Injectable Poloxamer Hydrogel Formulations for Intratympanic Delivery of Dexamethasone.

BACKGROUND: In this study, we prepared and evaluated an injectable poloxamer (P407) hydrogel formulation for intratympanic (IT) delivery of dexamethasone (DEX). METHODS: DEX-loaded P407 hydrogels were characterized in terms of thermogelation, drug loading capacities, particle size, and drug release. The in vivo toxicity and drug absorption of the DEX-loaded P407 formulation after IT injection were evaluated using an animal model by performing histopathological analysis and drug concentration measurements. RESULTS: The P407 hydrogel effectively solubilized hydrophobic DEX and demonstrated a sustained release compared to the hydrophilic DEX formulation. The in vivo study showed that the hydrogel formulation delivered considerable drug concentrations to the inner ear and displayed a favorable safety profile without apparent cytotoxicity or inflammation. CONCLUSION: P407 hydrogel can be useful as an injectable inner ear delivery formulation for hydrophobic drugs due to their biocompatibility, drug-solubilizing capacity, thermogelation, and controlled release.

Sex differences in hearing impairment due to diet-induced obesity in CBA/Ca mice.

BACKGROUND: Obesity is an independent risk factor for hearing loss. Although attention has focused on major obesity comorbidities such as cardiovascular disease, stroke, and type 2 diabetes, the impact of obesity on sensorineural organs, including the auditory system, is unclear. Using a high-fat diet (HFD)-induced obese mouse model, we investigated the impact of diet-induced obesity on sexual dimorphism in metabolic alterations and hearing sensitivity. METHODS: Male and female CBA/Ca mice were randomly assigned to three diet groups and fed, from weaning (at 28 days) to 14 weeks of age, a sucrose-matched control diet (10 kcal% fat content diet), or one of two HFDs (45 or 60 kcal% fat content diets). Auditory sensitivity was evaluated based on the auditory brainstem response (ABR), distortion product otoacoustic emission (DPOAE), and ABR wave 1 amplitude at 14 weeks of age, followed by biochemical analyses. RESULTS: We found significant sexual dimorphism in HFD-induced metabolic alterations and obesity-related hearing loss. Male mice exhibited greater weight gain, hyperglycemia, increased ABR thresholds at low frequencies, elevated DPOAE, and lower ABR wave 1 amplitude compared to female mice. The hair cell (HC) ribbon synapse (CtBP2) puncta showed significant sex differences. The serum concentration of adiponectin, an otoprotective adipokine, was significantly higher in female than in male mice; cochlear adiponectin levels were elevated by HFD in female but not male mice. Adiponectin receptor 1 (AdipoR1) was widely expressed in the inner ear, and cochlear AdipoR1 protein levels were increased by HFD, in female but not male mice. Stress granules (G3BP1) were significantly induced by the HFD in both sexes; conversely, inflammatory (IL-1ß) responses were observed only in the male liver and cochlea, consistent with phenotype HFD-induced obesity. CONCLUSIONS: Female mice are more resistant to the negative effects of an HFD on body weight, metabolism, and hearing. Females showed increased peripheral and intra-cochlear adiponectin and AdipoR1 levels, and HC ribbon synapses. These changes may mediate resistance to HFD-induced hearing loss seen in female mice.

Injectable glycol chitosan thermogel formulation for efficient inner ear drug delivery.

We prepared a new injectable thermogel to enhance the efficiency of inner ear delivery of dexamethasone (DEX). Hexanoyl glycol chitosan (HGC) was synthesized and evaluated as an amphiphilic thermogel (Tgel ~ 32 °C) for use as a solubilizing agent as well as an injectable carrier for intratympanic delivery of the hydrophilic and hydrophobic forms of DEX. Various thermogel formulations with different drug types and concentrations were prepared, and their physicochemical and thermogelling properties were characterized by 1H NMR, ATR-FTIR, and rheometer. They exhibited versatile release kinetics from several hours to more than 2 weeks, depending on drug type and concentration. Our formulations further showed good residual stability for more than 21 days without any cytotoxicity or inflammation in the middle and inner ear and could deliver a considerably high drug concentration into the inner ear. Therefore, HGC thermogel has great potential as an effective and safe formulation for inner ear drug delivery.

Junctional Modulation of Round Window Membrane Enhances Dexamethasone Uptake into the Inner Ear and Recovery after NIHL.

Delivery of substances into the inner ear via local routes is increasingly being used in clinical treatment. Studies have focused on methods to increase permeability through the round window membrane (RWM) and enhance drug diffusion into the inner ear. However, the clinical applications of those methods have been unclear and few studies have investigated the efficacy of methods in an inner ear injury model. Here, we employed the medium chain fatty acid caprate, a biologically safe, clinically applicable substance, to modulate tight junctions of the RWM. Intratympanic treatment of sodium caprate (SC) induced transient, but wider, gaps in intercellular spaces of the RWM epithelial layer and enhanced the perilymph and cochlear concentrations/uptake of dexamethasone. Importantly, dexamethasone co-administered with SC led to significantly more rapid recovery from noise-induced hearing loss at 4 and 8 kHz, compared with the dexamethasone-only group. Taken together, our data indicate that junctional modulation of the RWM by SC enhances dexamethasone uptake into the inner ear, thereby hastening the recovery of hearing sensitivity after noise trauma.

Hearing Impairment in a Mouse Model of Diabetes Is Associated with Mitochondrial Dysfunction, Synaptopathy, and Activation of the Intrinsic Apoptosis Pathway.

Although previous studies continuously report an increased risk of hearing loss in diabetes patients, the impact of the disease on the inner ear remains unexplored. Herein, we examine the pathophysiology of diabetes-associated hearing impairment and cochlear synaptopathy in a mouse model of diabetes. Male B6.BKS(D)-Leprdb/J (db/db, diabetes) and heterozygote (db/+, control) mice were assigned into each experimental group (control vs. diabetes) based on the genotype and tested for hearing sensitivity every week from 6 weeks of age. Each cochlea was collected for histological and biological assays at 14 weeks of age. The diabetic mice exerted impaired hearing and a reduction in cochlear blood flow and C-terminal-binding protein 2 (CtBP2, a presynaptic ribbon marker) expression. Ultrastructural images revealed severely damaged mitochondria from diabetic cochlea accompanied by a reduction in Cytochrome c oxidase subunit 4 (COX4) and CR6-interacting factor 1 (CRIF1). The diabetic mice presented significantly decreased levels of platelet endothelial cell adhesion molecule (PECAM-1), B-cell lymphoma 2 (BCL-2), and procaspase-9, but not procaspase-8. Importantly, significant changes were not found in necroptotic programmed cell death markers (receptor-interacting serine/threonine-protein kinase 1, RIPK1; RIPK3; and mixed lineage kinase domain-like pseudokinase, MLKL) between the groups. Taken together, diabetic hearing loss is accompanied by synaptopathy, microangiopathy, damage to the mitochondrial structure/function, and activation of the intrinsic apoptosis pathway. Our results imply that mitochondrial dysfunction is deeply involved in diabetic hearing loss, and further suggests the potential benefits of therapeutic strategies targeting mitochondria.

Mitochondrial Damage and Necroptosis in Aging Cochlea.

Age-related hearing loss (ARHL) is an irreversible, progressive neurodegenerative disorder and is presently untreatable. Previous studies using animal models have suggested mitochondrial damage and programmed cell death to be involved with ARHL. Thus, we further investigated the pathophysiologic role of mitochondria and necroptosis in aged C57BL/6J male mice. Aged mice (20 months old) exhibited a significant loss of hearing, number of hair cells, neuronal fibers, and synaptic ribbons compared to young mice. Ultrastructural analysis of aged cochleae revealed damaged mitochondria with absent or disorganized cristae. Aged mice also showed significant decrease in cochlear blood flow, and exhibited increase in gene expression of proinflammatory cytokines (IL-1ß, IL-6, and TNF-α), receptor-interacting serine/threonine-protein kinase 1 and 3 (RIPK1 and RIPK3) and the pseudokinase mixed-lineage kinase domain-like (MLKL). Immunofluorescence (IF) assays of cytochrome C oxidase I (COX1) confirmed mitochondrial dysfunction in aged cochleae, which correlated with the degree of mitochondrial morphological damage. IF assays also revealed localization and increased expression of RIPK3 in sensorineural tissues that underwent significant necroptosis (inner and outer hair cells and stria vascularis). Together, our data shows that the aging cochlea exhibits damaged mitochondria, enhanced synthesis of proinflammatory cytokines, and provides new evidence of necroptosis in the aging cochlea in in vivo.

Acoustic Trauma Modulates Cochlear Blood Flow and Vasoactive Factors in a Rodent Model of Noise-Induced Hearing Loss.

Noise exposure affects the organ of Corti and the lateral wall of the cochlea, including the stria vascularis and spiral ligament. Although the inner ear vasculature and spiral ligament fibrocytes in the lateral wall consist of a significant proportion of cells in the cochlea, relatively little is known regarding their functional significance. In this study, 6-week-old male C57BL/6 mice were exposed to noise trauma to induce transient hearing threshold shift (TTS) or permanent hearing threshold shift (PTS). Compared to mice with TTS, mice with PTS exhibited lower cochlear blood flow and lower vessel diameter in the stria vascularis, accompanied by reduced expression levels of genes involved in vasodilation and increased expression levels of genes related to vasoconstriction. Ultrastructural analyses by transmission electron microscopy revealed that the stria vascularis and spiral ligament fibrocytes were more damaged by PTS than by TTS. Moreover, mice with PTS expressed significantly higher levels of proinflammatory cytokines in the cochlea (e.g., IL-1ß, IL-6, and TNF-α). Overall, our findings suggest that cochlear microcirculation and lateral wall pathologies are differentially modulated by the severity of acoustic trauma and are associated with changes in vasoactive factors and inflammatory responses in the cochlea.

Cochlear Glucocorticoid Receptor and Serum Corticosterone Expression in a Rodent Model of Noise-induced Hearing Loss: Comparison of Timing of Dexamethasone Administration.

Glucocorticoid (GC) is a steroid hormone secreted from the adrenal cortex in response to stress, which acts by binding to cytoplasmic glucocorticoid receptors (GRs). Dexamethasone (DEX) is a synthetic GC exhibiting immunosuppressive effects in both human and rodent models of hearing loss. While clinical evidence has shown the effectiveness of DEX for treatment of various inner ear diseases, its mechanisms of action and the optimal timing of treatment are not well understood. In the present study, intergroup comparisons were conducted based on the time point of treatment with DEX: (1) pretreatment; (2) posttreatment; and (3) pre&post-noise. The pre&post DEX treatment group showed a significant improvement in threshold shift at 1 day post-noise exposure as compared to the TTS (transient threshold shift)-only group at 8 and 16 kHz. Both TTS and PTS (permanent threshold shift) significantly reduced cochlear GR mRNA expression and increased serum corticosterone and cochlear inflammatory cytokines. The pre&post DEX treatment group showed a significant decrease in serum corticosterone level as compared to other DEX treatment groups and TTS-treated group at 3 days after acoustic trauma. Our results suggest that the timing of DEX administration differentially modulates systemic steroid levels, GR expression and cochlear cytokine expression.

Tinnitus in the side with better hearing.

OBJECTIVES: We aimed to confirm the characteristics of patients with tinnitus in the better-hearing side. MATERIALS AND METHODS: Among the 778 patients who visited the tinnitus clinic complaining of unilateral tinnitus at a local university hospital between March 2014 and December 2017, we recruited 62 patients who showed tinnitus in the better-hearing side on pure-tone audiometry. The mean hearing threshold was calculated using the arithmetic mean of the pure tone thresholds at 1, 2, 3, and 4 kHz. In addition, patients' medical history, tinnitus questionnaires, and other audiologic test results were thoroughly analyzed together for diagnosis. RESULTS: Fluctuating hearing loss without vertigo or Ménière's disease were the most common etiologies (n = 16, 25.8%), followed by high-frequency hearing loss (n = 13, 21.0%), sudden idiopathic hearing loss (n = 6, 9.7%), and presbycusis (n = 6, 9.7%). Somatosensory tinnitus was also observed in seven patients. Neck pain was associated with tinnitus in five patients (8.1%), and two other patients (3.2%) experienced temporomandibular disorder in the same side as the tinnitus. CONCLUSION: Tinnitus was associated with deterioration of hearing even when it occurred in the better-hearing side. Among the possible etiologies, fluctuating hearing loss in the tinnitus side was the most common audiologic finding. Assessment of hearing level at each frequency was more effective in detecting high-frequency hearing loss rather than the use of the mean hearing level. In addition, somatosensory tinnitus should not be ignored.

Paper patching for patulous eustachian tube.

BACKGROUND: Paper patching, a method that places cigarette paper over the most mobile quadrants of the tympanic membrane, is one of the treatment options for patulous eustachian tube (PET). AIMS/OBJECTIVES: The objective of this study was to compare the outcomes of two different treatment strategies for PET. MATERIAL AND METHODS: Twenty-three patients underwent paper patching of the tympanic membrane and 16 patients were treated with nasal saline irrigation with or without ipratropium bromide nasal spray. Medical records were reviewed for resolution of PET symptoms as categorical variables (complete remission, partial remission, or no improvement) with a minimum follow-up of 3 months. RESULTS: Immediately after undergoing paper patching, 20 of the 23 patients (87.0%) reported complete remission (CR). The percentage of CR after paper patching was 82.6% at 1 month and 65.2% at 3 months. A greater percentage of patients reported CR of aural symptoms in the paper patching group than in the nasal irrigation group at both 1 and 3 months after treatment (p < .05). CONCLUSIONS AND SIGNIFICANCE: Repetitive paper patching resolves aural discomfort in most PET patients for at least 3 months and can be considered as a first-line treatment option for PET in the outpatient setting.

Audiometric Profiles in Patients With Normal Hearing and Bilateral or Unilateral Tinnitus.

HYPOTHESIS: Patients with subjective nonpulsatile tinnitus and a normal conventional audiogram have more objective audiologic evidence of hidden hearing loss and deafferentation-related pathology than patients without tinnitus. BACKGROUND: The aim of this study was to assess the epidemiologic characteristics and audiologic profiles, including auditory brainstem response (ABR), distortion product otoacoustic emission, and threshold-equalizing noise test results, in patients with tinnitus and a normal audiogram. METHODS: The test results for 20 patients complaining of nonpulsatile chronic tinnitus were compared with those of 91 subjects with normal hearing and no tinnitus. RESULTS: Patients with unilateral tinnitus had higher tinnitus handicap inventory scores than those with bilateral tinnitus (p < 0.05). Threshold-equalizing noise tests were normal in all study participants. In patients with unilateral tinnitus, the ABR and distortion product otoacoustic emission test results were similar to those of controls. In contrast, patients with bilateral tinnitus showed a shortening of latency in wave III of the ABR on the right (p = 0.047) and in wave V on the left (p = 0.024). Logistic regression analysis revealed that enhanced wave III/I (p = 0.018) and V/I (p = 0.012) ratios on the left and poorer pure-tone average on the right were significant risk factors for bilateral tinnitus. CONCLUSION: The mechanism involved in the development of tinnitus may depend on its laterality. Bilateral tinnitus may be associated with hyperactivity at the level of the cochlear nucleus whereas a higher-order cortical area may be involved in unilateral tinnitus.

Clinical Characteristics of Dizziness Associated with Acute Peripheral Facial Palsy.

BACKGROUND AND OBJECTIVES: We aimed to assess the clinical significance of dizziness associated with acute peripheral facial palsy (APFP). SUBJECTS AND METHODS: Medical records of patients who visited an otorhinolaryngology clinic at a university hospital and were admitted for treatment of APFP between 2014 and 2016 were thoroughly reviewed. RESULTS: In total, 15.3% (n=15) of patients had dizziness. Continuous, rotatory dizziness without exacerbating factors was most common and frequently accompanied by nausea/vomiting. Dizziness disappeared within 1 week during the hospitalization period. Patients with Ramsay Hunt syndrome (31.0%) had dizziness more frequently than those with Bell's palsy (8.7%). In addition, higher hearing thresholds and pain around the ear was reported more often in dizzy patients (p<0.05). Logistic regression analysis revealed that the initial House-Brackmann grade of facial paralysis was solely associated with final recovery, but dizziness was not associated with prognosis. CONCLUSIONS: Patients with APFP may have transient dizziness in the early stage, which may be more frequently accompanied by worse hearing thresholds and/or pain around the ear. However, these symptoms including dizziness seem to be unrelated to final prognosis.

Susceptibility of Diabetic Mice to Noise Trauma.

Diabetes can lead to many end-organ complications. However, the association between diabetes and hearing loss is not well understood. Here, we investigated the effect of noise exposure on diabetic mice compared with wild-type mice. Hearing threshold shifts, histopathologic changes in the cochlea, and inflammatory responses were evaluated over time. After noise exposure, more severe hearing threshold shifts, auditory hair cell loss, and synaptopathies were notable in diabetic mice compared with wild-type mice. Moreover, increased inflammatory responses and reactive oxygen species production were observed in the ears of diabetic mice. The results demonstrated that diabetic mice are more susceptible to noise trauma.

Effects of dexamethasone on intracochlear inflammation and residual hearing after cochleostomy: A comparison of administration routes.

Preservation of residual hearing after cochlear implant is an important issue with regards to hearing performance. Various methods of steroid administration have been widely used in clinical practice to reduce inflammation and preserve residual hearing. Here we compare the effect of different routes of dexamethasone administration on intracochlear inflammation and residual hearing in guinea pig ears. Dexamethasone was delivered into the guinea pigs either through intracochlear, intratympanic or systemic route. The intracochlear concentration of dexamethasone, residual hearing, inflammatory cytokines and histopathologic changes were evaluated over time. A higher intracochlear dexamethasone concentration was observed after intracochlear administration than through the other routes. Residual hearing was better preserved with local dexamethasone administration as was supported by the reduced inflammatory cytokines, more hair cell survival and less severe intracochlear fibrosis and ossification concurrently seen in the local delivery group than in the systemic group. The results demonstrate that local dexamethasone delivery can reduce intracochlear inflammation and preserve residual hearing better than in systemically administered dexamethasone.

Characteristics of patients with acute peripheral facial palsy showing Varicella zoster virus DNA in saliva.

Infection caused by Varicella zoster virus (VZV) is a common etiology of acute peripheral facial palsy (APFP). We aimed to assess the clinical significance of detecting VZV DNA from the saliva of patients with APFP. Saliva collected from 36 patients with unilateral APFP at initial visit was analyzed to detect VZV DNA by polymerase chain reaction. The House-Brackmann (HB) scale was used to evaluate FP severity on admission day, at week 2, and at weeks 10-12 after start of treatment. Among the 28 patients without rash, VZV DNA was detected in 3 patients (10.7%); 6 of 8 patients (75.0%) with rash showed VZV DNA (P = 0.001). VZV DNA-positive patients had worse hearing than VZV DNA-negative patients (P < 0.05). A significantly higher proportion of VZV DNA-negative patients (96.3%) showed complete recovery than VZV DNA-positive patients (55.6%) (P = 0.006). In conclusion, VZV DNA-positive patients had worse hearing and incomplete recovery compared to VZV DNA-negative patients, irrespective of the presence of rash. Tests for the detection of VZV DNA in saliva may be helpful for early differential diagnosis and choosing an appropriate medical treatment of APFP.

Phlorofucofuroeckol Improves Glutamate-Induced Neurotoxicity through Modulation of Oxidative Stress-Mediated Mitochondrial Dysfunction in PC12 Cells.

Stroke is a complex neurodegenerative disorder with a clinically high prevalence and mortality. Despite many efforts to protect against ischemic stroke, its incidence and related permanent disabilities continue to increase. In this study, we found that pretreatment with phlorofucofuroeckol (PFF), isolated from brown algae species, significantly increased cell viability in glutamate-stimulated PC12 cells. Additionally, glutamate-stimulated cells showed irregular morphology, but PFF pretreatment resulted in improved cell morphology, which resembled that in cells cultured under normal conditions. We further showed that PFF pretreatment effectively inhibited glutamate-induced apoptotic cell death in a caspase-dependent manner. Reactive oxygen species (ROS) induced by oxidative stress are closely associated with ischemia-induced neurological diseases. Exposure of PC12 cells to glutamate induced abundant production of intracellular ROS and mitochondrial dysfunction, which was attenuated by PFF in a dose-dependent manner. In vivo studies revealed that PFF-mediated prevention was achieved predominantly through inhibition of apoptosis and mitochondrial ROS generation. Taken together, these results suggest the possibility of PFF as a neuroprotective agent in ischemic stroke.

Lateral geniculate body evoked potentials elicited by visual and electrical stimulation.

PURPOSE: Blind individuals who have photoreceptor loss are known to perceive phosphenes with electrical stimulation of their remaining retinal ganglion cells. We proposed that implantable lateral geniculate body (LGB) stimulus electrode arrays could be used to generate phosphene vision. We attempted to refine the basic reference of the electrical evoked potentials (EEPs) elicited by microelectrical stimulations of the optic nerve, optic tract and LGB of a domestic pig, and then compared it to visual evoked potentials (VEPs) elicited by short-flash stimuli. METHODS: For visual function measurement, VEPs in response to short-flash stimuli on the left eye of the domestic pig were assessed over the visual cortex at position Oz with the reference electrode at Fz. After anesthesia, linearly configured platinum wire electrodes were inserted into the optic nerve, optic track and LGB. To determine the optimal stimulus current, EEPs were recorded repeatedly with controlling the pulse and power. The threshold of current and charge density to elicit EEPs at 0.3 ms pulse duration was about ±10 µA. RESULTS: Our experimental results showed that visual cortex activity can be effectively evoked by stimulation of the optic nerve, optic tract and LGB using penetrating electrodes. The latency of P1 was more shortened as the electrical stimulation was closer to LGB. The EEPs of two-channel in the visual cortex demonstrated a similar pattern with stimulation of different spots of the stimulating electrodes. We found that the LGB-stimulated EEP pattern was very similar to the simultaneously generated VEP on the control side, although implicit time deferred. CONCLUSIONS: EEPs and VEPs derived from visual-system stimulation were compared. The LGB-stimulated EEP wave demonstrated a similar pattern to the VEP waveform except implicit time, indicating prosthetic-based electrical stimulation of the LGB could be utilized for the blind to perceive vision of phosphenes.