Landmark‐Based Anatomical Dissection and Real‐Time Functional Assessment of the Mouse Eustachian Tube

ABSTRACT Objective Eustachian tube dysfunction (ETD) contributes to middle ear diseases, but practical methods for functional assessment in mice remain limited. We aimed to develop a landmark‐guided mouse Eustachian tube (ET) dissection protocol and a real‐time pressure‐based system for ET functional evaluation. Methods A detachable portable device was developed for real‐time measurement of intraluminal pressure changes in the mouse middle ear‐ET complex. The ET was isolated using the cochlea, auditory bulla, and styliform process as landmarks while preserving the mucosa and adjacent tissues. Acute ET inflammation was induced by intratympanic injection of lipopolysaccharide, heat‐inactivated Streptococcus pneumoniae , or Staphylococcus aureus ; PBS served as a control. ET function was assessed using active clearance of negative pressure (ACNP), passive opening pressure (POP), and passive closing pressure (PCP), followed by histologic validation. Results The pressure system recorded real‐time pressure changes and enabled repeated ACNP and POP measurements under the tested conditions. Compared with controls, ACNP was significantly reduced in all pathogen‐treated groups (all p < 0.05), with the greatest reduction in the S. aureus group. ACNP differed among pathogen‐treated groups, mainly driven by the S. aureus group ( p = 0.0078; η 2 = 0.802). POP increased significantly after inflammatory challenge but did not differ among pathogen‐treated groups ( p = 0.3132; η 2 = 0.282). Histology showed epithelial injury, ciliary loss, mucus retention, and mucosal thickening in treated groups, most prominently after S. aureus challenge. Conclusion We developed a portable pressure‐based system and a landmark‐guided dissection workflow for structural and functional evaluation of the mouse ET. These methods provide a practical methodological approach for studies using mouse models of acute inflammation‐associated ETD. Level of Evidence N/A.