Name: high-speed human temporal bone sectioning for the assessment of covid-19-associated middle ear pathology
Uploaded: 2022-05-18T14:00:00
Description: Watch this Scientific Journal Video about High-Speed Human Temporal Bone Sectioning for the Assessment of COVID-19-Associated Middle Ear Pathology at JoVE.com

我们感谢穆罕默德·莱哈尔对这个项目的协助。这项工作得到了美国国立卫生研究院（T32DC000027，NSA）的部分支持。

该协议是在IRB（IRB00250002）批准下开发的，并符合使用人体组织和感染材料的机构政策。每个颞骨捐献者在死亡前都提供了书面同意，或者是在死后从捐献者家属那里获得的同意。有关本协议中使用的所有材料、设备和软件的详细信息，请参阅材料 表 。
1. 颞骨采集
<ol><li>在使用本方案之前，获得当地机构审查委员会（IRB）委员会的批准，审查使用?...

<ol>
	<li>Nogueira, J. F., 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=A+brief+history+of+otorhinolaryngology:+Otology,+laryngology+and+rhinology.">A brief history of otorhinolaryngology: Otology, laryngology and rhinology.</a> Brazilian Journal of Otorhinolaryngology. 73 (5), 693-703 (2007).</li><li>Pappas, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Otology+through+the+ages.">Otology through the ages.</a> Otolaryngology-Head and Neck Surgery. 114 (2), 173-196 (1996).</li><li>Schuknecht, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Otopathology:+The+past,+present,+and+future.">Otopathology: The past, present, and future.</a> Auris Nasus Larynx. 23, 43-45 (1996).</li><li>Monsanto, R. D. 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S., 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=Insights+into+presbycusis+from+the+first+temporal+bone+laboratory+within+the+United+States.">Insights into presbycusis from the first temporal bone laboratory within the United States.</a> Otology &amp; Neurotology. 43 (3), 400-408 (2022).</li><li>Schuknecht, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Pathology of the Ear. , (1993).</li><li>Chole, 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=Labs+in+crisis:+Protecting+the+science--and+art--of+otopathology.">Labs in crisis: Protecting the science--and art--of otopathology.</a> Otology &amp; Neurotology. 31 (4), 554-556 (2010).</li><li>Nager, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Pathology of the Ear and Temporal Bone. , (1993).</li><li>. COVID-19 Personal Protective Equipment (PPE) Available from: <a target="_blank" href="https://www.cdc.gov/niosh/emres/2019_ncov_ppe.html">https://www.cdc.gov/niosh/emres/2019_ncov_ppe.html</a> (2022)</li><li>Essalmani, R., 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=Distinctive+roles+of+Furin+and+TMPRSS2+in+SARS-CoV-2+infectivity.">Distinctive roles of Furin and TMPRSS2 in SARS-CoV-2 infectivity.</a> Journal of Virology. 96 (8), 0012822 (2022).</li><li>Ueha, R., Kondo, K., Kagoya, R., Shichino, S., Yamasoba, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ACE2,+TMPRSS2,+and+Furin+expression+in+the+nose+and+olfactory+bulb+in+mice+and+humans.">ACE2, TMPRSS2, and Furin expression in the nose and olfactory bulb in mice and humans.</a> Rhinology. 59 (1), 105-109 (2021).</li><li>Frazier, K. M., Hooper, J. E., Mostafa, H. H., Stewart, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SARS-CoV-2+virus+isolated+from+the+mastoid+and+middle+ear:+Implications+for+COVID-19+precautions+during+ear+surgery.">SARS-CoV-2 virus isolated from the mastoid and middle ear: Implications for COVID-19 precautions during ear surgery.</a> JAMA Otolaryngology - Head &amp; Neck Surgery. 146 (10), 964-966 (2020).</li><li>Cunningham, C. D., Schulte, B. A., Bianchi, L. M., Weber, P. C., Schmiedt, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microwave+decalcification+of+human+temporal+bones.">Microwave decalcification of human temporal bones.</a> Laryngoscope. 111 (2), 278-282 (2001).</li><li>Stephenson, R., 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=Immunohistochemical+location+of+Na+,+K+-ATPase+&#945;1+subunit+in+the+human+inner+ear.">Immunohistochemical location of Na+, K+-ATPase &#945;1 subunit in the human inner ear.</a> Hearing Research. 400, 108113 (2021).</li><li>McCall, A. A., 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=Extralabyrinthine+manifestations+of+DFNA9.">Extralabyrinthine manifestations of DFNA9.</a> Journal of the Association for Research in Otolaryngology. 12 (2), 141-149 (2011).</li><li>Wu, P. Z., O'Malley, J. T., de Gruttola, V., Liberman, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+hearing+loss+is+dominated+by+damage+to+inner+ear+sensory+cells,+not+the+cellular+battery+that+powers+them.">Age-related hearing loss is dominated by damage to inner ear sensory cells, not the cellular battery that powers them.</a> The Journal of Neuroscience. 40 (33), 6357-6366 (2020).</li><li>Miller, M. E., Lopez, I. A., Linthicum, F. H., Ishiyama, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Connexin+26+immunohistochemistry+in+temporal+bones+with+cochlear+otosclerosis.">Connexin 26 immunohistochemistry in temporal bones with cochlear otosclerosis.</a> Annals of Otology, Rhinology &amp; Laryngology. 127 (8), 536-542 (2018).</li><li>Lopez, I. A., 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=Immunohistochemical+techniques+for+the+human+inner+ear.">Immunohistochemical techniques for the human inner ear.</a> Histochemistry and Cell Biology. 146 (4), 367-387 (2016).</li></ol>

人类颞骨研究对于研究内耳和中耳病理学至关重要，但仍然是一项时间和资源密集型工作。本文介绍了一种使用金刚石显微镜锯生成厚颞骨切片的技术，这些切片可以在进一步切片之前快速脱钙，从而将从组织收获到研究的时间从9-10个月减少到10-14天。该技术可以减少颞骨处理所需的资源，并促进时间敏感型研究，例如与COVID-19中耳和内耳病理学13相关的研究，这是开发该协议的...

人类颞骨研究为研究内耳和中耳的病理学和病理生理学提供了宝贵的资源。在19世纪之前，人们对耳科疾病1，2，3知之甚少。为了更好地了解耳科疾病和“从庸医手中抢救出耳外科手术”，约瑟夫·汤因比（1815-1866）开发了研究人类颞骨组织学切片的方法3。在19世纪余下的时间里，维也纳的亚当·波利策（Adam Politzer，1835-1920）和欧洲各地的其他人进一步推动了这项工作，他使用颞骨切片来描述影响耳朵的许多常见疾病的组织病理学2，3，4。
美国第一个人类颞骨实验室于1927年在约翰霍普金斯医院开业，Stacy Guild（1890-1966）在那里开发了颞骨切片方法5，6。Guild开发的方法包括9-10个月的过程，包括死后收获，固定，硝酸脱钙，乙醇脱水，细胞样蛋白包埋，切片，染色和安装。哈罗德·舒克内希特（1917-1996）后来对这种技术进行了修改7;然而，这一过程的基本组成部分基本上没有改变。
维持颞骨实验室所需的大量资源对颞骨研究提出了挑战，并可能导致其在过去30年中受欢迎程度的下降4，8。颞骨实验室资源的很大一部分必须用于9-10个月的颞骨制备过程。准备过程中最耗时的步骤之一是颞骨的脱钙，颞骨是人体中最密集的骨头。脱钙通常在硝酸或乙二胺四乙酸（EDTA）中进行，需要数周至数月，同时需要频繁更换溶液7，9。此外，对人耳进行时间敏感的研究，例如与COVID-19大流行相关的研究，可能会受到这种缓慢的准备过程的阻碍。本文介绍了一种高速颞骨切片技术，该技术使用金刚石显微镜锯生成厚切片，从而可以在颞骨收获后10-14天内进行快速脱钙和组织分析。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anti-ACE-2 Antibody (1:50 applied dilution)</td>
			<td>Novus Biologicals</td>
			<td>SN0754</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Furin Antibody (1:250 dilution)</td>
			<td>Abcam</td>
			<td>EPR 14674</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-TMPRSS2 Antibody (1:1,000 dilution)</td>
			<td>Novus Biologicals</td>
			<td>NBP1-20984</td>
			<td></td>
		</tr>
		<tr>
			<td>BX43 Manual System Microscope</td>
			<td>Olympus Life Science Solutions</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CBN/Diamond Hybrid Wafering Blade</td>
			<td>Pace Technologies</td>
			<td>WB-007GP</td>
			<td></td>
		</tr>
		<tr>
			<td>Collin Mallet - 8&#39;&#39;</td>
			<td>Surgical Mart</td>
			<td>SM1517</td>
			<td></td>
		</tr>
		<tr>
			<td>DS-Fi3 Microscope Camera</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dual Endogenous Enzyme Block (commercial blocking solution)</td>
			<td>Dako</td>
			<td>S2003</td>
			<td></td>
		</tr>
		<tr>
			<td>Eaosin Stain</td>
			<td>Sigma-Aldrich</td>
			<td>548-24-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Formalin solution, neutral buffered 10%</td>
			<td>Sigma-Aldrich</td>
			<td>HT501128</td>
			<td></td>
		</tr>
		<tr>
			<td>Formical-4 Decalcifier (formic acid decalcifying solution)</td>
			<td>StatLab</td>
			<td>1214-1 GAL</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin Stain</td>
			<td>Sigma-Aldrich</td>
			<td>H9627</td>
			<td></td>
		</tr>
		<tr>
			<td>HRP-Conjugated Anti-Rabbit Secondary Antibody (1:100 dilution)</td>
			<td>Leica Biosystems</td>
			<td>PV6119</td>
			<td></td>
		</tr>
		<tr>
			<td>ImmPRESS HRP Horse Anti-Goat igG Detection Kit, Peroxidase (1:100 dilution)</td>
			<td>Vector Laboratories</td>
			<td>MP-7405</td>
			<td></td>
		</tr>
		<tr>
			<td>Lambotte Osteotome</td>
			<td>Surgical Mart</td>
			<td>SM1553</td>
			<td></td>
		</tr>
		<tr>
			<td>Metallographic PICO 155P Precision Saw</td>
			<td>Pace Technologies</td>
			<td>PICO 155P</td>
			<td>microsaw</td>
		</tr>
		<tr>
			<td>NIS Elements Software Version 4.6</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraplast Plus</td>
			<td>Sigma-Aldrich</td>
			<td>P3683</td>
			<td>paraffin</td>
		</tr>
		<tr>
			<td>Positive Charged Microscope Slides with White Frosted End</td>
			<td>Walter Products</td>
			<td>1140B15</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Shandon Crytome FSE Cryostat Microtome</td>
			<td>New Life Scientific Inc.</td>
			<td>A78900104</td>
			<td>cryotome</td>
		</tr>
		<tr>
			<td>Triology Pretreatment Solution (commercial pretreatment solution)</td>
			<td>Sigma-Aldrich</td>
			<td>920P-05</td>
			<td></td>
		</tr>		
 <tr>
			<td>Xylene</td>
			<td>Sigma-Aldrich</td>
			<td>920P-05</td>
			<td></td>
		</tr>
	</tbody>
</table>

high-speed human temporal bone sectioning for the assessment of covid-19-associated middle ear pathology

人颞骨切片的组织病理学分析是研究内耳和中耳病理学的基本技术。颞骨切片通过死后颞骨采集、固定、脱钙、包埋和染色制备。由于颞骨的密度，脱钙是一个耗时且资源密集型的过程;完整的组织准备平均可能需要9-10个月。这减缓了耳病理学研究，阻碍了时间敏感的研究，例如与COVID-19大流行相关的研究。本文描述了一种快速制备和脱钙颞骨切片以加速组织处理的技术。
使用标准技术在死后收获颞骨，并固定在10%福尔马林中。使用带有双金刚石锯片的精密微锯将每个部分切割成三个厚的部分。然后将厚颞骨切片在脱钙溶液中脱钙7-10天，然后嵌入石蜡中，使用冷冻组切片成薄（10μm）切片，并安装在未充电的载玻片上。然后将组织样本脱蜡并重新水合以进行抗体染色（ACE2，TMPRSS2，呋喃蛋白）并成像。该技术将从收获到组织分析的时间从9-10个月缩短到10-14天。高速颞骨切片可能会提高耳病理学研究的速度，减少组织制备所需的资源，同时还可以促进时间敏感的研究，例如与COVID-19相关的研究。

中耳粘膜和咽鼓管的苏木精和曙红染色显示处理后中耳粘膜和粘膜下中耳组织保存（图1）。免疫组化图像显示ACE2，TMPRSS2和Furiin蛋白在中耳粘膜和咽鼓管内的表达（图1）。这些蛋白质在中耳内的存在提供了一条可能的途径，通过该途径SARS-CoV-2可能感染中耳内的呼吸道上皮11，12，13?...

Watch this Scientific Journal Video about High-Speed Human Temporal Bone Sectioning for the Assessment of COVID-19-Associated Middle Ear Pathology at JoVE.com

High-Speed Human Temporal Bone Sectioning for the Assessment of COVID-19-Associated Middle Ear Pathology

本文介绍了一种用于快速人颞骨切片的技术，该技术利用带有双金刚石锯片的微锯生成薄片，用于快速脱钙和分析颞骨免疫组织化学。

用于评估COVID-19相关中耳病变的高速人颞骨切片

Histopathologic analysis of human temporal bone sections is a fundamental technique for studying inner and middle ear pathology. Temporal bone sections ...

该方案将颞骨处理的时间从9至10个月减少到10至14天。该技术的主要优点是可以快速评估耳病理学，在我们的例子中，这与评估COVID-19相关的中耳病理学有关。这种技术可能对颞骨的时间敏感研究特别有帮助。将来，它可能会进一步优化用于研究内耳病理学。在尸检期间开始收获，并将颅神经急剧地分开到内耳道。做第一个骨切口，骨骼与颞骨的鳞状部分平行并仅内侧。然后，在颞骨内侧边界进行与第一个骨平行的第二个骨切口。在前方三到四厘米处做第三个切口，平行于岩脊。接下来，使第四个切口大致平行于第三个切口，位于岩脊后两厘米处。使用尖锐的解剖以释放沿标本下边缘附着的软组织并去除颞骨。收获后，立即将组织浸没在200至300毫升10%缓冲福尔马林中。然后，将带有双金刚石锯片的精密微锯设置为五毫米的距离。为了快速组织脱钙，将每个标本切成三个部分，使得颞骨的中心部分厚5毫米，两端的颞骨厚度为3至5毫米。将切片放入200至300毫升0.37摩尔EDTA中，在室温下放置7至10天以进行软化。中耳粘膜和咽鼓管的苏木精和曙红染色表明，使用该方案处理后组织被保存下来。免疫组织化学分析显示，这些组织中存在ACE2，跨膜蛋白酶丝氨酸和呋喃蛋白，为中耳呼吸道上皮中的SARS-CoV2感染提供了可能的根源。在钻石锯内适当定位颞骨组织非常重要。可以与这种方法结合使用的替代技术包括不同蛋白质的免疫组织化学，不同的组织染色和转录组学。这项技术使我们能够快速评估COVID-19大流行期间中耳内SARS-CoV2感染中是否含有蛋白质。

Research

JoVE Journal

Immunology and Infection

Bioenergetics and the Oxidative Burst: Protocols for the Isolation and Evaluation of Human Leukocytes and Platelets

生物能量和氧化突发：协议用于人类白细胞和血小板的隔离与评价

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and ...

科研

免疫与感染

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

一种新型<em&gt;在体外</em&gt;模型研究人类全血细胞间的相互作用

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways ...

Super-resolution Imaging of the Natural Killer Cell Immunological Synapse on a Glass-supported Planar Lipid Bilayer

的自然杀伤细胞免疫突触上的玻璃支承平面脂双层超分辨率成像

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has ...

Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells (MSCs)

Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells MSCs

人骨髓间充质干细胞（干细胞）的免疫调节性能评估

The immunomodulatory properties of multilineage human mesenchymal stem cells (MSCs) appear to be highly relevant for clinical use towards a wide-range of ...

Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

对于检测磁力搅拌器方法<em&gt;旋毛虫</em&gt;幼虫肌肉样品

Trichinellosis is a debilitating disease in humans and is caused by the consumption of raw or undercooked meat of animals infected with the nematode ...

A Flow Cytometry-Based Cytotoxicity Assay for the Assessment of Human NK Cell Activity

人外周血 NK 细胞活性评估流基于流式细胞仪检测细胞毒性检测

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, ...

Assessment of the Cytotoxic and Immunomodulatory Effects of Substances in Human Precision-cut Lung Slices

人精切肺切片中物质的细胞毒性及免疫调节作用的评价

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

全血流式细胞术分析人单核细胞亚群的表征

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...

Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue

人外周血和淋巴组织中原发性 T 细胞突触界面的评价

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of ...

Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection

微链 rna 的共聚焦成像和模式识别受体在负感 rna 病毒感染中的应用

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors ...