Ultrasound Microbubble-Facilitated Inner Ear Delivery of Gold Nanoparticles Involves Transient Disruption of the Tight Junction Barrier in the Round Window Membrane.

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.

Insonation of Systemically Delivered Cisplatin-Loaded Microbubbles Significantly Attenuates Nephrotoxicity of Chemotherapy in Experimental Models of Head and Neck Cancer.

The use of cisplatin (CDDP), the most common chemotherapy drug for head and neck cancer, is limited by its undesirable side effects, especially nephrotoxicity. We investigated ultrasound microbubbles (USMB) as a tool to increase the local intra-tumoral CDDP level while decreasing systemic CDDP cytotoxicity. We allowed CDDP to interact with human serum albumin and then sonicated the resulting CDDP‒albumin complex to generate CDDP-loaded MBs (CDDP-MBs). We then established a head-and-neck tumor-bearing mouse model by implanting FaDu-fLuc/GFP cells into severe combined immunodeficiency mice and used IVIS® bioluminescence imaging to determine the tumor xenograft formation and size. Twice weekly (until Day 33), we administered CDDP only, CDDP + MBs + US, CDDP-MBs, or CDDP-MBs + US intravenously by tail-vein injection. The US treatment was administered at the tumor site immediately after injection. The in vivo systemic distribution of CDDP indicated that the kidney was the most vulnerable organ, followed by the liver, and then the inner ear. However, CDDP uptake into the kidney and liver was significantly decreased in both the CDDP-MBs and CDDP-MBs + US groups, suggesting that MB binding significantly reduced the systemic toxicity of CDDP. The CDDP-MBs + US treatment reduced the tumor size as effectively as conventional CDDP-only chemotherapy. Therefore, the combination of CDDP-MBs with ultrasound is effective and significantly attenuates CDDP-associated nephrotoxicity, indicating a promising clinical potential for this approach.

Hypoxia Induces a Metabolic Shift and Enhances the Stemness and Expansion of Cochlear Spiral Ganglion Stem/Progenitor Cells.

Previously, we demonstrated that hypoxia (1% O2) enhances stemness markers and expands the cell numbers of cochlear stem/progenitor cells (SPCs). In this study, we further investigated the long-term effect of hypoxia on stemness and the bioenergetic status of cochlear spiral ganglion SPCs cultured at low oxygen tensions. Spiral ganglion SPCs were obtained from postnatal day 1 CBA/CaJ mouse pups. The measurement of oxygen consumption rate, extracellular acidification rate (ECAR), and intracellular adenosine triphosphate levels corresponding to 20% and 5% oxygen concentrations was determined using a Seahorse XF extracellular flux analyzer. After low oxygen tension cultivation for 21 days, the mean size of the hypoxia-expanded neurospheres was significantly increased at 5% O2; this correlated with high-level expression of hypoxia-inducible factor-1 alpha (Hif-1α), proliferating cell nuclear antigen (PCNA), cyclin D1, Abcg2, nestin, and Nanog proteins but downregulated expression of p27 compared to that in a normoxic condition. Low oxygen tension cultivation tended to increase the side population fraction, with a significant difference found at 5% O2 compared to that at 20% O2. In addition, hypoxia induced a metabolic energy shift of SPCs toward higher basal ECARs and higher maximum mitochondrial respiratory capacity but lower proton leak than under normoxia, where the SPC metabolism was switched toward glycolysis in long-term hypoxic cultivation.

Rodent models for investigating the dysregulation of immune responses in type 1 diabetes.

Type 1 diabetes (T1D) is an autoimmune disease mediated by T cells that selectively destroy the insulin-producing ß cells. Previous reports based on epidemiological and animal studies have demonstrated that both genetic factors and environmental parameters can either promote or attenuate the progression of autoimmunity. In recent decades, several inbred rodent strains that spontaneously develop diabetes have been applied to the investigation of the pathogenesis of T1D. Because the genetic manipulation of mice is well developed (transgenic, knockout, and conditional knockout/transgenic), most studies are performed using the nonobese diabetic (NOD) mouse model. This paper will focus on the use of genetically manipulated NOD mice to explore the pathogenesis of T1D and to develop potential therapeutic approaches.

Kinetics of adaptive immunity to Haemophilus influenzae type b meningitis in transgenic mice: evidence from diverse expression of double T1/T2 transgenes and Th1/Th2-related cytokines.

To investigate the kinetic changes in adaptive immunity during experimental Haemophilus influenzae type b (Hib) meningitis, we established a murine meningitis model based on T1/T2 doubly transgenic mice. These mice carry two transgenes that express two distinct cell-surface markers: a human Thy1 transgene (hThy1) under the control of the murine IFN-gamma promoter, and a murine Thy1.1 transgene (mThy1.1) under the control of the murine IL-4 promoter, designated T1 and T2, respectively. Mice infected with Hib displayed severest symptoms and lowest total splenocyte counts on day 3 after infection. Simultaneously, we examined the significantly low percentage of CD19+ B cells, the relatively high level of CD4+ T cells and significantly high percentage of CD8+ T cells in Hib-infected mice. Furthermore, we observed the early induction of both Th1 and Th2 responses, in terms of the augmentation of Th1 cells (IFN-gamma-producing CD4+ T cells) and Th2 cells (IL-4-producing CD4+ T cells) in Hib-infected mice. On day 7 after infection, the Th1 response gradually declined and the Th2 response rather sustained. Two weeks after infection, both Th1 and Th2 cells were barely detectable. Moreover, we demonstrated using an antigen-specific re-stimulation test to analyze the effector function of lymphocyte subsets that CD8+ T cells contributed to more predominantly production of IFN-gamma than CD4+ T cells did; and CD4+ T cells partly contributed to the secretion of IL-4 from flowcytometry of intracellular cytokine staining. Our results support that these transgenic mice provide an available model to dissect the complex kinetic change of adaptive immunity in bacterial infectious diseases.