CAMSAP3 facilitates basal body polarity and the formation of the central pair of microtubules in motile cilia.

Synchronized beating of cilia on multiciliated cells (MCCs) generates a directional flow of mucus across epithelia. This motility requires a "9 + 2" microtubule (MT) configuration in axonemes and the unidirectional array of basal bodies of cilia on the MCCs. However, it is not fully understood what components are needed for central MT-pair assembly as they are not continuous with basal bodies in contrast to the nine outer MT doublets. In this study, we discovered that a homozygous knockdown mouse model for MT minus-end regulator calmodulin-regulated spectrin-associated protein 3 (CAMSAP3), Camsap3tm1a/tm1a , exhibited multiple phenotypes, some of which are typical of primary ciliary dyskinesia (PCD), a condition caused by motile cilia defects. Anatomical examination of Camsap3tm1a/tm1a mice revealed severe nasal airway blockage and abnormal ciliary morphologies in nasal MCCs. MCCs from different tissues exhibited defective synchronized beating and ineffective generation of directional flow likely underlying the PCD-like phenotypes. In normal mice, CAMSAP3 localized to the base of axonemes and at the basal bodies in MCCs. However, in Camsap3tm1a/tm1a , MCCs lacked CAMSAP3 at the ciliary base. Importantly, the central MT pairs were missing in the majority of cilia, and the polarity of the basal bodies was disorganized. These phenotypes were further confirmed in MCCs of Xenopus embryos when CAMSAP3 expression was knocked down by morpholino injection. Taken together, we identified CAMSAP3 as being important for the formation of central MT pairs, proper orientation of basal bodies, and synchronized beating of motile cilia.

Increased cochlear otic capsule thickness and intracortical canal porosity in the oim mouse model of osteogenesis imperfecta.

Osteogenesis imperfecta (OI or brittle bone disease) is a group of genetic disorders of the connective tissues caused mainly by mutations in the genes encoding collagen type I. Clinical manifestations of OI include skeletal fragility, bone deformities, and severe functional disabilities, such as hearing loss. Progressive hearing loss, usually beginning in childhood, affects approximately 70% of people with OI with more than half of the cases involving the inner ear. There is no cure for OI nor a treatment to ameliorate its corresponding hearing loss, and very little is known about the properties of OI ears. In this study, we investigate the morphology of the otic capsule and the cochlea in the inner ear of the oim mouse model of OI. High-resolution 3D images of 8-week old oim and WT inner ears were acquired using synchrotron microtomography. Volumetric morphometric measurements were conducted for the otic capsule, its intracortical canal network and osteocyte lacunae, and for the cochlear spiral ducts. Our results show that the morphology of the cochlea is preserved in the oim ears at 8 weeks of age but the otic capsule has a greater cortical thickness and altered intracortical bone porosity, with a larger number and volume density of highly branched canals in the oim otic capsule. These results portray a state of compromised bone quality in the otic capsule of the oim mice that may contribute to their hearing loss.

Channel Interaction During Infrared Light Stimulation in the Cochlea.

BACKGROUND AND OBJECTIVES: The number of perceptually independent channels to encode acoustic information is limited in contemporary cochlear implants (CIs) because of the current spread in the tissue. It has been suggested that neighboring electrodes have to be separated in humans by a distance of more than 2 mm to eliminate significant overlap of the electric current fields and subsequent interaction between the channels. It has also been argued that an increase in the number of independent channels could improve CI user performance in challenging listening environments, such as speech in noise, tonal languages, or music perception. Optical stimulation has been suggested as an alternative modality for neural stimulation because it is spatially selective. This study reports the results of experiments designed to quantify the interaction between neighboring optical sources in the cochlea during stimulation with infrared radiation. STUDY DESIGN/MATERIALS AND METHODS: In seven adult albino guinea pigs, a forward masking method was used to quantify the interaction between two neighboring optical sources during stimulation. Two optical fibers were placed through cochleostomies into the scala tympani of the basal cochlear turn. The radiation beams were directed towards different neuron populations along the spiral ganglion. Optically evoked compound action potentials were recorded for different radiant energies and distances between the optical fibers. The outcome measure was the radiant energy of a masker pulse delivered 3 milliseconds before a probe pulse to reduce the response evoked by the probe pulse by 3 dB. Results were compared for different distances between the fibers placed along the cochlea. RESULTS: The energy required to reduce the probe's response by 3 dB increased by 20.4 dB/mm and by 26.0 dB/octave. The inhibition was symmetrical for the masker placed basal to the probe (base-to-apex) and the masker placed apical to the probe (apex-to-base). CONCLUSION: The interaction between neighboring optical sources during infrared laser stimulation is less than the interaction between neighboring electrical contacts during electrical stimulation. Previously published data for electrical stimulation reported an average current spread in human and cat cochleae of 2.8 dB/mm. With the increased number of independent channels for optical stimulation, it is anticipated that speech and music performance will improve. Lasers Surg. Med. © 2020 Wiley Periodicals LLC.

Using synchrotron X-ray phase-contrast micro-computed tomography to study tissue damage by laser irradiation.

BACKGROUND AND OBJECTIVE: The aim of this study was to determine if X-ray micro-computed tomography could be used to locate and characterize tissue damage caused by laser irradiation and to describe its advantages over classical histology for this application. STUDY DESIGN/MATERIALS AND METHODS: A surgical CO2 laser, operated in single pulse mode (100 milliseconds) at different power settings, was used to ablate different types of cadaveric animal tissues. Tissue samples were then harvested and imaged with synchrotron X-ray phase-contrast and micro-computed tomography to generate stacks of virtual sections of the tissues. Subsequently, Fiji (ImageJ) software was used to locate tissue damage, then to quantify volumes of laser ablation cones and thermal coagulation damage from 3D renderings of tissue image stacks. Visual comparisons of tissue structures in X-ray images with those visible by classic light microscopy histology were made. RESULTS: We demonstrated that micro-computed tomography could be used to rapidly identify areas of surgical laser ablation, vacuolization, carbonization, and thermally coagulated tissue. Quantification and comparison of the ablation crater, which represents the volume of ablated tissue, and the thermal coagulation zone volumes were performed faster than we could by classical histology. We demonstrated that these procedures can be performed on fresh hydrated and non-sectioned plastic embedded tissue. CONCLUSION: We demonstrated that the application of non-destructive micro-computed tomography to the visualization and analysis of laser induced tissue damage without tissue sectioning is possible. This will improve evaluation of new surgical lasers and their corresponding effect on tissues. Lasers Surg. Med. 48:866-877, 2016. © 2016 Wiley Periodicals, Inc.

Roles of the espin actin-bundling proteins in the morphogenesis and stabilization of hair cell stereocilia revealed in CBA/CaJ congenic jerker mice.

Hearing and vestibular function depend on mechanosensory staircase collections of hair cell stereocilia, which are produced from microvillus-like precursors as their parallel actin bundle scaffolds increase in diameter and elongate or shorten. Hair cell stereocilia contain multiple classes of actin-bundling protein, but little is known about what each class contributes. To investigate the roles of the espin class of actin-bundling protein, we used a genetic approach that benefited from a judicious selection of mouse background strain and an examination of the effects of heterozygosity. A congenic jerker mouse line was prepared by repeated backcrossing into the inbred CBA/CaJ strain, which is known for excellent hearing and minimal age-related hearing loss. We compared stereocilia in wild-type CBA/CaJ mice, jerker homozygotes that lack espin proteins owing to a frameshift mutation in the espin gene, and jerker heterozygotes that contain reduced espin levels. The lack of espins radically impaired stereociliary morphogenesis, resulting in stereocilia that were abnormally thin and short, with reduced differential elongation to form a staircase. Mean stereociliary diameter did not increase beyond ∼0.10-0.14 µm, making stereocilia ∼30%-60% thinner than wild type and suggesting that they contained ∼50%-85% fewer actin filaments. These characteristics indicate a requirement for espins in the appositional growth and differential elongation of the stereociliary parallel actin bundle and fit the known biological activities of espins in vitro and in transfected cells. The stereocilia of jerker heterozygotes showed a transient proximal-distal tapering suggestive of haploinsufficiency and a slowing of morphogenesis that revealed previously unrecognized assembly steps and intermediates. The lack of espins also led to a region-dependent degeneration of stereocilia involving shortening and collapse. We conclude that the espin actin-bundling proteins are required for the assembly and stabilization of the stereociliary parallel actin bundle.

Pupillometry to show stress release during equine sports massage therapy.

Anecdotal reports state that wellness treatments for horses, such as massage therapy, relaxes the treated animal. Massage therapists and horse owners typically report an "improvement" without verifying or quantifying the treatment results. This paper shows that the effect of wellness treatment and stress release can be measured with pupillometry. One of the horse's pupils was photographed at the beginning and end of the treatment to determine the changes in the balance between the sympathetic and parasympathetic system activities. The owners assigned horses to two experimental groups: animals receiving a massage (N = 18) and horses standing with a person next to the horse for the time of a massage in the stable lane (N = 10). Six animals in the experimental group were excluded from the analysis because the pupils could not be traced. We opened the images of the pupil with Fiji (ImageJ) and used the elliptical selection tool to measure the pupils' and iris' areas. The ratio between the pupils' aperture and the iris' area was a normalized measure for pupil size. At the end of the experiment, we compared the normalized size of the pupils with a two-tailed paired t-test within groups and a two-tailed t-test between groups. For the experimental group, it was before and after the treatment, and for the control group, before and after the horse was placed in the stable lane. Comparisons between the experimental and control groups were made at the procedure's beginning and end. The treatment significantly decreased the normalized pupil area in the experimental group, on average, by a factor of 0.78 ± 0.15 (P = 0.042). For the horses in the control group, the pupil size increased, on average, by a factor of 1.14 ± 018. Changes were statistically not significant (P = 0.19). The initial pupil size of the horses in the experimental group was 1.88 times larger than that in the control group. After the treatment, the difference was reduced to a factor of 1.25. At the beginning of the experiment, the horses in the experimental group had, on average, larger pupil sizes than the horses in the control group, indicating that the horses in the experimental group were more stressed before the treatment than the control animals. The observed changes in pupil size in the experimental group likely resulted from enhanced parasympathetic and diminished sympathetic activity resulting from the treatment. Observed changes in pupil size agree with the anecdotal horse owner reports and the therapist's treatment notes.

Statins protect mice from high-decibel noise-induced hearing loss.

No medical interventions for noise induced hearing loss (NIHL) are approved by the Food and Drug Administration (USA). Here, we evaluate statins in CBA/CaJ mice as potential drugs for hearing loss. Direct delivery of fluvastatin to the cochlea and oral delivery of lovastatin were evaluated. Baseline hearing was assessed using Auditory Brain Stem Responses (ABRs). For fluvastatin, a cochleostomy was surgically created in the basal turn of the cochlea by a novel, laser-based procedure, through which a catheter attached to a mini-osmotic pump was inserted. The pump was filled with a solution of 50 µM fluvastatin+carrier or with the carrier alone for continuous delivery to the cochlea. Mice were exposed to one octave band noise (8-16 kHz x 2 h x 110 dB SPL). In our past work with guinea pigs, fluvastatin protected in the contralateral cochlea. In this study in CBA/CaJ mice, hearing was also assessed in the contralateral cochlea 1-4 weeks after noise exposure. At two weeks post exposure, ABR thresholds at 4, 8, 12, 16, and 32 kHz were elevated, as expected, in the noise+carrier alone treated mice by approximately 9-, 17-, 41-, 29-, and 34-dB, respectively. Threshold elevations were smaller in mice treated with noise+fluvastatin to about 2-, 6-, 20-,12- and 12-dB respectively. Survival of inner hair cell synapses were not protected by fluvastatin over these frequencies. Lovastatin delivered by gavage showed lower threshold shifts than with carrier alone. These data show that direct and oral statin delivery protects mice against NIHL.

In-situ hearing threshold estimation using Gaussian process classification.

One in six Americans suffers from hearing loss. While treatment with amplification is possible for many, the acceptance rate of hearing aids is low. Poor device fitting is one of the reasons. The hearing aid fitting starts with a detailed hearing assessment by a trained audiologist in a sound-controlled environment, using standard equipment. The hearing aid is adjusted step-by-step, following well-described procedures based on the audiogram. However, for many patients in rural settings, considerable travel time to a hearing center discourages them from receiving a hearing test and treatment. We hypothesize that hearing assessment with the patient's hearing aid can reliably substitute the hearing test in the clinic. Over-the-counter hearing aids could be programmed from a distance and fine-tuned by the hearing aid wearer. This study shows that a patient-controlled hearing assessment via a hearing aid in a non-clinical setting is not statistically different from an audiologist-controlled hearing assessment in a clinical setting. The differences in hearing obtained with our device and the Gaussian Process are within 3 dB of the standard audiogram. At 250 Hz, the sound delivery with the hearing aid used in this study added an additional reduction of sound level, which was not compensated.

Treatments for hearing loss in osteogenesis imperfecta: a systematic review and meta-analysis on their efficacy.

About 70% of people with osteogenesis imperfecta (OI) experience hearing loss. There is no cure for OI, and therapies to ameliorate hearing loss rely on conventional treatments for auditory impairments in the general population. The success rate of these treatments in the OI population with poor collagenous tissues is still unclear. Here, we conduct a systematic review and meta-analysis on the efficacy of treatments addressing hearing loss in OI. This study conforms to the reporting standards of the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA). Data sources include published articles in Medline via PubMed, Web of Science, Scopus, and Embase, from their inception to November 2020. Studies included individuals with OI undergoing a hearing loss treatment, having pre- and postoperative objective assessment of hearing function at a specified follow-up length. Our search identified 1144 articles, of which 67 were reviewed at full-text screening. A random-effects meta-analysis was conducted on the selected articles (n = 12) of people with OI that underwent stapes surgery. Success was assessed as the proportion of ears with a postoperative Air-Bone Gap (ABG) ≤ 10 dB. A systematic review was conducted on the remaining articles (n = 13) reporting on other treatments. No meta-analysis was conducted on the latter due to the low number of articles on the topic and the nature of single case studies. The meta-analysis shows that stapes surgeries have a low success rate of 59.08 (95% CI 45.87 to 71.66) in the OI population. The systematic review revealed that cochlear implants, bone-anchored hearing aids, and other implantable hearing aids proved to be feasible, although challenging, in the OI population, with only 2 unsuccessful cases among the 16 reviewed single cases. This analysis of published data on OI shows poor clinical outcomes for the procedures addressing hearing loss. Further studies on hearing loss treatments for OI people are needed. Notably, the mechanisms of hearing loss in OI need to be determined to develop successful and possibly non-invasive treatment strategies.

Mammalian middle ear mechanics: A review.

The middle ear is part of the ear in all terrestrial vertebrates. It provides an interface between two media, air and fluid. How does it work? In mammals, the middle ear is traditionally described as increasing gain due to Helmholtz's hydraulic analogy and the lever action of the malleus-incus complex: in effect, an impedance transformer. The conical shape of the eardrum and a frequency-dependent synovial joint function for the ossicles suggest a greater complexity of function than the traditional view. Here we review acoustico-mechanical measurements of middle ear function and the development of middle ear models based on these measurements. We observe that an impedance-matching mechanism (reducing reflection) rather than an impedance transformer (providing gain) best explains experimental findings. We conclude by considering some outstanding questions about middle ear function, recognizing that we are still learning how the middle ear works.

Infrared photostimulation of the crista ampullaris.

The present results show that the semicircular canal crista ampullaris of the toadfish, Opsanus tau, is sensitive to infrared radiation (IR) applied in vivo. IR pulse trains (∼1862 nm, ∼200 µs pulse⁻¹) delivered to the sensory epithelium by an optical fibre evoked profound changes in phasic and tonic discharge rates of postsynaptic afferent neurons. Phasic afferent responses to pulsed IR occurred with a latency of <8 ms while tonic responses developed with a time constant (τ) of 7 ms to 10 s following the onset or cessation of the radiation. Afferents responded to direct optical radiation of the sensory epithelium but did not respond to thermal stimuli that generated nearly equivalent temperature increases of the whole organ. A subset of afferent neurons fired an action potential in response to each IR pulse delivered to the sensory epithelium, at phase-locked rates up to 96 pulses per second. The latency between IR pulses and afferent nerve action potentials was much greater than synaptic delay and spike generation, demonstrating the presence of a signalling delay interposed between the IR pulse and the action potential. The same IR stimulus applied to afferent nerve axons failed to evoke responses of similar magnitude and failed to phase-lock afferent nerve action potentials. The present data support the hypothesis that pulsed IR activates sensory hair cells, thus leading to modulation of synaptic transmission and afferent nerve discharge reported here.

Complete activation of thyroid hormone receptor ß by T3 is essential for normal cochlear function and morphology in mice.

BACKGROUND/AIMS: Thyroid hormones (THs) regulate many developmental processes, including the developmental onset of cochlear differentiation and function. TH action is mediated mostly by triiodothyronine (T3) bound to thyroid hormone nuclear receptors (TRs). At positive regulated genes and in the absence of THs, nuclear co-repressors are bound to TRs and decrease basal transcription rate. Ligand (T(3)) binding results in the dissociation of co-repressors and the recruitment of co-activators to the complex, which results in full transcriptional activation. METHODS: We measured cochlear function in two knock-in mouse models: TRß(E457A/E457A), with the TRß co-activator binding surface (AF-2) disrupted to prevent co-activator binding; and TRß(Δ337T/Δ337T), which is unable to bind T(3). Cochlear morphology and function were analyzed in 10-week-old normal and mutated mice. Cochlear function was determined by measuring auditory brainstem responses, cochlear tuning and compound action potential (CAP) thresholds. RESULTS: All TRß(Δ337T/Δ337T) and 85% of the TRß(E457A/E457A) mice presented elevated CAP thresholds (P < 0.05 or less). Five percent of the TRß(E457A/E457A) mice presented normal CAP thresholds with broadened cochlear tuning. TRß(E457A/E457A) and TRß(Δ337T/Δ337T) presented developmental defects that led to a decreased width (P < 0.01) and an increased thickness (P<0.01) of the tectorial membrane. In addition, TRß(Δ337T/Δ337T) animals showed an increased tectorial membrane area (P<0.01). CONCLUSION: Both mutations were deleterious to tectorial membrane development and led to important alterations in cochlear morphology and loss of cochlear function.

Near-infrared stimulation of the auditory nerve: A decade of progress toward an optical cochlear implant.

OBJECTIVES: We provide an appraisal of recent research on stimulation of the auditory system with light. In particular, we discuss direct infrared stimulation and ongoing controversies regarding the feasibility of this modality. We also discuss advancements and barriers to the development of an optical cochlear implant. METHODS: This is a review article that covers relevant animal studies. RESULTS: The auditory system has been stimulated with infrared light, and in a much more spatially selective manner than with electrical stimulation. However, there are experiments from other labs that have not been able to reproduce these results. This has resulted in an ongoing controversy regarding the feasibility of infrared stimulation, and the reasons for these experimental differences still require explanation. The neural response characteristics also appear to be much different than with electrical stimulation. The electrical stimulation paradigms used for modern cochlear implants do not apply well to optical stimulation and new coding strategies are under development. Stimulation with infrared light brings the risk of heat accumulation in the tissue at high pulse repetition rates, so optimal pulse shapes and combined optical/electrical stimulation are being investigated to mitigate this. Optogenetics is another promising technique, which makes neurons more sensitive to light stimulation by inserting light sensitive ion channels via viral vectors. Challenges of optogenetics include the expression of light sensitive channels in sufficient density in the target neurons, and the risk of damaging neurons by the expression of a foreign protein. CONCLUSION: Optical stimulation of the nervous system is a promising new field, and there has been progress toward the development of a cochlear implant that takes advantage of the benefits of optical stimulation. There are barriers, and controversies, but so far none that seem intractable. LEVEL OF EVIDENCE: NA (animal studies and basic research).

Infrared neural stimulation at different wavelengths and pulse shapes.

Neural stimulation with infrared radiation has been explored for brain tissue, peripheral nerves, and cranial nerves including the auditory nerve. Initial experiments were conducted at wavelengths between λ = 1850 and λ = 2140 nm and the radiant energy was delivered with square pulses. Water absorption of the infrared radiation at λ = 1860 nm is similar to absorption at wavelengths between λ = 1310 and λ = 1600 nm, which are in the radiation wavelength range used for the communication industry. Technology for those wavelengths has already been developed and miniaturized and is readily available. The possibility of the infrared light to evoke compound action potentials (CAP) in the cochlea at λ = 1,375, λ = 1,460, and λ = 1550 nm was explored and compared to that of λ = 1860 nm in guinea pigs. Furthermore, rise and fall times of the 100 µs long pulses were changed and four basic pulse shapes (square, triangular, ramp-up, and ramp-down) were explored in their ability to evoke a CAP. In animals with pure tone threshold averages (PTAs) above 70 dB SPL, the results show that the favorable wavelength is λ = 1460 nm to reach threshold for stimulation and λ = 1375 nm or λ = 1460 nm for obtaining maximum amplitude. The most favorable pulse shape is either ramp-up or triangular.

Are suspensory ligaments important for middle ear reconstruction?

As the resolution of 3D printing techniques improves, the possibility of individualized, 3-ossicle constructions adds a new dimension to middle ear prostheses. In order to optimize these designs, it is essential to understand how the ossicles and ligaments work together to transmit sound, and thus how ligaments should be replicated in a middle ear reconstruction. The middle ear ligaments are thought to play a significant role in maintaining the position of the ossicles and constraining axis of rotation. Paradoxically, investigations of the role of ligaments to date have shown very little impact on middle ear sound transmission. We explored the role of the two attachments in the gerbil middle ear analogous to human ligaments, the posterior incudal ligament and the anterior mallear process, severing both attachments and measuring change in hearing sensitivity. The impact of severing the attachments on the position of the ossicular chain was visualized using synchrotron microtomography imaging of the middle ear. In contrast to previous studies, a threshold change on the order of 20 dB across a wide range of frequencies was found when both ligaments were severed. Concomitantly, a shift in position of the ossicles was observed from the x-ray imaging and 3D renderings of the ossicular chain. These findings contrast with previous studies, demonstrating that these ligaments play a significant role in the transmission of sound through the middle ear. It appears that both mallear and incudal ligaments must be severed in order to impair sound transmission. The results of this study have significance for middle ear reconstructive surgery and the design of 3D-printed three-ossicle biocompatible prostheses.

Listening to speech with a guinea pig-to-human brain-to-brain interface.

Nicolelis wrote in his 2003 review on brain-machine interfaces (BMIs) that the design of a successful BMI relies on general physiological principles describing how neuronal signals are encoded. Our study explored whether neural information exchanged between brains of different species is possible, similar to the information exchange between computers. We show for the first time that single words processed by the guinea pig auditory system are intelligible to humans who receive the processed information via a cochlear implant. We recorded the neural response patterns to single-spoken words with multi-channel electrodes from the guinea inferior colliculus. The recordings served as a blueprint for trains of biphasic, charge-balanced electrical pulses, which a cochlear implant delivered to the cochlear implant user's ear. Study participants completed a four-word forced-choice test and identified the correct word in 34.8% of trials. The participants' recognition, defined by the ability to choose the same word twice, whether right or wrong, was 53.6%. For all sessions, the participants received no training and no feedback. The results show that lexical information can be transmitted from an animal to a human auditory system. In the discussion, we will contemplate how learning from the animals might help developing novel coding strategies.

CO(2) laser fiber soft cochleostomy: development of a technique using human temporal bones and a guinea pig model.

OBJECTIVE: A novel approach for creating a soft cochleostomy has been described using a handheld CO(2) laser hollow waveguide from termed in the following the handheld CO(2) laser fiber. The effects on cochlear function have been studied in an animal model and were compared to the effects of the micro drill. STUDY DESIGN: Combined human temporal bone and experimental animal study. MATERIALS AND METHODS: Four human temporal bones were used to describe the technique to create a cochleostomy using the handheld CO(2) laser fiber. A cochleostomy was made by thinning the cochlear wall with a motorized drill and by creating an opening with the CO(2) laser fiber. Eighteen guinea pigs were used to investigate the effects of the CO(2) laser and the drill on cochlear function. An electrode was placed in the round window niche to measure compound action potentials (CAPs). Baseline cochlear function was determined by recording CAP thresholds evoked by acoustic tone pips and was re-assessed during and after a cochleostomy was made. The protocol was repeated using a diamond-burr drill technique. RESULTS: The handheld CO(2) laser fiber is an ideal tool to create cochleostomies under 1 mm in diameter. In the guinea pig animal model, CAP thresholds showed little change after creating the cochleostomy at 4 W laser power setting and revealed focal threshold elevations averaging 32 dB at higher laser power settings. CONCLUSIONS: The human temporal bone study introduces a novel surgical approach for soft cochleostomies that uses both the drill and the CO(2) laser fiber. Threshold elevations, which were observed after making the cochleostomy with the laser, compared favorably to the diamond burr technique.

X-ray fluorescence microscopy: A method of measuring ion concentrations in the ear.

This technical note describes synchrotron x-ray fluorescence microscopy (XFM) as a method for measuring the concentrations of different elements in cross-sections of the ear at extremely high resolution. This method could be of great importance for addressing many open questions in hearing research. XFM uses synchrotron radiation to evoke emissions from many biologically relevant elements in the tissue. The intensity and wavelength of the emitted radiation provide a fingerprint of the tissue composition that can be used to measure the concentration of the elements in the sampled location. Here, we focus on energies that target biologically-relevant elements of the periodic table between magnesium and zinc. Since a highly focused x-ray beam is used, the spot size is well below 1 µm and the samples can be scanned at a nanometer lateral resolution. This study shows that measurement of the concentrations of different elements is possible in a mid-modiolar cross-section of a mouse cochlea. Images are presented that indicate potassium and chloride "hot spots" in the spiral ligament and the spiral limbus, providing experimental evidence for the potassium recycling pathway and showing the cochlear structures involved. Scans of a section obtained from the incus, one of the middle ear ossicles, in a developing mouse have shown that zinc is not uniformly distributed This supports the hypothesis that zinc plays a special role in the process of ossification. Although limited by sophisticated sample preparation and sectioning, the method provides ample exciting opportunities, to understand the role of genetics and epigenetics on hearing mechanisms in ontogeny and phylogeny.

Systemic application of honokiol prevents cisplatin ototoxicity without compromising its antitumor effect.

Cisplatin is a potent drug used in about 40% of cancer treatment but also leads to severe deafness in 60-80% of the cases. Although the mechanism is known to be related to the accumulation of reactive oxygen species (ROS), no drug or FDA approved treatment is currently available to prevent cisplatin ototoxicity. With this study, we show for the first time that honokiol (HNK), a pleiotropic poly-phenol prevents cisplatin-induced hearing loss. HNK also improves the wellbeing of the mice during the treatment, determined by the increase in the number of surviving animals. In a transgenic tumor mouse model, HNK does not hinder cisplatin's antitumor effect. The mechanism is related to the activation of sirtuin 3, a deacetylase in mitochondria essential for ROS detoxification. We expect a paradigm shift in cisplatin chemotherapy based on the current study and future clinical trials, where honokiol is applied to reduce side effects including hearing loss.

Hearing differences in Hartley guinea pig stocks from two breeders.

Across the world, dozens of outbred Hartley guinea pig stocks are used for auditory experiments. The genetic makeup of these different stocks will differ due to differences in breeding protocols, history and genetic drift. In fact, outbred breeding protocols are not intended to produce genetically identical animals, neither across breeders, nor across time. For this reason, it is unclear how reproducible experimental results are likely to be using animals from different stocks. We evaluated the consistency of cochlear function using both clicks and tones in Hartley guinea pigs as a function of breeder (Kuiper and Charles River) and sex using archival Auditory Brain Stem Response (ABR) data and tissue from our own laboratory. Sound levels required to reach baseline threshold for click-induced ABRs were similar between male Charles River and male Kuiper guinea pig stocks. However, the median and average thresholds after exposure to high level noise were larger in the Kuiper population than in the Charles River population with corresponding threshold shifts higher in the Kuiper than in the Charles River animals. We evaluated the relationship between pure-tone thresholds and sex, age, breeder stock, left or right cochleas, weight and 5 test frequencies before and after noise exposure using a linear mixed statistical model. Across all frequencies, the effect of breeder on baseline threshold is statistically significant, with effect sizes most pronounced at the lower frequencies before exposure to noise. After noise exposure, the differences are minimal in the model, indicating that differences in threshold shift are chiefly due to differences in initial baseline hearing. However, a contingency calculation comparing response/no response at the highest speaker output at 32 kHz gave a statistically significant difference between the stocks: 28% of Kuiper cochleas responded to the highest output of the speaker as compared with 71.4% of Charles River cochleas, indicating that noise exposure induced a larger threshold shift in a greater proportion of Kuiper animals. Using our archival cochlear tissue from these studies, we confirmed the sex of each animal by PCR, then compared males and females of the Kuiper stock. Across all baseline frequencies, the effect of sex on threshold is statistically significant, with no noticeable difference after exposure. The effect sizes for baseline thresholds are most pronounced at lower frequencies. These data demonstrate that Hartley guinea pig stocks from different breeders are not uniform in their auditory characteristics, and that due to these differences, results and conclusions can differ among laboratories. Moreover, within a single stock, males and females can provide different data, confirming that male and female animals must be individually evaluated in any auditory protocol.