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.
Mice ; Animals ; Transcriptome ; Aging/metabolism* ; Cochlea ; Stria Vascularis ; Presbycusis

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.
Animals ; Cochlea ; Endothelial Cells ; Mice ; Mice, Inbred C57BL ; Pericytes ; Permeability ; Stria Vascularis ; Tight Junctions

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.
Aging ; Animals ; Cochlea ; Endothelial Cells ; Large-Conductance Calcium-Activated Potassium Channels ; Mice ; Mice, Inbred C57BL ; Pericytes ; Stria Vascularis

Cochlea ; Humans ; Macrophages ; Stria Vascularis

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.
Animals ; Cochlea ; Cystic Fibrosis Transmembrane Conductance Regulator ; biosynthesis ; Dexamethasone ; pharmacology ; Ear, Inner ; Endolymphatic Hydrops ; drug therapy ; metabolism ; Glucocorticoids ; pharmacology ; Guinea Pigs ; Stria Vascularis

OBJECTIVETo 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.

METHODSSixty-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.

RESULTSCompared 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).

CONCLUSIONSPPTA 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.

3,4-Methylenedioxyamphetamine ; analogs & derivatives ; pharmacology ; Animals ; Cochlea ; blood supply ; metabolism ; pathology ; Female ; Guinea Pigs ; Interleukin-1beta ; genetics ; metabolism ; Male ; Neuroprotective Agents ; pharmacology ; Organ of Corti ; metabolism ; RNA, Messenger ; metabolism ; Random Allocation ; Reperfusion Injury ; metabolism ; Spiral Ganglion ; metabolism ; Stria Vascularis ; metabolism ; Tumor Necrosis Factor-alpha ; genetics ; metabolism ; fas Receptor ; metabolism

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.
Animals ; Auditory Threshold ; Blood Vessels ; Cochlea ; Endothelin-1 ; Endothelins ; Evoked Potentials, Auditory, Brain Stem ; Hand ; Hearing ; Homeostasis ; Immunohistochemistry ; Mice ; Neurons ; Noise ; Proteins ; Receptors, Endothelin ; RNA, Messenger ; Spiral Ganglion ; Spiral Ligament of Cochlea ; Stria Vascularis

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

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.
Aging ; Animals ; Apoptosis ; Cochlea ; Evoked Potentials, Auditory, Brain Stem ; Hair ; Hearing ; Hearing Loss ; Ligaments ; Mice ; Mice, Inbred CBA ; Models, Animal ; Organ of Corti ; Presbycusis ; Spiral Ganglion ; Stria Vascularis

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.
Animals ; Apoptosis ; Blotting, Western ; Cell Survival ; Cochlea ; Fluorescent Antibody Technique ; Hearing Loss ; Injections, Intraperitoneal ; Insulin-Like Growth Factor I ; Mice ; Nerve Growth Factors ; Polymerase Chain Reaction ; Reverse Transcription ; RNA, Messenger ; Somatomedins ; Stria Vascularis