Name: high-speed human temporal bone sectioning for the assessment of covid-19-associated middle ear pathology
Uploaded: 2022-05-18T14:00:00.000+00:00
Duration: 3 min 42 s
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 review board (IRB) board approval, review institutional policies for the use of human and infectious material, and obtain consent for tissue donation before utilizing this protocol.</li>
	<li>Put on proper personal protective equipment (PPE) before working with tissue from COVID-19-positive temporal bone donors. Ensure that the PPE consists of an N-95 respirator and face shield (or alternatively a positive pressure air purification system), gown, and two pairs of gloves. Review techniques for properly donning and doffing PPE before beginning this protocol10.</li>
	<li>Harvest temporal bone tissue within 12-24 h of donor death. If the body is not refrigerated, ensure that the harvest occurs within 8 h of death.
	<ol>
		<li>Harvest temporal bones during autopsy using the four-incision, block method by osteotome7.
		<ol>
			<li>Following craniotomy, remove the brain and divide the cranial nerves to the internal auditory canal sharply.</li>
			<li>Make the first bony incision with the osteotome parallel to and just medial to the squamous portion of the temporal bone.</li>
			<li>Perform the second bony incision parallel to the first at the medial border of the temporal bone.</li>
			<li>Then, make the third incision 3-4 cm anterior and parallel to the petrous ridge.</li>
			<li>Make the fourth incision roughly parallel to the third, 2 cm posterior to the petrous ridge. 
			NOTE: Ensure that all incisions extend through the skull base.</li>
			<li>Then, remove the temporal bone using sharp dissection to free the soft tissue attachment along the inferior edge of the specimen. If embalming is to be performed following temporal bone harvest, tie off the stump of the carotid artery.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Tissue fixation, decalcification, and sectioning
<ol>
	<li>Immediately place the tissue in 200-300 mL of 10% buffered formalin (formaldehyde) so that the tissue is fully submerged. Leave the tissue in formaldehyde for a minimum of 72 h at room temperature, changing the solutions daily. Store the tissue in an air-tight container and perform solution changes under a fume hood while wearing appropriate PPE to prevent virus spread. 
	NOTE: Following the 72 h fixation period, the specimens no longer need to be considered infectious.</li>
	<li>Use a precision microsaw with twin diamond blades to cut each specimen into three &#34;thick&#34; sections to allow for more rapid tissue decalcification. Set the twin diamond blades to a distance of 5 mm, so that the central section of temporal bone is 5 mm thick. Ensure that the sections of tissue on either end are 3-5 mm thick.</li>
	<li>Place the thick sections in 200-300 mL of 23% w/w formic acid decalcifying solution for 7-10 days at room temperature, until the tissue is soft enough for paraffin embedding. Change the solutions daily. Check for adequate tissue decalcification by palpating the specimen, which should feel soft. 
	NOTE: Decalcification may also be verified by x-ray.</li>
	<li>Rinse the specimens in running tap water for 24 h by placing them in a large beaker underneath a running faucet.</li>
	<li>Dehydrate the tissue in a series of alcohols of increasing concentration7. Submerge the tissue in 100-200 mL of 70% ethanol for 1.5 h, followed by three washes in 95% and 100% ethanol for 1.5 h each, respectively. Next, wash the tissue 3 x 1.5 h in Xylene.</li>
	<li>Embed the thick sections in paraffin, with four treatments of 45 min each.</li>
	<li>Use a cryotome to cut thin (10 &#181;m) sections. Position the tissue so that the tissue is cut in the axial plane. Cut thicker (20 &#181;m) sections if desired.</li>
</ol>
3. Immunohistochemistry and imaging
<ol>
	<li>If traditional hematoxylin and eosin staining is desired (not described here), perform staining before mounting the sections on glass slides7.</li>
	<li>Mount the sections on positively charged glass slides.</li>
	<li>Bake the slides on a hot plate at 60 &#176;C for 20 min.</li>
	<li>Place the slides in a holder and submerge them in a commercial pretreatment solution containing an organic solvent (diethanolamine in this case) and ethanol to deparaffinize, rehydrate, and unmask the tissue. 
	NOTE: This step is necessary to achieve satisfactory results with immunohistochemistry for formalin-fixed and paraffin-embedded tissue.</li>
	<li>Heat the slides in a pressure cooker on high pressure for 20 min.</li>
	<li>Place the slides in a humidified chamber and block the tissue with a commercial blocking solution.</li>
	<li>Probe the tissue sections with the primary antibody against angiotensin-converting enzyme 2 (ACE2; 1:50), transmembrane protease serine 2 (TMPRSS2, 1:1,000), and the Furin protease (1:250). 
	NOTE: The ACE2, TMPRSS2, and Furin antibodies are used because these proteins are believed to play a role in SARS-CoV-2 infectivity11,12, and this protocol sought to investigate the possible mechanisms by which SARS-CoV-2 may infect the middle ear13.</li>
	<li>Treat with HRP-conjugated anti-rabbit secondary antibody (ACE2, Furin; 1:100 dilution) or anti-goat (TMPRSS2, 1:100 dilution) secondary antibody.</li>
	<li>Counterstain the slides with 70% hematoxylin in water.</li>
	<li>Acquire images on a light microscope with a mounted digital camera. Obtain images of the middle ear mucosa and Eustachian tube at 20x and 10x magnification, respectively.</li>
</ol>

<ol>
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C., Pauna, H. F., Paparella, M. M., Cureoglu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Otopathology+in+the+United+States:+History,+current+situation,+and+future+perspectives.">Otopathology in the United States: History, current situation, and future perspectives.</a> Otology &amp; Neurotology. 39 (9), 1210-1214 (2018).</li><li>Crowe, S. J., Guild, S. R., Polvogt, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observations+on+the+pathology+of+high-tone+deafness.">Observations on the pathology of high-tone deafness.</a> Journal of Nervous and Mental Disease. 80, 480 (1934).</li><li>Andresen, N. 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>

The authors have no conflicts of interest to declare.

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 related to COVID-19 middle and inner ear pathology13, which was the motivation for developing this protocol. This protocol allowed for the visualization of ACE2, TMPRS22, and Furin expression within the middle ear and Eustachian tube, providing a likely mechanism by which SARS-CoV-2 may infect the middle ear by spreading from the nasopharynx through the Eustachian tube.
The primary innovation of this protocol is the use of a diamond saw that cuts the tissue before decalcification, which generates &#34;thick&#34; sections of temporal bone that may be rapidly decalcified by increasing the surface area of the tissue exposed to the decalcifying agent. This step allows for tissue decalcification to occur within 7-10 days instead of the 1-2 months that traditional protocols may require for decalcification. The &#34;thick&#34; sections of tissue created with the diamond saw may also be dehydrated more quickly than a standard block of temporal bone tissue, thereby reducing the time between decalcification and embedding. Further, this protocol utilizes paraffin as an embedding agent rather than celloidin, which takes approximately 3 months to harden7. While celloidin provides superior preservation of cochlear structures, paraffin hardens much quicker and facilitates immunostaining. With these modifications, temporal bone tissue can be processed in 10-14 days as opposed to the 9-10 months required in more traditional processing strategies7,9.
This protocol has several limitations. Cutting the tissue with the diamond saw before embedding risks damaging the organ of Corti (OOC). The OOC was severely damaged in most specimens during the development of this technique, which may limit the value of this technique for studying pathologies such as age-related hearing loss, where it is vital that the delicate structures of the inner ear are preserved. It may be possible to position the specimen so that the diamond saw does not directly cut through the otic capsule bone and preserves inner ear structures, but this remains an area of active investigation. Animal models may provide a valuable tool to further refine this protocol and increase the chance of preserving inner ear structures in these valuable human specimens. Tissue quality in this protocol is additionally limited by the use of the paraffin-embedding agent, which was selected due to time constraints. Celloidin embedding better maintains cellular integrity in otopathologic specimens but takes months to harden7. Last, this protocol still requires significant time and resource investment. The use of a diamond microsaw is an additional cost to the materials required for traditional temporal bone preparation, and the 7-10 days required for decalcification is still lengthy. In the future, it may be possible to combine these methods with microwave-assisted decalcification14 to further shorten the time required for processing.
Despite its limitations, the protocol for high-speed temporal bone processing described here offers another tool for studying middle ear, and potentially inner ear, pathology. This technique has been useful during the COVID-19 pandemic and may also reduce the costs associated with maintaining an active temporal bone laboratory by reducing the time and labor necessary for tissue processing. Temporal bone research has decreased in popularity within the United States, decreasing from 28 active laboratories in the 1980s to only 4 active laboratories at present4. This decline in the number of operating temporal bone laboratories may be related to the significant costs associated with harvesting and processing temporal bones4,8. Consequently, it is important that we aim to develop techniques that reduce the resources necessary for temporal bone processing and also those that allow for the study of temporal bone pathology in new contexts, such as the COVID-19 pandemic. Further, high-speed tissue processing, such as that outlined in this protocol, may aid the use of new biologic techniques (e.g., immunohistochemistry, transcriptomics) that allow for the assessment of middle and inner ear pathology at the molecular level15,16,17,18,19. We have barely scratched the surface in terms of utilizing molecular techniques to study human tissue in otologic disease, and they may provide important insights that cannot be provided by animal models.

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><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&#039;&#039;</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 epithelium within the middle ear11,12,13. Further, the expression of these proteins within the Eustachian tube may explain a route by which the virus enters the middle ear, traveling to the middle ear from the nasopharynx via the Eustachian tube.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64012/64012fig01.jpg" /> 
Figure 1: Example of stained middle ear tissue. The figure shows hematoxylin and eosin, ACE2, TMPRSS2, and Furin staining of the middle ear mucosa (top row, 20x magnification) and Eustachian tube (bottom row, 10x magnification). Scale bars = 50 &#181;m (top row); 100 &#181;m (bottom row). Abbreviations: H&#38;E = hematoxylin and eosin; angiotensin-converting enzyme 2; TMPRSS2 = transmembrane protease, serine 2. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/64012/64012fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

high-speed-human-temporal-bone-sectioning-for-assessment-covid-19

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

2.2K Views.  Johns Hopkins University School of Medicine. 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. 

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

A Protocol for Decellularizing Mouse Cochleae for Inner Ear Tissue Engineering

In mammals, mechanosensory hair cells that facilitate hearing lack the ability to regenerate, which has limited treatments for hearing loss. Current regenerative medicine strategies have focused on transplanting stem cells or genetic manipulation of surrounding support cells in the inner ear to encourage replacement of damaged stem cells to correct hearing loss. Yet, the extracellular matrix (ECM) may play a vital role in inducing and maintaining function of hair cells, and has not been well investigated. Using the cochlear ECM as a scaffold to grow adult stem cells may provide unique insights into how the composition and architecture of the extracellular environment aids cells in sustaining hearing function. Here we present a method for isolating and decellularizing cochleae from mice to use as scaffolds accepting perfused adult stem cells. In the current protocol, cochleae are isolated from euthanized mice, decellularized, and decalcified. Afterward, human Wharton&#39;s jelly cells (hWJCs) that were isolated from the umbilical cord were carefully perfused into each cochlea. The cochleae were used as bioreactors, and cells were cultured for 30 days before undergoing processing for analysis. Decellularized cochleae retained identifiable extracellular structures, but did not reveal the presence of cells or noticeable fragments of DNA. Cells perfused into the cochlea invaded most of the interior and exterior of the cochlea and grew without incident over a duration of 30 days. Thus, the current method can be used to study how cochlear ECM affects cell development and behavior.

The goal of this protocol is to demonstrate an effective method to decellularize and decalcify mouse cochleae for utilization as scaffolds for tissue engineering applications.

In mammals, mechanosensory hair cells that facilitate hearing lack the ability to regenerate, which has limited treatments for hearing loss. Current ...

Bioengineering

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a spiral shape and have a tonotopic gradient for sound detection. The mouse cochlea is approximately 6 mm long and often divided into three turns (apex, middle, and base) for analysis. To investigate cell loss, cell division, or mosaic gene expression, the whole mount or surface preparation of the cochlea is useful. This dissection method allows visualization of all cells within the organ of Corti when combined with immunostaining and confocal microscopy to image cells at different planes in the z-axis. Multiple optical cross-sections can also be obtained from these z-stack images. In addition, the whole mount dissection method can be used for scanning electron microscopy, although a different fixation method is needed. Here, we present a method to isolate the organ of Corti as three intact cochlear turns (apex, middle, and base). This method can be used for mice ranging from one week of age through adulthood and differs from the technique used for neonatal samples where calcification of the cochlea is incomplete. A slightly modified version can be used for dissection of the rat cochlea. We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies.

We present a method to isolate the adult organ of Corti as three intact cochlear turns (apex, middle, and base). We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies. Together these techniques allow the study of hair cells, supporting cells, and other cell types found in the cochlea.

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a ...

Neuroscience

The Mouse Round-window Approach for Ototoxic Agent Delivery: A Rapid and Reliable Technique for Inducing Cochlear Cell Degeneration

Investigators have utilized a wide array of animal models and investigative techniques to study the mammalian auditory system. Much of the basic research involving the cochlea and its associated neural pathways entails exposure of model cochleae to a variety of ototoxic agents. This allows investigators to study the effects of targeted damage to cochlear structures, and in some cases, the self-repair or regeneration of those structures. Various techniques exist for delivery of ototoxic agents to the cochlea. When selecting a particular technique, investigators must consider a number of factors, including the induction of inadvertent systemic toxicity, the amount of cochlear damage produced by the surgical procedure itself, the type of lesion desired, animal survivability, and reproducibility/reliability of results. Currently established techniques include parenteral injection, intra-peritoneal injection, trans-tympanic injection, endolymphatic sac injection, and cochleostomy with perilymphatic perfusion. Each of these methods has been successfully utilized and is well described in the literature; yet, each has various shortcomings. Here, we present a technique for topical application of ototoxic agents directly to the round window niche. This technique is non-invasive to inner ear structures, produces rapid onset of reliably targeted lesions, avoids systemic toxicity, and allows for an intra-animal control (the contra-lateral ear). Results stemming from this approach have helped deeper understanding of auditory pathophysiology, cochlear cell degeneration, and regenerative capacity in response to an acute injury. Future investigations may use this method to conduct interventional studies involving gene therapy and stem cell transplantation to combat hearing loss.

Various methods exist for introducing ototoxic agents to the cochleae of animal models. Presented is a surgical protocol for delivery of ototoxic agents to the round window niche. The procedure is reliable, creates targeted intra-cochlear lesions, and avoids mechanical damage to the microarchitecture. Examination of cochlear self-repair/regeneration is possible.

Investigators have utilized a wide array of animal models and investigative techniques to study the mammalian auditory system. Much of the basic research ...

Medicine

Morphological and Functional Evaluation of Ribbon Synapses at Specific Frequency Regions of the Mouse Cochlea

Cochlear inner hair cells (IHCs) transmit acoustic signals to spiral ganglion neurons (SGNs) through ribbon synapses. Several experimental studies have indicated that hair cell synapses may be the initial targets in sensorineural hearing loss (SNHL). Such studies have proposed the concept of cochlear &#34;synaptopathy&#34;, which refers to alterations in ribbon synapse number, structure, or function that result in abnormal synaptic transmission between IHCs and SGNs. While cochlear synaptopathy is irreversible, it does not affect the hearing threshold. In noise-induced experimental models, restricted damage to IHC synapses in select frequency regions is employed to identify the environmental factors that specifically cause synaptopathy, as well as the physiological consequences of disturbing this inner ear circuit. Here, we present a protocol for analyzing cochlear synaptic morphology and function at a specific frequency region in adult mice. In this protocol, cochlear localization of specific frequency regions is performed using place-frequency maps in conjunction with cochleogram data, following which the morphological characteristics of ribbon synapses are evaluated via synaptic immunostaining. The functional status of ribbon synapses is then determined based on the amplitudes of auditory brainstem response (ABR) wave I. The present report demonstrates that this approach can be used to deepen our understanding of the pathogenesis and mechanisms of synaptic dysfunction in the cochlea, which may aid in the development of novel therapeutic interventions.

This manuscript describes an experimental protocol for evaluating the morphological characteristics and functional status of ribbon synapses in normal mice. The present model is also suitable for noise-induced and age-related cochlear synaptopathy-restricted models. The correlative results of previous mouse studies are also discussed.

Cochlear inner hair cells (IHCs) transmit acoustic signals to spiral ganglion neurons (SGNs) through ribbon synapses. Several experimental studies have ...

Nasal Brushing Sampling and Processing Using Digital High Speed Ciliary Videomicroscopy &ndash; Adaptation for the COVID-19 Pandemic

Primary Ciliary Dyskinesia (PCD) is a genetic motile ciliopathy, leading to significant otosinopulmonary disease. PCD diagnosis is often missed or delayed due to challenges with different diagnostic modalities. Ciliary videomicroscopy, using Digital High-Speed Videomicroscopy (DHSV), one of the diagnostic tools for PCD, is considered the optimal method to perform ciliary functional analysis (CFA), comprising of ciliary beat frequency (CBF) and beat pattern (CBP) analysis. However, DHSV lacks standardized, published operating procedure for processing and analyzing samples. It also uses living respiratory epithelium, a significant infection control issue during the COVID-19 pandemic. To continue providing a diagnostic service during this health crisis, the ciliary videomicroscopy protocol has been adapted to include adequate infection control measures.
Here, we describe a revised protocol for sampling and laboratory processing of ciliated respiratory samples, highlighting adaptations made to comply with COVID-19 infection control measures. Representative results of CFA from nasal brushing samples obtained from 16 healthy subjects, processed and analyzed according to this protocol, are described. We also illustrate the importance of obtaining and processing optimal quality epithelial ciliated strips, as samples not meeting quality selection criteria do now allow for CFA, potentially decreasing the diagnostic reliability and the efficiency of this technique.

Nasal Brushing Sampling and Processing Using Digital High Speed Ciliary Videomicroscopy &#8211; Adaptation for the COVID-19 Pandemic

To guarantee a successful and high-quality ciliary functional analysis for PCD diagnosis, a precise and careful method for respiratory epithelium sampling and processing is essential. To continue providing PCD diagnostic service during the COVID-19 pandemic, the ciliary videomicroscopy protocol has been updated to include appropriate infection control measures.

Primary Ciliary Dyskinesia (PCD) is a genetic motile ciliopathy, leading to significant otosinopulmonary disease. PCD diagnosis is often missed or delayed ...

Endaural Endoscopic Atticoantrotomy (Retrograde Mastoidectomy) using a Constant Suction Bone-drilling Technique

Endoscopic middle ear surgery is a widely employed minimally invasive surgical technique to address middle ear and mastoid pathology. Bone drilling is the main technical challenge of endoscopic middle ear surgery. The accompanying video describes the detailed protocol of a constant-suction bone-drilling technique and the procedure of endaural exclusive endoscopic atticoantrotomy (retrograde mastoidectomy) using this technique. The main components of this bone-drilling technique include a soft and flexible suction tube, which is placed into the tympanic cavity to provide constant suction, and a soft sleeve, which is wrapped around the drill shaft to prevent the high-speed rotating shaft from damaging the lens of the endoscope. With these simple modifications, the traditional otological electrodrill can be used for drilling a tiny endaural incision in endoscopic middle ear surgery. Based on this bone-drilling technique, endaural endoscopic atticoantrotomy (retrograde mastoidectomy) can be successfully established for the removal of various amounts of bone, depending on the extent of the lesion. The short-term postoperative outcome seems promising.

Endaural Endoscopic Atticoantrotomy Retrograde Mastoidectomy using a Constant Suction Bone-drilling Technique

In this paper, we present the protocol and short-term outcomes of endaural exclusive endoscopic atticoantrotomy using a constant suction bone-drilling technique.

Endoscopic middle ear surgery is a widely employed minimally invasive surgical technique to address middle ear and mastoid pathology. Bone drilling is the ...

An Integrated (Microscopic/Endoscopic) Dissection Ear Surgery Course

Traditionally, otologic surgical training consisted of microscopic cadaveric dissections. However, during the last decades the endoscope has significantly changed the surgical perspective in the otologic field. Thus, the modern ear and lateral skull base surgeon should master the entire spectrum of endoscopic and microscopic approaches, with the aim of tailoring the procedure and guaranteeing the best possible functional outcome. This work proposes a step-by-step guided and illustrated dissection course, including indications for the setup of the cadaver lab and the integration of the microscope and endoscope to enhance the use of both instruments. The alternation of the endoscope and microscope allows the novice to train the correct handling of the instruments in the surgical field under both optical views. This aspect is of utmost importance since it is not advisable to start off a technique without practicing the other one, as both are important and complementary in the modern otologic surgery setting.

An Integrated Microscopic/Endoscopic Dissection Ear Surgery Course

Traditionally, otologic surgical training consisted of microscopic cadaveric dissections. However, during the last decades the endoscope has significantly ...

Dissection of the Auditory Bulla in Postnatal Mice: Isolation of the Middle Ear Bones and Histological Analysis

In most mammals, auditory ossicles in the middle ear, including the malleus, incus and stapes, are the smallest bones. In mice, a bony structure called the auditory bulla houses the ossicles, whereas the auditory capsule encloses the inner ear, namely the cochlea and semicircular canals. Murine ossicles are essential for hearing and thus of great interest to researchers in the field of otolaryngology, but their metabolism, development, and evolution are highly relevant to other fields. Altered bone metabolism can affect hearing function in adult mice, and various gene-deficient mice show changes in morphogenesis of auditory ossicles in utero. Although murine auditory ossicles are tiny, their manipulation is feasible if one understands their anatomical orientation and 3D structure. Here, we describe how to dissect the auditory bulla and capsule of postnatal mice and then isolate individual ossicles by removing part of the bulla. We also discuss how to embed the bulla and capsule in different orientations to generate paraffin or frozen sections suitable for preparation of longitudinal, horizontal, or frontal sections of the malleus. Finally, we enumerate anatomical differences between mouse and human auditory ossicles. These methods would be useful in analyzing pathological, developmental and evolutionary aspects of auditory ossicles and the middle ear in mice.

We present a protocol to isolate the auditory bulla, capsule, and ossicles from postnatal mice for whole mount and histological analysis.

In most mammals, auditory ossicles in the middle ear, including the malleus, incus and stapes, are the smallest bones. In mice, a bony structure called ...

Biology

Extracting the Cochlea from a Human Temporal Bone: A Cadaveric Protocol

The extraction of the cochlea from a cadaver human temporal bone may be required for different studies of the inner ear. For histological evaluations, the inner ear must be extracted from the temporal bone to facilitate histologic processing; likewise, some micro-computed tomography devices are too small to accommodate the complete temporal bones; additionally, the image quality can be enhanced when the cochlea is isolated.
The inner ear is located within the petrous part of the temporal bone. The inner ear can be divided into the osseous labyrinth or otic capsule and the membranous labyrinth inside the otic capsule. Furthermore, the inner ear can be divided into the vestibular system (the semicircular canals and the vestibule) and the cochlea. The appreciation of the location and orientation of the cochlea within the temporal bone is difficult, as it is embedded within bony structures and thus cannot be directly visualized. Nevertheless, there are distinct anatomical structures that can help guide the process to allow a reliable drill-out of the cochlea. The landmarks in the posterior parts of the cochlea are the facial nerve, semicircular canals, and the vestibule. In the middle, the inferior borders of the cochlea are identified by the round window and the basal turn of the cochlea. In the anterior border, one encounters the carotid artery; the landmark for the superior border is the genicular ganglion (GG) of the facial nerve. The medial structures are determined by the locations of the internal auditory canal, the superior semicircular canal, and the canal of the internal carotid artery.
In this article, we present a method for extracting the cochlea reliably out of the temporal bone by drill-out while following several anatomical landmarks.

This article presents a reliable method for extracting the human cochlea from the cadaver temporal bone by drill-out while following distinct anatomical landmarks.

The extraction of the cochlea from a cadaver human temporal bone may be required for different studies of the inner ear. For histological evaluations, the ...

Harvest of Vestibular End-Organs under Physiologic Conditions during Labyrinthectomy

The living human inner ear is challenging to study because it is encased within dense otic capsule bone that limits access to biological tissue. Traditional temporal bone histopathology methods rely on lengthy, expensive decalcification protocols that take 9-10 months and reduce the types of tissue analysis possible due to RNA degradation. There is a critical need to develop methods to access fresh human inner ear tissue to better understand otologic diseases, such as M&#233;ni&#232;re's disease, at the cellular and molecular level. This paper describes a technique for the harvest of human vestibular end organs from a living donor under physiologic conditions. An individual with M&#233;ni&#232;re's disease and 'drops attacks' that were refractory to intratympanic gentamicin injection underwent labyrinthectomy. A traditional mastoidectomy was first performed, and the horizontal and superior semicircular canals (SCC) were identified. The mastoid cavity was filled with a balanced salt solution so that the labyrinth could be opened under more physiologic conditions to preserve cellular integrity. A zero-degree endoscope fit with a lens-cleaning sheath irrigation system was used to visualize the submerged mastoid cavity, and a 2 mm diamond burr was used to skeletonize and open the horizontal and superior SCCs, followed by the vestibule. The ampullae and portion of the canal ducts for the superior and lateral SCCs were harvested. The utricle was similarly harvested. Harvested tissue was immediately placed in an ice-cold buffer and then fixed for one hour in 4% paraformaldehyde in phosphate-buffered saline (PBS). The tissue was rinsed several times in 1x PBS and stored for 48 h at 4 &#176;C. The tissue samples underwent immunostaining with a combination of primary antibodies against tenascin-C (Calyx), oncomodulin (streolar hair cells), calretinin (Calyx and Type II hair cells), synaptic vesicle protein 2 (efferent fibers and boutons), &#946;-tubulin 1 (Calyx and afferent boutons), followed by incubation with fluorophore-conjugated secondary antibodies. The tissue samples were then rinsed and mounted for confocal microscopy examination. Images revealed the presence of ampullar and macular hair cells and neural structures. This protocol demonstrates that it is possible to harvest intact, high-quality human inner ear tissue from living donors and may provide an important tool for the study of otologic disease.

Here, we describe a technique for harvesting human vestibular end-organs under physiologic conditions during labyrinthectomy and their analysis using immunostaining.