OBJECTIVE: To study the evidence of adenosine triphosphate (ATP) in the marginal cells of the stria vascularis in the neonatal rat cochlea, namely, whether ATP vesicles could be detected in the marginal cells and ATP release could be detected in the culture solution in vitro. METHOD: The marginal cells of 1-3 day old Sprague-Dawley rats were cultivated, purified and verified. ATP vesicles Were observed in the marginal cells under fluorescence microscope using quinacrine staining technique. The concentration of ATP released from the cells in the extracellular solution was determined through bioluminescence assay. RESULT: The marginal cells were verified in vitro by detecting antibodies of cytokeratin and vimentin using flow cytometry. A large number of anterior-like staining could be observed in cultured marginal cells that were incubated with quinacrine. The concentration of ATP released from the cells in the culture solution could be determined, and the concentration of ATP could be calculated by fluorescent intensity. CONCLUSION The ATP vesicles exist in the marginal cells and can release ATP.
Adenosine Triphosphate ; metabolism ; Animals ; Animals, Newborn ; Cells, Cultured ; Cochlea ; cytology ; Rats ; Rats, Sprague-Dawley ; Stria Vascularis ; cytology

OBJECTIVETo establish a mice model of cisplatin-induced ototoxicity, and to investigate the effect of cisplatin on apoptosis of spiral ganglion cell and expression of caspase-3 in mouse cochlea.

METHODSTerminal deoxynucleotidyl transferase-mediated nick end labeling method (TUNEL) was used to monitor the apoptosis of spiral ganglion cell. Envision method of immunohistochemistry was applied to detect the expression of caspase-3 in cochlea. Auditory brainstem response (ABR) was measured to observe the change of hearing.

RESULTSThe weight and hearing of mice in different dose of cisplatin groups were declined significantly as compared with those of control group (P < 0.05, P < 0.01), and the TUNEL positive cell number and expression of caspase-3 were greater remarkably with the more cisplatin injected.

CONCLUSIONA mouse model of cisplatin-induced ototoxicity can be established. Cisplatin can lead to the apoptosis of spiral ganglion cells, and caspase-3 has participated in this apoptosis process, which approves further that apoptosis might be one of the mechanisms of cisplatin ototoxicity.

Animals ; Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Cisplatin ; pharmacology ; Cochlea ; cytology ; drug effects ; metabolism ; Male ; Mice ; Mice, Inbred Strains ; Spiral Ganglion ; cytology ; drug effects ; metabolism

OBJECTIVE: To observe whether bFGF could cross the blood-labyrinth barrier (BLB) after intra-abdominal injection and to establish an experimental basis for its clinical applications. METHOD: Thirty guinea pigs were divided into three groups. Animals in group 1 were administered o I-bFGF, while animals in group 2 and 3 were administered 125 and saline, respectively, via intra-abdominal injection. The both cochlea, blood, liver, brain, thyroid gland and kidney were collected and weighted. A radioimmunoassay analyzer was employed to measure counts per minute (CPM) of each sample, and autoradiography was performed on both cochlea. RESULT: The CPM value of organ samples in the 125I group was higher than that in other groups, and radioactive grain was observed in cochlear samples of this group. In the 125I-bFGF group, blood demonstrated the highest CPM value, while cochlea and brain demonstrated the lowest CPM value, with no radioactive grain observed in cochlear samples. CONCLUSION bFGF has some difficulties in getting across BLB, so the way of bFGF application in clinics need further study.
Animals ; Autoradiography ; Cochlea ; cytology ; metabolism ; Fibroblast Growth Factor 2 ; administration & dosage ; Guinea Pigs ; Injections, Intraperitoneal ; Iodine Radioisotopes ; administration & dosage

OBJECTIVETo 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.

METHODSPostnatal 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.

RESULTSMath1 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.

CONCLUSIONSProbably 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.

Animals ; Basic Helix-Loop-Helix Transcription Factors ; genetics ; metabolism ; Cell Differentiation ; Cochlea ; cytology ; metabolism ; Epithelial Cells ; metabolism ; Gene Expression Regulation, Developmental ; Hair Cells, Auditory ; cytology ; metabolism ; Homeodomain Proteins ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Transcription Factor HES-1

The present study was aimed to investigate the effect of hydrogen peroxide (H₂O₂, oxygen free radical donator) on the current of large-conductance calcium-activated potassium channels (BK(Ca) channels) in isolated outer hair cells of old guinea pig cochlea, and to explore the underlying mechanism. Outer hair cells of old guinea pig cochlea were acutely enzyme-isolated, and currents were recorded by whole-cell patch clamp. The results showed that, rapid activation and non-deactivation electric currents with a string of large amplitude were recorded. Activation voltage of the current was above -40 - -30 mV. The amplitude of current was increased continuously with the rising of membrane potential. The current showed characteristics of outward rectification without "rundown" phenomenon. IbTX (100 nmol/L) could completely block the activity of channel, which confirmed BK(Ca) channel's current. BK(Ca) current amplitude and peak current density increased with the increment of H₂O₂ concentration (1, 2, 4 μmol/L), showing concentration-dependent activation by H₂O₂. Our results suggest that oxygen free radical/BK(Ca) pathway may be able to adjust the balance of intracellular calcium in outer hair cells.
Animals ; Calcium ; metabolism ; Cochlea ; cytology ; Guinea Pigs ; Hair Cells, Vestibular ; drug effects ; Hydrogen Peroxide ; pharmacology ; Large-Conductance Calcium-Activated Potassium Channels ; metabolism ; Membrane Potentials

BACKGROUNDOur previous studies have shown that both apoptosis and necrosis are involved in hair cell (HC) pathogenesis in aging cochleae. To better understand the biological mechanisms responsible for the regulation of HC death, we examined the activity of succinate dehydrogenase (SDH), a mitochondrial bioenergetic enzyme, in the HCs of aging cochleae.

METHODSThe auditory brainstem response thresholds elicited by tone bursts at 4, 10 and 20 kHz were measured in both young (2-3 months) and aging (22-23 months) Wistar rats. SDH activity was evaluated with a colorimetric assay using nitroblue tetrazolium monosodium salt. The SDH-labeled organs of Corti were double stained with propidium iodide, a DNA intercalating fluorescent probe for illustration of HC nuclei. All the specimens were examined with fluorescence microscopy and confocal microscopy.

RESULTSAging rats exhibited a significant elevation of ABR thresholds with threshold shifts being 34 dB at 20 kHz, 28 dB at 10 kHz, and 25 dB at 4 kHz. Consistent with the reduction in the cochlear function, aging cochleae exhibited the reduction of SDH staining intensity in the apical and the basal ends of the cochleae, where a large number of apoptotic, necrotic, and missing HCs were evident. The reduction in SDH staining appeared in a cell-death-mode dependent fashion. Specifically, SDH labeling remained in apoptotic HCs. In contrast, SDH staining was markedly reduced or absent in necrotic HCs.

CONCLUSIONSIn the aging cochlea, SDH activity is preserved in HCs undergoing apoptosis, but is substantially reduced in necrosis. These results suggest that mitochondrial energetic function is involved in the regulation of cell death pathways in the pathogenesis of aging cochleae.

Aging ; metabolism ; Animals ; Apoptosis ; physiology ; Cochlea ; cytology ; enzymology ; Female ; Hair Cells, Auditory ; enzymology ; Male ; Necrosis ; physiopathology ; Rats ; Rats, Wistar ; Succinate Dehydrogenase ; genetics ; metabolism

The aims of this study were to evaluate the expression of enhanced green fluorescent protein (EGFP) driven by 6 different promoters, including cytomegalovirus IE enhancer and chicken beta-actin promoter (CAG), cytomegalovirus promoter (CMV), neuron-specific enolase promoter (NSE), myosin 7A promoter (Myo), elongation factor 1alpha promoter (EF-1alpha), and Rous sarcoma virus promoter (RSV), and assess the dose response of CAG promoter to transgene expression in the cochlea. Serotype 1 adeno-associated virus (AAV1) vectors with various constructs were transduced into the cochleae, and the level of EGFP expression was examined. We found the highest EGFP expression in the inner hair cells and other cochlear cells when CAG promoter was used. The CMV and NSE promoter drove the higher EGFP expression, but only a marginal activity was observed in EF-1alpha promoter driven constructs. RSV promoter failed to driven the EGFP expression. Myo promoter driven EGFP was exclusively expressed in the inner hair cells of the cochlea. When driven by CAG promoter, reporter gene expression was detected in inner hair cells at a dose as low as 3 x 10(7) genome copies, and continued to increase in a dose- dependent manner. Our data showed that individual promoter has different ability to drive reporter gene expression in the cochlear cells. Our results might provide important information with regard to the role of promoters in regulating transgene expression and for the proper design of vectors for gene expression and gene therapy.
Animals ; Cochlea/cytology/*metabolism ; Dependovirus/*genetics ; Dose-Response Relationship, Drug ; Female ; Genetic Vectors/*genetics ; Green Fluorescent Proteins/metabolism ; Humans ; Mice ; Mice, Inbred C57BL ; Promoter Regions, Genetic/*genetics ; *Transgenes

OBJECTIVE: To investigate the influence of overexpression of manganese superoxide dismutase (MnSOD) of stria marginal cells (MCs) of the rat cochlea by the recombinant adeno-associated viruses of the serotypes 2 (AAV2) mediated gene-delivery for hydrogen peroxide-induced oxidative stress in vitro. METHOD: Primary cultures of MCs were infected using rAAV2-MnSOD-EGFP at dosage of multiplicity of infection (MOI) 10(1)v x /cell and using rAAV2-EGFP as control. The expression of MnSOD in MCs was examined using western blot and the activity of MnSOD was determinated by colorimetric assays. Oxidative stress was induced in MCs by exposing them to H2O2 (400 micromol/L) for 2 hour and preculturing them in normal medium. After 24 h the amount of the lipid peroxidation production malondialdehyde (MDA) was detected. Apoptosis was assessed by flow cytometry by Propidium oidium staining. The expression of the cleaved Caspase-3 was assessed by Western blot. RESULT: (1) EGFP expression in MCs could not be detected until 4 days after rAAV2- MnSOD-EGFP infection and reached fastigium after 10 days and lasted over a month. The MnSOD level in the rAAV2- MnSOD-EGFP group was higher than that in the control group. (2) After being exposed to H2O2, the amounts of MDA in rAAV2-MnSOD-EGFP group, control group and normal group were 0.464 +/- 0.049, 1.103 +/- 0.033 and 0.185 +/- 0.005 (nmol/mg prot), respectively. The expression of the cleaved-caspase-3 in rAAV2-MnSOD-EGFP group was lower than that in control group and the number of apoptotic cells decreased significantly. CONCLUSION The results demonstrate that the rAAV2-MnSOD-EGFP can effectively transfect cultured MCs, and the transgenic cells show a high expression of MnSOD which can protect the MCs against oxidative challenge. The role of overexpression MnSOD in MCs apoptosis induced by oxidative injury may be associated with suppressing the activation of caspase-3.
Animals ; Apoptosis ; Caspase 3 ; metabolism ; Cells, Cultured ; Cochlea ; metabolism ; Dependovirus ; genetics ; Oxidative Stress ; Rats ; Rats, Wistar ; Stria Vascularis ; cytology ; Superoxide Dismutase ; biosynthesis ; Transfection

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.
Animals ; Basic Helix-Loop-Helix Transcription Factors ; genetics ; metabolism ; Cell Transdifferentiation ; genetics ; Cochlea ; cytology ; Ear, Inner ; Green Fluorescent Proteins ; genetics ; metabolism ; Guinea Pigs ; Hair Cells, Auditory ; cytology ; Labyrinth Supporting Cells ; cytology

In order to elucidate the mechanism underlying the attenuation of streptomycin ototoxicity by tetramethylpyrazine (TMP), the present study investigated the effect of TMP on the outward K(+) current in the outer hair cells of guinea pig cochlea. Sixty guinea pigs were divided into 6 groups randomly. Auditory brainstem response (ABR) was used to observe the change in thresholds and to evaluate ototoxicity induced by streptomycin. Whole-cell patch-clamp technique was used to observe the effect of TMP on outward K(+) current in isolated outer hair cells. The results showed that TMP attenuated the threshold shift caused by streptomycin and increased the amplitudes of Ca(2+)-sensitive K(+) current [I(K(Ca))] in the outer hair cells. The present data suggest that TMP displays anti-ototoxicity induced by streptomycin. The augmented amplitudes of I(K(Ca)) of the outer hair cells induced by TMP may be one of the mechanisms underlying its ototoxicity-attenuating effect.
Animals ; Auditory Threshold ; Cochlea ; cytology ; Evoked Potentials, Auditory, Brain Stem ; Guinea Pigs ; Hair Cells, Auditory, Outer ; drug effects ; Patch-Clamp Techniques ; Potassium Channels ; metabolism ; Pyrazines ; Streptomycin ; toxicity