Baechinger, D. | bioRxiv Science

bioRxiv2025

Integrated Histology and Molecular Profiling of Postmortem Human Auditory and Vestibular Organs via a Poly(Methyl Methacrylate)-Based Workflow

Hearing and balance disorders are the most prevalent sensory impairments, affecting hundreds of millions worldwide, yet their underlying cellular and molecular pathologies remain poorly understood. This knowledge gap stems from the inaccessibility of the ears sensory organs--embedded within the temporal bone (TB), the hardest bone in the body--which cannot be biopsied in living patients without causing irreversible damage. Conventional histopathology workflows rely on postmortem en bloc extraction of TBs, followed by lengthy decalcification, celloidin embedding, and manual serial sectioning of these large specimens--a process that takes one to two years, is labor- and cost-intensive, and lacks compatibility with most modern protein, DNA, and RNA assays. Here, we present a rapid, reversible polymethyl methacrylate (rPMMA) workflow that enables advanced molecular histopathology studies on formalin-fixed, calcified TBs. Our protocol uses low-temperature (-40 {degrees}C to +4 {degrees}C) resin embedding, precision near-serial sectioning (10-50 {micro}m) via femtosecond laser microtomy or precision diamond wire sawing, and subsequent deacrylation to fully restore tissue accessibility for high-fidelity histomorphology, multiplexed immunofluorescence, whole-genome sequencing, and in situ mRNA detection (RNAscope) assays. Compared to the gold-standard celloidin workflow, our method reduces processing time and costs by approximately 90% while integrating equivalent histomorphology with advanced molecular assays, providing a new benchmark for multidimensional studies in human hearing and balance pathologies.

pathology↗

bioRxiv2025

Hyperplastic Growth, Not Hydrostatic Distension, in Endolymphatic Hydrops in Humans Challenges the Classic View of Meniere's Disease

Menieres disease (MD), a degenerative inner ear disorder, is characterized by debilitating episodic vertigo "attacks" and hearing fluctuations, progressing to permanent sensory impairment. The prevailing dogma attributes these symptoms to an abnormal inner ear fluid buildup--known as endolymphatic hydrops (EH)--with concomitant rise of fluid pressure and repetitive microtrauma to sensory epithelia. However, this pressure-based mechanism lacks direct experimental evidence and fails to explain key clinical aspects of MD--exposing a critical gap in our disease understanding. To revisit the fundamental nature of EH, we performed 3D reconstructive, machine-learning-enhanced histological analyses and immunohistochemistry on human postmortem inner ear specimens. Contrary to the classic theory, EH-affected epithelia showed no signs of pressure-induced change. Instead, we observed an up to four-to seven-fold increase in epithelial cell number (hyperplasia) in both early and advanced EH stages. Quantification of the hyperplastic epithelial surface area, as well as immunohistochemical localization of key fluid homeostasis-associated proteins in the hyperplastic epithelium suggest this epithelial hyperplasia may actively compensate for cell loss in the endolymphatic sac, a key site of MD pathology. These findings challenge the conventional view of EH as solely a pathological pressure phenomenon, instead revealing an unexpected massive cellular expansion of these epithelia, consistent with a coordinated compensatory cellular response aimed at preserving inner ear fluid homeostasis and function in a compromised environment. This paradigm shift introduces dual beneficial and detrimental roles for EH, and suggests new therapeutic avenues for MD focused on promoting compensatory tissue repair while preventing maladaptive remodeling. Significance StatementFor over a century, the leading explanation for Menieres disease--a debilitating inner ear disorder causing vertigo and hearing loss--has been a buildup of fluid and pressure in the inner ear, analogous to conditions like glaucoma. However, this long-held theory has never been directly proven, and treatments based on reducing this supposed pressure have shown limited success. Our research challenges this traditional view, revealing that the expansion of endolymphatic spaces is not primarily a fluid pressure problem, but secondary to a complex cellular response. This fundamental shift in understanding Menieres disease opens new avenues for developing effective therapies to prevent and treat hearing loss and vertigo attacks.

neuroscience↗