ZBTB20 is essential for cochlear maturation and hearing in mice.

The mammalian cochlear epithelium undergoes substantial remodeling and maturation before the onset of hearing. However, very little is known about the transcriptional network governing cochlear late-stage maturation and particularly the differentiation of its lateral nonsensory region. Here, we establish ZBTB20 as an essential transcription factor required for cochlear terminal differentiation and maturation and hearing. ZBTB20 is abundantly expressed in the developing and mature cochlear nonsensory epithelial cells, with transient expression in immature hair cells and spiral ganglion neurons. Otocyst-specific deletion of Zbtb20 causes profound deafness with reduced endolymph potential in mice. The subtypes of cochlear epithelial cells are normally generated, but their postnatal development is arrested in the absence of ZBTB20, as manifested by an immature appearance of the organ of Corti, malformation of tectorial membrane (TM), a flattened spiral prominence (SP), and a lack of identifiable Boettcher cells. Furthermore, these defects are related with a failure in the terminal differentiation of the nonsensory epithelium covering the outer border Claudius cells, outer sulcus root cells, and SP epithelial cells. Transcriptome analysis shows that ZBTB20 regulates genes encoding for TM proteins in the greater epithelial ridge, and those preferentially expressed in root cells and SP epithelium. Our results point to ZBTB20 as an essential regulator for postnatal cochlear maturation and particularly for the terminal differentiation of cochlear lateral nonsensory domain.

CD-MOF-1 Growth on Polysaccharide Gels through Only C2-OH/C3-OH or C5-O/C6-OH Group Formed Four-Coordinated K+ Ions for Developing Porous Biogels.

The γ-cyclodextrin (γ-CD) metal-organic frameworks (CD-MOF-1) consist of γ-CD and potassium (K+) ions through coordinating an eight-coordinated K+ ion with two C5-linked oxygen and C6-linked hydroxyl (C5-O/C6-OH) groups in the primary faces of adjacent γ-CD units and two C2- and C3-linked hydroxyl (C2-OH/C3-OH) groups in the secondary faces. Herein, we found polysaccharide gels with only C2-OH/C3-OH or C5-O/C6-OH groups in pyranoid rings can form four-coordinated K+ ions and then coordinate γ-CD in a KOH solution for CD-MOF-1 growth. Exposure of C2-OH/C3-OH or C5-O/C6-OH groups in polysaccharide gels is important to form active four-coordinated K+ ions. Mechanism supporting this work is that four-coordinated K+ ion sites are first formed after coordinating C2-OH/C3-OH groups in pectin and then coordinating C5-O/C6-OH groups in the primary faces of γ-CD units. Alternatively, four-coordinated K+ ions with C5-O/C6-OH groups in chitosan can coordinate the C2-OH/C3-OH groups in the secondary faces of γ-CD units. Mechanism of CD-MOF-1 growing on pectin and chitosan gels through the proposed four-coordinated K+ ions is also universally applicable to other polysaccharide gels with similar C2-OH/C3-OH or C5-O/C6-OH groups such as alginate gel. Based on this mechanism, we developed pectin and chitosan gel-based CD-MOF-1 composites and exemplified applications of them in antibacterial and organic dye removal. To help future research and applications of this mechanism, we share our theoretical assumption for further investigations that any matrices with an ortho-hydroxyl carbon chain or ortho-hydroxyl ether structures may form four-coordinated K+ ions for CD-MOF-1 growth. The proposed mechanism will broaden the development of novel CD-MOF-1 composites in various fields.

Protective Mechanisms of Various Active Substances on Cell DNA Damage and Apoptosis Induced by Deoxynivalenol.

Deoxynivalenol (DON) is a secondary metabolite of fungi that is harmful to humans and animals. This study examined the protective effects of natural substances, including resveratrol, quercetin, vitamin E, vitamin C, and microbe-derived antioxidants (MA), on both human gastric mucosal cells (GES-1) and pig small intestinal epithelial cells (IPEC-1) when induced by DON. Cells were incubated with active substances for 3 h and then exposed to DON for 24 h. The oxidative stress index, cell cycle, and apoptosis were measured. As compared to cells treated only with DON, pretreatment with active substances improved the balance of the redox status in cells caused by DON. Specifically, quercetin, vitamin E, vitamin C, and MA showed the potential to alleviate the G2 phase cell cycle arrest effect that was induced by DON in both kinds of cells. It was observed that vitamin E and vitamin C can alleviate DON-induced apoptosis and the G2 phase cycle arrest effect mediated via the ATM-Chk 2-Cdc 25C and ATM-P53 signaling pathways in GES-1 cells. In IPEC-1 cells, vitamin C and MA can alleviate both DON-induced apoptosis and the G2 phase cycle arrest effect via the ATM-Chk 2-Cdc 25C signaling pathway. Different bioactive substances utilize different protective mechanisms against DON in interacting with different cells. The proper addition of vitamin E and vitamin C to food can neutralize the toxic effect of DON, while the addition of vitamin C and MA to animal feed can reduce the harm DON does to animals.

Math1 gene transfer based on the delivery system of quaternized chitosan/Na-carboxymethyl-beta-cyclodextrin nanoparticles.

Mammalian cochlear hair cells don't regenerate naturally after injury, which usually leave permanent hearing loss. Math1 gene is a positive regulator of hair cell differentiation during cochlear development and was proved to be very critical in hair cell regeneration in deaf animals. Generating new cochlear hair cells by forced Math1 expression may be a cure for hearing loss. However, satisfying gene delivering vectors in gene therapy are not available. We combined quaternized chitosan (QCS) with Na-carboxymethyl-beta-cyclodextrin (CM-beta-CD) as novel non-viral vector, which adsorbs pRK5-Math1-EGFP perfectly at the mass ratio of 4:1. In vitro cell transfection can reach a 40% transfect efficiency and relatively low cytotoxity than liposomes. These results suggest that QCS/CM-beta-CD nanoparticle complexes could be a novel non-viral gene carrier in further clinical application.

Isolation, growth and differentiation of hair cell progenitors from the newborn rat cochlear greater epithelial ridge.

Mammalian cochlear hair cell loss is irreversible and leads to permanent hearing loss. To restore hearing physiologically, it is necessary to generate new functional hair cells either from endogenous cells or from exogenously transplanted hair cells/progenitors. Previous studies suggest that cochlear greater epithelial ridge (GER) and lesser epithelial ridge (LER) cells are capable of differentiating into hair cells. While it was recently possible to obtain and culture pure LER progenitors, isolation of pure GER progenitors has not been reported. Here we describe a method that allows isolation of pure GER cells from neonatal rat cochleae. The cochlear epithelial sheet (CES) containing GER progenitor cells was mechanically separated from the underlying mesenchymal tissue after digestion with thermolysin. The GER area could then be dissected following mechanical removal of organ of Corti as well as all the lateral area. The isolated GER cells showed significant proliferation and expressed markers for GER cells but not markers for hair cells or LER. When the GER cells were cultured in serum-free medium containing epidermal growth factor, spheres were formed where they continued to proliferate. Furthermore, when GER cells were induced to express Hath1 or co-cultured with mesenchymal cells prepared from neonate rat cochleae, they showed the potential to differentiate into hair cell-like cells. Successful isolation, culture and differentiation of GER hair cell progenitors will shed additional light on the mechanism of hair cell differentiation and potential hair cell replacement.

Development of Dual Quantitative Lateral Flow Immunoassay for the Detection of Mycotoxins.

Lateral flow immunoassays have been widely used in recent years for detection of toxins, heavy metals, and biomarkers. To improve the efficiency of individual lateral flow immunoassays, multiplex analytical strips play an important role in the detection of several important analytes. In this chapter, development of a dual lateral flow immunoassay is presented for detection of a variety of low molecular weight molecules. Various buffers, additives, and materials are introduced and evaluated. Depending on the analyte to be tested, the technique allows for selection of optimum buffers, additives, and other materials.

Screening of single-stranded DNA (ssDNA) aptamers against a zearalenone monoclonal antibody and development of a ssDNA-based enzyme-linked oligonucleotide assay for determination of zearalenone in corn.

A hypotoxic immunosorbent assay for the detection of zearalenone (ZEN) was developed, by identifying a single-stranded DNA (ssDNA) aptamer with high specificity and affinity for a ZEN monoclonal antibody (mAb-ZEN). ssDNA aptamers, which could mimic ZEN epitopes, were identified using the modified systematic evolution of ligands by an exponential enrichment (SELEX) technique. The purified mAb-ZEN was coated on microtiter plates as a target recognized by the random oligonucleotide ssDNA library. The binding affinity between the aptamers and mAb-ZEN during each round was measured by the biotin­streptavidin­horseradish peroxidase system. During 15 rounds of screening, an increasing binding affinity was observed. The enriched ssDNA library binding to mAb-ZEN with high affinity was cloned, sequenced, and analyzed. One aptamer (number 46), which displays the highest affinity and specificity for the mAb-ZEN, was used to establish an indirect competition enzyme-linked oligonucleotide assay (ELONA) to measure the ZEN concentration in corn. Under optimal conditions, the regression equation for quantification of ZEN was y = −0.0778x + 0.713 (R2 = 0.9981). The detection limit and IC50 were 0.01 and 0.2 ng/mL, respectively, with a working range of 0.03­2.5 ng/mL. The recovery rates of the spiked samples in the ELONA ranged from 95 to 105%. Aptamers, which can mimic many types of low-weight analytes in agricultural products, could serve as surrogates for the development of hypotoxic, environmentally friendly immunological detection methods.

Rapid simultaneous quantification of zearalenone and fumonisin B1 in corn and wheat by lateral flow dual immunoassay.

A lateral flow dual immunoassay (LFDIA) was developed for rapid quantitative detection of zearalenone (ZEN) and fumonisin B1 (FB1) in corn and wheat samples on a single test strip. Two test lines and the control line on the nitrocellulose membrane were coated with ZEN and FB1 conjugates and goat anti-mouse IgG, respectively. Colloidal gold nanoparticles were conjugated with monoclonal antibodies against ZEN or FB1. The intensity of the test lines was analyzed by a photometric strip reader to determine the concentrations of ZEN and FB1 based on the calibration curves of known concentrations versus intensity readings. Test parameters such as types of buffers, ratio of the two gold-labeled antibodies, and dilution of the sample extracts and the gold-labeled antibodies were optimized. The detection limit was 0.35 and 5.23 ng/mL for ZEN and FB1, respectively, and the corresponding detection ranges were 0.94-7.52 and 9.34-100.45 ng/mL, respectively. Spiked and natural samples were analyzed using both LFDIA and liquid chromatography-tandem mass spectrometry. The two methods had a good correlation (R(2) = 0.96). The dual quantitative LFDIA is sensitive, rapid, and easy-to-use for on-site testing of a large number of samples.

Novel chemiluminescence immunoassay for the determination of zearalenone in food samples using gold nanoparticles labeled with streptavidin-horseradish peroxidase.

A novel highly sensitive chemiluminescence immunoassay (CLIA) was developed to detect zearalenone in food samples by using both biotinylated zearalenone conjugates and gold (Au) nanoparticles labeled with streptavidin-horseradish peroxidase for signal amplification. Biotinylated zearalenone-ovalbumin conjugates and Au nanoparticles labeled with streptavidin-horseradish peroxidase were synthesized separately. The concentrations of immunoreagents and the reaction times of these immunoreagents were optimized to improve the performances of analytical methods. For the CLIA based on biotinylated zearalenone conjugates and Au nanoparticles labeled with streptavidin-horseradish peroxidase, the limit of detection was 0.008 ng/mL and the IC50 was 0.11 ng/mL. The linear working range was 0.02-0.51 ng/mL. The cross-reactivities with the zearalenone analogues (α-zearalanol, zearalanone, α-zearalenol, ß-zearalanol, and ß-zearalenol) were 32, 17, 12, 0.3, and 0.1%, respectively. The recovery rates in spiked food samples were 97-117%, and the intraday and interday relative standard deviations were both <10%. Parallel analysis of natural food samples showed a good correlation between this novel CLIA and liquid chromatography-tandem mass spectrometry. This method provides a rapid, accurate, and highly sensitive method to determine levels of zearalenone in food samples.

Simultaneous quantitative determination of multiple mycotoxins in cereal and feedstuff samples by a suspension array immunoassay.

Mycotoxins produced by different species of fungi may coexist in single cereal and feedstuff samples, which could become highly toxic for humans and animals. In order to quantify four mycotoxins (zearalenone, fumonisin B1, deoxynivalenol, and aflatoxin B1) in cereal and feedstuff samples simultaneously, a new suspension array immunoassay was developed. Antimycotoxin monoclonal antibodies were conjugated to the surface of different encoding microspheres (19#, 37#, 39#, and 49#), and mycotoxin-protein conjugates were then coupled with biotin. Using streptavidin-phycoerythrin as a signal reporter protein, this direct competition multiple suspension array immunoassay was optimized. The results showed that the detection limits for zearalenone, fumonisin B1, deoxynivalenol, and aflatoxin B1 were 0.51, 6.0, 4.3, and 0.56 ng/mL, respectively, with detection ranges of 0.73-6.8, 11.6-110.3, 8.6-108.1, and 1.1-14.1 ng/mL, respectively. For the detection of the spiked samples, the recovery rates were between 92.3% and 115.5%. This method also shows a good correlation coefficient (r = 0.99, P < 0.01) with liquid chromatography-tandem mass spectrometry in the detection of toxins in commercial cereal and feedstuff samples. This suspension array immunoassay was high-throughput and accurate for the rapid quantitative detection of multiple mycotoxins in commercial cereal and feedstuff samples.

[Math1 gene therapy for kanamycin and furosemide-induced deaf guinea pigs].

OBJECTIVE: To observe the morphology and function changes of cochlear hair cells before and after math1 gene injection into the cochlea of deaf guinea pigs which were induced by kanamycin and furosemide. To explore the feasibility of Math1 gene for medicine-induced deafness therapy. METHODS: Kanamycin (500 mg/kg) and furosemide (50 mg/kg) were given to the healthy adult guinea pigs intramuscularly and intravenously to establish the deafness model. The guinea pigs whose auditory brainstem response (ABR) threshold > 95 dB SPL were randomly divided into five groups. Blank control group (without any treatment, n = 3), operation control group (right ear scala tympani operation, n = 3), artificial perilymph group (right ear scala tympani injection artificial perilymph, n = 3), virus vector group [right ear scala tympani injection adenovirus which carrying enhanced green fluorescent protein (EGFP) gene (Ad. EGFP) , n = 4], Math1 gene therapy group [right ear scala tympani injection adenovirus which carrying Math1 and EGFP gene (Ad. Math1-EGFP), n = 6]. Each animal received ABR test before and after injection. The cochlear tissue was observed by scanning electronic microscopy. RESULTS: The ABR thresholds of tone burst( 4, 8, 16, 20 kHz ) were not statistically significant in different groups (P > 0.05). The number of hair cells increased in some of severe deaf guinea pigs after the injection of Ad. Math1-EGFP gene. However, there was no obvious difference with morphology and numbers of cochlea hair cells in other groups. CONCLUSIONS: The injection of Math1 gene to cochlea can regenerate or repair the hair cells of medicine-induced deaf guinea pigs, but there was no improvement on the hearing loss.

Migration and differentiation of mouse embryonic stem cells transplanted into mature cochlea of rats with aminoglycoside-induced hearing loss.

CONCLUSION: Mouse embryonic stem cells (ESCs) transplanted into the scala tympani are able to migrate in the cochlea of rats deafened with aminoglycoside and partly restore the structure of sensory epithelia of the inner ear. OBJECTIVES: To explore the migration and differentiation of enhanced green fluorescence protein (EGFP)-expressing ESCs by transplanting them into the scala tympani of rats with amikacin sulfate-induced hearing loss. METHODS: Adult Sprague-Dawley (SD) rats were deafened with amikacin sulfate. Mouse ESCs expressing EGFP (EGFP-ESCs) were transplanted into the scala tympani. The migration and differentiation were observed at different time points. RESULTS: EGFP-ESCs transplanted into normal cochlea did not migrate, but those in the amikacin-damaged cochlea could survive and migrate into the scala media and the vestibular cisterna. For the first time, we observed that the EGFP-ESCs migrated into the scala media, took the place of the organ of Corti, and formed a structure just like the cochlear tunnel. Some grafted stem cells even expressed myosin VIIa, the molecular marker of hair cells. Some nerve fibers reached to the bottom of the hair cell-like cells. The ESCs migrated into the vestibule and restored the sensory epithelia of the ampullary crest. The number of the transplanted ESCs reduced over the 6 week period of the study.

[Histological changes of peripheral vestibular organs in the inner ears of Smad4 conditional knockout mice].

OBJECTIVE: To investigate the histological changes in the vestibular endorgans of Smad4 gene conditional knockout mice and to explore the influence of the Smad4 gene on vestibular development. METHODS: Histological changes of periphery vestibular organs in inner ear of Smad4 conditional knockout mice were investigated by frozen sections, immunofluorescence, confocal microscopy, scanning electron microscopy and transmission electron microscopy. RESULTS: There was no Smad4 expression in the inner ear cartilage capsule of Smad4-/- mice. In Smad4+/- mice, Smad4 expression in the same cartilage capsule was positive, and it was strong positive in Smad4+/+ mice. Smad4 expression in vestibular sense epithelium, crista ampullaris and macula, was positive. And no difference was found among these three genotypes. Studying at scanning electron microscopy and transmission electron microscopy levels and anti-filament immunofluorescence showed that no pathological changes were observed in all the three genotype mice. CONCLUSION: Although the Smad4 gene was knockout effectively in the auricular cartilage capsule of Smad4 conditional knockout mice,the histological changes of Smad4 conditional knockout mice in vestibulum auris internal were slightly.

Regeneration of stereocilia of hair cells by forced Atoh1 expression in the adult mammalian cochlea.

The hallmark of mechanosensory hair cells is the stereocilia, where mechanical stimuli are converted into electrical signals. These delicate stereocilia are susceptible to acoustic trauma and ototoxic drugs. While hair cells in lower vertebrates and the mammalian vestibular system can spontaneously regenerate lost stereocilia, mammalian cochlear hair cells no longer retain this capability. We explored the possibility of regenerating stereocilia in the noise-deafened guinea pig cochlea by cochlear inoculation of a viral vector carrying Atoh1, a gene critical for hair cell differentiation. Exposure to simulated gunfire resulted in a 60-70 dB hearing loss and extensive damage and loss of stereocilia bundles of both inner and outer hair cells along the entire cochlear length. However, most injured hair cells remained in the organ of Corti for up to 10 days after the trauma. A viral vector carrying an EGFP-labeled Atoh1 gene was inoculated into the cochlea through the round window on the seventh day after noise exposure. Auditory brainstem response measured one month after inoculation showed that hearing thresholds were substantially improved. Scanning electron microscopy revealed that the damaged/lost stereocilia bundles were repaired or regenerated after Atoh1 treatment, suggesting that Atoh1 was able to induce repair/regeneration of the damaged or lost stereocilia. Therefore, our studies revealed a new role of Atoh1 as a gene critical for promoting repair/regeneration of stereocilia and maintaining injured hair cells in the adult mammal cochlea. Atoh1-based gene therapy, therefore, has the potential to treat noise-induced hearing loss if the treatment is carried out before hair cells die.

Type I hair cell regeneration induced by Math1 gene transfer following neomycin ototoxicity in rat vestibular sensory epithelium.

CONCLUSION: In the current study, hair cells of vestibular terminal organs in rats were completely eliminated with trans-scala vestibuli injection of neomycin, and then the Math1 gene was transferred. It was shown that type I vestibular hair cells were regenerated and synapses were formed. OBJECTIVES: The objective of this study was to identify the cell type of the regenerated vestibular hair cells and relative innervation and synaptic linkage after hair cells of vestibular terminal organs in rats were completely eliminated. METHODS: Neomycin injection was used to eliminate all the vestibular terminal organs, and then the animals were treated with an injection of Ad-Math1-EGFP in the scala vestibuli of the cochlea. RESULTS: Math1 gene transfer into the inner ear induced type I hair cell regeneration and synaptic formation. However, neither the number nor the appearance of the hair cells was normal.

The differentiation of mesenchymal stem cells into inner ear hair cell-like cells in vitro.

CONCLUSION: Bone marrow mesenchymal stem cells (MSCs) have the ability to differentiate into hair cells, and this method of culturing MSCs provides a useful tool for studies on mammalian cochlear hair cell regeneration. OBJECTIVE: To investigate a method to induce bone marrow MSCs to differentiate into inner ear hair cells. METHODS: Rat bone marrow MSCs were isolated from healthy rats and cultured in vitro. To make sure that the cultured cells were bone marrow MSCs, the expression of MSC markers such as SH2, CD31, CD34, and CD44 genes on the cultured cells was assessed by RT-PCR. Adipogenic cells and osteogenic cells were induced by the differentiation of the cultured cells, respectively, suggesting that the cultured cells have the characteristic of pluripotent differentiation. Then they were induced to differentiate into neural stem cells and hair cell progenitor cells. Immunohistochemistry experiments were carried out to detect the expression of molecular markers. Scanning electron microscope samples were prepared for observation of the morphology of the cells. RESULTS: Rat bone marrow MSCs were successfully isolated, purified, cultured, and identified in vitro. They were also successfully induced to differentiate into neural progenitor cells and then hair cell-like cells that expressed myosin VIIa.

The role of Smad4 in vestibular development in mice.

The regulation of the bone morphogenetic protein (BMP) signal transduction pathway is important in the development of the inner ear and vestibular system. We reported previously that small mothers against decapentaplegic homolog-4 (Smad4) is required for inner ear cochlear development and normal auditory function in mammals; however, the distribution and functional mechanisms of Smad4 at various stages of vestibular development remained unclear. To investigate the relationship between the Smad4 gene and vestibular organ development, we measured changes in the expression of BMP4 and Smad4 during vestibular development in C57BL/6 mice. In addition, vestibular structures, pathologic changes, and the vestibular function of chondrocyte-specific Smad4 knockout mice were compared to those of the control group. We found that the expression of Smad4 in the inner ear was delayed compared with that of BMP4. Moreover, chondrocyte-specific Smad4 knockout homozygous mice showed stunted growth and partial vestibular deformities, but it showed less histologic changes in the vestibular end-organs and saccule dysfunction. These results suggest that Smad4 participates in late-stage shaping of the configuration of the vestibule and development of vestibular functional, but a Smad4-independent pathway for the inner ear vestibular BMP4 signal transduction could not be rule out.

Effects of DAPT and Atoh1 overexpression on hair cell production and hair bundle orientation in cultured Organ of Corti from neonatal rats.

BACKGROUND: In mammals, hair cells do not undergo spontaneous regeneration when they are damaged and result in permanent hearing loss. Previous studies in cultured Organ of Corti dissected from neonatal animals have shown that both DAPT (r-secretase inhibitor in the Notch signal pathway) treatment and Atoh1 overexpression can induce supernumerary hair cells. The effects of simultaneous DAPT treatment and Atoh1 over expression in the cells of cultured Organ of Corti from neonatal rats are still obscure. PRINCIPAL FINDINGS: In this study, we set out to investigate the interaction of DAPT treatment and Atoh1 overexpression as well as culture time and the location of basilar fragment isolated form neonatal rat inner ear. Our results showed that DAPT treatment induced more hair cells in the apical turn, while Atoh1 overexpression induced more extra hair cells in the middle turn of the cultured Organ of Corti. When used together, their effects are additive but not synergistic. In addition, the induction of supernumerary hair cells by both DAPT and Atoh1 overexpression is dependent on the treatment time and the location of the cochlear tissue. Moreover, DAPT treatment causes dramatic changes in the orientation of the stereociliary bundles of hair cells, whereas Atoh1 overexpression didn't induce drastic change of the polarity of stereociliary bundles. CONCLUSIONS/SIGNIFICANCE: Taken together, these results suggest that DAPT treatment are much more potent in inducing supernumerary hair cells than Atoh1 overexpression and that the new hair cells mainly come from the trans-differentiation of supporting cells around hair cells. The orientation change of stereociliary bundle of hair cells may be attributed to the insertion of the newly formed hair cells. The immature hair bundles on the newly formed hair cells may also contribute to the overall chaos of the stereociliary bundle of the sensory epithelia.

Death mode-dependent reduction in succinate dehydrogenase activity in hair cells of aging rat cochleae.

BACKGROUND: Our previous studies have shown that both apoptosis and necrosis are involved in hair cell (HC) pathogenesis in aging cochleae. To better understand the biological mechanisms responsible for the regulation of HC death, we examined the activity of succinate dehydrogenase (SDH), a mitochondrial bioenergetic enzyme, in the HCs of aging cochleae. METHODS: The auditory brainstem response thresholds elicited by tone bursts at 4, 10 and 20 kHz were measured in both young (2-3 months) and aging (22-23 months) Wistar rats. SDH activity was evaluated with a colorimetric assay using nitroblue tetrazolium monosodium salt. The SDH-labeled organs of Corti were double stained with propidium iodide, a DNA intercalating fluorescent probe for illustration of HC nuclei. All the specimens were examined with fluorescence microscopy and confocal microscopy. RESULTS: Aging rats exhibited a significant elevation of ABR thresholds with threshold shifts being 34 dB at 20 kHz, 28 dB at 10 kHz, and 25 dB at 4 kHz. Consistent with the reduction in the cochlear function, aging cochleae exhibited the reduction of SDH staining intensity in the apical and the basal ends of the cochleae, where a large number of apoptotic, necrotic, and missing HCs were evident. The reduction in SDH staining appeared in a cell-death-mode dependent fashion. Specifically, SDH labeling remained in apoptotic HCs. In contrast, SDH staining was markedly reduced or absent in necrotic HCs. CONCLUSIONS: In the aging cochlea, SDH activity is preserved in HCs undergoing apoptosis, but is substantially reduced in necrosis. These results suggest that mitochondrial energetic function is involved in the regulation of cell death pathways in the pathogenesis of aging cochleae.

Chondrocyte-specific Smad4 gene conditional knockout results in hearing loss and inner ear malformation in mice.

Smad4 is the central intracellular mediator of transforming growth factor-beta (TGF-beta) signaling, which plays crucial roles in tissue regeneration, cell differentiation, embryonic development, and regulation of the immune system. Conventional Smad4 gene knockout results in embryonic lethality, precluding its use in studies of the role of Smad4 in inner ear development. We used chondrocyte-specific Smad4 knockout mice (Smad4Co/Co) to investigate the function of Smad4 in inner ear development. Smad4Co/Co mice were characterized by a smaller cochlear volume, bone malformation, and abnormalities of the osseous spiral lamina and basilar membrane. The development of the hair cells was also abnormal, as evidenced by the disorganized stereocilia and reduced density of the neuronal processes beneath the hair cells. Auditory function tests revealed the homozygous Smad4Co/Co mice suffered from severe sensorineural hearing loss. Our results suggest that Smad4 is required for inner ear development and normal auditory function in mammals.