Name: three-dimensional imaging of nociceptive intraepidermal nerve fibers in human skin biopsies
Uploaded: 2013-04-29T13:00:00
Description: Watch this Scientific Journal Video about Three-dimensional Imaging of Nociceptive Intraepidermal Nerve Fibers in Human Skin Biopsies at JoVE.com

这项工作是由美国国立卫生研究院资助的支持K08 NS061039-01A2，神经学研究发现，和A.阿尔弗雷德·陶布曼医学研究所在密歇根大学的计划。这项工作用了密歇根州的糖尿病研究和培训中心，由国家卫生部授予5P90 DK-20572研究院从国家糖尿病，消化道和肾脏疾病研究所资助的形态和图像分析核心。笔者想感谢罗宾逊的Singleton和戈登史密斯（犹他州大学）的慷慨捐款支持初始伤害性的生物标志物免疫组化技术的发展，人类皮肤样本。

A部分：免疫组化 96 - 孔板的下游和预防的背景染色打孔皮肤活检从人类受试者收集孵育12-24小时，在固定液（用0.75M的L-赖氨酸溶液（pH7.4）中的2％的多聚甲醛和0.05 mM的高碘酸钠）在4℃下，如前面所述。样本，然后cryoprotected的在磷酸盐缓冲盐水（PBS），在4℃的20％甘油1周，嵌入安装媒体最佳的切削温度（OCT），然后划分成50微米厚的部分在低温恒温器中。下面描述的?...

<ol>
	<li>Lauria, G., Holland, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+innervation:+changes+with+aging,+topographic+location,+and+in+sensory+neuropathy.">Epidermal innervation: changes with aging, topographic location, and in sensory neuropathy.</a> J. Neurol. Sci. 164 (2), 172-178 (1999).</li><li>Sullivan, K. A., Hayes, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+diabetic+neuropathy.">Mouse models of diabetic neuropathy.</a> Neurobiol. Dis. 28 (3), 276-285 (2007).</li><li>McArthur, J. C., Stocks, E. A., Hauer, P., Cornblath, D. R., Griffin, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+nerve+fiber+density:+normative+reference+range+and+diagnostic+efficiency.">Epidermal nerve fiber density: normative reference range and diagnostic efficiency.</a> Arch. Neurol. 55 (12), 1513-1520 (1998).</li><li>Griffin, J. W., McArthur, J. C., Polydefkis, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+cutaneous+innervation+by+skin+biopsies.">Assessment of cutaneous innervation by skin biopsies.</a> Curr. Opin. Neurol. 14 (5), 655-659 (2001).</li><li>Cheng, H. T., Dauch, J. R., Hayes, J. M., Yanik, B. M., Feldman, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nerve+growth+factor/p38+signaling+increases+intraepidermal+nerve+fiber+densities+in+painful+neuropathy+of+type+2+diabetes.">Nerve growth factor/p38 signaling increases intraepidermal nerve fiber densities in painful neuropathy of type 2 diabetes.</a> Neurobiol. Dis. 45 (1), 280-287 (2012).</li><li>Lauria, G., Lombardi, R., Camozzi, F., Devigili, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+biopsy+for+the+diagnosis+of+peripheral+neuropathy.">Skin biopsy for the diagnosis of peripheral neuropathy.</a> Histopathology. 54 (3), 273-285 (2009).</li><li>Vlckova-Moravcova, E., Bednarik, J., Dusek, L., Toyka, K. V., Sommer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+validity+of+epidermal+nerve+fiber+densities+in+painful+sensory+neuropathies.">Diagnostic validity of epidermal nerve fiber densities in painful sensory neuropathies.</a> Muscle Nerve. 37 (1), 50-60 (2008).</li><li>Casanova-Molla, J., Morales, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+fluorescence+quantitation+provides+a+new+approach+to+assess+cutaneous+innervation.">Axonal fluorescence quantitation provides a new approach to assess cutaneous innervation.</a> J. Neurosci. Methods. 200 (2), 190-198 (2011).</li><li>Wang, L., Hilliges, M., Jernberg, T., Wiegleb-Edstrom, D., Johansson, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+gene+product+9.5-immunoreactive+nerve+fibres+and+cells+in+human+skin.">Protein gene product 9.5-immunoreactive nerve fibres and cells in human skin.</a> Cell Tissue Res. 261 (1), 25-33 (1990).</li><li>Lauria, G., Morbin, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+capsaicin+receptor+immunoreactivity+in+human+peripheral+nervous+system+and+in+painful+neuropathies.">Expression of capsaicin receptor immunoreactivity in human peripheral nervous system and in painful neuropathies.</a> J. Peripher. Nerv. Syst. 11 (3), 262-271 (2006).</li><li>Penna, G., Fibbi, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+benign+prostatic+hyperplasia+stromal+cells+as+inducers+and+targets+of+chronic+immuno-mediated+inflammation.">Human benign prostatic hyperplasia stromal cells as inducers and targets of chronic immuno-mediated inflammation.</a> J. Immunol. 182 (7), 4056-4064 (2009).</li><li>Lentz, S. I., Edwards, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+DNA+(mtDNA)+Biogenesis:+Visualization+and+Duel+Incorporation+of+BrdU+and+EdU+Into+Newly+Synthesized+mtDNA+In+Vitro.">Mitochondrial DNA (mtDNA) Biogenesis: Visualization and Duel Incorporation of BrdU and EdU Into Newly Synthesized mtDNA In Vitro.</a> J. Histochem. Cytochem. 58 (2), 207-218 (2010).</li><li>Polydefkis, M., Hauer, P., Griffin, J. W., McArthur, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+biopsy+as+a+tool+to+assess+distal+small+fiber+innervation+in+diabetic+neuropathy.">Skin biopsy as a tool to assess distal small fiber innervation in diabetic neuropathy.</a> Diabetes Technol. Ther. 3 (1), 23-28 (2001).</li><li>Lauria, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+fibre+neuropathies.">Small fibre neuropathies.</a> Curr. Opin. Neurol. 18 (5), 591-597 (2005).</li><li>Sorensen, L., Molyneaux, L., Yue, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relationship+among+pain,+sensory+loss,+and+small+nerve+fibers+in+diabetes.">The relationship among pain, sensory loss, and small nerve fibers in diabetes.</a> Diabetes Care. 29 (4), 883-887 (2006).</li><li>Lauria, G., Morbin, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+swellings+predict+the+degeneration+of+epidermal+nerve+fibers+in+painful+neuropathies.">Axonal swellings predict the degeneration of epidermal nerve fibers in painful neuropathies.</a> Neurology. 61 (5), 631-636 (2003).</li><li>Herrmann, D. N., McDermott, M. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+nerve+fiber+density,+axonal+swellings+and+QST+as+predictors+of+HIV+distal+sensory+neuropathy.">Epidermal nerve fiber density, axonal swellings and QST as predictors of HIV distal sensory neuropathy.</a> Muscle Nerve. 29 (3), 420-427 (2004).</li><li>Navarro, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+27:+Neural+plasticity+after+nerve+injury+and+regeneration.">Chapter 27: Neural plasticity after nerve injury and regeneration.</a> Int. Rev. Neurobiol. 87, 483-505 (2009).</li></ol>

测量IENFD已被广泛用于周围神经病变13,14的程度来决定。目前，最常用的协议仅用于测量神经纤维的密度，穿透基底膜的表皮，它并没有考虑到轴突分枝和/或神经的形态变化。此外，还没有被证明电流IENFD分析与疼痛的存在PN 15相关联IENFD的。 我们以前报道，都与在db / db小鼠的疼痛行为，2型糖尿病的小鼠模型增加的痛觉IENFD的数字。在该报告中，我们首先采用?...

目前，通常的做法是从皮肤打孔活检，可用于诊断小纤维神经病6-8医师量化表皮内神经纤维密度，（IENFD）。活检取自小腿远端（DL），10厘米以上外踝，和大腿近端（PT），20厘米以下的髂前上棘9。 ，所有IENF标记蛋白基因产物9.5（PGP），泛神经标记10-12。目前，标准做法是小纤维神经病诊断确定由PGP染色与明视场显微镜6 IENFD。此外，一些研究小组已经使用PGP免疫组化免疫协议7-9。小纤维神经病变通常伴有神经性疼痛。为了进一步了解必不可少的疼痛处理IENF的作用，我们制定了技术合作标签总IENF的纤维，产生疼痛。 Nocicep略去IENF，特别是Aδ和C纤维，可以研究通过使用PGP IENF标签和痛觉标记，原肌球蛋白受体激酶A（TRK）5。蒂尔克是伤害性的发展是必不可少的神经生长因子的高亲和力受体。 A-阳性的TRK痛觉神经纤维肽纤维快递P物质（SP）和降钙素基因相关肽（CGRP）。此前，劳里亚和他的同事们采用双标记技术，研究PN，共同标签PGP阳性IENF与疼痛标记10。在我们以前的研究中，我们证实蒂尔克A-阳性IENF，但不是蒂尔克A-负面IENF的，上调糖尿病痛性神经病变的动物模型。这个共同标签技术提供了比较量化痛觉IENFD的，总IENFD和学习能力与PN相关的形态学变化的能力。可视的痛觉IENF和COMPA能力，重新量化总IENFD到痛觉IENFD的疼痛的存在可以提供客观的证据，并有可能洞察疼痛的严重程度与PN。这种技术也适用于皮肤的动物模型。在以往的研究相比，目前的协议描述为3D图像分析的方法，创造了机会，以避免错误可能发生在2D图像分析。

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>10X PBS</td>
			<td>Fisher Scientific</td>
			<td>BP399-4</td>
			<td>To make up 1X PBS</td>
		</tr>
		<tr>
			<td>Image-IT FX Signal</td>
			<td>Invitrogen</td>
			<td>I36933</td>
			<td>Image-IT</td>
		</tr>
		<tr>
			<td>Protein Gene Product 9.5 (Polyclonal rabbit)</td>
			<td>AbD Serotec</td>
			<td>7863-0504</td>
			<td>PGP</td>
		</tr>
		<tr>
			<td>Tropomyosin Related-Kinase A (Polyclonal goat)</td>
			<td>R&amp;D Systems</td>
			<td>AF1056</td>
			<td>Trk A</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 donkey &alpha;-rabbit</td>
			<td>Invitrogen</td>
			<td>A21206</td>
			<td>AF488 donkey &alpha;-goat</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 donkey &alpha;-goat</td>
			<td>Invitrogen</td>
			<td>A21447</td>
			<td>AF647 donkey &alpha;-goat</td>
		</tr>
		<tr>
			<td>Albumin, from Bovine Serum</td>
			<td>Sigma-Aldrich</td>
			<td>A7906-100</td>
			<td>BSA</td>
		</tr>
		<tr>
			<td>Triton X- 100</td>
			<td>Sigma-Aldrich</td>
			<td>T9284</td>
			<td>TX-100</td>
		</tr>
		<tr>
			<td>Non-calibrated Loop</td>
			<td>LeLoop</td>
			<td>MP 199025</td>
			<td>inoculating Loop</td>
		</tr>
		<tr>
			<td>96-well assay plate</td>
			<td>Corning Incorporated</td>
			<td>3603</td>
			<td>Well plate</td>
		</tr>
		<tr>
			<td>Prolong Gold antifade reagent with DAPI</td>
			<td>Invitrogen</td>
			<td>P36931</td>
			<td>DAPI</td>
		</tr>
		<tr>
			<td>Microscope Cover Glass 22x22 mm</td>
			<td>Fisher Scientific</td>
			<td>12-541-B</td>
			<td>Coverslips</td>
		</tr>
		<tr>
			<td>Superfrost Plus Microscope Slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
			<td>Microscope Slides</td>
		</tr>
		<tr>
			<td>Olympus Fluoview Laser Scanning Confocal Microscope</td>
			<td>Olympus</td>
			<td>FV500</td>
			<td>Confocal Microscope</td>
		</tr>
		<tr>
			<td>Optimum Cutting Temperature</td>
			<td>Sakura</td>
			<td>4583</td>
			<td>OCT</td>
		</tr>
		<tr>
			<td>Leica cryostat</td>
			<td>Leica</td>
			<td>CM1850</td>
			<td>Cryostat</td>
		</tr>
	</tbody>
</table>

three-dimensional imaging of nociceptive intraepidermal nerve fibers in human skin biopsies

一拳的皮肤活检是常用的量化表皮内神经纤维密度（IENFD）的周边多发性神经病变的诊断1,2。目前，通常的作法是，以收集从小腿远端（DL）和近端大腿长度依赖多神经病的评价3（PT）中的3毫米的皮肤活组织切片检查。然而，由于多向性质IENFs，它是具有挑战性的研究通过分析重叠的神经结构的二维（2D）成像。另外，三维（3D）成像，可以提供一个更好的解决方案，这种困境。 在目前的报告中，我们提出了应用三维成像的方法研究痛性神经病变（PN）。为了拣选IENFs的，皮肤样本进行处理的蛋白基因产物9.5（PGP），锅的神经元的标记物的免疫荧光分析。目前，标准做法是诊断小纤维神经病使用IENFD阻止使用PGP免疫组化开采的明视场显微镜4。在目前的研究中，我们采用双重免疫分析识别总IENFD，使用PGP，痛觉IENF，通过使用抗体，承认原肌球蛋白受体激酶A（TRK），高亲和力受体的神经生长因子5。 PGP和TRK A抗体共同染色IENF与优势，有利于PN明确PGP阳性，痛觉纤维染色的研究。这些荧光信号可以被量化，以确定的伤害IENFD及形态学的影响IENF与PN。荧光共聚焦显微镜图像采集和3D分析处理。 3D成像提供了旋转的能力，以进一步分析与PN相关的形态学变化。两者合计，荧光染色，共聚焦成像和3D分析明显受益PN的研究。

我们套用了目前的协议与PN患者在PT和DL皮肤活检研究的形态IENF。犹他州立大学的皮肤，从三个科目，收集展示的病理形态学与PN。主要内容包括：案例1：一名51岁的男性与2型糖尿病（持续时间：14个月;疼痛得分：51）的PN历史案例2：一个56岁的男性与PN的历史2型糖尿病（工期：108个月;疼痛得分：47）;案例3：一个66岁的男性与PN 2型糖尿病（持续时间：42个月;疼痛得分：46）的历史。神经系统病史和?...

Watch this Scientific Journal Video about Three-dimensional Imaging of Nociceptive Intraepidermal Nerve Fibers in Human Skin Biopsies at JoVE.com

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

为了研究伤害表皮内神经纤维（IENFs）在痛苦神经病（PN）的变化，我们开发的协议，可以直接检查观察伤害IENFs的三维形态变化。三维分析IENFs的有潜力评估的形态变化IENF 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. ...

Research

JoVE Journal

Medicine

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

人类神经内分泌肿瘤细胞株为研究人类神经内分泌肿瘤治疗的三维模型

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

科研

医学

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

三维组织培养模型研究人类原发性骨髓和恶性肿瘤

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Three-Dimensional 3D Tumor Spheroid Invasion Assay

三维（3D）肿瘤球体侵袭试验

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells

人垂体腺瘤细胞的三维海藻酸钠珠文化

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from ...

Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

通过轻型显微镜对小鼠急性心肌炎模型中白细胞浸润的定量可视化

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently ...

Objective Nociceptive Assessment in Ventilated ICU Patients: A Feasibility Study Using Pupillometry and the Nociceptive Flexion Reflex

应用 Pupillometry 和伤害性屈曲反射的可行性研究--通气 ICU 患者的客观伤害评估

The concept of objective nociceptive assessment and optimal pain management have gained increasing attention. Despite the known negative short- and ...

Three-Dimensional Printing of a Complex Aortic Anomaly

复杂主动脉异常的三维打印

Complex congenital aortic anomalies include diverse types of malformations that may be clinically asymptomatic or present with respiratory or esophageal ...

Refined CLARITY-Based Tissue Clearing for Three-Dimensional Fibroblast Organization in Healthy and Injured Mouse Hearts

在健康和受伤的老鼠心脏中为三维纤维细胞组织进行精炼的清晰组织清除

Cardiovascular disease is the most prevalent cause of mortality worldwide and is often marked by heightened cardiac fibrosis that can lead to increased ...

High-Resolution Three-Dimensional Imaging of the Footpad Vasculature in a Murine Hindlimb Gangrene Model

小鼠后肢坏死模型中脚垫脉管系统的高分辨率三维成像

Peripheral arterial disease (PAD) is a significant cause of morbidity resulting from chronic exposure to atherosclerotic risk factors. Patients suffering ...

Guided Endodontics: Three-Dimensional Planning and Template-Aided Preparation of Endodontic Access Cavities

引导牙髓病学：牙髓通路腔的三维规划和模板辅助制备

Pulp canal obliterations (PCO) are often a consequence of dental trauma, such as luxation injuries. Even though dentin apposition is a sign of vital pulp, ...