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Objective: To investigate the regulatory effect of miR-181a with abnormal expression on the expression of c-fos in cochlear hair cells undergoing oxidative damage., Methods: House Ear Institute-Organ of Corti1 (HEI-CO1) cells were assigned to 50, 100, and 200 µmol/L tert-butyl hydroperoxide (t-BHP) exposure groups and control group. The HEI-CO1 cells in the exposure groups were exposed to 50, 100, or 200 µmol/L t-BHP for 12 h. Then, total RNA and total protein were extracted from the HEI-CO1 cells, and the expression of miR-181a/-181d was measured by qPCR. The miR-181a with abnormal expression was selected as the subject of study. The putative miR-181a target sequence in the 3' untranslated region (3'-UTR) of c-fos was predicted by searching on a bioinformatics website. The HEI-CO1 cells were transfected with miR-181a mimics by lipofection, and the transfection efficiency was measured by qPCR. The mRNA and protein expression of c-fos was measured by qPCR and Western blot. The pGL3-c-fos-3'UTR-WT plasmid was constructed, and the luciferase activity of the plasmid in the case of high miR-181a expression was measured using the Dual-Luciferase Reporter Assay System., Results: Compared with those in the control group, the expression of miR-181a in 100 and 200 µmol/L t-BHP exposure groups was significantly decreased, with expression ratios of 0.744 and 0.766 (P < 0.01), while the expression of miR-181d in 50 µmol/L t-BHP exposure group was significantly increased, with an expression ratio of 1.29 (P < 0.01). There was no significant difference in miR-181a expression between the 100 and 200 µmol/L t-BHP exposure groups (P > 0.05). The predication results revealed that c-fos was regulated by miR-181a in humans and mice, with complete complementarity to the seed region of miR-181a, and there was high degree of target sequence conservation across species. The expression of miR-181a in the HEI-OC1 cells transfected with miR-181a mimics was elevated 892.979 times at 24 hours after transfection. As compared with those of controls, the mRNA and protein expression levels of c-fos in the transfected HEI-OC1 cells were significantly increased (P < 0.05 and P < 0.01). The luciferase activity of pGL3-c-fos-3'UTR-WT plasmid was not suppressed but increased in the case of high miR-181a expression., Conclusion: miR-181a has no direct inhibitory effect on the mRNA and protein expression of c-fos, which may not be the target gene of miR-181a. Bioinformatic prediction might produce false-positive results.

Objective: To explore whether the Ad5-atoh1/EGFP could transdifferent the supporting cells into the new hair cells in young adult guinea pigs cochlea in vivo., Method: Twelve healthy pigmented guinea pigs weighted 200-250 g were included in this experiment. 5 ul of Ad5-E1/E3 defected-atoh1/EGFP were infused into the scala media through a hole made on the lateral wall of the cochlea. Six of the 12 animal were killed 2 weeks after the infusion operation. The others were killed 4 weeks after the operation. The whole mount of the basal membranes were directly observed under the fluorescence microscope for the expression of the EGFP (enhance green fluorescent protein) or for the expression of the hair cellspecific marker and nuclear after staining with myosin VIIa rabbit polyclonal antibody and Dapi dye., Result: New cells with big nuclear, ellipse body and expressed with EGFP were found in the region near to the outmost row of the outer hair cells in 2 animal 2 weeks after the infusion. Moreover there were 3 animals with specific morphologic new cells in the location where ever been located by the outer hair cells and the region as 2 weeks animals 4 weeks after the infusion. Those cells were stained by myosin VIIa antibody., Conclusion: Atoh1 gene could transdifferent some supporting cells in the basal membrane into hair cell like cells in young adult guinea pigs in vivo. These supporting cells locate in the region of outer hair cells and the basal membrane which do not belong to the region of outer hair cells.

Objective: To explore the protective effects of N-acetylcysteine (NAC) on cochlear damage occurring in irradiated guinea pigs., Methods: Seventy-two guinea pigs were randomly divided into 4 groups (n = 18 each). Control group received neither NAC nor irradiation, irradiation group received total cranium irradiation of 70 Gy, irradiation & saline group cranium irradiation of 70 Gy and saline solution through a round window and NAC group cranium irradiation of 70 Gy and NAC through a round window. The right ear received radiation. The animals were sacrificed at Day 14 post-irradiation. The specimens were dehydrated, embeded in paraffin and serially cut into 5-µm slices. Sections were stained with immunohistochemistry and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL). The cochlear basal membranes were observed for malondialdehyde (MDA) and superoxide dismutase (SOD) with scanning electron microscope., Results: The cilium of hair cells had no clear loss and apoptotic number of spiral ganglion cells decreased in NAC group. The average optical density value of Caspase 3 in spiral ganglion in NAC group significantly decreased versus the irradiation group (0.08 ± 0.02 vs 0.10 ± 0.01, P < 0.01). The level of MDA of NAC group also decreased versus the irradiation group (0.33 ± 0.05 vs 0.84 ± 0.13, P < 0.05). The level of SOD in the NAC group increased versus the irradiation group (10.7 ± 3.0 vs 8.7 ± 1.3, P < 0.05). The ratio of apoptotic cell in SGC in the NAC group at Day 14 (7.8% ± 1.8%) decreased versus the irradiation group (32.0% ± 8.7%) at Day 14., Conclusion: MDA and SOD may be involved in the pathogenesis of cochlear cell damage. And NAC protects the irradiated cochlear cell.

Aminoglycoside antibiotics, due to their strong antibacterial effects and broad antimicrobial spectra, have been very commonly used in clinical practice in the past half century. However, aminoglycoside antibiotics manifest severe ototoxicity and nephrotoxicity, and are one of top factors in hearing loss. In this study, three members of the aminoglycoside antibiotics family, gentamycin, neomycin and streptomycin, were chosen as the representatives to be investigated for their toxicity to the embryonic development and the larva hair cells in zebrafish, and also to their target genes associated with hearing-related genes. The results showed that: (1) the lethal effect of all three drugs demonstrated a significant dependence on concentration, and the severity order of the lethal effect was streptomycin > neomycin > gentamycin; (2) all the three drugs caused the larva trunk bending in resting state at 5 dpf (day past fertilization), probably due to their ototoxicity in the physical imbalance and postural abnormalities; (3) impairment and reducing of the hair cells were observed in all three cases of drug treatment; (4) four genes, eya1, val, otx2 and dlx6a, which play an important role in the development of hearing organs, showed differential and significant decrease of gene expression in a drug concentration-dependent manner. This study for the first time reports the relevance between the expression of hearing genes and the three ototoxic antibiotics and also proved the feasibility of establishing a simple, accurate, intuitive and fast model with zebrafish for the detection of drug ototoxicity.

Objective: To investigate the effects of erlong zuoci (ELZC) pills and its disassembled prescriptions (Shudi-huang-Zexie group and Zexie group) on the enzymatic activity and protein expression changes of the key apoptosis molecules in the gentamycin injured hair cells., Method: The model of gentamycin induced ototoxicity in mice cochlear primary cultures was copied. Cochlear organotypic cultures of postnatal day 3-5 (P3-P5) mice were treated with gentamycin alone or in combination with ELZC pills, Shudihuang-Zexie group or Zexie group respectively. The enzymatic activity of Caspase-9 and Caspase-3 was determined by means of fluorescence staining in situ. The protein expression of Bcl-2 and Bax in the hair cell area was examined by immunofluorescence in normal and treated specimens., Result: Average optical density analysis indicated that, compared to the normal group, 0.03 mmol x L(-1) gentamycin could significantly activate Caspase-9 and Caspase-3, downregulate the ratio of Bcl-2 and Bax protein expression. Compared to the gentamycin model group, ELZC pills significantly inhibited the enzymatic activity of Caspase-9 and upregulated the ratio of Bcl-2 and Bax protein expression, showing inhibition trend toward the enzymatic activity of Caspase-3. Both Shudihuang-Zexie group and Zexie group could effectively inhibit the enzymatic activity of Caspase-9 and Caspase-3, upregulate the ratio of Bcl-2 and Bax protein expression., Conclusion: ELZC pills, Shudihuang-Zexie group and Zexie group can effectively protect hair cells from gentamycin by correcting the abnormal changes of the mitochondrion-dependent signal transduction pathway.

Objective: To investigate the ototoxicity of co-administration of kanamycin and furosemide in mouse and establish a reliable model to induce a sensorineural hearing loss., Methods: CBA/J mice strain was selected, with the age around 3-4 weeks old, to be received a single subcutaneous injection of kanamycin at dose of 1 g/kg and another single intraperitoneal injection of furosemide at dose of 0.4 g/kg 30 - 45 min afterward. The auditory brainstem response (ABR) threshold shift was tested. The series of experimental methods including propidium iodide, phalloidin staining, semithin section toluidine blue staining, TUNEL, scanning electron microscopy and transmission electron microscopy were applied to observe the characteristics of the lesion of cochlea and hair cells. The time course was set as following: before injection, 12, 24, 48 hours and 1, 2, 4, 12 weeks after injections, respectively., Results: The ABR threshold shift was firstly presented a significant increase at 12 h after injection at 2, 4, 8 kHz, then the ABR threshold kept going up during next 36 h until it was presented a stable level around 90 dB. Pathological examination showed an absence of outer hair cells at basal turn rapidly since 12 h after treatment, and then by 48 h the most commonly observed lesion, where all outer hair cells throughout the length of the cochlea were killed, in the contrast, however, the inner hair cells loss were delayed and mild. TUNEL-positive nuclei demonstrated that most hair cells died via an apoptotic pathway. In scanning electron microscopy abundance of necrotic outer hair cells were detected by 24 h after treatment, in which reticular lamina were collapsed. Then all outer hair cells were replaced by expansion of heads of supporting cells. At 48 h after treatment, marginal cells presented a swollen and some of them were observed to be fused. In addition, spherical cell extrusion appeared to leak out from some marginal cells. By 2 weeks, nearly all microvillus were lost and marginal cells presented a shape of stone-like change. A significant and progressive decrease in strial vascularis thickness was found, of which the reason probably related with a reduction in volume of marginal cells., Conclusion: This systemic protocol eliminates hair cells extensively in vivo, and it could be a reliable model to examine different aspects of cochlear pathology in transgenic or mutant mice strains.

Objective: To investigate the generative or regenerative hair cell and studies of molecular and genetic in the inner ear, the long-term culture systems of utricular sensory epithelial cell of the rats were established., Method: Utricular sensory epithelial of postnatal day 1 rats was isolated by mechanical dissociation. The explants were digested by thermolysin,then transferred to an aliquot containing trypsin and collagenase for incubation to harvest the pure utricular sensory epithelial cell. USEC wes cultured in Dulbecco modified Eagle medium (DMEM) and passaged. USEC of the 25 passages observed by inverted microscope and ultrastructural examination with transmission electron microscope. Immunocytochemical method with cytokeratin 18, vimentin, Brn3. a and Calretinin; reverse transcription PCR with mRNA of AchRa9 and Myosin VII a. The markers of hair cells were used identify the characterization of USEC of the 25 passages., Result: USEC have been cultured for more than 6 months and passaged 25 passages. USEC of the 25 passages showed a large,flat,polygonal epithelial morphotype with big,round nuclei. The cells grew into monolayer,"cobblestone-like" appearance and showed "Dome" formation. The cells expressed cytokeratin 18, not expressed vimentin, has rich microvilli and complex tight junction, which indicated the epithelial origination of USEC. Coexpressed of the hair cell characteristic markers Brn3. a, Calretinin and mRNA of AchRa9, Myosin VIIa was identified the culture cell that may represent rat progenitor hair cell., Conclusion: The long-term culture systems of utricular sensory epithelial cell of the rats were successfully established. The long-term culture USEC coexpressed the characteristic markers of the hair cell ,which identified the culture cell may represent progenitor cell of rats hair cell. It may provide valuable cell sources for in-depth investigation the mechanisms of hair cell generation or regeneration and studies of molecular and genetic in the inner ear.

Objective: To evaluate if caspase pathway was involved in streptomycin-induced cell apoptosis in cochlear hair cells., Methods: F344 rats at postnatal day 3 or 4 were used for the study in cochlear organotypic cultures. The cochlear basilar membrane was micro-dissected out and cultured overnight, and then treated with 1 mmol/L streptomycin for 24 hours. Before the termination, the activity of caspase-8, 9 or 6 were detected with FAM-peptide-FMK labeled caspase-8, 9 or 6, respectively. The stereocilia and cuticular plate of hair cells were stained with TRITC conjugated phalloidin, and the nuclei were stained with Topro-3 DNA probe. The specimens were observed and photographed under confocal fluorescent microscope., Results: Streptomycin with 1 mmol/L causes about 80% cochlear hair cells missing in the basal turn and 10% hair cell loss in the apex. After streptomycin treatment, the nuclear shrinkage and fragmentation were found in most cochlear hair cells, and the caspase-8, caspase-9 and caspase-6 were greatly activated., Conclusions: Apoptosis is involved in the cochlear hair cells death induced by Streptomycin in vitro. The caspase activities in upstream and downstream are maybe the major apoptotic pathway.

Objective: To construct an universal recombinate adeno-associated virus (AAV), rAAV-NT4-ADNF-9, and to detect the ability of transfection of the rAAV vector into cochlea in vitro., Method: pSSVHG-CMV-ADNF-9 plasmid was introduced into 293 cell by method of Ca3 (PO4)2 using three plasmids of pSSHG-CMV-NT4-ADNF-9, pFG140 and pAAV/Ad. The recombinate adeno-associated virus (rAAV) was harvested, and the titrations of the rAAV concentrated was detected by dot-blot test. Isolate and culture the cochlear hair cell of SD rats newly born in vitro. The rAAV-NT4-ADNF-9 was added to the medium while plating. Cochlear were collected 24 h after cultivation for RT-PCR to detect the transfection of rAAV-NT4-ADNF-9., Result: The titration of rAAV stock produced 2 x 10(16) total particles/L, which showed that rAAV-NT4-ADNF-9 was constructed successfully. The cochlear hair cell of SD rats newly born was isolated and cultured in vitro successfully. It certified that rAAV-NT4-ADNF-9 was able to transfect into cochlear and express secretory NT4-ADNF-9 peptide by RT-PCR., Conclusion: The rAAV vector constructed in this paper, rAAV-NT4-ADNF-9, can transfer into cochlear cultured in vitro, which layed a foundation of further research for gene therapy.

Objective: To investigate the degeneration mechanics of outer hair cells in guinea pig., Method: The mechanics of outer hair cells isolated by enzyme were observed under inverted microscope for 6-8 h continuously., Result: Over half of living outer hair cells could keep good conditions in 6 hours. During the degeneration there was always a longitudinal fold line from tip to base. Presence or absence of calcium, as well as lossing of stereociliary bundle, couldn't change the conditions of out hair cells., Conclusion: Neither calcium nor stereociliary bundle is the decisive cause in keeping outer hair cells alive, and its degeneration may be basically related with something surrounding the cell.

Objective: In order to meet the needs of electrophysiology outer hair cells, we investigate a method to separate outer hair cells simply and effectively., Method: The basal membrane was dissected combined with spiral ligament and axis of cochlea, then incubated with enzyme, and then mechanically triturated by micropipette., Result: A large number of living outer hair cells were obtained. Of 80% cells could keep good condition in 6 hours., Conclusion: If increasing the enzyme concentration slightly and the large part of cochlea without outer bone was enzymes, we can get living outer hair cells easily.

Objective: To construct an adenoviral vector that codes for both human NT3 and EGFP, to confirm the transduction efficiency in rat cochlear cultures and to assess the protection of NT3 on SGNs survival., Method: PAdeasy-1 and pAdTrack CMV were used to constructed Ad/NT3 adenovirus and then to transfer postnatal day 3 rat cochlear cultures. The transduction efficiency was determined by microscope observation. The amounts of SGNs were counted to evaluated protection of Ad/NT3 on SGNs survival., Result: EGFP positive cells were observed in all cochlear turns. There was approximately 49% in outer sulcus cells and 27% in the interdental cells; less than 2% of the hair cells and SGN. The amounts of SGN of treated Ad/NT3 adenovirus are more than cochlea SGN only Ad/EGFP adenovirus after cultured for 15 days., Conclusion: Ad/NT3 adenovirus could transduce EGFP and NT3 in large number of supporting cells, but few hair cells or SGNs. The putative release of NT3 from these supporting cells could enhance cell survival and promote neurite outgrowth from SGNs.

Objective: To investigate the effect of Hath1 (human atonal homolog 1) overexpression on greater epithelial ridge (GER) cells from postnatal rat cochlea in vitro., Methods: GER cells were isolated by using a combinatorial approach of enzymatic digestion and mechanical separation from P1 rat cochlear. The GER cell cultures were infected by adenovirus containing Hath1 and enhanced green fluorescent protein (ad-Hath1-EGFP), while transfecting EGFP(ad-EGFP) was as controls. Immunostaining were performed at different time points after adenovirus infection., Results: Some of the infected GER cells became myosin VIIa-positive following ad-Hath1-EGFP infection. The earliest time point to see induction of hair cell differentiation (hair cell marker expression) by ad-Hathl was 5 days post-infection. In contrast, infection of the GER sheet cultures with ad-EGFP control virus did not show any myosin VIIa-positive cells at 3-12 days post-infection in all cultures examined., Conclusions: GER cells may potentially serve as hair cell progenitors and they are capable of differentiating hair cell-like cells when forced to express Hath1.

Objective: To establish in vitro culture systems of greater epithelial ridge (GER) cells from rat cochlear and to investigate the characterization, growth pattern and ultrastructure of GER cells., Methods: Using a combinatorial approach of enzymatic digestion and mechanical separation to allow isolation and culture of GER cells from P1 rat cochleae. The dissociated GER cells were cultured in serum-free or 10% fetal bovine serum DMEM respectively. BrdU, phalloidin, ZO1, calretinin and myosin VIIa immunostaining and scanning electron microscope observation were performed in GER cell cultures., Results: The dissociated GER cell cultures showed positive to ZO1, phalloidin and BrdU staining, but negative to myosin VIIa and calretinin. They assumed a polygonal morphology which was similar to epithelial cells and grew in islands-like patches in medium containing 10% fetal bovine serum while forming spheres in serum-free medium. The GER cells presented significant ability to proliferate in both conditions. Scanning electron microscope showed that there was microvillus and centre bodies but not hair cell specific stereociliary bundles on the surface of GER cultures., Conclusions: The GER cell cultures showed significant ability to proliferate and grew in islands-like patches in medium containing 10% fetal bovine serum while forming spheres in serum-free medium. The dissociated GER cells expressed epithelial cell specific marker but not marker of hair cells.

Objective: To compare the influences of stapedectomy and small fenestra stapedotomy on the hearing of guinea pigs., Methods: Twenty-four (48 ears) guinea pigs were randomized equally into two groups, and the left ears were subjected to stapedectomy and total stapes replacement with a prosthesis, or sham operation (12 ears) to expose the footplate of the stapes and the round window. Each guinea pig was tested by ABR perioperatively. Four guinea pigs were chosen randomly from each group and decapitated for morphological examination by light microscopy and scanning electron microscopy after ABR test., Results: In the sham operation group, the post-operative latencies of each wave, the intervals and the hearing threshold exhibited no significant changes other than prolonged latency of wave I. In stapedectomy group, the hearing threshold increased to 23.75-/+3.77 dBSPL 1 h after operation with significantly prolonged post-operative latencies of all the waves and intervals but for III-IV interval, which was shortened. The latencies of each wave (especially waves I and III) in the stapedectomy group were increased by a greater magnitude than those in the sham operation group, but the intervals were comparable between the two groups. No significant difference was noted in the parameters of ABR either 1 h or 1 day after the operation between the two groups, in which the architecture of cochleas remained intact with similar number of spiral ganglion cells. The stereocilia of the outer hearing cells (OHC) were normal in the sham operation group while in stapedectomy group, slight stereocilia disorder occurred but became normal 1 day after operation. No obvious changes were found in the stereocilia of the inner hearing cell (IHC) in either groups., Conclusion: Stapedectomy can induce mild hearing loss without seriously damaging the function of the cochlea in guinea pigs.

Objective: To further investigate the mechanisms of hair cell generation or regeneration, the primary culture systems of cochlear sensory epithelial cell (CSEC) of rats were established., Method: Cochlear sensory epithelial (containing the organ of Corti) of postnatal day 1 (P1) rats was isolated by mechanical dissociation. The explants were grown on sterilized disposable plastic dishes,cultured in Dulbecco modified Eagle medium(DMEM), and observed daily by inverted microscopy. The culture medium was changed twice a week. The pure CSEC was harvested by the limiting dilution method. CSEC was identified by immunocytochemical method with cytokeratin 18, vimentin, Brn3. a, Calretinin, BrdU and ultrastructural examination with transmission electron microscopy. The markers of epithelial cell and the markers of hair cell were used to identify the origin and character of CSEC. CSEC mitotic division was detected by BrdU staining of nuclear DNA., Result: The fresh explants were light yellow. The morphology of CSEC couldn't be seen clearly under the inverted microscope because of the complex structures of cochlear sensory epithelial. CSEC grew out of the explants usually at 2nd culture day. Fibroblast like cells (FLC) around the CSEC grew faster than CSEC, but could easily be excluded. Pure CSEC may grow into monolayer with 'cobblestone-like' appearance and show a large, flat, polygonal epithelial morphotype with big, round nuclei. Some cells showed 'Dome' formation, probably due to fluid collection underneath the cell monolayer. The culture CSEC coexpressed cytokeratin 18 and vimentin has rich microvilli and complex tight junction,which indicated the epithelial origination of CSEC. Coexpressed of the Brn3. a and Calretinin of the hair cell characteristic markers suggested the culture cell may represent rat progenitor hair cell. BrdU staining showed CSEC produced new hair cell-like cell (progenitor hair cell) by the mitotic division., Conclusion: The primary culture systems of cochlear sensory epithelial cell of rats were successfully established in vitro. CSEC coexpressed the characteristic markers of the immature hair cell,which identified the culture cell may represent progenitor cell of rats hair cell. It may be a suitable model for in-depth investigation the molecular mechanisms of hair cell generation or regeneration.

Objective: To explore whether there could be proliferative cells in the cochlea of the newborn rat or not and what kinds of cells should be differentiated from the proliferative cells while to study the effect of the growth factors on the proliferative cells and the ultrastructure of the proliferative cells., Methods: The Corti's organ were dissected from the cochlea of newborn SD rats and cultured. The proliferative condition of cells was tested by infusing the 5-bromo-2-deoxyuridine (BrdU) into the culture medium. And the variety of the spheres and differentiated cells were identified by immunohistochemistry. Corti's organ from forty-eight surface preparations was randomly divided into 4 groups: control group; epidermal growth factor (EGF) group; basic fibroblast growth factor (bFGF) group and EGF + bFGF group, with each group including 12 Corti's organ, and then the number of cell spheres of each Corti's organ was counted. The data was statistically analysed with ANOVE. Finally, the proliferative cells were observed under scanning electron microscope and transmission electron microscope., Results: (1) The cell spheres can be observed in the cell culture of the Corti's organ. In present experiment, 90.1% of cells in spheres were labeled by BrdU, while nestin of spheres, the marker of hair cells--myosin 7A, espin, and phalloidin of the differentiated cells were positive. The marker of neuron-microfilament-M was also positive, and some differentiated cells were labeled by myosin 7A and BrdU, espin and BrdU, NF-M and BrdU at the same time. (2) The average number (x +/- s) of spheres from single Corti's organ was: 45.3 +/- 23.00 in control group, 86.2 +/- 34.1 in EGF group, 96.5 +/- 33.6 in bFGF group and 131.2 +/- 47.00 in EGF + bFGF group. There were significant differences between other groups respectively (P < 0.05) but there was no significant differences between EGF group and bFGF group (P > 0.05). (3) Scanning electron microscopy and transmission electron microscopy showed that cells of the spheres were round and had the same size and many short and thin microvilli on the surface of these cells. The cytoplasm were rich of organellae such as endoplasmic reticulum, mitochondrion, and cytoskeleton such as microfilament, microtube, et al. Tight junction, desmosomes and gap junctions between two adjacent cells were seen., Conclusions: The proliferative cells are observed in the cochlea of the newborn rats and proliferative cells could differentiated into hair cells with bundles-like structure and neuron. Both EGF and bFGF possess the promoting effects for proliferation on the proliferative cells while the proliferative cells have characters of earlier immature cells.

Objective: To investigate the effect of water soluble extracts of traditional Chinese herbs on growth of mouse hair follicles and hair bulb cells in vitro., Methods: Mouse hair follicles and hair bulb cells were cultured in Williams E medium with (experimental groups) or without (control group) water soluble extracts of Chinese herbs; the experimental group was further divided into mixture and single herb groups. Hair growth was observed by microscopy and growth activity of hair bulb cells was detected by MTT colorimetric assay., Result: On day 7 of culture, the hair growth in the mixture groups was faster than that in the control group (P<0.05). On day 3 and 5 of culture, the cell growth activity in the mixture groups was greater than that in the control group (P<0.05). While the hair growth and the cell growth activity between the single herb groups and the control group were not significantly different., Conclusion: The water soluble extracts of mixed traditional Chinese medicines can promote the growth of mouse hair in vitro and stimulate the proliferation of hair bulb cells; while those of the single traditional Chinese herb have no effect.

Aim: To study the antagonistic action of acanthopanax senticosus injection (ASS) on gentamicin ototoxicity., Methods: Guinea pigs were randomly divided into control group, GM group, ASS group, and ASS + GM group. The changes of hearing threshold, cochlear morphology, expression of caspases-3 were determined by ABR, TEM, and Western blot, respectively., Results: The ABR threshold in GM group increased markedly. There was no significant difference in ABR threshold between ASS group and control group, but the ABR threshold in ASS group was much lower than that both in GM group and ASS + GM group. Severely injured hair cells with morphological characteristics of apoptosis were found under TEM in GM group, and the hair cells were less injured in ASS + GM group. The results of Western blot showed that the expression of caspase-3 increased markedly in GM group, but it increased slightly in ASS + GM group., Conclusion: ASS may antagonize the GM ototoxicity by inhibiting the expression of caspases-3.

Aim: To investigate the mechanism of protection by sound conditioning from acoustic trauma., Methods: Sound conditioning experimental model of animals was established. The expression of CaM, HSP70 and F-actin in hair cells were examined with the method of immunohistochemistry. Free calcium concentration in hair cells was observed by LSCM at the same time. Quantitative investigation was devised to assess the changes of F-actin, CaM, HSP70 and intracellular calcium concentration in hair cells., Results: The expression of CaM, HSP70 and F-actin all showed an increased trend after noise exposure. HSP70 and F-actin expressed significantly more in group CH than that expressed in group H. Compared with group H, the expression of CaM showed an increased trend in group CH. Elevation of intracellular calcium concentration could be resulted from noise exposure. The calcium concentration in group H was significantly higher than that in group C and group CH., Conclusion: A suitable sound conditioning can make the auditory system of guinea pig more resistant to noise trauma. The strengthened cytoskeleton system and the intracellular calcium homeostasis play a critical role in the protective mechanism of sound conditioning.

Objective: To investigate whether apoptosis is involved in the pathogenesis of the immune response of inner ear, and relation between signal transportation of Caspase-3 and apoptosis., Method: Forty-five healthy, female white guinea pigs were employed in this study. Sensitized systematically with keyhole limpet hemocyanin (KLH), the KLH-immunized animals were inoculated with the same antigen, and the control animals were injected PBS through cochlea basal turn. All animals were sacrificed respectively at 1st, 3rd, 5th, 7th and 14th day after inner ear vaccination. The auditory brainstem response (ABR) was measured before the KLH injection to the inner ear and just before sacrificing the animal. Paraffin sections of cochleas from animals were stained using a TUNEL assay to identify inner ear cells undergoing apoptosis,and immunohistochemistry method was used to detect the expression of Caspase-3 in the inner ears., Result: After immunization, the ABR thresholds of the KLH-challenged ears were significantly elevated by comparing with the preimmunization (P < 0.05), but after injection, there was no significant difference in the ABR thresholds of the PBS-injected ears compared with preinjection. TUNEL-positive cells were found in the KLH immunized inner ears but no TUNEL-positive cells were observed in the control inner ears except for a few positive cells in the supporting cells, the stria vascularis cells and the spiral ganglion cells. The positive cells are the outer hair cells in Cortis organ, and the marginal cells in the stria vascularis and the neurons in the spiral ganglion. And, under morphological analysis by light microscope, these cells have the features characteristic of apoptosis. Apparent Caspase-3 immunoreactivity expression could be detected in Corti's organ, the lateral wall and the neurons of the spiral ganglion in the KLH-immunized inner ears from 5th day after inner ear vaccination, but no cells staining positive for Caspase-3 were found in the control inner ears., Conclusion: Apoptosis is induced by the immune response of inner ear; Caspase-3 cascade has important effect on the course of apoptosis.

Objective: To investigate the character of damaged vestibular hair cells caused by gentamycin, and to identify whether apoptosis and phosphorylation of JNK occurs in the damaged cells., Method: Healthy guinea pigs were divided into experimental group and control group randomly with 15 in each group, animals in experimental and control group received systemic injection of gentamycin [100 mg/(kg x d)] and saline respectively for 7 consequent days. All the animals were sacrificed on the 8th day, the crista vestibular of each were observed by preparation and frozen dissection. The apoptosis of hair cell were detected by TUNEL method and phosphorylation of JNK were detected by immunochemistry., Result: The lesion and apoptosis of hair cells could be seen in the experimental group,and the phosphorylation could be observed too; the hair cells of control group showed no sign of apoptosis or phosphorylation of JNK., Conclusion: Gentamycin cause vestibular hair lesion by inducing apoptosis and activation of JNK plays an important role in the procedure of apoptosis.

Objective: To detect the expression of Math1, Hes1 and Hes5 in greater epithelial ridge (GER) cells of rat cochlear and explore their influence on hair cell differentiation., Methods: Postnatal day 0 (P0), day 1 (P1) , day 3 (P3) day 4 (P4) and day 5 (P5) rat cochlear were dissected respectively and then pure GER cells were separated by a combinatorial approach of attachment and mechanical separation. The total RNA of GER cells was extracted by Trizol one step method and the expression of Math1, Hes1 and Hes5 in GER cells was detected with reverse transcription polymerase chain reaction., Results: Math1 was expressed in P0 - P5 rat GER cells and Hes1 was expressed only in PO - P3 rat GER cells, while there was no expression of Hes5 in P0 - P5 rat GER cells., Conclusions: Probably only when the expression of Math1 reaches a certain level can it induce GER cells to differentiate into hair cells. Meanwhile this process might controlled by Hes1 to some extent.

Hair cells were dissociated from basila papilla of 7 to 10 week post-hatched chicks (n = 15) by collagenase digestion followed by trituration. Morphological measurements of 215 viable hair cells from 5 chicks were performed. The average dimensions of the cells were: length 20.22 +/- 3.32 microns, width 9.93 +/- 2.01 microns of tall hair cells (n = 164); length 10.97 +/- 2.29 microns, width 14.8 +/- 2.29 microns of short hair cells (n = 30); length = width = 11.93 +/- 1.74 microns of intermediate hair cells (n = 21). Using fluorescein diacetate and propidium iodide, viability of isolated hair cells from another 5 chicks was determined. With excitatory light, fluorescence of viable cells were bright green in 3 hours, while the fluorescence of live cells were attenuated to such an extent that light green shadows were showed up to 5 hours after chicks were decapitated. The results suggested that the criterion of morphological observation and dying of cells should be combined to determine viability of isolated hair cells.

Does the ongoing proliferation of sensory cells occur in the postnatal avian inner ear? This question still awaits further investigation. 5-bromodeoxyuridine (BrdU) is a thymidine analogue that is incorporated into DNA during S phase of the cell cycle. Chickens were given intraperitoneal injections of BrdU at 100 ml/kg body weight. Six hours after injection, they were sacrificed and the temporal bones were removed and prepared for BrdU immunohistochemistry to examine mitotic activity in the inner ear. A small number of labeled hair cells and supporting cells were seen in the basilar papilla (BP). There is a low rate of ongoing production in the postnatal avian vestibular epithelium. The ongoing, postnatal proliferation of vestibular epithelial cell suggests that this epithelium has the potential for repair after degeneration, trauma or ototoxic damage.

Young guinea pigs (250-300 g) were rapidly decapitated and the temporal bones were immediately removed. Under dissecting microscope, the crista ampullaris was removed and incubated in Hank's balanced salt solution containing 0.125 mg/ml collagenase (Sigma IV) at 37 degrees C for 50-60 min. After mechanical dissociation, the isolated cells were transferred onto the coverslips coated with poly-lysine. The cells were left 10-15 min to settle down on the coverslips. Afterward, the collagenase containing solution was replaced by Hank's solution. Observations were made under converted phase-contrast microscope. The results were as follows: 1. Vestibular hair cells are classified into two types. Type I cell was flask-shaped with sensory hairs, a cuticular plate, a long neck and a round bottom containing a large nucleus. The length of type I cell was 18.10 +/- 2.05 microns (mean +/- s n = 32). Type II cells were round shaped or cylindrically shaped without neck but with sensory hairs, a cuticular plate. The longitudinal axis of cylindrically shaped cell was 14.00 +/- 3.16 microns and horizontal axis 9.88 +/- 2.01 microns (mean +/- s n = 29). The diameter of the round cell was 10.31 +/- 1.98 microns (mean +/- s n = 10). 2. The proportion of type I and type II cells was 61/39. 3. An average of 500-700 vestibular hair cells were obtained from each ampulla. 4. Generally, vestibular hair cells could live in Hank's solution for 7-8 hours. The earliest sign of viability degression was the appearance of granules in the cytoplasm.

Isolated live hair cells are important models for studying the electrophysiology, pathology and pharmacology of the hair cell. Using mechanical isolation method after papain treatment 70 +/- 27 hair cells, including 0 to 4 inner hair cells, could be obtained from each cochlea of 4 guinea pigs. The criteria for a good viability of isolated cochlear hair cells were: 1. a smooth hair cell membrane; 2. hair cells not swollen; 3. the nucleus in the normal position; 4. the cytoplasm in a state of semitransparency with a halo at the periphery (birefringence) and 5. no Brownian movement of the organelles within the cytoplasm. With short-term culturing at room temperature, approximately 90% of the isolated hair cells retained a good viability at the end of two hours. Subsequently the hair cells gradually degenerated but still, at the end of five hours, about 40% of them appeared intact. The degeneration patterns have been carefully observed and described.