A de novo silencer causes elimination of MITF-M expression and profound hearing loss in pigs.

BACKGROUND: Genesis of novel gene regulatory modules is largely responsible for morphological and functional evolution. De novo generation of novel cis-regulatory elements (CREs) is much rarer than genomic events that alter existing CREs such as transposition, promoter switching or co-option. Only one case of de novo generation has been reported to date, in fish and without involvement of phenotype alteration. Yet, this event likely occurs in other animals and helps drive genetic/phenotypic variation. RESULTS: Using a porcine model of spontaneous hearing loss not previously characterized we performed gene mapping and mutation screening to determine the genetic foundation of the phenotype. We identified a mutation in the non-regulatory region of the melanocyte-specific promoter of microphthalmia-associated transcription factor (MITF) gene that generated a novel silencer. The consequent elimination of expression of the MITF-M isoform led to early degeneration of the intermediate cells of the cochlear stria vascularis and profound hearing loss, as well as depigmentation, all of which resemble the typical phenotype of Waardenburg syndrome in humans. The mutation exclusively affected MITF-M and no other isoforms. The essential function of Mitf-m in hearing development was further validated using a knock-out mouse model. CONCLUSIONS: Elimination of the MITF-M isoform alone is sufficient to cause deafness and depigmentation. To our knowledge, this study provides the first evidence of a de novo CRE in mammals that produces a systemic functional effect.

Functional mutation of SMAC/DIABLO, encoding a mitochondrial proapoptotic protein, causes human progressive hearing loss DFNA64.

SMAC/DIABLO is a mitochondrial proapoptotic protein that is released from mitochondria during apoptosis and counters the inhibitory activities of inhibitor of apoptosis proteins, IAPs. By linkage analysis and candidate screening, we identified a heterozygous SMAC/DIABLO mutation, c.377C>T (p.Ser126Leu, refers to p.Ser71Leu in the mature protein) in a six-generation Chinese kindred characterized by dominant progressive nonsyndromic hearing loss, designated as DFNA64. SMAC/DIABLO is highly expressed in human embryonic ears and is enriched in the developing mouse inner-ear hair cells, suggesting it has a role in the development and homeostasis of hair cells. We used a functional study to demonstrate that the SMAC/DIABLO(S71L) mutant, while retaining the proapoptotic function, triggers significant degradation of both wild-type and mutant SMAC/DIABLO and renders host mitochondria susceptible to calcium-induced loss of the membrane potential. Our work identifies DFNA64 as the human genetic disorder associated with SMAC/DIABLO malfunction and suggests that mutant SMAC/DIABLO(S71L) might cause mitochondrial dysfunction.

Analysis of the heteroplasmy level and transmitted features in hearing-loss pedigrees with mitochondrial 12S rRNA A1555G mutation.

BACKGROUND: Mitochondrial cytopathies are characterized by a large variability of clinical phenotypes and severity. The amount of mutant mitochondrial DNA (mtDNA) in a cell, called the heteroplasmy level, is an important determinant of the degree of mitochondrial dysfunction and therefore disease severity. Understanding the distribution of heteroplasmy levels across a group of offspring is an important step in understanding the inheritance of diseases. Recently, the mtDNA A1555G mutation was found to be associated with non-syndromic and drug-induced hearing loss. RESULTS: Here, we report five pedigrees with multiple members having the A1555G mutation and showing diverse clinical manifestations and different heteroplasmy levels. Clinical evaluations revealed that the hearing impairment phenotypes varied with respect to the severity of hearing loss, age of onset of hearing loss, and pattern of audiometric configuration. These five Chinese pedigrees had different penetrance of hearing loss, ranging from 10-52%. A molecular study showed that the average heteroplasmy rates of the five pedigrees were 31.98% (0-91.35%), 78.28% (32.8-96.08%), 87.99% (82.32-94.65%), 93.34% (91.02-95.05%), and 93.57% (91.38-94.24%). There was no gradual tendency of heteroplasmy to increase or decrease along with transmission. A study of the relationship between clinical features and genetic background found that the percentage of deafness was 0 when the heteroplasmy level was less than 50%, 25% when the heteroplasmy level was 50-80%, 47.06% when the heteroplasmy level was 80-90%, and 57.58% when the heteroplasmy level exceeded 90%. The risk of deafness rose with the heteroplasmy level. CONCLUSIONS: The results suggest that there are large random shifts in the heteroplasmy level between mothers and offspring with the A1555G mutation; heteroplasmy could disappear randomly when the heteroplasmy level of the pedigree was low enough, and no regular pattern was found. The heteroplasmy level may be one of the factors influencing the penetrance of deafness caused by the mtDNA A1555G mutation.

Mitochondria toxin-induced acute cochlear cell death indicates cellular activity-correlated energy consumption.

The different cell types within the cochlea may have a specific contribution to the pathological changes during metabolism failure, which may provide clues for developing novel strategies for inner ear therapy. In order to evaluate activity-correlated cell death during metabolism failure in the cochlea, 3-nitropropionic acid was used to irreversibly inhibit the respiratory chain. Dose-response of the cochlear cells to 3-nitropropionic acid was analyzed in vitro. 3-Nitropropionic acid was administered onto the round window of guinea pigs. Cell death was identified by terminal transferase labeling the free 3'OH breaks in the DNA strands in vivo and propidium iodide nuclear permeation in vitro. As a result, 23.6 and 96.3 % cell death were induced by 10 and 100 mM 3-nitropropionic acid, respectively, in vitro. In the guinea pigs, 500 mM 3-nitropropionic acid induced vestibular dysfunction and severe to profound hearing losses. The cells that are the most sensitive to 3-nitropropionic acid treatment include the stria marginal and intermediate cells, epithelial cells of the Reissner's membrane, and spiral ligament fibrocytes (types II and V). Moderate sensitive cells were satellite fibrocytes of the spiral limbic central zone, osteocytes of the cochlear shell, hair cells, and spiral ganglion cells. Reduction of neurofilament in the soma and periphery processes of spiral ganglion cells occurred after the exposure. These results may be relevant to the mechanisms of injury in sudden onset sensorineural hearing loss and hazardous substance exposure-induced hearing loss.

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.

Comparison of frequency-specific hearing outcomes after endoscopic and microscopic tympanoplasty.

BACKGROUND: Hearing results of endoscopic and microscopic tympanoplasty have been compared using the average pure tone threshold which could conceal subtle differences at a specific frequency. OBJECTIVES: To compare frequency-specific hearing outcomes of endoscopic and microscopic tympanoplasty. MATERIAL AND METHODS: The study included 42 patients who underwent endoscopic or microscopic type I tympanoplasty. The medical charts of these patients were reviewed retrospectively. We evaluated the pure tone audiometry at 250, 500, 1000, 2000 and 4000 Hz, including bone conduction (BC), air conduction (AC) and air-bone gap (ABG) before and after the surgery. The main outcome measures were frequency-specific pre- and post-operative hearing thresholds and the corresponding changes. We also assessed the graft success rate and surgical complications. RESULTS: BC revealed a significant aggravation at 4000 Hz in microscopic tympanoplasty group, but no significant differences between the two groups at any frequencies. Both groups showed improvements in AC and ABG at all frequencies, without significant differences between the two groups at any single frequency. The maximum improvement of AC and ABG was found at 250 Hz. The graft success rate and operative complications were also similar. CONCLUSIONS AND SIGNIFICANCE: The frequency-specific hearing outcomes of endoscopic and microscopic tympanoplasty are similar.

Acid stimulation-induced semi-pluripotent characteristics in human somatic cells.

BACKGROUND: Clinical trials of cell-based therapies using induced pluripotent stem (iPS) cells have already been started for several neurological diseases. OBJECTIVE: The purpose of the present study was to explore the characteristics and differentiation of somatic cells in vitro undergoing a low pH treatment, so as to provide new therapeutic strategies for treating sensorineural hearing loss. METHODS: Somatic cells were treated with low pH solution to alter their characteristics. In addition, a mouse model of the cochlear lesion was constructed using bilirubin. Subsequently, the characteristics and therapeutic effect of somatic cells undergoing low pH treatment were examined by morphology, alkaline phosphatase (AKP) activity, immunofluorescence assay and q-PCR. RESULTS: The cells in the experimental group grew better than those in the control group. The AKP activity in the experimental group was higher than that in the control group. The expression of Nanog and Oct4 was both positive in the two groups. When the cells were changed to neurobasal medium, the marker of nestin was positive. CONCLUSION: The human somatic cells undergoing a low pH treatment showed the similar characteristics as those of iPS cells, although the functions and therapeutic effect of these altered human somatic cells need to be further studied.

Mutations at position 7445 in the precursor of mitochondrial tRNA(Ser(UCN)) gene in three maternal Chinese pedigrees with sensorineural hearing loss.

We report here the clinical, genetic and molecular characterization of three Chinese pedigrees with nonsyndromic bilateral hearing loss. Clinical and genetic evaluations revealed the variable severity and age-of-onset in hearing impairment in these families. Strikingly, there were extremely low penetrances of hearing impairment in these Chinese families. Sequence analysis of the complete mitochondrial DNA (mtDNA) showed the known A7445C mutation in two pedigrees and the novel A7445T mutation in another pedigree, in addition to distinct sets of mtDNA polymorphisms belong to Asian haplogroups D4j and F4. Indeed, the A7445C or A7445T mutation in the CO1 and the precursor of tRNA(Ser(UCN)) genes was present in homoplasmy only in the maternal lineage of those pedigrees but not other members of these families and 164 Chinese controls. In fact, the A7445C or A7445T mutation results in a read-through of the stop condon AGA of the CO1 message on the H strand of mtDNA, thereby adding three amino acids (Ser-Gln-Lys) to the C-terminal of the polypeptide. However, the mutated polypeptide may retain a partial function. Alternatively, the A7445C or A7445T mutation is adjacent to the site of 3' end endonucleolytic processing of L-strand RNA precursor, spanning tRNA(Ser(UCN)) and ND6 mRNA. Thus, the A7445C or A7445T mutation may also cause a defect in the processing of the L-strand RNA precursor, thus causing mitochondrial dysfunctions. Furthermore, the occurrence of the mutations at position 7445 in these genetically unrelated subjects affected by hearing impairment strongly indicates that mutations at the position 7445 are involved in the pathogenesis of hearing impairment.

Aging and the peripheral vestibular system.

Whereas much has been learned about age-related auditory changes in the inner ear, relatively little is known about the aging effects on the vestibular part of the inner ear-the peripheral vestibular system. Here we review relevant literature with regard to the prevalence of vestibular dysfunction, vestibular functional and structural changes in the elderly. The prevalence of vestibular dysfunction increases with age. Functionally, as age increases, VEMP amplitudes decrease, VEMP thresholds increase, VOR gain of HIT decreases. Due to the complexity of the vestibular system, variations in subject age and measurement techniques, findings in VEMP latency and caloric tests are conflicting. To address this, a direct measure of the peripheral vestibular system should be applied. Structurally, age-related loss in vestibular ganglion and otoconia have been noted; hair cell changes are not well defined; while subcellular changes remain to be explored. Defining how the onset of vestibular dysfunction correlates with structural degeneration will offer insights into the mechanisms underlying vestibular aging.

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.

[Genetic counseling and intervention for families with deaf-mute patients based on genetic testing: analysis of 5 families].

OBJECTIVE: To analyze the molecular genetic mechanisms of pathogenesis of deafness in the families with deaf-mute patients and analyze the strategies of genetic counseling and intervention for these families. METHODS: Peripheral blood samples were collected from the probands with deaf-muteness and their parents of five families and genetic tests were conducted to analyze the GJB2, SLC26A4 (PDS), and mitochondrial DNA (mtDNA) A 1555G genes for the existence of mutation. Families 1-3 had one child with hearing loss each while the parents had normal hearing and the mothers had been pregnant for 6-18 weeks. Both parents of family 4 were deaf-mute, and the wife of family 5 was deaf-mute while her husband had normal hearing. RESULTS: The proband from family 1 was proven to carry compound GJB2 mutations while his parents carried a single GJB2 mutation; prenatal testing showed that the fetus only carried the paternal mutation. The proband from family 2 was proven to carry compound SLC26A4 (PDS) mutations while his parents carried a single SLC26A4 (PDS) mutation; prenatal testing showed that the fetus only carried the paternal mutation. The proband from family 3 and his parents didn't carry any GJB2, SLC26A4 and mtDNA A1555G mutation. Observation showed that the new born babies of these three families all had normal hearing revealed by new born hearing screening and ABR test. The husband from family 4 was homozygous GJB2 235delC while his wife was mtDNA A1555G positive. This couple was advised to strictly avoid the administration of aminoglycoside antibiotics to their future offspring. In family 5, the wife carried compound SLC26A4 (PDS) mutations while her husband carried a single SLC26A4 (PDS) mutation; and they were told about the 50% risk of their offspring's suffering from enlarged vestibular aqueduct syndrome. CONCLUSION: Genetic testing with prenatal testing and relevant intervention for the families with deaf-mute patients can be applied to prevent another deaf-mute member from being born.

[Expression of liposome-mediated PEDF-GFP gene in the retina of the BN rats through different gene delivery route].

OBJECTIVE: Transducing PEDF-GFP plasmid to the retina of the BN rats with cationic liposome through different gene delivery route, then observe the expression and location of the PEDF-GFP. METHODS: PEDF-GFP plasmid with cationic liposome was delivered to the retina of the BN rat through subretinal injection and intravitreal injection. The expression of GFP was observed under fluorescence microscope, and the mRNA of PEDF gene was detected by RT-PCR. RESULTS: Green fluorescence was emitted from the total retina include RPE cell under fluorescent microscope after 24 h in two gene delivery route, The fluorescence intensity was stronger with time changing. Gradual fluorescence increase in the retina and RPE cells occurred and lasted 4 weeks. The expression of PEDF mRNA was also detected by RT-PCR after 24 h, and maintained stable 4 weeks after infection. CONCLUSIONS: Cationic liposome can mediate PEDF-GFP gene into the retina of the BN rat effectively; subretinal injection and intravitreal injection both are effective gene delivery route; and their stable expression can maintain 4 weeks after transfection.

Molecular mechanisms underlying the protective effects of hydrogen-saturated saline on noise-induced hearing loss.

OBJECTIVES: This study aimed to explore the molecular mechanism of the protective effects of hydrogen-saturated saline on NIHL. METHODS: Guinea pigs were divided into three groups: hydrogen-saturated saline; normal saline; and control. For saline administration, the guinea pigs were given daily abdominal injections 3 d before and 1 h before noise exposure. ABR were tested to examine cochlear physiology changes. The changes of 8-hydroxy-desoxyguanosine (8-HOdG), interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-10 (IL-10), tumor necrosis factor-α (TNF-α), intercellular cell adhesion molecule-1 (ICAM-1) and high mobility group box-1 protein (HMGB1) in the cochlea were also examined. RESULTS: The results showed that pre-treatment with hydrogen-saturated saline could significantly attenuate noise-induced hearing loss. The concentration of 8-HOdG was also significantly decreased in the hydrogen-saturated saline group compared with the normal saline group. After noise exposure, the concentrations of IL-1, IL-6, TNF-α, and ICAM-1 in the cochlea of guinea pigs in the hydrogen-saturated saline group were dramatically reduced compared to those in the normal saline group. The concentrations of HMGB-1 and IL-10 in the hydrogen-saturated saline group were significantly higher than in those in the normal saline group immediately and at 7 d after noise exposure. CONCLUSIONS: This study revealed for the first time the protective effects of hydrogen-saturated saline on noise-induced hearing loss (NIHL) are related to both the anti-oxidative activity and anti-inflammatory activity.

Transplantation of human umbilical cord mesenchymal stem cells in cochlea to repair sensorineural hearing.

To examine if transplantation of human umbilical cord mesenchymal stem cells (UMSC) into cochlea can be used to repair sensorineural hearing. Here we transplanted the fifth and sixth generations of UMSCs through the subarachnoid cavity of congenital deaf albino pigs. Auditory brainstem responses (ABR) were measured before and after UMSC transplantation. Cochlear samples were collected at 2 hrs, 3 days, 1, 2, 3, 4 and 8 weeks after transplantation. Immunohistochemistry was used to detect the proliferated cell nuclear antigen (PCNA). The UMSCs were found in different regions of the cochlea, including the stria vascularis, the basal membrane and the spiral ganglions, 3 days to 4 weeks after the transplantation. UMSCs and their DNA were found also in the areas of the brain, the heart, the liver, the kidney and the lung. ABR tests displayed a new waveform in the congenital deaf albino pigs after the UMSCs transplantation. We conclude that human UMSCs injected into the subarachnoid space can migrate into the inner ear, the central nervous system and the periphery organs. The presence of UMSCs in the cochlea maybe associated with changes of ABR waveforms in the congenital deaf albino pigs.

[Meta-analysis on autogenous fat injection for unilateral vocal cord paralysis].

OBJECTIVE: This study conduct a qualitative synthesis and quantitative meta-analysis of VFAFI, aimed to study whether it is a useful treatment for UVCP. METHOD: Electronic databases PubMed, YZ365. COM, WANFANG DATA, CMJD, CHKD,CNKI were searched using relevant keywords. Reported treatment outcomes were clustered into three categories,i. e. subjective, perceptual,acoustic,aerodynamic,and stroboscopic. Meta-analyses were performed on studies with numerical results using random effects model. RESULT: Five articles were identified with a total of 404 patients. All the studies reported significant improvements or decrease after VFAFI in each category of outcome measurements. Meta-analysis demonstrated a significant increase or decrease in all categories. Adverse effects include slight inflammatory reponse can resolve spontaneously within 1 month. The recurrence rate after VFAFI was high due to the self absorption. NNE and Jitter of post-operation is lower than pre-operation,there is no significantly change between the control group and experimental group; F0, Shimmer and MPT of post-operation is higher than pre-operation, there is no significantly change between the control group and experimental group. CONCLUSION: The invasiveness and morbidity of VFAFI are low and the side effects are self-limited. Meta-analyses demonstrated significant improvements or decreased from both objective and subjective measurements. Further controlled studies with longer follow-up periods and more person were included may evaluate the effectiveness of VFAFI more reliably.

[Meta-analysis on effectiveness of prelingually deaf patients at different ages following cochlear implantation].

OBJECTIVE: To assess the clinical effeetiveness of prelingually deaf children after cochlear implantation at different ages so as to provide reasonable expectations for the patients and guidance for the clinical treatment. METHOD: Electronic databases PubMed, YZ365. COM, WANFANG DATA, CMJD, CHKD, CNKI were searched using relevant keywords. Extracted data included author, year of publication, diagnosis, et al. Reported treatment outcomes were clustered into speech discrimination and hearing abilities. Meta-analyses were performed on studies with numerical results using random or fixed effects model. RESULT: There were eight randomized control studies including 442 patients. Comparing speech perception of prelingually deaf children after cochlear implantation younger than three years old (experimental group) and 3-6 years old (control group), three and six months after operation showed that experimental group performed significantly worse than control group; 12 months after operation showed that experimental group performed significantly better than control group. Comparing hearing abilities, three and six months after operation showed that experimental group performed significantly worse than control group; 12 months after operation showed showed that experimental group performed significantly better than control group. Comparing speech perception of younger or older than 4. 5 years old children showed that after 1.5-2 years of operation children implanted younger than 4.5 years of age performed significantly better than children implanted older than 4.5 years old. Comparing speech perception of 7-12 years old children showed that after 3, 6, 12 months of operation patients of 7-12 years old performed significantly better than those children older than 12 years old. Comparing speech perception of implantation younger or older than 18 years old (7-14 yeas old was group A, > 14-18 yeas old was group B, older than 18 yeas old was group C) showed that after one and four years of operation A > B > C, and there were significant differences among them. Comparing warble tone threshold average (WTA) showed that after one year of operation A < B < C, and there were significant differences among them. However, after four years of operation, there was no significant difference among them. CONCLUSION: Prelinguistically deafened patients younger than three years old with cochlear implantation, insisting on scienctific rehabilitation training for a long period of time can receive the optimal recovery effect. The older patients are suggested as early as possible receiving cochlear implantation. The longer they are implanted, the better results they will receive. Moreover, the younger age they are implanted, the faster postoperative language progress they will receive. Further controlled studies with longer follow-up periods and more person included may make the effectiveness of cochlear implantaion more reliable.

Mutations in the mitochondrial 12S rRNA gene in elderly Chinese people.

CONCLUSION: Our data indicate that the mitochondrial 12S rRNA gene, and particularly the A827G mutation, may be associated with susceptibility to age-related hearing loss. OBJECTIVE: Hearing loss associated with aging is common among elderly persons. In all genetic backgrounds, mitochondrial DNA (mtDNA) mutations may be one of the most important factors contributing to aging and age-related hearing loss. The mitochondrial 12S rRNA is a hot spot for deafness-associated mutations in Chinese populations. The purpose of the present study was to elucidate the relationship of 12S rRNA gene polymorphisms and age-related hearing loss. METHODS: The 12S rRNA gene polymorphisms were detected by direct sequencing. Statistical analyses were performed to assess the associations between age-related hearing loss and 12S rRNA gene variants. RESULTS: We report here a systematic mutational screening of the mitochondrial 12S rRNA gene in 662 elderly subjects from the general population with various hearing threshold levels (211 controls and 451 age-related hearing loss subjects). Mutational screening of the mitochondrial 12S rRNA gene identified 55 nucleotide changes, including 4 mutations localized at highly conserved sites and 51 known variants. Of the known deafness-associated mutations in the mitochondrial 12S rRNA gene, the incidence of the A1555G mutation was 0.15%, A827G was 4.38%, T1095C was 0.45%, and T1005C was 3.78%. The incidence of the other known variants was 0.15-99.85%. We found statistically significant differences in the proportions of subjects with the A827G mutation among the various age-related hearing loss groups and normal controls.

Cartilage engineering using chondrocyte cell sheets and its application in reconstruction of microtia.

The imperfections of scaffold materials have hindered the clinical application of cartilage tissue engineering. The recently developed cell-sheet technique is adopted to engineer tissues without scaffold materials, thus is considered being potentially able to overcome the problems concerning the scaffold imperfections. This study constructed monolayer and bilayer chondrocyte cell sheets and harvested the sheets with cell scraper instead of temperature-responsive culture dishes. The properties of the cultured chondrocyte cell sheets and the feasibility of cartilage engineering using the chondrocyte cell sheets was further investigated via in vitro and in vivo study. Primary extracellular matrix (ECM) formation and type II collagen expression was detected in the cell sheets during in vitro culture. After implanted into nude mice for 8 weeks, mature cartilage discs were harvested. The morphology of newly formed cartilage was similar in the constructs originated from monolayer and bilayer chondrocyte cell sheet. The chondrocytes were located within evenly distributed ovoid lacunae. Robust ECM formation and intense expression of type II collagen was observed surrounding the evenly distributed chondrocytes in the neocartilages. Biochemical analysis showed that the DNA contents of the neocartilages were higher than native human costal cartilage; while the contents of the main component of ECM, glycosaminoglycan and hydroxyproline, were similar to native human costal cartilage. In conclusion, the chondrocyte cell sheet constructed using the simple and low-cost technique is basically the same with the cell sheet cultured and harvested in temperature-responsive culture dishes, and can be used for cartilage tissue engineering.

Clinical observation on hearing conditions of centenarians in northern district of China.

CONCLUSION: The hearing conditions of the centenarians were quite poor as regards hearing thresholds and speech detection ability. OBJECTIVE: To investigate hearing conditions of centenarians. METHODS: A total of 54 centenarians in Rizhao and Linyi Districts in Shandong Province were investigated to assess hearing conditions of centenerians comprehensively by questionnaire investigation, pure-tone audiometry, acoustic immitance, intelligence evaluation, and speech detection scores. Also, 135 individuals were recruited as controls and divided into four groups according to their age: 45-59 years, 60-69 years, 70-79 years, and 80-89 years. RESULTS: The hearing thresholds of the centenarians were dramatically higher than those of the control group (p < 0.05) and all centenarians suffered moderate to profound hearing loss according to the World Health Organization (WHO) criteria. Few centenarians had normal level of speech detection scores. All centenarians showed descending hearing curve, and the hearing threshold of the male centenarians at 8000 Hz was higher than that of the females (p = 0.047). There was a significant air-bone conduction gap in the centenarians (p < 0.05).

Effects of alpha-tocopherol on noise-induced hearing loss in guinea pigs.

Preventing noise-induced hearing loss (NIHL) by antioxidants is based on the hypothesis that generation of reactive oxygen species is one of the causes of NIHL. alpha-Tocopherol is a naturally occurring antioxidant with no noticeable side effects. In this study, we attempted to protect guinea pigs from developing NIHL by administering alpha-tocopherol. Pigmented male guinea pigs were exposed to a noise (4 kHz octave band, 100 dB SPL), 8 h/day for 3 days consecutively. alpha-Tocopherol (10 mg/kg or 50 mg/kg daily) was given by intraperitoneal injection from 3 days before through 3 days after the noise exposure. Auditory evoked brainstem response (ABR) thresholds at 2, 4 and 8 kHz were recorded prior to the experiment, immediately post-noise, 2 and 8 days post-noise. On day 8 post-noise, after the ABR recording, guinea pigs were decapitated and the cochleae were removed for cochlear surface preparations and scanning electron microscope (SEM) study. ABR threshold shifts of groups receiving alpha-tocopherol were significantly smaller than those of groups not receiving alpha-tocopherol at all frequencies and all time points tested except that of group 3 at 8 kHz 8 days post-noise. No hair cell loss was seen on the surface preparations, but stereocilia loss was found by SEM study. The noise-induced stereocilia loss was significantly decreased by alpha-tocopherol. These results indicate that alpha-tocopherol can attenuate the noise-induced cochlear damage. Further investigations on the preventive effect of alpha-tocopherol on NIHL in noise-exposed workers are necessary.