Author(s)
Maia Smith, BSA MS
Annika Nambiar, BS MPH
Noor Souman, BS
Marina Saito, DVM PhD
Rebecca Cook, BS
Tomoko Makishima, MD PhD
Affiliation(s)
University of Texas Medical Branch Galveston
Abstract:
Educational Objective: At the conclusion of this presentation, the participants should be able to recognize how three dimensional (3D) lightsheet fluorescence microscopy enhances visualization of inner ear anatomy compared to traditional two dimensional (2D) microscopy in mice.
Objectives: To compare 2D whole mount immunostaining and 3D lightsheet fluorescence microscopy of the temporal bone in Casp-3 transgenic mice and identify architectural differences in cochlear and vestibular anatomy, with emphasis on the planar orientation of vestibular organs.
Study Design: Basic science research.
Methods: Temporal bones from Casp-3 wild type (WT), heterozygous (Het), and knockout (KO) mice were dissected, decalcified, and tissue cleared using the urea based Sca/e S protocol (Hama et al. Nat Neurosci. 18(10):1518-29). Ears were immunostained with markers for hair cells (Myo6, Phalloidin), endothelial cells (CD31), and neuronal cells (NFH) and imaged with either 2D fluorescence or 3D lightsheet microscopy.
Results: Whole mount microscopy demonstrated ampullar fusion, misshapen ampullae, and reduced saccular hair cell density in KO mice. Lightsheet microscopy enabled visualization of vestibular spatial relationships, revealing asymmetric left-right utricle-saccule (?L-R) angle difference in KO mice (14.0°+/=9.4°) compared to WT (7.96°+/=5.5°, p=0.084). KO mice showed different degrees of inner and outer hair cell loss, while Het and WT genotypes showed normal cochlear hair cell organization.
Conclusions: Whole mount imaging provides high cellular resolution, whereas lightsheet microscopy preserves 3D spatial context, enabling quantitative assessment of vestibular orientation. Together, these methods reveal vestibular organ asymmetry in Casp-3 KO mice and highlight the utility of 3D imaging for identifying spatial phenotypes in audiovestibular dysfunction models.