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。下面描述的协议，是专为8内的皮肤，皮肤切片的最大数量未能经过自由浮动的免疫组化实验在一个96 - 孔板。 第1天： 1。防止非特异性免疫反应的角质层<ol><li>出版商96 -孔板，如在图1中示出。 </lI&gt; <li>图像IT FX信号（图像），有效阻断背景染色11,12，第1行中的96孔板，每孔加入150μl。 </li><li>行2和3的96 - 孔板中，在每孔中加入150μl的PBS洗涤。 </li><li>收购两项每名患者的50微米的部分：一节从DL活检，活检采取PT的一个部分。接种环（LeLoop）是用来传输从一个部分到下一个冲洗，以避免形态破坏。轻轻将每50微米的部分，用接种环，进入个人的第1行，包含图像。孵育30分钟，在RT上平坦的摇臂部分在图像。 </li><li>冲洗部分用1X PBS（行2和3）的两倍，在RT下10分钟。将漂洗之间的一台摇臂孔板。 </li></ol> 2。 5％BSA封闭液和1％的清​​洗液的制备<ol><li>虽然活检切片图像IT孵化，准备5％封闭的解决方案重刑（5％BSA，0.3％TX-100，0.1 M的PBS涡BSA解决方案，直到完全溶解）。 </li><li>进入第4行每口井的5％BSA封闭液加入150μl。 </li><li>用接种环，部分转移到个别井的5％BSA封闭液。为1-2小时，在RT下孵育5％牛血清白蛋白封闭溶液中的部分，在平坦的摇臂。 </li></ol> 3。 1％BSA冲洗液和稀释初级抗体的制备<ol><li>虽然部分5％BSA封闭溶液中孵育，制备1％冲洗液（1％BSA，0.3％TX-100，0.1M PBS涡的BSA溶液，直至完全溶解）。 </li><li>虽然部分5％BSA封闭溶液中孵育，稀释第一抗体在1％漂洗液。 <ol><li>对于八个部分（8×150微升= 1200微升）共1,200微升是必要的。第一抗体是由在1500微升（约20％的额外体积）。 </li><li>稀释的主要抗体：PGP，1:500; TRKÅ，1:500在含1％BSA的冲洗液。 </li></ol></li></ol> 4。孵化的切片活检小学抗体<ol><li>到指定的96 - 孔板的孔中（第5行），加入150μl稀释的第一抗体。 </li><li>传输部分从封锁5％的溶液（第4行）到指定的初级抗体井（第5行）。 </li><li>用封口膜和铝箔密封的96 - 孔板，以避免干燥和曝光灯。 </li><li>一台摇臂上，孵育96孔板O / N在4℃。 </li></ol>第2天： 5。在1％BSA的冲洗液冲洗穿刺活检<ol><li>到第6行中的各孔，7，和8中，加入150μl含1％BSA的冲洗液。 </li><li>冲洗部分三次，用1％BSA（行6，7，和8），在RT 1小时，每次冲洗液。盖孔板铝箔和冲洗平面之间的摇杆。 </li></ol> 6。稀释二级抗体 &lt;醇&gt; <li>当章节被培养在含1％BSA的冲洗液在最后一次漂洗（第8行），1％BSA的冲洗液中稀释的二抗： <ol><li>对于八个部分（8×150微升= 1200微升）共1,200微升是必要的。该次级抗体是由在1500微升（约20％的额外体积）。 </li><li>的二级抗体稀释：PGP（的Alexa Fluor 488驴抗兔1:250），蒂尔克的Alexa Fluor 647驴抗山羊，1:250），A（1％BSA冲洗液中。 </li></ol></li></ol> 7。孵化的切片活检中学抗体<ol><li>到他们的指定的96 - 孔板的孔中（行9）加入150μl稀释的二级抗体。 </li><li>从1％BSA封闭溶液（第8行），在进入第二抗体以及（9行）传输部分。 </li><li>用封口膜和铝箔密封的96 - 孔板，以避免干燥和曝光灯。 </li><li>孵育部分在指定的二级抗体Ø/ N在4°C一台摇臂上。 </li></ol> 8。在1％BSA的冲洗液冲洗穿刺活检<ol><li>加入150μl含1％BSA的冲洗液到每口井的行10，11，和12。 </li><li>冲洗部分三次，用1％BSA漂洗溶液（行10，11，和12），在RT每次1小时。盖以及用铝箔板和一台摇臂之间冲洗。 </li></ol> 9。显微镜玻片制备和安装切片活检<ol><li>虽然活检切片孵育1％BSA冲洗液（第12行）在最后一次漂洗时，准备显微镜幻灯片。 </li><li>将50μl的1％BSA漂洗溶液一次一张幻灯片上。 </li><li>删除部分从1％BSA的冲洗液（第12行），并将其放置在1％BSA漂洗指定的显微镜载片上的解决办法，将50μl滴。优化部分的位置后，用鼓起的玻璃吸管，去除多余的1％BSA冲洗液，采取预防措施以避免触及标本。 </li></ol> 注意：请确保部分不折叠;，试样应反对表面的显微镜幻灯片持平。 <ol start="4"><li>将1滴延长Gold抗淬灭试剂用DAPI附近的活检安装在显微镜幻灯片。一个22x22毫米显微镜的盖玻片，轻轻把它放在活检和一滴延长Gold抗淬灭试剂DAPI下降。重复的每个部分。 </li><li>让显微镜玻片干燥RT在黑暗O / N。 </li></ol> 注：删除任何气泡，用枪头。擦去多余的延长黄金抗淬灭剂。 B部分：共聚焦成像 10。共聚焦成像<ol><li>荧光图像信号采用奥林巴斯FluoView的500激光扫描共聚焦显微镜40X的油浸目标和变焦两次与FluoView 5.0版软件（1.3 NA）。 </LI&gt; <li>使用的Alexa Fluor 488和647的Alexa Fluor 543-nm的绿色氦氖激光和633 nm的氦氖红色激光，分别以激发。的Alexa Fluor 488的信号表示由绿色的查找表（LUT）的Alexa Fluor 647由红色LUT。 </li><li>每个检测器的共焦的孔设置在400微米，以提高信号。应采取循序扫描，分辨率为1024×1024，以最大限度地提高信号分离。 </li><li>使用1.2μm的z轴步进间隔（基于计算由FluoView软件的最佳扫描单元）与卡尔曼的平均（两帧）捕获三维的z系列。 </li></ol> C部分：三维可视化与动画 11。三维（3D）的可视化和动画<ol><li> Imaris里X64软件（版本7.3，位平面AG）打开Fluoview文件，以可视化的三维图像集。 </li><li>调整显示根据需要，以提高特定的神经信号的对比度。 </li><li>创建一个SUrface可视化真皮表皮边界。使用轮廓工具绘制模式绘制边界。 </li><li>分配一个半透明的表面的边界，以便查看相关的荧光信号。 </li><li>捕获静止图像的3D荧光信号来创建快照映像的3D电影。 </li><li>使用动画功能来创建一个3D的电影。图像可以360度旋转几次，以显示每个信号分开和合并后的（ 图2，图3，和4）。设置动画创建由每秒15帧200帧动画电影。作为一个原始的AVI文件保存每部电影。 </li><li>压缩最终的AVI电影的VirtualDub软件（版本1.9.4）。 </li></ol>

<ol>
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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. 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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>

没有利益冲突的声明。

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. ...

three-dimensional-imaging-nociceptive-intraepidermal-nerve-fibers

Research

JoVE Journal

Medicine

13.0K 次观看.  University of Michigan. 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。

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

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

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 malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer 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

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.

In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue.

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

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

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

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from brown sea algae. Briefly, the tumor tissue is cut into small pieces and submitted to an enzymatic digestion with collagenase and trypsin. Next, a cell suspension is obtained. The tumor cell suspension is mixed with 1.2% sodium alginate and dropped into a CaCl2 solution, and the alginate/cell suspension is gelled on contact with the CaCl2 to form spherical beads. The cells embedded in the alginate beads are supplied with nutrients provided by the culture media enriched with 20% FBS. Three-dimensional culture in alginate beads maintains the viability of adenoma cells for long periods of time, up to four months. Moreover, the cells can be liberated from the alginate by washing the beads with sodium citrate and seeded on glass coverslips for further immunocytochemical analyses. The use of a cell culture model allows for the fixation and visualization of the actin cytoskeleton with minimal disorganization. In summary, alginate beads provide a reliable culture system for the maintenance of pituitary adenoma cells.

Three-dimensional culture in alginate beads, due to its simplicity and reproducibility, was chosen to maintain the pituitary adenoma cells. The procedures included an initial enzymatic digestion and mechanical dissociation of the tumor tissue, and the subsequent cell suspension was encapsulated in alginate beads.

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

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently labelled structures in large biological specimens, and is down to cellular resolution. Meanwhile, the constant refinement of underlying protocols and the enhanced availability of specialized commercial systems enable us to investigate the microstructure of whole mouse organs and even allow for the characterization of cellular behavior in various live-cell imaging approaches. Here, we describe a protocol for the spatial whole-mount visualization and quantification of the CD45+ leukocyte population in inflamed mouse hearts. The method employs a transgenic mouse strain (CD11c.DTR)that has recently been shown to serve as a robust, inducible model for the study of the development of fulminant fatal myocarditis, characterized by lethal cardiac arrhythmias. This protocol includes myocarditis induction, intravital antibody-mediated cell staining, organ preparation, and LSFM with subsequent computer-assisted image post-processing. Although presented as a highly-adapted method for our particular scientific question, the protocol represents the blueprint of an easily adjustable system that can also target completely different fluorescent structures in other organs and even in other species.

Here, we describe a light-sheet microscopy approach to visualize the cardiac CD45+ leukocyte infiltrate in a murine model of aseptic fulminant myocarditis, which is induced by the intratracheal diphtheria toxin treatment of CD11c.DTR mice.

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

The concept of objective nociceptive assessment and optimal pain management have gained increasing attention. Despite the known negative short- and long-term consequences of unresolved pain or excessive analgosedation, adequate nociceptive monitoring remains challenging in non-communicative, critically ill adults. In the intensive care unit (ICU), routine nociceptive evaluation is carried out by the attending nurse using the Behavior Pain Scale (BPS) in mechanically ventilated patients. This assessment is limited by medication use (e.g., neuromuscular blocking agents) and the inherent subjective character of nociceptive evaluation by third parties.
Here, we describe the use of two nociceptive reflex testing devices as tools for objective pain evaluation: the pupillary dilation reflex (PDR) and nociception flexion reflex (NFR). These measurement tools are non-invasive and well tolerated, providing clinicians and researchers with objective information regarding two different nociceptive processing pathways: (1)&#160;the pain-related autonomic reactivity&#160;and (2) the ascending component of the somatosensory system. The use of PDR and NFR measurements are currently limited to specialized pain clinics and research institutions because of impressions that these are technically demanding or time-consuming procedures, or even because of a lack of knowledge regarding their existence.
By focusing on the two abovementioned nociceptive reflex assessments, this study evaluated their feasibility as a physiological pain measurement method in daily practice. Pursuing novel technologies for evaluating the analgesia level in unconscious patients may further improve individual pharmacological treatment and patient related outcome measures. Therefore, future research must include large well-designed clinical trials in a real-life environment.

Pain assessment in anesthetized patients who cannot communicate with the outside world in any way remains challenging despite the development of innovative objective pain evaluation tools. In this project, the pupillary dilation reflex and the nociception flexion reflex are assessed in critically ill, mechanically ventilated adult patients.

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

Complex congenital aortic anomalies include diverse types of malformations that may be clinically asymptomatic or present with respiratory or esophageal symptoms. These anomalies may be associated with other congenital heart diseases. It is hard to identify the accurate anatomic vessel location from two-dimensional imaging data, such as computed tomography. As an additive manufacturing method, three-dimensional (3-D) printing can covert the acquired imaging data into 3-D physical models. This protocol describes the procedure for modeling the volumetric DICOM imaging into 3-D data and printing it as an anatomically realistic 3-D model. Using this model, surgeons can identify the vessel location of complex aortic anomalies, which is helpful for pre-operative planning and intra-operative guidance.

Here, we present a protocol to use three dimensional printed models for pre-operative planning and intra-operative reorganization of complicated vascular locations when handling a congenital 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

Cardiovascular disease is the most prevalent cause of mortality worldwide and is often marked by heightened cardiac fibrosis that can lead to increased ventricular stiffness with altered cardiac function. This increase in cardiac ventricular fibrosis is due to activation of resident fibroblasts, although how these cells operate within the 3-dimensional (3-D) heart, at baseline or after activation, is not well understood. To examine how fibroblasts contribute to heart disease and their dynamics in the 3-D heart, a refined CLARITY-based tissue clearing and imaging method was developed that shows fluorescently labeled cardiac fibroblasts within the entire mouse heart. Tissue resident fibroblasts were genetically labeled using Rosa26-loxP-eGFP florescent reporter mice crossed with the cardiac fibroblast expressing Tcf21-MerCreMer knock-in line. This technique was used to observe fibroblast localization dynamics throughout the entire adult left ventricle in healthy mice and in fibrotic mouse models of heart disease. Interestingly, in one injury model, unique patterns of cardiac fibroblasts were observed in the injured mouse heart that followed bands of wrapped fibers in the contractile direction. In ischemic injury models, fibroblast death occurred, followed by repopulation from the infarct border zone. Collectively, this refined cardiac tissue clarifying technique and digitized imaging system allows for 3-D visualization of cardiac fibroblasts in the heart without the limitations of antibody penetration failure or previous issues surrounding lost fluorescence due to tissue processing.

A refined method of tissue clearing was developed and applied to the adult mouse heart. This method was designed to clear dense, autofluorescent cardiac tissue, while maintaining labeled fibroblast fluorescence attributed to a genetic reporter strategy.

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

Peripheral arterial disease (PAD) is a significant cause of morbidity resulting from chronic exposure to atherosclerotic risk factors. Patients suffering from its most severe form, chronic limb-threatening ischemia (CLTI), face substantial impairments to daily living, including chronic pain, limited walking distance without pain, and nonhealing wounds. Preclinical models have been developed in various animals to study PAD, but mouse hindlimb ischemia remains the most widely used. There can be significant variation in response to ischemic insult in these models depending on the mouse strain used and the site, number, and means of arterial disruption. This protocol describes a unique method combining femoral artery and vein electrocoagulation with the administration of a nitric oxide synthase (NOS) inhibitor to reliably induce footpad gangrene in Friend Virus B (FVB) mice that resembles the tissue loss of CLTI. While traditional means of assessing reperfusion such as laser Doppler perfusion imaging (LDPI) are still recommended, intracardiac perfusion of the lipophilic dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) is used to label the vasculature. Subsequent whole-mount confocal laser scanning microscopy allows for high-resolution, three-dimensional (3D) reconstruction of footpad vascular networks that complements traditional means of assessing reperfusion in hindlimb ischemia models.

The present protocol describes a unique, clinically relevant model of peripheral arterial disease that combines femoral artery and vein electrocoagulation with the administration of a nitric oxide synthase inhibitor to induce hindlimb gangrene in FVB mice. Intracardiac DiI perfusion is then used for high-resolution, three-dimensional imaging of the footpad vasculature.

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

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, pulpitis or apical periodontitis may develop in the long term. Root canal treatment of teeth with severe PCO and pulpal or periapical pathosis is challenging for general practitioners and even for well-equipped endodontic specialists. To ensure detection of the calcified root canal and avoid excessive loss of tooth structure or root perforation, static navigation using templates ("Guided Endodontics") was introduced a few years ago. The general workflow includes three-dimensional imaging using cone-beam computed tomography (CBCT), a digital surface scan, and superimposition of both in a planning software. This is followed by virtual planning of the access cavity and the design of a template that will guide the drill to the desired target point. To do this, a true-to-scale virtual image of the drill must be placed in a way that the tip of the drill reaches the orifice of the calcified root canal. Once the template has been fabricated using computer-aided design and computer-aided manufacturing (CAD/CAM) or a 3D printer, guided preparation of the access cavity can be performed clinically. For research purposes, a postoperative CBCT image can be used to quantify the accuracy of the access cavity performed. This work aims to present the technique of static guided endodontics from imaging to clinical implementation.

Guided endodontics describes a template-aided approach for access cavity preparation. The procedure requires cone-beam computed tomography and a surface scan to produce a template. An incorporated sleeve guides the drill to the target point. This allows the preparation of minimally invasive endodontic access cavities in calcified teeth.

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, ...