Mortui vivos docent: a modern revival of temporal bone plug harvests

Abstract

Human temporal bones (HTBs) are invaluable resources for the study of otologic disorders and for evaluating novel treatment approaches. Given the high costs and technical expertise required to collect and process HTBs, there has been a decline in the number of otopathology laboratories. Our objective is to encourage ongoing study of HTBs by outlining the necessary steps to establish a pipeline for collection and processing of HTBs. In this methods manuscript, we: (1) provide the design of a temporal bone plug sawblade that can be used to collect specimens from autopsy donors; (2) establish that decalcification time can be dramatically reduced from 9 to 3 months if ethylenediaminetetraacetic acid is combined with microwave tissue processing and periodic bone trimming; (3) show that serial sections of relatively-rapidly decalcified HTBs can be successfully immunostained for key inner ear proteins; (4) demonstrate how to drill down a HTB to the otic capsule within a few hours so that subsequent decalcification time can be further reduced to only weeks. We include photographs and videos to facilitate rapid dissemination of the developed methods. Collected HTBs can be used for many purposes, including, but not limited to device testing, imaging studies, education, histopathology, and molecular studies. As new technology develops, it is imperative to continue studying HTBs to further our understanding of the cellular and molecular underpinnings of otologic disorders.

Keywords: histopathology; human temporal bone; inner ear; microwave decalcification; otic capsule; otology; temporal bone plug cutter.

Figure 1

Autopsy saw and bone plug sawblade. The autopsy saw is used to drive a custom-designed bone plug sawblade for extraction of temporal bone plugs.

Figure 2

Steps of temporal bone plug extraction. (A) The skull base is labeled with key anatomic landmarks. (B) The bone plug sawblade is centered over the arcuate eminence with the internal auditory canal included in the posteromedial edge of the blade margin. Blade should be advanced in a plane perpendicular to the middle cranial fossa. Once a change in resistance is encountered, the sawblade should be removed. (C) Bone plug will remain in place due to inferior soft tissue attachments. These attachments can be cut using surgical scissors or a scalpel. (D) Bone plug has been removed from the skull base. Careful attention should be paid to minimize cosmetic defects based on final preferences of the donor and/or donor’s family. A video depicting this process can be found through the linked (Supplementary Video 1).

Figure 3

Progression of microwave and EDTA decalcification in extracted temporal bone plug. Micro computed tomography (micro-CT) scans of extracted temporal bone plugs were performed at regular intervals during decalcification. Top row: external auditory canal (*) and tympanic membrane (arrow) are labeled for orientation. Full decalcification was achieved at 23 weeks (D). Bottom row: the cochlea is largely decalcified by the 7 weeks timepoint (B) and is completely decalcified by 14 weeks (C). Calcified bone surrounding the semicircular canal (SC) is persistent through 14 weeks (C) with full decalcification achieved at 23 weeks (D). EDTA, ethylenediaminetetraacetic acid; IAC, internal auditory canal; IM joint, incudomalleolar joint; SC, semicircular canal; BL, baseline. All scales = 5 mm.

Figure 4

Trimming of a temporal bone plug to the otic capsule. (A) Full bone plug labeled with relevant anatomy following fixation in 10% neutral buffered formalin. (B) Fully trimmed bone plug down to the otic capsule. Inlet image in lower right shows otic capsule size in comparison to a penny. CN, cranial nerve.

Figure 5

Hematoxylin and eosin stained mid-modiolar cochlear cross section. Donor bone was collected from a 75 years-old male at a 44 h post-mortem interval and was decalcified using ethylenediaminetetraacetic acid (EDTA), the microwave, and by trimming. The bone was embedded in paraffin and cut in 5 μm sections to achieve a mid-modiolar cross section. (A) Cochlear turns are seen spiraling around the modiolus (MOD) which contains spiral ganglion neuronal cell bodies and axons of the auditory nerve. Tectorial membrane (arrow) and basilar membrane (*) are clearly visualized, as are the scala vestibuli (SV) and scala tympani (ST). Scale bar = 1 mm. (B) Higher magnification image of organ of Corti. Hair cells and supporting cells present, but morphology limited likely due to long post-mortem interval. Tectorial membrane (arrow) and basilar membrane (*) visualized. Scale bar = 100 μm.

Figure 6

Immunohistochemistry of the organ of Corti. Donor bone was collected from an 87 years-old male at a 7 h post-mortem interval and was decalcified using ethylenediaminetetraacetic acid (EDTA), the microwave, and by trimming. The bone was embedded in paraffin and cut in 8 μm sections. Immunohistochemistry using anti-myosin VIIa antibodies of a mid-modiolar section revealed an organ of Corti with an intact inner hair cell, three outer hair cells, and the tunnel of Corti. IHC, inner hair cell; OHCs, outer hair cells; TC, tunnel of Corti.

Figure 7

Processing pipeline and downstream applications for human temporal bones. The different processing steps for human temporal bone (HTB) analysis are displayed along with potential applications at each step of the pathway. TPFM, two-photon fluorescence microscopy; SR-PCI, synchrotron radiation phase contrast imaging; μCT, micro-computed tomography; μOCT, micro-optical coherence tomography; H&E, hematoxylin and eosin; IHC, immunohistochemistry; ICP-MS, induction coupled plasma mass spectrometry.