Multichannel cochlear implant for selective neuronal activation and chronic use in the free-moving Mongolian gerbil.

BACKGROUND: Animal models for chronic multichannel cochlear implant stimulation and selective neuronal activation contribute to a better understanding of auditory signal processing and central neural plasticity. NEW METHOD: This paper describes the design and surgical implantation of a multichannel cochlear implant (CI) system for chronic use in the free-moving gerbil. For chronic stimulation, adult-deafened gerbils were connected to a multichannel commutator that allowed low resistance cable rotation and stable electric connectivity to the current source. RESULTS: Despite the small scale of the gerbil cochlea and auditory brain regions, final electrophysiological mapping experiments revealed selective and tonotopically organized neuronal activation in the auditory cortex. Contact impedances and electrically evoked auditory brainstem responses were stable over several weeks demonstrating the long-term integrity of the implant and the efficacy of the stimulation. COMPARISON WITH EXISTING METHODS: Most animal models on multichannel signal processing and stimulation-induced plasticity are limited to larger animals such as ferrets, cats and primates. Multichannel CI stimulation in the free-moving rodent and evidence for selective neuronal activation in gerbil auditory cortex have not been previously reported. CONCLUSIONS: Overall, our results show that the gerbil is a robust rodent model for selective and tonotopically organized multichannel CI stimulation. We anticipate that this model provides a useful tool to develop and test both passive stimulation and behavioral training strategies for plastic reorganization and restoration of degraded unilateral and bilateral central auditory signal processing in the hearing impaired and deaf central auditory system.

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.

Validation of minimally invasive, image-guided cochlear implantation using Advanced Bionics, Cochlear, and Medel electrodes in a cadaver model.

PURPOSE: Validation of a novel minimally invasive, image-guided approach to implant electrodes from three FDA-approved manufacturers-Medel, Cochlear, and Advanced Bionics-in the cochlea via a linear tunnel from the lateral cranium through the facial recess to the cochlea. METHODS: Custom microstereotactic frames that mount on bone-implanted fiducial markers and constrain the drill along the desired path were utilized on seven cadaver specimens. A linear tunnel was drilled from the lateral skull to the cochlea followed by a marginal, round window cochleostomy and insertion of the electrode array into the cochlea through the drilled tunnel. Post-insertion CT scan and histological analysis were used to analyze the results. RESULTS: All specimens ([Formula: see text]) were successfully implanted without visible injury to the facial nerve. The Medel electrodes ([Formula: see text]) had minimal intracochlear trauma with 8, 8, and 10 (out of 12) electrodes intracochlear. The Cochlear lateral wall electrodes (straight research arrays) ([Formula: see text]) had minimal trauma with 20 and 21 of 22 electrodes intracochlear. The Advanced Bionics electrodes ([Formula: see text]) were inserted using their insertion tool; one had minimal insertion trauma and 14 of 16 electrodes intracochlear, while the other had violation of the basilar membrane just deep to the cochleostomy following which it remained in scala vestibuli with 13 of 16 electrodes intracochlear. CONCLUSIONS: Minimally invasive, image-guided cochlear implantation is possible using electrodes from the three FDA-approved manufacturers. Lateral wall electrodes were associated with less intracochlear trauma suggesting that they may be better suited for this surgical technique.

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.

Vascular trauma during cochlear implantation: a contributor to residual hearing loss?

OBJECTIVE: This study was performed to investigate the vascular structures of the cochlea that are potentially vulnerable to mechanical trauma during cochlear implant surgery. BACKGROUND: Despite improvements in surgical technique and electrode design, residual hearing is lost in a significant percentage of cochlear implant patients. Although a variety of factors may contribute, it is widely believed that mechanical trauma plays an important role. This study focused on the vasculature of scala tympani and its potential susceptibility to injury during implant surgery. METHODS: Anatomic study of normal human temporal bones prepared by either conventional cross sectioning or by microdissection for examination by light or scanning electron microscopy. RESULTS: Blood vessels located at or near the perilymphatic surface of scala tympani are predominately of the venous type, and they are situated so as to be at risk for trauma during implantation. Those vessels include veins on the lateral wall and floor of scala tympani, the modiolar wall and the undersurface of the osseous lamina and basilar membrane. CONCLUSION: Injury or occlusion of blood vessels associated with scala tympani may adversely affect inner ear function, potentially contributing to hearing loss following cochlear implantation.

Cochlear implantation updates: the Dallas Cochlear Implant Program.

This report provides an overview of many research projects conducted by the Dallas Cochlear Implant Program, a joint enterprise between the University of Texas at Dallas, the University of Texas Southwestern Medical Center, and Children's Medical Center. The studies extend our knowledge of factors influencing communication outcomes in users of cochlear implants. Multiple designs and statistical techniques are used in the studies described including both cross sectional and longitudinal analyses. Sample sizes vary across the studies, and many of the samples represent large populations of children from North America. Multiple statistical techniques are used by the team to analyze outcomes. The team has provided critical information regarding electrode placement, signal processing, and communication outcomes in users of cochlear implants.

Development and evaluation of the modiolar research array--multi-centre collaborative study in human temporal bones.

OBJECTIVE: Multi-centre collaborative study to develop and refine the design of a prototype thin perimodiolar cochlear implant electrode array and to assess feasibility for use in human subjects. STUDY DESIGN: Multi-centre temporal bone insertion studies. MATERIALS AND METHODS: The modiolar research array (MRA) is a thin pre-curved electrode that is held straight for initial insertion with an external sheath rather than an internal stylet. Between November 2006 and February 2009, six iterations of electrode design were studied in 21 separate insertion studies in which 140 electrode insertions were performed in 85 human temporal bones by 12 surgeons. These studies aimed at addressing four fundamental questions related to the electrode concept, being: (1) Could a sheath result in additional intra-cochlear trauma? (2) Could a sheath accommodate variations in cochlea size and anatomies? (3) Could a sheath be inserted via the round window? and (4) Could a sheath be safely removed once the electrode had been inserted? These questions were investigated within these studies using a number of evaluation techniques, including X-ray and microfluoroscopy, acrylic fixation and temporal bone histologic sectioning, temporal bone microdissection of cochlear structures with electrode visualization, rotational tomography, and insertion force analysis. RESULTS: Frequent examples of electrode rotation and tip fold-over were demonstrated with the initial designs. This was typically caused by excessive curvature of the electrode tip, and also difficulty in handling of the electrode and sheath. The degree of tip curvature was progressively relaxed in subsequent versions with a corresponding reduction in the frequency of tip fold-over. Modifications to the sheath facilitated electrode insertion and sheath removal. Insertion studies with the final MRA design demonstrated minimal trauma, excellent perimodiolar placement, and very small electrode dimensions within scala tympani. Force measurements in temporal bones demonstrated negligible force on cochlear structures with angular insertion depths of between 390 and 450°. CONCLUSION: The MRA is a novel, very thin perimodiolar prototype electrode array that has been developed using a systematic collaborative approach. The different evaluation techniques employed by the investigators contributed to the early identification of issues and generation of solutions. Regarding the four fundamental questions related to the electrode concept, the studies demonstrated that (1) the sheath did not result in additional intra-cochlear trauma; (2) the sheath could accommodate variations in cochlea size and anatomies; (3) the sheath was more successfully inserted via a cochleostomy than via the round window; and (4) the sheath could be safely removed once the electrode had been inserted.

Automatic Identification of Cochlear Implant Electrode Arrays for Post-Operative Assessment.

Cochlear implantation is a procedure performed to treat profound hearing loss. Accurately determining the postoperative position of the implant in vivo would permit studying the correlations between implant position and hearing restoration. To solve this problem, we present an approach based on parametric Gradient Vector Flow snakes to segment the electrode array in post-operative CT. By combining this with existing methods for localizing intra-cochlear anatomy, we have developed a system that permits accurate assessment of the implant position in vivo. The system is validated using a set of seven temporal bone specimens. The algorithms were run on pre- and post-operative CTs of the specimens, and the results were compared to histological images. It was found that the position of the arrays observed in the histological images is in excellent agreement with the position of their automatically generated 3D reconstructions in the CT scans.

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.

Round window insertion of precurved perimodiolar electrode arrays: how successful is it?

HYPOTHESIS: The goal of this human temporal bone study was to assess the safety and efficacy of round window insertion of the Cochlear Contour Advance electrode and identify any anatomic obstacles that may affect its insertion. BACKGROUND: Minimizing intracochlear trauma during electrode insertion reduces the risk of losing residual hearing. The Contour Advance electrode is designed for insertion via an anteroinferior cochleostomy, in which drilling causes a degree of inner ear trauma. If consistently successful round window insertions are possible with this electrode, it would allow for better perimodiolar positioning in the basal turn and greater likelihood of hearing preservation. METHODS: A total of 16 human temporal bone round window insertions with the Contour Advance electrode were performed at the University of Melbourne and Southwestern Medical Center, Dallas. Surgeons' observations, fluoroscopy, histology and anatomic dissections were analyzed. RESULTS: Successful full insertions of the electrode with minimal resistance and good perimodiolar placement were achieved in less that 50% of cases in the Melbourne series and in two-thirds of cases in the Dallas series. A common finding by both centers was obstruction of electrode entry at the anteroinferior margin of the round window, which prevented optimal scala tympani positioning and often resulted in intracochlear trauma. CONCLUSION: The anteroinferior region of the round window bony margin influences the trajectory of insertion of the relatively large Contour Advance electrode as well as physically obstructing and distorting the array. A pure round window insertion is not predictable and reliable enough to be a recommended approach for this electrode.

Anatomic verification of a novel method for precise intrascalar localization of cochlear implant electrodes in adult temporal bones using clinically available computed tomography.

OBJECTIVES/HYPOTHESIS: We have previously described a novel, automated, nonrigid, model-based method for determining the intrascalar position of cochlear implant (CI) electrode arrays within human temporal bones using clinically available, flat-panel volume computed tomography (fpVCT). We sought to validate this method by correlating results with anatomic microdissection of CI arrays in cadaveric bones. STUDY DESIGN: Basic science. METHODS: Seven adult cadaveric temporal bones were imaged using fpVCT before and after electrode insertion. Using a statistical model of intracochlear anatomy, an active shape model optimization approach was employed to identify the scalae tympani and vestibuli on the preintervention fpVCT. The array position was estimated by identifying its midline on the postintervention scan and superimposing it onto the preintervention images using rigid registration. Specimens were then microdissected to demonstrate the actual array position. RESULTS: Using microdissection as the standard for ascertaining electrode position, automatic identification of the basilar membrane coupled with postintervention fpVCT for electrode position identification accurately depicted the array location in all seven bones. In four specimens, the array remained within the scala tympani; in three, the basilar membrane was breached. CONCLUSIONS: We have anatomically validated this automated method for predicting the intrascalar location of CI arrays using CT. Using this algorithm and pre- and postintervention CT, rapid feedback regarding implant location and expected audiologic outcomes could be obtained in clinical settings.

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.

Usher syndrome IIIA gene clarin-1 is essential for hair cell function and associated neural activation.

Usher syndrome 3A (USH3A) is an autosomal recessive disorder characterized by progressive loss of hearing and vision due to mutation in the clarin-1 (CLRN1) gene. Lack of an animal model has hindered our ability to understand the function of CLRN1 and the pathophysiology associated with USH3A. Here we report for the first time a mouse model for ear disease in USH3A. Detailed evaluation of inner ear phenotype in the Clrn1 knockout mouse (Clrn1(-/-)) coupled with expression pattern of Clrn1 in the inner ear are presented here. Clrn1 was expressed as early as embryonic day 16.5 in the auditory and vestibular hair cells and associated ganglionic neurons, with its expression being higher in outer hair cells (OHCs) than inner hair cells. Clrn1(-/-) mice showed early onset hearing loss that rapidly progressed to severe levels. Two to three weeks after birth (P14-P21), Clrn1(-/-) mice showed elevated auditory-evoked brainstem response (ABR) thresholds and prolonged peak and interpeak latencies. By P21, approximately 70% of Clrn1(-/-) mice had no detectable ABR and by P30 these mice were deaf. Distortion product otoacoustic emissions were not recordable from Clrn1(-/-) mice. Vestibular function in Clrn1(-/-) mice mirrored the cochlear phenotype, although it deteriorated more gradually than cochlear function. Disorganization of OHC stereocilia was seen as early as P2 and by P21 OHC loss was observed. In sum, hair cell dysfunction and prolonged peak latencies in vestibular and cochlear evoked potentials in Clrn1(-/-) mice strongly indicate that Clrn1 is necessary for hair cell function and associated neural activation.

Anatomy of the middle-turn cochleostomy.

OBJECTIVE: Middle-turn cochleostomies are occasionally used for cochlear implant electrode placement in patients with labyrinthitis ossificans. This study evaluates the anatomic characteristics of the middle-turn cochleostomy and its suitability for placement of implant electrodes. METHODS: Ten cadaveric human temporal bones were dissected using a facial recess approach. A middle-turn cochleostomy was drilled 2 mm anterior to the oval window and just inferior to the cochleariform process. The preparations were then stained with osmium tetroxide and microdissections were performed. The location of the cochleostomy on the cochlear spiral and its path through the various cochlear compartments were evaluated in all 10 specimens. A Cochlear Corporation depth gauge was inserted in five of the specimens and insertion trauma, number of contact rings, and depth of insertion were recorded. RESULTS: Eight of the 10 cochleostomies were placed at approximately 360 degrees on the cochlear spiral, near the transition between the basal and middle turns. In one case, the cochleostomy was found to enter the cochlear apex and in another it entered scala vestibuli of the proximal basal turn. The cochleostomy entered scala media in six bones and scala vestibuli in four specimens. A depth gauge was inserted in five specimens. The number of contacts placed within the cochlear lumen ranged from four to nine. There was evidence of insertional trauma to the lateral wall of the cochlear duct, basilar membrane, and Reissner's membrane, but no evidence of fractures to the osseous spiral lamina or modiolus. CONCLUSION: This study demonstrates that electrodes inserted via a middle-turn cochleostomy are likely to enter scala vestibuli and have access to the middle- and apical-cochlear turns. It is also possible that the electrode could be directed into the descending portion of the basal turn depending on cochleostomy orientation. Middle-turn cochleostomy seems to be a viable alternative for electrode placement when preservation of residual hearing is not a concern.

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 implant electrode insertion: the round window revisited.

OBJECTIVE: To examine aspects of round window (RW) anatomy that are relevant to its use as a portal for atraumatic insertion of cochlear implant electrodes. STUDY DESIGN: Anatomic study using human cadaveric temporal bones. METHODS: A series of 30 temporal bones was dissected to permit microscopic study of the RW region. RESULTS: The bony overhangs of the RW niche limit visibility of the RW membrane during surgery. Measurements of RW membrane area visible through a facial recess opening before and after drilling the overhangs in 15 temporal bones showed that RW membrane visibility is typically increased by a factor of 1.5 to 3 times after drilling and by as much as 13 times when the opening of the RW niche is relatively small. Observations from within the scala tympani in 15 cochlear dissections showed substantial variability in size of the RW opening available for electrode insertion. Area measurements of the portion of the RW covered by the vertical segment of the RW membrane ranged from 0.8 to 1.75 mm2 in these specimens. In addition, irregularities in contour of the RW margin may make insertion challenging, which may necessitate drilling the anterior-inferior margin of the RW. Drilling in this region should be approached with care because of the close proximity of the cochlear aqueduct opening. CONCLUSION: RW insertion can be performed in a manner that is potentially less traumatic than the standard cochleostomy insertion. It may therefore be advantageous in cases in which hearing preservation is the goal.

Surgical aspects of cochlear implantation: mechanisms of insertional trauma.

The development of hybrid electroacoustic devices has made conservation of residual hearing an important goal in cochlear implant surgery. Our laboratory has recently conducted anatomical studies directed toward better understanding mechanisms underlying loss of residual hearing associated with electrode insertion. This paper provides an overview of observations based on microdissection, scanning electron microscopy and temporal bone histology relating to inner ear injury that may occur during implant surgery. Trauma to cochlear structures including lateral wall tissues, the basilar membrane, the osseous spiral lamina and the modiolus is considered in relation to the implications of specific types of injury for hearing preservation. These findings are relevant to the design of future implant devices and to various important issues regarding the surgical technique used for implantation, including the possible use of the round window as a portal of entry for electrode insertion.

Gene expression analysis of distinct populations of cells isolated from mouse and human inner ear FFPE tissue using laser capture microdissection--a technical report based on preliminary findings.

Laser Capture Microdissection (LCM) allows microscopic procurement of specific cell types from tissue sections that can then be used for gene expression analysis. We first tested this method with sections of adult mouse inner ears and subsequently applied it to human inner ear sections. The morphology of the various cell types within the inner ear is well preserved in formalin fixed paraffin embedded (FFPE) sections, making it easier to identify cell types and their boundaries. Recovery of good quality RNA from FFPE sections can be challenging, however, recent studies in cancer research demonstrated that it is possible to carry out gene expression analysis of FFPE material. Thus, a method developed using mouse FFPE tissue can be applied to human archival temporal bones. This is important because the majority of human temporal bone banks have specimens preserved in formalin and a technique for retrospective analysis of human archival ear tissue is needed. We used mouse FFPE inner ear sections to procure distinct populations of cells from the various functional domains (organ of Corti, spiral ganglion, etc.) by LCM. RNA was extracted from captured cells, amplified, and assessed for quality. Expression of selected genes was tested by RT-PCR. In addition to housekeeping genes, we were able to detect cell type specific markers, such as Myosin 7a, p27(kip1) and neurofilament gene transcripts that confirmed the likely composition of cells in the sample. We also tested the method described above on FFPE sections from human crista ampullaris. These sections were approximately a year old. Populations of cells from the epithelium and stroma were collected and analyzed independently for gene expression. The method described here has potential use in many areas of hearing research. For example, following exposure to noise, ototoxic drugs or age, it would be highly desirable to analyze gene expression profiles of selected populations of cells within the organ of Corti or spiral ganglion cells rather than a mixed population of cells from whole inner ear tissue. Also, this method can be applied for analysis of human archival ear tissue.

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.