cdh23 affects congenital hearing loss through regulating purine metabolism.

Introduction: The pathogenic gene CDH23 plays a pivotal role in tip links, which is indispensable for mechanoelectrical transduction in the hair cells. However, the underlying molecular mechanism and signal regulatory networks that influence deafness is still largely unknown. Methods: In this study, a congenital deafness family, whole exome sequencing revealed a new mutation in the pathogenic gene CDH23, subsequently; the mutation has been validated using Sanger sequencing method. Then CRISPR/Cas9 technology was employed to knockout zebrafish cdh23 gene. Startle response experiment was used to compare with wide-type, the response to sound stimulation between wide-type and cdh23-/-. To further illustrate the molecular mechanisms underlying congenital deafness, comparative transcriptomic profiling and multiple bioinformatics analyses were performed. Results: The YO-PRO-1 assay result showed that in cdh23 deficient embryos, the YO-PRO-1 signal in inner ear and lateral line neuromast hair cells were completely lost. Startle response experiment showed that compared with wide-type, the response to sound stimulation decreased significantly in cdh23 mutant larvae. Comparative transcriptomic showed that the candidate genes such as atp1b2b and myof could affect hearing by regulating ATP production and purine metabolism in a synergetic way with cdh23. RT-qPCR results further confirmed the transcriptomics results. Further compensatory experiment showed that ATP treated cdh23-/- embryos can partially recover the mutant phenotype. Conclusion: In conclusion, our study may shed light on deciphering the principal mechanism and provide a potential therapeutic method for congenital hearing loss under the condition of CDH23 mutation.

Progressive hearing loss and degeneration of hair cell stereocilia in taperin gene knockout mice.

The TPRN gene encodes taperin, which is prominently present at the taper region of hair cell stereocilia. Mutations in TPRN have been reported to cause autosomal recessive nonsyndromic deafness 79(DFNB 79). To investigate the role of taperin in pathogenesis of hearing loss, we generated TPRN knockout mice using TALEN technique. Sanger sequencing confirmed an 11 bp deletion at nucleotide 177-187 in exon 1 of TPRN, which results in a truncated form of taperin protein. Heterozygous TPRN+/- mice showed apparently normal auditory phenotypes to their wide-type (WT) littermates. Homozygous TPRN-/- mice exhibited progressive sensorineural hearing loss as reflected by auditory brainstem response to both click and tone burst stimuli at postnatal days 15 (P15), 30 (P30), and 60 (P60). Alex Fluor-594 phalloidin labeling showed no obvious difference in hair cell numbers in the cochlea between TPRN-/- mice and WT mice under light microscope. However, scanning electronic microscopy revealed progressive degeneration of inner hair cell stereocilia, from apparently normal at postnatal days 3 (P3) to scattered absence at P15 and further to substantial loss at P30. The outer hair cell stereocilia also showed progressive degeneration, though much less severe, Collectively, we conclude that taperin plays an important role in maintenance of hair cell stereocilia. Establishment of TPRN knockout mice enables further investigation into the function of this gene.

Taurine enhances excitability of mouse cochlear neural stem cells by selectively promoting differentiation of glutamatergic neurons over GABAergic neurons.

Taurine is a sulfur-containing amino acid present in high concentrations in mammalian tissues, and has been implicated in several processes involving brain development and neurotransmission. However, the role of taurine in inner ear neural development is still largely unknown. Here we report that taurine enhanced the viability and proliferation of in vitro mouse cochlear neural stem cell culture, as well as improved neurite outgrowth. Moreover, prolonged taurine treatment also increased the neural electrical activity by escalating changes of intracellular calcium concentration, the number of spontaneous Ca(2+) oscillations in cells, and the frequencies of Ca(2+) spikes. Most importantly, we found that this escalated neural excitability by taurine was due to combined effect of increase in the population of excitatory glutamatergic neuron and decrease in inhibitory GABAergic neuron population. This is the first report on the effect of taurine to selectively promote neural stem cell differentiation by altering neuron type commitment. Our study has supported the potential of taurine as treatment against hearing loss caused by neuron degeneration, or even as an agent to improve sensitivity of hearing by increasing overall excitability of auditory nervous system.

Composite scaffolds of nano-hydroxyapatite and silk fibroin enhance mesenchymal stem cell-based bone regeneration via the interleukin 1 alpha autocrine/paracrine signaling loop.

Composite scaffolds of nano-hydroxyapatite (nHAp) and silk fibroin (SF) have been reported to promote bone regeneration mainly through signaling pathways associated with cell-biomaterial interaction. However, it is unclear whether soluble factors also play a role in osteoinduction with nHAp-SF. In this study, we confirmed the biocompatibility and superior osteoinductivity of nHAp-SF scaffolds versus SF scaffolds both in vitro and on a calvarial defect model in vivo. This was followed by further analysis with microarray assay. The cDNA microarray results identified 247 differentially expressed genes in bone marrow mesenchymal stem cells (BMSCs) cultured on SF-nHAp scaffolds versus SF scaffolds. The greatest disparity in gene expression levels were observed with Il1α and Ilr2. Real-time PCR assay validated the results. The addition of IL-1α into cultures of BMSCs with SF significantly increased both Bmp2 and Ilr2 expression. However, with BMSCs alone, the Il1r2 expression increased substantially, whereas Bmp2 expression exhibited a decrease rather than increase. These data suggested that nHAp may exert osteoinductive effects on BMSCs via the secretion of IL-1α in an autocrine/paracrine fashion, and IL-1α activity could be regulated through the synthesis of IL1R2 by BMSCs upon interaction with nHAp. These results complemented our understanding of the underlying mechanisms of biomaterial osteoinductivity.

Imaging assessment of profound sensorineural deafness with inner ear anatomical abnormalities.

OBJECTIVE: To explore the value of a combined computed tomography (CT) and magnetic resonance imaging (MRI) in evaluating profound sensorineural deafness patients before cochlear implant (CI) surgery. METHODS: A retrospective analysis of 1012 cases of profound sensorineural deafness that received CI was performed. RESULTS: A total of 96 cases were diagnosed with inner ear abnormalities including large vestibular aqueduct syndrome (LVAS, n = 61), Michel deformity (n = 3), cochlear incomplete partition I (n = 2), cochlear incomplete partition II (n = 6), cochlear hypoplasia with vestibular malformation (n = 3), cochlear ossification (n = 3), bilateral internal auditory canal obstruction (n = 5) and internal auditory canal stenosis (n = 2). CONCLUSION: High resolution CT (HRCT) can display bony structures while MRI can image the membranous labyrinth in preoperative evaluation for cochlear implantation. The combination of these two modalities provides reliable anatomical information regarding the bony and membranous labyrinths, as well as the auditory nerve.

Bumetanide hyperpolarizes madin-darby canine kidney cells and enhances cellular gentamicin uptake by elevating cytosolic Ca(2+) thus facilitating intermediate conductance Ca(2+)--activated potassium channels.

Loop diuretics such as bumetanide and furosemide enhance aminoglycoside ototoxicity when co-administered to patients and animal models. The underlying mechanism(s) is poorly understood. We investigated the effect of these diuretics on cellular uptake of aminoglycosides, using Texas Red-tagged gentamicin (GTTR), and intracellular/whole-cell recordings of Madin-Darby canine kidney (MDCK) cells. We found that bumetanide and furosemide dose-dependently enhanced cytoplasmic GTTR fluorescence by ~60 %. This enhancement was suppressed by La(3+), a non-selective cation channel (NSCC) blocker, and by K(+) channel blockers Ba(2+) and clotrimazole, but not by tetraethylammonium (TEA), 4-aminopyridine (4-AP) or glipizide, nor by Cl(-) channel blockers diphenylamine-2-carboxylic acid (DPC), niflumic acid (NFA), and CFTRinh-172. Bumetanide and furosemide hyperpolarized MDCK cells by ~14 mV, increased whole-cell I/V slope conductance; the bumetanide-induced net current I/V showed a reversal potential (V r) ~-80 mV. Bumetanide-induced hyperpolarization and I/V change was suppressed by Ba(2+) or clotrimazole, and absent in elevated [Ca(2+)]i, but was not affected by apamin, 4-AP, TEA, glipizide, DPC, NFA, or CFTRinh-172. Bumetanide and furosemide stimulated a surge of Fluo-4-indicated cytosolic Ca(2+). Ba(2+) and clotrimazole alone depolarized cells by ~18 mV and reduced I/V slope with a net current V r near -85 mV, and reduced GTTR uptake by ~20 %. La(3+) alone hyperpolarized the cells by ~-14 mV, reduced the I/V slope with a net current V r near -10 mV, and inhibited GTTR uptake by ~50 %. In the presence of La(3+), bumetanide-caused negligible change in potential or I/V. We conclude that NSCCs constitute a major cell entry pathway for cationic aminoglycosides; bumetanide enhances aminoglycoside uptake by hyperpolarizing cells that increases the cation influx driving force; and bumetanide-induced hyperpolarization is caused by elevating intracellular Ca(2+) and thus facilitating activation of the intermediate conductance Ca(2+)-activated K(+) channels.

Effect of mitomycin on normal dermal fibroblast and HaCat cell: an in vitro study.

OBJECTIVE: To evaluate the effects of mitomycin on the growth of human dermal fibroblast and immortalized human keratinocyte line (HaCat cell), particularly the effect of mitomycin on intracellular messenger RNA (mRNA) synthesis of collagen and growth factors of fibroblast. METHODS: The normal dermal fibroblast and HaCat cell were cultured in vitro. Cell cultures were exposed to 0.4 and 0.04 mg/ml of mitomycin solution, and serum-free culture medium was used as control. The cellular morphology change, growth characteristics, cell proliferation, and apoptosis were observed at different intervals. For the fibroblasts, the mRNA expression changes of transforming growth factor (TGF)-ß1, basic fibroblast growth factor (bFGF), procollagen I, and III were detected by reverse transcription polymerase chain reaction (RT-PCR). RESULTS: The cultured normal human skin fibroblast and HaCat cell grew exponentially. A 5-min exposure to mitomycin at either 0.4 or 0.04 mg/ml caused marked dose-dependent cell proliferation inhibition on both fibroblasts and HaCat cells. Cell morphology changed, cell density decreased, and the growth curves were without an exponential phase. The fibroblast proliferated on the 5th day after the 5-min exposure of mitomycin at 0.04 mg/ml. Meanwhile, 5-min application of mitomycin at either 0.04 or 0.4 mg/ml induced fibroblast apoptosis but not necrosis. The apoptosis rate of the fibroblast increased with a higher concentration of mytomycin (p<0.05). A 5-min exposure to mitomycin at 0.4 mg/ml resulted in a marked decrease in the mRNA production of TGF-ß1, procollagen I and III, and a marked increase in the mRNA production of bFGF. CONCLUSIONS: Mitomycin can inhibit fibroblast proliferation, induce fibroblast apoptosis, and regulate intracellular protein expression on mRNA levels. In addition, mitomycin can inhibit HaCat cell proliferation, so epithelial cell needs more protecting to avoid mitomycin's side effect when it is applied clinically.

Evaluation of intratympanic dexamethasone for treatment of refractory sudden sensorineural hearing loss.

OBJECTIVE: To observe and compare the efficacy of intratympanic application of dexamethasone (DXM) for the treatment of refractory sudden sensorineural hearing loss (SSNHL), the DXM was given in three different ways: by tympanic membrane injection, by drip through a ventilation tube, and by perfusion through a round window catheter. METHODS: We conducted a nonrandomized retrospective clinical trial involving 55 patients with refractory SSNHL. For 21 patients (the perfusion group), DXM (2.5 mg/0.5 ml) was perfused transtympanically through a round window catheter using an infusion pump for 1 h twice a day for 7 d giving a total amount of 35.0 mg. For 23 patients (the injection group), DXM (2.5 mg/time) was injected by tympanic membrane puncture at intervals of 2 d on a total of four occasions giving a total amount of 10.0 mg. For 11 patients (the drip group), DXM (2.5 mg/0.5 ml) was dripped via a ventilation tube placed by myringotomy, once on the first day and twice a day for the remaining 6 d giving a total amount of 32.5 mg. Thirty-two patients with refractory SSNHL who refused to undertake further treatments were defined as the control group. Hearing recovery and complications were compared among the groups. Hearing results were evaluated based on a four-frequency (0.5, 1.0, 2.0, 4.0 kHz) pure tone average (PTA). RESULTS: Post-treatment audiograms were obtained one month after treatments were completed. The improvements in average PTA for the perfusion, injection, and drip groups were 9.0, 8.6, and 1.7 dB, respectively. Hearing improvement was significantly greater in the perfusion and injection groups than in the control group (1.4 dB) (P<0.05). In the perfusion group, 8 out of 21 patients (38.1%) had a PTA improvement of 15‒56 dB (mean 29.8 dB); in the injection group, 8 out of 23 patients (34.8%) had a PTA improvement of 16‒54 dB (mean 24.9 dB); in the drip group, 1 of 11 patients (9.1%) had a PTA improvement of 26.0 dB; in the control group, 3 out of 32 patients (9.4%) had a PTA improvement of 15‒36 dB (mean 14.9 dB). CONCLUSIONS: Topical intratympanic application of DXM is a safe and effective method for the treatment of SSNHL cases that are refractory to conventional therapies.

[Aberrant expression pattern of a novel mutation in connexin 26 gene resulting in autosomal recessive deafness].

OBJECTIVE: To report a novel deafness-causing mutation c.465T>A, p.Y155X in connexin 26 (CX26) (also called gap junction protein beta-2, GJB2), and perform functional analysis of the mutated protein p.Y155X in Hela cells to explore the underlying mechanism on deafness. METHODS: Mutations in CX26 gene of the proband in an autosomal recessive inherited deafness family were tested by direct DNA sequencing method. Mutant p.Y155X, which was found in the deafness family, and wild type CX26 (wtCX26), were directionally subcloned into the pEGFP-N1 plasmid to construct the recombinant fusion protein expression vector of CX26 p.Y155X-EGFP and wtCX26-EGFP, followed by transfecting into HeLa cells. The expression of the mutated and wild type proteins was analyzed using Western blot analysis. The intracellular localization of proteins and the formation of gap junction-like plaques at plasma membrane were observed under confocal microscope. Gap junction coupling was tested by calcein-AM dye transfer experiment. RESULTS: A novel nonsense mutation c.465T>A, p.Y155X in the CX26 gene was found in the autosomal recessive deafness family. The molecular weight of protein p.Y155X was smaller than that of wtCX26 in transiently expressed HeLa cells. The mutated protein failed to reach the cell surface to form gap junction plaques, and displayed cytoplasmic accumulation. Also, no calcein-AM dye was transferred from the donor cells to the recipient cells when both were transfected with CX26 p.Y155X. The wtCX26 protein localized at the cell membrane to form gap junction plaques with permeability to fluorescent dye calcein AM. CONCLUSION: CX26 p.Y155X could not be targeted to the plasma membrane and there was no formation of gap junction channels between the adjacent cells. The mutation c.465T>A, p.Y155X in CX26 gene was responsible for the autosomal recessive hearing impairment in this family.

[Effect of brain-derived neurotrophic factor gene transfected bone-marrow mesenchymal stem cells on damaged cochlear spiral ganglion cells of guinea pigs].

OBJECTIVE: To investigate the protective role of brain-derived neurotrophic factor (BDNF) gene transfected bone-marrow mesenchymal stem cells (BMSC) on cochlear spiral ganglion cells (SGC) impaired by aminoglycoside antibiotics (AmAn). METHODS: The differentiation of BMSC transfected by BDNF gene (BDNF-BMSC) were detected with immunohistochemical examination of Nestin, neuron-specific enolase (NSE), and glial fibrillary acid protein (GFAP) antibody in vitro. BDNF gene transfected BMSC were transplanted into the cochleae of guinea pigs deafened by amikacin, while the control groups were designed in which artificial perilymphatic fluid (APF), BMSC or BDNF gene was injected into cochleae alone. The cochleae were obtained on the week 1, 2 and 4 after injection, respectively, paraffin-embedded, and cut in a paramodiolar plane subsequently. The histopathological changes of cochleae were observed, the density of SGC was calculated by staining with HE, and the corresponding optical density (COD) was calculated with immunohistochemical staining using NSE antibody. And the protective role of various groups on the cochlear SGC were compared. RESULTS: The positive staining rate of BDNF gene transfected BMSC with Nestin, NSE and GFAP antibody were all higher than that of BMSC in vitro (P < 0.01). After transplantation into cochleae, the differences of SGC density and COD among various groups were all significant on the same time points (P < 0.05). The SGC density and COD of the BDNF gene transfected BMSC group were the highest. The SGC density and COD of various groups on week 4 were all obviously decreased than those on week 1 and 2 (P < 0.05). CONCLUSION: AmAn-induced SGC damage could be depressed by BMSC, BDNF gene or BDNF gene transfected BMSC transplantation into cochleae, while BDNF gene transfected BMSC showed the best protective role.

[Expression of brain-derived neurotrophic factor modified bone marrow mesenchymal stem cells in the cochlea of drug deafened guinea pigs and its protection role].

OBJECTIVE: To study the expression of brain-derived neurotrophic factor (BDNF) gene modified bone marrow mesenchymal stem cells (MSC) in the cochlea of drug-deafened guinea pigs and its protection to spiral ganglion cells (SGC). METHODS: Guinea pigs deafened by subcutaneous injection of amikacin were randomly divided into two groups, BDNF gene modified bone marrow MSC were injected into the cochlea through fenestration of scala tympani in the experimental group, while artificial perilymphatic fluid were injected in the control group. Experimental animals were executed at 7 and 28 days post-operation. Expression of BDNF mRNA was examined by quantitate real time RT-PCR, histological images of cochlear sections were analyzed to calculate the cellular density of the SGC, and terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) was used to identify the apoptotic neurons. RESULTS: The BDNF expressive level in experimental group was higher than in the control group at 7 d and 28 d post-operation, whose differences were both statistically significant (P < 0.01). And, It showed a higher abundance of ganglion cell numbers, as well as a decreased apoptotic index in experimental group compared with the control group at 7 d and 28 d post-operation, whose differences were all statistically significant (P < 0.01). CONCLUSION: BDNF gene modified MSC could maintain expression for at least 28 days after transplantation into cochlea of drug deafened guinea pigs, and protect SGC.

[Expression of stathmin mRNA and protein in laryngeal squamous cell carcinoma and its clinical implication].

OBJECTIVE: To investigate the expressions of stathmin gene and its coding protein in laryngeal squamous cell carcinoma, and to explore the relationship between stathmin gene and the biological behaviors of laryngeal squamous cell carcinoma for understanding the tumorigenicity and development of laryngeal squamous cell carcinoma. METHODS: Laryngeal carcinoma tissues (studying group) in the tumors center and laryngeal normal tissues (control group) parted from 1.0 cm of the safe borderline of the tumors were took from 38 patients with laryngeal squamous cell carcinoma while they were in operation. Semiquantitative method of reverse transcriptase polymerase chain reaction (RT-PCR) was used to analyze the expression level of stathmin mRNA, and immunohistochemical staining (frozen section) was used to detect the expressions of stathmin protein, in laryngeal carcinoma tissues and laryngeal normal tissues of 38 cases, respectively. RESULTS: mRNA of stathmin gene was all positively expressed in laryngeal carcinoma tissues and in laryngeal normal tissues of 38 cases by RT-PCR. However, stathmin mRNA was obviously overexpressed in laryngeal carcinoma tissues than that in laryngeal normal tissues (t = 9.655, P < 0.05). Immunohistochemical staining showed stathmin protein was positively expressed in laryngeal carcinoma tissues of 26 cases (26/38, 68.4%), and mild-positively expressed in laryngeal normal tissues in 13 cases (13/38, 34.2%). There was significant difference between the expression rate of stathmin protein in laryngeal carcinoma tissues and in laryngeal normal tissues (chi2 = 8.901, P < 0.05). Meanwhile, the expression level of stathmin mRNA and the positive-expressed rate of stathmin protein in laryngeal carcinoma tissues of the advanced stage patients group (III stage and IV stage) were significantly higher than these in laryngeal carcinoma tissues of I and II stage patients group (t = 6.284, chi2 = 5.810, P < 0.05), and they were also significantly higher in laryngeal carcinoma tissues of the patients group with cervical lymph node metastasis than in laryngeal carcinoma tissues of the patients group without cervical lymph node metastasis (t = 9.350, chi2 = 6.923, P < 0.05). CONCLUSIONS: The expression levels of stathmin gene and protein were significantly higher in laryngeal squamous cell carcinoma than these in laryngeal normal tissues, the levels are also significantly higher in advanced stage patients group (III stage and IV stage) than in the early stage patients group (I and II), and they are also related to the cervical lymph node metastasis of carcinoma. Stathmin gene may play an important role in the pathogenesis and development of laryngeal carcinoma and may be related to its prognosis.

[Reconstructive methods for hypopharynx and cervical esophagus].

OBJECTIVE: To review the surgical treatment for reconstructing hypopharynx and cervical esophagus after hypopharyngo-oesophagectomy, and to evalue its efficacy. METHODS: Different methods were adopted to reconstruct the hypopharynx and cervical esophagus among 25 cases, including 14 cases of carcinoma of the hypopharynx and 11 of carcinoma of hypopharynx and cervical esophagus. In accordance with the standard of the International Union Against Cancer in 1997, the 25 cases were divided into different clinic stages, among which 5 were in T(2)N(0), 2 in T(2)N(1), 4 in T(3)N(0), 3 in T(3)N(1), 7 in T(4)N(1) and 3 in T(4)N(2). Treatment protocol was as follow: Pure operation for 5 cases, re-operation after radiotherapy for 2 cases, operation plus radiotherapy for 18 cases, laryngeal conservation operation for 8, and neck dissection for 21 cases. Reconstruction was done by using free jejunal transplantation, gastric pull-up, the laryngotracheal flap, and myocutaneous flap. RESULTS: After the reconstruction, 3 cases of free jejunal graft and gastric pull-up, 4 of laryngotracheal flap recovered oral fleeding within 2 weeks. No serious complications occurred. After 18 cases underwent the myocutaneous flap reconstruction, no complications occurred in 10 patients, but there were different complications in 8 cases, including pharyngocutaneous fistula (6 cases), haryngoesphageal stenosis (7 cases), and pectoralis major myocutaneous flap necrotic (1 case). The 3-year survival rate was 38.9% (7/18). CONCLUSION: Reconstruction with free jejunal graft, gastric pull-up, and laryngotracheal flap constitutes is a safe and reliable method to restore the continuity of the upper digestive tract after pharyngo-laryngo-oesophagectomy. After the reconstruction with myocutaneous flap, there is high incidence of pharyngocutaneous fistula and haryngoesophageal stenosis.

[Functional interaction of the C-terminal of Nogo protein with connexin 26 and the expression of Nogo's mRNA in the murine inner ear].

OBJECTIVE: To screen and identify the proteins that interact with connexin 26 (CX26) and to analyze the expressions of these proteins in cochlea so as to explore the proteins that relate to the trafficking, assembly, localizing and gap junction functions of CX26. METHODS: The whole coding region of GJB2 (CX26) gene was amplified from normal human genomic DNA by polymerase chain reaction (PCR) and then directionally subcloned into the vector pGBKT7 plasmid of the Match Maker Ga14 Two-Hybrid System 3 as a target to screen the interactive proteins of CX26 from the human fetal brain cDNA library by the yeast two hybrid technique. The false positive clones were discarded from the preys by repeated yeast two hybrid method between CX26 and everyone of the preys respectively. The DNAs of the insert of the identified positive clone were sequenced and BLAST analyzed against the GenBank. Lastly, the mRNA of the gene encoding the identified protein was analyzed in the murine inner ear by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The insert of one positive clone contained 867 bp with the former 525 bp being coding region. The DNA sequence and the open reading frame of the insert were identical to the 525 bp before the stop codes (including the stop codes) and the 238 bp after the stop codes of RTN4 gene which encoded Nogo protein. And the 174 amino acid residues encoded by the insert were those of the C-terminal of Nogo protein: Nogo-A, Nogo-B and Nogo-C. RTN4 mRNA expressed in the murine inner ear was confirmed by RT-PCR method. CONCLUSION: The C-terminal of Nogo protein interacts with CX26. Nogo protein expresses in the inner ear and may take part in the trafficking of CX26 or CX26 gap junction function.

[Orientation of three lysosomal enzymes in the mouse inner ear and hearing loss in enzyme gene deficiency].

OBJECTIVE: To determine the distribution and influence of lysosomal neuraminidase (Neul), protective protein/cathepsin A (PPCA) and beta-galactosidase (beta-gal) in the inner ear of the mouse, and to observe their auditory alterations in enzyme deficiency. METHODS: Six wild type (2 months postnatal) (Neu1+/+, PPCA+/+ and beta-gal+/+) mice were used, and Neu1, PPCA and beta-gal homozygous (Neu1-/-, PPCA-/- and beta-gal-/-) mice at the same age used as control in this experiment. The auditory thresholds were examined through the auditory brainstem responses (ABR) to click, which tone pips were 8, 16, and 32 kHz. The mice were intracardically perfused with 4% paraformaldehyde. The bulla were further fixed in 4% paraformaldehyde, processed and sectioned with paraffin embedded method. Immunohistochemistry was used to determine the cellular localizations of Neu1, PP-CA, and beta gal in the inner ear. RESULTS: There was a similar distributive pattern of Neu1, PPCA and betagal in the inner ear. Neu1 intense staining was observed in the cochlear spiral ganglion cells, spiral limbus, spiral ligament, vestibular ganglion cells, cristae, maculae hair cells, and weak staining in inner hair cells, outer hair cells, supplying cells of the organ of Corti and stria vascularis. The intense staining of PPCA and beta-gal were observed in the spiral ganglion and vestibular ganglion cells, and weak staining in the spiral limbus, spiral ligament, stria vascularis and organ of Corti. The inner ear exhibited no staining when Neul, PPCA and beta-gal were deficient, respectively. A positive staining of PPCA and beta-gal was presented in Neu1-/- mice, and as well as Neu1 and PPCA in beta-gal-/- mice. However, the staining of Neu1 was not presented, and only very weak staining of beta-gal in PPCA-/- mice. The auditory thresholds of Neul, PPCA, and beta-gal mice were elevated for 60-69 dB, 40-48 dB, and 7-10 dB above those of wildtype littermates, respectively. CONCLUSION: Neu1 PPCA and beta-gal are distributed in the inner ear of mouse, and the three enzymes also form a lysosomal multi-enzyme complex in the inner ear. The respective enzyme deficiencies can induce the hearing the loss of different levels.

[Clinical observation of sensorineural hearing loss in patients suffering from nasopharyngeal carcinoma after radiotherapy].

OBJECTIVE: To study the extent and incidence of sensorineural hearing loss (SNHL) after radiotherapy (RT). METHODS: Twenty-eight patients with diagnosed nasopharyngeal carcinoma (NPC) were selected. The pure-tone audiography, auditory brain stem evoked response (ABR), impedance audiometry and evoked otoacoustic emissions (EOAE) recordings were performed before RT, 1 month, 1, 2 and 5 years after RT. RESULTS: At 1 month after RT, there were 7.1 and 25.7 dB increased mean bone conduction (BC) thresholds at speech (0.5 - 4.0 kHz) and at high frequency (8.0 kHz), and their BC thresholds were statistically significant increase than those before RT, respectively (P < 0.001). At 1 year after RT, there were 17.6 and 28.1 dB increased respectively, and their thresholds were statistically significant increase than those at pre-irradiation (P < 0.001). There were also significant increases in thresholds than those at 1 month of post-irradiation (P <0.001 or P < 0.05). At 2 years after RT, 21 and 27.4 dB were increased at respective those two frequencies, and there was a statistically difference only at speech frequencies when compared with those at 1 year after RT (P < 0.05). At 5 years after RT, 26.7 and 35.8 dB were increased at these two frequencies, and there were significant increases in threshold than those before, 1 month, 1 and 2 years after RT, respectively (P < 0.001). From 1 month to 5 years after RT, 37. 5% to 94. 7% of ears had a BC hearing threshold of at least 15 dB losses at speech frequency, whereas the percentage at high frequency was 85.4 to 97.4%. Up to 63.2% and 73.7% of ears had 30 dB SNHL at least at speech and high frequency, respectively. Furthermore, the degree of mean threshold loss was greater at high frequency than at speech frequency. The mean value of wave I, III and V latency, and I -V interpeak latency intervals of ABR had no significant difference between at 1 month after RT and before RT (P > 0.05). The wave I , III and V latency, and I - V interpeak latency intervals at 1 year and 2 years were significantly prolonged when compared with those before and 1 month after RT (P < 0.05), but there were no significant difference between 1 year and 2 years after RT (P > 0.05). The wave I, III and V latency, and I -V interpeak latency intervals at 5 years after RT were also significantly longer than those before RT (P < 0.001). There were significant difference in wave I , III and V latency (P < 0.05), and no significant difference in wave I - V interpeak latency intervals (P > 0. 05) between 5 years after RT and 1 year or 2 years after RT. Seven of 10 ears at 1 year after RT and 4 of 7 ears at 5 years after RT had normal EOAE, but they all had abnormal ABR response. CONCLUSIONS: SNHL in NPC patients start soon after completion of RT, especially more commonly in high frequency. The incidence and the extent of hearing loss are increased with time of follow-up. The hearing impairment could occur in the cochlea and/or the retrocochlear auditory pathway, which show that the sensitivity of radiation damage may be different in different patient and anatomic site of auditory system.

[Morphological and functional alterations of ear in lysosomal neuraminidase gene deficient mouse].

OBJECTIVE: To observe the alterations of the auditory function and morphology of the ear in the mouse sialidosis models which has been generated by targeted deletion of lysosomal neuraminidase gene (Neul) and closely resembled the phenotypes in corresponding human conditions, and to explore pathophysiological mechanisms of hearing impairment. METHODS: Neul homozygous (Neul -/-) mice at 3 weeks, 2 and 4 months of age, and their wildtype littermates (Neu1 +/+) were examined for auditory thresholds through auditory brainstem responses (ABR) to click, 8, 16, and 32 kHz stimuli. Morphological analyses in ears were performed by series temporal bone section and light microscopy. RESULTS: Neul -/- mice at 3 weeks of age showed an elevated ABR threshold, 50-55 dB above those of Neul +/+ mice. Up to 2 and 4 months of age, their thresholds were further elevated for 60-68 dB. There were distinct pathological changes of middle and inner ear of 3 weeks of age in Neul -/- mice, especially at 2-4 months of age there were significant cerumen occlusion in the external auditory canal and severe otitis media. Vacuolation associated with lysosomal storage was observed within ossicles and cochlear bone cells, stria vascularis cells, spiral ganglion neurons and macrophages, spiral limbus, spiral prominence, Reissner's membrane cells, and the mesothelial cells of the perilymphatic scala and basilar membrane, but not within the organ of Corti. Vestibular ganglion neurons, hair cells and supporting cells in cristae and maculae also showed vacuolation. CONCLUSIONS: The deficiency of lysosomal neuraminidase may result in a serious hearing loss and morphological alterations of ear. The external auditory canal obstruction, otitis media and ossicle changes may cause conductive hearing loss, and the defects in lysosomal storage of neurons, stria vascularis, spiral limbus, Reissner's membrane and basilar membrane cells may contribute to sensorineural deafness.

[Mutation screening in selected exons of myosin 7a gene in prelingual non-syndromic hearing impairment patients].

OBJECTIVE: To ascertain the frequency and characteristics of myosin 7a gene mutations in Chinese with prelingual nonsyndromic hearing impairment. METHODS: Most of cases were collected within Hunan province, including 31 sporadic congenital deaf patients and 65 patients from 34 hereditary prelingual deafness families, and 100 health individuals were used as control. Genomic DNA was extracted from the patients and subjected to the PCR to amplify selected exons of myosin 7a gene, and then the amplified products were screened for base variations by single strand conformational polymorphismanalysis (SSCP). The bands with abnormal conformation were sequenced to confirm the mutation. RESULTS: G to A substitution was detected at nucleotide 617 in exon 7 as hetrozygous state in two patients and was not found in unaffected members in their family. This mutation caused Arg206Gln within a highly conserved heptapeptide sequence of myosin 7a protein, and was close relevant to the prelingual nonsyndromic deafness. CONCLUSIONS: The Arg206Gln mutation in exon 7 of myosin 7a is possibly a novel mutation to cause prelingual nonsyndromic hearing impairment. Our results provide the evidence that exon 7 of Myosin 7a is a mutational hotspot region in genetic deafness.

GJB2 (Cx26) gene mutations in Chinese patients with congenital sensorineural deafness and a report of one novel mutation.

BACKGROUND: Mutations in GJB2 gene are a major cause of autosomal recessive congenital hearing loss and the cause in some rare cases of the autosomal dominant form. The purpose of this study was to investigate the frequency and the features of GJB2 mutations in the Chinese patients with congenital sensorineural deafness. METHODS: Using PCR amplifying the entire coding region of GJB2 gene and direct DNA sequencing to analyze mutations in this gene among unrelated 69 cases with autosomal recessive congenital nonsyndromic deafness and 27 cases of dominant congenital deafness and 35 sporadic cases. We also detected mutations in GJB2 in 100 control subjects with normal hearing. RESULTS: 17.4% (12/69) of the probands in the autosomal recessive, 7.4% (2/27) of dominant families and 5.7% (2/35) of the sporadic congenital deafness patients had deafness-causing mutations in GJB2, respectively. Nine types of the mutations in GJB2 were detected in the recessive and sporadic group. They consisted of five types of polymorphism, and four types of deafness-causing mutation with homozygous 35delG in 1 sporadic (1/35), and 235delC frameshift mutation in 1 sporadic (homozygotes) and 10 recessive patients (2 heterozygotes and 8 homozygotes), and homozygous 442G-->A missense mutation and homozygous 465T-->A nonsense mutation in 1 different recessive proband, respectively. The 465T-->A that related to recessive deafness was a novel mutation found by this study. The homozygous (10/69, 14.5%) and the heterozygous (2/69, 2.9%) GJB2 mutation in the recessive patients (12/69, 17.4%) and the homozygotes in the sporadic patient (2/35, 5.7%) all had congenital severe to profound sensorineural hearing loss. 511G-->A missense mutation and 299-300delAT frameshift mutation were found in two autosomal dominant congenital deafness families (2/27, 7.4%). The total mutation frequency of GJB2 was 12.2% (16/131) in the Chinese patients with congenital sensorineural deafness and 235delC was the most common deafness-causing mutation. Six types of mutation-5 types of polymorphism and 1 type of heterozygous deletion (235delC) mutation were found in the 100 control subjects. The carry rate of the most frequent type of mutation 235delC was 0.5% in the controls (1/200 alleles). 109G-->A was the most frequent (15/100, 15%) and 79G-->A was the second common (8/100, 8%) polymorphism in this population. CONCLUSIONS: The general mutation rate of GJB2 is 12.2% (16/131) and the 235delC is the most common type of deafness-causing mutation in Chinese patients with congenital hearing loss. 465T-->A nonsense mutation that is associated to autosomal recessive deafness is a novel mutation found by this screening. 511G-->A and 299-300delAT mutations contribute to autosomal dominant hearing loss. The study further supports the view that the common types of mutation in GJB2 according to different ethnic background and that the mutation prevalence in the East Asian deafness population is lower than that in the white population.

[Subunit of voltage-dependent calcium channels in the murine spiral ganglion cells].

OBJECTIVE: To investigate subunit type of voltage-dependent calcium channels in the spiral ganglion cells of the mouse. METHODS: The spiral ganglion cells were dissected from cochleae of neonatal mice and cultured for 24 h. Total RNA was extracted from cultured spiral ganglion cells. After reverse transcription, resulting cDNA was amplified by polymerase chain reaction (PCR) with primers targeted to nucleotide sequences corresponding to 7 different calcium channel subunits. The types of calcium channel subunits were identified by PCR analysis and nucleotide sequencing. RESULTS: Reverse transcription (RT)-PCR products representing subunit gene expression were strongly and consistently amplified for alpha1 D, alpha1 E, alpha2/delta, beta1 and beta3. Nucleotide sequencing confirmed the identity of mouse cochlear subunit cDNAs. CONCLUSIONS: alpha1D, alpha1E, alpha2/delta, beta1 and beta3 subunits are expressed in spiral ganglion cells. And the coexpression of alpha1D and alpha1 E demonstrate the presence of L-type and R-type calcium channels in mammalian spiral ganglion cells.