Harvest of Vestibular End-Organs under Physiologic Conditions during Labyrinthectomy

Summary

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

Abstract

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 Ménière'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 Ménière'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 °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), β-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.

Introduction

The living human inner ear is challenging to study due to its location within the dense otic capsule bone of the temporal bone. Consequently, access to human inner tissue has been limited, and researchers have mainly relied upon post-mortem tissue harvest. Post-mortem temporal bone histopathology (TBH) has been a critical tool for understanding human otologic disease for over 100 years^1,2,3. Tissue for TBH is prepared by the post-mortem harvest of the temporal bone, a lengthy (9-10 month) decalcification and tissue preparation process, followed by hematoxylin and eosin staini....

Protocol

This protocol was developed with the approval of the institutional review board (IRB) of Johns Hopkins University School of Medicine (IRB00203441) and per institutional policies for using human tissue and potentially infectious material. Tissue collection was performed during labyrinthectomy, which is part of standard clinical care for recalcitrant Ménière's disease with drop attacks.

1. Labyrinthectomy and tissue harvest

1. Obtain local institutional re.......

Discussion

This paper describes a new technique for the underwater harvesting of vestibular end organs in BSS using endoscopes and their analysis using immunofluorescent imaging. Here, we demonstrate the harvest of intact vestibular end-organs with intact vestibular hair cells and sufficient tissue quality for successful immunolabeling. The hair cell density in our specimen was similar to those obtained in other studies from live organ donors¹³. To our knowledge, this is the first report of an underwater tec.......

Materials

Name	Company	Catalog Number	Comments
10x Phosphate Buffered Saline Stock	Sigma-Aldrich	P5493
32% Paraformaldehyde Stock Solution	ThermoFisher Scientific	50-980-495
Alexa Fluor 488 Anti-Rabbit Secondary Antibody	Jackson Immunoresearch	111545144
Alexa Fluor 568 Anti-Mouse Secondary Antibody	Jackson Immunoresearch	115575146
Alexa Fluor 647 Anti-Goat Secondary Antibody	Jackson Immunoresearch	705607003
Balanced Salt Solution	ThermoFisher Scientific	14040117
Bovine Serum Albumin	Sigma-Aldrich	10711454001
Confocal microscope	Nikon A1	A1
Cover glass (18 mm x 18 mm, thickness #1.5 )	Corning	2850-18
Endo-Scrub 2 Lens Cleaning Sheath	Medtronic	IPCES2SYSKIT
Ethylenediaminetetraacetic (EDTA) Acid Solution	Sigma-Aldrich	E8008
Goat Anti-oncomodulin Antibody	R&D Systems	AF6345
Hopkins 0 Degree Telescope	Karl Storz
Mouse Anti-calretinin Antibody	BD Biosciences	610908
ProLong Gold antifade reagent	Invitrogen	P10144
Rabbit Anti-tenascin C Antibody	Millipore	AB19013
Triton X-100	Sigma-Aldrich	9036-19-5