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 staining. While TBH will remain an essential tool for revealing new information about the healthy and diseased human inner ear, lengthy post-mortem times and long and harsh tissue processing methods limit its utility for certain purposes, necessitating adjunct methods to study human inner ear tissue. High-resolution magnetic resonance imaging can visualize inner ear organs but lacks sufficient resolution to view structures at the cellular or molecular level^4,5. Due to these challenges, many human inner ear diseases remain poorly understood.

An alternative approach is to harvest inner ear tissue during surgery. During labyrinthectomy or translabyrinthine vestibular schwannoma resection, the inner ear tissues are intentionally sacrificed. Utricles harvested from patients during translabyrinthine vestibular schwannoma resection have been used to characterize vestibular hair cell morphology^6,7,8 and study hair cell regeneration^9,10. More recently, techniques have been developed to harvest inner ear organs from organ donors using a transcanal approach that can be used to remove the utricle and potentially other vestibular end organs through a widened oval window with minimal tissue trauma^11,12. Using this technique, it has been possible to characterize single-cell transcriptomic profiles for the human utricle¹³. However, these techniques expose the inner ear organs to non-physiologic conditions during harvest. Specifically, inner ear organs may be exposed to the absence of perilymphatic fluid and submersion in normal saline drill irrigation, which has a substantially different ion composition than perilymphatic fluid. Further, the dehydrated membranous labyrinth is difficult to visualize, even with maximal magnification of the operating microscope, which makes atraumatic surgical dissection challenging. Mechanical trauma may further damage tissue, and our anecdotal experience suggests that surgical tissue is often of insufficient quality of immunostaining due to mechanical damage and cellular degeneration. There is a need for new techniques to atraumatically harvest human inner ear tissue for biological studies that may elucidate poorly understood human inner ear diseases. Here we describe an underwater technique for harvesting human vestibular end-organs under more physiologic conditions during labyrinthectomy and their analysis using immunostaining.

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 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. Perform tissue harvest during surgical labyrinthectomy or translabyrinthine approach to the internal auditory canal, which is part of standard clinical care.
2. Prepare the patient.
   1. Before the induction of anesthesia, discuss with the anesthesia team the avoidance of long-acting paralysis so that the facial nerve may be monitored.
   2. Once general anesthesia is induced, intubate the patient and rotate the operating table 180°. Place facial nerve monitoring electrodes and ensure that pre-operative antibiotic prophylaxis is administered.
   3. Inject 1% lidocaine with 1:100,000 epinephrine into the external auditory canal and postauricular area. Prepare the ear in the standard fashion using betadine.
3. Use a #15 blade to create a standard (5-6 cm) postauricular curvilinear incision 1 cm posterior to the postauricular fold.
4. Lift the postauricular soft tissue.
   1. Identify the temporalis fascia superiorly and elevate the postauricular skin flap anteriorly towards the ear canal.
   2. Create a standard 7-shaped periosteal incision and elevate the periosteum anteriorly until the spine of Henle and the bony ear canal are identified. Place self-retraining retractors.
5. Perform mastoidectomy.
   1. Perform a complete mastoidectomy, identifying the tegmen superiorly, sigmoid sinus posteriorly, and thinning the external auditory canal anteriorly.
   2. Identify the horizontal semicircular canal and the short process of the incus.
      NOTE: The mastoid should not be saucerized to ensure it remains a vessel capable of holding liquid for underwater tissue collection.
6. Identify the semicircular canals (SC).
   1. Under the operating microscope (2-4x magnification), use a 3 mm coarse diamond burr to remove the perilabyrinthine air cells lateral to the superior SC, the subarcuate air cells, and retro-labyrinthine air cells behind the posterior SC, extending superiorly to the common crus.
7. Submerge the labyrinth.
   1. Submerge the mastoid cavity in a balanced salt solution (BSS) and visualize the labyrinth using a zero-degree endoscope with a lens-cleaning sheath irrigation system.
   2. Use the lens-cleaning sheath irrigation system to irrigate the mastoid cavity with BSS, washing away blood and allowing improved visualization of the labyrinth.
8. Blue-line' SCs.
   1. While maintaining an adequate level of BSS in the mastoid cavity and using the endoscope for visualization, use a 3 mm diamond burr to 'blue line' all three semicircular canals by carefully drilling away the otic capsule bone until the semicircular canal appears as a bluish line when viewed with the endoscope.
   2. Irrigate intermittently with BSS solution using the irrigation system to wash away blood and achieve adequate visualization, as illustrated in Figure 1.
9. Harvest SC ampullae.
   1. Under BSS, enter the dome of the lateral semicircular canal and follow this anteriorly until its ampullae are identified. Enter the superior SC and follow this medially towards its ampullae, which may be harvested with the ampullae of the superior SC. Cut the lateral SC duct sharply to facilitate removal.
   2. Elevate the horizontal and superior SC ampullae off the crista using a Rosen needle and separate the afferent fibers from the epithelia and membranous labyrinth. Then, remove these structures.
   3. Transect the dome of the posterior SC and follow this inferiorly and anteriorly to its ampullae. Similarly, harvest the posterior SC ampullae and place all tissue in BSS on ice.
10. Harvest macula.
    1. Remove the bone between the horizontal and posterior SC ampullae to expose the vestibule. The membranous walls of the utricle will have been torn with the removal of the ampullae. So long as a fluid level is maintained, the maculae will remain attached anteriorly; elevate and remove it.
    2. The saccule sits within the spherical recess; sharply elevate and remove it from the recess. Similarly, place the tissue samples in BSS on ice.
11. Transport tissue samples on ice from the operating room to the laboratory for fixation within 1 h of harvesting.

2. Immunohistochemistry and imaging

1. Fix the sample.
   1. Place the tissue sample in 4% paraformaldehyde (PFA) in 1x phosphate-buffered saline (1x PBS) at room temperature (RT) with gentle rocking or orbital shaking (100 rpm) for 1 h.
      ​NOTE: 4% PFA solution (for 5 mL total): 625 mL of 32% PFA stock solution + 500 mL of 10xPBS + 3875 mL of ddH₂O.
2. Transfer tissue samples to 1x PBS solution and rinse 3x 15 min (or longer if necessary) at RT with gentle rocking or orbital shaking.
3. Remove excess connective tissue under the dissecting microscope in 1x PBS at RT.
4. Decalcify the sample.
   1. Transfer tissue samples to 5% ethylenediaminetetraacetic acid (EDTA) in 1x PBS solution to clear the sensory epithelia from fine bone debris and otoconia. Run incubation for 15-30 min at RT, with gentle rocking or orbital shaking.
5. Again, transfer tissue samples to 1x PBS solution and rinse 3x 15 min (or longer if necessary) at RT with gentle rocking or orbital shaking.
6. Apply 125-200 mL of 2x blocking buffer (2x BB) to each sample and incubate for 1.5-5 h at RT or overnight (ON) at 4 °C. Run the incubation under constant orbital shaking.
   ​NOTE: 2x Blocking buffer (2x BB): 1 g of bovine serum albumin (BSA) dissolved in up to 5 mL of 1x PBS supplemented with 0.5% Triton X-100. The BSA serves as the blocking agent for non-specific antigen binding sites.
7. Stain using primary antibodies.
   1. Apply 120-150 mL of a combination of primary antibodies dissolved in 1x BB to the desired dilution. Apply rabbit anti-tenascin-C antibodies at 1:200 dilution (2.5 μg/mL), goat anti-oncomodulin antibodies at 1:200 dilution (2.5 μg/mL), mouse anti-calretinin at 1:300 dilution (2.2 μg/mL), and DAPI at 1:1000 dilution (1 μg/mL). Then, transfer the sample (covered) to 4 °C (fridge or cold room) under gentle orbital shaking for ON incubation.
      ​NOTE: 1x BB: Mix equal volume of 2x BB and 1x PBS supplemented with 0.5% Triton X-100.
8. Rinse tissue samples in 1x PBS solution, 3x 15 min (or longer if necessary), at RT with gentle rocking.
9. Stain using secondary antibody.
   1. Apply 120-150 mL of a combination of fluorophore-conjugated secondary antibodies dissolved in 1x BB at 1:2000 dilution (1 μg/mL). Apply 488 nm conjugated anti-rabbit secondary antibody, 568 nm anti-mouse secondary antibody, and 647 nm anti-goat secondary antibody.
   2. Then, incubate the sample (covered) at RT under gentle orbital shaking for 1.5-2 h (or ON at 4 °C).
10. Rinse tissue samples in 1x PBS solution, 3x 15 min (or longer if necessary), at RT with gentle rocking.
11. Perform microdissection and mounting.
    1. Under the dissecting microscope, remove excess tissue and membranes from around the sensory vestibular epithelia. Use 70% ethanol to clean the surface of a glass histology slide, air dry, and apply a 55 mL drop of the appropriate mounting medium.
    2. Transfer the sensory tissue samples to the mounting medium. Use fine forceps to gently orient the samples with the hair bundle side up in the mounting medium.
    3. Gently lay a cover glass of the appropriate size onto the mounting medium containing the samples, trying to limit air bubbles. Ensure that the cover glass is of #1.5 thickness grade required for confocal microscopy.
    4. Place the slides in a dark area(e.g., a bench drawer) to let the mounting medium set. Seal the cover glass to the slide using clear nail polish and let it dry in a dark area before examining it with the confocal microscope. Afterward, store the sealed slides in the dark (slide box of slide folder) at 4° until imaging.
12. Use a confocal microscope at 40x-100x magnification with 488 nm and 657 nm channels to image the specimen.

Results

Using this technique the human utricle and lateral and superior canal ampullae were harvested intact with minimal trauma (Figure 2). As can be seen in Figure 2, the ampullae can be harvested with a substantial portion of the membranous duct. Immunofluorescent labeling with anti-tenascin-C (extracellular matrix protein) and anti-oncomodulin (small calcium-binding protein of the parvalbumin protein family) showed intact type 1 vestibular hair cells (

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