OBJECTIVE: To establish a practical model for Wistar rat cochlea organ culturing in vitro, and to observe the growing status in hypoxia of the spiral ganglion cell and nerve fiber. METHOD: We used an in vitro hypoxia model and dissociated cultures of the basal membrane from the cochlea of 3-day-old Wistar rats. And put them in incubator (37 degrees C, 90% N2, 5% CO2, 5% O2) to hypoxia culture for different times. The culture were Immunofluorescence dyed and count the number of the spiral ganglion cell and the cell density in unit area (24 mm x 36 mm), and observe the morph of nerve fiber under the confocal microscope, the results were compared with controls. RESULT: Hypoxia early (6 h) nerve fiber appear edema, spiral ganglion cell didn't change compared with controls; nerve fiber appear break and disintegration and the spiral ganglion cell decrease in 12 hours culturing, and the cell density in unit area had remarkable difference compared with control (P < 0.01). Hypoxia leads to the cell density decrease in a time-dependent manner, the longer of cultures times in hypoxia, the heavier of damage in spiral ganglion cell and nerve fiber. Twelve hours culturing, and the cell density in unit area had remarkable difference compared with control (P < 0.01). Hypoxia leads to the cell density decrease in a time-dependent manner, the longer of cultures times in hypoxia, the heavier of damage in spiral ganglion cell and nerve fiber. CONCLUSION The study findings suggest that hypoxia makes the spiral ganglion cell and nerve fiber damage of culturing in vitro, and nerve fiber more susceptible than spiral ganglion cell for hypoxia.
Animals ; Cell Count ; Cell Survival ; Female ; Hair Cells, Auditory, Inner ; cytology ; pathology ; Hypoxia ; pathology ; In Vitro Techniques ; Male ; Nerve Fibers ; pathology ; Rats ; Rats, Wistar ; Spiral Ganglion ; cytology ; pathology

OBJECTIVE: To evaluate the protective effect of low dose ouabain on injury of cultured spiral ganglion neurons evoked by trophic factors deprived and to explore the mechanism. METHOD: Spiral ganglion neurons were cultured in vitro for 7 days, and then exposed to Neurobasal medium + B27 supplement, Neurobasal medium only or Neurobasal medium + 10 nmol/L ouabain, respectively. After 48 h exposed to different medium, spiral ganglion neurons were stained by FITC labeled Annexin-V and PI, then apoptosis index were calculated using fluorescent microscope and flow cytometry, respectively. In addition, spiral ganglion tissues were cultured for 48 h to evaluate dendrite growth of spiral ganglion neurons in each group. Immunocytochemistry were performed to detect the level of Bcl-2 in each group at 6 h and 12 h. RESULT: Spiral ganglion neurons exposed to Neurobasal medium +10 nmol/L ouabain have a similar apoptosis index compare with that of Neurobasal medium + B27 supplement, but a much lower apoptosis index than that of Neurobasal medium only. In addition, dendrite growth of spiral ganglion neurons exposed to Neurobasal medium +10 nmol/L ouabain was much longer than that of Neurobasal medium only. Bcl-2 level increased in spiral ganglion neurons exposed to Neurobasal medium + 10 nmol/L ouabain at 6 h. CONCLUSION Low dose of ouabain protects injury of spiral ganglion neurons evoked by trophic factors deprived in vitro. This effect may mediated by increasing the level of Bcl-2.
Animals ; Apoptosis ; drug effects ; Cell Survival ; drug effects ; Cells, Cultured ; Ouabain ; administration & dosage ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Spiral Ganglion ; drug effects ; pathology

BACKGROUND AND OBJECTIVES: Auditory neuropathy is a hearing disorder characterized by the absence or the marked impairment of the auditory brainstem responses with the preservation of the cochlear microphonics (CMs) and otoacoustic emissions. This suggests that outer hair cell (OHC) function is normal but proximal auditory function to OHCs is impaired. It is assumed that the lesion is localized at the level of the inner hair cells (IHCs), auditory nerve fibers, or the synapse between them. This study was aimed to observe the change of hearing threshold and pathology of spiral ganglion cell induced by ouabain application, and present basic data to explain the auditory neuropathy. MATERIALS AND METHOD: Twenty ears of twenty normal hearing cats were used in this study. Cats were treated with 100 microL ouabain (1 mM) applied on the round window. After three days, compound action potential (CAP) and CM were measured and the cochlea was obtained. Pathologic change of spiral ganglion cell was evaluated under light microscope after H&E stain. Normal saline was injected for the control group. RESULTS: In the ouabain group, CAP threshold was increased in all tested frequencies (p<0.001) and the difference of CM threshold was not significant in all frequencies (p>0.05). There was significant difference between CAP and CM threshold shift (p<0.001). In the control group, there was no significant difference in CAP and CM thresholds. Light microscopic findings show that the condensed chromatin and nuclear fragments of spiral ganglion cells of an ear was exposed to ouabain, and outer hair cell and inner hair cell were not damaged. CONCLUSION: This study shows that the CAP threshold was significantly increased but the CM threshold was not changed in the ouabain group. Ouabain induced damage of spiral ganglion cells. This study is not sufficient to explain auditory neuropathy because threshold shift of CAP is not obvious, but it would be helpful to explain that selective damage of spiral ganglion cell would be the mechanism of auditory neuropathy.
Action Potentials ; Animals ; Cats* ; Chromatin ; Cochlea ; Cochlear Nerve ; Ear ; Evoked Potentials, Auditory, Brain Stem ; Hair ; Hearing ; Hearing Disorders ; Ouabain* ; Pathology ; Spiral Ganglion* ; Synapses

The aim of the present study was to assess the ototoxicity of kanamycin sulfate (KM) in adult rats and its underlying mechanism. Forty male Sprague-Dawley rats (6-7 weeks old) were randomly divided into the experimental group and the control group. The animals in the experimental group were injected subcutaneously with KM (500 mg/kg per day) for two weeks, and the control group received equal volume of normal saline. To assess the ototoxicity of KM, the auditory brainstem response (ABR) was recorded to monitor the changes in hearing thresholds, and the density of spiral ganglion cells (SGCs) and morphology of cochlea were observed using surface preparations and frozen sections of cochlea. The results showed that the hearing threshold of rats in the experimental group was elevated by more than 60 dB across all the frequencies two weeks after the first administration of KM. And in the experimental group, the density of SGCs became lower, and organ of Corti suffered loss of hair cells. The loss of outer hair cells (OHCs) was more severe than that of inner hair cells (IHCs), correlated with the density decrease of SGCs. We conclude that the ototoxicity of KM in the adult rats was apparent and the underlying mechanism is associated with the loss of SGCs and hair cells.
Animals ; Cochlea ; drug effects ; pathology ; Evoked Potentials, Auditory, Brain Stem ; drug effects ; Hair Cells, Auditory, Outer ; cytology ; drug effects ; pathology ; Hearing Loss ; chemically induced ; physiopathology ; Kanamycin ; toxicity ; Male ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Spiral Ganglion ; pathology ; physiology ; ultrastructure

The aim of this study was to determine the effects of transplanted neural differentiated human mesenchymal stem cells (hMSCs) in a guinea pig model of auditory neuropathy. In this study, hMSCs were pretreated with a neural-induction protocol and transplanted into the scala tympani of the guinea pig cochlea 7 days after ouabain injury. A control model was made by injection of Hanks balanced salt solution alone into the scala tympani of the guinea pig cochlea 7 days after ouabain injury. We established the auditory neuropathy guinea pig model using 1 mM ouabain application to the round window niche. After application of ouabain to the round window niche, degeneration of most spiral ganglion neurons (SGNs) without the loss of hair cells within the organ of Corti and increasing the auditory brain responses (ABR) threshold were found. After transplantation of neural differentiated hMSCs, the number of SGNs was increased, and some of the SGNs expressed immunoreactivity with human nuclear antibody under confocal laser scanning microscopy. ABR results showed mild hearing recovery after transplantation. Based on an auditory neuropathy animal model, these findings suggest that it may be possible to replace degenerated SGNs by grafting stem cells into the scala tympani.
Animals ; Cardiotonic Agents/toxicity ; Cochlea/drug effects/pathology ; Disease Models, Animal ; Female ; Guinea Pigs ; Hearing Loss, Central/chemically induced/pathology/*therapy ; Humans ; *Mesenchymal Stem Cell Transplantation ; Mesenchymal Stem Cells/*cytology ; Neurogenesis ; Ouabain/toxicity ; Spiral Ganglion/pathology ; Transplantation, Heterologous

To present a summary of current knowledge regarding acute kanamycin sulfate-induced deafness in guinea pig, by reviewing the published literature. Animal model of acute deafness induced by a single dose of kanamycin sulfate in combination with ethacrynic acid or furosemide in guinea pig was usually used to investigate the mechanism of cochlear cell degeneration. There were different time sequences of cell degeneration of spiral ganglion cell and hair cell in different studies. The findings may result from different doses, order of two drugs administration or time point chosen. There remains scope for further research in chronic kanamycin-induced deafness, which more replicates the type of exposure to people than acute deafness.
Animals ; Anti-Bacterial Agents ; administration & dosage ; adverse effects ; Cochlea ; Deafness ; chemically induced ; Disease Models, Animal ; Ethacrynic Acid ; adverse effects ; Guinea Pigs ; Hair Cells, Auditory ; pathology ; Humans ; Kanamycin ; administration & dosage ; adverse effects ; Neurons ; Spiral Ganglion ; drug effects ; pathology

BACKGROUND AND OBJECTIVES: Deafness is the most common sensory deficit and hereditary defect in human populations. The present study investigated the causative gene in circling mice using the complementation test. In addition, the phenotypes and histopathologic findings in circler mice, spinner mice, and compound heterozygote mice were analyzed to elucidate the mechanism of causative gene in inner ear deafness. MATERIALS AND METHOD: In order to analyze inner ear pathology in time sequence for the circler mice, spinner mice, and compound heterozygote, five groups of the homozygous mutants of different ages were used: 10, 18, 21, 35, and 90 days old. The organs of Corti and spiral ganglion neurons in the basal and middle turns were included for quantification. For the preparation of genomic DNA, tail tissues were used. RESULTS: The hair cells in the organ of Corti degenerated in a time-dependent manner. In the basal and middle turns, the volume ratio of spiral ganglion neurons significantly decreased as the mutant aged. RT-PCR analysis indicated that transmembrane inner ear (Tmie) was absent in the case of circler mice, similar to spinner mouse of which is defective Tmie gene. Therefore the variations may be a result from strain-specific allelic differences of the Chr 9 Tmie gene itself (allelic heterogeneity). CONCLUSION: The cir mutant is a suitable mouse model for neuroepithelial defects. PCR and RT-PCR analyses suggest that the Tmie transcript is absent in circler mice. This model represents another candidate for human genetic hearing loss.
Animals ; Deafness* ; DNA ; Ear, Inner ; Genes, Recessive ; Genetic Complementation Test ; Hair ; Hearing Loss ; Heterozygote ; Humans ; Mice* ; Models, Animal ; Neurons ; Organ of Corti ; Pathology ; Phenotype ; Polymerase Chain Reaction ; Spiral Ganglion ; Tail

OBJECTIVE: To research advanced glycation end-product receptors (RAGE), NF-kappaB, p21 expressions in C57BL/6j mice cochlea spiral ganglion cells(SGC) ,and then to investigate the presbycusis pathogenesis. METHOD: To take C57BL/6J mice:2 month 25,and 10 month 25. Histological sections were observed the SGC. RAGE, NF-kappaB, p21 were immunohistochemical in the SGC,with IPP6 to IOD. RESULT: (1) The count SGCs of 10 month-old was obviously decreased comparing to 2 month-old, the count of 2 month SGC is 39 +/- 5, 10 month group is 20 +/- 6, P < 0.01; (2) RAGE, NF-kappaB, p21 expressed in spiral ganglion cell,different place with different age,and the means optical density in the 10 month are higher than the 2 month, respectively. The IOD of RAGE expression in 2 month SGC: 0.179 +/- 0.025, 10 month IOD: 0.308 +/- 0.050; The IOD of NF-kappaB expression in 2 month SGC: 0.181 +/- 0.045, 10 month IOD: 0.335 +/- 0.120; The IOD of p21 expression in 2 month SGC: 0.160 +/- 0.023, 10 month IOD: 0.365 +/- 0.031, compare with 2 group, respectively, P < 0.05, and the difference has statistics sense. CONCLUSION RAGE,NF-kappaB, p21 expressions are in SGCs and increases with the aging of SGCs, suppose RAGE, NF-kappaB, p21 may participate in the process of presbycusis pathogenesis.
Aging ; Animals ; Male ; Mice ; Mice, Inbred C57BL ; NF-kappa B ; metabolism ; Neurons ; metabolism ; Presbycusis ; pathology ; Proto-Oncogene Proteins p21(ras) ; metabolism ; Receptor for Advanced Glycation End Products ; Receptors, Immunologic ; metabolism ; Spiral Ganglion ; metabolism

The aim of the present study was to investigate the role of glutamate receptors in the damage of spiral ganglion neurons (SGNs) induced by acute acoustic noise. This investigation included in vivo and in vitro studies. In vivo, kynurenic acid (KYNA), a broad-spectrum antagonist of glutamate receptors, was applied to the round window of guinea pigs, and its protective effect was observed. The animals were divided into three groups: control (saline, 0.9%, 10 microL), saline (0.9%, 10 microL) + noise and KYNA (5 mmol/L, 10 microL) + noise. Saline and KYNA were applied to the round window membrane with a microsyringe. The animals were exposed to 110 dB SPL of white noise for 1 h. Hearing thresholds for auditory brainstem responses (ABRs) and compound action potentials (CAPs) in all animals were measured before and after treatment. The amplitudes of III waveform of ABR and N1 waveform of CAP and the latency of N1 waveform at different stimulation levels (intensity-amplitude and intensity-latency functions) were also measured. The cochleas were then dissected for transmission electron microscopy (TEM) after final electrophysiological measurement. In vitro, the SGNs of the normal guinea pigs were isolated and glutamate (100 micromol/L or 1 000 micromol/L) was added into the medium. The morphology of the SGNs was examined by light microscopy. In vivo results showed that the hearing function and morphology of the inner ear including hair cells and SGNs in the control group were normal. Compared with that in the control group the thresholds for ABR and CAP (click and tone burst) in saline + noise group were elevated significantly. The input-output functions showed that the amplitudes of III waveform of ABR and N1 waveform of CAP decreased and the latency of N1 waveform increased obviously. There was significant difference in the amplitude and latency between saline + noise group and KYNA + noise group (P<0.05). TEM indicated obvious swelling and vacuoles at the terminate of dendrites of SGNs in NS + noise group. On the contrary, the afferent dendrites in KYNA + noise group showed normal appearance without swelling and vacuoles. In vitro experiment showed that the isolated SGNs of guinea pigs obviously swelled and even died after application of 100 micromol/L or 1 000 micromol/L glutamate. These results suggest that noise exposure causes hearing impairment, damage of hair cells and hair cell/afferent synapse and death of SGNs. The antagonist of glutamate receptors provides protective effects against hearing loss and SGN damage. It is inferred that excessive release of glutamate from the inner hair cells induced by noise may be responsible for these damages. Glutamate receptors are involved in the degeneration and death of SGNs.
Action Potentials ; physiology ; Animals ; Evoked Potentials, Auditory, Brain Stem ; physiology ; Excitatory Amino Acid Antagonists ; pharmacology ; Guinea Pigs ; Hearing Loss, Noise-Induced ; metabolism ; pathology ; physiopathology ; Kynurenic Acid ; pharmacology ; Male ; Neurons ; pathology ; Noise ; adverse effects ; Random Allocation ; Receptors, Glutamate ; metabolism ; Spiral Ganglion ; pathology

OBJECTIVETo investigate the protein and mRNA expression patterns of apoptosis-related genes, together with evidence of apoptosis, in relation to experimental autoimmune inner ear disease (AIED).

METHODSMale C57BL/6 mice at 4 weeks age (n = 80) were randomly assigned to one of the five group (n = 16). The inbred mice were given a single subcutaneous injection of diluted solution of pertussis and an emulsion containing equal parts of complete Freund adjuvant (CFA) and inner ear antigens (IEAg) extracted form guinea pig. The animals were sacrificed for inner ear examination at a defined time after the immunization (7, 14, 21 or 28 days). An autoimmune inner ear diseases model was established. Apoptosis was detected by terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (d-UTP) nick end-laying (TUNEL) method. Using immunohistochemical techniques and reverse transcriptase polymerase chain reaction to clarify the profile of Fas, FasL, and bcl-2.

RESULTSUnder normal conditions, no TUNEL-positive cell was observed in the cochlea except for a few positive cells in the supporting cells of Corti's organ and macula sacculi. Inner ear antigens administration induced TUNEL-positive reactions in a wide variety of cells such as inner hair cells, supporting cells, stria vascularis and spiral ligament fibrocytes. No positive staining was evident in outer hair cells, spiral ganglion cells and Scarpa's ganglion cells during the whole period. Fas proteins were expressed in a wide range of cells in inner ear. The levels of Fas mRNA were no significant differences between normal and AIED mice. FasL and bcl-2 proteins could be detected in spiral ganglion cells and Scarpa's ganglion cells both in normal and AIED mice. FasL positive cells increased in number in inner ear of AIED mice. bcl-2 positive cells were not detectable in inner hair cells, stria vascularis and spiral ligament both in normal and AIED mice. The mRNA of three kinds of apoptosis-related genes was detectable in the normal and AIED mice. FasL mRNA was expressed at low levels in normal, being maximal at 14 d post inoculation and decreased gradually to steady levels by 2 weeks. The levels of bcl-2 mRNA increased significantly during the period of AIED.

CONCLUSIONApoptosis mediated by Fas/FasL signal system may play a role in the initiation and maintenance of AIED. bcl-2 has a crucial role in the regulation of the process of apoptosis in the inner ear of AIED mice.

Animals ; Apoptosis ; Autoimmune Diseases ; genetics ; immunology ; metabolism ; pathology ; Fas Ligand Protein ; genetics ; metabolism ; Guinea Pigs ; In Situ Nick-End Labeling ; Labyrinth Diseases ; genetics ; immunology ; metabolism ; pathology ; Male ; Mice ; Mice, Inbred C57BL ; Proto-Oncogene Proteins ; genetics ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; Spiral Ganglion ; metabolism ; fas Receptor ; genetics ; metabolism