(0929) Evaluating Autophagy in Double Transgenic GFP-LC3 Mice Crossed With mCherry-LC3 Mice After Noise Exposure

Introduction: Autophagy is a cellular process that enables the degradation and recycling of dysfunctional cellular components. While there is evidence to suggest that autophagy plays a role in hair cell (HC) damage, the underlying mechanisms are not fully understood. The use of transgenic mice expressing GFP-LC3 has proven to be an effective tool for the systemic detection of autophagosomes. The aim of this study was to employ double transgenic mice expressing both GFP-LC3 and mCherry-LC3 to investigate autophagosome dynamics in HC damage following noise exposure.

Methods: This study involved 12 mice aged 5-6 weeks, divided into two groups. Each group comprised 4 double transgenic GFP-LC3/mCherry-LC3 mice and 2 C57Bl/6 mice serving as controls. In each group, 2 transgenic and one C57Bl/6 mouse were subjected to 100 dB SPL noise for 2 hours under anesthesia, while the remaining 3 mice were not exposed to noise. Perfusion fixation was performed 24 hours post-noise exposure, followed by the dissection of the organ of Corti and immunostaining with Myosin VII, DAPI, and LC3 A/B antibodies.

Results: Cytological analysis indicated a significant increase in the number and size of L3 puncta in the perinuclear and cuticular regions of the inner hair cells of noise-exposed mice, suggesting enhanced autophagic activity in these areas. In contrast whole mount staining with anti-LC3 antibodies did not reveal noise-induced autophagosome formation in comparison to the double transgenic GFP-LC3/mCherry-LC3 mice.

Conclusions: There may be varying dominant damage mechanisms across different regions of the cochlea.
The double transgenic GFP-LC3/mCherry-LC3 mouse model is a promising tool for further investigation into the mechanisms of noise-induced hearing loss.