No abstract available.
Stria Vascularis*

Cochlea ; Humans ; Macrophages ; Stria Vascularis

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 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.
Animals ; Cell Culture Techniques ; methods ; Cell Division ; Female ; Rats ; Rats, Wistar ; Stria Vascularis ; cytology

The endolymphatic secretory epithelium are stria vascularis in cochlear and dark cell in vestibule which are regulated by Na+-K+ ATPase. It is important that we study intracytoplasmic Na+-K+ ATPase for the physiologic research of inner ear. Recently cerium-based method for stain of Na+-K+ ATPase was developed. This study was underkaken to investigate the morphologic changes and Na+-K+ ATPase activity in stria vascularis and vestibular dark cell of mongolian gerbil after systemic intramuscular injection(200mg/kg or 300mg/kg) for 7days or local infiltration of streptomycin through round window. The results are as follows. 1) The strong Na+-K+ ATPase activity was seen at basolateral infoldings of marginal cell in stria vascularis but weak Na+-K+ ATPase activity in dark cell near transitional area. 2) There was no change of Na+-K+ ATPase activity in the stria vascularis and dark cell by systemic injection of streptomycin. The decrease of Na+-K+ ATPase activity in stria vascularis was seen at destruction site of infoldings by local infiltration of streptomycin but no changes in dark cell. 3) The ultrastructural changes of marginal cell by local infiltration of streptomycin were intracytoplasmic vacuole, partial loss of cytoplasmic infoldings, edema, and increase of melanin particle. but, there was no change of ultrastructure in dark cell except increase of melanin particle. The changes of ultrastructure of stria vascularis was variable by systemic streptomycin injection and there was no dark cell change except increased melanin particle. From the above results, the changes of ultrastructure and Na+-K+ ATPase were more severe by local infiltration of streptomycin through round window than systemic injection of streptomycin. The local infiltration of streptomycin through round window may be suitable method for the induction of inner ear damage.
Adenosine Triphosphatases* ; Cytoplasm ; Ear, Inner ; Edema ; Epithelium* ; Gerbillinae ; Melanins ; Streptomycin* ; Stria Vascularis ; Vacuoles

BACKGROUND AND OBJECTIVES: Cisplatin is frequently used in the treatment of various forms of malignancies. It's therapeutic efficacy, however, is limited due to the occurrence of sensorineural hearing loss. Little is known about the course of hearing loss after cessation of cisplatin administration. We observed the cochlear duct morphology with normal and cisplatin treated animals. MATERIALS AND METHODS: Healthy rats (strain Sprague-Dawly, weighting 80-100 mg) were used for all experiments. Total 15 rats were selected. They were divided into two groups, a treated group and a control. Four survival groups (n=3, respectively) were assigned as the treated group. After treatment with cisplatin, each survival group was sacrificed 1, 4, 10, 20 days. Except for the normal control (n=3), twelve animals were treated with cisplatin by daily I.P. injection of 1.5 mg/kg for 8 consecutive days. RESULTS: One day after cessation of cisplatin administration, outer hair cells (OHCs) loss and stria vascularis were degenerated especially in the basal turn. At 4, 10, 20 days, the OHCs and stria vascularis morphology of the survival group were similar to those of the survival group at one day. CONCLUSION: Our findings suggest cisplatin ototoxicity is predominantly involved in the basal turn of cochlear duct. This finding was significantly correlated with high frequency hearing loss of cisplatin ototoxicity.
Animals ; Cisplatin* ; Cochlear Duct ; Hair ; Hearing Loss ; Hearing Loss, Sensorineural ; Rats ; Stria Vascularis

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

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.
Animals ; Gene Expression ; Genetic Vectors ; Plasmids ; Rats ; Rats, Sprague-Dawley ; Stria Vascularis ; cytology ; Superoxide Dismutase ; genetics ; Transfection

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