Comprehensive Behavioral Analysis of Opsin 3 (Encephalopsin)-Deficient Mice Identifies Role in Modulation of Acoustic Startle Reflex.

Opsin-3 (Opn3, encephalopsin) was the first nonvisual opsin gene discovered in mammals. Since then, several Opn3 functions have been described, and in two cases (adipose tissue, smooth muscle) light sensing activity is implicated. In addition to peripheral tissues, Opn3 is robustly expressed within the central nervous system, for which it derives its name. Despite this expression, no studies have investigated developmental or adult CNS consequences of Opn3 loss-of-function. Here, the behavioral consequences of mice deficient in Opn3 were investigated. Opn3-deficient mice perform comparably to wild-type mice in measures of motor coordination, socialization, anxiety-like behavior, and various aspects of learning and memory. However, Opn3-deficient mice have an attenuated acoustic startle reflex (ASR) relative to littermates. This deficit is not because of changes in hearing sensitivity, although Opn3 was shown to be expressed in auditory and vestibular structures, including cochlear outer hair cells. Interestingly, the ASR was not acutely light-dependent and did not vary between daytime and nighttime trials, despite known functions of Opn3 in photoreception and circadian gene amplitude. Together, these results demonstrate the first role of Opn3 on behavior, although the role of this opsin in the CNS remains largely elusive.

Role of cochlear synaptopathy in cytomegalovirus infected mice and in children.

OBJECTIVES: Determine whether a murine model of cytomegalovirus (CMV) and CMV- infected children show evidence of synaptopathy. STUDY DESIGN: Murine model of CMV infection and case series. SUBJECTS AND METHODS: C57 BL/6 mice were inoculated with murine-CMV (mCMV). Auditory function was assessed using Auditory Brainstem Response (ABR) and distortion product otoacoustic emission (DPOAE) testing. Temporal bones from mCMV-infected mice were used for both ribbon synapse and hair cell quantification. Four groups of children (non-CMV normal hearing, non-CMV hearing impaired, CMV normal hearing and CMV hearing impaired) underwent ABRs between 2014 and 2018. The outcomes included raw amplitude, wave I:V amplitude ratio, absolute latency, and interpeak latency. RESULTS: Mice at 8 weeks post mCMV infection had higher ABR and DPOAE (P < 0.05) thresholds and increased outer hair cell loss compared to uninfected mice and mCMV-infected mice at 4 and 6 weeks post infection, indicating progressive hearing loss. A reduction in the wave I amplitude and synaptic counts were noted earlier at 4 weeks in CMV-infected mice (P < 0.05). The human data indicated that the wave I:V amplitude ratio was lower on average in CMV-infected groups when compared to the uninfected cohorts. The wave I:V amplitude ratio for the click and 4k stimuli were not significantly different between the congenital CMV-infected and uninfected children with normal or with hearing loss. CONCLUSION: This study suggests mCMV infection results in a synaptopathy before hair cell damage. Additional studies need to be performed to determine whether this effect is also observed in CMV-infected children. LEVEL OF EVIDENCE: Animal studies and basic science- NA; human studies: level 4.

Oncolytic virotherapy: a potential therapeutic approach for cholesteatoma.

PURPOSE OF REVIEW: To review the principles of oncolytic virotherapy and summarize the recent preliminary evidence on the efficacy of oncolytic virotherapy for cholesteatoma (CHST) treatment in vitro in human CHST cells and in a gerbil CHST model. RECENT FINDINGS: The use of oncolytic virotherapy for nonmalignant lesions is innovative. In-vitro results showed that oncolytic herpes simplex virus 1 (oHSV) selectively targets and kills CHST cells. In a gerbil model of CHST, local oHSV injections were associated with a decrease in CHST volume and modulation of bony changes. SUMMARY: Surgical treatment options for CHST are limited by high morbidity and recidivism, emphasizing the need for developing treatment alternatives. Preliminary results support the potential therapeutic effect of oncolytic virotherapy on CHST, yet further research is needed to evaluate this novel approach.

Epidermal growth factor receptor deficiency: Expanding the phenotype beyond infancy.

Transmembrane tyrosine kinase receptors represent a fundamental mechanism for transducing extracellular signals into the activation of signaling cascades responsible for intercellular communication, embryogenesis and tissue integrity. The epidermal growth factor receptor (EGFR) is a canonical member of this family, regarded for its dysregulated function in various malignancies. Here, we describe a young female born prematurely with friable and immature skin who developed chronic diarrhea, recurrent gastrointestinal and respiratory infections, as well as an ichthyotic and inflammatory papulopustular rash accompanied by alopecia. Whole-exome sequencing revealed a constitutional homozygous variant in EGFR (NM_005228.3:c1283G>A; p.[G428D]), identified as a pathogenic loss-of-function variant in three patients with EGFR deficiency. These patients succumbed to early mortality; however, the proposita's condition has stabilized, despite only supportive interventions, with dermatological improvements and reduced frequency of infections at 8 years. This report provides a clinical phenotyping of the longest surviving individual with EGFR deficiency and substantiates our understanding of the natural history of this multisystemic dermatological disorder.

Engineered oncolytic virus for the treatment of cholesteatoma: A pilot in vivo study.

OBJECTIVE: Determine if oncolytic herpes simplex virus (oHSV) can eradicate cholesteatoma (CHST) in a gerbil model. METHODS: An in vivo model of CHST was developed in Mongolian gerbils by combining Pseudomonas aeruginosa inoculation with double ligation of the external auditory canal (EAC). CHST size and bone thickness were measured using morphometric and volumetric quantification techniques via micro-computed tomography (micro-CT). The CHST induction and quantification techniques were then used in an additional group of 10 gerbils (n = 20 ears) to determine the within-group treatment efficacy of oHSV against CHST in vivo. Treated animals received either one, two, or three intrabullar injections of oHSV between 2 and 6 weeks postinduction of CHST. RESULTS: The P. aeruginosa inoculation plus double EAC ligation technique successfully induced a range of CHST growth in 100% of the ears in the model-development group. Osteolytic effects of CHST were observed in 6% of ears whereas osteoblastic effects were observed in 31% of ears. CHST volume decreased by 50% or more in 12 of the 20 ears in the oHSV-treatment groups. An apparent reversal of osteoblastic effects was also observed in three out of four ears 6 weeks following the third oHSV injection. CONCLUSIONS: P. aeruginosa inoculation plus double EAC ligation reliably induces CHST formation in gerbil. CT-based volumetric measures are significantly more accurate than single-slice morphometric area measures for quantification of CHST size. Treatment with oHSV appears to be efficacious for reducing CHST volume by as much as 77% with as few as one treatment. LEVEL OF EVIDENCE: NA.

Auditory system dysfunction in Brazilian gasoline station workers.

Objective: To examine the auditory system of Brazilian gasoline station workers using an extensive audiological test battery. Design: This was a cross-sectional study. The audiological evaluation included a questionnaire, pure-tone audiometry, acoustic immittance tests, transient-evoked otoacoustic emissions (TEOAEs), distortion product otoacoustic emissions (DPOAEs), auditory brainstem response (ABR) and P300 auditory-evoked potentials. Study sample: A total of 77 Brazilian gasoline station workers were evaluated, and their results were compared with those of 36 participants who were not exposed to chemicals or noise at work. The gasoline station employees worked in 18 different gas stations, and the noise area measurements from all gas stations revealed time-weighted averages below 85 dBA. Results: Of the 77 gasoline station workers evaluated, 67.5% had audiometric results within the normal range, but 59.7% reported difficulties in communication in noisy places. Gasoline station workers showed significantly poorer results than non-exposed control participants in one or more conditions of each of the audiological tests used, except P300. Conclusions: The results suggest that the gasoline station workers have both peripheral and central auditory dysfunctions that could be partly explained by their exposure to gasoline.

Evidence for Oncolytic Viral Eradication of Cholesteatoma In Vitro.

Cholesteatomas (CHSTs) are congenital or acquired lesions of the temporal bone that are associated with significant morbidity. We hypothesized that an oncolytic herpes simplex virus (oHSV) could preferentially eradicate primary human CHST cells in vitro and that this virus will replicate selectively and efficiently in CHST cells when compared with control cells. In this work, primary human CHST cells were cultured from surgically collected tissue. Cholesteatomas and control cells were grown and infected by oncolytic oHSV. More than 80% CHST cells versus <5% control cells were killed by oHSV. The oHSV showed a significant enhanced cytotoxic effect against CHST cells in a time- and dose-dependent manner. Therefore, this novel therapy has promise as a future treatment to minimize the spread and recurrence of CHST.

Use of noise cancellation earphones in out-of-booth audiometric evaluations.

OBJECTIVE: To assess the utility of noise cancelation earphones (NCE) in audiometric evaluations. DESIGN: Degree of noise reduction of Bose QuietComfort 15 NCE was assessed through probe-microphone measures and sound-field audiometry. Occlusion effects from NCE were assessed for potential effects on bone-conduction thresholds. STUDY SAMPLE: Twenty participants were tested to determine average occlusion effect values during bone-conduction testing with and without NCE. Noise reduction values of the NCE were assessed on a single subject through probe-microphone measures and sound-field testing. RESULTS: NCE sufficiently reduced ambient noise to levels acceptable for air-conduction testing as well as for bone-conduction testing for most patients when adding minimal adjustment to acceptable levels as outlined by the ANSI S3.1-1999 standard. In addition, NCE did not create a clinically significant change in the occlusion effect for bone-conduction testing. CONCLUSION: NCE placed over insert earphones provide a sound pressure level at the tympanic membrane that is below ANSI standards for routine air-conduction testing and result in sufficient ambient noise reduction for bone-conduction testing with most patients. There is no clinically significant occlusion effect from NCE during routine bone-conduction audiometry. These findings support the utility of using NCE for offsite audiometric testing.

Predicting the location of missing outer hair cells using the electrical signal recorded at the round window.

The electrical signal recorded at the round window was used to estimate the location of missing outer hair cells. The cochlear response was recorded to a low frequency tone embedded in high-pass filtered noise conditions. Cochlear damage was created by either overexposure to frequency-specific tones or laser light. In animals with continuous damage along the partition, the amplitude of the cochlear response increased as the high-pass cutoff frequency increased, eventually reaching a plateau. The cochlear distance at the onset of the plateau correlated with the anatomical onset of outer hair cell loss. A mathematical model replicated the physiologic data but was limited to cases with continuous hair cell loss in the middle and basal turns. The neural contribution to the cochlear response was determined by recording the response before and after application of Ouabain. Application of Ouabain eliminated or reduced auditory neural activity from approximately two turns of the cochlea. The amplitude of the cochlear response was reduced for moderate signal levels with a limited effect at higher levels, indicating that the cochlear response was dominated by outer hair cell currents at high signal levels and neural potentials at low to moderate signal levels.

Analysis of the cochlear microphonic to a low-frequency tone embedded in filtered noise.

The cochlear microphonic was recorded in response to a 733 Hz tone embedded in noise that was high-pass filtered at 25 different frequencies. The amplitude of the cochlear microphonic increased as the high-pass cutoff frequency of the noise increased. The amplitude growth for a 60 dB SPL tone was steeper and saturated sooner than that of an 80 dB SPL tone. The growth for both signal levels, however, was not entirely cumulative with plateaus occurring at about 4 and 7 mm from the apex. A phenomenological model of the electrical potential in the cochlea that included a hair cell probability function and spiral geometry of the cochlea could account for both the slope of the growth functions and the plateau regions. This suggests that with high-pass-filtered noise, the cochlear microphonic recorded at the round window comes from the electric field generated at the source directed towards the electrode and not down the longitudinal axis of the cochlea.

Mapping auditory nerve firing density using high-level compound action potentials and high-pass noise masking.

Future implementation of regenerative treatments for sensorineural hearing loss may be hindered by the lack of diagnostic tools that specify the target(s) within the cochlea and auditory nerve for delivery of therapeutic agents. Recent research has indicated that the amplitude of high-level compound action potentials (CAPs) is a good predictor of overall auditory nerve survival, but does not pinpoint the location of neural damage. A location-specific estimate of nerve pathology may be possible by using a masking paradigm and high-level CAPs to map auditory nerve firing density throughout the cochlea. This initial study in gerbil utilized a high-pass masking paradigm to determine normative ranges for CAP-derived neural firing density functions using broadband chirp stimuli and low-frequency tonebursts, and to determine if cochlear outer hair cell (OHC) pathology alters the distribution of neural firing in the cochlea. Neural firing distributions for moderate-intensity (60 dB pSPL) chirps were affected by OHC pathology whereas those derived with high-level (90 dB pSPL) chirps were not. These results suggest that CAP-derived neural firing distributions for high-level chirps may provide an estimate of auditory nerve survival that is independent of OHC pathology.

Predicting auditory nerve survival using the compound action potential.

OBJECTIVE: Advances in cochlear hair-cell regeneration, neural regeneration, and genetic therapy encourage continued development of diagnostic tests that can accurately specify the appropriate target within the cochlea and auditory nerve for delivery of therapeutic agents. In this study, we test the hypothesis that the morphology of the acoustically evoked compound action potential (CAP) may reflect the condition of the auditory nerve in individuals with sensorineural hearing loss. DESIGN: CAPs to tone burst stimuli at octave frequencies from 1 to 16 kHz were recorded at low- to high-stimulus levels from sedated Mongolian gerbils with partial lesions of the auditory nerve (n = 10). Distortion-product otoacoustic emissions were measured to ensure preservation of normal outer hair-cell function. CAPs were analyzed with conventional measures of N1 latency and amplitude and by fitting the CAPs with a mathematical model that includes a parameter (N) representing the number of nerve fibers contributing to the CAP and a parameter (f) representing the oscillation frequency of the CAP waveform. Nerve fiber density and percent normal nerve area were estimated from cross-sections of the auditory nerve bundle. RESULTS: Despite substantial lesions in the auditory nerve, CAP thresholds remained within normal or were only moderately elevated and were not correlated with histological measures of nerve fiber density and normal nerve area. At high-stimulus levels, the model parameter N was strongly correlated with nerve fiber density for three of the five test frequencies and with normal nerve area for all five test frequencies. Correlations between N1 amplitude measures at high-stimulus levels and our histological measures were also significant for the majority of test frequencies, but they were generally weaker than the correlations for the model parameter N. The model parameter f, at low- and high-stimulus levels, was also positively correlated with measures of normal nerve area. CONCLUSIONS: Consistent with previous findings, physiological measures of threshold were not correlated with partial lesions of the auditory nerve. The model parameter N at high-stimulus levels was strongly correlated with normal nerve area suggesting, that it is a good predictor of auditory nerve survival. The model parameter N also seemed to be a better predictor of the condition of the auditory nerve than the conventional measure of N1 amplitude. Because the model parameter f was correlated with normal nerve area at low- and high-stimulus levels, it may provide information on the functional status of the auditory nerve.

Comparison of transient evoked otoacoustic emissions and distortion product otoacoustic emissions when screening hearing in preschool children in a community setting.

OBJECTIVE: The first purpose of this study was to compare transient evoked otoacoustic emissions (TEOAE) with distortion product otoacoustic emissions (DPOAE) to determine if they resulted in equivalent signal-to-noise ratios (SNRs) when used for hearing screening in a preschool population in a community setting. The second purpose was to determine if the OAE methods would result in equivalent pass/refer rates. The third purpose was to determine the agreement between the pass/refer rates from a tympanometric screening and the pass/refer rates from each OAE method. METHODS: Thirty-three preschool children ages 4 months to 4 years, 4 months were tested using DPOAE and TEOAE. The frequencies 800-4000Hz were compared. The tympanometric gradient was obtained from a tympanogram done on each ear. A multivariate statistic was used to compare the emission SNR from both methods. A chi(2) statistic was used to compare the pass/refer rates from both methods. The agreement between the pass/refer rates from the OAE screens and from the tympanometric gradient were compared. RESULTS: TEOAE and DPOAE SNRs were significantly different in the low frequency however, there were no significant differences found in the high frequencies. There were no significant pass/refer differences found between the methods at any frequency. When comparing the agreement between the OAE methods with the tympanometry, both methods produced nearly equivalent agreement with tympanometric gradient. However, the overall correspondence between OAE findings and tympanometry was not perfect. CONCLUSIONS: Both methods are effective and especially equivalent in the high frequencies and can be recommended for use in a preschool population in the field. Tympanometric gradient disagreed with both OAE screening results about 25% of the time. Finally, our study also found that higher refer rates can be expected when young (<3 years) preschool children are included in the screen.