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

Summary

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.

Abstract

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 µm) 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.

Introduction

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 disease^1,2,3. To better understand otologic disease and "rescue aural surgery from the hands of quacks," 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 ....

Protocol

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's family. See the Table of Materials for details about all materials, equipment, and software used in this protocol.

1. Temporal bone harvest

1. 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 befo....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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