Conventional temporal bone imaging approaches, including computed tomography (CT) and magnetic resonance imaging, reveal gross anatomic defects but provide limited resolution of intracochlear microanatomy due to the inner ear's encasement in the dense otic capsule. The gold standard for study of auditory pathology has long been post-mortem histology of temporal bones (1). This approach is time-intensive and requires several processing steps including fixation, decalcification, embedding, sectioning, and staining. These steps can introduce artifacts and limit the utility of sectioned temporal bones for other applications which require their three-dimensional structure. Since human temporal bones are precious specimens collected from autopsy, it is important to maximize their multi-utility (2). Therefore, a non-destructive imaging approach which provides structural information of the inner ear is invaluable in the study of human SNHL while allowing the imaged specimen to be used for additional downstream applications such as device development.

Recently, synchrotron-radiation phase-contrast imaging (SR-PCI) has been established as a high-resolution technique that can be used in non-decalcified, unsectioned, and unstained human temporal bones (3). While this method offers several advantages over traditional histology, access to SR-PCI infrastructure is limited and often tightly regulated (4). MicroCT, which affords low micrometer level spatial resolution, is a more widely available technology. MicroCT images have been used to develop high-resolution cochlear atlases from which lower resolution clinical CT images have been segmented into different cochlear regions (5, 6). Furthermore, microCT resolution can be improved with the use of contrast agents, of which iodine solutions, osmium tetroxide (OsO₄), and phosphotungstic acid (PTA) are most commonly used (7, 8). Iodine staining is advantageous as it is not permanent and can be removed via leaching or chemical destaining with sodium thiosulfate (STS) (8). Combining the ease of access of microCT with improved resolution from iodine staining could provide a reversible and non-destructive imaging method for the study of temporal bones. We compare microCT images obtained with three different contrast agents and demonstrate the feasibility of reversible iodine staining for the first time in human temporal bones.

Mid-modiolar virtual two-dimensional (2D) sections through non-dehydrated, non-decalcified specimens were compared across each of the contrast agents at different time points (Figure 1). Additionally, contrast ratios were calculated for the AN relative to bone and fluid-filled spaces (Table 2). I₂KI staining effectiveness peaked between 48 and 96 h after which soft tissue became saturated as evidenced by bright spots of iodine buildup. In I₂KI stained bone, the basilar membrane (BM), Reissner's membrane (RM), and spiral ligament (SL) were visualized in the basal, middle, and apical turns of the cochlea. Clear distinctions between the scala tympani (ST) and scala vestibuli (SV) were also appreciated. Furthermore, the AN and its branching structure is seen. PTA staining, conversely, showed poorer resolution of intracochlear structures and the AN than I₂KI staining at all time points. Faint contrast is visible at 96 h, at which point the BM can be partially visualized. Contrast enhancement did not improve appreciably at later time points. OsO₄ stained bone was imaged at a single 48 h timepoint following the protocol adapted from Glueckert et al., 2018. In OsO₄ stained bone, the BM is visualized in all cochlear turns, but the RM is only weakly observed in the basal turn. The stria vascularis (StV) and SL are well defined in the basal and middle turns, and the AN trunk is clearly visualized.

Chemical destaining with STS was effective in removing I₂KI from temporal bone specimens (Figure 2). The structural details of the cochlear interior and AN were no longer appreciated in the mid-modiolar virtual 2D sections after 48 h of treatment with STS.

Virtual whole mount of I₂KI stained bone displayed the full cytoarchitecture of the organ of the Corti including the modiolar core, hair cells, and AN bundles. None of these features were visualized following destaining with STS.

Virtual 3D mid-modiolar section of I₂KI stained bone provided distinct boundaries between the ST, SV, and scala media (SM) (Figure 3A). The SL and the AN are also well defined. 3D reconstruction of the I₂KI stained temporal bone shows the anatomical relationship between the superior and inferior vestibular nerve, facial nerve, and AN in the internal auditory meatus (Figure 3B).

Human temporal bones are invaluable for the study of diseases of the auditory and vestibular systems, for research involving new devices and for training of surgeons and investigators alike. Nevertheless, resources devoted to the study of temporal bones have decreased over the years. In fact, only three federally-sponsored active temporal bone research laboratories remain today in the United States due to the high costs associated with collecting and processing these specimens (10). Moreover, collection and study of temporal bones from patients with well documented otologic disease and/or prior surgical procedures is limited. Of the nearly 200 deafness causing genes, histopathologic analysis has only been reported for 22 (11). This lack of reported histopathology extends to those who have undergone surgical procedures such as labyrinthectomies or cochlear implantation (10).

The scarcity of temporal bones characterized at the otopathological level can partially be attributed to the challenges of traditional histological processing. This approach is technically rigorous, and processing steps take many months. Additionally, histological processing can introduce artifacts into tissue which can confound findings (12). Furthermore, the 3D relationships between structures cannot easily be appreciated with sectioned temporal bone slides. These factors underly the need for alternative methods of studying auditory pathology that are faster, more accessible and cost-effective while facilitating multi-utility of the same specimen.

In this study, we demonstrate the feasibility of a non-destructive approach to studying human temporal bone histopathology using microCT technology coupled with iodine contrast. MicroCT is a readily available imaging modality that has been used extensively in the study of human temporal bones (13–15). Given comparable staining outcomes to OsO₄, I₂KI affords the additional benefit of reversibility. We have demonstrated that chemical destaining with STS returns I₂KI stained bone to a near original state with regards to imaging properties. It is important to note, however, that destaining does not return a specimen to its initial chemical state as colorless iodine remains in the sample. Even so, the structural integrity of the specimen is left intact allowing for continued use of these bones for additional analyses.

The current study does have limitations inherent to the use of microCT imaging. Specifically, non-contrast microCT affords poor soft tissue visibility due to weak absorption of x-rays, as was evident in our unstained temporal bones. However, visualization of intracochlear structures was improved with contrast agents OsO₄ and I₂KI. A limitation of our methodology is that direct injection into the RW might cause injury to the cochlear cytoarchitecture, despite the injections being done in a controlled fashion by a skilled investigator. Finally, to maximize resolution provided by the microCT system, temporal bones need to be drilled down to the level of the otic capsule, which adds time prior to imaging.

Temporal bones have been used in applications ranging from uncovering molecular and cellular mechanisms of audiovestibular dysfunction (11, 16–18), development of devices for inner ear stimulation and monitoring (19, 20), and validation of novel imaging strategies (3, 21–24). Therefore, the value of each bone cannot be understated. While we await the development of methods for investigation of temporal bones which preclude the need for processing, reversible I₂KI staining with microCT can be used as an alternative approach for the study of the inner ear which preserves three-dimensional structure.