A missense mutation in Kcnc3 causes hippocampal learning deficits in mice.

Although a wide variety of genetic tools has been developed to study learning and memory, the molecular basis of memory encoding remains incompletely understood. Here, we undertook an unbiased approach to identify novel genes critical for memory encoding. From a large-scale, in vivo mutagenesis screen using contextual fear conditioning, we isolated in mice a mutant, named Clueless, with spatial learning deficits. A causative missense mutation (G434V) was found in the voltage-gated potassium channel, subfamily C member 3 (Kcnc3) gene in a region that encodes a transmembrane voltage sensor. Generation of a Kcnc3G434V CRISPR mutant mouse confirmed this mutation as the cause of the learning defects. While G434V had no effect on transcription, translation, or trafficking of the channel, electrophysiological analysis of the G434V mutant channel revealed a complete loss of voltage-gated conductance, a broadening of the action potential, and decreased neuronal firing. Together, our findings have revealed a role for Kcnc3 in learning and memory.

No Reduction in the 226-Hz Probe Tone Acoustic Reflex Amplitude Following Severe Inner Hair Cell Loss in Chinchillas.

The relationship between the middle ear acoustic reflex (AR) and inner hair cell (IHC) loss is currently unknown. Given that IHC are believed to convey nearly all acoustic information to the central auditory nervous system, it has been assumed that loss of IHC would significantly impact the AR. To evaluate this relationship, we assessed the presence and amplitude of the AR in chinchillas before and after treatment with carboplatin, an anticancer drug that reliably and selectively destroys IHC in this species. Baseline measures of hearing sensitivity, including auditory brainstem response (ABR) thresholds and distortion product otoacoustic emissions (DPOAE), were assessed and then re-evaluated following carboplatin treatment. Post-carboplatin ABR thresholds and DPOAE were found to be unchanged or slightly elevated; results were consistent with published reports. Our main hypothesis was that loss of IHC would abolish the reflex or significantly reduce its amplitude. Contrary to our hypothesis, the ipsilateral 226-Hz AR continued to be reliably elicited following carboplatin treatment. Post-mortem histological analysis confirmed significant IHC loss (65-85 %), but no measurable loss of outer hair cells (OHCs). Given that loss of IHC alone does not significantly reduce the 226-Hz AR, our results suggest that few IHC are needed to maintain the 226-Hz AR response. These results suggest additional studies are needed to better understand the role of IHC in the reflex arc, present opportunities to further study the reflex pathway, and could change how we use the clinical AR as a potential diagnostic tool for IHC dysfunction, including those related to IHC synaptopathy.

Effect of Selective Carboplatin-Induced Inner Hair Cell Loss on Temporal Integration in Chinchillas.

Integration of acoustic information over time is essential for processing complex stimuli, such as speech, due to its continuous variability along the time domain. In both humans and animals, perception of acoustic stimuli is a function of both stimulus intensity and duration. For brief acoustic stimuli, as duration increases, thresholds decrease by approximately 3 dB for every doubling in duration until stimulus duration reaches 500 ms, a phenomenon known as temporal integration. Although hearing loss and damage to outer hair cells (OHC) have been shown to alter temporal integration in some studies, the role of cochlear inner hair cells (IHC) on temporal integration is unknown. Because IHC transmit nearly all acoustic information to the central auditory system and are believed to code both intensity and timing information, these sensory cells likely play a critical role in temporal integration. To test the hypothesis that selective IHC loss degrades the temporal integration function, behaviorally trained chinchillas were treated with carboplatin, a drug known to selectively destroy IHC with little to no effect on OHC in this species. Pure-tone thresholds were assessed across frequencies (1, 2, 4, 8, 12 kHz) as a function of signal duration (500, 100, 50, 10, and 5 ms). Baseline testing showed a significant effect of duration on thresholds. Threshold decreased as a function of increasing duration, as expected. Carboplatin treatment (75 mg/kg) produced a moderate to severe loss of IHC (45-85%) with little-to-no loss of OHC. Contrary to our hypothesis, post-carboplatin temporal integration thresholds showed no significant differences from baseline regardless of stimulus duration or frequency. These data suggest that few IHC are necessary for temporal integration of simple stimuli. Temporal integration may be sensitive to loss of OHC and loss of cochlear non-linearities but does not appear to be sensitive to selective IHC loss.

Audiogenic Seizures in the Fmr1 Knock-Out Mouse Are Induced by Fmr1 Deletion in Subcortical, VGlut2-Expressing Excitatory Neurons and Require Deletion in the Inferior Colliculus.

Fragile X syndrome (FXS) is the most common form of inherited intellectual disability and the leading monogenetic cause of autism. One symptom of FXS and autism is sensory hypersensitivity (also called sensory over-responsivity). Perhaps related to this, the audiogenic seizure (AGS) is arguably the most robust behavioral phenotype in the FXS mouse model-the Fmr1 knock-out (KO) mouse. Therefore, the AGS may be considered a mouse model of sensory hypersensitivity. Hyperactive circuits are hypothesized to underlie dysfunction in a number of brain regions in patients with FXS and Fmr1 KO mice, and the AGS may be a result of this. But the specific cell types and brain regions underlying AGSs in the Fmr1 KO are unknown. We used conditional deletion or expression of Fmr1 in different cell populations to determine whether Fmr1 deletion in those cells was sufficient or necessary, respectively, for the AGS phenotype in males. Our data indicate that Fmr1 deletion in glutamatergic neurons that express vesicular glutamate transporter 2 (VGlut2) and are located in subcortical brain regions is sufficient and necessary to cause AGSs. Furthermore, the deletion of Fmr1 in glutamatergic neurons of the inferior colliculus is necessary for AGSs. When we demonstrate necessity, we show that Fmr1 expression in either the larger population of VGlut2-expressing glutamatergic neurons or the smaller population of inferior collicular glutamatergic neurons-in an otherwise Fmr1 KO mouse-eliminates AGSs. Therefore, targeting these neuronal populations in FXS and autism may be part of a therapeutic strategy to alleviate sensory hypersensitivity.SIGNIFICANCE STATEMENT Sensory hypersensitivity in fragile X syndrome (FXS) and autism patients significantly interferes with quality of life. Audiogenic seizures (AGSs) are arguably the most robust behavioral phenotype in the FXS mouse model-the Fmr1 knockout-and may be considered a model of sensory hypersensitivity in FXS. We provide the clearest and most precise genetic evidence to date for the cell types and brain regions involved in causing AGSs in the Fmr1 knockout and, more broadly, for any mouse mutant. The expression of Fmr1 in these same cell types in an otherwise Fmr1 knockout eliminates AGSs indicating possible cellular targets for alleviating sensory hypersensitivity in FXS and other forms of autism.

Ephrins and Ephs in cochlear innervation and implications for advancing cochlear implant function.

OBJECTIVES/HYPOTHESIS: Determine if the neuronal pathfinding cues resulting from Eph/ephrin interaction in the inner ear play a role in establishing the tonotopic innervation of the cochlea. STUDY DESIGN: Protein expression of Ephs and ephrins was evaluated in the inner ear of mice and chicks. Subsequently, in vitro, in vivo, and functional electrophysiologic studies were performed to indicate that Ephs and ephrins play a role regulating the normal innervation patterns in the mouse inner ear. METHODS: Eph and ephrin protein expression was identified in the inner ear by western blotting and localized by fluorescence immunohistochemistry and X-gal staining. Eph/ephrin effects on neurite outgrowth was assessed via co-culture with EphB2 expressing COS-1 cells. Anatomic effects of disrupting Eph/ephrin signaling on cochlear innervation were determined with lipophilic dye tracing and functional effects with auditory brainstem response (ABR). RESULTS: Expression of several different Ephs and ephrins were found in the inner ear of chicks and mice. The changes in ephrin-A2 immunoreactivity after gentamicin ototoxicity coincide with the spatio-temporal pattern of hair cell loss and regeneration in the chick cochlea. EphB2 inhibited outgrowth of spiral ganglion cell neurites. Knockout mice with null function of EphB1, EphB2, and EphB3 demonstrated abnormal inner ear innervation and elevated ABR thresholds, indicating hearing loss. CONCLUSIONS: Ephrin-A2 may be involved in the guidance of ganglion cells to hair cells in the chick. Disruption of Eph/ephrin signaling results in abnormal innervation and hearing loss, suggesting that these proteins play a role in establishing normal innervation patterns in the mouse cochlea. LEVEL OF EVIDENCE: NA

Hearing loss is an early consequence of Npc1 gene deletion in the mouse model of Niemann-Pick disease, type C.

Niemann-Pick disease, type C1 (NPC1) is a rare lysosomal lipidosis that is most often the result of biallelic mutations in NPC1, and is characterized by a fatal neurological degeneration. The pathophysiology is complex, and the natural history of the disease is poorly understood. Recent findings from patients with NPC1 and hearing loss suggest that multiple steps along the auditory pathway are affected. The current study was undertaken to determine the auditory phenotype in the Npc1 (nih) mutant mouse model, to extend analyses to histologic evaluation of the inner ear, and to compare our findings to those reported from human patients. Auditory testing revealed a progressive high-frequency hearing loss in Npc1 (-/-) mice that is present as early as postnatal day 20 (P20), well before the onset of overt neurological symptoms, with evidence of abnormalities involving the cochlea, auditory nerve, and brainstem auditory centers. Distortion product otoacoustic emission amplitude and auditory brainstem response latency data provided evidence for a disruption in maturational development of the auditory system in Npc1 (-/-) mice. Anatomical study demonstrated accumulation of lysosomes in neurons, hair cells, and supporting cells of the inner ear in P30 Npc1 (-/-) mice, as well as increased numbers of inclusion bodies, myelin figures, and swollen nerve endings in older (P50-P70) mutant animals. These findings add unique perspective to the pathophysiology of NPC disease and suggest that hearing loss is an early and sensitive marker of disease progression.

Ototopic applications of povidone iodine/dexamethasone in the rat.

HYPOTHESIS: Repeated applications of low-concentration povidone iodine (PI) combined with dexamethasone (Dex) through a tympanic membrane ventilation tube will not cause ototoxic changes in the rat. BACKGROUND: Otitis externa (OE) and acute otitis media (AOM) are 2 of the most common otologic disorders requiring outpatient antibiotic treatment. The development of topical treatments that are easy to administer would help to limit systemic exposure to antibiotics in these patients. Topical formulations containing Dex and low-dose PI were designed to provide both antimicrobial and anti-inflammatory effects for the treatment of OE and AOM. Treatment with PI alone has shown mixed results in studies designed to determine PI. Low concentrations of PI combined with Dex should yield less ototoxicity while maintaining effectiveness. METHODS: We performed tympanostomies on rats, inserting a ventilation tube to administer 1% or 2% PI, plus 0.1% Dex over a period of 7 days. Hearing was accessed via auditory brainstem response (ABR) testing over the duration of the study and histologic analysis was performed 15 days after the initial application to determine the effect of administration of PI/Dex on middle and inner ear structures. CONCLUSION: The preparations used in the present investigation were formulated to allow repeated applications to both the external and middle ear, without risk to hearing or equilibrium. Neither of the PI/Dex formulations tested caused pathologic changes in the ear that significantly affected equilibrium, hearing function or morphology.

Cochlear implant device failure secondary to cholesterol granuloma-mediated cochlear erosion.

STUDY DESIGN: Case report. SETTING: A tertiary care pediatric hospital. PATIENT: A 7-year-old boy complained of retroauricular pain 3.5 years after cochlear implantation. Temporal bone computed tomographic scan revealed a soft tissue density filling the mastoid and middle ear space. There was extensive osseous erosion involving the cochlear promontory that resulted in lateral displacement electrode array into the middle ear space. INTERVENTION: Mastoidectomy with removal of cochlear implant. RESULTS: Pathologic examination of the soft tissue filling the mastoid cavity was consistent with a cholesterol granuloma. The cultures of the mastoid cavity demonstrated no growth. Examination of the explanted device revealed a cell layer covering the length of the electrode. The stylet tract was filled with cells that grew out through the silicone component. No birefringent cholesterol crystals were detected in the tissue surrounding the electrode. Biofilms were not identified. CONCLUSION: The authors describe a unique case of a cholesterol granuloma of the middle ear and mastoid in a cochlear implant recipient causing erosion of the otic capsule and displacement of the cochlear implant electrode array into the middle ear space.

Effects of cochlear drilling with Piezosurgery Medical device in rats.

OBJECTIVES/HYPOTHESIS: Drilling on the otic capsule for cochleostomy should be less traumatic to the cochlea with the Piezosurgery Medical device (PZ) than with a standard diamond drill (DD). "Soft" cochleostomy is used for preservation of residual hearing in cochlear implant patients. PZ drilling can be used for accurate cochleostomy placement with minimal soft-tissue damage and may be superior for atraumatic drilling on the cochlea, as compared with a DD. This study compared inner ear effects after drilling the rat otic capsule with the PZ versus the DD. STUDY DESIGN: Prospective animal study using rats. METHODS: Otic capsule drilling was performed on the left ear with the DD (n = 5) or the PZ (n = 5), while otic capsule temperature was monitored. Contralateral ears served as controls. The animals were sacrificed after 1 week. Organ of Corti damage was morphologically evaluated and compared between groups. RESULTS: Basal turn hair cell loss was observed in all ears in the PZ group, regardless of drilling depth. However, no cochlear damage was found in any ears in the DD group. CONCLUSIONS: Otic capsule drilling with the PZ results in greater trauma to the rat inner ear than drilling using conventional methods.

Comparison of systemic and otic administration of ofloxacin.

OBJECTIVES: To assess the feasibility of delivering ofloxacin across the intact tympanic membrane; to compare middle ear bioavailability of ofloxacin after otic and systemic administrations; to determine distribution of otically delivered ofloxacin to other tissues. STUDY DESIGN: A prospective, controlled animal study. METHODS: Rats underwent surgery wherein the middle ear cavity was opened and filled with saline. An equivalent amount of ofloxacin was delivered intraperitoneally or into the external ear canal. Saline within the middle ear was sampled and completely replaced in 15-minute intervals for 3 hours. Blood was collected twice after the initial application of ofloxacin for high-performance liquid chromatography (HPLC). Animals were sacrificed 3 hours after the initial addition of ofloxacin; the temporal bones were harvested for histological analysis; urine and colon mucosa were collected for HPLC analysis. RESULTS: Both systemic and otic applications led to a comparable accumulation of ofloxacin in the middle ear over the 3-hour period after the initial administration. The pharmacokinetics of ofloxacin penetration into the middle ear was sporadic and subject-dependent. Both methods of administration led to drug accumulation in blood serum, urine, and colonic mucosa. CONCLUSIONS: Topical application of ofloxacin to the intact tympanic membrane allows for drug penetration into the middle ear space. Similar middle ear ofloxacin levels could be achieved with systemic and topic applications, but drug concentrations were inconsistent. The accumulation of ofloxacin in other tissues suggests applications designed to be ototopical may also result in systemic absorption.

Ototoxicity of topical azithromycin solutions in the guinea pig.

OBJECTIVE: To investigate possible ototoxic effects of topical azithromycin (AZ) in the guinea pig. DESIGN: A prospective, controlled animal study. SETTING: The University of Texas Southwestern Medical Center at Dallas. PARTICIPANTS: Twenty-three pigmented guinea pigs were given single, unilateral middle ear applications of a solution containing 3% (n = 3), 2% (n = 5), 1% (n = 5), or 0.5% (n = 5) AZ or saline (n = 5). The contralateral ear served as the untreated control. MAIN OUTCOME MEASURES: The animals were observed for behavioral changes for 2 weeks and then humanely killed. The ears were processed for anatomical evaluation. Morphologic changes were analyzed by quantitation of middle ear changes and cochlear inner and outer hair cell loss. Statistical analysis was performed to examine effects by dose. RESULTS: Analysis revealed extensive middle and inner ear changes associated with all formulations of AZ. Moderate correlation was found between the extent of middle ear changes and AZ concentration (r(2) = 0.59), whereas a strong correlation was seen between inner ear damage and AZ concentration (r(2) = 0.94). Both inner and outer hair cells were affected, with inner hair cell damage consistently greater than outer hair cell damage. CONCLUSIONS: The results of this study demonstrate that ototopical AZ can cause middle ear changes and significant hair cell loss in the guinea pig. This finding, together with previous clinical reports, indicates that topical AZ should be used with caution in the clinical setting.

Controlled release of hepatocyte growth factor from a bovine acellular scaffold for vocal fold reconstruction.

A bovine acellular scaffold was found to facilitate tissue remodeling in a rat model of vocal fold injury, whereas hepatocyte growth factor (HGF) has been shown to have an antiscarring effect in the larynx. This study examined the loading and release kinetics of HGF in vitro, and the potential of the acellular scaffold as a timed-release system for the delivery of HGF in vivo. Bilateral wounds were created in the posterior vocal folds of 20 rats, with HGF-loaded acellular scaffolds implanted into the wounds unilaterally, and scaffolds without HGF implanted into the contralateral vocal folds as control. The rats were humanely sacrificed after 3, 7, 30, and 90 days and their larynges were examined histologically and immunohistochemically. Expressions of key matrix proteins in the vocal fold coronal sections were quantified by digital image analysis. Results demonstrated a gradual, sustained release of HGF for at least 7 days in vitro, consistent with the detection of glycosaminoglycans inherent of the scaffold. In rat vocal folds implanted with HGF-loaded scaffolds, apparently fewer inflammatory cells were observed 3 days after surgery when compared to the control. The mean relative densities of collagen III and hyaluronic acid were significantly lower than those of the control 7 days after surgery. Scaffold implants were apparently degraded by 3 months in all animals, with no evidence of fibrosis or calcification. These data suggested that the bovine acellular scaffold could be promising for the exogenous delivery of select growth factors in vivo.

A bovine acellular scaffold for vocal fold reconstruction in a rat model.

With a rat model of vocal fold injury, this study examined the in vivo host response to an acellular xenogeneic scaffold derived from the bovine vocal fold lamina propria, and the potential of the scaffold for constructive tissue remodeling. Bilateral wounds were created in the posterior vocal folds of 20 rats, and bovine acellular scaffolds were implanted into the wounds unilaterally, with the contralateral vocal folds as control. The rats were humanely sacrificed after 3 days, 7 days, 1 month, and 3 months, and the coronal sections of their larynges were examined histologically. Expressions of key matrix proteins including collagen I, collagen III, elastin, fibronectin, hyaluronic acid, and glycosaminoglycans (GAGs) were quantified with digital image analysis. Significant infiltration of host inflammatory cells and host fibroblasts in the scaffold implant was observed in the acute stage of wound repair (3 days and 7 days postsurgery). The mean relative densities of collagen I, collagen III, and GAGs in the implanted vocal folds were significantly higher than those in the control after 3 days, followed by gradual decreases over 3 months. Histological results showed that the scaffolds were apparently degraded by 3 months, with no fibrotic tissue formation or calcification. These preliminary findings suggested that the bovine acellular scaffold could be a potential xenograft for vocal fold regeneration.

Biofilm presence in humans with chronic suppurative otitis media.

OBJECTIVE: To study the presence of biofilm formation in humans with chronic suppurative otitis media (CSOM). STUDY DESIGN: Cross-sectional study. SETTING: Tertiary academic hospital. SUBJECTS AND METHODS: Patients undergoing middle ear surgery between July 2006 and March 2008. Tissue samples were obtained from 25 patients, of which 20 specimens were successfully processed for this study. The remaining samples were not analyzed due to sample damage or loss during preparation. Of the 20 specimens studied, 10 were harvested as the experimental group from patients with CSOM and the other 10 harvested as controls from patients undergoing otologic surgery for acoustic neuroma, cochlear implant, or routine tympanoplasty. Ages ranged from 26 to 74 years (mean 45 yrs). Male-to-female ratio was 2:3. Scanning electron microscopy and confocal laser scanning microscopy were used to identify the presence of biofilms. Live-dead staining was used to assess whether bacteria present were viable. The outcome measured was the presence of adherent biofilms on middle ear mucosa. RESULTS: Biofilms were present in six of 10 samples (60%) from the CSOM group, but only in one of 10 control samples (10%). Comparative analysis revealed a statistically significant difference (P < 0.05) in the presence of biofilms in specimens from the CSOM group versus the control group. CONCLUSION: Biofilms were statistically more common in patients with CSOM compared with control patients.

A mouse model with postnatal endolymphatic hydrops and hearing loss.

Endolymphatic hydrops (ELH), hearing loss and neuronal degeneration occur together in a variety of clinically significant disorders, including Meniere's disease (MD). However, the sequence of these pathological changes and their relationship to each other are not well understood. In this regard, an animal model that spontaneously develops these features postnatally would be useful for research purposes. A search for such a model led us to the Phex Hyp-Duk mouse, a mutant allele of the Phex gene causing X-linked hypophosphatemic rickets. The hemizygous male (Phex Hyp-Duk/Y) was previously reported to exhibit various abnormalities during adulthood, including thickening of bone, ELH and hearing loss. The reported inner-ear phenotype was suggestive of progressive pathology and spontaneous development of ELH postnatally, but not conclusive. The main focuses of this report are to further characterize the inner ear phenotype in Phex Hyp-Duk/Y mice and to test the hypotheses that (a) the Phex Hyp-Duk/Y mouse develops ELH and hearing loss postnatally, and (b) the development of ELH in the Phex Hyp-Duk/Y mouse is associated with obstruction of the endolymphatic duct (ED) due to thickening of the surrounding bone. Auditory brainstem response (ABR) recordings at various times points and histological analysis of representative temporal bones reveal that Phex Hyp-Duk/Y mice typically develop adult onset, asymmetric, progressive hearing loss closely followed by the onset of ELH. ABR and histological data show that functional degeneration precedes structural degeneration. The major degenerative correlate of hearing loss and ELH in the mutants is the primary loss of spiral ganglion cells. Further, Phex Hyp-Duk/Y mice develop ELH without evidence of ED obstruction, supporting the idea that ELH can be induced by a mechanism other than the blockade of longitudinal flow of endolymphatic fluid, and occlusion of ED is not a prerequisite for the development of ELH in patients.

Development of outer hair cells in Ames waltzer mice: mutation in protocadherin 15 affects development of cuticular plate and associated structures.

The Ames waltzer (av) mouse mutant harbors a mutation in the protocadherin 15 gene (Pcdh15) and is a model for deafness in Usher syndrome 1F and nonsyndromic deafness DFNB23. Mutation in Pcdh15 affects stereocilia morphogenesis and polarity. Disruptions of apical cellular components in outer hair cells have also been described in av mutants. Organization of stereocilia and cell polarization may be dependent on proper orientation of structural components residing in the apical portion of the cell during development. We used electron and immunofluorescent microscopy to examine structural maturation of outer hair cells in av3J mice with emphasis on the fonticulus, basal body/centriole complex, actin mesh, and the microtubule network during initiation of bundle organization, between embryonic day (E) 16.5 and postnatal day 5 (P5). We found major ultrastructural rearrangements near the hair cell surface in av3J mice. Earliest changes were in kinocilia, basal body, and stereocilia positioning and microtubule arrangement once the kinocilia had lateralized to the side of the cell (between E16.5 and postnatal day [P] 0, before cuticular plate formation and stereocilia elongation). By P0, the developing fonticulus in av mice appeared enlarged, with a normal vesicle density. Stereocilia bundle disorganization increased after P0, with disruptions of the actin mesh within the cuticular plate. These observations support the hypothesis that mutations in Pcdh15 in av3J mice adversely affect coordinated maturation of apical cell components, resulting in disturbed stereocilia bundle polarity in av mice.

Cochlear neuronal tracing for frequency mapping with DiI, NeuroVue, and Golgi methods.

CONCLUSIONS: Labeling experiments using NeuroVue Red dye allowed us to demonstrate individual afferent fiber tracks in the cochlea from the synaptic region of the inner hair cell in the organ of Corti (OC) to the spiral ganglion in Rosenthal's canal. Further optimization is necessary to obtain 3-dimensional (3D) neural distribution in the apical region for frequency mapping. OBJECTIVES: We intend to develop a method by which the radial fibers of the spiral ganglion (SG) can be individually visualized and tracked in 3D from the base to the apex of the cochlea. The combined trajectories of fibers from each cochlea could then be calculated for modeling of the 3D relationship of OC and SG in cochlear implant studies to assist in the optimization of cochlear implants for music and speech perception in noise. MATERIALS AND METHODS: We tested three different methods to visualize cochlear nerve fibers from OC to SG. Adult rat and mouse ears were stained with DiI dye, modified Golgi-Cox method or NeuroVue dye, sectioned or whole-mounted, and viewed with confocal or standard light microscope. RESULTS: In DiI staining, spacial resolution and the number of neurons to be stained are too low to utilize this method to create a characteristic frequency map of the cochlea. The Golgi method mainly stained efferent nerve fibers, resulting in less information on cochlear nerve distribution. NeuroVue Red dye allowed clear tracking of individual fibers when combined with DAPI counterstaining.

Progression of inner ear pathology in Ames waltzer mice and the role of protocadherin 15 in hair cell development.

The Ames waltzer (av) mouse mutant exhibits auditory and vestibular abnormalities resulting from mutation of protocadherin 15 (Pcdh15). Ames waltzer has been identified as an animal model for inner ear pathology associated with Usher syndrome type 1F. Studies correlating anatomical phenotype with severity of genetic defect in various av alleles are providing better understanding of the role played by Pcdh15 in inner ear development and of sensorineural abnormalities associated with alterations in Pcdh15 protein structure as a result of gene mutation. In this work we present new findings on inner ear pathology in four alleles of av mice with differing mutations of Pcdh15 as well as varying alterations in inner ear morphology. Two alleles with in-frame deletion mutations (Pcdh15 (av-J) and Pcdh15 (av-2J)) and two presumptive functional null alleles (Pcdh15 (av-3J) and Pcdh15 (av-Tg)) were studied. Light and electron microscopic observations demonstrated that the severity of cochlear and vestibular pathology in these animals correlates positively with the extent of mutation in Pcdh15 from embryonic day 18 (E18) up to 12 months. Electron microscopic analysis of immature ears indicated early abnormalities in the arrangement of stereocilia and the inner and outer hair cell cuticular plates, stereocilia rootlets, and the actin meshwork within the cuticular plate. In severe cases, displacement of the kinocilium and alterations in the shape of the cuticular plate was also observed. Mice harboring in-frame deletion mutations showed less disorganization of stereocilia and cuticular plates in the organ of Corti than the presumptive functional null alleles at P0-P10. A slower progression of pathology was also seen via light microscopy in older animals with in-frame deletions, compared to the presumptive functional null mutations. In summary, our results demonstrate that mutation in Pcdh15 affects the initial formation of stereocilia bundles with associated changes in the actin meshwork within the cuticular plate; these effects are more pronounced in the presumed null mutation compared to mutations that only affect the extracellular domain. The positive correlation of severity of effects with extent of mutation can be seen well into adulthood.

Bacterial biofilm formation on a human cochlear implant.

OBJECTIVE: To report the characteristics of a bacterial biofilm from the surface of a cochlear implant. BACKGROUND: Bacterial biofilm formation on implanted devices causes intractable infections and device extrusions necessitating device removal, with loss of function. More information is needed about biofilm characteristics and interactions with the implant surface before better treatments can be designed. STUDY DESIGN: A retrospective case review was combined with a descriptive histological study of the surface of an otologic device. METHODS: The receiver/stimulator device removed from a cochlear implant patient because of intractable infection and partial device extrusion was fixed and processed for microscopic examination. Its surface and the material present on its surface were analyzed using light and electron microscopy, focusing on surface texture, cell types, and bacteria species and extracellular polymeric substances present within the biofilm. RESULTS: Stereomicroscopic examination revealed extracellular polymeric substances, pinkish yellow in color, with spheres of uniform size scattered throughout, indicative of a biofilm containing Staphylococcus aureus. Biofilm density was greatest in depressions on the surface of the implant. Cross-sectional analysis revealed bacteria interspersed with polymorphonuclear leukocytes. Scanning electron microscopic examination demonstrated an amorphous layer of extracellular polymeric substances containing small filaments, bacteria, and inflammatory cells. Only Staphylococcus aureus was detected. CONCLUSION: Cochlear implant material can provide a surface for bacterial biofilm formation. Impressions can provide an environment conducive to biofilm establishment and growth, ultimately necessitating device removal, with loss of implant function. Biofilm characterization should aid in design of cochlear implant devices less susceptible to biofilm formation.

Characterization of vestibular dysfunction in the mouse model for Usher syndrome 1F.

The deaf-circling Ames waltzer (av) mouse harbors a mutation in the protocadherin 15 (Pcdh15) gene and is a model for inner ear defects associated with Usher syndrome type 1F. Earlier studies showed altered cochlear hair cell morphology in young av mice. In contrast, no structural abnormality consistent with significant vestibular dysfunction in young av mice was observed. Light and scanning electron microscopic studies showed that vestibular hair cells from presumptive null alleles Pcdh15(av-Tg) and Pcdh15(av-3J) are morphologically similar to vestibular sensory cells from control littermates, suggesting that the observed phenotype in these alleles might be a result of a central, rather than peripheral, defect. In the present study, a combination of physiologic and anatomic methods was used to more thoroughly investigate the source of vestibular dysfunction in Ames waltzer mice. Analysis of vestibular evoked potentials and angular vestibulo-ocular reflexes revealed a lack of physiologic response to linear and angular acceleratory stimuli in Pcdh15 mutant mice. Optokinetic reflex function was diminished but still present in the mutant animals, suggesting that the defect is primarily peripheral in nature. These findings indicate that the mutation in Pcdh15 results in either a functional abnormality in the vestibular receptor organs or that the defects are limited to the vestibular nerve. AM1-43 dye uptake has been shown to correlate with normal transduction function in hair cells. Dye uptake was found to be dramatically reduced in Pcdh15 mutants compared to control littermates, suggesting that the mutation affects hair cell function, although structural abnormalities consistent with significant vestibular dysfunction are not apparent by light and scanning electron microscopy in the vestibular neuroepithelia of young animals.