Massachusetts Eye and Ear/ Harvard Initiative for the NIDCD National Human Ear Resource Network - Project Summary This proposal to join the NIDCD National Human Ear Resource Network pools the skills of an international consortium of researchers with decades of experience in the histopathological study of acquired and hereditary disorders of hearing and balance, and in the curation of one of the largest collections of human temporal-bone (TB) specimens in the world. Because the ear cannot be biopsied, and non-invasive imaging cannot yet provide cellular level resolution, insight into the pathologies underlying deafness and balance disorders derives from the study of TBs removed at autopsy, which classically have been decalcified, plastic-embedded and serially sectioned, with a subset then stained with hematoxylin and eosin (H&E) for pathological study. In Aim 1, we continue to accrue and process human TBs, document the histopathology and medical/otologic history, and upload the de-identified data to a searchable database, along with digitized images of all the H&E-stained slide sets, to jumpstart more widespread collaborations in the study of human otopathology. We will also continue to distribute unstained sections from our TB archives to researchers for additional morphological, molecular biological or genetic studies of particular disorders. In Aim 2, we enhance techniques for human TB study in three key ways: first (Aim 2a), by perfecting a novel approach to embedding (methyl methacrylate) and sectioning (laser microtomy) that is cheaper and faster than celloidin, allows morphological and chemical analysis of undecalcified bone and otoconia, and is less destructive to protein and RNA/DNA; second (Aim 2b), by pursuing the ex vivo imaging of intact cochleas via high-resolution x-ray tomography, enhanced by coupling celltype-specific immunomarkers to heavy metals and by developing software to automate extraction of quantitative histopathological data from these 3D image stacks; and third (Aim 2c), by developing and applying techniques for DNA extraction and whole-exome sequencing from donor tissue samples and/or celloidin TBs, to identify the pathogenic variants responsible for the numerous hereditary hearing loss cases in our archives that were not genotyped during life. In Aim 3, we strive to accelerate the pace of discovery in human otopathology by sponsoring fellowships, organizing national symposia and by creating and disseminating training materials, via hands-on courses, online tutorials, technical manuals on the classic and novel techniques needed for the procurement, processing and histopathological analysis of human temporal bones.
