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

We thank Mohamed Lehar for his assistance with this project. This work was partially supported by the National Institutes of Health (T32DC000027, NSA).

This protocol was developed with IRB (IRB00250002) approval and in accordance with institutional policies for the use of human tissue and infectious material. Each temporal bone donor provided written consent before death, or consent was obtained posthumously from the donor&#39;s family. See the Table of Materials for details about all materials, equipment, and software used in this protocol.
1. Temporal bone harvest
<ol>
	<li>Obtain local institutional revi...

<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. 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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>

Human temporal bone research is critical for studying inner and middle ear pathology but remains a time- and resource-intensive endeavor. This paper describes a technique that uses a diamond microsaw to generate thick temporal bone sections that can be rapidly decalcified before further sectioning so that the time from tissue harvest to study can be reduced from 9-10 months to 10-14 days. This technique may reduce the resources required for temporal bone processing and facilitate time-sensitive studies, such as those rel...

Human temporal bone research provides an invaluable resource to study the pathology and pathophysiology of the inner and middle ear. Before the 19th century, little was known regarding otologic disease1,2,3. To better understand otologic disease and &#34;rescue aural surgery from the hands of quacks,&#34; Joseph Toynbee (1815-1866) developed methods to study histologic sections of the human temporal bone3. This work was furthered by Adam Politzer (1835-1920) in Vienna and others across Europe during the remainder of the 19th century, who used temporal bone sections to describe the histopathology of many common conditions affecting the ear2,3,4.
The first human temporal bone laboratory in the United States was opened in 1927 at Johns Hopkins Hospital, where Stacy Guild (1890-1966) developed methods for temporal bone sectioning5,6. The methods developed by Guild consisted of a 9-10 month process that included postmortem harvest, fixation, decalcification in nitric acid, dehydration in ethanol, celloidin embedding, sectioning, staining, and mounting. Modifications to this technique were later made by Harold Schuknecht (1917-1996)7; however, the basic components of this process remain essentially unchanged.
The significant resources required to maintain a temporal bone laboratory have presented a challenge for temporal bone research and likely contributed to its declining popularity over the past 30 years4,8. A significant portion of temporal bone laboratory resources must be devoted to the 9-10 month process of temporal bone preparation. One of the most time-consuming steps in preparation is the decalcification of the temporal bone, which is the densest bone in the human body. Decalcification is typically performed in nitric acid or ethylenediaminetetraacetic acid (EDTA) and takes weeks to months while requiring the frequent changing of solutions7,9. Further, time-sensitive studies of the human ear, such as those related to the COVID-19 pandemic, may be hindered by this slow preparation process. This paper describes a technique for high-speed temporal bone sectioning that uses a diamond microsaw to generate thick sections that allow for rapid decalcification and tissue analysis within 10-14 days of temporal bone harvest.

<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

Histopathologic analysis of human temporal bone sections is a fundamental technique for studying inner and middle ear pathology. Temporal bone sections are prepared by postmortem temporal bone harvest, fixation, decalcification, embedding, and staining. Due to the density of the temporal bone, decalcification is a time-consuming and resource-intensive process; complete tissue preparation may take 9-10 months on average. This slows otopathology research and hinders time-sensitive studies, such as those relevant to the COVID-19 pandemic. This paper describes a technique for the rapid preparation and decalcification of temporal bone sections to speed tissue processing.
Temporal bones were harvested postmortem using standard techniques and fixed in 10% formalin. A precision microsaw with twin diamond blades was used to cut each section into three thick sections. Thick temporal bone sections were then decalcified in decalcifying solution for 7-10 days before being embedded in paraffin, sectioned into thin (10 &#181;m) sections using a cryotome, and mounted on uncharged slides. Tissue samples were then deparaffinized and rehydrated for antibody staining (ACE2, TMPRSS2, Furin) and imaged. This technique reduced the time from harvest to tissue analysis from 9-10 months to 10-14 days. High-speed temporal bone sectioning may increase the speed of otopathology research and reduce the resources necessary for tissue preparation, while also facilitating time-sensitive studies such as those related to COVID-19.

Hematoxylin and eosin staining of the middle ear mucosa and Eustachian tube showed preservation of the middle ear mucosa and submucosal middle ear tissue following processing (Figure 1). Immunohistochemical images showed expression of the ACE2, TMPRSS2, and Furin proteins within the middle ear mucosa and Eustachian tube (Figure 1). The presence of these proteins within the middle ear provides a possible route by which SARS-CoV-2 may infect the respiratory epithe...

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

This article describes a technique for rapid human temporal bone sectioning that utilizes a microsaw with twin diamond blades to generate thin slices for rapid decalcification and analysis of temporal bone immunohistochemistry.

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

This protocol reduces the time for temporal bone processing from 9 to 10 months to 10 to 14 days. The main advantage of this technique is it allows for the rapid assessment of otopathology, which in our case is relevant for the assessment of COVID-19-associated middle ear pathology. This technique may be particularly helpful for time-sensitive studies of the temporal bone.In the future, it may be further optimized for use studying inner ear pathology. Begin the harvest during autopsy and divide the cranial nerves sharply to the internal auditory canal. Make the first bony incision with the osteotome parallel to and just medial to the squamous portion of the temporal bone.Then, perform the second bony incision parallel to the first at the medial border of the temporal bone. Make the third incision at three to four centimeters anterior and parallel to the petrous ridge. Next, make the fourth incision roughly parallel to the third at two centimeters posterior to the petrous ridge.Use sharp dissection to free the soft tissue attached along the inferior edge of the specimen and remove the temporal bone. After the harvest, immediately submerge the tissue in 200 to 300 milliliters of 10%buffered formalin. Then, set a precision microsaw with twin diamond blades to a distance of five millimeters.For rapid tissue decalcification, cut each specimen into three sections such that the central section of the temporal bone is five millimeters thick and those on either end are three to five millimeters thick. Place the sections in 200 to 300 milliliters of 0.37-molar EDTA for seven to 10 days at room temperature for softening. Hematoxylin and eosin staining of the middle ear mucosa and eustachian tube showed that the tissue is preserved after processing using this protocol.Immunohistochemical analyses showed the presence of ACE2, transmembrane protease serine, and furin proteins within these tissues, providing a possible root for SARS-CoV2 infection in the respiratory epithelium of the middle ear. It is important to appropriately position the temporal bone tissue within the diamond saw. Alternative techniques that can be combined with this method include immunohistochemistry for different proteins, different tissue stains, and transcriptomics.This technique allowed us to rapidly assess for the presence of the proteins involved in SARS-CoV2 infection within the middle ear during the COVID-19 pandemic.

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