这项工作是支持青少年糖尿病研究基金会国际（批17-2008-1031）和美国国立卫生研究院（授予1R01DK077903 - 01A1）。

简介: 周围神经病变的准确定量是很重要的定义处于危险的患者，预期恶化，并评估新的治疗方法。常规方法评估神经功能缺损和电生理和定量感觉测试的量化功能的改变，检测神经病变。然而，最早的损害，出现细小纤维，但这些测试主要是评估大纤维功能障碍，表现出的再生和修复能力有限。只允许直接检查的​​无髓神经纤维的损伤和修复技术，电子显微镜和皮肤打孔活检腓肠神经活检。然而，无论是侵入性的程序和需要长时间的实验程序和相当的专业知识。角膜共焦显微镜是一种非侵入性的临床技术，它提供了角膜神经纤维在体内成像。 HRT的III -罗斯托克角膜模块（RCM） 操作手册 1。相机的制备在考试的最初步骤，使用HRT的物镜尖的准备。 <ol><li>首先，在激光扫描相机的客观管，设置12屈光度的折射，然后相机调整到最低位置。 </li><li>套用一个大的同质性，泡沫自由，豌豆大小，镜头上的尖端，涉及的下降。 </li><li>从无菌容器中取出一个TomoCap，它安装在镜头一角，凝胶，形成了一个半月板之间的物镜和第。尽可能在持有人可能推。小心不要触摸TomoCap前表面在安装过程中。 <img src="/files/ftp_upload/2194/2194fig1.jpg" alt="图1" /> 图1。物镜尖（顶）申请viscotear凝胶（底部）应用TomoCap准备。 </li><li>移动激光扫描相机尽量向后摄像机安装可能。 </li><li> TomoCap的内外表面都出现一个明亮的激光反射。应调整焦平面调整轮，直到光明的反映，是观察，表明该镜头集中在前面的第的。在重点位置显示显示深度值现在应该-150微米，150微米之间。 </li><li>将深度设置重置为零。 <img src="/files/ftp_upload/2194/2194fig2.jpg" alt="图2" /> 图2。焦平面调整。 </li></ol> 2。准备病人拍摄主体的眼睛是麻醉使用下降0.4％benoxinate盐酸，"活视康"人造眼泪水润滑眼睛的前面。 坐在舒适的主题是，与下巴的下巴上休息，并要求坚决按他们的额头对额头栏。 <img src="/files/ftp_upload/2194/2194fig3.jpg" alt="图3" /> 图3准备病人。 3。对齐相机它使考试更容易，如果您指示病人与没有被检查眼外固定光修复。显微镜头小巧的尺寸意味着主体的对侧眼不遮挡，使使用距离目标。 <img src="/files/ftp_upload/2194/2194fig4.jpg" alt="图4" /> 图4。角膜模块的物镜。 <ol><li> CCD相机等，其光轴垂直的激光扫描摄像机的光轴的位置。相机应该对病人的右侧时，成像的右眼，反之亦然。 在这个位置上，你应该看到TomoCap前表面的CCD相机实时图像的中心。 </li><li>移动的激光扫描对病人的相机，直到病人的角膜在一个约5至10毫米的TomoCap距离是移动的激光扫描相机上/下和左/右使用摄像机安装上的黑色旋钮，直至在TomoCap是在患者的角膜的中心位置。在这个阶段，您可以检查红色激光灯是在角膜中心和微调，观察角膜，这是在CCD摄像机的图像中可见的激光束的反射。这种反思必须完全发生在角膜前极。 <img src="/files/ftp_upload/2194/2194fig5.jpg" alt="图5" /> 图5。对准捕捉角膜中央图像角膜的前极的激光束。 </li><li>让患者尽可能广泛的他/她的眼睛打开。慢慢地向前移动的激光扫描相机对病人直到TomoCap接触病人的角膜。如果它是必需正确的激光扫描摄像机的位置，然后移动相机远离病人第一，做了校正，然后将它再次接触角膜。 <img src="/files/ftp_upload/2194/2194fig6.jpg" alt="图6" /> 图6。TomoCap和角膜之间的薄露桥的示范。 </li><li>相机和角膜之间的最小的接触是不够的。在优化调整，薄露TomoCap和角膜之间的桥梁上的CCD相机实时图像是可见的。 </li></ol>如果按TomoCap或移动相机太接近角膜，然后你会看到一个通过CCD相机实时图像的外观扁平角膜在角膜上的压力。也得到了线的图像显示会出现的压力，因此并不适用于角膜的压力太大。一旦接触已经建立，不要移动headrestto避免角膜上TomoCap滑动的位置。 <img src="/files/ftp_upload/2194/2194fig7.jpg" alt="图7" /> 图7（左）一个straie由于对角膜的TomoCap压力过大应用程序的外观;（右）与正常的外观没有多余的压力形象。 4。检查病人视野已被选定后，被打开的HRT III激光相机和图像采集窗口出现在屏幕上。使用CCD图像TomoCap和对病人的角膜激光灯的位置。然后可以开始使用所选择的模式的考试。 <img src="/files/ftp_upload/2194/2194fig8.jpg" alt="图8" /> 图8。查看监视器上的图像采集过程中的模式。 采集模式在部分模式中，一个单一的形象收购和储存您每次按下脚踏开关。我们使用这种模式下，捕获图像从整个角膜层包括鲍曼的一层角膜中心。然后捕获足够数量的图像后，可以尝试其他模式，包括顺序和数量。 <img src="/files/ftp_upload/2194/2194fig9.jpg" alt="图9" /> 图9显示不同的图像采集方式与选择。 与序列扫描，可以获取一个可调节的帧速率高达100张图像的序列。您可以选择之间的帧速率30帧和1帧每秒（fps）和基于这种模式，它可以让你获得为期3至100秒的影片。 在容积扫描模式下，相机会自动获得一个系列40个连续的焦平面图像。一个85微米的总深度，可以扫描"视野400微米"场镜和连续两个图像之间的焦距，将约2.1微米。 经过几次扫描镜头聚焦平面是可以改变的，相对的角膜上皮细胞层，将成为关注的焦点，然后按一下复位按钮深度值设置为0表面的角膜细胞层的深度来衡量的。 对于我们目前研究的目的，我们得到从不同深度的角膜中央的图像。因此，我们不走动角膜激光扫描相机的水平或垂直的，我们只是向前或向后移动，并捕获在同一角膜点鲍曼的层深处的图像。每幅图像之间的距离大约是1微米，因此，如果鲍曼的深度为10μm，我们将有10张。然而，归根结底，我们选择约2-3微米它们之间的距离的图像。 检查后完成收购窗口中按一下相机的电源按钮关闭相机。使病人意识到，他或她不应该擦他或她的眼睛，直到局部麻醉消失。作为一项预防措施，你不妨在考试后执行一个病人的角膜裂隙灯检查。 从相机中取出TomoCap，处理它，然后用棉花清洁显微镜镜头棉签蘸蒸馏水。 5。考试分析 A.选择帧审查和分析现有的考试，选择合适的病人记录（S）必须在数据库窗口。图像浏览窗口，提供了一个现有的角膜检查概述选定的病人。在不同的日子进行的考试都存储在单独的访问选项卡。三种不同的扫描类型，代表三种不同的图标，在HRT三角膜访问"选项卡，喜的图像浏览窗口delberg眼科资源管理器。 调查角膜图像，单击相应的图像图标和"显示"功能打开下面的窗口: <img src="/files/ftp_upload/2194/2194fig10.jpg" alt="图10" /> 图10显示拍摄的图像后，检查所有。 <img src="/files/ftp_upload/2194/2194fig11.jpg" alt="图11" /> 图11出口前眼与CCD相机视图显示正确对齐选定的图像显示。 要导出相应的图像作进一步的分析，点击工具栏上的"导出"，然后选择你要保存的图像文件夹。 我们获取和为每一个病人的角膜层捕获的图像，但这项研究的主要焦点是鲍曼的层和角膜神经，只有这一层的图像进行分析。我们选择从鲍曼的每个科目在不同深度的角膜层5-6帧。 <img src="/files/ftp_upload/2194/2194fig12.jpg" alt="图12" /> 图12显示，从一个健康的主题所有角膜层。 在鲍曼的角膜中心层在不同深度的平均5-6帧是随机选取的定量测量和分析。 B.分析图像鲍曼的角膜中央与HRT的第三层，从拍摄的图像进行分析，我们使用专门的软件称为"CCM的图像分析工具v 0.6"本集团内已开发的。 角膜神经的四个主要参数的测量，用这个软件: <ul><li>神经纤维密度（NFD），这是定义为主要神经/帧的数量。 </li><li>神经分支密度（NBD）的主要分支/帧。 </li><li>神经纤维长度（NFL），这是所有神经/帧的总长度。 </li><li>神经纤维迂曲（NFT）的主要神经/帧。 </li></ul>要打开CCM的形象，去文件&gt;打开。 <ul><li>一旦加载图像，其大小和类型显示在控制面板的左上角手端的图像信息框。 </li><li>默认的规模是显示在编辑行的图像信息。如果规模是不同的，修改在编辑行的数量。 </li><li>要开始分析的图像，选择你想测量从在控制面板的右上角方公制中的参数。 </li><li>在结果框在页面的NFD的底部，NBD的实际数量，NFL作为一个曲折系数（TC）毫米和迂曲。 </li><li>然而，之后的结果导出到Excel工作表，结果将没有/ NFD的NBD和毫米/ 毫米 2为NFL 2毫米。 </li><li>为此，整理后的测量点击添加测量，然后转到文件&gt;另存为，然后用Excel打开保存的结果。 </li></ul><img src="/files/ftp_upload/2194/2194fig13.jpg" alt="图13" /> 图13。角膜神经图像显示与跟踪分析工具使用"CCM图像分析工具"。 6。代表性的成果: 我们已经证明了一个渐进的角膜神经变性日益增加1和糖尿病神经病变在糖尿病早期神经损伤，先在皮肤活检表皮内神经纤维的损失 2 以下 3胰腺移植对糖尿病患者神经性的严重程度和修复分层的严重性。我们也证明了神经损伤特发性小纤维神经病4 Fabry病5。 <img src="/files/ftp_upload/2194/2194fig14.jpg" alt="图14" /> 图14。HRT的角膜鲍曼的第三层（一）控制主体（b）与神经病变和严重的神经损伤患者相比角膜共聚焦显微镜图像。

<ol>
	<li>Abbott, C. A., Kallinikos, P., Marshall, A., Finnigan, J., Morgan, P., Efron, N., Boulton, A. J. M., Malik, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+Confocal+Microscopy:+A+Novel+Non-invasive+Test+to+Diagnose+and+Assess+Progression+of+Human+Diabetic+Neuropathy.">Corneal Confocal Microscopy: A Novel Non-invasive Test to Diagnose and Assess Progression of Human Diabetic Neuropathy.</a> Diabetes Care. 33, 1792-1797 (2010).</li><li>Quattrini, C., Tavakoli, M., Jeziorska, M., Kallinikos, P., Tesfaye, S., Marshall, A., Finnigan, J., Boulton, A. J. M., Efron, N., Malik, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surrogate+Markers+of+Small+Fiber+Damage+in+Human+Diabetic+Neuropathy.">Surrogate Markers of Small Fiber Damage in Human Diabetic Neuropathy.</a> Diabetes. 56, 2148-2154 (2007).</li><li>Mehra, S., Tavakoli, M., Efron, N., Boulton, A. J. M., Augustine, T., Malik, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+Confocal+Microscopy+Detects+Early+Nerve+Regeneration+After+Pancreas+Transplantation+in+Patients+with+Type+1+Diabetes.">Corneal Confocal Microscopy Detects Early Nerve Regeneration After Pancreas Transplantation in Patients with Type 1 Diabetes.</a> Diabetes Care. 30, 2608-2612 (2007).</li><li>Tavakoli, M., Marshall, A., Pitceathly, R., Fadavi, H., Gow, D., Roberts, M. E., Efron, N., Boulton, A. J. M., Malik, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+confocal+microscopy:+A+novel+means+to+detect+nerve+fibre+damage+in+patients+with+impaired+glucose+tolerance+and+idiopathic+small+fibre+neuropathy.">Corneal confocal microscopy: A novel means to detect nerve fibre damage in patients with impaired glucose tolerance and idiopathic small fibre neuropathy.</a> Experimental Neurology. 223, 245-250 (2010).</li><li>Tavakoli, M., Marshall, A., Thompson, L., Kenny, M., Waldek, S., Efron, N., Malik, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+confocal+microscopy:+A+novel+technique+to+detect+small-fibre+neuropathy+in+patients+with+Fabry+disease.">Corneal confocal microscopy: A novel technique to detect small-fibre neuropathy in patients with Fabry disease.</a> Muscle &#38; Nerve. 40, 976-984 (2009).</li></ol>

角膜共聚焦显微镜（CCM）是一种无创的临床技术，可用于检测早期神经损伤小纤维病变和周围神经病变，包括糖尿病，Fabry病和特发性小纤维神经病（ISFN）量化。眼前的这个技术的临床影响，在我们的能力诊断，后续的进展和评估糖尿病的神经病变和其他周围神经病变患者的治疗反应和患者的治疗效果将有一个直接的影响方面的一个巨大的飞跃。

<table><tbody><tr><td>HRT III CCM Machine - The Rostock Cornea Module</td><td></td><td></td><td> Heidelberg Engineering</td></tr><tr><td>CCM image analysis software v0.6 </td><td></td><td></td><td>University of Manchester</td></tr></tbody></table>

corneal confocal microscopy: a novel non-invasive technique to quantify small fibre pathology in peripheral neuropathies

周围神经病变的准确定量是很重要的定义处于危险的患者，预期恶化，并评估新的治疗方法。常规方法评估神经功能缺损和电生理和定量感觉测试的量化功能的改变，检测神经病变。然而，最早的损害，出现细小纤维，但这些测试主要是评估大纤维功能障碍，表现出的再生和修复能力有限。只允许直接检查的​​无髓神经纤维的损伤和修复技术，电子显微镜和皮肤打孔活检腓肠神经活检。然而，无论是侵入性的程序和需要长时间的实验程序和相当的专业知识。角膜共焦显微镜是一种非侵入性的临床技术，它提供了角膜神经纤维在体内成像。我们已经证明了早期神经损伤，先于皮肤活检表皮内的神经纤维与神经性的严重程度和修复以下胰腺移植对糖尿病患者分层损失。我们也证明了在特发性小纤维神经病变和法布里病的神经损害。

Watch this Scientific Journal Video about Corneal Confocal Microscopy: A Novel Non-invasive Technique to Quantify Small Fibre Pathology in Peripheral Neuropathies at JoVE.com

Corneal Confocal Microscopy: A Novel Non-invasive Technique to Quantify Small Fibre Pathology in Peripheral Neuropathies

角膜共焦显微镜是一种非侵入性的临床技术，可用于量化C纤维损伤与神经性的严重程度增加患者的诊断和分层。

角膜共聚焦显微镜:一种新型的非侵入性技术来量化小纤维病变周围神经病变

The accurate quantification of peripheral neuropathy is important to define at risk patients, anticipate deterioration, and assess new therapies. ...

corneal-confocal-microscopy-novel-non-invasive-technique-to-quantify

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

26.4K 次观看.  University of Manchester. 角膜共焦显微镜是一种非侵入性的临床技术，可用于量化C纤维损伤与神经性的严重程度增加患者的诊断和分层。

视频: Corneal Confocal Microscopy: A Novel Non-invasive Technique to Quantify Small Fibre Pathology in Peripheral Neuropathies

Three-dimensional Imaging of Nociceptive Intraepidermal Nerve Fibers in Human Skin Biopsies

A punch biopsy of the skin is commonly used to quantify intraepidermal nerve fiber densities (IENFD) for the diagnosis of peripheral polyneuropathy 1,2. At present, it is common practice to collect 3 mm skin biopsies from the distal leg (DL) and the proximal thigh (PT) for the evaluation of length-dependent polyneuropathies 3. However, due to the multidirectional nature of IENFs, it is challenging to examine overlapping nerve structures through the analysis of two-dimensional (2D) imaging. Alternatively, three-dimensional (3D) imaging could provide a better solution for this dilemma.
In the current report, we present methods for applying 3D imaging to study painful neuropathy (PN). In order to identify IENFs, skin samples are processed for immunofluorescent analysis of protein gene product 9.5 (PGP), a pan neuronal marker. At present, it is standard practice to diagnose small fiber neuropathies using IENFD determined by PGP immunohistochemistry using brightfield microscopy 4. In the current study, we applied double immunofluorescent analysis to identify total IENFD, using PGP, and nociceptive IENF, through the use of antibodies that recognize tropomyosin-receptor-kinase A (Trk A), the high affinity receptor for nerve growth factor 5. The advantages of co-staining IENF with PGP and Trk A antibodies benefits the study of PN by clearly staining PGP-positive, nociceptive fibers. These fluorescent signals can be quantified to determine nociceptive IENFD and morphological changes of IENF associated with PN. The fluorescent images are acquired by confocal microscopy and processed for 3D analysis. 3D-imaging provides rotational abilities to further analyze morphological changes associated with PN. Taken together, fluorescent co-staining, confocal imaging, and 3D analysis clearly benefit the study of PN.

In order to study the changes of nociceptive intraepidermal nerve fibers (IENFs) in painful neuropathies (PN), we developed protocols that could directly examine three-dimensional morphological changes observed in nociceptive IENFs. Three-dimensional analysis of IENFs has the potential to evaluate the morphological changes of IENF in PN.

三维成像伤害表皮内神经纤维在人体皮肤活检

A punch biopsy of the skin is commonly used to quantify intraepidermal nerve fiber densities (IENFD) for the diagnosis of peripheral polyneuropathy 1,2. ...

科研

医学

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 ...

神经科学

The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. Structural changes at the NMJ can result in neurotransmission failure, resulting in weakness, atrophy and even death of the muscle fiber. Many studies have investigated how genetic modifications or disease can alter the structure of the mouse NMJ. Unfortunately, it can be difficult to directly compare findings from these studies because they often employed different parameters and analytical methods. Three protocols are described here. The first uses maximum intensity projection confocal images to measure the area of acetylcholine receptor (AChR)-rich postsynaptic membrane domains at the endplate and the area of synaptic vesicle staining in the overlying presynaptic nerve terminal. The second protocol compares the relative intensities of immunostaining for synaptic proteins in the postsynaptic membrane. The third protocol uses Fluorescence Resonance Energy Transfer (FRET) to detect changes in the packing of postsynaptic AChRs at the endplate. The protocols have been developed and refined over a series of studies. Factors that influence the quality and consistency of results are discussed and normative data are provided for NMJs in healthy young adult mice.

The neuromuscular junction (NMJ) is altered in a variety of conditions that can sometimes culminate in synaptic failure. This report describes fluorescence microscope-based methods to quantify such structural changes.

神经肌肉接头处:测量突触大小，碎片和改变突触蛋白密度利用共聚焦荧光显微镜

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. ...

Three-dimensional Imaging and Analysis of Mitochondria within Human Intraepidermal Nerve Fibers

The goal of this protocol is to study mitochondria within intraepidermal nerve fibers. Therefore, 3D imaging and analysis techniques were developed to isolate nerve-specific mitochondria and evaluate disease-induced alterations of mitochondria in the distal tip of sensory nerves. The protocol combines fluorescence immunohistochemistry, confocal microscopy and 3D image analysis techniques to visualize and quantify nerve-specific mitochondria. Detailed parameters are defined throughout the procedures in order to provide a concrete example of how to use these techniques to isolate nerve-specific mitochondria. Antibodies were used to label nerve and mitochondrial signals within tissue sections of skin punch biopsies, which was followed by indirect immunofluorescence to visualize nerves and mitochondria with a green and red fluorescent signal respectively. Z-series images were acquired with confocal microscopy and 3D analysis software was used to process and analyze the signals. It is not necessary to follow the exact parameters described within, but it is important to be consistent with the ones chosen throughout the staining, acquisition and analysis steps. The strength of this protocol is that it is applicable to a wide variety of circumstances where one fluorescent signal is used to isolate other signals that would otherwise be impossible to study alone.

This protocol uses three-dimensional (3D) imaging and analysis techniques to visualize and quantify nerve-specific mitochondria. The techniques are applicable to other situations where one fluorescent signal is used to isolate a subset of data from another fluorescent signal.

人 Intraepidermal 神经纤维内线粒体三维成像及分析

The goal of this protocol is to study mitochondria within intraepidermal nerve fibers. Therefore, 3D imaging and analysis techniques were developed to ...

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 ...

Full-Field Optical Coherence Microscopy for Histology-Like Analysis of Stromal Features in Corneal Grafts

The quality of donor corneal stroma, which makes up about 90% of total corneal thickness, is likely to be one of the main, if not the major, limiting factor(s) for success of deep anterior lamellar and penetrating keratoplasty. These are surgical procedures that involve replacing part or all of the diseased corneal layers, respectively, by donated tissue, the graft, taken from a recently deceased individual. However, means to evaluate stromal quality of corneal grafts in eye banks are limited and lack the capability of high-resolution quantitative assessment of disease indicators. Full-field optical coherence microscopy (FF-OCM), permitting high-resolution 3D imaging of fresh or fixed ex vivo biological tissue samples, is a non-invasive technique well suited for donor cornea assessment. Here we describe a method for the qualitative and quantitative analysis of corneal stroma using FF-OCM. The protocol has been successfully applied to normal donor corneas and pathological corneal buttons, and can be used to identify healthy and pathologic features on both the macroscopic and microscopic level, thereby facilitating the detection of stromal disorders that could compromise the outcome of keratoplasty. By improving the graft quality control, this protocol has the potential to result in better selection (and rejection) of donor tissues and hence decreased graft failure.

We describe use of full-field optical coherence microscopy as a method for high quality assessment of corneal donor stroma. This protocol can be used to identify features indicative of health or disease, and is aimed at improving the screening and selection of donor tissues, and hence the results of keratoplasty.

全场光学相干显微镜，用于角膜移植物基质特征的组织学样分析

The quality of donor corneal stroma, which makes up about 90% of total corneal thickness, is likely to be one of the main, if not the major, limiting ...

免疫与感染

Characterization of Neuromuscular Junctions in Mice by Combined Confocal and Super-Resolution Microscopy

Neuromuscular junctions (NMJs) are highly specialized synapses between lower motor neurons and skeletal muscle fibers that play an essential role in the transmission of molecules from the nervous system to voluntary muscles, leading to contraction. They are affected in many human diseases, including inherited neuromuscular disorders such as Duchenne muscular dystrophy (DMD), congenital myasthenic syndromes (CMS), spinal muscular atrophy (SMA), and amyotrophic lateral sclerosis (ALS). Therefore, monitoring the morphology of neuromuscular junctions and their alterations in disease mouse models represents a valuable tool for pathological studies and preclinical assessment of therapeutic approaches. Here, methods for labeling and analyzing the three-dimensional (3D) morphology of the pre- and postsynaptic parts of motor endplates from murine teased muscle fibers are described. The procedures to prepare samples and measure NMJ volume, area, tortuosity and axon terminal morphology/occupancy by confocal imaging, and the distance between postsynaptic junctional folds and acetylcholine receptor (AChR) stripe width by super-resolution stimulated emission depletion (STED) microscopy are detailed. Alterations in these NMJ parameters are illustrated in mutant mice affected by SMA and CMS.

This protocol describes a method for morphometric analysis of neuromuscular junctions by combined confocal and STED microscopy that is used to quantify pathological changes in mouse models of SMA and ColQ-related CMS.

通过联合共聚焦和超分辨率显微镜表征小鼠的神经肌肉接头

Neuromuscular junctions (NMJs) are highly specialized synapses between lower motor neurons and skeletal muscle fibers that play an essential role in the ...

High-frequency Ultrasonography of the Digital and Palmar Nerve Branches of the Hand in Peripheral Nerve Diseases

Peripheral nerve ultrasound is a well-established imaging technique to evaluate certain peripheral nerve pathologies. However, there is a poor correlation between ultrasound abnormalities of peripheral nerves and electrodiagnostic or clinical evidence of axonal loss. This is a significant limitation of peripheral nerve ultrasound, as many peripheral nerve diseases encountered in clinical settings are related to axonal loss. Furthermore, clinical and electrodiagnostic evidence of axonal loss directly correlates with disability in all peripheral nerve diseases. However, due to the floor effects often encountered in electrodiagnostic studies, these correlations, as well as definitive diagnoses, are often challenging. Thus, imaging techniques that correlate with axonal loss are essential for expanding the utility of peripheral nerve ultrasound as a potential biomarker for peripheral nerve diseases. With new technological advancements and the ever-increasing imaging capabilities of high-frequency ultrasound, the palmar and digital nerve branches of the hand can be imaged with exceptionally high resolution even using point-of-care ultrasound devices. Their superficial and distal-most anatomic locations are ideal for evaluating polyneuropathies, as these branches degenerate earliest during axonal loss. However, no studies have systematically evaluated these nerve branches to determine if they can be reproducibly measured with ultrasound. The current protocol was adapted for the systematic assessment of cross-sectional areas of the median and ulnar nerves in the palmar surface and digits of the hand. This protocol provides reference data for a subset of nerves that demonstrate high intraclass correlation coefficients between three separate ultrasonographers. Finally, as a proof of concept and to demonstrate the clinical applications of this protocol, representative data from individuals with genetically confirmed inherited polyneuropathies are compared with established normative data to examine cross-sectional area differences.

The present protocol describes high-frequency neuromuscular ultrasonography of the digital and palmar branches of the median and ulnar nerve, which can aid in localizing peripheral nerve diseases and be adapted to evaluate digital nerve injuries.

Peripheral nerve ultrasound is a well-established imaging technique to evaluate certain peripheral nerve pathologies. However, there is a poor correlation ...

Serial Block-Face Scanning Electron Microscopy (SBF-SEM) of Biological Tissue Samples

Serial block-face scanning electron microscopy (SBF-SEM) allows for the collection of hundreds to thousands of serially-registered ultrastructural images, offering an unprecedented three-dimensional view of tissue microanatomy. While SBF-SEM has seen an exponential increase in use in recent years, technical aspects such as proper tissue preparation and imaging parameters are paramount for the success of this imaging modality. This imaging system benefits from the automated nature of the device, allowing one to leave the microscope unattended during the imaging process, with the automated collection of hundreds of images possible in a single day. However, without appropriate tissue preparation cellular ultrastructure can be altered in such a way that incorrect or misleading conclusions might be drawn. Additionally, images are generated by scanning the block-face of a resin-embedded biological sample and this often presents challenges and considerations that must be addressed. The accumulation of electrons within the block during imaging, known as "tissue charging," can lead to a loss of contrast and an inability to appreciate cellular structure. Moreover, while increasing electron beam intensity/voltage or decreasing beam-scanning speed can increase image resolution, this can also have the unfortunate side effect of damaging the resin block and distorting subsequent images in the imaging series. Here we present a routine protocol for the preparation of biological tissue samples that preserves cellular ultrastructure and diminishes tissue charging. We also provide imaging considerations for the rapid acquisition of high-quality serial-images with minimal damage to the tissue block.

Serial Block-Face Scanning Electron Microscopy SBF-SEM of Biological Tissue Samples

This protocol outlines a routine method for using serial block-face scanning electron microscopy (SBF-SEM), a powerful 3D imaging technique. Successful application of SBF-SEM hinges on proper fixation and tissue staining techniques, as well as careful consideration of imaging settings. This protocol contains practical considerations for the entirety of this process.

生物组织样本的串行块面扫描电子显微镜 （SBF-SEM）

Serial block-face scanning electron microscopy (SBF-SEM) allows for the collection of hundreds to thousands of serially-registered ultrastructural images, ...

生物学

<meta charset="utf8"></head><body>评估眼表疾病中角膜神经的健康状况

Corneal Sensitivity Testing Procedure for Ophthalmologic and Optometric Patients

The cornea is the most densely innervated structure in the human body, making it one of the most sensitive tissues. Changes in corneal nerve sensitivity can be observed in several ocular surface diseases. Nerve sensitivity may be increased, as is often seen in patients with a neuropathic component to ocular pain, or decreased, as is seen in patients with neurotrophic keratitis. Corneal sensitivity testing involves assessing a patient's reaction to brief corneal stimulation, yields insight into the health of the corneal nerves, and provides diagnostic value for evaluating the health of the nerves and the interplay with the ocular surface. Currently, there is limited published guidance on how to conduct corneal sensitivity testing in a clinical setting. This article presents a protocol for testing corneal sensitivity using easy-to-use, low-cost materials that are readily accessible to eye care providers (either a cotton swab, a piece of dental floss, or a finely tapered tissue). This protocol allows for qualitative assessment of corneal sensitivity in which responses to corneal stimulation are rated from 0 (no response) to 3 (hypersensitive response). This test can be performed quickly (in approximately 30 s). Given its diagnostic value and accessibility, corneal sensitivity testing should be included as part of the standard eye examination for any patient undergoing an ocular surface examination.

<meta charset="utf8"></head><body> 作者聚焦:在临床环境中建立实用且经济高效的角膜敏感性检测方案

Corneal sensitivity testing provides insight into the health of the corneal nerves and helps diagnose ocular surface diseases. We present a concise protocol to qualitatively assess corneal sensitivity that can be readily used by eye care providers across clinical settings.

眼科和验光患者的角膜敏感性测试程序

The cornea is the most densely innervated structure in the human body, making it one of the most sensitive tissues. Changes in corneal nerve sensitivity ...

角膜共聚焦显微镜:一种新型的非侵入性技术来量化小纤维病变周围神经病变

摘要

探索更多视频

此视频中的章节

三维成像伤害表皮内神经纤维在人体皮肤活检

在皮肤中的单神经末梢体内双光子显微镜

神经肌肉接头处:测量突触大小，碎片和改变突触蛋白密度利用共聚焦荧光显微镜

人 Intraepidermal 神经纤维内线粒体三维成像及分析

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

全场光学相干显微镜，用于角膜移植物基质特征的组织学样分析

通过联合共聚焦和超分辨率显微镜表征小鼠的神经肌肉接头

High-frequency Ultrasonography of the Digital and Palmar Nerve Branches of the Hand in Peripheral Nerve Diseases

生物组织样本的串行块面扫描电子显微镜（SBF-SEM）

眼科和验光患者的角膜敏感性测试程序

角膜共聚焦显微镜:一种新型的非侵入性技术来量化小纤维病变周围神经病变

摘要

探索更多视频

此视频中的章节

三维成像伤害表皮内神经纤维在人体皮肤活检

在皮肤中的单神经末梢体内双光子显微镜

神经肌肉接头处:测量突触大小，碎片和改变突触蛋白密度利用共聚焦荧光显微镜

人 Intraepidermal 神经纤维内线粒体三维成像及分析

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

全场光学相干显微镜，用于角膜移植物基质特征的组织学样分析

通过联合共聚焦和超分辨率显微镜表征小鼠的神经肌肉接头

High-frequency Ultrasonography of the Digital and Palmar Nerve Branches of the Hand in Peripheral Nerve Diseases

生物组织样本的串行块面扫描电子显微镜 （SBF-SEM）

眼科和验光患者的角膜敏感性测试程序

生物组织样本的串行块面扫描电子显微镜（SBF-SEM）