Search

The ototoxic mechanisms of cisplatin on the organ of Corti and spiral ganglion neurons have been extensively studied, while few studies have been focused on the stria vascularis (SV). Herein, we verified the functional and morphological impairment in SV induced by a single injection of cisplatin (12 mg/kg, I.P.), represented by a reduction in Endocochlear Potentials (EP) and strial atrophy, and explored underlying mechanisms. Our results revealed increased extravasation of chromatic tracers (Evans blue dye and FITC-dextran) around microvessels after cisplatin exposure. The increased vascular permeability could be attributed to changes of pericytes (PCs) and perivascular-resident macrophage-like melanocytes (PVM/Ms) in number or morphology, as well as the enhanced level of HIF-1α and downstream VEGF. This capillary leakage led to a high accumulation of cisplatin in the perivascular space in SV, and disrupted the integrity of blood-labyrinth barrier (BLB). Also, tight junction (ZO-1) loosening and Na + , K + -ATPase damage was considered to be other critical contributors of BLB breakdown, which resulted in EP drop and consequent hearing loss. This study explored the role of stria vascularis in cisplatin-induced ototoxicity in terms of BLB hyperpermeability and pointed to a novel therapeutic target for the prevention of cisplatin-related hearing loss., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

It is widely accepted that the stria vascularis (SV) in cochlea plays a critical role in the generation of endocochlear potential (EP) and the secretion of the endolymph. 17β-estradiol (E2) is the most potent and abundant endogenous estrogen during the premenopausal period, thus, considered as the reference estrogen. This study aimd to investigate the protective effect of E2 by promoting the expression of vascular endothelial growth factor (VEGF) and thus promoting the vascular regeneration of the SV in elderly mice. After being treated with E2 either in vivo or in vitro, the hearing threshold changes of C57BL/6J elder mice continuously reduced, endothelial cell morphology improved, the number of endothelial cells (ECs) tubular nodes increased significantly, the ability of tubular formation enhanced significantly and the expression of VEGF increased. In vitro, cell model in conjunction with in vivo ovariectomized model was established to demonstrate for the first time that E2 promotes angiogenesis by promoting the secretion of VEGF through the phosphatidylinositol 3-kinase (PI3K)/AKT pathway (PI3K/AKT). In conclusion, E2 demonstrated potent angiogenesis properties with significant protection against Age-Related Hearing Loss (ARHL), which provides a new idea for the improvement of ARHL., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

In 129 Sv autosomal Alport mice, the strial capillary basement membranes (SCBMs) progressively thicken between 5 and 9 weeks of age resulting in a hypoxic microenvironment with metabolic stress and induction of pro-inflammatory cytokines and chemokines. These events occur concomitant with a drop in endocochlear potential and a susceptibility to noise-induced hearing loss under conditions that do not permanently affect age/strain-matched littermates. Here we aimed to gain an understanding of events that occur before the onset of SCBM thickening. Alport stria has normal thickness and shows levels of extracellular matrix (ECM) molecules in the SCBMs commensurate with wild-type mice. Hearing thresholds in the 3-week Alport mice do not differ from those of wild-type mice. We performed RNAseq analysis using RNA from stria vascularis isolated from 3-week Alport mice and wild type littermates. Data was processed using Ingenuity Pathway Analysis software and further distilled using manual procedures. RNAseq analysis revealed significant dysregulation of genes involved in cell adhesion, cell migration, formation of protrusions, and both actin and tubulin cytoskeletal dynamics. Overall, the data suggested changes in the cellular architecture of the stria might be apparent. To test this notion, we performed dual immunofluorescence analysis on whole mounts of the stria vascularis from these same animals stained with anti-isolectin gs-ib4 (endothelial cell marker) and anti-desmin (pericyte marker) antibodies. The results showed evidence of pericyte detachment and migration as well as the formation of membrane ruffling on pericytes in z-stacked confocal images from Alport mice compared to wild type littermates. This was confirmed by TEM analysis. Earlier work from our lab showed that endothelin A receptor blockade prevents SCBM thickening and ECM accumulation in the SCBMs. Treating cultured pericytes with endothelin-1 induced actin cytoskeletal rearrangement, increasing the ratio of filamentous to globular actin. Collectively, these findings suggest that the change in type IV collagen composition in the Alport SCBMs results in cellular insult to the pericyte compartment, activating detachment and altered cytoskeletal dynamics. These events precede SCBM thickening and hearing loss in Alport mice, and thus constitute the earliest event so far recognized in Alport strial pathology., Competing Interests: Declaration of competing interest The authors of this work have no conflicts of interest to disclose., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Objective The endocytosis of cationized feritin (CF) via a clathrin-mediated pathway is regulated by a signaling network. Marginal cells showed the active endocytosis of CF via a clathrin-mediated pathway. The internalization of receptors through this clathrin-mediated pathway is an important regulatory event in signal transduction. Numerous kinases are involved in endocytosis, and each endocytic route is subjected to high-order regulation by cellular signaling mechanisms. In this study, we investigated whether ROCK and MLCK signaling cascades and G-proteins regulate the endocytosis of CF in marginal cells of the stria vascularis. Methods CF was infused into the cochlear duct with pertussis toxin (PTX),Clostridium botulinum C3 toxin (BTX), guanosine(g-thio)-triphosphate (GTP-γS), ML-7, Y-27632. Endocytic activity was measured at 30 min after the start of infusion under an electron microscope. Results In marginal cells, CF was internalized via a clathrin-mediated pathway that depends on F-actin and microtubules (MT). Its processes were controlled by myosin light chain kinase (MLCK) and Rho-associated kinase (ROCK), but not affected by G-protein-coupled receptor (GPCR) or the RhoA signaling cascade. Conclusion Our previous study showed that the main endocytotic pathway of microperoxidase (MPO) did not depend on the Rho/ROCK molecular switch or actin/myosin motor system, but was mainly regulated by the RhoA signaling cascade. The present study results indicate that these signaling cascades regulating CF internalization completely differ from the cascades for MPO internalization., (Copyright © 2019. Published by Elsevier B.V.)

Objective: Tsumura Suzuki Obese Diabetes (TSOD) mice exhibit early age-associated hearing loss. Histopathological analysis of these mice shows narrowing of capillaries in the stria vascularis and chronic reduction of blood flow in the cochlea. In this study, we investigated the effect of oral administration of a herbal medicine or calorie restriction on hearing in TSOD mice., Methods: TSOD mice were divided into 4 groups: CR (calorie restriction), BF and DS (treated with the herbal medicines, Bofutsushosan and Daisaikoto, respectively), and the control group. Body weight, blood glucose levels, and auditory brainstem responses (ABRs) were measured. The cochleae were excised and evaluated histopathologically., Results: Blood glucose levels were suppressed in the CR, BF, and DS groups. In addition, the elevation of ABR thresholds was inhibited in the CR, BF, and DS groups. Cochlear blood vessels remained wide in the three treatment groups compared with the control group. These results suggested that the administration of these herbal medicines improved glucose tolerance and yielded results similar to those on calorie restriction., Conclusion: Oral administration of 2 herbal medicines can prevent hearing function disorder in a model mouse of diabetes. The results may clarify the possibility of clinical application., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Objectives: This study aimed to evaluate the effect of quercetin on cochlear function and morphology, and its possible protective effect against acute cisplatin-induced ototoxicity in rats., Materials and Methods: This prospective and controlled animal study was conducted on Wistar albino rats divided into four groups. Otoacoustic emission measures were performed three days after the first infiltration in Group 1 (saline), 2 (cisplatin), and 3 (quercetin). This interval was five days for Group 4 (cisplatin+quercetin). At the end of the study, the rats were decapitated with deep anesthesia, and histological changes in the cochleas were observed by light microscopy., Results: Group 2 (cisplatin) revealed significant differences between the first and second measures in all frequencies. When compared to other group, the difference of the changes in Group 2 statistically significantly decreased, especially in higher frequencies. Morphologically, there were no acute changes in Group 1 and Group 3. Outer hair cell loss and the degeneration of stria vascularis and spiral ganglion were observed in both Groups 2 and 4; the damages in the latter were lesser., Conclusion: Quercetin does not have negative effect on cochlea, and it has protective effect on cisplatin-induced ototoxicity.

The present study was designed to investigate the expression and function of transmembrane protein 16 (TMEM16A), a calcium‑activated chloride channel (CaCC), in the stria vascularis (SV) of the cochlea of guinea pigs at different ages, and to understand the role of CaCCs in the pathogenesis of presbycusis (age‑related hearing loss), the most common type of sensorineural hearing loss that occurs with natural aging. Guinea pigs were divided into the following groups: 2 weeks (young group), 3 months (youth group), 1 year (adult group), D‑galactose intervention (D‑gal group; aging model induced by subcutaneous injection of D‑galactose) and T16Ainh‑A01 (intraperitoneal injection of 50 µg/kg/day TMEM16A inhibitor T16Ainh‑A01 for 2 weeks). Differences in the hearing of guinea pigs between the various age groups were analyzed using auditory brainstem response (ABR), and immunofluorescence staining was performed to detect TMEM16A expression in the SV and determine the distribution. Reverse transcription‑quantitative PCR and western blot analyses were conducted to detect the mRNA and protein levels of TMEM16A in SV in the different age groups. Morris water maze behavior analysis demonstrated that spatial learning ability and memory were damaged in the D‑gal group. Superoxide dismutase activity and malondialdehyde content assays indicated that there was oxidative stress damage in the D‑gal group. The ABR thresholds gradually increased with age, and the increase in the T16Ainh‑A01 group was pronounced. Immunofluorescence analysis in the cochlear SV of guinea pigs in different groups revealed that expression of TMEM16A increased with increasing age (2 weeks to 1 year); fluorescence intensity was reduced in the D‑gal model of aging. As the guinea pigs continued to mature, the protein and mRNA contents of TMEM16A in the cochlea SV increased gradually, but were decreased in the D‑gal group. The findings indicated that CaCCs in the cochlear SV of guinea pigs were associated with the development of hearing in guinea pigs, and that downregulation of TMEM16A may be associated with age‑associated hearing loss.

Can damaged or degenerated vessels be regenerated in the ear? The question is clinically important, as disruption of cochlear blood flow is seen in a wide variety of hearing disorders, including in loud sound-induced hearing loss (endothelial injury), ageing-related hearing loss (lost vascular density), and genetic hearing loss (e.g., Norrie disease: strial avascularization). Progression in cochlear blood flow (CBF) pathology can parallel progression in hair cell and hearing loss. However, neither new vessel growth in the ear, nor the role of angiogenesis in hearing, have been investigated. In this study, we used an established ex vivo tissue explant model in conjunction with a matrigel matrix model to demonstrate for the first time that new vessels can be generated by activating a vascular endothelial growth factor (VEGF-A) signal. Most intriguingly, we found that the pattern of the newly formed vessels resembles the natural 'mesh pattern' of in situ strial vessels, with both lumen and expression of tight junctions. Sphigosine-1-phosphate (S1P) in synergy with VEGF-A control new vessel size and growth. Using transgenic neural/glial antigen 2 (NG2) fluorescent reporter mice, we have furthermore discovered that the progenitors of "de novo" strial vessels are NG2-derived cells. Taken together, our data demonstrates that damaged strial microvessels can be regenerated by reprogramming NG2-derived angiogenic cells. Restoration of the functional vasculature may be critical for recovery of vascular dysfunction related hearing loss., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

The study of strial pericytes has gained great interest as they are pivotal for the physiology of stria vascularis. To provide an easily accessible in vitro model, here we described a growth medium-based approach to obtain and cultivate primary bovine cochlear pericytes (BCP) from the stria vascularis of explanted bovine cochleae. We obtained high-quality pericytes in 8-10 days with a > 90% purity after the second passage. Immunocytochemical analysis showed a homogeneous population of cells expressing typical pericyte markers, such as neural/glial antigen 2 (NG2), platelet-derived growth factor receptorβ (PDGFRβ), α-smooth muscle actin (α-SMA), and negative for the endothelial marker von Willebrand factor. When challenged with tumor necrosis factor or lipopolysaccharide, BCP changed their shape, similarly to human retinal pericytes (HRPC). The sensitivity of BCP to ototoxic drugs was evaluated by challenging with cisplatin or gentamicin for 48 hr. Compared to human retinal endothelial cells and HRPC, cell viability of BCP was significantly lower ( p < 0.05) after the treatment with gentamicin or cisplatin. These data indicate that our protocol provides a simple and reliable method to obtain highly pure strial BCP. Furthermore, BCP are suitable to assess the safety profile of molecules which supposedly exert ototoxic activity, and may represent a valid alternative to in vivo tests., (© 2018 Wiley Periodicals, Inc.)

Lead exposure causes or aggravates hearing damage to human or animal, but the detailed effects of lead exposure on auditory system including injury sites of the cochlea in mammal remain controversy. To investigate the effect of chronic lead exposure on auditory system, 40 adult guinea pigs with normal hearing were randomly divided into five groups. They were fed 2 mmol/L lead acetate in drinking water for 0, 15, 30, 60, and 90 days (n = 8), respectively. Lead concentrations in blood, cochlea, and brainstem were measured. Auditory function was measured by auditory brainstem response (ABR) and distortion product otoacoustic emission (DPOAE). The morphology of cochlea and brainstem was observed, and expression of autophagy-related protein in brainstem was also assessed. The blood lead concentration reached a high level at the 15th day and kept stable, but the lead level in brainstem and cochlear tissue increased obviously at the 60th day and 90th day of lead exposure, respectively. There was no significant difference in the morphology of hair cells and stria vascularis (SV) among these five groups, but the number of spiral ganglion neuron (SGN) gradually decreased after 60 days. The differences of ABR thresholds and DPOAE amplitudes were not statistically significant among each group, but I wave latency, III latency, and I-III wave interval of ABR were delayed with the prolonging of time of lead exposure. The expressions of autophagy-related protein ATG5, ATG6, and LC3B in brainstem were increased after 30 days. These results suggest that the key target of lead toxicity was the auditory nerve conduction pathway including SGNs and brainstem, rather than cochlear hair cells and SV. Autophagy may play a very important role in lead toxicity to auditory nervous system.

Cisplatin is an anti-cancer drug that causes oxotoxic side effects such as impairment of inner ear function and hearing loss. We aimed to investigate the effects of allicin against cisplatin-induced stria vascularis damage in mice, and to clarify the mechanism underlying the protective effects of allicin against ototoxicity. Stria vascularis injury was induced in mice by intraperitoneal injection of cisplatin, which was significantly prevented by pretreatment with allicin. Allicin not only reduced cisplatin-activated expression of cleaved caspase-3 in marginal cells, PVM/Ms (perivascular resident macrophage-like melanocytes), and basal cells of the stria vascularis, but also decreased the expression of poly(ADP-ribose) polymerase-1 (PARP-1) and apoptosis-inducing factor (AIF) nuclear translocation in the stria vascularis cells. Our results demonstrate that allicin plays an effective role in protecting stria vascularis injury induced by cisplatin by inhibiting caspase-dependent, as well as caspase-independent PARP-1-AIF-mediated apoptotic pathways. Therefore, allicin may be useful in preventing cisplatin-induced ototoxicity., (© 2019 S. Karger AG, Basel.)

Cisplatin, a small platinum-containing molecule, is a widely used, highly effective anticancer drug. However, severe side effects have been found in cancer patients treated with cisplatin, including nephrotoxicity, neurotoxicity, and ototoxicity. These cisplatin-induced side effects can have a major impact on patient quality of life, including social development problems in pediatric patients that develop hearing loss. Previous studies have suggested that the major cause of cisplatin-induced ototoxicity is abnormal accumulation of reactive oxygen species (ROS) and oxidative stress. Alpha-lipoic acid (ALA), one of the most effective antioxidants, is known to be involved in the cellular antioxidant system and may have a protective effect on cisplatin-induced ototoxicity. However, the therapeutic effect of ALA on damaged hearing function and its detailed mechanism of action are not fully understood. This study focused on determining whether ALA has a potential as a protective and/or therapeutic agent for cisplatin-induced ototoxicity. Histological and physiological analyses were performed using cisplatin-treated mouse cochlea and HEI-OC1 culture cells in pre- and post-treatment with ALA in vitro and in vivo. We found that ALA contributes to protecting mitochondrial function by preventing ROS accumulation and inhibiting apoptotic cell death. Importantly, post-treatment with ALA consistently showed an almost equal restorative effect to pretreatment, in vitro and in vivo, supporting the possible use of ALA as a therapeutic agent for cisplatin-induced ototoxicity. This study is the first report on a strong therapeutic potential of ALA to rescue ototoxic hearing loss caused by cisplatin, and our data provide key evidence that ALA may act as a reducing agent for glutathione disulfide to increase glutathione levels on behalf of glutathione reductase. This result was consistent in both cultured cells and the mouse model, which improves the clinical value of ALA for therapy of cisplatin-induced ototoxicity.

The present study was designed to investigate the electrophysiological properties of strial pericytes and the effect of aspirin on pericyte K+ channels. Pericytes were identified by determining their morphological characteristics and using pericyte‑associated immunofluorescence techniques. The electrophysiological properties of strial pericytes were observed with a whole‑cell patch‑clamp technique. Alterations in the outward current of cochlear pericytes in the stria vascularis of guinea pigs were examined following the application of K+ channel retardants. The effects of aspirin on pericyte K+ channels were also evaluated with the whole‑cell patch‑clamp technique. The results demonstrated that pericytes were desmin positive, and their nuclei were large and surrounded by a small proportion of the cytoplasm. Cytoplasmic processes gradually declined in size as branches grew parallel to the capillary axis. Thus, capillaries were surrounded by tips. The electrophysiological properties of the cochlear pericytes in the stria vascularis of guinea pigs were also determined. The membrane capacitance of the pericytes was 5.9±0.3 pF, while the membrane resistance and resting potential were 2.2±0.3 GΩ and ‑30.9±1.2 mV, respectively. The current densities of the pericytes (pA/pF) were 3.2±0.7, 10.6±1.0, 15.7±0.9 and 21.3±1.2 at command voltages of 0, +20, +40, and +60 mV, respectively. The K+ channels were activated when the pericytes were within the range of ‑20 mV to +20 mV, particularly at 0 mV. The inhibition rates of the outward current of cochlear pericytes in the stria vascularis of the guinea pigs were determined by administering iberiotoxin (IBTX) and IBTX + 4‑aminopyridine. Once the background leakage current was removed, the following inhibition rates were obtained with 3, 10, 30, 300 and 1,000 µmol/l aspirin: 20.8±4.8, 34.1±6.9, 48.2±6.7, 63.6±7.1 and 65.7±8.1%, respectively. The outward current of the cochlear pericytes in the stria vascularis was inhibited by aspirin with a half maximal inhibitory concentration of 24.5±4.5 µmol/l. The membranes of the pericytes in the stria vascularis are characterized by high‑conductance calcium‑activated K+ (BKCa) and voltage‑dependent K+ (KV) channels. The outward current of the cochlear pericytes in the stria vascularis of guinea pigs was inhibited by aspirin in a concentration‑dependent manner. In addition, BKCa and KV channels were inhibited by aspirin.

The aim of this study is to investigate the effect of rectal ozone and intratympanic ozone therapy on cisplatin-induced ototoxicity in rats. Eighteen female Wistar albino rats were included in our study. External auditory canal and tympanic membrane examinations were normal in all rats. The rats were randomly divided into three groups. Initially, all the rats were tested with distortion product otoacoustic emissions (DPOAE), and emissions were measured normally. All rats were injected with 5-mg/kg/day cisplatin for 3 days intraperitoneally. Ototoxicy had developed in all rats, as confirmed with DPOAE after 1 week. Rectal and intratympanic ozone therapy group was Group 1. No treatment was administered for the rats in Group 2 as the control group. The rats in Group 3 were treated with rectal ozone. All the rats were tested with DPOAE under general anesthesia, and all were sacrificed for pathological examination 1 week after ozone administration. Their cochleas were removed. The outer hair cell damage and stria vascularis damage were examined. In the statistical analysis conducted, a statistically significant difference between Group 1 and Group 2 was observed in all frequencies according to the DPOAE test. In addition, between Group 2 and Group 3, a statistically significant difference was observed in the DPOAE test. However, a statistically significant difference was not observed between Group 1 and Group 3 according to the DPOAE test. According to histopathological scoring, the outer hair cell damage score was statistically significantly high in Group 2 compared with Group 1. In addition, the outer hair cell damage score was also statistically significantly high in Group 2 compared with Group 3. Outer hair cell damage scores were low in Group 1 and Group 3, but there was no statistically significant difference between these groups. There was no statistically significant difference between the groups in terms of stria vascularis damage score examinations. Systemic ozone gas therapy is effective in the treatment of cell damage in cisplatin-induced ototoxicity. The intratympanic administration of ozone gas does not have any additional advantage over the rectal administration.

Ototoxicity is one of the most important adverse effects of cisplatin chemotherapy. As a common treatment of acute sensorineural hearing loss, systemic administration of steroids was demonstrated ineffective against cisplatin-induced hearing loss (CIHL) in published studies. The current study aimed to evaluate the potential protective effect of dexamethasone (DEX) encapsulated in polyethyleneglycol-coated polylactic acid (PEG-PLA) nanoparticles (DEX-NPs) against cisplatin-induced hearing loss following systemic administration. DEX was fabricated into PEG-PLA nanoparticles using emulsion and evaporation technique as previously reported. DEX or DEX-NPs was administered intraperitoneally to guinea pigs 1h before cisplatin administration. Auditory brainstem response (ABR) threshold shifts were measured at four frequencies (4, 8, 16, and 24kHz) 1 day before and three days after cisplatin injection. Cochlear morphology was examined to evaluate inner ear injury induced by cisplatin exposure. A single dose of DEX-NPs 1h before cisplatin treatment resulted in a significant preservation of the functional and structural properties of the cochlea, which was equivalent to the effect of multidose (3 days) DEX injection. In contrast, no significant protective effect was observed by single dose injection of DEX. The results of histological examination of the cochleae were consistent with the functional measurements. In conclusion, a single dose DEX-NPs significantly attenuated cisplatin ototoxicity in guinea pigs after systemic administration at both histological and functional levels indicating the potential therapeutic benefits of these nanoparticles for enhancing the delivery of DEX in acute sensorineural hearing loss., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

We confirmed that ATP is released from cochlear marginal cells in the stria vascular but the cell organelle in which ATP stores was not identified until now. Thus, we studied the ATP-containing cell organelles and suggest that these are lysosomes. Primary cultures of marginal cells of Sprague-Dawley rats aged 1-3 days was established. Vesicles within marginal cells stained with markers were identified under confocal laser scanning microscope and transmission electron microscope (TEM). Then ATP release from marginal cells was measured after glycyl-L-phenylalanine-ß- naphthylamide (GPN) treatment using a bioluminescent assay. Quinacrine-stained granules within marginal cells were labeled with LysoTracker, a lysosome tracer, and lysosomal-associated membrane protein 1(LAMP1), but not labeled with the mitochondrial tracer MitoTracker. Furthermore, LysoTracker-labelled puncta showed accumulation of Mant-ATP, an ATP analog. Treatment with 200 μM GPN quenched fluorescently labeled puncta after incubation with LysoTracker or quinacrine, but not MitoTracker. Quinacrine-labeled organelles observed by TEM were lysosomes, and an average 27.7 percent increase in ATP luminescence was observed in marginal cells extracellular fluid after GPN treatment. ATP-containing vesicles in cochlear marginal cells of the stria vascular from neonatal rats are likely lysosomes. ATP release from marginal cells may be via Ca(2+)-dependent lysosomal exocytosis.

Hearing loss secondary to diabetes remains under debate. In our study, we used Zucker Diabetic Fatty (ZDF) rats as an animal model of type 2 diabetes to investigate whether (1) hearing ability impairment and structural alterations of the inner ear occur in diabetes and (2) an angiotensin II receptor blocker (losartan) can protect rats from diabetic damage. Homozygous mutants were treated with a placebo or losartan and heterozygous animals served as non-diabetic controls. All animals underwent immunohistochemical and electronmicroscopical analysis. Functional testing of hearing ability was performed by click-evoked auditory brainstem responses. The present study showed significant sensorineural hearing impairment in placebo-treated diabetic rats (hearing threshold, 45.0 ± 2.1 dB SPL) compared to both non-diabetic controls (34.7 ± 4 dB SPL) and losartan-treated diabetic rats (36.1 ± 7.4 dB SPL). Concurrently, the functional decline in the placebo-treated rats was associated with significant morphological abnormalities, particularly in the intermediate cells of the stria vascularis and with strial dysfunction. These degenerative changes were indicated by the down-regulation of several pumps, ionic and cellular channels, which are involved in the cycling of K(+) and the maintenance of the endocochlear potential essential for the hearing process. Thus, the inner ear can be regarded as a target organ during hyperglycemic disorders and a metabolically induced "diabetic otopathy" may be added to angiopathy, nephropathy and neuropathy as a specific complication of diabetes mellitus. Blockade of the angiotensin II receptor can prevent this "diabetic otopathy" despite hyperglycemic serum levels.

Noise-induced hearing loss is at least in part due to disruption of endocochlear potential, which is maintained by various K(+) transport apparatuses including Na(+), K(+)-ATPase and gap junction-mediated intercellular communication in the lateral wall structures. In this study, we examined the changes in the ion-trafficking-related proteins in the spiral ligament fibrocytes (SLFs) following in vivo acoustic overstimulation or in vitro exposure of cultured SLFs to 4-hydroxy-2-nonenal, which is a mediator of oxidative stress. Connexin (Cx)26 and Cx30 were ubiquitously expressed throughout the spiral ligament, whereas Na(+), K(+)-ATPase α1 was predominantly detected in the stria vascularis and spiral prominence (type 2 SLFs). One-hour exposure of mice to 8 kHz octave band noise at a 110 dB sound pressure level produced an immediate and prolonged decrease in the Cx26 expression level and in Na+, K(+)-ATPase activity, as well as a delayed decrease in Cx30 expression in the SLFs. The noise-induced hearing loss and decrease in the Cx26 protein level and Na(+), K(+)-ATPase activity were abolished by a systemic treatment with a free radical-scavenging agent, 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl, or with a nitric oxide synthase inhibitor, N(ω)-nitro-L-arginine methyl ester hydrochloride. In vitro exposure of SLFs in primary culture to 4-hydroxy-2-nonenal produced a decrease in the protein levels of Cx26 and Na(+), K(+)-ATPase α1, as well as Na(+), K(+)-ATPase activity, and also resulted in dysfunction of the intercellular communication between the SLFs. Taken together, our data suggest that disruption of the ion-trafficking system in the cochlear SLFs is caused by the decrease in Cxs level and Na(+), K(+)-ATPase activity, and at least in part involved in permanent hearing loss induced by intense noise. Oxidative stress-mediated products might contribute to the decrease in Cxs content and Na(+), K(+)-ATPase activity in the cochlear lateral wall structures.

Objective: This study was conducted to evaluate the relationship between hearing and cochlear histopathology after arginine vasopressin administration in rats., Methods: A total of 30 Wistar rats were injected with either 0.02 unit/g of arginine vasopressin or the same amount of isotonic saline solution. The initial auditory brain stem response threshold was recorded and additional measurements were made at 10, 30, 60, and 90 min after injection of arginine vasopressin or isotonic saline solution. The threshold for each timepoint was compared with the initial threshold. Histological quantitative assessment of endolymphatic hydrops in the cochlea was performed using light microscopy and assessment of the basal, intermediate, and marginal cells of the stria vascularis was performed with electron microscopy., Results: The auditory brain stem threshold 60 min after arginine vasopressin injection increased significantly in comparison with the initial threshold (P<0.05). Although the index for endolymphatic hydrops in rats administered arginine vasopressin was not different from that in controls (P>0.05), vacuoles in the intermediate cells were increased significantly in the treated rats (P<0.01)., Conclusion: Hearing impairment was detected without endolymphatic hydrops in rats administered arginine vasopressin. An increase of vacuoles in the intermediate cells may account for the hearing impairment induced by arginine vasopressin injection., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

This study tests the hypothesis that pegylated nanoparticles (NPs) could be taken up by the cochlear cells [House Ear Institute-organ of Corti 1 (HEI-OC1) and Stria vascularis K-1 (SVK-1)], through endocytic pathways. Furthermore, the in vitro drug release and the cytotoxicity of Furosemide (FUR)-loaded NPs on these two cochlear cells are investigated. FUR-loaded pegylated NPs are prepared by the emulsion-solvent diffusion method without surfactant. The NPs are characterized for particle mean diameter, polydispersity index (PDI), morphology, percent drug encapsulation efficiency (EE%), and FUR release kinetics. The methyl tetrazolium salt (MTS) and lactate dehydrogenase (LDH) bioassays are used to evaluate in vitro, the cytotoxicity of FUR-loaded NPs and native FUR. The NPs uptake is investigated using confocal microscopy, microplate reader/fluorimetry, and flow cytometry. Spherical NPs with a mean diameter range of 133-210 nm and PDI values varying from 0.037 to 0.41 are produced. The FUR EE% is 86% and the drug is released from the NPs according to the zero-order and Higuchi models. After treatment with blank NPs, the percentage of cell viability and cell death are 95.96% and 8.95%, in HEI-OC1 cells, respectively. The NPs are internalized by HEI-OC1 cells through a clathrin-dependent pathway. In addition, results show that NPs can be taken up via clathrin and cytoskeleton mediated pathways in SVK-1 cells. The internalization of the pegylated NPs can enhance the drug toxicity by necrosis in a dose-dependent and sustained release manner. The formulated NPs provide a promising template for a targeted drug delivery system to the inner ear., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Ototoxicity is a common side effect of cisplatin chemotherapy. This study was undertaken to determine the potential protective effects of a systemic administration of dexamethasone against cisplatin-induced ototoxicity. A prospective controlled trial conducted in an animal model. The setting was Animal care research facilities of the Montreal Children's Hospital Research Institute. An experimental guinea pig model was used. The animals were divided as follows: group 1 (n = 10): 12 mg/kg intraperitoneal (IP) cisplatin, group 2 (n = 14): 15 mg/kg/day dexamethasone IP for 2 days followed by cisplatin 12 mg/kg IP, group 3 (n = 14): 10 mg/kg/day dexamethasone IP for 2 days, on day 3, they received cisplatin 12 mg/kg IP followed by 20 mg/kg/day dexamethasone for 2 days and group 4 (n = 5): 10 ml of saline IP twice a day for 3 days. Auditory brainstem response (ABR) threshold shifts were measured at four frequencies (8, 16, 20 and 25 kHz) for groups 1, 2 and 3. Histological changes in the organ of Corti, the stria vascularis, the spiral ligament and the spiral ganglion neurons as well as scanning electron microscopy for outer hair cells were completed. Immunohistochemistry for tumour necrosis factor-alpha (TNF-α) was performed. ABR threshold shifts were similar in all groups. Histological and scanning electron findings demonstrate that dexamethasone has greater protective effect on the stria vascularis. Systemic dexamethasone administration in a guinea pig model did not provide significant protection against cisplatin-induced ototoxicity. Dexamethasone may be useful in future applications as a complementary treatment.

The aim of this study was to investigate the effectiveness of the oral administration of pomegranate extract (PE) as a protective agent against cisplatin-induced ototoxicity. The study included a prospective, controlled animal study Group 1 (n = 6), received no cisplatin or PE, and group 2 (n = 6) received cisplatin at 8 mg/kg/day for 3 consecutive days. Group 3 (n = 6) received not only cisplatin at 8 mg/kg/day for 3 consecutive days, but also received PE (100 μL/day) via gavage for 5 days prior to the cisplatin injection and for 3 days concomitantly with the cisplatin injections. To measure cisplatin ototoxic effects, "distortion product otoacoustic emissions" (DPOAE) were analyzed 3 days before and after the cisplatin injections. Histological changes in the cochleas were observed by light microscopy. Compared with group 3, the DPOAE amplitudes of group 2 decreased significantly. Among the groups, there was a statistically significant difference in basal and mid turn external ciliated cells (ECC) number, but there was no statistically significant difference in apical turn. Differences in stria vascularis (SV) changes were statistically significant between the groups, and the median score for SV injury was significantly greater in group 2 than in group 3. Differences in the median scores for SGC changes being significantly greater in group 2 than in group 3. In conclusion, these results indicated that oral administration of PE afforded statistically significant protection to the cochlea in rats from cisplatin toxicity, and thus, oral experimental dose of PE administration may have a protective effect against cisplatin ototoxicity in rats.

It is well known that ethacrynic acid (EA) can potentiate the ototoxicity of aminoglycoside antibiotics (AmAn) such as kanamycin (KM), if they were applied at the same time. Currently, to create the model of EA-KM-induced cochlear lesion in rats, adult rats received a single injection of EA (75 mg/kg, intravenous injection), or followed immediately by KM (500 mg/kg, intramuscular injection). The hearing function was assessed by auditory brainstem response (ABR) measurement in response to click and/or tone bursts at 4, 8, 12, 16, 20, 24, and 32 kHz. The static microcirculation status in the stria vascularis after a single EA injection was evaluated with eosin staining. The pathological changes in cochlear and vestibular hair cells were also quantified after co-administration of EA and KM. After a single EA injection, blood flow in vessels supplying the stria vascularis rapidly diminished. However, the blood supply to the cochlear lateral wall partially recovered 5 h after EA treatment. Threshold changes in ABR were basically parallel to the microcirculation changes in stria vascularis after single EA treatment. Importantly, disposable co-administration of EA and KM resulted in a permanent hearing loss and severe damage to the cochlear hair cells, but spared the vestibular hair cells. Since the cochlear lateral wall is the important part of the blood-cochlea barrier, EA-induced anoxic damage to the epithelium of stria vascularis may enhance the entry of KM to the cochlea. Thus, experimental animal model of selective cochlear damage with normal vestibular systems can be reliably created through co-administration of EA and KM.

Research in mammalian hair cell regeneration is hampered by a lack of in vivo model of adult mouse inner ear injury. In the present study we investigated the effects of a combination of a single dose of aminoglycoside followed by a loop diuretic in adult mice. The auditory brainstem response threshold shift, extent and defining characteristics of the cochlear lesion were assessed and verified at different time points post-treatment. Our data indicated that this drug combination caused the rapid and extensive death of outer hair cells (OHCs). OHC death presented throughout the cochlea that commenced in the basal turn by 24 h and progressed apically. In contrast, inner hair cell (IHC) loss was delayed and mild. Terminal deoxynucleotidyl transferase dUTP nick end labelling-positive nuclei demonstrated that the majority of OHCs died via an apoptotic pathway. Auditory threshold shifts of up to 90 dB SPL indicated a profound hearing loss. In addition, the endocochlear potential (EP) in the drug-treated animals displayed a significant decline at 12 h post-treatment followed by recovery by 48 h post-treatment. Despite this recovery, there was a significant and progressive decrease in strial vascularis thickness, which was predominantly due to atrophy of marginal cells. The present study reproduced an adult mouse model of aminoglycoside-induced hearing loss. The mechanism underlying the recovered EP in the model with extensive hair cell death is discussed.

Aminoglycosides enter inner ear hair cells via apical endocytosis, or mechanoelectrical transduction channels, implying that, in vivo, aminoglycosides enter hair cells from endolymph prior to exerting their cytotoxic effect. If so, circulating aminoglycosides likely cross the strial blood-labyrinth barrier and enter marginal cells prior to clearance into endolymph. We characterized the competitive antagonism of unconjugated aminoglycosides on the uptake of fluorescent gentamicin (GTTR) in the stria vascularis and kidney cells at an early time point. In mice, uptake of GTTR by kidney proximal tubule cells was competitively antagonized by gentamicin at all doses, but only weakly by kanamycin (mimicking in vitro data). GTTR fluorescence was approximately 100-fold greater in proximal tubule cells than in the stria vascularis. Furthermore, only high molar ratios of aminoglycosides significantly reduced strial uptake of GTTR. Thus, gentamicin antagonism of GTTR uptake is more efficacious in proximal tubules than in the stria vascularis. Competitive antagonism of GTTR uptake is indicative of specific cell-regulatable uptake mechanisms (e.g., ion channels, transporters) in the kidney. Strial uptake mechanisms have lower specific affinity for gentamicin, and/or density (compared to the kidney), yet may be critical to transport gentamicin across the strial blood-labyrinth barrier into marginal cells., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

The use of the effective antineoplastic agent cisplatin is limited by its serious side effects, such as oto- and nephrotoxicity. Ototoxicity is a problem of special importance in children, because deafness hampers their language and psychosocial development. Recently, organic cation transporters (OCTs) were identified in vitro as cellular uptake mechanisms for cisplatin. In the present study, we investigated in an in vivo model the role of OCTs in the development of cisplatin oto- and nephrotoxicity. The functional effects of cisplatin treatment on kidney (24 hours excretion of glucose, water, and protein) and hearing (auditory brainstem response) were studied in wild-type and OCT1/2 double-knockout (KO) mice. No sign of ototoxicity and only mild nephrotoxicity were observed after cisplatin treatment of knockout mice. Comedication of wild-type mice with cisplatin and the organic cation cimetidine protected from ototoxicity and partly from nephrotoxicity. For the first time we showed that OCT2 is expressed in hair cells of the cochlea. Furthermore, cisplatin-sensitive cell lines from pediatric tumors showed no expression of mRNA for OCTs, indicating the feasibility of therapeutic approaches aimed to reduce cisplatin toxicities by competing OCT2-mediated cisplatin uptake in renal proximal tubular and cochlear hair cells. These findings are very important to establish chemotherapeutical protocols aimed to maximize the antineoplastic effect of cisplatin while reducing the risk of toxicities.

Objective: Water homeostasis is essential for the inner ear to maintain the function of hearing and equilibrium. Bumetanide inhibits Na(+)-K(+)-2Cl(-) co-transporter (NKCC), which is expressed in the basolateral membrane of stria vascularis marginal cell, and it causes the enlargement of intrastrial space. Aquaporin (AQP) 2 is expressed in the perilymph side of stria vascularis basal cell, and the vasopressin type 2 antagonist OPC-31260 produces downregulation of AQP2 mRNA levels in the inner ear. The aim of this study is to investigate the influence of OPC-31260 on experimentally induced enlargement of the intrastrial space caused by bumetanide., Materials and Methods: Wistar rats were divided into two groups, a BUM group and an OPC-BUM group. The BUM group was exposed to bumetanide, and the OPC-BUM group was exposed to bumetanide after being premedicated with OPC-31260. The specimens of the stria vascularis were observed using transmission electron microscopy and analyzed quantitatively and statistically., Results: Morphological changes of intrastrial space enlargement occurred in both the BUM and OPC-BUM groups. The ratio of the areas of the intrastrial space area to the stria vascularis were calculated, and the OPC-BUM group mean showed a minimal difference from the BUM-group mean. However, there is no statistical difference., Conclusions: Premedication of rats with OPC-31260 tended to reduce bumetanide-induced enlargement of the intrastrial space. This may indicate that the effect of bumetanide on the stria vascularis is much stronger than that of OPC-31260.

In the stria vascularis (SV), it is known that the Na(+)-K(+)-ATPase is expressed abundantly and its activity in the basolateral membrane of marginal cells is high. Ouabain, an inhibitor of the Na(+)-K(+)-ATPase, causes not only a decline in the endocochlear DC potential but also acute vacuolar formation in marginal cells. We studied the ionic mechanisms underlying the ouabain-induced vacuolar formation in marginal cells using perilymphatic perfusion in guinea pigs. Perilymphatic perfusion with 1 mM ouabain dissolved in the artificial perilymph for 50 min caused many vacuoles of a wide range of sizes in the apical cytoplasm of marginal cells, the bulging of marginal cells into the scala media and strial volume increase. Removal of K(+) from the perilymph reduced the proportion of vacuoles and strial thickening, but the bulging of marginal cells remained. In contrast, the sizes of vacuoles were drastically reduced and extrusion of marginal cells into the scala media could not be observed in the absence of perilymphatic Na(+). Furthermore, the total volume of SV was obviously reduced in comparison with the control. These results indicate that perilymphatic Na(+) and K(+) are responsible for these morphological changes caused by ouabain, and that perilymphatic Na(+) plays an important role in the cellular volume regulation in SV in the presence of ouabain. It is supposed that the transport system of perilymphatic Na(+) and K(+) into marginal cells may contribute to vacuolar formation when ouabain is administered. Regarding Na(+), we hypothesize two possibilities for the perilymphatic Na(+) transporting pathway as follows. Na(+) in the perilymph could enter the endolymph via Reissner's membrane or the basilar membrane; Na(+) in the endolymph would then be taken up by marginal cells via the apical membrane and secreted into the intrastrial space by Na(+)-K(+)-ATPase in the basolateral membrane. Another, less likely, possibility is that Na(+) in the perilymph is transported into basal cells or fibrocytes in the spiral ligament, then into intermediate cells via gap junctions, and finally secreted into the intrastrial space via Na(+)-K(+)-ATPase of intermediate cells. Regarding K(+), it is expected that the K(+) recycling pathway plays a role in ouabain-induced vacuolar formation in marginal cells.

Conclusion: The intrastrial space was enlarged remarkably at 20 min after vasopressin (VP) injection, and this enlargement of the intrastrial space was reduced by administration of OPC-31260 before VP injection. These results suggest that VP increases the influx of water from the perlymph to the basal cells via aquaporin (AQP) 2 and causes the formation of endolymphatic hydrops., Objectives: To investigate a time course of changes of the stria vascularis after VP injection and the influence of OPC-31260 on experimentally induced enlargement of the intrastrial space caused by VP injection., Materials and Methods: In the time course study, Wistar rats were injected with 50 microg/kg of VP subcutaneously. The stria vascularis specimens were harvested at 10, 20, 30, and 60 min after VP injection. For OPC-31260 administration, animals were administered 100 mg/kg of OPC-31260 orally 1 h before receiving 50 microg/kg of VP subcutaneously. The specimens were harvested 20 min after VP injection. These specimens were observed using transmission electron microscopy., Results: In the time course study, the incidence of intrastrial space enlargement was 50%, 100%, 25%, and 0% for 10, 20, 30, and 60 min, respectively. In the study with OPC-31260 administration, the stria vascularis showed no morphological changes.

Aminoglycosides enter inner ear hair cells across their apical membranes via endocytosis, or through the mechanoelectrical transduction channels in vitro, suggesting that these drugs enter cochlear hair cells from endolymph to exert their cytotoxic effect. We used zebrafish to determine if fluorescently tagged gentamicin (GTTR) also enters hair cells via apically located calcium-sensitive cation channels and the cytotoxicity of GTTR to hair cells. We then examined the serum kinetics of GTTR following systemic injection in mice and which murine cochlear sites preferentially loaded with systemically administered GTTR over time by confocal microscopy. GTTR is taken up by, and is toxic to, wild-type zebrafish neuromast hair cells. Neuromast hair cell uptake of GTTR is attenuated by high concentrations of extracellular calcium or unconjugated gentamicin and is blocked in mariner mutant zebrafish, suggestive of entry via the apical mechanotransduction channel. In murine cochleae, GTTR is preferentially taken up by the stria vascularis compared to the spiral ligament, peaking 3 h after intra-peritoneal injection, following GTTR kinetics in serum. Strial marginal cells display greater intensity of GTTR fluorescence compared to intermediate and basal cells. Immunofluorescent detection of gentamicin in the cochlea also revealed widespread cellular labeling throughout the cochlea, with preferential labeling of marginal cells. Only GTTR fluorescence displayed increasing cytoplasmic intensity with increasing concentration, unlike the cytoplasmic intensity of fluorescence from immunolabeled gentamicin. These data suggest that systemically administered aminoglycosides are trafficked from strial capillaries into marginal cells and clear into endolymph. If so, this will facilitate electrophoretically driven aminoglycoside entry into hair cells from endolymph. Trans-strial trafficking of aminoglycosides from strial capillaries to marginal cells will be dependent on as-yet-unidentified mechanisms that convey these drugs across the intra-strial electrical barrier and into marginal cells.

Previous studies have indicated the importance of the spiral ligament (SL) in the pathogenesis of sensorineural hearing loss. The aim of this study was to establish a mouse model for SL degeneration as the basis for the development of new strategies for SL regeneration. We injected 3-nitropropionic acid (3-NP), an inhibitor of succinate dehydrogenase, at various concentrations into the posterior semicircular canal of adult C57BL/6 mice. Saline-injected animals were used as controls. Auditory function was monitored by measurements of auditory brain stem responses (ABRs). On postoperative day 14, cochlear specimens were obtained after the measurement of the endocochlear potential (EP). Animals that were injected with 5 or 10 mM 3-NP showed a massive elevation of ABR thresholds along with extensive degeneration of the cochleae. Cochleae injected with 1 mM 3-NP exhibited selective degeneration of the SL fibrocytes but alterations in EP levels and ABR thresholds were not of sufficient magnitude to allow for testing functional recovery after therapeutic interventions. Animals injected with 3 mM 3-NP showed a reduction of around 50% in the EP along with a significant loss of SL fibrocytes, although degeneration of spiral ganglion neurons and hair cells was still present in certain regions. These findings indicate that cochleae injected with 3 mM 3-NP may be useful in investigations designed to test the feasibility of new therapeutic manipulations for functional SL regeneration.

Conclusion: It is suggested that transient receptor potential vanilloid (TRPV)-1 and -2 may be of pathological significance for sensory cells and ganglions, while TRPV-3 and -4 may play an important part in neuroprotection of the inner ear., Objective: Changes in the expression of TRPV-1, -2, -3, and -4 in gentamicin (GM)-treated mouse inner ear were studied., Materials and Methods: CBA/J mice were used in this study. The localization of TRPV-1, -2, -3, and -4 in the inner ear of both untreated and GM-treated CBA/J animals (intratympanic injection of 5 mg GM) was investigated by immunohistochemistry., Results: TRPV-1, -2, and -3 were co-expressed in the inner ear sensory and ganglion cells, while TRPV-4 was also expressed in the stria vascularis and vestibular dark cells. Following GM treatment, the intensity of immunofluorescent reaction to TRPV-1 and TRPV-2 increased, while that to TRPV-3 and TRPV-4 decreased.

Conclusions: That the endolymphatic sac (ES) reacts to changes in inner ear blood flow may be important for homeostasis of the inner ear fluid volume and pressure., Objectives: To elucidate the effect of changes in inner ear blood flow on the ES and to learn more about the volume and pressure regulatory function of the ES., Materials and Methods: Epinephrine or sodium nitroprusside (SNP) was injected into the middle ear cavity of adult CBA/J mice. The ES were analyzed morphologically by light microscopy., Results: Epinephrine reduced the luminal size of the ES leading to an accumulation of intraluminal homogeneous substance. Injection of SNP increased the size of the ES lumen, accompanied by a collapse of the lateral intercellular space (LIS) and dense perisaccular tissue. These changes were almost reversed 4 h after injection.

We fed DBA mice with alpha-lipoic acid (100 mg/kg body weight/day), beginning 2, 4, or 8 weeks after birth. Hearing thresholds were measured weekly. At 12 weeks after birth, control mice not fed with alpha-lipoic acid showed significant hearing decreases at all frequencies. In contrast, mice fed with alpha-lipoic acid beginning at 2 weeks after birth showed significantly better hearing at all frequencies. Mice fed with alpha-lipoic acid beginning at 4 and 8 weeks after birth also showed significantly better hearing than control mice after they were fed with alpha-lipoic acid. The stria vascularis of mice fed with alpha-lipoic acid showed reduced 8-oxoguanine residues in DNA and cytoplasm compared with that of control mice. Western blotting showed that the level of hypoxia-inducible factor-1alpha was lower in mice fed with alpha-lipoic acid than in control mice. From these results, we suggest that alpha-lipoic acid prevented early-onset hearing impairment in DBA mice.

Conclusion: The concentration of gadolinium (Gd) used clinically showed no remarkable effects on the stria vascularis; however, a higher concentration had adverse effects. The concentration of Gd must be borne in mind when injecting Gd into the tympanic cavity., Objective: Endolymphatic hydrops has been visualized using high resolution MRI with the intratympanic administration of Gd in patients with Meniere's disease. We attempted to investigate the effects of Gd on the stria vascularis., Materials and Methods: Gd hydrate diluted eightfold with saline or non-diluted Gd or saline was injected into the tympanic cavity of guinea pigs. To investigate the effects of Gd on the stria vascularis, we measured endocochlear DC potential (EP) and observed the stria vascularis using transmission electron microscopy., Results: Intratympanic injections of Gd hydrate diluted eightfold with saline (1/8 Gd) and saline did not cause apparent changes in the EP. Moreover, the amplitude of the EP decreased significantly 60 min after non-diluted Gd was injected. Transmission electron micrographs of the stria vascularis revealed no significant morphological difference between the ears injected with 1/8 Gd and those injected with saline. There was significant morphological change in the ear injected with non-diluted Gd. The intercellular spaces were markedly enlarged.

Objective: This study aimed to elucidate the potential inner-ear effects of fotemustine, a chemotherapeutic agent which crosses the blood-brain barrier and is used in the treatment of primary and metastatic brain tumours and metastatic melanoma., Methods: This study utilised distortion product otoacoustic emissions and transmission electron microscopy in order to conduct electrophysiological and morphological assessments, using a rat experimental model. Twelve ears of six male rats were examined two months following intraperitoneal slow infusion of fotemustine (100 mg/m2 or 7.4 mg/kg). Pre- and post-treatment measurements were compared. Finally, electron microscopy was performed on three rat temporal bones., Results: After infusion of fotemustine, distortion product otoacoustic emissions revealed a significant reduction in signal-to-noise ratios only at 3600 Hz (from 11.95 +/- 7.52 to -0.26 +/- 9.45 dB) and at 3961 Hz (from 18.09 +/- 7.49 to 6.74 +/- 12.11 dB) (referenced to 2f1 - f2). Transmission electron microscopy of the temporal bone revealed ultrastructural changes in the outer hair cells, stria vascularis and cochlear ganglion at the cochlear basal turn. The ganglion cell perikarya were unaffected., Conclusions: Fotemustine was administered via intraperitoneal slow infusion in a rat experimental model. Twelve ears of six survivors, from 10 rats, were evaluated at the second month. Fotemustine was determined to have a potential for ototoxicity at 3600 and 3961 Hz. Three randomly chosen rats underwent electron microscopy for morphological analysis. Morphological effects in the cochlear basal turn were observed. Oedematous intracytoplasmic spaces and perivascular areas of the stria vascularis, as well as distorted chromatin content, were detected, thereby suggesting potential ototoxic effects for this agent. Further experimental and clinical studies are required in order to determine whether the effect seen in this pilot study is reversible, and to analyse effects in humans.

Conclusion: This study indicates that epithelial Na(+)-selective channels (ENaC) recycle Na(+) via clathrin-mediated endocytosis in the marginal cells of the stria vascularis and that clathrin-independent endocytosis appeared to be modulated by the amount of Na(+) transported. These results suggest the presence of ENaC in the luminal membrane of marginal cells and that ENaC are an efficient pathway for the uptake of Na(+) from the endolymph., Objective: The ENaC found in many transporting epithelia play a key role in the regulation of salts and water homeostasis, cellular pH, cell volume, and cell function. Both biochemical and physiological approaches have been used to identify, characterize, and quantify this important channel, but its location in the marginal cells of the stria vascularis has not been fully clarified. The aim of this study was to determine the localization and regulation of ENaC., Materials and Methods: Forty healthy female guinea pigs were used: 20 for the control experiment, 10 for the amiloride experiment, and 10 for the aldosterone experiment. We perfused cationized ferritin (CF) and microperoxidase (MPO) as tracers for clathrin-mediated and clathrin-independent endocytosis, respectively, into the cochlear duct. After 30 min of endolymphatic perfusion, the tissues were fixed and CF- and MPO-loaded endosomes within the marginal cell were observed by transmission electron microscopy. The numbers of CF- and MPO-loaded endosomes were compared between the three groups., Results: In the amiloride group, the numbers of CF- and MPO-loaded endosomes decreased in comparison with the control. In the aldosterone group, the numbers of CF- and MPO-loaded endosomes decreased and increased, respectively. Recently, it has been reported that ENaC are endocytosed via clathrin-mediated endosomes and aldosterone decreases the rate of endocytosis of ENaC. In this study, the results of the aldosterone experiment were consistent with those of recent studies.

Conclusion: A new murine model for the study of Ménière's disease has been developed by treatment with both lipopolysaccharide (LPS) and aldosterone. Induction of vestibular dysfunction in the hydropic animal model may entail additional stress such as reduced inner ear blood flow, and sudden acute changes in endolymph volume and/or pressure., Objective: The purpose of this study was to develop a more suitable animal model, showing closer resemblance to the pathophysiological process in Ménière's disease., Materials and Methods: Adult CBA/J mice were treated by intratympanic injection of LPS, intraperitoneal injection of aldosterone, or injection of both LPS and aldosterone. Morphological analyses were performed in the cochlea and endolymphatic sac., Results: All experimental animals showed mild to moderate endolymphatic hydrops. Those treated with both LPS and aldosterone showed reversible vestibular dysfunction after the intratympanic injection of epinephrine.

Using immunohistochemical and electrophysiological methods, we investigated the role of L-type Ca(2+) channels in the regulation of the endocochlear potential (EP) of the endolymphatic surface cells (ESC) of the guinea pig stria vascularis. The following findings were made: (1) Administration of 30 microg/ml nifedipine via a vertebral artery significantly suppressed the transient asphyxia-induced decrease in the EP (TAID) and the transient asphyxia-induced increase in the Ca(2+), referred to as TAIICa, concentration in the endolymph ([Ca](e)). (2) The endolymphatic administration of 1 microg/ml nifedipine significantly inhibited the TAID as well as the TAIICa. The endolymphatic administration of nifedipine (0.001-10 microg/ml) inhibited the TAID in a dose-dependent manner. (3) The endolymphatic administration of (+)-Bay K8644, an L-type Ca(2+) channel closer, significantly inhibited the TAID, whereas (-)-Bay K8644, an L-type Ca(2+) channel opener, caused a large decrease in the EP from approximately +75 mV to approximately +20 mV at 10 min after the endolymphatic administration. (4) By means of immunohistochemistry, a positive staining reaction with L-type Ca(2+) channels was detected in the marginal cells of the stria vascularis. (5) Under the high [Ca](e) condition, we examined the mechanism of the TAIICa and hypothesized that the TAIICa might have been caused by the decrease in the EP through a shunt pathway in the ESC. (6) The administration of nifedipine to the endolymph significantly inhibited the Ba(2+)-induced decrease in the EP. These findings support the view that L-type Ca(2+) channels in the marginal cells regulate the EP, but not directly the TAIICa.

Ethacrynic acid (EA) significantly enhances the ototoxic effects of cisplatin. To gain insights into the mechanisms underlying Cis/EA ototoxicity, cochleas were labeled with several apoptotic markers. Cis/EA treatment caused extensive outer hair cell (OHC) and inner hair cell (IHC) damage; OHC lesions decreased from the base towards apex of the cochlea whereas the IHC lesion was relatively constant (25-60%) along the length of the cochlea. Propidium iodide labeled OHC nuclei appeared relatively normal at 6h post-treatment, were condensed and fragmented at 12h post-treatment and were frequently missing 48 h post-treatment. Initiator caspase 8, associated with membrane death receptors, and TRADD, a protein that recruits caspase 8, were present in OHC at 6h post-treatment. Caspase 8 labeling increased from 6 to 24h, but was largely absent at 48 h post-treatment. Executioner caspase 3 and caspase 6, which lie downstream of caspase 8, were expressed in OHC 12-24h post-treatment. Initiator caspase 9, associated with mitochondrial damage, was only expressed at low levels at 48 h post-treatment. These results suggest that the rapid onset of Cis/EA induced programmed cell death is initiated by membrane death receptors associated with TRADD and caspase 8.

The antineoplastic drug cisplatin is known to cause a reduction in endocochlear potential. The hypothesis to be tested was whether a single high dose of cisplatin affects the melanocytes by altering the expression of melanin. Pigmented guinea pigs received a bolus injection of cisplatin (8 mg/kg as a 15-second intravenous infusion). Auditory brainstem response (ABR) thresholds and morphological analysis of the hair cells and the stria vascularis were made 96 h after injection. ABR thresholds were elevated (15-40 dB) at 12-30 kHz and a significant loss of outer hair cells in the more basal regions was found. Cisplatin caused a significantly lower density of melanin in the intermediate cells in the basal region without any signs of apoptosis. Changes in melanin content were not noted in the middle or apical cochlear regions. Significant correlations were found between melanin density, ABR threshold shifts and outer hair cell loss in the region corresponding to 30 kHz. The findings reported here further support the multiple cytotoxic effect of cisplatin on the inner ear., (Copyright 2007 S. Karger AG, Basel.)

Objectives: The vibration caused by drills used for middle ear surgery is considered one of the causes of postoperative sensorineural deafness. Seki et al reported that when drill-induced damage was created in the auditory ossicles of guinea pigs, permeability across the capillary vessels in the stria vascularis increased significantly with the duration of drill-induced vibration. The present study was undertaken to examine changes in permeability across the stria vascularis capillaries following vibration in experimental animals pretreated with steroids, with the goal of developing a method of preventing a vibration-induced increase in permeability across these capillaries., Methods: After an intravenous dose of hydrocortisone and horseradish peroxidase, the auditory ossicles of guinea pigs were vibrated with a drill for 60 seconds., Results: Intravenous steroid administration before vibration reduced the leakage of horseradish peroxidase from the stria vascularis capillaries after vibration., Conclusions: The findings suggested that steroids suppress the increase in permeability across the stria vascularis capillaries that results from drill-induced vibration.

Extracellular ATP decreases K+ secretion in strial marginal cells via apical P2Y4 receptors. We investigated the effect of reactive blue 2 (RB-2), an antagonist of rat P2Y4, on rat strial marginal cells using a voltage-sensitive vibrating probe. The application of RB-2 increased K+ secretion in a dose-dependent manner, and this increase was characterized as a peak followed by a partial relaxation to a steady-state. Moreover, this response was similar to that caused by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). Suramin had no similar effect, except at high concentration. Thus, we tested the effects of these chemicals on P2Y4 receptors in strial marginal cells. Both RB-2 and DIDS had antagonistic activities at P2Y4, and the antagonist potency at P2Y4 paralleled the potency of K+ secretion. Interestingly, 2'- and 3'-O-(4-benzoyl-benzoyl)adenosine 5'-triphosphate (BzATP) exhibited an agonistic effect at P2Y4 receptor, which was blocked by RB-2, but not by pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS). Based on these results, we speculate that direct and/or indirect inhibitory mechanisms between P2Y4 and KENQ1/KCNE1 K+ channels exist in strial marginal cell.

Ototoxicity is a typical dose-limiting side effect of cancer chemotherapy with cisplatin but much less so with carboplatin. To elucidate the underlying molecular pathological mechanisms, we have measured the formation and persistence of drug-induced DNA adducts in the nuclei of inner ear cells of guinea pigs after short-term exposure to either cisplatin or carboplatin using immunofluorescence staining and quantitative image analysis. After application of carboplatin, all cells of the cochlea exhibited a similar burden of guanine-guanine intrastrand cross-links in DNA. In contrast, we observed a pronounced 3- to 5-fold accumulation of this cytotoxic adduct exclusively in the marginal cells of the stria vascularis between 8 and 48 h after treatment with cisplatin. In the kidney, the other critical target tissue of cisplatin toxicity, a similar high preferential formation of cytotoxic DNA adducts was measured in the tubular epithelial cells but not in other renal cell types. As for the ear, this excessive formation of DNA damage in a particular cell type was seen in animals treated with cisplatin but not those treated with carboplatin. Because cisplatin ototoxicity is often attributed to oxidative stress mediated by the generation of radical oxygen species (ROS), we have measured in parallel the levels of the lead DNA oxidation product 8-oxoguanine (8-oxoG) in cochlear cryosections. Compared with basal levels in untreated control cochleas, no additional formation of 8-oxoG was detectable up to 48 h after cisplatin treatment in the DNA of either inner-ear cell type. This suggests that the generation of ROS may be a secondary event in cisplatin ototoxicity.

Background: After establishing a murine model of aminoglycoside antibiotic (AmAn) induced ototoxicity, the sensitivity of AmAn induced ototoxicity in three murine strains and the effect of kanamycin on the expression of Na-K-2Cl cotransporter-1 (NKCC1) in stria vascularis were investigated., Methods: C57BL/6J, CBA/CaJ, NKCC1(+/-) mice (24 of each strain) were randomly divided into four experimental groups: A: kanamycin alone; B: kanamycin plus 2, 3-dihydroxybenzoate; C: 2, 3-dihydroxybenzoate alone; and D: control group. Mice were injected with kanamycin or/and 2, 3-dihydroxybenzoate twice daily for 14 days. Auditory brainstem response (ABR) was measured and morphology of cochlea delineated with succinate dehydrogenase staining. Expression of NKCC1 in stria vascularis was detected immunohistochemically., Results: All three strains in groups A and B developed significant ABR threshold shifts (P < 0.01), which were accompanied by outer hair cell loss. NKCC1 expression in stria vascularis was the weakest in group A (A cf D, P < 0.01) and the strongest in groups C and D (P < 0.05). CBA/CaJ mice had the highest sensitivity to AmAn., Conclusions: Administration of kanamycin established AmAn induced ototoxicity. Kanamycin inhibited the expression of NKCC1 in stria vascularis. 2, 3-dihydroxybenzoate attenuated AmAn induced ototoxicity-possibly by enhancing the expression of NKCC1. Age related hearing loss did not show additional sensitivity to AmAn induced ototoxicity in murine model.

Objective: To investigate the effect of 5-fluorouracil (5-FU) ointment on the inner ear of guinea pigs., Study Design and Setting: In group A (n = 7), 5-FU ointment was applied into the left external auditory canal. In group B (n = 10), 5-FU ointment was applied to the left middle ear through myringotomy. In both groups, the right ear served as a control. One week later the endocochlear DC potential (EP) was measured and morphology of the cochleae was examined using scanning electron microscopy (SEM) and light microscopy., Results: In group A, there was no significant difference between the EP values of the experimental side and the control side. In group B, there was a statistically significant difference between them (P < 0.05). Morphologic findings showed no damage., Conclusion: 5-FU ointment application to the external ear seems to be safe but its application to the middle ear may pose some risk of ototoxicity.

Objective: To study the feasibility of application of rat-tail collagen in the primary culture of rats marginal cells of stria vascularis., Method: The effect of self-made rat-tail collagen on the primary culture of rats marginal cells of stria vascularis was observed and estimated., Result: When cochlear stria vascularis fragment was isolated and cultured for 24 h, a few culture cells appeared around the fragments. About 48 to approximately 72 h later, clusters of culture cells could be seen,these cells showed the cobblestone shape under the microscope. The immunohistochemistry and the transmission electron microscopy showed the epithelial origination of these cells., Conclusion: It is feasible to the primarily culture the rats marginal cells of stria vascularis with self-made rat-tail collagen.

Cisplatin ototoxicity has at least three major targets in the cochlea: the stria vascularis, the organ of Corti, and the spiral ganglion. This study aims to differentiate between these three targets. In particular, we address the question of whether the effects at the level of the organ of Corti and spiral ganglion are mutually dependent or whether they develop in parallel. This question was approached by studying the ototoxic effects while they develop electrophysiologically and comparing these to earlier presented histological data [Van Ruijven et al., 2004. Hear. Res. 197, 44-54]. Guinea pigs were treated with intraperitoneal injections of cisplatin at a dose of 2 mg/kg/day for either 4, 6, or 8 consecutive days. This time sequence has not revealed any evidence of one ototoxic process triggering another. Therefore, we have to stay with the conclusion of Van Ruijven et al. (2004) that both processes run in parallel.

We investigated whether an acyclic polyisoprenoid antiulcer drug, geranylgeranylacetone (GGA), induces the expression of HSP70 in the rat cochlea. Immunoblotting revealed upregulation of HSP70 in the cochlea at 12 h after transtympanic (local) or oral (systemic) administration of GGA, and this increased at 24 h after administration. Positive immunohistochemical staining of HSP70 was observed in the hair cells, the spiral ganglion, the stria vascularis, the spiral ligament, and the perivascular portion of modiolar vessels. We therefore subsequently studied the effects of GGA as an HSP-inducer on inner ear trauma due to inflammation. Damage to the lateral wall due to inflammation induced by lipopolysaccharide inoculation was protected against by pretreatment with GGA, as assessed physiologically by measurement of cochlear blood flow and morphologically by electron microscopy. The results of the present study suggest that GGA can protect the cochlea against other injuries including those induced by noise, ototoxic drugs, and ischemia by upregulating HSP70.

Alport syndrome results from mutations in genes encoding collagen alpha3(IV), alpha4(IV), or alpha5(IV) and is characterized by progressive glomerular disease associated with a high-frequency sensorineural hearing loss. Earlier studies of a gene knockout mouse model for Alport syndrome noted thickening of strial capillary basement membranes in the cochlea, suggesting that the stria vascularis is the primary site of cochlear pathogenesis. Here we combine a novel cochlear microdissection technique with molecular analyses to illustrate significant quantitative alterations in strial expression of mRNAs encoding matrix metalloproteinases-2, -9, -12, and -14. Gelatin zymography of extracts from the stria vascularis confirmed these findings. Treatment of Alport mice with a small molecule inhibitor of these matrix metalloproteinases exacerbated strial capillary basement membrane thickening, demonstrating that alterations in basement membrane metabolism result in matrix accumulation in the strial capillary basement membranes. This is the first demonstration of true quantitative analysis of specific mRNAs for matrix metalloproteinases in a cochlear microcompartment. Further, these data suggest that the altered basement membrane composition in Alport stria influences the expression of genes involved in basement membrane metabolism.