Single-cell transcriptomic atlas of mouse cochlear aging

Progressive functional deterioration in the cochlea is associated with age-related hearing loss (ARHL). However, the cellular and molecular basis underlying cochlear aging remains largely unknown. Here, we established a dynamic single-cell transcriptomic landscape of mouse cochlear aging, in which we characterized aging-associated transcriptomic changes in 27 different cochlear cell types across five different time points. Overall, our analysis pinpoints loss of proteostasis and elevated apoptosis as the hallmark features of cochlear aging, highlights unexpected age-related transcriptional fluctuations in intermediate cells localized in the stria vascularis (SV) and demonstrates that upregulation of endoplasmic reticulum (ER) chaperon protein HSP90AA1 mitigates ER stress-induced damages associated with aging. Our work suggests that targeting unfolded protein response pathways may help alleviate aging-related SV atrophy and hence delay the progression of ARHL.

The effect of large-conductance calcium-activated potassium channels on the migration of pericytes in the mice of senile cochlear stria vascularis / 中华耳鼻咽喉头颈外科杂志

Objective: To investigate whether large conductance calcium-activated potassium channel (BK(Ca)) was involved in the migration of pericytes (PC) in the mice of senile cochlear stria vascularis capillaries PC. Methods: C57BL/6J mice were divided into 3-month (n=10) and 12-month groups (n=10). Auditory brainstem response (ABR) was used to test the hearing threshold of each group. The immunofluorescence was used to detect the expression changes of osteopontin (OPN) and β-BK(Ca) channels on cochlear stria vascularis PC. The morphological changes of perivascular cells in cochlea were observed by transmission electron microscope (TEM). Cell experiment: The PC, which were in the stria vascularis of the cochlea were primary cultured and identified. A cell senile model was made with D-gal. The appropriate intervention concentration of low galactose (D-gal) was determined by CCK8. β-galactosidase (SA-β-gal) staining was used to evaluate the cell decrept level. The change of BK(Ca) channels current on PC were recorded by whole cell patch clamp technique. The expression of BK(Ca) channels on PC was detected by immunofluorescence. The migration and invasion ability of two groups were detected by using Scratch test and Transwell. The levels of OPN and β-BK(Ca) channels were detected by Western blot. SPSS 22.0 software was used to analyze the data. Results: The ABR threshold in the 12-month group was higher than 3-month group (t=12.66, P<0.01). In the 12-month group, the expression of β-BK(Ca) channel was lower and the expression of OPN was increased (t=14.64, P<0.01; t=20.73, P<0.01). In TEM, cochlear stria vascularis PC were tightly connected to endothelial cells in 3-month group, while PC were loosely connected to endothelial cells or PC soma were separated from the capillary in 12-month group. Cell experiment: The positive rate of PC in the primary cultured cochlear stria vascularis is above 95%. Compared with the SA-β-gal stained cells in the control group, the positive rate of 15 mg/ml D-gal intervention PC was 85% (t=36.90, P<0.01). Whole cell patch clamp BK(Ca) channels current decreased in the D-gal group compared with the young group PC (t=12.18, P<0.05). The OPN expression in the senile group was higher than control group (t=16.30, P<0.01), while the β-BK(Ca) channels expression was decreased (t=11.98, P<0.01; t=15.72, P<0.05), and migration ability raised (t=7.91, P<0.01;t=7.59, P<0.01). After intervened of BK(Ca) channels specific blocker IBTX in the D-gal group, the expression of OPN and migration were increased (t=4.26, P<0.05; t=5.88, P<0.01; t=21.97, P<0.01). Conclusion: PC migration capacity were increased during the senile period, and the expression of β-BK(Ca) channel was decreased. The administration of IBTX, a specific blocker of BK(Ca) channel, at the cell level could increase the migration capacity, suggesting that BK(Ca) might be involved in the migration of PC in the stria vascularis of the aging cochlea.

Effect of microvascular pericytes of cochlear stria vascularis on endothelial cell permeability in C57BL/6J mice / 中华耳鼻咽喉头颈外科杂志

Objective: To study the changes in the permeability of the blood labyrinth barrier of the aging cochlea in mice, and to establish a non-contact co-culture model of endothelial cells (EC) and pericytes (PC) to furtherly investigate the cochlear stria vascularis microvascular pericytes impact on the permeability of endothelial cells. Methods: C57BL/6J mice were divided into two groups, three months old as young group, 12 months old as senile group. Cell experiment was divided into four groups, EC group, EC+PC co-culture group, D-gal+EC group and D-gal+EC+PC co-culture group. Auditory brainstem response (auditory brain response, ABR) was used to detect the auditory function of the two groups of mice. Evans blue staining was applied to detect the permeability of the cochlear blood labyrinth barrier of the two groups of mice. Transmission electron microscopy was used to observe the ultrastructure of blood labyrinth barrier endothelial cells, pericytes and tight junctions in the two groups of mice. Immunohistochemistry was used to detect the expression levels of tight junction proteins in the stria vascularis of the cochlea of the two groups of mice. Transwell chamber was used to detect the permeability of endothelial cells. Western blot and immunofluorescence technology were used to detect the expression level of tight junction protein on endothelial cells. SPSS 20.0 software was used to analyze the data. Results: Compared with the young group, the ABR threshold of the aging group was significantly increased, the latency of wave I was prolonged (t=10.25, P<0.01;t=5.61, P<0.05), the permeability of the cochlear blood labyrinth barrier was increased and the expression of tight junction protein on the vascular stria was decreased (P<0.05). The cochlear ultrastructure showed that the cochlear vascular stria microvascular lumen was deformed, the basement membrane thickened and the tight junction gap between endothelium enlarged. The positive rate of ECs and PCs in primary culture was more than 95%. The cells induced by 15 g/L D-gal were determined to be senescent cells. Compared with EC group, the expression of tight junction protein in endothelial cells of D-gal+EC group decreased（t=7.42，P<0.01；t=13.19，P<0.05）and the permeability increased (t=11.17, P<0.01). In the co-culture group, the expression of tight junction protein between endothelial cells in EC+PC co-culture group and D-gal+EC+PC co-culture group increased and the permeability decreased. Conclusions: In aging mice, the permeability of cochlear blood labyrinth barrier will increase and the level of tight junction protein will decrease; in aging state, cochlear vascular stria microvascular pericytes may affect endothelial cell permeability by regulating the expression of tight junction protein.

The Effects of Riluzole on Cisplatin-induced Ototoxicity

Abstract Introduction Riluzole (2-amino-6-trifluoromethoxy benzothiazole) is known as a neuroprotective, antioxidant, antiapoptotic agent. It may have beneficial effects on neuronal cell death due to cisplatin-induced ototoxicity. Objective To evaluate the effect of riluzole on cisplatin-induced ototoxicity in guinea pigs. Methods Twenty-four guinea pigs, studied in three groups, underwent auditory brainstem response evaluation using click and 8 kHz tone burst stimuli. Subsequently, 5 mg/kg of cisplatin were administered to all animals for 3 days intraperitoneally (i.p.) to induce ototoxicity. Half an hour prior to cisplatin, groups 1, 2 and 3 received 2 ml of saline i.p., 6 mg/kg of riluzole hydrochloride i.p., and 8 mg/kg of riluzole hydrochloride i.p., respectively, for 3 days. The auditory brainstem responses were repeated 24 hours after the last drug administration. The cochleae were analyzed by transmission electron microscopy (TEM). Results After drug administiration, for 8,000 Hz stimulus, group 1 had significantly higher threshold shifts when compared with groups 2 (p < 0.05) and 3 (p < 0.05), and there was no significant difference in threshold shifts between groups 2 and 3 (p > 0.05). Transmission electron microscopy findings demonstrated the protective effect of riluzole on the hair cells and the stria vascularis, especially in the group treated with 8 mg/kg of riluzole hydrochloride. Conclusion We can say that riluzolemay have a protective effect on cisplatin- induced ototoxicity. However, additional studies are needed to confirm these results and the mechanisms of action of riluzole.

Age-related expression of plasma membrane Ca(2+) -ATPase isoform 2 in the cochleas of C57BL/6J mice / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To investigate the location and distribution of plasma membrane Ca²⁺ -ATPase isoform 2(PMCA2) in the cochleas of C57BL/6J mice at various ages (4w, 14w, 22w, 45w), and to reveal the relationship of PMCA2 and age-related hearing loss (AHL).</p><p><b>METHODS</b>The distribution of PMCA2 in the cochleas of C57BL/6J mice was detected by immunohistochemistry at various ages (4w, 14w, 22w, 45w). Real-time polymerase chain reaction (Rt-PCR) was used to detect the level of PMCA2 mRNA in the cochleas of C57BL/6J mice at the ages of 4, 14, 22 and 45 weeks old respectively. Using SPSS17.0 software for statistical analysis.</p><p><b>RESULTS</b>PMCA2 was mainly located in the hear cells, stria vascularis, and spiral ganglion cells. Faint labeling of PMCA2 was also observed in spiral ligament. Hair cells missed and the number of spiral ganglion cells reduced with age. Expression of PMCA2 in the cochleas of C57BL/6J mice also showed age-related decreasing. The results of Rt-PCR demonstrated the expression of mRNA of gene (Atp2b2) at 14 weeks age was significantly less than 4 week-old mice cochlears (P<0.05). The expression of mRNA of gene (Atp2b2) at 22 weeks age was significantly less than 14 week-old mice cochlears (P<0.05). The expression of mRNA of gene (Atp2b2) at 45 weeks age was significantly less than 14 week-old mice cochlears (P<0.01).</p><p><b>CONCLUSIONS</b>PMCA2 is mainly located in the hear cells, stria vascularis, and spiral ganglion cells. Faint labeling of PMCA2 is also observed in spiral ligament. The expression of PMCA2 demonstrates an age-related decrease with age. The mRNA expression level of PMCA2 gene(Atp2b2) in the cochleas of C57BL/6J mice displayed an age-related decrease. PMCA2 transporters may play a critical role in maintaining the normal morphology of the inner ear and it may be related to AHL.</p>

Effects of intratympanic injection of dexamethasone on endolymphatic hydrops and changes in guinea pigs inner ear CFTR expression / 临床耳鼻咽喉头颈外科杂志

OBJECTIVE@#To observe the effect of tympanic injection of dexamethasone in the guinea pig endolymphatic hydrops and the change CFTR expression, to explore the effect of glucocorticoid treatment endolymphatic and its possible mechanism.@*METHOD@#Thirty guinea pigs were divided into three groups: hormone group, water group, control group. The animals(hormone group, water group) in study were injected DDAVP 4 μg/kg in the first 7 d, and will increase to 6 μg/kg in the second 3 d. The control group was given normal saline, continuous 10 d. After twelfth days, the hormone group transtympanic injection of dexamethasone (5 mg/ml, 0.5 ml), and water group, control group transtympanic given normal saline (0.5 ml), continuous injection 5 d. Using immuno- histochemistry and Western blot to detect the cystic fibrosis transmembrane conductance regulator cochlear factor (CFTR) expression.@*RESULT@#The water group ABR thresholds was significantly higher than that before the experiment (P < 0.01), and the water group was significantly higher than the rest of the groups (P < 0.01); Hormone group compared with the control group increased threshold value (P < 0.05). The control group had no endolym- phatic hydrops, the water group showed varying degrees of endolymphatic hydrops, cochlear duct and vestibular plus cochlear duct area ratio compared with the control group, hormone group was significantly higher (P < 0.01). hormone group area ratio was higher than the control group (P < 0.05). CFTR was primarily expressed in the stria vascularis, Corti's, spiral ligament, basilar membrane, cochlear ganglion,etc . The expression of CFTR in the water group was increased than that in the control group, and the hormone group (P < 0.01); the expression of hormone group increased compared with the control group (P < 0.05).@*CONCLUSION@#Tympanic injection of dexa- methasone can alleviate the desmopressin acetatein guinea pigs caused by membranous labyrinth, and the improve- ment of the hearing; Tympanic injection of dexamethasone can make the endolymphatic hydrops cochlea of guinea pigs decreased CFTR expression, indicating that the expression and possible mechanisms of CFTR intratympanic steroids reduce endolymphatic hydrops changes.

Expression Changes of Endothelin-1, Endothelin Receptor A and B in the Cochlea after Noise Exposure / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: There are several evidences of reduced cochlea blood flow after noise exposure in the cochlea. However, the pathophysiology of blood flow change is still obscure, and endothelins, proteins that constrict blood vessels and play a key role in vascular homeostasis using its receptors may have importance in this respect. In this study, we investigated the expression changes of endothelin-1 (ET-1), endothelin receptor A (ETAR) and B (ETBR) according to auditory threshold change after noise exposure. MATERIALS AND METHOD: Mice were exposed to different noise to generate transient (group 2) and permanent threshold shift (group 3), respectively. Auditory threshold shifts were evaluated with auditory brainstem response and expression changes of ET-1, ETAR and ETBR after noise exposure were evaluated by immunohistochemistry and real time RT-PCR. RESULTS: After noise exposure, the increased ET-1, ETAR and ETBR immunoreactivities were observe in stria vascularis, spiral ligament and spiral ganglion neuron. ET-1 mRNA expressions increased after noise exposure in both group 2 and group 3 compared to those of the control group. At 2 weeks after noise exposure, however, the ET-1 mRNA expressions in group 3 increased compared to that of the control but decreased compared to that of group 2. On the other hand, ETAR mRNA expression increased at 2 weeks after noise exposure in both groups, just after noise exposure in group 2 and at 2 weeks after noise exposure in group 3. CONCLUSION: These results suggest that expression changes of ET-1, ETAR and ETBR might be associated with hearing threshold shift and recovery after noise exposure in the cochlea.

Effect of piperphentonamine hydrochloride on expressions of interleukin-1β and TNF-α mRNA and Fas protein in guinea pigs with cochlear ischemia/reperfusion injury / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the relationship between IL-1β and TNF-α mRNA and Fas protein expressions and cochlear ischemia reperfusion injury and investigate the protective mechanism of PPTA against cochlear reperfusion injury.</p><p><b>METHODS</b>Sixty-four guinea pigs were randomly divided into normal control group, blank control group, ischemia/reperfusion (by clamping the bilateral vertebral artery and right common carotid artery for 1 h) control group, and ischemia/reperfusion with PPTA treatment group. In PPTA group, PPTA was injected via the femoral vein immediately after reperfusion, and ischemia/reperfusion control group received saline injection. In 6 guinea pigs from each group, the cochlear tissues were removed after 24 h of reperfusion for examination of expressions of IL-1β and TNF-α mRNA by real-time PCR, and the rest animals were used for immunohistochemical detection of Fas protein.</p><p><b>RESULTS</b>Compared with those of normal group and blank control group, the expressions of IL-1β and TNF-β mRNA increased significantly after cochlear ischemia/reperfusion (P<0.001), but were lowered significantly by PPTA (P<0.001). Positive expression of Fas protein expression was detected in the Corti organ, spiral ganglion and stria vascularis in ischemia/reperfusion control group with significantly higher IOD values than those of the other 3 groups (P<0.05). The IOD value showed no significant difference between PPTA-treated group, normal control group, and blank control group (P>0.05).</p><p><b>CONCLUSIONS</b>PPTA can suppress the expression of Fas protein and IL-1β and TNF-β mRNAs in the cochlea of guinea pigs with cochlear ischemia/reperfusion. The protective effect of PPTA against cochlear ischemia/reperfusion is mediated probably by inhibition of inflammatory responses and cell apoptosis.</p>

Mechanism of ATP release from cultured marginal cells of stria vascularis in neonatal rat / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To further confirm release of adenosine triphosphate (ATP) from cultured marginal cells in vitro of stria vascularis in neonatal rat, and to explore the mechanism of ATP release from marginal cells.</p><p><b>METHODS</b>Isolation and in vitro culture of marginal cells of neonatal rats' cochlea. ATP released by marginal cells in extracellular fluid were detected using bioluminescence assay when add regain separately as follow: bafilomycin A(1), didecyl adipate (DDA), extracellular K(+), thapsigargin, extracellular Ca(2+), U73122, and aristolochic acid.</p><p><b>RESULTS</b>The concentrations of ATP in the extracellular fluid significantly and gradually decreased along with increasing concentrations of bafilomycin A(1). The concentrations of ATP in the extracellular fluid were in linear increased with DDA was added to marginal cell suspensions. ATP concentrations increased as the concentration of extracellular K(+) was increased, and reached the peak with a K(+) concentration of 9.15 mmol/L. At higher K(+) concentrations, ATP concentrations decreased. With the addition of increasing concentrations of thapsigargin to test marginal cells, ATP concentrations were significantly decreased. When extracellular Ca(2+) was completely chelated, marginal cells continued to release ATP. Moreover, as extracellular Ca(2+)increased, the release of ATP decreased. However, the amount of ATP releas remained to a baseline when extracellular concentration of Ca(2+) reached 1.25 mmol/L or above. When concentrations of U73122 remained within the range of 0.25 to 1.25 µmol/L, as U73122 increased, the release of ATP decreased. When concentrations of aristolochic acid ranging from 12.5 to 100.0 µmol/L were added to the marginal cells suspension, the release of ATP was significantly decreased. However, with concentrations of aristolochic acid less than 100.0 µmol/L, the release of ATP tended to be not significantly different from the amount of ATP released by control group.</p><p><b>CONCLUSIONS</b>ATP could be release from marginal cells cultured in vitro of vascular stria in neonatal rats. ATP release from marginal cells has relevant with calcium pump, K(+) channel state and signaling pathway related enzymes.</p>

The recombinant adeno-associated viruses of the serotypes 2 mediated overexpression of MnSOD protects against oxidation-induced apoptosis in stria marginal cells of rat cochlea / 临床耳鼻咽喉头颈外科杂志

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.

Evidence for the existence of ATP in the marginal cells of the neonatal rat cochlea / 临床耳鼻咽喉头颈外科杂志

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.

Expression and Distribution of Tumor Necrosis Factor-Alpha in Mice Cochlea Exposed to Noise / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: Recent studies have shown that inflammatory responses occur in the inner ear under various damaging conditions including noise-overstimulation.Identification of time-dependent expression patterns of pro-inflammatory cytokines during the response initiation should lead to rational therapeutic strategies that block the response and reduce the damaging sequelae. MATERIALS AND METHOD: We evaluated the time-dependent expression pattern of pro-inflammatory cytokines in noise-exposed mouse cochlea (white noise, 120 dB SPL, 3 hours) using immunohistochemistry and reverse transcriptase-polymerase chain reaction(RT-PCR). RESULTS: The most potent pro-inflammatory cytokine, the tumor necrosis factor-alpha (TNF-alpha), was up-regulated after noise exposure. Immunohistochemical analyses showed that the TNF-alpha expression was distinctively induced within the spiral ganglion and stria vascularis. RT-PCR showed that TNF-alpha was induced shortly after noise exposure and persisted upto seven days following noise exposure. CONCLUSION: Taken together, acoustic trauma induces cochlear inflammation and the data suggest that TNF-alpha may have some role in cochlea damage that occur following noise exposure.

Expression of Insulin-like Growth Factors in a Mouse Model of Salicylate Ototoxicity

OBJECTIVES: Insulin-like growth factors (IGFs) are known to be neurotrophic factors, and they efficiently signal to cells to grow, differentiate and survive. The purpose of study was to identify the expressions of IGFs in mice with salicylate ototoxicity, which is a typical reversible hearing loss model. METHODS: The mice were given intraperitoneal injections of salicylate (400 mg/kg) and about a 30 dB threshold shift was achieved at 3 hours. The expressions of IGF-1 and 2 were confirmed by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blotting. Localization of IGFs was confirmed using confocal immunofluorescence imaging. For in-vitro study on the HEI-OC1 auditory cells, the cell viability was calculated and the apoptotic features of the nuclei were observed with Hoechst staining. RESULTS: The expressions of the IGFs mRNA and protein were significantly increased in the salicylate ototoxicity groups compared with that of the normal control group. Salicylate induced apoptosis and decreased viability of the HEI-OC1 auditory cells in a time- and dose-dependent manner. The expressions of IGFs were localized in the stria vascularis, and these IGFs play a protective role in the in-vivo condition of salicylate ototoxicity. CONCLUSION: IGFs were highly expressed in the mice with salicylate ototoxicity, and this expression was mainly focused in the stria vascularis in the salicylate intoxicated mice. The systemic action of IGFs, which were expressed in the vascular-rich stria vascularis, can act as a major protective mechanism in a mouse model of salicylate ototoxicity.

Comparison of Cochlear Morphology and Apoptosis in Mouse Models of Presbycusis

OBJECTIVES: Morphological studies on presbycusis, or age-related hearing loss, have been performed in several different strains of mice that demonstrate hearing loss with auditory pathology. The C57BL/6 (C57) mouse is a known model of early onset presbycusis, while the CBA mouse is characterized by relatively late onset hearing loss. We performed this study to further understand how early onset hearing loss is related with the aging process of the cochlea. METHODS: We compared C57 cochlear pathology and its accompanying apoptotic processes to those in CBA mice. Hearing thresholds and outer hair cell functions have been evaluated by auditory brainstem response (ABR) recordings and distortion product otoacoustic emission (DPOAE). RESULTS: ABR recordings and DPOAE studies demonstrated high frequency hearing loss in C57 mice at P3mo of age. Cochlear morphologic studies of P1mo C57 and CBA mice did not show differences in the organ of Corti, spiral ganglion, or stria vascularis. However, from P3mo and onwards, a predominant early outer hair cell degeneration at the basal turn of the cochlea in C57 mice without definitive degeneration of spiral ganglion cells and stria vascularis/spiral ligament, compared with CBA mice, was observed. Additionally, apoptotic processes in the C57 mice also demonstrated an earlier progression. CONCLUSION: These data suggest that the C57 mouse could be an excellent animal model for early onset 'sensory' presbycusis in their young age until P6mo. Further studies to investigate the intrinsic or extrinsic etiologic factors that lead to the early degeneration of organ of Corti, especially in the high frequency region, in C57 mice may provide a possible pathological mechanism of early onset hearing loss.

Expression of Aquaporin-2 in the Inner Ear of Water-Deprived Guinea Pigs / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: Although the role of aquaporin-2 (AQP2) in the kidney has been well defined, its role in the inner ear remains to be determined. The present study was to investigate the effect of water deprivation on the expression of AQP2 in the inner ear. MATERIALS AND METHOD: Healthy male guinea pigs weighing 250 g were used. The experimental group underwent water restriction and the control underwent water loading with sucrose-containing water for 3 days. Concentrations of plasma arginine-vasopressin (AVP) were determined and electrocochleography (ECoG) recordings were made. An RT-PCR, real-time PCR and Westernblotting analysis were used for quantitative analysis of AQP2 mRNA and AQP2 protein expression. Immunohistochemistry was also used to evaluate the distribution of AQP2 water channel proteins in the inner ear. RESULTS: AQP2 was mainly expressed in the epithelium of endolymphatic sac, spiral limbus, spiral ligament and stria vascularis of scala media. The concentrations of plasma AVP were 9.2+/- 0.8 pg/mL in the experimental group and 0.78+/-0.3 pg/mL in the control. The summation potential/ action potential (SP/AP) ratio in ECoG was markedly increased in the experimental group (0.55 in the experimental and 0.29 in the control). RT-PCR and real time PCR as well as Western blot analysis showed that the level of AQP2 mRNA and protein in the cochlea and endolymphactic sac of the water-deprived group was significantly higher than those in the control group. CONCLUSION: These data suggest that AQP2 is one of the important water channels in fluid homeostasis in the inner ear. Moreover, the volume of endolymphatic space can be increased via AVP-AQP2 system in response to water deprivation.

Oxidative stress experimental model of rat with stria vascularis marginal cells injury induced by hydrogen peroxide in vitro / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To set up the oxidative stress experimental model of rat cochlea with stria vascularis marginal cells injury induced by hydrogen peroxide in vitro.</p><p><b>METHODS</b>Cultured marginal cells of rat were treated by 200, 300, 400, 600 and 800 micromol/L hydrogen peroxide (H(2)O(2)) for 0.5, 1, 2, 4, 16 and 24 hours, respectively. Cell viability was assessed by the CCK-8 assay. The content of the lipid peroxidation production malondialdehyde (MDA) were detected in H(2)O(2) induced marginal cells injury with different concentration H(2)O(2). Apoptosis was assessed by flow cytometry by propidium sodium staining. The expression of the cleaved-caspase-3 was assessed by Western blot.</p><p><b>RESULTS</b>Being exposed to H(2)O(2), marginal cells displayed nuclear pyknosis and margination, cytoplasmic condensation, cell shrinkage and formation of membrane and bounded apoptotic bodies. A time-dependent and dose-dependent decrease of cellular viability was detected with the treatment of H(2)O(2). Cellular maleic dialdehyde was generated in proportion to the concentration of H(2)O(2) at 2 hours and the number of apoptotic cells increased significantly (P < 0. 05). Western blot showed the expression of the cleaved-caspase-3 increased when 200 micromol/L, 300 micromol/L and 400 micromol/L H(2)O(2) treated cultured marginal cells. Thereafter the expression of the cleaved-caspase-3 decreased with 600 micromol/L H(2)O(2) and with 800 micromol/L H(2)O(2) the expression of cleaved-caspase-3 was weak.</p><p><b>CONCLUSIONS</b>The findings indicated that the experimental model can be established successfully using cultured cells exposed to H(2)O(2) and activation of caspase-3 is associated with hydrogen peroxide induced rat marginal cells the oxidative stress injury.</p>

Expression Patterns of KCNJ10 K+ Channel in the Cochlear Lateral Wall After Acoustic Trauma / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: It is well known that noise exposure leads to the sensory hair cell loss and other neuronal damage in the cochlea. But recently it has been reported that noise exposure could also damage lateral wall of cochlea such as stria vascularis and spiral ligament. K+ is the major cation in endolymph and important to maintain homeostasis within the cochlea. We have investigated the expression patterns of KCNJ10 K+ channel in noise induced cochlear damage. MATERIALS AND METHOD: Twenty adult male guinea pigs (300-350 g) were included in this study. In experimental group (n=16), acoustic trauma was induced by continuous broad band noise for 2 hr to 115 dB SPL and broad band noise for 6 hr to 120 dB SPL with 3 consecutive days. After noise exposure, auditory brainstem response threshold shift and hair cell loss were evaluated. A study for KCNJ10 K+ channel expression was examined by immunohistochemical staining. RESULTS: After noise exposure, auditory brainstem response showed transient threshold shift (TTS) and permanent threshold shift (PTS) in accordance with noise exposure. The expression patterns of CKNJ10 K+ channel were changeable in TTS group. But there were no change of expression patterns in PTS group. CONCLUSION: In the cochlear lateral wall, KCNJ10 K+ channel expressions were affected with noise exposure and these changes might be associated with the regulation of homeostasis in the cochlea lateral wall.

Regional Distribution of 5-HT 1A, 1B, and 1D Receptors in Rat Vestibular Nuclei (Vn) and Inner Ear

Migraine and anxiety disorders are frequently co-morbid with balance disorders. Potential mechanisms for migrainous vertigo include sites of action of 5-HT (serotonin) 1B and 1D receptor agonists such as rizatriptan, which attenuate motion sickness in migraineurs. Selective serotonin reuptake inhibitors (SSRIs) are also known to be efficacious in the treatment of vertigo. Relative distribution of the 5-HT receptor subtypes and their functional roles in the vestibular nuclei and inner ear is still unknown. Using 5-HT1A, 1B, AND 1D receptors-specific antibody, we have demonstrated a differential distribution of these receptor subtypes within the rat vestibular nuclei and inner ear. For 5-HT receptor subtypes expression in the vestibular and auditory periphery, most ganglion cells in the vestibular ganglion showed immunoreactivity for 5-HT1A, 5-HT1B and 5-HT1D receptors. In addition, 5-HT1B and 1D receptors immunopositive reactivities were associated with endothelial cells of small blood vessels in the vestibular ganglion and nerve, endothelial cells in both the spiral ligament deep to the spiral prominence and stria vascularis and endothelial cells on blood vessels along the margins of the spiral ganglion. For 5-HT receptor subtypes expression in the vestibular nuclei (VN), the 5-HT1A, 1B and 1D receptors were expressed differentially in the VN. Fine varicose axons in the periventricular plexus showed intense 5-HT1A receptor expression in the medial VN (MVN) and extended into the superior VN (SVN). By contrast, 5-HT1B receptors were not expressed the ventricular plexus axons. Rather, 5-HT1B and 1D receptors immunopositive cell bodies and neuronal processes were dense in rostral MVN, dorsal SVN, lateral VN (LVN) and ventral aspect of nucleus prepositus hypoglossi (NPH). In the present study, inner ear and vestibular nuclei showed distinct distributions of 5- HT1A, 1B and 1D receptors expressions that are parallel to their distribution in peripheral and central nociceptive pathways. These differentially distributed 5-HT receptor subtypes are potential targets to explain the efficacy of SSRIs and triptans in treating migraine and migrainous vertigo.

Establishment of marginal cell culture from stria vascularis explants of adult rat / 临床耳鼻咽喉头颈外科杂志

OBJECTIVE@#To establish a cell culture system of marginal cells(MCs) of the adult rat cochlea stria vascularis.@*METHOD@#The cochlea strial vascularised were isolated under a dissecting microscopy as explants, then culture in vitro to get the monolayer epithelial cells. Immunocytochemistry method and transmission electron microscopy could be used to identify the origin and characterization of the cultured cells. RT-PCR was used to detect the expression of IsK, an unique marker of MCs.@*RESULT@#When grown on plastic dishes, cultured MCs showed a polygonal "cobblestone-like" appearance and dome formation, composed of several hundreds to thousands of cells. The characterized microvilli on the cultured MCs surface could be seen under the TEM. For immunochemistry, over 95% of the cultured cells showed positive reaction to cytokeratin antigen. RT-PCR detected the expression of the mRNA of IsK protein in the cultured cells.@*CONCLUSION@#The results show that a cell culture system of cochlear strial marginal cells of adult rat has been successfully established which can provide a stable source of MCs for ongoing physiological, biochemical and molecular characterization.

Construction of recombinant plasmid pEGFP-N1/MnSOD and its express in vitro / 临床耳鼻咽喉头颈外科杂志

OBJECTIVE@#To construct eukaryotic expression plasmid of rat Mn-superoxide dismutase (MnSOD) gene pEGFP-N1/MnSOD and express it in primary culture system of marginal cells (MC) of the rats.@*METHOD@#The aimed segments were obtained from rat myocardial tissue and were inserted into a eukaryotic expression plasmid pEGFP-N1/MnSOD. The recombinant expression plasmid pEGFP-N1/MnSOD was transfected into MC by lipofectamine 2000-mediated gene transfer method, which were observed through co-Focus Fluorescence microscopy. The percentage of transfection was determined by fluorescence activated call sorting (FACS) analysis.@*RESULT@#Rat MnSOD cDNA eukaryotic expression plasmid pEGFP-N1/MnSOD have been successfully constructed. Green fluorescent protein(GFP) and MnSOD protein could be contacted in the transfected MC 48 hours after transfection. The percentage of transfection was 23.47%.@*CONCLUSION@#Rat MnSOD cDNA eukaryotic expression plasmid pEGFP-N1/MnSOD have been successfully constructed and expressed successfully in MC. The research paved the way for antioxidant gene therapy of inner ear.