or left undisturbed, for 15 min, three times per day throughout the perinatal period. To measure the neuroplastic changes related to this positive early experience, Golgi-Cox staining of brain tissue is utilized. Owing to the fact that Golgi-Cox impregnation stains a discrete number of neurons rather than all of the cells, staining of the rodent brain with Golgi-Cox solution permits the visualization of entire neuronal elements, including the cell body, dendrites, axons, and dendritic spines. The staining procedure is carried out over several days and requires that the researcher pay close attention to detail. However, once staining is completed, the entire brain has been impregnated and can be preserved indefinitely for ongoing analysis. Therefore, Golgi-Cox staining is a valuable resource for studying experience-dependent plasticity.

This paper describes the procedures for tactile stimulation of rat pups and subsequent Golgi-Cox staining of neuronal morphology. Tactile stimulation is a positive experience that is administered in the perinatal period by stroking pups with a household duster. Golgi-cox staining is a reliable procedure permitting the visualization of entire neurons.

可视化的一个积极的早期经验，触觉刺激的影响，对树突状形态和突触连接与高尔基染色考克斯

To generate longer-term changes in behavior, experiences must be producing stable changes in neuronal morphology and synaptic connectivity. Tactile ...

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly controlled formation and maintenance of complex neuronal networks are not completely understood thus far. The open questions concerning neurons in health and disease are diverse and reaching from understanding the basic development to investigating human related pathologies, e.g.,&#160;Alzheimer&#39;s disease and&#160;Schizophrenia. The most detailed analysis of neurons can be performed in vitro. However, neurons are demanding cells and need the additional support of astrocytes for their long-term survival. This cellular heterogeneity is in conflict with the aim to dissect the analysis of neurons and astrocytes. We present here a cell-culture assay that allows for the long-term cocultivation of pure primary neurons and astrocytes, which share the same chemically defined medium, while being physically separated. In this setup, the cultures survive for up to four weeks and the assay is suitable for a diversity of investigations concerning neuron-glia interaction.

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

This protocol describes the indirect neuron-astrocyte coculture for compartmentalized analysis of neuron-glia interactions.

间接神经元的星形胶质细胞共培养试验:一个<em&gt;体外</em&gt;设置 - 为神经元的神经胶质细胞相互作用的详细调查

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly ...

Targeting Alpha Synuclein Aggregates in Cutaneous Peripheral Nerve Fibers by Free-floating Immunofluorescence Assay

To date, for most neurodegenerative diseases only a post-mortem histopathological definitive diagnosis is available. For Parkinson&#39;s disease (PD), the diagnosis still relies only on clinical signs of motor involvement that appear later on in the disease course, when most of the dopaminergic neurons are already lost. Hence, there is a strong need for a biomarker that can identify patients at the beginning of disease or at the risk of developing it. Over the last few years, skin biopsy has proved to be an excellent research and diagnostic tool for peripheral nerve diseases such as small fiber neuropathy. Interestingly, a small fiber neuropathy and alpha synuclein (&#945;Syn) neural deposits have been shown by skin biopsy in PD patients. Indeed, skin biopsy has the great advantage of being an easily accessible, minimally invasive and painless procedure that allows the analysis of peripheral nervous tissue prone to the pathology. Moreover, the possibility of repeating the skin biopsy in the course of the follow-up of the same patient allows studying the longitudinal correlation with the disease progression. We set up a standardized reliable protocol to investigate the presence of &#945;Syn aggregates in skin nerve fibers of the PD patient. This protocol involves few short fixation steps, a cryotome sectioning and then a free-floating immunofluorescence double-staining with two specific antibodies: anti Protein Gene Product 9.5 (PGP9.5) to mark the cutaneous nerve fibers and anti 5G4 for detecting &#945;Syn aggregates. It is a versatile, sensitive and easy to perform protocol that can also be applied for targeting other proteins of interest in skin nerves. The ability to mark &#945;Syn aggregates is another step forward to the use of skin biopsy as a tool for establishing a pre-mortem histopathological diagnosis of PD.

Here, we present a protocol for a free-floating indirect immunofluorescence assay on skin biopsy sections that allows for the identification of disease specific conformation variants of alpha synuclein involved in Parkinson disease and multiple proteins of the peripheral nervous system.

通过自由浮动免疫荧光测定,在皮外神经纤维中定位阿尔法核糖核酸聚合体

To date, for most neurodegenerative diseases only a post-mortem histopathological definitive diagnosis is available. For Parkinson&#39;s disease (PD), the ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of sciatic nerve injury rodent models. In this protocol, we describe a novel kinematic analysis method that uses a three-dimensional (3D) motion capture apparatus for functional evaluations using a rat sciatic nerve crush injury model. First, the rat is familiarized with treadmill walking. Markers are then attached to the designated bone landmarks and the rat is made to walk on the treadmill at the desired speed. Meanwhile, the posterior limb movements of the rat are recorded using four cameras. Depending on the software used, marker tracings are created using both automatic and manual modes and the desired data are produced after subtle adjustments. This method of kinematic analysis, which uses a 3D motion capture apparatus, offers numerous advantages, including superior precision and accuracy. Many more parameters can be investigated during the comprehensive functional evaluations. This method has several shortcomings that require consideration: The system is expensive, can be complicated to operate, and may produce data deviations due to skin shifting. Nevertheless, kinematic analysis using a 3D motion capture apparatus is useful for performing functional anterior and posterior limb evaluations. In the future, this method may become increasingly useful for generating accurate assessments of various traumas and diseases.

We introduce a kinematic analysis method that uses a three-dimensional motion capture apparatus containing four cameras and data processing software for performing functional evaluations during fundamental research involving rodent models.

坐骨神经粉碎损伤大鼠模型中功能评估的3D运动学分析

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...

Preparation of Peripheral Nerve Stimulation Electrodes for Chronic Implantation in Rats

Peripheral nerve cuff electrodes have long been used in the neurosciences and related fields for stimulation of, for example, vagus or sciatic nerves. Several recent studies have demonstrated the effectiveness of chronic VNS in enhancing central nervous system plasticity to improve motor rehabilitation, extinction learning, and sensory discrimination. Construction of chronically implantable devices for use in such studies is challenging due to rats&#8217; small size, and typical protocols require extensive training of personnel and time-consuming microfabrication methods. Alternatively, commercially available implantable cuff electrodes can be purchased at a significantly higher cost. In this protocol, we present a simple, low-cost method for construction of small, chronically implantable peripheral nerve cuff electrodes for use in rats. We validate the short and long-term reliability of our cuff electrodes by demonstrating that VNS in ketamine/xylazine anesthetized rats produces decreases in breathing rate consistent with activation of the Hering-Breuer reflex, both at the time of implantation and up to 10 weeks after device implantation. We further demonstrate the suitability of the cuff electrodes for use in chronic stimulation studies by pairing VNS with skilled lever press performance to induce motor cortical map plasticity.

Existing approaches for constructing chronically implantable peripheral nerve cuff electrodes for use in small rodents often require specialized equipment and/or highly trained personnel. In this protocol we demonstrate a simple, low-cost approach for fabricating chronically implantable cuff electrodes, and demonstrate their effectiveness for vagus nerve stimulation (VNS) in rats.

大鼠慢性植入的外周神经刺激电极的制备

Peripheral nerve cuff electrodes have long been used in the neurosciences and related fields for stimulation of, for example, vagus or sciatic nerves. ...

Automated Gait Analysis to Assess Functional Recovery in Rodents with Peripheral Nerve or Spinal Cord Contusion Injury

Peripheral and central nerve injuries are mostly studied in rodents, especially rats, given the fact that these animal models are both cost-effective and a lot of comparative data has been published in the literature. This includes a multitude of assessment methods to study functional recovery following nerve injury and repair. Besides evaluation of nerve regeneration by means of histology, electrophysiology, and other in vivo and in vitro assessment techniques, functional recovery is the most important criterion to determine the degree of neural regeneration. Automated gait analysis allows recording of a vast quantity of gait-related parameters such as Paw Print Area and Paw Swing Speed as well as measures of inter-limb coordination. Additionally, the method provides digital data of the rats&#8217; paws after neuronal damage and during nerve regeneration, adding to our understanding of how peripheral and central nervous injuries affect their locomotor behavior. Besides the predominantly used sciatic nerve injury model, other models of peripheral nerve injury such as the femoral nerve can be studied by means of this method. In addition to injuries of the peripheral nervous systems, lesions of the central nervous system, e.g., spinal cord contusion can be evaluated. Valid and reproducible data assessment is strongly dependent on meticulous adjustment of the hard- and software settings prior to data acquisition. Additionally, proper training of the experimental animals is of crucial importance. This work aims to illustrate the use of computerized automated gait analysis to assess functional recovery in different animal models of peripheral nerve injury as well as spinal cord contusion injury. It also emphasizes the method&#8217;s limitations, e.g., evaluation of nerve regeneration in rats with sciatic nerve neurotmesis due to limited functional recovery. Therefore, this protocol is thought to help researchers interested in peripheral and central nervous injuries to assess functional recovery in rodent models.

Automated gait analysis is a feasible tool to evaluate functional recovery in rodent models of peripheral nerve injury and spinal cord contusion injury. While it requires only one setup to assess locomotor function in various experimental models, meticulous hard- and soft-ware adjustment and training of the animals is highly important.

自动步态分析，评估带外周神经或脊髓挫伤的啮齿动物的功能恢复

Peripheral and central nerve injuries are mostly studied in rodents, especially rats, given the fact that these animal models are both cost-effective and ...