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

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/cm 2 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.

Development of thermosensitive poloxamer 407-based microbubble gel with ultrasound mediation for inner ear drug delivery

“…US triggers cavitation of MBs, including inertial and stable cavitation, to produce microstreaming and shock waves in the middle ear to facilitate drug diffusion through RWM. RWM permeability is immediately increased following USMB treatment, and the enhancement of RWM permeability can persist for 72 h after USMB treatment (Shih et al, 2013 ; Lin et al, 2019 ). Electron microscopic observation of the RWM revealed heterogeneous pore-like openings and perforation on the outer epithelial layer of the RWM, indicating an acoustic cavitation-induced sonoporation effect on the RWM (Lin et al, 2019 ).…”

“…RWM permeability is immediately increased following USMB treatment, and the enhancement of RWM permeability can persist for 72 h after USMB treatment (Shih et al, 2013 ; Lin et al, 2019 ). Electron microscopic observation of the RWM revealed heterogeneous pore-like openings and perforation on the outer epithelial layer of the RWM, indicating an acoustic cavitation-induced sonoporation effect on the RWM (Lin et al, 2019 ). The disruptions in the outer epithelial layer were effectively repaired, and the RWM was completely recovered within 1 month post-USMB (Lin et al, 2019 ).…”

“…Electron microscopic observation of the RWM revealed heterogeneous pore-like openings and perforation on the outer epithelial layer of the RWM, indicating an acoustic cavitation-induced sonoporation effect on the RWM (Lin et al, 2019 ). The disruptions in the outer epithelial layer were effectively repaired, and the RWM was completely recovered within 1 month post-USMB (Lin et al, 2019 ). Moreover, we demonstrated that the efficiency of DEX delivery into the inner ear can be enhanced by USMB.…”

Ultrasound‐microbubble cavitation facilitates adeno‐associated virus mediated cochlear gene transfection across the round‐window membrane

“…The integrity of the RWM in response to this USMB treatment method was reported more recently in a separate paper by the same group. 35…”

“…In other study, RWM damage by USMB was observed with electron microscopy with information how long the wound will be recovered. 35 In this study, we developed a new ultrasound probe with a considerably smaller diameter (1.5 mm). By using this small probe, we were able to create intense, focalized damage to the RWM with a lower ultrasound power and a smaller amount of MB solution.…”