Hair Cells Of Guinea Pig Research Articles

Zinc-finger protein A20 protects hair cells from damage made by high-power microwave

Abstract Inner ear hair cells are important for maintaining hearing. Irreversible damage to hair cells is an important cause of sensorineural deafness. Electromagnetic radiation, especially high-power microwave, is an important threat to human health in modern society and war. However, it is not clear whether high-power microwave has an effect on cochlea hair cells. This study aimed to assess the effects of high-power microwave on cochlear hair cells in guinea pigs, and investigate the potential protection of these cells against high-power microwave-induced damage by recombinant adenovirus A20. Based on experimental results, a 65 W/cm2 irradiation density applied to guinea pigs in this study to establish a high-power microwave inner ear injury model. In addition, pAdEeay-1/A20 was injected via a round window into experimental guinea pig cochlea, whereas artificial perilymph was injected into the control group. Auditory function was assessed by testing the auditory brainstem response threshold, and damage to cochlear hair cells was investigated by cell counting and scanning electron microscopy observations of the basilar membrane. Inner ear injury was observed 6hours after 65 W/cm2 of irradiation and the auditory brainstem response threshold was significantly higher in the irradiation group (P < 0.05) compared with other groups. Propidium iodide staining and scanning electron microscopy results indicated that significant morphological changes occurred after radiation, especially to inner hair cells, which exhibited remarkable damage and the presence of several unknown spherical substances. Auditory brainstem response threshold was decreased in the pAdEeay-1/A20 group compared with the artificial perilymph group; moreover, damage to hair cells was milder in the pAdEeay-1/A20 group compared with the control group (P < 0.01). Thus, high-power microwave can cause damage to cochlear hair cells, as well as hearing loss with prolonged exposure and/or high dosage. In this regard, 65 W/cm2 of irradiation for 6hours is a reliable target dose for observation of damage. The zinc finger protein A20 can protect cochlear hair cells from high-power microwave-induced damage and prevent further hearing loss. This study was approved by the Laboratory Animal Welfare and Ethics Committee of the Third Military Medical University, China on April 18, 2017.

Establishment of a cochlear injury model using bone-conducted ultrasound irradiation in guinea pigs and investigation on peripheral coding and recognition of ultrasonic signals

The cochlea of guinea pigs was irradiated with different frequencies of bone-conducted ultrasound (BCU) at a specific dose to induce cochlear hair cell-specific injuries, in order to establish frequency-related cochlear hair cell-specific injury models. Cochlear near-field potentials were then evoked using BCU of different frequencies and intensities to explore the peripheral coding and recognition of BCU by the cochlea. The inner ears of guinea pigs were irradiated by 30 kHz at 100 db and 80 kHz at100 db BCU for 6h to create frequency-related, ultrasound-specific cochlear injury models. Then, 30 kHz and 80 kHz BCU of different intensities were used to evoke auditory brainstem response (ABR) thresholds, compound action potential (CAP) thresholds, and action potential (AP) intensity-amplitude input-output curves in the normal control group and the ultrasonic cochlear injury group. This allowed us to explore the coding and recognition of BCU frequencies and intensities by cochlear hair cells. Immunofluorescence assay of outer hair cell (OHC) Prestin and inner hair cell (IHC) Otofelin was performed to verify the injury models. Irradiation of guinea pig inner ears by 30 kHz and 80 kHz BCU at a specific dose induced hair cell injuries at different sites. Irradiation with low frequency BCU mainly induced OHC injury, whereas irradiation with high frequency BCU induced IHC injury; moreover, IHC injury was more serious than OHC injury. The 30 kHz-evoked ABR threshold was significantly higher in the 30 kHz ultrasonic cochlear injury group compared to the normal control group. The 30 kHz-evoked ABR threshold was significantly higher in the 30 kHz ultrasonic cochlear injury group compared to the 80 kHz ultrasonic cochlear injury group. The difference in the 80 kHz-evoked ABR thresholds were not significant between the 30 kHz and 80 kHz ultrasonic cochlear injury groups. The click- and 30 kHz-evoked AP intensity-amplitude curves for the 30 kHz ultrasonic cochlear injury group indicate that the AP amplitude evoked at the same intensity was higher in the 30 kHz-evoked group than the click-evoked group. The spatial positions of cochlear hair cells in guinea pigs had a coding function for the frequencies of low-frequency ultrasound. OHCs have an amplification effect on the coding of low-frequency ultrasonic intensities. The peripheral perception of high frequency BCU may not require the participation of cochlear hair cells.

The effect of acupuncture at Jǐngjiājǐ (颈夹脊) on the repair and regeneration of cochlear hair cells of rats with sensorineural deafness

ObjectiveTo observe the effect of acupuncture at Jǐngjiājǐ (颈夹脊) on the repair and regeneration of cochlear hair cells of guinea pigs with sensorineural deafness. MethodsSixty healthy guinea pigs were selected, 20 guinea pigs were randomly assigned to the normal control group (group A), and other guinea pigs were randomly divided into model control group (group B) and acupuncture treatment group (group C) after injection with gentamicin sulfate in order to induce deafness. No intervention was given to the guinea pigs in group A and group B, and acupuncture at Jǐngjiājǐ was given to the guinea pigs in group C for 30 days. ABR threshold, DPOAE amplitudes and hair cells counting of guinea pigs in each group were recorded after intervention for 30 days. ResultsAfter intervention for 30 days, ABR threshold in group C was significantly lower than that in group B (38.46 ± 7.36 vs 82.94 ± 6.47, P<0.01), and the DPOAE amplitudes in group C were obviously higher than that in group B (28.06 ± 5.64 vs 25.23 ± 5.38, P<0.01). The number of cochlear hair cells in group C increased significantly, over 50% of the hair cells survived, accounting for 66.67% of the observation cases. The number of cochlear hair cells in the 3rd and 4th gyri was close to the normal level, and plenty of proliferous sustentacular cells can be seen. Compared with group B, the number of cochlear outer hair cells in each gyrus in group C significantly increased (36.76 ± 1.97 vs 28.59 ± 2.24, P<0.01), indicating that acupuncture at Jǐngjiājǐ can promote the repair and regeneration of cochlear hair cells. ConclusionAcupuncture at Jǐngjiājǐ can promote the repair and regeneration of cochlear hair cells, thus improving the hearing of guinea pigs with deafness.

Identification and analysis of the proteins interacted with Prestin in cochlear outer hair cells of guinea pig

Objective: To explore the regulation and mechanism of Prestin protein by identifying the proteins interacted with Prestin in cochlear outer hair cell(OHC) and analyzing their biological function. Methods: Co-immunoprecipitation combined mass spectrometry technology was used to isolate and identify the proteins interacted with Prestin protein of OHC, bioinformatics was used to construct Prestin protein interaction network. The proteins interacted with Prestin in OHC of guinea pig were determined by matching primary interaction mass spectrometry with protein interaction network, and annotated their functions. Results: The results of co-immunoprecipitation combined with mass spectrometry showed that 116 kinds of credible proteins could interact with Prestin. By constructing Prestin protein interaction network, matching the results of mass spectrometry and analyzing of sub-cellular localization, eight kinds of proteins were confirmed that they interacted with Prestin directly, namely EEF2, HSP90AB1, FN1, FLNA, EEF1A1, HSP90B1, ATP5A1, and ERH, respectively, which were mainly involved in the synthesis and transportation, transmembrane folding and localization, structural stability and signal transduction of Prestin protein. Conclusion: EEF2, HSP90AB1, FN1, FLNA, EEF1A1, HSP90B1, ATP5A1 and ERH provide molecular basis for sensory amplification function of OHCs by participating in biotransformation, transmembrane folding and localization, signal transduction and other biological processes of Prestin protein.

Existence of nicotinic receptors in a subset of type I vestibular hair cells of guinea pigs

In mammals, vestibular hair cells (VHCs) are classified as type I and II according to morphological criteria. Acetylcholine (ACh) is identified as the primary efferent neurotransmitter. To date, cholinergic activities have been reported in mammalian type II VHCs, but similar activities in type I VHCs have not been pursued presumably because the body of type I VHCs were suggested to be totally surrounded by afferent nerve calyces. A few reports showed that part of type I VHCs were incompletely surrounded by calyces and received contact from the efferent nerve endings in the mammals studied. The possibility of the expression of cholinergic receptors, their subunit composition, and their function in mammals’ type I VHCs are still unclear. In this study, nicotinic responses were investigated by the whole-cell patch clamp technique in isolated type I VHCs of guinea pigs. Of the cells, 7.3% were sensitive to cholinergic agonists and showed an excitatory current at −40 mV which was not sensitive to nifedipine, iberiotoxin (IBTX), and apamin. The main carriers of this current were Na+ and K+. The rank order of activation potency was nicotine > 1,1-dimethyl-4-phenyl-piperazinium (DMPP) > ACh. These nicotinic ACh receptors (nAChRs) were not blocked by strychnine and α-bungarotoxin (α-BTX), but sensitive to d-tubocurarine (dTC) and mecamylamine (Mec). The findings provide physiological evidence that some subtypes of nAChRs may be located in a subset of type I VHCs, which were different from α9α10 nAChRs.

A single dose of dexamethasone encapsulated in polyethylene glycol-coated polylactic acid nanoparticles attenuates cisplatin-induced hearing loss following round window membrane administration.

This study aimed to investigate the sustained drug release properties and hearing protection effect of polyethylene glycol-coated polylactic acid (PEG-PLA) stealth nanoparticles loaded with dexamethasone (DEX). DEX was fabricated into PEG-PLA nanoparticles using an emulsion and evaporation technique, as previously reported. The DEX-loaded PEG-PLA nanoparticles (DEX-NPs) had a hydrodynamic diameter of 130±4.78 nm, and a zeta potential of −26.13±3.28 mV. The in vitro release of DEX from DEX-NPs lasted 24 days in phosphate buffered saline (pH 7.4), 5 days in artificial perilymph (pH 7.4), and 1 day in rat plasma. Coumarin 6-labeled NPs placed onto the round window membrane (RWM) of guinea pigs penetrated RWM quickly and accumulated to the organs of Corti, stria vascularis, and spiral ganglion cells after 1 hour of administration. The DEX-NPs locally applied onto the RWM of guinea pigs by a single-dose administration continuously released DEX in 48 hours, which was significantly longer than the free DEX that was cleared out within 12 hours after administration at the same dose. Further functional studies showed that locally administrated single-dose DEX-NPs effectively preserved outer hair cells in guinea pigs after cisplatin insult and thus significantly attenuated hearing loss at 4 kHz and 8 kHz frequencies when compared to the control of free DEX formulation. Histological analyses indicated that the administration of DEX-NPs did not induce local inflammatory responses. Therefore, prolonged delivery of DEX by PEG-PLA nanoparticles through local RWM diffusion (administration) significantly protected the hair cells and auditory function in guinea pigs from cisplatin toxicity, as determined at both histological and functional levels, suggesting the potential therapeutic benefits in clinical applications.

Memantine’s action against aminoglycoside-induced ototoxicity

The objectives of this study were (1) to assess the protective role of NMDA antagonists against the ototoxic effects of aminoglycosides, (2) to provide any possible evidence between ototoxicity due to aminoglycosides and excitotoxicity. An animal experiment was conducted. Twenty-eight, 3-month-old female New Zealand rabbits, weighing 1,000-1,500g, were studied prospectively for 28days after intramuscular administration of amikacin (15mg/kg/day divided into two equal doses) for 14days. Twenty-one rabbits were categorized into three equal treatment groups and seven animals received no medication and served as the control group. The animals of A, B and C groups were injected, intramuscularly, with amikacin 15mg/kg/day, divided into two equal doses every day for 14days. Animals of group A received in parallel memantine (per os) and those of group B received p.o. the same volume of placebo solution. The rabbits of the third group (group C) received on the 15th day and every 2days for the next 2weeks, until the day 28, memantine of the same quantity as the members of group A. Differences in DPOAE amplitudes, and therefore in cochlear activity, between group A and group B were revealed. DPOAE amplitudes of group B were further reduced compared to the respective amplitudes in rabbits of group A. No improvement was observed in DPOAE measurements performed after the discontinuation of injections. The findings in group C should be examined separately. The measurements showed apparent reversal ototoxic effects in four of the animals. The development of aminoglycoside otoprotective strategies is a primary goal in ototoxicity research. The administration of NMDA antagonists has been shown to prevent, at least to some extent, toxic damage to hair cells in guinea pigs, treated with aminoglycoside antibiotics.

Two Distinct Channels Mediated by m2mAChR and α9nAChR Co-Exist in Type II Vestibular Hair Cells of Guinea Pig

Acetylcholine (ACh) is the principal vestibular efferent neurotransmitter among mammalians. Pharmacologic studies prove that ACh activates a small conductance Ca2+-activated K+ channels (KCa) current (SK2), mediated by α9-containing nicotinic ACh receptor (α9nAChR) in mammalian type II vestibular hair cells (VHCs II). However, our studies demonstrate that the m2 muscarinic ACh receptor (m2mAChR) mediates a big conductance KCa current (BK) in VHCs II. To better elucidate the correlation between these two distinct channels in VHCs II of guinea pig, this study was designed to verify whether these two channels and their corresponding AChR subtypes co-exist in the same VHCs II by whole-cell patch clamp recordings. We found that m2mAChR sensitive BK currents were activated in VHCs II isolated by collagenase IA, while α9nAChR sensitive SK2 currents were activated in VHCs II isolated by trypsin. Interestingly, after exposing the patched cells isolated by trypsin to collagenase IA for 3 min, the α9nAChR sensitive SK2 current was abolished, while m2mAChR-sensitive BK current was activated. Therefore, our findings provide evidence that the two distinct channels and their corresponding AChR subtypes may co-exist in the same VHCs II, and the alternative presence of these two ACh receptors-sensitive currents depended on isolating preparation with different enzymes.

Effect of JNK signal transduction pathway in intense noise-induced apoptosis of vestibular hair cells in guinea pigs

To investigate the mechanism of intense noise-induced apoptosis of vestibular hair cells in guinea pigs and the effect of phosphorylated c-Jun N-terminal kinase (JNK) signal transduction pathway in intense noise-induced apoptosis of vestibular hair cells. Thirty-two guinea pigs were randomly and equally divided into 1, 5, and 15 d experimental groups and control group. The guinea pigs in the experimental groups were exposed to 4 kHz narrow-band noise at 120 dB SPL for 4 h and then subjected to measurement of auditory brainstem response at 1, 5, or 15 d after noise exposure. In each group, four guinea pigs were used to prepare paraffin sections of vestibular hair cells, and the rest for extraction of total protein from vestibular hair cells. The apoptosis of vestibular hair cells was detected by terminal deoxynucleotidyl transferase (TdT)-mediated d-UTP nick-end labeling (TUNEL). The expression levels of p-JNK and pc-Jun were measured by immunohistochemistry and Western blot. TUNEL-positive cells were found in the vestibular hair cells in the experimental groups, most in the 1 d experimental group and least in the 15 d experimental group, but no positive cells were found in the control group. The immunohistochemical results showed that p-JNK and pc-Jun were detected in the cell nuclei in the experimental groups, but no p-JNK- and pc-Jun-positive cells were found in the control group. The Western blot showed that p-JNK and pc-Jun were increased and activated quickly at 1d after noise exposure, reached the peak levels at 5 d after noise exposure, and were then decreased gradually, but they were still at relatively high levels at 15 d after noise exposure. Intense noise can cause injury to vestibular hair cells by inducing cell apoptosis, and p-JNK marks the activation of JNK signal transduction pathway, suggesting that JNK signal transduction pathway plays an important role in intense noise-induced apoptosis of vestibular hair cells in guinea pigs.

Muscarinic acetylcholine receptor subtype expression in type vestibular hair cells of guinea pigs

Recent studies have demonstrated that five subtypes (M1-M5) of muscarinic acetylcholine receptor (mAChR) are expressed in the vestibular periphery. However, the exact cellular location of the mAChRs is not clear. In this study, we investigated whether there is the expression of M1-M5 muscarinic receptor mRNA in isolated type II vestibular hair cells of guinea pig by using single-cell RT-PCR. In vestibular end-organ, cDNA of the expected size was obtained by RT-PCR. Moreover, mRNA was identified by RT-PCR from individually isolated type II vestibular hair cells (single-cell RT-PCR). Sequence analysis confirmed that the products were M1-M5 mAChR. These results demonstrated that M1-M5 mAChR was expressed in the type II vestibular hair cells of the guinea pig, which lends further support for the role of M1-M5 mAChR as a mediator of efferent cholinergic signalling pathway in vestibular hair cells.

Morphological Observation of Hair Cells in Guinea Pigs on the Recovery Course after Acoustic Stimuration

Morphological Observation of Hair Cells in Guinea Pigs on the Recovery Course after Acoustic Stimuration

Noise exposure induced cochlear hair cell death pathways in guinea pig

ObjectiveTo understand the mechanism of noise exposure induced outer hair cells (OHCs) death pathways. MethodsThirty two guinea pigs were used in this study. The animals were either exposed for 4 h/day to broadband noise at 122 dB SPL(A–weighted) for 2 consecutive days or perfused with MNNG. After auditory test, the cochleae of animals were dissected. Propidium iodide (PI), a DNA intercalating fluorescent probe, was used to trace morphological changes in OHC nuclei. F–actin staining was used to determine missing OHCs. Caspase–3 was detected in living organ of Corti whole mounts using the fluorescent probe. The single strand DNA(ssDNA) in apoptotic OHCs in guinea pigs and apoptosis inducing factor(AIF) in hair cells in guinea pigs were examined by immunohistology method. Whole mounts of organ of Corti were prepared. Morphological and fluorescent changes were examined under a confocal microscope. Results(1) Both apoptotic and necrotic hair cells appeared following noise exposure. (2) Noise exposure induced single strand DNA in apoptotic OHCs but not in the normal OHCs. (3) Either after noise exposure or after MNNG perfusion, apoptotic OHCs were featured by nuclear condensation or fragmentation with caspase–3 activation, whereas necrotic OHCs were characterized by nuclear swelling without caspase–3 activation. (4) In normal organ of Corti, AIF was located in the mitochondria areas. After noise exposure, AIF was translocated from mitochondria in apoptotic and necrotic OHCs. ConclusionThese findings indicate that noise exposure damages DNA in the OHC, which triggers action of Caspase–3. Subsequently, AIF is translocated to the nucleus, leading to DNA damage and OHCs death.

Acetylcholine–induced calcium oscillation in isolated outer hair cells in guinea pig

ObjectiveThis study is to explore the relationship between acetylcholine (ACh)-induced calcium release from intracellular Ca2+stores and function of outer hair cell(OHC) motors, in an attempt to elucidate the mechanism of OHC electromotility at resting state. MethodsOHCs were isolated from adult guinea pig (200-300 g) cochlea and loaded with Fluo-3/AM. The cells were treated with ACh/dHBSS, ACh/HBSS, dHBSS only or HBSS only. Intracellular [Ca2+]i variations in cells under the four treatments were observed using an Ar-Kr laser scan confocal microscope. ResultsCa2+]i oscillations were recorded in five OHCs treated with ACh/dHBSS but not in other cells. This is the first time that Ach-excited [Ca2+i oscillations are reported in guinea pig OHCs independent of extracellular calcium. ConclusionsACh-excited [Ca2+]i oscillations in OHCs originates from intracellular calcium release and may play a crucial role in maintaining active mechanical motility of the OHC at resting and modulating OHC electromotility.

The fate of outer hair cells after acoustic or ototoxic insults

In epithelial sheets, clearance of dead cells may occur by one of several routes, including extrusion into the lumen, phagocytic clearance by invading lymphocytes, or phagocytosis by neighboring cells. The fate of dead cochlear outer hair cells is unclear. We investigated the fate of the “corpses” of dead outer hair cells in guinea pigs and mice following drug or noise exposure. We examined whole mounts and plastic sections of normal and lesioned organ of Corti for the presence of prestin, a protein unique to outer hair cells. Supporting cells, which are devoid of prestin in the normal ear, contained clumps of prestin in areas of hair cell loss. The data show that cochlear supporting cells surround the corpses and/or debris of degenerated outer hair cells, and suggest that outer hair cell remains are phagocytosed by supporting cells within the epithelium.

Taurine modulates calcium influx through L-type voltage-gated calcium channels in isolated cochlear outer hair cells in guinea pigs

Taurine has been proposed to play a role in calcium modulation. To explore the effect of taurine on intracellular calcium homeostasis of isolated cochlear outer hair cells and on the gentamycin-induced inhibition of calcium influx evoked by high K + depolarization, we employed fluo-3 imaging of intracellular calcium ([Ca 2+] i) via confocal laser scanning microscopy to measure real-time changes of [Ca 2+] i. We found that the sole application of taurine (5, 10, 20 mM) induced a transient [Ca 2+] i increase in a concentration-dependent manner, which was inhibited either by the application of an L-type calcium-channel blocker nifedipine or a calcium-free medium. Pre-incubation with 1 mM gentamicin induced inhibition of [C a 2+] i elevation evoked by high K +. Short-term (10 min) exposure with a high level of taurine (20 mM) prevented this inhibition. These results indicated that taurine at a high concentration was able to promote calcium influx through L-type calcium channels in isolated outer hair cells and antagonize gentamycin-induced inhibition of calcium elevation evoked by high K + by its calcium homeostatic effect.

Effects of cinnarizine on calcium and pressure-dependent potassium currents in guinea pig vestibular hair cells

In vestibular hair cells, K+ currents induced by rises in hydrostatic pressure have recently been demonstrated. These currents are inhibited by charybdotoxin, a blocker of Ca2+-dependent K+ channels. On the other hand, cinnarizine is a blocker of voltage-gated Ca2+ currents in hair cells and is used as a drug in conditions with vestibular vertigo. Our aim was to test in patch-clamp experiments (conventional whole-cell mode) whether cinnarizine, by reducing Ca2+ influx, inhibited Ca2+ and pressure-sensitive K+ currents in vestibular type-II hair cells of guinea pigs. A quantitatively similar inhibition of K+ currents was evoked by extracellular Ca2+ removal, cinnarizine (0.5 microM), and the L-type Ca2+ channel blocker nifedipine (3 microM). Cinnarizine abrogated increases of K+ currents induced by increases in the hydrostatic pressure (from 0.2 to 0.5 cm H2O). At a higher concentration (1 microM), cinnarizine elicited K+ current inhibitions larger than those elicited by Ca2+ removal. Moreover, it reduced K+ currents in the absence of Ca2+, in contrast to nifedipine. However, charybdotoxin abolished these effects of cinnarizine. We thus conclude that cinnarizine inhibits, by two mechanisms, pressure-induced currents that are sensitive to charybdotoxin and Ca2+. It reduces Ca2+ influx and exerts a Ca2+-independent inhibition, with a lower IC50 than that required for Ca2+ channel blockade. These two actions may importantly contribute to its therapeutic effects.

Taicatoxin inhibits the calcium-dependent slow motility of mammalian outer hair cells

The effects of taicatoxin on the slow motility of isolated outer hair cells of guinea pig were studied in the experiments. Pretreatment with taicatoxin (0.19 μM) was able to prevent both the cell shortening induced by high K + (50 mM), and the cell elongation induced by ionomycin (10 μM). These effects of taicatoxin can be mimicked by pretreatment of cells with Ca 2+-free medium on the slow motility in response to ionomycin or high K +. Pretreatment with neither calcium channel blockers such as nifedipine (L-type blocker), ω-conotoxin GVIA (N-type blocker), and ω-agatoxin IVA (P-type blocker); nor potassium channel blockers, such as tetraethylammonium chloride (TEA) and 3,4-diaminopyridine (3,4-DAP) can antagonize the cell shortening effect induced by high K + and cell elongation induced by ionomycin. The calcium-imaging experiment indicated that taicatoxin, but not nifedipine, did prevent an increase of intracellular Ca 2+ level significantly induced by high K +. These results demonstrate that the effect of taicatoxin was to block the calcium entry through calcium channels of cell membrane, without relative to its properties of potassium channel blockers. We conclude that taicatoxin-sensitive-calcium channels at least impart, play a significant role in the slow motility of outer hair cell.

Calpain and Caspase Inhibitors Protect Vestibular Sensory Cells from Gentamicin Ototoxicity

Objective--Apoptosis may play an important role in the mechanism of aminoglycoside ototoxicity. Caspases and calpains are regarded to be important factors in the regulation of cell death in the inner ear. We hypothesized that caspase or calpain inhibitors would protect hair cells from aminoglycoside ototoxicity. Material and Methods--In order to test this hypothesis we carried out a pilot study to determine if gentamicin (GM) would induce caspase and calpain immunolabeling in guinea pig hair cells. Having confirmed this we carried out the main experiment using guinea pig vestibular organ culture to determine if caspase and calpain would protect the hair cells from GM ototoxicity. Results--Immunoreactivity for caspase-3 and m-calpain was detected in the vestibular sensory cells and ganglia after GM treatment. Both caspase and calpain inhibitors protected hair cells against gentamicin ototoxicity. Conclusion--It is suggested that inhibition of apoptosis is essential in order to block aminoglycoside ototoxicity.

Transgene expression in mature guinea pig cochlear cells in vitro

Inoculation of adenovirus vectors in vivo has induced transgene expression in a variety of cochlear cells, but not hair cells or supporting cells in most previous studies. Specific hair cell inoculation by viral vectors has not been demonstrated in the mature guinea pig cochlea in vitro. We injected an Adex1CAlacZ into the mature guinea pig cochlear explants, which were incubated for 24–72 h. We found many lacZ-positive cells in a variety of tissues including the spiral ganglion and stria vascularis. Transgene expression was also found in the outer hair cells and supporting cells, such as the Deiters cells and pillar cells. These findings indicate that adenovirus vectors can be transfected into mature guinea pig hair cells and supporting cells in culture.

Cochlear function and transgene expression in the guinea pig cochlea, using adenovirus- and adeno-associated virus-directed gene transfer.

Development of a viral vector that can infect hair cells of the cochlea without producing viral-associated ototoxic effects is crucial for utilizing gene replacement therapy as a treatment for certain forms of hereditary deafness. In the present study, cochlear function was monitored using distortion-product otoacoustic emissions (DPOAEs) in guinea pigs that received infusions of either (E1(-), E3(-)) adenovirus, or adeno-associated virus (AAV), directly into the scala tympani. Replication-deficient (E1(-), E3(-)) adenovirus-directed gene transfer, using the cytomegalovirus (CMV) promoter, drove transgene expression to inner hair cells and pillar cells of the cochlea. AAV transduction was tested with several promoters, such as platelet-derived growth factor (PDGF), neuron-specific enolase (NSE), and elongation factor 1alpha (EF-1alpha) promoters; which drove transgene expression to cochlear blood vessels, nerve fibers, and certain spiral limbus cells, respectively. AAV transgene expression was visualized by green fluorescent protein immunostaining. Immunocytochemistry to heparan sulfate confirmed the absence of proteoglycans in guinea pig hair cells, indicating that the receptor for AAV was not present on these cells. However, the heparan sulfate proteoglycan expression pattern mimicked the AAV transduction pattern. An overall finding was that cochlear function was not altered throughout the infection period using AAV titers as high as 5 x 10(8) IP/infused cochlea. In contrast, cochlear function was severely compromised by 8 days postinfection with adenoviral titers of 5 x 10(8) PFU/infused cochlea, and outer hair cells were eliminated. Thus, cochlear hair cells are amenable to in vivo gene transfer using a replication-deficient (E1(-), E3(-)) adenovirus. However, replication-defective or gutted adenovirus vectors must be employed to overcome the ototoxic effects of (E1(-), E3(-)) adenovirus vectors.