Mitigation of Hearing Damage With Liraglutide Treatment in Chinchillas After Repeated Blast Exposures at Mild-TBI.

INTRODUCTION: Although hearing protection devices (HPDs) have been widely used during training and combat, over one million veterans experience service-connected hearing loss. Hearing damage has been reported to be associated with blast-induced mild traumatic brain injury (mTBI) and there is a lack of understanding and treatment. Liraglutide is a glucagon-like peptide-1 receptor agonist and a potential treatment for TBI-induced memory deficits. This study aims to investigate the function of the liraglutide to prevent damage and facilitate hearing restoration in chinchillas exposed to multiple high-intensity, mTBI-level blasts. MATERIALS AND METHODS: Chinchillas were divided into three treatment groups: blast control, pre-blast drug treatment, and post-blast drug treatment. On day 1, the chinchilla ears were protected by HPDs and exposed to three blasts with peak pressure levels of 15-25 psi. The auditory brainstem response (ABR), distortion product otoacoustic emission (DPOAE), and middle latency response (MLR) were recorded pre- and post-blast on day 1 and on days 4, 7, 14, and 28. RESULTS: Substantial acute damage was observed and progressively recovered in chinchillas after the blast exposures. The pre-blast treatment group exhibited the lowest elevation of the ABR threshold and reduction of the wave I amplitude on day 1 after blasts. The liraglutide treatment insignificantly facilitated the recovery of the DPOAE levels and ABR thresholds on days 14 and 28. The pre-blast treatment chinchillas showed reduced MLR amplitudes on days 4 and 7. CONCLUSIONS: This study indicated that the pre-blast liraglutide administration provided damage protection against blasts in addition to the HPDs. Current evidence suggests that the effect of liraglutide is more prominent in the early phase of the experiment.

3D Finite Element Model of Human Ear with 3-Chamber Spiral Cochlea for Blast Wave Transmission from the Ear Canal to Cochlea.

Blast-induced auditory trauma is a common injury in military service members and veterans that leads to hearing loss. While the inner ear response to blast exposure is difficult to characterize experimentally, computational models have advanced to predict blast wave transmission from the ear canal to the cochlea; however, published models have either straight or spiral cochlea with fluid-filled two chambers. In this paper, we report the recently developed 3D finite element (FE) model of the human ear mimicking the anatomical structure of the 3-chambered cochlea. The model consists of the ear canal, middle ear, and two and a half turns of the cochlea with three chambers separated by the Reissner's membrane (RM) and the basilar membrane (BM). The blast overpressure measured from human temporal bone experiments was applied at the ear canal entrance and the Fluent/Mechanical coupled fluid-structure interaction analysis was conducted in ANSYS software. The FE model-derived results include the pressure in the canal near the tympanic membrane (TM) and the intracochlear pressure at scala vestibuli, the TM displacement, and the stapes footplate (SFP) displacement, which were compared with experimentally measured data in human temporal bones. The validated model was used to predict the biomechanical response of the ear to blast overpressure: distributions of the maximum strain and stress within the TM, the BM displacement variation from the base to apex, and the energy flux or total energy entering the cochlea. The comparison of intracochlear pressure and BM displacement with those from the FE model of 2-chambered cochlea indicated that the 3-chamber cochlea model with the RM and scala media chamber improved our understanding of cochlea mechanics. This most comprehensive FE model of the human ear has shown its capability to predict the middle ear and cochlea responses to blast overpressure which will advance our understanding of auditory blast injury.

Real-time measurement of stapes motion and intracochlear pressure during blast exposure.

Blast-induced auditory injury is primarily caused by exposure to an overwhelming amount of energy transmitted into the external auditory canal, the middle ear, and then the cochlea. Quantification of this energy requires real-time measurement of stapes footplate (SFP) motion and intracochlear pressure in the scala vestibuli (Psv). To date, SFP and Psv have not been measured simultaneously during blast exposure, but a dual-laser experimental approach for detecting the movement of the SFP was reported by Jiang et al. (2021). In this study, we have incorporated the measurement of Psv with SFP motion and developed a novel approach to quantitatively measure the energy flux entering the cochlea during blast exposure. Five fresh human cadaveric temporal bones (TBs) were used in this study. A mastoidectomy and facial recess approach were performed to identify the SFP, followed by a cochleostomy into the scala vestibuli (SV). The TB was mounted to the "head block", a fixture to simulate a real human skull, with two pressure sensors - one inserted into the SV (Psv) and another in the ear canal near the tympanic membrane (P1). The TB was exposed to the blast overpressure (P0) around 4 psi or 28 kPa. Two laser Doppler vibrometers (LDVs) were used to measure the movements of the SFP and TB (as a reference). The LDVs, P1, and Psv signals were triggered by P0 and recorded simultaneously. The results include peak values for Psv of 100.8 ± 51.6 kPa (mean ± SD) and for SFP displacement of 72.6 ± 56.4 µm, which are consistent with published experimental results and finite element modeling data. Most of the P0 input energy flux into the cochlea occurred within 2 ms and resulted in 10-70 µJ total energy entering the cochlea. Although the middle ear pressure gain was close to that measured under acoustic stimulus conditions, the nonlinear behavior of the middle ear was observed from the elevated cochlear input impedance. For the first time, SFP movement and intracochlear pressure Psv have been successfully measured simultaneously during blast exposure. This study provides a new methodology and experimental data for determining the energy flux entering the cochlea during a blast, which serves as an injury index for quantifying blast-induced auditory damage.

Hearing protection and damage mitigation in Chinchillas exposed to repeated low-intensity blasts.

Repeated exposures to blast overpressure (BOP) introduce hearing complaints in military service members even with the use of hearing protection devices (HPDs). Although epidemiology and animal studies have been performed to investigate the damage formation mechanism of blast-induced hearing damage, there is still a lack of understanding and therapeutic solutions, especially for HPD-protected ears. Recent studies revealed the potential therapeutic function of liraglutide, a glucagon-like peptide-1 receptor agonist, to facilitate post-blast hearing restoration in chinchillas. This study is a continuation and summary of the previous studies performed by Jiang et al. (2021, 2022) to investigate the damage mitigation function of liraglutide treatment in chinchillas with open and protected ears after repeated low-intensity blast exposures within 28 days of observation. Chinchillas were divided into six experimental groups: pre-blast treatment, post-blast treatment, and blast control with ears open or protected by earplug (EP). All animals were exposed to six consecutive blasts at the level of 3-5 psi (21-35 kPa) on Day 1. Hearing function tests including auditory brainstem response (ABR), distortion product otoacoustic emission (DPOAE), and middle latency response (MLR) were performed on Day 1 (pre- and post-blast) and Days 4, 7, 14, and 28 after blast exposure. Results indicated that the damage mitigation function of the liraglutide treatment in the open-ear chinchillas was reflected by the significantly lower ABR threshold shifts in the drug treatment groups than in the blast controls. In EP groups, the higher ABR wave I/V ratio and lower MLR amplitude observed in the drug-treated chinchillas suggested that the post-blast hyperactivities in the auditory system might be potentially ameliorated by the liraglutide treatment. The 28-day-long experiment showed the effect of liraglutide treatment increased with time in both open and EP groups. This study demonstrated that the use of HPDs prevented the blast-induced complications in the middle ear and reduced the damage caused in the central auditory system. The liraglutide treatment showed an effect increasing with time and different outcomes in open and EP chinchillas. This innovation in the animal model of chinchilla provides insights to investigate subtle changes in the higher-level structures of the auditory system.

Mitigation of Hearing Damage After Repeated Blast Exposures in Animal Model of Chinchilla.

High-intensity sound or blast-induced hearing impairment is a common injury for Service members. Epidemiology studies revealed that the blast-induced hearing loss is associated with the traumatic brain injury (TBI), but the mechanisms of the formation and prevention of auditory injuries require further investigation. Liraglutide, a glucagon-like peptide-1 receptor (GLP-1R) agonist, has been reported as a potential treatment strategy for TBI-caused memory deficits; however, there is no study on therapeutics of GLP-1R for blast-induced hearing damage. This paper reports our current study on progressive hearing damage after repeated exposures to low-level blasts in the animal model of chinchilla and the mitigation of hearing damage using liraglutide. Chinchillas were divided into three groups (N = 7 each): blast control, pre-blast treatment, and post-blast treatment. All animals were exposed to six consecutive blasts at the level of 3-5 psi (21-35 kPa) on Day 1. The auditory brainstem response (ABR) was measured on Day 1 (pre- and post-blast) and Days 4, 7, and 14 after blast exposure. Upon the completion of the experiment on Day 14, the brain tissues of animals were harvested for immunofluorescence studies. Significant damage was revealed in blast-exposed chinchillas by increased ABR thresholds, decreased ABR wave I amplitudes, and cell apoptosis in the inferior colliculus in the blast control chinchillas. Treatment with liraglutide appeared to reduce the severity of blast-induced hearing injuries as observed from the drug-treated chinchillas comparing to the blast controls. This study bridges the gap between TBI and hearing impairment and suggests a possible intervention for blast-induced hearing loss for Service members.

A 3D Printed Human Ear Model for Standardized Testing of Hearing Protection Devices to Blast Exposure.

Hypothesis: A 3D printed human temporal bone (TB) that is anatomically accurate would cost-effectively reproduce the responses observed in blast testing of human cadaveric TBs with and without passive hearing protection devices (HPDs). Background: HPDs have become critical personal protection equipment against auditory damage for service members. Acoustic test fixtures and human TBs have been used to test and develop HPDs; however, the lack of a cost-effective, standardized model impedes the improvement of HPDs. Methods: In this study, the 3D printed TB model was printed with flexible and rigid polymers and consisted of the ear canal, tympanic membrane (TM), ossicular chain, middle ear suspensory ligaments/muscle tendons, and middle ear cavity. The TM movement under acoustic stimulation was measured with laser Doppler vibrometry. The TB model was then exposed to blasts with or without HPDs and pressures at the ear canal entrance (P0) and near the TM in the ear canal (P1) were recorded. All results were compared with that measured in human TBs. Results: Results indicated that in the 3D printed TB, the attenuated peak pressures at P1 induced by HPDs ranged from 0.92 to 1.06 psi (170-171 dB) with blast peak pressures of 5.62-6.54 psi (186-187 dB) at P0, and measured results were within the mean and SD of published data. Vibrometry measurements also followed a similar trend as the published results. Conclusions: The 3D printed TB model accurately evaluated passive HPDs' protective function during blast and the potential for use as a model for acoustic transmission was investigated.

Central and peripheral auditory abnormalities in chinchilla animal model of blast-injury.

Exposure to blast overpressure or high-intensity sound can cause injuries to the auditory system, which leads to hearing loss or tinnitus. In this study, we examined the involvement of peripheral auditory system (PAS), and central auditory system (CAS) changes after exposure to blast overpressure (15-25 psi) on Day 1 and additionally during 7 days of post blast time period in chinchillas. Auditory brainstem response (ABR), distortion product otoacoustic emission (DPOAE), and cochlear hair cell changes were measured or identified in post-blast period within 7 days to detect injuries in the PAS. In the CAS, changes in NMDAR1 (excitatory receptor) and GABAA (inhibitory receptor) as well as changes in serotonin (5-HT2A) and acetylcholine (AChR) receptors were examined in different brain regions: auditory cortex (AC), geniculate body (GB), inferior colliculus (IC) and amygdala by immunofluorescence staining. We observed the PAS abnormalities of increased ABR threshold and decreased DPOAE response in animals after blast exposure with hearing protection devices (e.g., earplug). Blast exposure also caused a reduction in both NMDAR1 and GABAA receptor levels in acute condition (post-blast or Day 1) in AC and IC, while serotonin and acetylcholine receptor levels displayed a biphasic response at Day 1 and Day 7 post-exposure. Results demonstrate that the earplug can protect the tympanic membrane and middle ear against structural damage, but the hearing level, cochlear outer hair cell, and the central auditory system (levels of excitatory and inhibitory neurotransmitter receptors) were only partially protected at the tested blast overpressure level. The findings in this study indicate that blast exposure can cause both peripheral and central auditory dysfunctions, and the central auditory response is independent of peripheral auditory damage. The CAS dysfunction is likely mediated by direct transmission of shockwaves in all the regions of central nervous system (CNS), including nerves and surrounding tissues along the auditory pathways. Hence, targeting central auditory neurotransmitter abnormalities may have a therapeutic benefit to attenuate blast-induced hearing loss and tinnitus.

Dual-laser measurement of human stapes footplate motion under blast exposure.

Hearing damage is one of the most frequently observed injuries in Service members and Veterans even though hearing protection devices (HPDs, e.g. earplugs) have been implemented to prevent blast-induced hearing loss. However, the formation and prevention mechanism of the blast-induced hearing damage remains unclear due to the difficulty for conducting biomechanical measurements in ears during blast exposure. Recently, an approach reported by Jiang et al. (2019) used two laser Doppler vibrometers (LDVs) to measure the motion of the tympanic membrane (TM) in human temporal bones during blast exposure. Using the dual laser setup, we further developed the technology to detect the movement of the stapes footplate (SFP) in ears with and without HPDs while under blast exposure. Eight fresh human cadaveric temporal bones (TBs) were involved in this study. The TB was mounted in a "head block" after performing a facial recess surgery to access the SFP, and a pressure sensor was inserted near the TM in the ear canal to measure the pressure reaching the TM (P1). The TB was exposed to a blast overpressure measuring around 7 psi or 48 kPa at the entrance of the ear canal (P0). Two LDVs were used to measure the vibrations of the SFP and TB (as a reference). The exact motion of the SFP was determined by subtracting the TB motion from the SFP data. Results included a measured peak-to-peak SFP displacement of 68.7 ± 31.6 µm (mean ± SD) from all eight TBs without HPDs. In five of the TBs, the insertion of a foam earplug reduced the SFP displacement from 48.3 ± 6.3 µm to 21.8 ± 10.4 µm. The time-frequency analysis of the SFP velocity signals indicated that most of the energy spectrum was concentrated at frequencies below 4 kHz within the first 2 ms after blast and the energy was reduced after the insertion of HPDs. This study describes a new methodology to quantitatively characterize the response of the middle ear and the energy entering the cochlea during blast exposure. The experimental data are critical for determining the injury of the peripheral auditory system and elucidating the damage formation and prevention mechanism in an ear exposed to blast.

Prevention of Blast-induced Auditory Injury Using 3D Printed Helmet and Hearing Protection Device - A Preliminary Study on Biomechanical Modeling and Animal.

INTRODUCTION: Repeated blast exposures result in structural damage to the peripheral auditory system (PAS) and the central auditory system (CAS). However, it is difficult to differentiate injuries between two distinct pathways: the mechanical damage in the PAS caused by blast pressure waves transmitted through the ear and the damage in the CAS caused by blast wave impacts on the head or traumatic brain injury. This article reports a preliminary study using a 3D printed chinchilla "helmet" as a head protection device associated with the hearing protection devices (e.g., earplugs) to isolate the CAS damage from the PAS injuries under repeated blast exposures. MATERIALS AND METHODS: A finite element (FE) model of the chinchilla helmet was created based on micro-computed tomography images of a chinchilla skull and inputted into ANSYS for FE analysis on the helmet's protection against blast over pressure. The helmet was then 3D printed and used for animal experiments. Chinchillas were divided into four cases (ears open, with earplug only, with both earplug and helmet, and with helmet only) and exposed to three blasts at blast over pressure of 15 to 20 psi. Hearing function tests (e.g., auditory brainstem response) were performed before and after blast on Day 1 and Days 4 and 7 after blasts. RESULTS: The FE model simulation showed a significant reduction in intracranial stress with the helmet, and the animal results indicated that both earplug and helmet reduced the severity of blast-induced auditory injuries by approximately 20 dB but with different mechanisms. CONCLUSIONS: The biomechanical modeling and animal experiments demonstrated that this four-case study in chinchillas with helmet and hearing protection devices provides a novel methodology to investigate the blast-induced damage in the PAS and CAS.

Surface Motion Changes of Tympanic Membrane Damaged by Blast Waves.

Eardrum or tympanic membrane (TM) is a multilayer soft tissue membrane located at the end of the ear canal to receive sound pressure and transport the sound into the middle ear and cochlea. Recent studies reported that the TM microstructure and mechanical properties varied after the ear was exposed to blast overpressure. However, the impact of such biomechanical changes of the TM on its movement for sound transmission has not been investigated. This paper reports the full-field surface motion of the human TM using the scanning laser Doppler vibrometry in human temporal bones under normal and postblast conditions. An increase of the TM displacement after blast exposure was observed in the posterior region of the TM in four temporal bone samples at the frequencies between 3 and 4 kHz. A finite element model of human TM with multilayer microstructure and orthogonal fiber network was created to simulate the TM damaged by blast waves. The consistency between the experimental data and the model-derived TM surface motion suggests that the tissue injuries were resulted from a combination of mechanical property change and regional discontinuity of collagen fibers. This study provides the evidences of surface motion changes of the TM damaged by blast waves and possible fiber damage locations.

Progressive hearing damage after exposure to repeated low-intensity blasts in chinchillas.

Hearing damage caused by blast waves is a frequent and common injury for Service members. However, most studies have focused on high-intensity blast exposures that are known to cause moderate to severe traumatic brain injury (TBI), and fewer studies have investigated the progressive hearing damage caused by low-intensity blast exposures (below mild TBI). In this paper, we report our recent study in chinchillas to investigate the auditory function changes over the time course after repetitive exposures to low-intensity blast. Two groups of chinchillas (N = 7 each) were used in this study. Group 1 was for an acute study with 2 blasts and Group 2 for progressive study with 3 blasts on Day 1 and observed for 7 days. Animals in both groups were exposed to blast overpressures of 21-35 kPa (3-5 psi or 180-185 dB SPL) at which the eardrum was usually not ruptured. One ear was left open while another ear was protected with an earplug. Blast overpressures were monitored at the entrance of the ear canal (P0) and near the eardrum in the canal (P1). Auditory brainstem responses (ABRs), distortion product otoacoustic emissions (DPOAEs), and middle latency responses (MLRs) were measured after each blast series in the acute group and on Days 1, 4, and 7 in the progressive group. Results show that hearing damage was induced in both ears after blast exposure on Day 1 and more damage was observed in open ears than plugged ears. Seven days after the three-blast series, the ABR threshold in open ears was still 7-20 dB higher on average than prior to the blasts. The MLR wave amplitude shifts were observed in both open and protected ears, which indicated central auditory damage. With the protection of an earplug, hearing thresholds had recovered to the pre-blast level by Day 7. Using this chinchilla blast model, acute and progressive hearing damages were quantified in both open and protected ears following repeated low-intensity blast exposures.

Dual-laser measurement and finite element modeling of human tympanic membrane motion under blast exposure.

Hearing damage is one of most prevalent injuries in military personnel and civilians exposed to a blast. However, the mechanism of how the blast overpressure interacts with the tympanic membrane (TM) and impairs the peripheral auditory system still remains unclear. A 3D finite element (FE) model of the human ear has been developed to predict the blast overpressure transmission through the ear (Leckness et al., 2018), but the model needs to be further validated in TM response to blast pressure. This paper reports the first-ever approach using two laser Doppler vibrometers (LDVs) to measure the motion of the TM when the ear was exposed to a blast. Five fresh human temporal bones were used in this study with a pressure sensor inserted near the TM to measure the pressure reaching the TM (P1). The temporal bone was mounted in a "head block" and exposed to blast at the overpressure around 35 kPa measured at the entrance of the ear canal (P0). The movements of the TM at the umbo and the "head block" were measured simultaneously by two LDVs and the exact motion of the TM was determined by subtracting the head block motion from the TM data. Results include that the maximum TM velocity was 12.62 ±â€¯3.63 m/s (mean ±â€¯SD) and the displacement was 0.78 ±â€¯0.26 mm. The peak-to-peak displacement normalized by the P0 pressure was 22.9 ±â€¯6.6 µm/kPa. The frequency domain analysis indicated that the spectrum peaks were located at frequencies below 3 kHz. The TM motion was then compared with that calculated from the FE model of the human ear with the measured P0 pressure wave applied at the ear canal entrance. The FE model-derived TM displacement under blast overpressure was consistent with the experimental results. This study provides a new methodology to determine the behavior of the middle ear in response to blast overpressure. The experimental data are critical for validating the FE model of the human ear for blast wave transduction and understanding the TM damage induced by blast exposure.

Dynamic properties of human incudostapedial joint-Experimental measurement and finite element modeling.

The incudostapedial joint (ISJ) is a synovial joint connecting the incus and stapes in the middle ear. Mechanical properties of the ISJ directly affect sound transmission from the tympanic membrane to the cochlea. However, how ISJ properties change with frequency has not been investigated. In this paper, we report the dynamic properties of the human ISJ measured in eight samples using a dynamic mechanical analyzer (DMA) for frequencies from 1 to 80 Hz at three temperatures of 5, 25 and 37 °C. The frequency-temperature superposition (FTS) principle was used to extrapolate the results to 8 kHz. The complex modulus of ISJ was measured with a mean storage modulus of 1.14 MPa at 1 Hz that increased to 3.01 MPa at 8 kHz, and a loss modulus that increased from 0.07 to 0.47 MPa. A 3-dimensional finite element (FE) model consisting of the articular cartilage, joint capsule and synovial fluid was then constructed to derive mechanical properties of ISJ components by matching the model results to experimental data. Modeling results showed that mechanical properties of the joint capsule and synovial fluid affected the dynamic behavior of the joint. This study contributes to a better understanding of the structure-function relationship of the ISJ for sound transmission.

Morphological changes in the round window membrane associated with Haemophilus influenzae-induced acute otitis media in the chinchilla.

OBJECTIVE: The round window membrane (RWM) encloses the round window, the opening into the scala tympani (ST) from the middle ear. During the course of acute otitis media (AOM), structural changes of the RWM can occur that potentially affect sound transmission into and out of the cochlea. The relationship between such structural changes and conductive hearing loss during AOM has remained unclear. The focus of the current study was to compare the thickness distribution across the RWM surface between normal ears and those with AOM in the chinchilla. We assessed the occurrence of AOM-associated histological changes in this membrane compared to uninfected control animals after AOM of two relatively short durations. MATERIAL AND METHODS: AOM was induced by transbullar injection of the nontypeable Haemophilus influenzae strain 86-028NP into two groups of adult chinchillas (n = 3 each). Bullae were obtained from the two infected groups, at 4 days or 8 days post challenge. Structures and thickness of these RWMs were compared between the two infected treatment groups and to RWMs from uninfected control animals (n = 3) at seven different RWM locations. RESULTS: RWM thickness in infected chinchillas increased significantly at locations along the central line on the 4th day post bacterial challenge compared to values found for uninfected control animals. Lymphocyte infiltration and edema were the primary contributors to these thickness increases. No significant further increases in RWM thickness were observed when RWMs from chinchillas ears infected for 4 and 8 days were compared. Thickness and structural changes at the RWM lateral and medial areas were less visually obvious and not statistically significant among the three treatment groups. These latter RWM regions clearly were less affected during AOM than the central areas. CONCLUSIONS: This histological study establishes that H. influenzae-induced AOM causes significant acute changes in chinchilla RWM structure that are characterized by region-specific increases in thickness. Our new morphological findings comparing normal and diseased chinchilla RWMs identify yet another biomechanical mechanism by which nontypeable H. influenzae may contribute to hearing loss in AOM.

Morphological changes in the tympanic membrane associated with Haemophilus influenzae-induced acute otitis media in the chinchilla.

OBJECTIVE: The tympanic membrane (TM) couples sound waves entering the outer ear canal to mechanical vibrations of the ossicular chain in the middle ear. During acute otitis media (AOM), dynamic structural changes in the TM can occur, which potentially affect sound transmission. It has remained unclear whether TM changes contribute significantly to the conductive hearing loss associated with human AOM. Studies that systematically and quantitatively assess the impact of morphological and mechanical characteristics of the TM on hearing in animal models of AOM have been few in number and lack detail. Our current study focused on the identification of quantitative morphological changes in the TM of the adult chinchilla. METHOD: AOM was produced by transbullar injection of the nontypeable (acapsular) Haemophilus influenzae strain 86-028NP into two treatment groups of chinchillas: one 4 days (4D) post bacterial challenge, and a second treatment group after 8 days (8D) post challenge. Structure and thickness were examined histologically at nine locations over the TM in untreated controls and in animals from both AOM treatment groups. RESULTS: TM thickness was found to have increased significantly (110-150%) at all measured locations of H. influenzae-infected ears when compared with uninfected (normal) TMs at 4D post bacterial challenge. Cellular proliferation and infiltration in the outer epithelial layer were primary contributors to this thickening. In ears infected for 8D, the TM was substantially thicker, a 200-300% increase from uninfected control values, due to edema and cell proliferation in both the outer and inner epithelial layers. In both 4D and 8D ears, thickening of the TM was more prominent in the superior-anterior quadrant. CONCLUSION: This study provides unequivocal structural evidence that significant TM thickness increases are associated with AOM induced by a well characterized H. influenzae human clinical isolate of low passage number. These and additional thickness data from early and later stages in middle ear infection will be used to derive the mechanical properties of the TM in a future study from our laboratory.