Ultrastructural Changes Associated With the Enhanced Permeability of the Round Window Membrane Mediated by Ultrasound Microbubbles

Yi-Chun Lin; Hsin-Chien Chen; Hang-Kang Chen; Yuan-Yung Lin; Chao-Yin Kuo; Hao Wang; Chia-Lien Hung; Cheng-Ping Shih; Chih-Hung Wang

doi:10.3389/fphar.2019.01580

Ultrastructural Changes Associated With the Enhanced Permeability of the Round Window Membrane Mediated by Ultrasound Microbubbles

Front Pharmacol. 2020 Jan 28:10:1580. doi: 10.3389/fphar.2019.01580. eCollection 2019.

Authors

Yi-Chun Lin¹, Hsin-Chien Chen², Hang-Kang Chen³, Yuan-Yung Lin^{1

2}, Chao-Yin Kuo², Hao Wang², Chia-Lien Hung³, Cheng-Ping Shih², Chih-Hung Wang^{1

2

4}

Affiliations

¹ Graduate Institute of Medical Sciences, National Defense Medical Center, Taipei, Taiwan.
² Department of Otolaryngology-Head and Neck Surgery, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan.
³ Teaching and Research Section, Taichung Armed Forces General Hospital, Taichung, Taiwan.
⁴ Taichung Armed Forces General Hospital, Taichung, Taiwan.

Abstract

The round window membrane (RWM) is the most common entryway for local drug and gene delivery into the inner ear, but its permeability can change the treatment outcome. We previously demonstrated a feasible and highly efficient approach using ultrasound-aided microbubble (USMB) cavitation to enhance the permeability of the RWM. Here, we investigated the safety of USMB exposure and the association between temporal changes in RWM permeability and ultrastructure. Experimental guinea pigs were divided into two treatment groups: a control group receiving round window soaking (RWS) with MBs and treatment (USM) groups undergoing 3 (USM-3) or 5 (USM-5) consecutive USMB exposures (1 min/exposure) at an acoustic intensity of 3 W/cm² and 1 MHz frequency. The trans-RWM delivery efficiency of biotin-fluorescein isothiocyanate conjugates, used as permeability tracers, revealed a greater than 7-fold higher delivery efficiency for the USM groups immediately after 3 or 5 exposures than for the RWS group. After 24 h, the delivery efficiency was 2.4-fold higher for the USM-3 group but was 6.6-fold higher for the USM-5 group (and 3.7-fold higher after 48 h), when compared to the RWS group. Scanning electron microscopy images of the RWM ultrastructure revealed USMB-induced sonoporation effects that could include the formation of heterogeneous pore-like openings with perforation diameters from 100 nm to several micrometers, disruption of the continuity of the outer epithelial surface layer, and loss of microvilli. These ultrastructural features were associated with differential permeability changes that depended on the USMB exposure course. Fourteen days after treatment, the pore-like openings had significantly decreased in number and the epithelial defects were healed either by cell expansion or by repair by newly migrated epithelial cells. The auditory brainstem response recordings of the animals following the 5-exposure USMB treatment indicated no deterioration in the hearing thresholds at a 2-month follow-up and no significant hair cell damage or apoptosis, based on scanning electron microscopy, surface preparations, and TUNEL assays. USMBs therefore appear to be safe and effective for inner ear drug delivery. The mechanism of enhanced permeability may involve a disruption of the continuity of the outer RWM epithelial layer, which controls transmembrane transport of various substances.

Keywords: inner ear; microbubble; permeability; round window membrane (RWM); scanning electron microscope (SEM); transmission electron microscope (TEM); ultrasound; ultrastructure.