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

Abstract

Purpose: Hearing and balance disorders are among the most prevalent sensory impairments globally, yet their cellular and molecular basis remains poorly understood. This gap stems from the inaccessibility of the inner ear, which is encased in the temporal bone (TB)-the hardest bone in the body-and cannot be biopsied in living patients. Conventional histopathology workflows, particularly the century-old celloidin method, are time-consuming, labor-intensive, and incompatible with modern molecular analyses. We aimed to develop a faster, more versatile histology workflow for human TBs that preserves both morphology and molecular integrity.

Methods: We developed a reversible polymethyl methacrylate (rPMMA) embedding protocol for formalin-fixed, calcified TBs using low-temperature resin infiltration (-40 to +4 °C). Precision near-serial sections (10-50 µm) were generated via femtosecond laser microtomy or precision diamond wire sawing. Deacrylation was performed to restore tissue accessibility for histological staining, multiplex immunofluorescence, whole-genome sequencing, and in situ RNA detection (RNAscope™).

Results: Compared to the celloidin workflow, our method reduced processing time and costs by over 90% while preserving histomorphology of comparable quality (n = 7 human TBs). It maintained antigenicity for multiplexed immunofluorescence, preserved native tissue-implant interfaces in implant-containing specimens, yielded high-quality DNA suitable for whole-genome sequencing (mean coverage 7.4 × in specimens with postmortem intervals < 24 h), and enabled mRNA detection at single-cell resolution. Celloidin-embedded controls consistently failed to support these molecular assays.

Conclusion: This rPMMA-based workflow combines gold-standard histomorphology with full compatibility for advanced molecular analyses. With dramatically reduced time and cost, it offers a new benchmark for integrated, spatially resolved studies of human hearing and balance pathologies.