Research Application of Laser-induced Shock Wave for Studying Blast-induced Cochlear Injury.

The ear is the organ most susceptible to explosion overpressure, and cochlear injuries frequently occur after blast exposure. Blast exposure can lead to sensorineural hearing loss (SNHL), which is an irreversible hearing loss that negatively affects the quality of life. Detailed blast-induced cochlear pathologies, such as the loss of hair cells, spiral ganglion neurons, cochlear synapses, and disruption of stereocilia, have been previously documented. However, determining cochlear sensorineural deterioration after a blast injury is challenging because animals exposed to blast overpressure usually experience tympanic membrane perforation (TMP), which causes concurrent conductive hearing loss. To evaluate pure sensorineural cochlear dysfunction, we developed an experimental animal model of blast-induced cochlear injury using a laser-induced shock wave. This method avoids TMP and concomitant systemic injuries and reproduces the functional decline in the SNHL component in an energy-dependent manner after LISW exposure. This animal model could be a platform for elucidating the pathological mechanisms and exploring potential treatments for blast-induced cochlear dysfunction.

Behavioral and Histopathological Impairments Caused by Topical Exposure of the Rat Brain to Mild-Impulse Laser-Induced Shock Waves: Impulse Dependency.

Although an enormous number of animal studies on blast-induced traumatic brain injury (bTBI) have been conducted, there still remain many uncertain issues in its neuropathology and mechanisms. This is partially due to the complex and hence difficult experimental environment settings, e.g., to minimize the effects of blast winds (tertiary mechanism) and to separate the effects of brain exposure and torso exposure. Since a laser-induced shock wave (LISW) is free from dynamic pressure and its energy is spatially well confined, the effects of pure shock wave exposure (primary mechanism) solely on the brain can be examined by using an LISW. In this study, we applied a set of four LISWs in the impulse range of 15-71 Pa·s to the rat brain through the intact scalp and skull; the interval between each exposure was ~5 s. For the rats, we conducted locomotor activity, elevated plus maze and forced swimming tests. Axonal injury in the brain was also examined by histological analysis using Bodian silver staining. Only the rats with exposure at higher impulses of 54 and 71 Pa·s showed significantly lower spontaneous movements at 1 and 2 days post-exposure by the locomotor activity test, but after 3 days post-exposure, they had recovered. At 7 days post-exposure, however, these rats (54 and 71 Pa·s) showed significantly higher levels of anxiety-related and depression-like behaviors by the elevated plus maze test and forced swimming test, respectively. To the best of the authors' knowledge, there have been few studies in which a rat model showed both anxiety-related and depression-like behaviors caused by blast or shock wave exposure. At that time point (7 days post-exposure), histological analysis showed significant decreases in axonal density in the cingulum bundle and corpus callosum in impulse-dependent manners; axons in the cingulum bundle were found to be more affected by a shock wave. Correlation analysis showed a statistically significant correlation between the depression like-behavior and axonal density reduction in the cingulum bundle. The results demonstrated the dependence of behavior deficits and axonal injury on the shock wave impulse loaded on the brain.

Specificity of tuberculin skin test improved by BCG immunization schedule change in Japan.

INTRODUCTION: Tuberculin skin test (TST) has been used to diagnose tuberculosis (TB) and latent tuberculosis infection (LTBI). However, in Bacillus Calmette-Guérin (BCG) vaccinated patients, TST tends to produce false-positive results. According to the previous vaccination schedule, Japanese people were mandated to receive up to three doses of BCG-vaccine. The vaccination schedule was changed in 2003 and as per the new schedule, only infants are administered a dose of BCG vaccine. Our hypothesis is that this change can lead to a reduction in the cross-reaction to TST. METHODS: We evaluated the TST results obtained from 1097 recruits from six defense camps and 667 recruits from an air base. These TST data were divided into two groups according to the date of birth: a new group and an old group according to the BCG immunization schedule. We then analyzed positive and negative reaction of TST and erythema sizes. RESULTS: We confirmed that the change in BCG-vaccination schedule significantly decreased TST false-positive reaction (Pmeta = 1.4 × 10-18; risk ratio = 0.83; 95% confidence interval: 0.80-0.87) and erythema size (Pmeta = 1.1 × 10-4; mean difference = 6.6 mm; 95% confidence interval: 3.2 mm-9.9 mm). CONCLUSIONS: We showed the reduction in BCG cross-reaction to TST, in the new BCG vaccination schedule group, compared to the old group, we also have extracted information on the improvement in the specificity of TST for LTBI and TB diagnosis, which resulted from BCG schedule change.

Tinnitus rat model generated by laser-induced shock wave; a platform for analyzing the central nervous system after tinnitus generation.

OBJECTIVE: Tinnitus is a phantom auditory sensation, which is mainly triggered by dysfunction of the peripheral auditory organ, such as cochlear disorders. Additionally, the central nervous system, specifically the limbic system, plays a crucial role in the generation and exacerbation of tinnitus. Therefore, to analyze the hypothesis that tinnitus has strong and specific association with the plastic changes in the limbic system, we assessed the neuronal plastic changes in the limbic system, including the hippocampus and the amygdala, in rats with single-sided tinnitus. METHODS: The cochlear damage was achieved by irradiating the cochlea with laser-induced shock wave (LISW). While both hearing loss and tinnitus were confirmed after exposure of rats to LISW, the degree of tinnitus was objectively measured using gap detection behavioral tests. Following the generation of hearing loss and tinnitus, plastic changes in the neurons of the limbic system were confirmed using a molecular marker (activity regulated cytoskeleton-associated protein; Arc). RESULTS: While the expression level of Arc-positive cells in the hippocampal CA1 showed an obvious increase in the hearing loss and tinnitus groups, a significant difference was found between the tinnitus and the control groups. In the dentate gyrus, although the largest number of Arc-positive cells was observed in the tinnitus group, there were no significant differences between the numbers of cells in the hearing loss and tinnitus groups compared to that in the control group. CONCLUSION: Although a significant increase of Arc-positive cells in the hippocampal CA1 was observed between the tinnitus group and control, no obvious tendencies of Arc-positive cells in the limbic system were observed between the rats with and without tinnitus behavior.

Pathophysiology of the inner ear after blast injury caused by laser-induced shock wave.

The ear is the organ that is most sensitive to blast overpressure, and ear damage is most frequently seen after blast exposure. Blast overpressure to the ear results in sensorineural hearing loss, which is untreatable and is often associated with a decline in the quality of life. In this study, we used a rat model to demonstrate the pathophysiological and structural changes in the inner ear that replicate pure sensorineural hearing loss associated with blast injury using laser-induced shock wave (LISW) without any conductive hearing loss. Our results indicate that threshold elevation of the auditory brainstem response (ABR) after blast exposure was primarily caused by outer hair cell dysfunction induced by stereociliary bundle disruption. The bundle disruption pattern was unique; disturbed stereocilia were mostly observed in the outermost row, whereas those in the inner and middle rows stereocilia remained intact. In addition, the ABR examination showed a reduction in wave I amplitude without elevation of the threshold in the lower energy exposure group. This phenomenon was caused by loss of the synaptic ribbon. This type of hearing dysfunction has recently been described as hidden hearing loss caused by cochlear neuropathy, which is associated with tinnitus or hyperacusis.

The beneficial effect of Hangesha-shin-to (TJ-014) in gentamicin-induced hair cell loss in the rat cochlea.

OBJECTIVE: Ototoxic damage caused by aminoglycosides (AG) leads to the loss of cochlear hair cells (HCs). In mammals, mature cochlear HCs are unable to regenerate, and their loss results in permanent hearing deficits. Our objective was to protect the inner ear from damage after an AG challenge. The generation of reactive oxygen species (ROS), one of the earliest events in the process of AG ototoxicity, is considered to play a key role in the initiation of HC death. We examined whether Hangesha-shin-to (TJ-014), a traditional Japanese Kampo medicine considered to be a potent antioxidant, protects HCs from gentamicin (GM)-induced damage. METHODS: Organ of Corti explants removed from postnatal day 3-5 rats were maintained in tissue culture and exposed to 50µM GM for up to 48h. The effects of TJ-014 on GM-induced ototoxicity were assessed by HC counts and immunohistochemistry against cleaved caspase-3, 8-hydroxy-2'-deoxyguanosine (8-OHdG), and a probe reacting to mitochondrial function changes. RESULTS: TJ-014 treatments significantly reduced GM-induced HC loss and immunoreactivities for cleaved caspase-3 and 8-OHdG; these effects were correlated with increasing TJ-014 concentrations. Moreover, TJ-014 protected the mitochondrial membrane potential from GM ototoxicity. CONCLUSION: These findings indicate the potential of TJ-014 to prevent GM-induced cochlear damage involving ROS.

ERK2 mediates inner hair cell survival and decreases susceptibility to noise-induced hearing loss.

Extracellular signal-regulated kinase (ERK) is a member of the family of mitogen-activated protein kinases (MAPKs) and coordinately regulates a multitude of cellular processes. In response to a variety of extracellular stimuli, phosphorylation of both threonine and tyrosine residues activates ERK. Recent evidence indicates that ERK is activated in response to cellular stress such as acoustic trauma. However, the specific role of ERK isoforms in auditory function is not fully understood. Here, we show that the isoform ERK2 plays an important role in regulating hair cell (HC) survival and noise-induced hearing loss (NIHL) in mice (C57BL/6J). We found that conditional knockout mice deficient for Erk2 in the inner ear HCs had hearing comparable to control mice and exhibited no HC loss under normal conditions. However, we found that these knockout mice were more vulnerable to noise and had blunted recovery from NIHL compared to control mice. Furthermore, we observed a significantly lower survival rate of inner hair cells in these mice compared to control mice. Our results indicate that ERK2 plays important roles in the survival of HC in NIHL.

Low-level laser therapy for prevention of noise-induced hearing loss in rats.

Noninvasive low-level laser therapy (LLLT) is neuroprotective, but the mechanism of this effect is not fully understood. In this study, the use of LLLT as a novel treatment for noise-induced hearing loss (NIHL) is investigated. Sprague-Dawley rats were exposed to intense noise and their right ears were irradiated with an 808nm diode laser at an output power density of 110 or 165mW/cm(2) for a 30min period for 5 consecutive days. Measurement of the auditory brainstem response revealed an accelerated recovery of auditory function in the groups treated with LLLT compared with the non-treatment group at days 2, 4, 7 and 14 after noise exposure. Morphological observations also revealed a significantly higher outer hair cell survival rate in the LLLT groups. Immunohistochemical analyses for inducible nitric oxide synthase (iNOS) and cleaved caspase-3 were used to examine oxidative stress and apoptosis. Strong immunoreactivities were observed in the inner ear tissues of the non-treatment group, whereas these signals were decreased in the LLLT group at 165mW/cm(2) power density. Our findings suggest that LLLT has cytoprotective effects against NIHL via the inhibition of iNOS expression and apoptosis.

Characteristics of laser-induced shock wave injury to the inner ear of rats.

Recently, the number of blast injuries of the inner ear has increased in the general population. In blast-induced inner ear injury, a shock wave (SW) component in the blast wave is considered to play an important role in sensorineural hearing loss. However, the mechanisms by which an SW affects inner ear tissue remain largely unknown. We aimed to establish a new animal model for SW-induced inner ear injury by using laser-induced SWs (LISWs) on rats. The LISWs were generated by irradiating an elastic laser target with 694-nm nanosecond pulses of a ruby laser. After LISW application to the cochlea through bone conduction, auditory measurements revealed the presence of inner ear dysfunction, the extent of which depended on LISW overpressure. A significantly lower survival rate of hair cells and spiral ganglion neurons, as well as severe oxidative damage, were observed in the inner ear exposed to an LISW. Although considerable differences in the pressure characteristics exist between LISWs and SWs in real blast waves, the functional and morphological changes shown by the present LISW-based model were similar to those observed in real blast-induced injury. Thus, our animal model is expected to be useful for laboratory-based research of blast-induced inner ear injury.

Inhaled hydrogen gas therapy for prevention of noise-induced hearing loss through reducing reactive oxygen species.

Reactive oxygen species (ROS) that form in the inner ear play an important role in noise-induced hearing loss (NIHL). Recent studies have revealed that molecular hydrogen (H2) has great potential for reducing ROS. In this study, we examined the potential of hydrogen gas to protect against NIHL. We tested this hypothesis in guinea pigs with 0.5%, 1.0% and 1.5% H2 inhalation in air for 5h a day after noise exposure, for five consecutive days. All animals underwent measurements for auditory brainstem response after the noise exposure; the results revealed that there was a better improvement in the threshold shift for the 1.0% and 1.5% H2-treated groups than the non-treated group. Furthermore, outer hair cell (OHC) loss was examined 7 days after noise exposure. A significantly higher survival rate of OHCs was observed in the 1.0% and 1.5% H2-treated group as compared to that of the non-treated group in the basal turn. Immunohistochemical analyses for 8-hydroxy-2'-deoxyguanosine (8-OHdG) were performed to examine the amount of oxidative DNA damage. While strong immunoreactivities against 8-OHdG were observed of the non-treated group, the H2-treated group showed decreased immunoreactivity for 8-OHdG. These findings strongly suggest that inhaled hydrogen gas protects against NIHL.

Activated protein C rescues the cochlea from noise-induced hearing loss.

Activated protein C (APC) is a serine/threonine protease and a physiological anticoagulant that exerts anti-inflammatory and anti-apoptotic effects. Although recent studies have revealed that APC has the potential to protect endothelial cells from apoptosis, the mechanisms of its cytoprotective effect are not fully understood. We examined the potential of APC to protect against noise-induced hearing loss (NIHL) and investigated phosphorylation of serine-threonine kinase (Akt) and inhibition of apoptosis as possible cytoprotective mechanisms. We administered intraperitoneal injections of APC (150, 300 U/kg) or normal saline to rats 30 min before exposure to a sound pressure level (SPL) of 126 dB and 4-kHz octave band noise for 5h. The auditory brainstem response (ABR) and distortion product otoacoustic emissions (DPOAE) were measured before and after exposure. ABR and DPOAE measurements revealed greater improvement in the APC group than in the control group 28 days after exposure. Our examination of outer hair cells (OHCs) at 28 days after noise exposure revealed a significantly higher OHC survival rate in the APC group than in the control group. Immunohistochemical analyses for cleaved-caspase 3, phospho-p38 (p-p38), TUNEL, and phospho-Akt (p-Akt) revealed strong immunoreactivities against cleaved-caspase 3, p-p38, and TUNEL in the inner ear tissues of the control group; however, these signals were decreased in the APC group. Moreover, APC significantly induced activation of p-Akt in the cochlea. These findings suggest that APC has a novel protective effect on the cochlea against NIHL that is mediated by p-Akt and the anti-apoptotic signaling pathway.

Minimally invasive surgery of sialolithiasis using sialendoscopy.

OBJECTIVE: The introduction of minimally invasive surgical procedures using sialendoscopy has significantly reduced the rate of major salivary gland removal due to sialolithiasis. The present study assessed the utility of sialendoscopy and identified potential factors influencing successful sialendoscopic salivary stone retrieval. METHODS: Medical records of sialendoscopic procedures performed at the Department of Otolaryngology of the National Defense Medical College in Japan from November 2007 to January 2014 were retrospectively reviewed. We identified 78 patients diagnosed with sialolithiasis and treated with sialendoscopy (SE). Factors analyzed included stone location, size, symptom duration, surgical methods, and complications. RESULTS: The mean age at presentation was 41 years (range, 11-76 years) with a male-to-female gender ratio of 1:1.89. In total, 73 submandibular and 5 parotid endoscopies were performed. Stone size ranged from 2 to 20mm in diameter. Submandibular stones were removed either by SE alone (9.6%), by transoral stone removal (19.2%), or a combined approach (57.5%). Only 13.7% (10/73) of the cases required submandibular gland removal. Stone size and shape were significant predictors for successful endoscopic stone removal, and stone size and location were significant predictors for submandibular gland removal. CONCLUSION: Sialendoscopy is a reasonable, minimally invasive treatment option for sialolithiasis that avoids salivary gland removal. The present results indicate that sialendoscopy is the first treatment of choice for submandibular gland sialolithiasis. Complete surgical excision is becoming uncommon as a first-line treatment, but it remains indispensable in certain cases.

Comparison of voice quality after laser cordectomy with that after radiotherapy or chemoradiotherapy for early glottic carcinoma.

PURPOSE OF THE STUDY: The present study aimed to compare the voice quality after each type of cordectomy with that after radiotherapy (RT) or chemoradiotherapy (CRT) for early glottic carcinoma. PROCEDURES: The GRBAS perceptive scale [consisting of 5 domains: grade (G), roughness (R), breathiness (B), asthenia (A), and strain (S)], aerodynamic tests and acoustic analyses, and the Voice Handicap Index questionnaire were evaluated for 58 laser cordectomy cases and 40 RT or CRT cases. Multiple comparison tests were conducted between each type of cordectomy and RT or CRT. RESULTS: No statistical difference was found between type I/II cordectomy and RT for T1 glottic carcinoma (T1RT), whereas T1RT showed a significantly better outcome than type III cordectomy (G score: p = 0.016, maximum phonation time: p < 0.01, mean flow rate: p < 0.01). Type IV cordectomy was equivalent to RT or CRT for T2 glottic carcinoma (T2RT/CRT), while types V and VI showed a worse voice quality than T2RT/CRT (G score: p = 0.038 to type VI, B score: p = 0.025 to type V and p = 0.032 to type VI, A score: p = 0.017 to type VI). CONCLUSIONS: Voice quality after laser cordectomy differs according to the type of cordectomy. Surgeons should inform patients about the expected voice quality after each treatment modality.

Ecotoxicity by the biodegradation of alkylphenol polyethoxylates depends on the effect of trace elements.

The bacteria Sphingomonas sp. strain BSN22, isolated from bean fields, degraded octylphenol polyethoxylates (OPEO(n)) to octylphenol (OP) under aerobic conditions. This biodegradation mechanism proceeded by the following two-step degradation process: (1) degradation of OPEO(n) to octylphenol triethoxylate (OPEO(3)), (2) degradation from OPEO(3) to OP via octylphenoxy acetic acid (OPEC(1)). The chemical structure of OPEC(1) was confirmed by analysis using (18)O-labeled water. Quantitative studies revealed that magnesium (Mg(2+)) and calcium (Ca(2+)) ions were essential for the biodegradation of OPEO(n). Furthermore, the rate of biodegradation was especially accelerated by ferric ions (Fe(3+)), and the accumulated amounts of endocrine active chemicals, such as OP, OPEO(1), and OPEC(1), significantly increased to the concentration of 22.8, 221.7, and 961.1 microM in the presence of 37.0 microM Fe(3+), respectively. This suggests that environmental elements significantly influence the resultant ecotoxicity as well as the rate of their biodegradation in the environment. This study on the mechanism of OPEO(n) biodegradation may play an important role in understanding and managing environmental safety, including drinking water safety.