Correction: Song et al. Protective Effect of Resveratrol in an Experimental Model of Salicylate-Induced Tinnitus. Int. J. Mol. Sci. 2022, 23, 14183.

In the original publication [...].

Protective Effect of Resveratrol in an Experimental Model of Salicylate-Induced Tinnitus.

To date, the effect of resveratrol on tinnitus has not been reported. The attenuative effects of resveratrol (RSV) on a salicylate-induced tinnitus model were evaluated by in vitro and in vivo experiments. The gene expression of the activity-regulated cytoskeleton-associated protein (ARC), tumor necrosis factor-alpha (TNFα), and NMDA receptor subunit 2B (NR2B) in SH-SY5Y cells was examined using qPCR. Phosphorylated cAMP response element-binding protein (p-CREB), apoptosis markers, and reactive oxygen species (ROS) were evaluated by in vitro experiments. The in vivo experiment evaluated the gap-prepulse inhibition of the acoustic startle reflex (GPIAS) and auditory brainstem response (ABR) level. The NR2B expression in the auditory cortex (AC) was determined by immunohistochemistry. RSV significantly reduced the salicylate-induced expression of NR2B, ARC, and TNFα in neuronal cells; the GPIAS and ABR thresholds altered by salicylate in rats were recovered close to their normal range. RSV also reduced the salicylate-induced NR2B overexpression of the AC. These results confirmed that resveratrol exerted an attenuative effect on salicylate-induced tinnitus and may have a therapeutic potential.

Quantitative measurement of optical properties and Hb concentration in a rodent model of inflammatory Meibomian gland dysfunction using spatial frequency domain imaging.

Herein, to investigate a new diagnostic method for Meibomian gland dysfunction (MGD) induced by eyelid inflammation, optical properties and deoxy-hemoglobin (Hb) concentrations in rodent eyelid tissues, including Meibomian glands(MGs), were measured using spatial frequency domain imaging (SFDI). Complete Freund's adjuvant solutions were injected into the eyelid margins of Sprague-Dawley rats to induce MGD. After three weeks, the optical properties and Hb of the MG and non-MG regions of the eyelids were measured ex-vivo using an SFDI system. The comparison of Hb showed that the MGD group exhibited significantly higher values than those of the control group in both regions. The optical properties at 730 and 850 nm for the MG regions in the MGD group were significantly different from those in the control group. In addition, the 630 nm absorption coefficients of both regions were significantly higher in the MGD group than in the control group. Thus, the SFDI technique can detect the increased Hb concentration and changes in the optical properties of the eyelids due to inflammatory MGD in a noncontact manner and has the potential to be used as a novel quantitative diagnostic method for the occurrence of MGD.

The application of SFDI and LSI system to evaluate the blood perfusion in skin flap mouse model.

The aim of this study is to evaluate whether the blood perfusion to tissues for detecting ischemic necrosis can be quantitatively monitored by spatial frequency domain imaging (SFDI) and laser speckle imaging (LSI) in a skin flap mouse model. Skin flaps were made on Institute of Cancer Research (ICR) mice. Using SFDI and LSI, the following parameters were estimated: oxyhemoglobin (HbO2), deoxyhemoglobin (Hb), total hemoglobin (THb), tissue oxygen saturation (StO2), and speckle flow index (SFI). Histologically, epithelium thickness, collagen deposition, and blood vessel count of skin flap tissues were analyzed. Then, the correlation of SFDI and histological results was assessed by application of Spearman rank correlation method. As the result, the number of blood vessels and the percentage of collagen areas showed significant difference between the necrotic tissue group and the non-necrotic one. Especially, the necrotic tissue had a complete epithelial loss and loses its normal structure. We identified that SFDI/LSI parameters were significantly different between non-necrotic and necrotic tissue groups. Especially, all SFDI and LSI parameters measured on the 1st day after surgery showed significant difference between necrotic tissue and non-necrotic tissue. In addition, the number of blood vessel and percentage of collagen area were positively correlated with HbO2 and StO2 among SFDI/LSI parameters. Meanwhile, the number of blood vessel and percentage of collagen area showed the negative correlation with Hb. By applying SFDI and LSI simultaneously to the skin flap, we could quantitatively monitor the blood perfusion and the tissue condition which can help us to detect ischemic necrosis objectively in early stage.

Evidence of Tinnitus Development Due to Stress: An Experimental Study in Rats.

OBJECTIVES/HYPOTHESIS: Tinnitus can develop due to, or be aggravated by, stress in a rat model. To investigate stress as a possible causal factor in the development of tinnitus, we designed an animal study that included tinnitus behavior and excitatory/inhibitory neurotransmitter expression after noise exposure as well as restraint stress. STUDY DESIGN: An experimental animal study. METHODS: Wistar rats were grouped according to single or double exposure to noise and restraint stress. The noise exposure (NE) group was subjected to 110 dB sound pressure level (SPL) of 16 kHz narrow-band noise (NBN) for 1 hour, and the restraint stress (RS) group was restrained for 1 hour with or without noise exposure. Gap prepulse inhibition of the acoustic startle (GPIAS) reflex was measured at an NBN of 16 kHz to investigate tinnitus development. Various immunohistopathologic and molecular biologic studies were undertaken to evaluate possible mechanisms of tinnitus development after noise and/or restraint stress. RESULTS: The RS-only group showed a reduced GPIAS response, which is a reliable sign of tinnitus development. In the double-stimulus groups, more tinnitus-development signs of reduced GPIAS responses were observed. The expression of γ-aminobutyric acid A receptor α1 (GABAAR α1) in the hippocampus decreased in the NE│RS group. Increased N-methyl-d-aspartate receptor1 intensities in the NE│RS group and decreased GABAAR α1 intensities in the RS and NE│RS groups were observed in the CA3 region of the hippocampus. CONCLUSIONS: Tinnitus appeared to develop after stress alone in this animal study. An imbalance in excitatory and inhibitory neurotransmitters in the hippocampus may be related to the development of tinnitus after acute NE and/or stress. LEVEL OF EVIDENCE: NA Laryngoscope, 131:2332-2340, 2021.

Role of microRNA­375­3p­mediated regulation in tinnitus development.

Changes in the dorsal cochlear nucleus (DCN) following exposure to noise play an important role in the development of tinnitus. As the development of several diseases is known to be associated with microRNAs (miRNAs/miRs), the aim of the present study was to identify the miRNAs that may be implicated in pathogenic changes in the DCN, resulting in tinnitus. A previously developed tinnitus animal model was used for this study. The study consisted of four stages, including identification of candidate miRNAs involved in tinnitus development using miRNA microarray analysis, validation of miRNA expression using reverse transcription­quantitative PCR (RT­qPCR), evaluation of the effects of candidate miRNA overexpression on tinnitus development through injection of a candidate miRNA mimic or mimic negative control, and target prediction of candidate miRNAs using mRNA microarray analysis and western blotting. The miRNA microarray and RT­qPCR analyses revealed that miR­375­3p expression was significantly reduced in the tinnitus group compared with that in the non­tinnitus group. Additionally, miR­375­3p overexpression via injection of miR­375­3p mimic reduced the proportion of animals with persistent tinnitus. Based on mRNA microarray and western blot analyses, connective tissue growth factor (CTGF) was identified as a potential target for miR­375­3p. Thus, it was inferred that CTGF downregulation by miR­375­3p may weaken with the decrease in miRNA expression, and the increased pro­apoptotic activity of CTGF may result in more severe neuronal damage, contributing to tinnitus development. These findings are expected to contribute significantly to the development of a novel therapeutic approach to tinnitus, thereby bringing about a significant breakthrough in the treatment of this potentially debilitating condition.

Neuroprotective Effect of Valproic Acid on Salicylate-Induced Tinnitus.

High-dose salicylate induces temporary moderate hearing loss and the perception of a high-pitched tinnitus in humans and animals. Previous studies demonstrated that high doses of salicylate increase N-methyl-d-aspartate (NMDA) receptor levels, resulting in a rise in Ca2+ influx and induction of excitotoxicity. Glutamate excitotoxicity is associated with failure in the maintenance of calcium homeostasis, mitochondrial dysfunction, and production of reactive oxygen species (ROS). Valproic acid (VPA) is widely used for the management of bipolar disorder, epilepsy, and migraine headaches, and is known to regulate NMDA receptor activity. In this study, we examined the beneficial effects of VPA in a salicylate-induced tinnitus model in vitro and in vivo. Cells were pretreated with VPA followed by salicylate treatment. The expression levels of NMDA receptor subunit NR2B, phosphorylated cAMP response element-binding protein-an apoptosis marker, and intracellular levels of ROS were measured using several biochemical techniques. We observed increased expression of NR2B and its related genes TNFα and ARC, increased intracellular ROS levels, and induced expression of cleaved caspase-3. These salicylate-induced changes were attenuated in the neuronal cell line SH-SY5Y and rat cortical neurons after VPA pretreatment. Together, these results provide evidence of the beneficial effects of VPA in a salicylate-induced temporary hearing loss and tinnitus model.

A Gap Prepulse with a Principal Stimulus Yields a Combined Auditory Late Response.

BACKGROUND AND OBJECTIVES: The gap prepulse inhibition of the acoustic startle response has been used to screen tinnitus in an animal model. Here, we examined changes in the auditory late response under various conditions of gap prepulse inhibition. SUBJECTS AND METHODS: We recruited 19 healthy adults (5 males, 14 females) and their auditory late responses were recorded after various stimuli with or without gap prepulsing. The N1 and P2 responses were selected for analysis. The gap prepulse inhibition was estimated to determine the optimal auditory late response in the gap prepulse paradigm. RESULTS: We found that the gap per se generated a response that was very similar to the response elicited by sound stimuli. This critically affected the gap associated with the maximal inhibition of the stimulus response. Among the various gap-stimulus intervals (GSIs) between the gap and principal stimulus, the GSI of 150 ms maximally inhibited the response. However, after zero padding was used to minimize artifacts after a P2 response to a gap stimulus, the differences among the GSIs disappeared. CONCLUSIONS: Overall, the data suggest that both the prepulse inhibition and the gap per se should be considered when using the gap prepulse paradigm to assess tinnitus in humans.

Axonal sprouting in the dorsal cochlear nucleus affects gap­prepulse inhibition following noise exposure.

One of the primary theories of the pathogenesis of tinnitus involves maladaptive auditory­somatosensory plasticity in the dorsal cochlear nucleus (DCN), which is assumed to be due to axonal sprouting. Although a disrupted balance between auditory and somatosensory inputs may occur following hearing damage and may induce tinnitus, examination of this phenomenon employed a model of hearing damage that does not account for the causal relationship between these changes and tinnitus. The present study aimed to investigate changes in auditory­somatosensory innervation and the role that axonal sprouting serves in this process by comparing results between animals with and without tinnitus. Rats were exposed to a noise­inducing temporary threshold shift and were subsequently divided into tinnitus and non­tinnitus groups based on the results of gap prepulse inhibition of the acoustic startle reflex. DCNs were collected from rats divided into three sub­groups according to the number of weeks (1, 2 or 3) following noise exposure, and the protein levels of vesicular glutamate transporter 1 (VGLUT1), which is associated with auditory input to the DCN, and VGLUT2, which is in turn primarily associated with somatosensory inputs, were assessed. In addition, factors related to axonal sprouting, including growth­associated protein 43 (GAP43), postsynaptic density protein 95, synaptophysin, α­thalassemia/mental retardation syndrome X­linked homolog (ATRX), growth differentiation factor 10 (GDF10), and leucine­rich repeat and immunoglobulin domain­containing 1, were measured by western blot analyses. Compared to the non­tinnitus group, the tinnitus group exhibited a significant decrease in VGLUT1 at 1 week and a significant increase in VGLUT2 at 3 weeks post­exposure. In addition, rats in the tinnitus group exhibited significant increases in GAP43 and GDF10 protein expression levels in their DCN at 3 weeks following noise exposure. Results from the present study provided further evidence that changes in the neural input distribution to the DCN may cause tinnitus and that axonal sprouting underlies these alterations.

Proposal of conditional random inter-stimulus interval method for unconstrained enclosure based GPIAS measurement systems.

Gap prepulse inhibition of acoustic startle (GPIAS) method has been used effectively for the objective assessment of tinnitus in animals. Among two types of enclosures for the GPIAS, the unconstrained type carries less risk of animal death due to the absence of binding stress in the enclosure, and lack of need for alteration to animal size variation as it grows. However, animals' voluntary movements, which have no relation to the startles evoked by acoustic stimuli, are problematic, as they cannot be excluded in the case of the unconstrained enclosure based GPIAS measurement system. In order to discount voluntary movements which are not associated with external acoustic stimuli, we propose the conditional random inter-stimulus interval (CR ISI) method for unconstrained enclosure based GPIAS measurement. With the proposed ISI method, the unconstrained enclosure based acoustic startle response measurement system has been implemented in this paper. As a result, the effectiveness of the proposed CR ISI method has been verified and compared with those of conventional ISI methods through animal experiments using SD-rats. The experimental results showed that abnormal startle responses and invalid GPIAS values caused by motion were prevented when our proposed CR ISI method was applied to our implemented system. It was also verified that our proposed CR ISI method is advantageous in reducing the total experimental time for acquiring normal startle responses and valid GPIAS values, compared to conventional ISI methods, since our proposed CR ISI can begin the acoustic stimulation only when the animal gets stable and motionless.

The relationship between classical item characteristics and item response time on computer-based testing.

PURPOSE: This study investigated the relationship between the item response time (iRT) and classic item analysis indicators obtained from computer-based test (CBT) results and deduce students' problem-solving behavior using the relationship. METHODS: We retrospectively analyzed the results of the Comprehensive Basic Medical Sciences Examination conducted for 5 years by a CBT system in Dankook University College of Medicine. iRT is defined as the time spent to answer the question. The discrimination index and the difficulty level were used to analyze the items using classical test theory (CTT). The relationship of iRT and the CTT were investigated using a correlation analysis. An analysis of variance was performed to identify the difference between iRT and difficulty level. A regression analysis was conducted to examine the effect of the difficulty index and discrimination index on iRT. RESULTS: iRT increases with increasing difficulty index, and iRT tends to decrease with increasing discrimination index. The students' effort is increased when they solve difficult items but reduced when they are confronted with items with a high discrimination. The students' test effort represented by iRT was properly maintained when the items have a 'desirable' difficulty and a 'good' discrimination. CONCLUSION: The results of our study show that an adequate degree of item difficulty and discrimination is required to increase students' motivation. It might be inferred that with the combination of CTT and iRT, we can gain insights about the quality of the examination and test behaviors of the students, which can provide us with more powerful tools to improve them.

Effects of transient auditory deprivation during critical periods on the development of auditory temporal processing.

OBJECTIVES: The central auditory pathway matures through sensory experiences and it is known that sensory experiences during periods called critical periods exert an important influence on brain development. The present study aimed to investigate whether temporary auditory deprivation during critical periods (CPs) could have a detrimental effect on the development of auditory temporal processing. MATERIALS AND METHODS: Twelve neonatal rats were randomly assigned to control and study groups; Study group experienced temporary (18-20 days) auditory deprivation during CPs (Early deprivation study group). Outcome measures included changes in auditory brainstem response (ABR), gap prepulse inhibition of the acoustic startle reflex (GPIAS), and gap detection threshold (GDT). To further delineate the specific role of CPs in the outcome measures above, the same paradigm was applied in adult rats (Late deprivation group) and the findings were compared with those of the neonatal rats. RESULTS: Soon after the restoration of hearing, early deprivation study animals showed a significantly lower GPIAS at intermediate gap durations and a larger GDT than early deprivation controls, but these differences became insignificant after subsequent auditory inputs. Additionally, the ABR results showed significantly delayed latencies of waves IV, V, and interpeak latencies of wave I-III and wave I-V in study group. Late deprivation group didn't exhibit any deterioration in temporal processing following sensory deprivation. CONCLUSION: Taken together, the present results suggest that transient auditory deprivation during CPs might cause reversible disruptions in the development of temporal processing.

Multimodal sensor-based weight drop spinal cord impact system for large animals.

BACKGROUND CONTEXT: A conventional weight drop spinal cord (SC) impact system for large animals is composed of a high-speed video camera, a vision system, and other things. However, a camera with high speed at over 5,000 frames per second (FPS) is very expensive. In addition, the use of the vision system involves complex pattern recognition algorithms and accurate arrangement of the camera and the target. PURPOSE: The purpose of this study was to develop a large animal spinal cord injury (SCI) modeling system using a multimodal sensor instead of a high-speed video camera and vision system. Another objective of this study was to demonstrate the possibility of the developed system to measure the impact parameters in the experiments using different stiffness materials and an in vivo porcine SC. STUDY DESIGN: A multimodal sensor-based SCI impact system was developed for large animals. The experiments to measure SC impact parameters were then performed using three different stiffness materials and a Yucatan miniature pig to verify the performance of system developed. METHODS: A comparative experiment was performed using three different stiffness materials such as high-density (HD) sponge, rubber, and clay to demonstrate the system and perform measurement for impact parameters such as impact velocity, impulsive force, and maximally compressed displacement reflecting physical properties of materials. In the animal experiment, a female Yucatan miniature pig of 60-kg weight was used. Impact conditions for all experiments were fixed at freefalling object mass of 50 g and height of 20 cm. RESULTS: In the impact test, measured impact velocities were almost the same for the three different stiffness materials at 1.84±0.0153 m/s. Impulsive forces for the three materials of rubber, HD sponge, and clay were 50.88 N, 32.35 N, and 6.68 N, respectively. Maximally compressed displacements for rubber, HD sponge, and clay were 1.93 mm, 3.35 mm, and 15.01 mm, respectively. In the pig experiment, impact velocity, impulsive force, and maximally compressed dural displacement were measured at 1.84 m/s, 13.35 N, and 3.04 mm, respectively. After 3 days from the experiment, paralysis was confirmed for the lower half body of the experimental pig. CONCLUSIONS: Through experiments, it was verified that our proposed system could be used to measure the SC impact parameters and induce SCI for large animals.

Compressed single pixel imaging in the spatial frequency domain.

We have developed compressed sensing single pixel spatial frequency domain imaging (cs-SFDI) to characterize tissue optical properties over a wide field of view ( 35 ?? mm × 35 ?? mm ) using multiple near-infrared (NIR) wavelengths simultaneously. Our approach takes advantage of the relatively sparse spatial content required for mapping tissue optical properties at length scales comparable to the transport scattering length in tissue ( l tr ? 1 ?? mm ) and the high bandwidth available for spectral encoding using a single-element detector. cs-SFDI recovered absorption ( ? a ) and reduced scattering ( ? s ? ) coefficients of a tissue phantom at three NIR wavelengths (660, 850, and 940 nm) within 7.6% and 4.3% of absolute values determined using camera-based SFDI, respectively. These results suggest that cs-SFDI can be developed as a multi- and hyperspectral imaging modality for quantitative, dynamic imaging of tissue optical and physiological properties.

Functional mapping of the auditory tract in rodent tinnitus model using manganese-enhanced magnetic resonance imaging.

Animal models of salicylate-induced tinnitus have demonstrated that salicylate modulates neuronal activity in several brain structures leading to neuronal hyperactivity in auditory and non-auditory brain areas. In addition, these animal tinnitus models indicate that tinnitus can be a perceptual consequence of altered spontaneous neural activity along the auditory pathway. Peripheral and/or central effects of salicylate can account for neuronal activity changes in salicylate-induced tinnitus. Because of this ambiguity, an in vivo imaging study would be able to address the peripheral and/or central involvement of salicylate-induced tinnitus. Therefore, in the present study, we developed a novel manganese-enhanced magnetic resonance imaging (MEMRI) method to map the in vivo functional auditory tract in a salicylate-induced tinnitus animal model by administrating manganese through the round window. We found that acute salicylate-induced tinnitus resulted in higher manganese uptake in the cochlea and in the central auditory structures. Furthermore, serial MRI scans demonstrated that the manganese signal increased in an anterograde fashion from the cochlea to the cochlear nucleus. Therefore, our in vivo MEMRI data suggest that acute salicylate-induced tinnitus is associated with higher spontaneous neural activity both in peripheral and central auditory pathways.

Parameter optimization for applying the prepulse gap paradigm to humans.

BACKGROUND AND OBJECTIVES: Turner and colleagues introduced a new method that can detect tinnitus in animals. The stimulus is composed of a small background noise that is identical to the pitch of the tinnitus and a large pulse noise that can evoke a startle response. In normal rats, the gap decreases the startle reflex. However, in tinnitus rats, the gap does not decrease the startle reflex. The goal of this study was to optimize the stimulation paradigm so that the prepulse inhibition of N1-P2 amplitude would be maximized in the normal human subjects. SUBJECTS AND METHODS: Seven normal control subjects without tinnitus were recruited. The stimulus was composed of two different sounds: the softer background noise and the louder pulse noise. A 50 msec silent gap was inserted before the pulse noise as the gap condition (G condition) but not in the no-gap condition (N condition). The averaged amplitude of the N1-P2 cortical response was recorded for the G and N conditions. RESULTS: The G/N ratio was the smallest when the gap was 20 msec prior to the pulse noise. The G/N ratio was 84.8±16.8% with the Hanning window and 78.5±5.9% without the window. The G/N ratio was 91.1±24.9%, 78.0±5.4%, and 79.0±18.1% when the intensity of the background noise was 10, 20, and 32 dB SL, respectively. When the intensity of the background noise was 20 and 32 dB SL, the N1-P2 amplitude of the G condition was significantly smaller than that of the N condition. CONCLUSIONS: The optimal stimulus should be composed of the 1 kHz pulse noise without Hanning window. The intensity of the background noise should be 20 dB HL and the location of the gap should be 20 msec prior to the pulse noise. It seems that with these optimized parameters we could expect a 78.0% inhibition of N1-P2 amplitude in normal subjects without tinnitus.

Comparisons of electromagnetic and piezoelectric floating-mass transducers in human cadaveric temporal bones.

Electromagnetic floating-mass transducers for implantable middle-ear hearing devices (IMEHDs) afford the advantages of a simple surgical implantation procedure and easy attachment to the ossicles. However, their shortcomings include susceptibility to interference from environmental electromagnetic fields, relatively high current consumption, and a limited ability to output high-frequency vibrations. To address these limitations, a piezoelectric floating-mass transducer (PFMT) has recently been developed. This paper presents the results of a comparative study of these two types of vibration transducer developed for IMEHDs. The differential electromagnetic floating-mass transducer (DFMT) and the PFMT were implanted in two different sets of three cadaveric human temporal bones. The resulting stapes displacements were measured and compared on the basis of the ASTM standard for describing the output characteristics of IMEHDs. The experimental results show that the PFMT can produce significantly higher equivalent sound pressure levels above 3 kHz, due to the flat response of the PFMT, than can the DFMT. Thus, it is expected that the PFMT can be utilized to compensate for high-frequency sensorineural hearing loss.

Discovery of Maritrema jebuensis n. sp. (Digenea: Microphallidae) from the Asian Shore Crab, Hemigrapsus sanguineus, in Korea.

Maritrema spp. (Digenea: Microphallidae) are parasites of birds, but have not been found in the Republic of Korea. In this study, metacercariae of Maritrema sp. were discovered in the Asian shore crab, Hemigrapsus sanguineus, caught in the mud-flats of Jebu-do, Hwasung-gun, Gyeonggi-do, and the adult flukes were confirmed by experimental infection into mice. Based on the symmetric ribbon-like vitellarium, adult flukes of Maritrema sp. were identified, but did not belong to previously described species in terms of the following morphologic characteristics: ceca reaching to the lateral wall at the anterior border of the ovary; ventral sucker larger than oral sucker; a prominent metraterm; and vitellarium forming a complete ring. Hence, we named this microphallid M. jebuensis n. sp. after the island where the second intermediate hosts were collected. From this study, it has been shown that Maritrema sp. is distributed in Korea and transmitted by the Asian shore crab, H. sanguineus.

Tegumental ultrastructure of adult Gynaecotyla squatarolae (Digenea: Microphallidae).

Gynaecotyla squatarolae (Digenea: Microphallidae) adult flukes were recovered from experimental chicks at day 4-6 post-infection and their tegumental ultrastructure was observed with a scanning electron microscopy. They were pyriform in shape, and their anterior halves were concaved ventrally. The whole body surface was covered with tegumental spines, which were wide and 16-17 digitated between oral and ventral suckers. The density of spines and number of digits decreased posteriorly. The oral sucker was subterminal and the excretory pore was at the posterior end of the worm. Two ventral suckers were similar in appearance and protruded near midline of the worm. The genital atrium was dextral to the small ventral sucker. The dorsal surface was covered with tegumental spines, but the spines were sparser than on the ventral surface. On the middle portion of the dorsal surface, a small opening presumed to be the Laurer's canal was seen. From these findings, it has been confirmed that the adult G. squatarolae has unique characteristics in the surface ultrastructure.