Visualizing Collagen Fibrils in the Cochlea's Tectorial and Basilar Membranes Using a Fluorescently Labeled Collagen-Binding Protein Fragment.

PURPOSE: A probe that binds to unfixed collagen fibrils was used to image the shapes and fibrous properties of the TM and BM. The probe (CNA35) is derived from the bacterial adhesion protein CNA. We present confocal images of hydrated gerbil TM, BM, and other cochlear structures stained with fluorescently labeled CNA35. A primary purpose of this article is to describe the use of the CNA35 collagen probe in the cochlea. METHODS: Recombinant poly-histidine-tagged CNA35 was expressed in Escherichia coli, purified by cobalt-affinity chromatography, fluorescence labeled, and further purified by gel filtration chromatography. Cochleae from freshly harvested gerbil bullae were irrigated with and then incubated in CNA35 for periods ranging from 2 h - overnight. The cochleae were fixed, decalcified, and dissected. Isolated cochlear turns were imaged by confocal microscopy. RESULTS: The CNA35 probe stained the BM and TM, and volumetric imaging revealed the shape of these structures and the collagen fibrils within them. The limbal zone of the TM stained intensely. In samples from the cochlear base, intense staining was detected on the side of the TM that faces hair cells. In the BM pectinate zone, staining was intense at the upper and lower boundaries. The BM arcuate zone was characterized by a prominent longitudinal collagenous structure. The spiral ligament, limbus and lamina stained for collagen, and within the spiral limbus the habenula perforata were outlined with intense staining. CONCLUSION: The CNA35 probe provides a unique and useful view of collagenous structures in the cochlea.

Asymmetry and Microstructure of Temporal-Suppression Patterns in Basilar-Membrane Responses to Clicks: Relation to Tonal Suppression and Traveling-Wave Dispersion.

The cochlea's wave-based signal processing allows it to efficiently decompose a complex acoustic waveform into frequency components. Because cochlear responses are nonlinear, the waves arising from one frequency component of a complex sound can be altered by the presence of others that overlap with it in time and space (e.g., two-tone suppression). Here, we investigate the suppression of basilar-membrane (BM) velocity responses to a transient signal (a test click) by another click or tone. We show that the BM response to the click can be reduced when the stimulus is shortly preceded or followed by another (suppressor) click. More surprisingly, the data reveal two curious dependencies on the interclick interval, Δt. First, the temporal suppression curve (amount of suppression vs. Δt) manifests a pronounced and nearly periodic microstructure. Second, temporal suppression is generally strongest not when the two clicks are presented simultaneously (Δt = 0), but when the suppressor click precedes the test click by a time interval corresponding to one to two periods of the best frequency (BF) at the measurement location. By systematically varying the phase of the suppressor click, we demonstrate that the suppression microstructure arises from alternating constructive and destructive interference between the BM responses to the two clicks. And by comparing temporal and tonal suppression in the same animals, we test the hypothesis that the asymmetry of the temporal-suppression curve around Δt = 0 stems from cochlear dispersion and the well-known asymmetry of tonal suppression around the BF. Just as for two-tone suppression, BM responses to clicks are most suppressed by tones at frequencies just above the BF of the measurement location. On average, the frequency place of maximal suppressibility of the click response predicted from temporal-suppression data agrees with the frequency at which tonal suppression peaks, consistent with our hypothesis.

D-Galactose-induced oxidative stress and mitochondrial dysfunction in the cochlear basilar membrane: an in vitro aging model.

The cochlear basilar membrane (CBM) contains inner hair cells and outer hair cells that convert sound waves into electrical signals and transmit them to the central auditory system. Cochlear aging, the primary reason of age-related hearing loss, can reduce the signal transmission capacity. There is no ideal in vitro aging model of the CBM. In this study, we cultured the CBM, which was dissected from the cochlea of the C57BL/6 mice 5 days after birth, in a medium containing 20 mg/mL, 40 mg/mL, or 60 mg/mL D-galactose (D-gal). Compared with the control group, the levels of senescence-associated ß-galactosidase were increased in a concentration-dependent manner in the CBM of the D-gal groups. In addition, levels of the mitochondrial superoxide and patterns of an age-related mitochondrial DNA3860-bp deletion were significantly increased. The ATP levels and the membrane potential of the mitochondrial were significantly decreased in the CBM of the D-gal groups compared with the control group. Furthermore, in comparison with the control group, damaged hair cell stereocilia and a loss of inner hair cell ribbon synapses were observed in the CBM of the D-gal groups. A loss of hair cells and activation of caspase-3-mediated outer hair cell apoptosis were also observed in the CBM of the high-dose D-gal group. These insults induced by D-gal in the CBM in vitro were similar to the ones that occur in cochlear natural aging in vivo. Thus, we believe that this is a successful in vitro aging model using cultured CBM. These results demonstrate the effects of mitochondrial oxidative damage on presbycusis and provide a reliable aging model to study the mechanisms of presbycusis in vitro.

Expression Patterns and Implications of LaminB1 in Rat Cochleae.

LaminB1, a major component of the nuclear lamina, is a potent regulator of cellular proliferation and senescence and also known to be essential for neuronal migration and brain development. However, the expression patterns of LaminB1 in the rat cochleae are still not fully revealed. Utilizing immunofluorescence, Western blotting, and quantitative real-time PCR, we identified the distribution and expression of LaminB1 in the rat cochleae. Immunofluorescence staining indicated that LaminB1 was mainly localized in the auditory hair cells (HCs), spiral ganglion cells (SGC), stria vascularis (STV, including spiral ligament), Reissner's membrane (RM), and limbus laminae spiralis (LLS). Western blotting analysis illustrated that the distribution of LaminB1 in rat cochleae was characterized by tissue specificity. The LaminB1 protein was expressed more in SGC and basilar membrane (BM) than in STV. Meanwhile, the mRNA expression of LaminB1 displayed difference in cochlear tissues. These observations preliminarily revealed the expression patterns of LaminB1, providing a theoretical basis for further study on the role of LaminB1 in auditory function.

Differential Expression of LaminB1 in the Developing Rat Cochlea.

OBJECTIVES: To explore the temporal expression pattern of LaminB1 in the cochlea of postnatal rat, and whether LaminB1 is associated with cochlear development. MATERIALS AND METHODS: Sprague-Dawley rats ranging from postnatal day 0 (p0) to 21 (p21) were used. The tissues of stria vascularis (STV) including spiral ligament, spiral ganglion cell (SGC), and basilar membrane (BM), including the organ of Corti, were dissected, respectively. Immunofluorescence, quantitative real-time polymerase chain reaction, and western blot were applied to detect the expression of LaminB1 in individual cochlear tissues at both mRNA and protein levels. RESULTS: Immunofluorescence revealed that LaminB1 was localized in the outer hair cells, inner hair cells, Kolliker's organ, Reissner's membrane, SGC, STV, and spiral ligament. The intensity of staining surrounding the scala media decreased during cochlear development. The expression of LaminB1 mRNA and protein in STV, SGC, and BM was at a maximum level at p0 but gradually declined to a minimum level at p21. CONCLUSION: Our research provided direct evidence that LaminB1 was expressed in the developing cochlea and developmentally regulated in cochlear tissues, suggesting a possible role of LaminB1 in cochlear development. Our result provided a theoretical basis for further study about the physiological function of LaminB1 in the peripheral auditory system.

Decreased expression of TERT correlated with postnatal cochlear development and proliferation reduction of cochlear progenitor cells.

Cochlear progenitor cells are considered as one of the best candidates for hair cell regeneration, thus, the regulation of cochlear progenitor cell proliferation has become a focus in this field. Several genes expressed in the inner ear during postnatal development have been demonstrated to be involved in maintaining the proliferative potential of progenitor cells, but the mechanism for regulating the proliferation and differentiation of cochlear progenitor cells remains poorly understood. Telomerase reverse transcriptase (TERT) has rate limiting telomerase activity and the overexpression of TERT has been shown to promote cell proliferation in series of cell lines. The aim of the present study was to evaluate the expression of TERT in the postnatal development of the cochlea and progenitor cells. The results demonstrated that TERT was expressed in the basilar membranes during the first postnatal week. In vitro, TERT expression in progenitor cells reached a maximum at day 4 after culture and decreased as the culture time prolonged or the cell passage number increased. These results led us to hypothesize that TERT may be involved in the development of the cochlea and in maintaining the proliferation ability of progenitor cells.

Loss of sestrin 2 potentiates the early onset of age-related sensory cell degeneration in the cochlea.

Sestrin 2 (SESN2) is a stress-inducible protein that protects tissues from oxidative stress and delays the aging process. However, its role in maintaining the functional and structural integrity of the cochlea is largely unknown. Here, we report the expression of SESN2 protein in the sensory epithelium, particularly in hair cells. Using C57BL/6J mice, a mouse model of age-related cochlear degeneration, we observed a significant age-related reduction in SESN2 expression in cochlear tissues that was associated with early onset hearing loss and accelerated age-related sensory cell degeneration that progressed from the base toward the apex of the cochlea. Hair cell death occurred by caspase-8 mediated apoptosis. Compared to C57BL/6J control mice, Sesn2 KO mice displayed enhanced expression of proinflammatory genes and activation of basilar membrane macrophages, suggesting that loss of SESN2 function provokes the immune response. Together, these results suggest that Sesn2 plays an important role in cochlear homeostasis and immune responses to stress.

Amplification mode differs along the length of the mouse cochlea as revealed by connexin 26 deletion from specific gap junctions.

The sharp frequency tuning and exquisite sensitivity of the mammalian cochlea is due to active forces delivered by outer hair cells (OHCs) to the cochlear partition. Force transmission is mediated and modulated by specialized cells, including Deiters' cells (DCs) and pillar cells (PCs), coupled by gap-junctions composed of connexin 26 (Cx26) and Cx30. We created a mouse with conditional Cx26 knock-out (Cx26 cKO) in DCs and PCs that did not influence sensory transduction, receptor-current-driving-voltage, low-mid-frequency distortion-product-otoacoustic-emissions (DPOAEs), and passive basilar membrane (BM) responses. However, the Cx26 cKO desensitizes mid-high-frequency DPOAEs and active BM responses and sensitizes low-mid-frequency neural excitation. This functional segregation may indicate that the flexible, apical turn cochlear partition facilitates transfer of OHC displacements (isotonic forces) for cochlear amplification and neural excitation. DC and PC Cx26 expression is essential for cochlear amplification in the stiff basal turn, possibly through maintaining cochlear partition mechanical impedance, thereby ensuring effective transfer of OHC isometric forces.

A connexin30 mutation rescues hearing and reveals roles for gap junctions in cochlear amplification and micromechanics.

Accelerated age-related hearing loss disrupts high-frequency hearing in inbred CD-1 mice. The p.Ala88Val (A88V) mutation in the gene coding for the gap-junction protein connexin30 (Cx30) protects the cochlear basal turn of adult CD-1Cx30A88V/A88V mice from degeneration and rescues hearing. Here we report that the passive compliance of the cochlear partition and active frequency tuning of the basilar membrane are enhanced in the cochleae of CD-1Cx30A88V/A88V compared to CBA/J mice with sensitive high-frequency hearing, suggesting that gap junctions contribute to passive cochlear mechanics and energy distribution in the active cochlea. Surprisingly, the endocochlear potential that drives mechanoelectrical transduction currents in outer hair cells and hence cochlear amplification is greatly reduced in CD-1Cx30A88V/A88V mice. Yet, the saturating amplitudes of cochlear microphonic potentials in CD-1Cx30A88V/A88V and CBA/J mice are comparable. Although not conclusive, these results are compatible with the proposal that transmembrane potentials, determined mainly by extracellular potentials, drive somatic electromotility of outer hair cells.

Dynamic activation of basilar membrane macrophages in response to chronic sensory cell degeneration in aging mouse cochleae.

In the sensory epithelium, macrophages have been identified on the scala tympani side of the basilar membrane. These basilar membrane macrophages are the spatially closest immune cells to sensory cells and are able to directly respond to and influence sensory cell pathogenesis. While basilar membrane macrophages have been studied in acute cochlear stresses, their behavior in response to chronic sensory cell degeneration is largely unknown. Here we report a systematic observation of the variance in phenotypes, the changes in morphology and distribution of basilar membrane tissue macrophages in different age groups of C57BL/6J mice, a mouse model of age-related sensory cell degeneration. This study reveals that mature, fully differentiated tissue macrophages, not recently infiltrated monocytes, are the major macrophage population for immune responses to chronic sensory cell death. These macrophages display dynamic changes in their numbers and morphologies as age increases, and the changes are related to the phases of sensory cell degeneration. Notably, macrophage activation precedes sensory cell pathogenesis, and strong macrophage activity is maintained until sensory cell degradation is complete. Collectively, these findings suggest that mature tissue macrophages on the basilar membrane are a dynamic group of cells that are capable of vigorous adaptation to changes in the local sensory epithelium environment influenced by sensory cell status.

SMAD4 Defect Causes Auditory Neuropathy Via Specialized Disruption of Cochlear Ribbon Synapses in Mice.

More than 100 genes have been associated with deafness. However, SMAD4 is rarely considered a contributor to deafness in humans, except for its well-defined role in cell differentiation and regeneration. Here, we report that a SMAD4 defect in mice can cause auditory neuropathy, which was defined as a mysterious hearing and speech perception disorder in human for which the genetic background remains unclear. Our study showed that a SMAD4 defect induces failed formation of cochlear ribbon synapse during the earlier stage of auditory development in mice. Further investigation found that there are nearly normal morphology of outer hair cells (OHCs) and post-synapse spiral ganglion nerves (SGNs) in SMAD4 conditional knockout mice (cKO); however, a preserved distortion product of otoacoustic emission (DPOAE) and cochlear microphonic (CM) still can be evoked in cKO mice. Moreover, a partial restoration of hearing detected by electric auditory brainstem response (eABR) has been obtained in the cKO mice using electrode stimuli toward auditory nerves. Additionally, the ribbon synapses in retina are not affected by this SMAD4 defect. Thus, our findings suggest that this SMAD4 defect causes auditory neuropathy via specialized disruption of cochlear ribbon synapses.

Activation of the antigen presentation function of mononuclear phagocyte populations associated with the basilar membrane of the cochlea after acoustic overstimulation.

The immune response is an important component of the cochlear response to stress. As an important player in the cochlear immune system, the basilar membrane immune cells reside on the surface of the scala tympani side of the basilar membrane. At present, the immune cell properties in this region and their responses to stress are not well understood. Here, we investigated the functional role of these immune cells in the immune response to acoustic overstimulation. This study reveals that tissue macrophages are present in the entire length of the basilar membrane under steady-state conditions. Notably, these cells in the apical and the basal sections of the basilar membrane display distinct morphologies and immune protein expression patterns. Following acoustic trauma, monocytes infiltrate into the region of the basilar membrane, and the infiltrated cells transform into macrophages. While monocyte infiltration and transformation occur in both the apical and the basal sections of the basilar membrane, only the basal monocytes and macrophages display a marked increase in the expression of major histocompatibility complex (MHC) II and class II transactivator (CIITA), a MHC II production cofactor, suggesting the site-dependent activation of antigen-presenting function. Consistent with the increased expression of the antigen-presenting proteins, CD4(+) T cells, the antigen-presenting partner, infiltrate into the region of the basilar membrane where antigen-presenting proteins are upregulated. Further pathological analyses revealed that the basal section of the cochlea displays a greater level of sensory cell damage, which is spatially correlated with the region of antigen-presenting activity. Together, these results suggest that the antigen-presenting function of the mononuclear phagocyte population is activated in response to acoustic trauma, which could bridge the innate immune response to adaptive immunity.

Role of p19ink4d in the pathogenesis of hearing loss.

This study aimed to investigate the p19 expression in cisplatin-treated rats and the role of p19 in the degeneration of inner ear cells. It also searched for p19 gene alterations in patients with profound sensorineural deafness. P19ink4d is essential for the postmitotic maintenance of hair cells. It is presumed that a mutation in the functional homolog of p19 or a disturbance in its regulated expression can be the underlying cause of hearing loss. Experiments were conducted on male and female Sprague-Dawley rats (aged 6-7 weeks, 280-320 g) with thresholds of auditory brainstem responses <30 dB in the sound pressure level, and signs of middle ear infection were used for the experiment. For clinical evaluation, 400 children (age less than 13 years) from unrelated families with severe or profound sensorineural hearing loss (SNHL) were recruited at the second Xiangya Hospital of Central South University between 2005 and 2013, and genomic DNA for deafness gene analysis was obtained from peripheral blood samples of the patients and their lineal relatives. It was found that the p19 expression increased over time in the inner ear cells after cisplatin administration, but the p19 mRNA and protein levels significantly decreased in rats with manifested hearing loss induced by cisplatin. However, no mutation existed within the coding exons of p19 in the patients with profound sensorineural deafness. To conclude, the results support the concept that p19 may play an important role in the ototoxic effects of cisplatin and is probably involved in the pathogenesis of hearing loss.

Retinoic acid signalling regulates the development of tonotopically patterned hair cells in the chicken cochlea.

Precise frequency discrimination is a hallmark of auditory function in birds and mammals and is required for distinguishing similar sounding words, like 'bat,' 'cat' and 'hat.' In the cochlea, tuning and spectral separation result from longitudinal differences in basilar membrane stiffness and numerous individual gradations in sensory hair cell phenotypes, but it is unknown what patterns the phenotypes. Here we used RNA-seq to compare transcriptomes from proximal, middle and distal regions of the embryonic chicken cochlea, and found opposing longitudinal gradients of expression for retinoic acid (RA)-synthesizing and degrading enzymes. In vitro experiments showed that RA is necessary and sufficient to induce the development of distal-like hair cell phenotypes and promotes expression of the actin-crosslinking proteins, Espin and Fscn2. These and other findings highlight a role for RA signalling in patterning the development of a longitudinal gradient of frequency-tuned hair cell phenotypes in the cochlea.

Trichostatin A protects against cisplatin-induced ototoxicity by regulating expression of genes related to apoptosis and synaptic function.

OBJECTIVE: Although inhibition of histone deacetylases (HDACs) has been shown to protect against cisplatin-induced hearing loss, the underlying mechanism is still poorly understood. In the present study, we aim to investigate the protective effect of trichostatin A (TSA), a specific inhibitor of HDACs, on cisplatin-induced ototoxicity and to determine the differentially expressed genes involved in this process. METHODS: The basilar membrane of the cochlea was isolated from 3-day newborn Wistar rats. Organotypic cultures were treated with 150 µM cisplatin or 200 nM TSA. For combination treatment, cells were pre-incubated with TSA for 1h, followed by TSA plus cisplatin treatment. Rhodamine-phalloidin staining was used to label hair cells, and immunocytochemistry with an anti-neurofilament-200 antibody was applied to label spiral ganglion neurons (SGNs). Global expression profile microarray analysis was used to identify differentially expressed genes. Molecular function and signal pathway analysis were performed using a protein analysis through evolutionary relationships (PANTHER) classification system. Real-time quantitative PCR (qPCR) was carried out for data validation. RESULTS: Severe loss of hair cells and SGNs occurred after 48 h of cisplatin incubation, while TSA significantly increased the number of hair cells and SGNs in the combination treatment group (P<0.05). Compared with control, expression of 71 genes were up-regulated and 383 genes were down-regulated upon cisplatin treatment. Addition of TSA induced the up-regulation of 1387 genes and down-regulation of 1226 genes as compared with cisplatin administration alone. After cisplatin treatment, we observed significant down-regulation of mRNA for several genes related to synaptic function genes, including Camk2a, Camk2b, Vglut1, Snap25 and Rab3b, whereas pretreatment with TSA elevated mRNA levels of these genes. TSA greatly decreased expression of genes related to the calcium signaling pathway (Capn1 and Capn2) and apoptosis signaling pathway (Tnfrsf1a and Tp53), while addition of TSA significantly reduced levels of Tnfrsf1a and Tp53 compared with cisplatin alone (P<0.01). CONCLUSIONS: Our results suggested that TSA might protect against cisplatin-induced ototoxicity via mediating expression of genes responsible for regulating apoptosis, intracellular calcium homeostasis, neurotransmitter synthesis and release, and synaptic plasticity.

Quantitative polarized light microscopy of unstained mammalian cochlear sections.

Hearing loss is the most common sensory deficit in the world, and most frequently it originates in the inner ear. Yet, the inner ear has been difficult to access for diagnosis because of its small size, delicate nature, complex three-dimensional anatomy, and encasement in the densest bone in the body. Evolving optical methods are promising to afford cellular diagnosis of pathologic changes in the inner ear. To appropriately interpret results from these emerging technologies, it is important to characterize optical properties of cochlear tissues. Here, we focus on that characterization using quantitative polarized light microscopy (qPLM) applied to unstained cochlear sections of the mouse, a common animal model of human hearing loss. We find that the most birefringent cochlear materials are collagen fibrils and myelin. Retardance of the otic capsule, the spiral ligament, and the basilar membrane are substantially higher than that of other cochlear structures. Retardance of the spiral ligament and the basilar membrane decrease from the cochlear base to the apex, compared with the more uniform retardance of other structures. The intricate structural details revealed by qPLM of unstained cochlear sections ex vivo strongly motivate future application of polarization-sensitive optical coherence tomography to human cochlea in vivo.

Contribution of active hair-bundle motility to nonlinear amplification in the mammalian cochlea.

The cochlea's high sensitivity stems from the active process of outer hair cells, which possess two force-generating mechanisms: active hair-bundle motility elicited by Ca(2+) influx and somatic motility mediated by the voltage-sensitive protein prestin. Although interference with prestin has demonstrated a role for somatic motility in the active process, it remains unclear whether hair-bundle motility contributes in vivo. We selectively perturbed the two mechanisms by infusing substances into the endolymph or perilymph of the chinchilla's cochlea and then used scanning laser interferometry to measure vibrations of the basilar membrane. Blocking somatic motility, damaging the tip links of hair bundles, or depolarizing hair cells eliminated amplification. While reducing amplification to a lesser degree, pharmacological perturbation of active hair-bundle motility diminished or eliminated the nonlinear compression underlying the broad dynamic range associated with normal hearing. The results suggest that active hair-bundle motility plays a significant role in the amplification and compressive nonlinearity of the cochlea.

The spatial pattern of cochlear amplification.

Sensorineural hearing loss, which stems primarily from the failure of mechanosensory hair cells, changes the traveling waves that transmit acoustic signals along the cochlea. However, the connection between cochlear mechanics and the amplificatory function of hair cells remains unclear. Using an optical technique that permits the targeted inactivation of prestin, a protein of outer hair cells that generates forces on the basilar membrane, we demonstrate that these forces interact locally with cochlear traveling waves to achieve enormous mechanical amplification. By perturbing amplification in narrow segments of the basilar membrane, we further show that a cochlear traveling wave accumulates gain as it approaches its peak. Analysis of these results indicates that cochlear amplification produces negative damping that counters the viscous drag impeding traveling waves; targeted photoinactivation locally interrupts this compensation. These results reveal the locus of amplification in cochlear traveling waves and connect the characteristics of normal hearing to molecular forces.

Modulation of outer hair cell electromotility by cochlear supporting cells and gap junctions.

Outer hair cell (OHC) or prestin-based electromotility is an active cochlear amplifier in the mammalian inner ear that can increase hearing sensitivity and frequency selectivity. In situ, Deiters supporting cells are well-coupled by gap junctions and constrain OHCs standing on the basilar membrane. Here, we report that both electrical and mechanical stimulations in Deiters cells (DCs) can modulate OHC electromotility. There was no direct electrical conductance between the DCs and the OHCs. However, depolarization in DCs reduced OHC electromotility associated nonlinear capacitance (NLC) and distortion products. Increase in the turgor pressure of DCs also shifted OHC NLC to the negative voltage direction. Destruction of the cytoskeleton in DCs or dissociation of the mechanical-coupling between DCs and OHCs abolished these effects, indicating the modulation through the cytoskeleton activation and DC-OHC mechanical coupling rather than via electric field potentials. We also found that changes in gap junctional coupling between DCs induced large membrane potential and current changes in the DCs and shifted OHC NLC. Uncoupling of gap junctions between DCs shifted NLC to the negative direction. These data indicate that DCs not only provide a physical scaffold to support OHCs but also can directly modulate OHC electromotility through the DC-OHC mechanical coupling. Our findings reveal a new mechanism of cochlear supporting cells and gap junctional coupling to modulate OHC electromotility and eventually hearing sensitivity in the inner ear.

Polyethylenimine-mediated cochlear gene transfer in guinea pigs.

OBJECTIVE: To demonstrate and compare polycationic-mediated cochlear gene transfer with linear polyethylenimine (PEI) via cochleostomy and osmotic pump infusion method. DESIGN: A dissociated cochlear culture was used to select the optimum nitrogen to phosphate ratio of PEI/DNA complexes to be used in vivo. The PEI-enhanced green fluorescent protein reporter gene DNA complex was introduced with single inoculation (cochleostomy) or with sustained delivery (osmotic pump method) into guinea pig cochleas and examined for transgene expression. SUBJECTS: Male Albino Hartley guinea pigs (250-350 g). RESULTS: The relatively low transfection efficiency of PEI limits its potential when compared with viral counterparts; however, sustained release of the vector solution was able to improve PEI's transfection efficiency. The PEI-infused cochleas maintained intact cellular and tissue architecture with absence of inflammation. Transfection confined to the perilymphatic space highlights the need to target the gene vector into the scala media if transfection is targeted at cells within the organ of Corti. CONCLUSION: These findings indicate that PEI is able to transfect the cochlea in vivo with sustained delivery and present an alternative for nonviral cochlear gene therapy.