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.

The application of insulin-like growth factor 1 (IGF-1) to the round window membrane (RWM) is an emerging treatment for inner ear diseases. RWM permeability is the key factor for efficient IGF-1 delivery. Ultrasound microbubbles (USMBs) can increase drug permeation through the RWM. In the present study, the enhancing effect of USMBs on the efficacy of IGF-1 application and the treatment effect of USMB-mediated IGF-1 delivery for noise-induced hearing loss (NIHL) were investigated. Forty-seven guinea pigs were assigned to three groups: the USM group, which received local application of recombinant human IGF-1 (rhIGF-1, 10 µg/µL) following application of USMBs to the RWM; the RWS group, which received IGF-1 application alone; and the saline-treated group. The perilymphatic concentration of rhIGF-1 in the USM group was 1.95- and 1.67- fold of that in the RWS group, 2 and 24 h after treatment, respectively. After 5 h of 118 dB SPL noise exposure, the USM group had the lowest threshold shift in auditory brainstem response, least loss of cochlear outer hair cells, and least reduction in the number of synaptic ribbons on postexposure day 28 among the three groups. The combination of USMB and IGF-1 led to a better therapeutic response to NIHL. Two hours after treatment, the USM group had significantly higher levels of Akt1 and Mapk3 gene expression than the other two groups. The most intense immunostaining for phosphor-AKT and phospho-ERK1/2 was detected in the cochlea in the USM group. These results suggested that USMB can be applied to enhance the efficacy of IGF-1 therapy in the treatment of inner ear diseases.

The application of ultrasound microbubbles (USMBs) enhances the permeability of the round window membrane (RWM) and improves drug delivery to the inner ear. In this study, we investigated the efficiency of USMB-aided delivery of chitosan-coated gold nanoparticles (CS-AuNPs) and the mechanism of USMB-mediated enhancement of RMW permeability. We exposed mouse inner ears to USMBs at an intensity of 2 W/cm2 and then filled the tympanic bulla with CS-AuNPs or fluorescein isothiocyanate-decorated CS-AuNPs (FITC-CS-AuNPs). The membrane uptake of FITC-CS-AuNPs and their depth of permeation into the three-layer structure of the RWM, with or without prior USMB treatment, were visualized by z-stack confocal laser scanning microscopy. Ultrastructural changes in the RWM due to USMB-mediated cavitation appeared as sunburn-like peeling and various degrees of depression in the RWM surface, with pore-like openings forming in the outer epithelium. This disruption of the outer epithelium was paralleled by a transient reduction in tight junction (TJ)-associated protein levels in the RWM and an enhanced delivery of FITC-CS-AuNPs into the RWM. Without prior USMB exposure, the treatment with CS-AuNPs also caused a noticeable reduction in TJ proteins of the RWM. Our findings indicated that the combined treatment with USMBs and CS-AuNPs represents a promising and efficient drug and gene delivery vehicle for a trans-RWM approach for inner ear therapy. The outer epithelial layer of the RWM plays a decisive role in controlling the transmembrane transport of substances such as CS-AuNPs following the administration of USMBs. Most importantly, the enhanced permeation of AuNPs involved the transient disruption of the TJ-created paracellular barrier in the outer epithelium of the RWM.

Purpose: To assess the association between serum testosterone (T) and metabolic syndrome (MS) in different age groups in Taiwanese men. Materials and Methods: Male participants, regardless of age or any underlying disease, were identified from MJ Health Screening Center in Taiwan from 2007 to 2016 for this crosssectional study. They were divided into three groups according to age, and further classified according to MS diagnosis. Basic patient characteristics with relevant parameters were obtained. One-way ANOVA of mean T values between different numbers of measures that exceeds the cut-off values of MS components was performed to assess the relationship of T and MS. Logistic regression analysis was also used to estimate the risk for MS with each increment in T, age, and BMI. Results: A total of 4,931 men were included. The MS group had significantly lower serum T levels compared to the non-MS group in each age group. The one-way ANOVA found the mean value of T was significantly higher in patients without MS component (6.19±2.12 ng/ mL) than those with 1-5 MS components (with one MS component: 5.48±2.13 ng/mL, two MS components: 4.93±2.03 ng/mL, three MS components: 4.37±1.60 ng/mL, four MS components: 4.13±2.89 ng/mL, five MS components: 3.74±1.27 ng/mL, and P<0.001). There was no significant difference between the patients with three components and the patients with four or five components. Logistic regression models with age stratification showed T with lower odds ratio (OR) for MS after adjusting for BMI in those ≥65 years old (OR=0.693; 95% CI=0.559-0.858; P<0.001); 50-64 years old (OR=0.868; 95% CI=0.802-0.940; P<0.001) and <50 years old (OR=0.810; 95% CI=0.758-0.865; P<0.001). Conclusion: Lower serum T was strongly associated with MS, with the predictive value increasing with age in Taiwanese men.

Gout is a common systemic inflammatory disease with a male predominance. This study aimed to determine the relationship between serum total testosterone level and hyperuricemia. Data on 1899 men, collected from 2007 to 2017, were included in the analysis. Serum testosterone and urate (SU) were measured on enrolment. The primary endpoints were SU levels ≥ 7 mg/dL and ≥9 mg/dL. On enrolment, participants had a mean age of 45.6 years and mean total testosterone and SU levels of 510 ng/dL and 6.6 mg/dL, respectively. The mean total testosterone levels were 533 and 470 ng/dL in patients with SU levels < 7 mg/dL and ≥7 mg/dL, respectively (p < 0.001); and 515 and 425 ng/dL in patients with SU levels < 9 mg/dL and ≥9 mg/dL, respectively (p < 0.001). After adjusting for age, body mass index, creatinine, serum lipid, fasting blood glucose, systolic blood pressure, and diastolic blood pressure, low testosterone level (<400 ng/dL) was significantly associated with an SU level ≥ 7 mg/dL (hazard ratio: 1.182, 95% confidence interval: 1.005–1.39) and ≥9 mg/dL (hazard ratio: 1.905, 95% confidence interval: 1.239–2.928). In men, a low testosterone level may be associated with an increased risk of hyperuricemia.