Cisplatin is retained in the cochlea indefinitely following chemotherapy.

Cisplatin chemotherapy causes permanent hearing loss in 40-80% of treated patients. It is unclear whether the cochlea has unique sensitivity to cisplatin or is exposed to higher levels of the drug. Here we use inductively coupled plasma mass spectrometry (ICP-MS) to examine cisplatin pharmacokinetics in the cochleae of mice and humans. In most organs cisplatin is detected within one hour after injection, and is eliminated over the following days to weeks. In contrast, the cochlea retains cisplatin for months to years after treatment in both mice and humans. Using laser ablation coupled to ICP-MS, we map cisplatin distribution within the human cochlea. Cisplatin accumulation is consistently high in the stria vascularis, the region of the cochlea that maintains the ionic composition of endolymph. Our results demonstrate long-term retention of cisplatin in the human cochlea, and they point to the stria vascularis as an important therapeutic target for preventing cisplatin ototoxicity.

The protective role of tetramethylpyrazine against cisplatin-induced ototoxicity.

OBJECTIVES: The aim of the present study was to investigate the protective effect of tetramethylpyrazine (TMP) on cisplatin-induced ototoxicity in rats. METHODS: Forty healthy, female, 24-week-old, Sprague-Dawley rats (n = 40) were randomly assigned to four groups as follows: group one (n = 10) received intraperitoneal (i.p.) physiological saline at daily doses of 3 mg/kg for seven days; group two (n = 10) received a single dose of i.p. 15 mg/kg cisplatin; group three (n = 10) received i.p. 140 mg/kg TMP daily for seven days plus a single dose of i.p. 15 mg/kg cisplatin on the fourth day; group four (n = 10) received i.p. 140 mg/kg TMP daily for seven days. Auditory brainstem response (ABR) and distortion product otoacoustic emission (DPOAE) measurements were obtained from the animals (40 rats, 80 ears) under general anesthesia before and after drug administration. The temporal bulla of animals were bilaterally removed for immunohistopathological examination. RESULTS: In group two, DPOAE and ABR values were significantly deteriorated after drug administration, whereas there was no statistically significant difference between the pre- and posttreatment DPOAE and ABR values for all frequencies for groups one, three and four. The mean scores for external ciliated cells (ECCs), stria vascularis (SV) and spiral ganglion (SG) injuries in hematoxylin and eosin (H&E) staining, and also caspase-3 immunoreactivity were significantly higher in group two than in the other groups. CONCLUSION: In the present study, the protective effect of TMP on cisplatin ototoxicity was demonstrated through studies of electrophysiology and immunohistopathology. Co-administration of TMP may have potential protective effects against cisplatin-induced ototoxicity.

[Study on the electrophsiological properties in the stria vascularis pericytes in cochlear of guinea pig].

OBJECTIVE: The purpose of this paper was to study the electrophysiological properties and the type of potassium channels on cell membrane in the stria vascularis pericytes in cochlear of guinea pig. METHODS: Firstly examined the expression of the stria vascularis pericytes by desmin, a marker of pericytes, in cochlear of guinea pig with immunofluorescent method. Using whole-cell patch clamp recording techniques to observe electrophysiological properties in the cochlear pericytes in stria vascularis of guinea pig. RESULTS: Pericytes were predominately distributed in the capillaries of cochlea.The average membrane capacitance, resistance, and potential of a single pericyte in stria vascularis were(5.9±0.3)pF, (2.2±0.3)GΩ and (-30.9±1.2)mV, respectively by using patch clamp technique. In addition, the average current density of cochlear pericyte was voltage-sensitive (Vh from 0 to + 60 mV, in 20 mV steps). The pericytes exhibited outward current and this property could be blocked by TEA (tetraethylammonium) 1 mmol/L, a large-conductance calcium-activated potassium channel(BKCa)inhibitor and 4-AP (4-aminopyridine) 1 mmol/L, a voltage-dependent K(+) channels(KV) channel blocker. TEA blocked the outward current from (296.2±35.9)pA to (163.7±16.8)pA and 4-AP blocked the outward current from (248.7±39.8)pA to (158.0±38.0)pA. CONCLUSION: These results suggest that pericytes in stria vascularis have BKCa and KV channels.

Novel expression patterns of connexin 30.3 in adult rat cochlea.

Mutations of the GJB4 gene, encoding connexin 30.3 (CX30.3), are associated with skin disorders. Recently, this gene was also detected in deaf individuals without skin disorders. However, the functional roles of CX30.3 in the cochlea remain unclear. A primary step toward understanding the role of CX30.3 in hearing and its dysfunction is the documentation of its cellular and sub-cellular locations within the cochlea. In the present study, we located and determined the cellular expression of Cx30.3 within the rat cochlea by using a polyclonal anti-Cx30.3 antibody. Expression of the Cx30.3 protein was detected in the spiral limbus, spiral ligament, spiral ganglion, and stria vascularis by immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR) analyses. Our results indicate the presence and localization of Cx30.3 in the rat cochlea. Knowledge of the spatial distribution of Cx30.3 will provide important insights into its role in the cochleae and normal auditory function.

Expression of aquaporins and vasopressin type 2 receptor in the stria vascularis of the cochlea.

Recently, considerable evidence has been accumulated to support the novel view that water homeostasis in the inner ear is regulated via the vasopressin-aquaporin 2 (VP-AQP2) system in the same fashion as in the kidney. Indeed, multiple subtypes of AQPs including AQP-2 are reported to be expressed in the cochlea. However, the mechanism that underlies VP-AQP-2 mediated water homeostasis remains to be elucidated. In the present study, the localizations of AQP-1, -2, -3, -4, -5, -7, -8, -9, and vasopressin type 2 receptor (V2-R) in the stria vascularis (SV) were molecular biologically and immunohistochemically examined to evaluate the role of the AQP water channel system in water homeostasis of the SV. A RT-PCR study revealed that AQPs and V2-R mRNA are expressed in the cochlea. As for their immunohistochemical localization, the AQP-2 protein is expressed on the basal side of the basal cells of the SV, and proteins of AQP-3 and V2-R are expressed on the apical side of the basal cells. AQP-7 and -9 proteins are expressed on the apical side of marginal cells. AQP-4, -5, and -8 protein expressions could not be detected in the lateral wall of the cochlea. From the present results, water flux in the SV is thought to be regulated at the level of the basal cells by vasopressin. Furthermore, such a distribution of AQP-2, -3, and V2-R suggests that VP-AQP-2 mediated water transport might work actively in the basal cells from perilymph towards endolymph containing AQP-1, -7 and -9.

MRNA expression of members of the IGF system in the organ of Corti, the modiolus and the stria vascularis of newborn rats.

We analyzed the mRNA expression of the insulin-like growth factor (IGF) family genes and of selected downstream pathway genes using the Affymetrix microarray system and confirmatory RT-PCR in the freshly prepared organ of Corti (OC), modiolus (MOD) and stria vascularis (SV) from neonatal rats (3-5 days old) and after 24h in culture. Among the seven members of the IGF family analyzed in this paper, IGF1, IGF2 and IGF-binding protein (IGFBP2) had the highest basal expression in all regions. Preparatory stress and culture increased the expression of IGF2, IGFBP2, IGFBP3, IGFBP5, glucose transporterl (GLUT1), signal transducer, and activator of transcription3 (STAT3), phosphoinositide-3-kinase regulatory subunit (Pik3r1), Jun oncogene (c-jun) and decreased that of mitogen-activated protein kinases MAPK3 and MAPK14 in all regions. Region-specific changes were observed in OC (GLUT1), MOD (IGFBP3 and c-jun) and SV (IGF2 and IGFBP2).

Comparison of the quantity of cochlear melanin in young and old C57BL/6 mice.

OBJECTIVE: To elucidate the functional relationship between cochlear melanin and aging. DESIGN: Melanin has been described in the cochlear labyrinth and has been suggested to protect the cochlea from various types of trauma. The quantity of melanin has been shown to change with aging in several organs; however, to our knowledge, aging changes in the cochlea have not been documented. Therefore, we chemically quantified cochlear eumelanin and pheomelanin contents and compared these in young and old C57BL/6 mice using high-performance liquid chromatography. Because melanin deposits in the cochlea present most extensively in the stria vascularis, we morphologically examined the stria using transmission electron microscopy. SUBJECTS: Cochleae from an inbred strain of C57BL/6 male and female mice; 6 at the age of 10 weeks and 5 at the age of 100 weeks were studied. RESULTS: The quantities of cochlear eumelanin and pheomelanin were 421 and 480 ng per cochlea in young mice, and 2060 and 765 ng per cochlea in old mice, respectively. Under transmission electron microscopy, the number of pigmented granules seemed to be greater in older mice compared with younger mice, especially in marginal cells. CONCLUSION: To our knowledge, our findings are the first quantitative evidence to show an age-related overexpression of cochlear melanin and an alteration in the proportion of eumelanin and pheomelanin with aging, suggesting a possible otoprotective function of eumelanin against age-related cochlear deterioration.

FXYD6 is a novel regulator of Na,K-ATPase expressed in the inner ear.

The exquisite sensitivity of the cochlea, which mediates the transduction of sound waves into nerve impulses, depends on the endolymph ionic composition and the endocochlear potential. A key protein in the maintenance of the electrochemical composition of the endolymph is the Na,K-ATPase. In this study, we have looked for the presence in the rat inner ear of members of the FXYD protein family, recently identified as tissue-specific modulators of Na,K-ATPase. Only FXYD6 is detected at the protein level. FXYD6 is expressed in various epithelial cells bordering the endolymph space and in the auditory neurons. FXYD6 co-localizes with Na,K-ATPase in the stria vascularis and can be co-immunoprecipitated with Na,K-ATPase. After expression in Xenopus oocytes, FXYD6 associates with Na,K-ATPase alpha1-beta1 and alpha1-beta2 isozymes, which are preferentially expressed in different regions of the inner ear and also with gastric and non-gastric H,K-ATPases. The apparent K(+) and Na(+) affinities of alpha1-beta1 and alpha1-beta2 isozymes are different. Association of FXYD6 with Na,K-ATPase alpha1-beta1 isozymes slightly decreases their apparent K(+) affinity and significantly decreases their apparent Na(+) affinity. On the other hand, association with alpha1-beta2 isozymes increases their apparent K(+) and Na(+) affinity. The effects of FXYD6 on the apparent Na(+) affinity of Na,K-ATPase and the voltage dependence of its K(+) effect are distinct from other FXYD proteins. In conclusion, this study defines the last FXYD protein of unknown function as a modulator of Na,K-ATPase. Among FXYD protein, FXYD6 is unique in its expression in the inner ear, suggesting a role in endolymph composition.

Aminoglycoside ototoxicity in three murine strains and effects on NKCC1 of stria vascularis.

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.

Collagen changes in the cochlea of aged Fischer 344 rats.

Hearing function in the Fischer 344 (F344) albino inbred strain of rats deteriorates with aging faster than in other strains, in spite of the small hair cell loss in old F344 animals [Popelar, J., Groh, D., Pelanova, J., Canlon, B., Syka, J., 2005. Age-related changes in cochlear and brainstem auditory function. Neurobiol. Aging, in press.]. This study was aimed at elucidating the structural changes in the inner ear of this rat strain during aging. Cochlear histopathology was examined in 20-24-month-old F344 rats and compared with that of young F344 rats (4 months) and of old rats of the Long-Evans (LE) strain. Hematoxylin/eosin staining in aged F344 rats showed degenerative changes in the organ of Corti, consisting of a damaged layer of marginal cells, reduced vascularization of the stria vascularis and a distorted tectorial membrane detached from the organ of Corti. Age-related changes in collagen distribution were observed with Masson's trichrome staining in the spiral ligament of old F344 rats. The results of immunohistochemical staining for type II collagen revealed a marked decrease in collagen fibers in the area connecting the spiral ligament and stria vascularis and a decrease in area IV fibrocytes in old F344 but not in LE rats. These findings may contribute to an explanation of the substantial hearing loss found in old F344 rats.

Transient receptor potential channels in the inner ear: presence of transient receptor potential channel subfamily 1 and 4 in the guinea pig inner ear.

CONCLUSION: The results of this study indicate that transient receptor potential subfamily 1 (TRPV1) may play a functional role in sensory cell physiology and that TRPV4 may be important for fluid homeostasis in the inner ear. OBJECTIVE: To analyze the expression of TRPV1 and -4 in the normal guinea pig inner ear. MATERIAL AND METHODS: Albino guinea pigs were used. The location of TRPV1 and -4 in the inner ear, i.e. cochlea, vestibular end organs and endolymphatic sac, was investigated by means of immunohistochemistry. RESULTS: Immunohistochemistry revealed the presence of TRPV1 in the hair cells and supporting cells of the organ of Corti, in spiral ganglion cells, sensory cells of the vestibular end organs and vestibular ganglion cells. TRPV4 was found in the hair cells and supporting cells of the organ of Corti, in marginal cells of the stria vascularis, spiral ganglion cells, sensory cells, transitional cells, dark cells in the vestibular end organs, vestibular ganglion cells and epithelial cells of the endolymphatic sac.

Multivariate search for differentially expressed gene combinations.

BACKGROUND: To identify differentially expressed genes, it is standard practice to test a two-sample hypothesis for each gene with a proper adjustment for multiple testing. Such tests are essentially univariate and disregard the multidimensional structure of microarray data. A more general two-sample hypothesis is formulated in terms of the joint distribution of any sub-vector of expression signals. RESULTS: By building on an earlier proposed multivariate test statistic, we propose a new algorithm for identifying differentially expressed gene combinations. The algorithm includes an improved random search procedure designed to generate candidate gene combinations of a given size. Cross-validation is used to provide replication stability of the search procedure. A permutation two-sample test is used for significance testing. We design a multiple testing procedure to control the family-wise error rate (FWER) when selecting significant combinations of genes that result from a successive selection procedure. A target set of genes is composed of all significant combinations selected via random search. CONCLUSIONS: A new algorithm has been developed to identify differentially expressed gene combinations. The performance of the proposed search-and-testing procedure has been evaluated by computer simulations and analysis of replicated Affymetrix gene array data on age-related changes in gene expression in the inner ear of CBA mice.

Expression and immunohistochemical localization of adrenomedullin in the mouse cochlea.

Adrenomedullin (AM) is a novel hypotensive and vasodilator peptide. It has been shown that AM is biosynthesized in various organs and cells and is suggested to play multiple roles including electrolyte homeostasis and body fluid control. The present study describes for the first time the presence of mRNA for AM and AM-like immunoreactivity in the cochlea. A reverse transcription-polymerase chain reaction (RT-PCR) performed with gene-specific AM primer on total RNA extracted from mouse cochlea revealed a PCR product of the expected size. Immunohistochemical examination showed positive immunostaining in vascular smooth muscle cells of the modiolar arterioles and in the stria vascularis of the cochlea. These results suggest that AM may play a role in the regulation not only of cochlear hemodynamics but also of cochlear fluid dynamics.

Immunolocalization of P2Y4 and P2Y2 purinergic receptors in strial marginal cells and vestibular dark cells.

K+ secretion by strial marginal cell and vestibular dark cell epithelia is regulated by UTP and ATP at both the apical and basolateral membranes, suggesting control by P2Y2 and/or P2Y4 purinergic receptors. Immunolocalization was used to determine the identity and distribution of these putative receptors. Membrane proteins from gerbil brain, gerbil vestibular labyrinth and gerbil stria vascularis were isolated and analyzed by Western blot. P2Y2 antibody stained one band at 42 kDa for each tissue, whereas P2Y4 antibody stained 3 bands on gerbil brain (75, 55 and 36 kDa), one band on gerbil stria vascularis (55 kDa) and two bands on vestibular labyrinth (42 and 56 kDa). All bands were absent when the antibodies were blocked with their respective antigenic peptide. P2Y4 was immunolocalized by fluorescence confocal microscopy to only the apical membrane of strial marginal cells and vestibular dark cells and was similar to apical immunostaining of KCNE1 in the same cells. By contrast, P2Y2 was observed on the basolateral but not the apical membrane of dark cells. Similarly, in the stria vascularis P2Y2 was observed in the basolateral region but not the apical membrane of marginal cells. Additional staining was observed in the spiral ligament underlying the stria vascularis. These findings identify the molecular bases of the regulation of K+ secretion by apical and basolateral UTP in the inner ear.

A role of glutamate in drug-induced ototoxicity: in vivo microdialysis study combined with on-line enzyme fluorometric detection of glutamate in the guinea pig cochlea.

The time course of the changes in perilymphatic glutamate was determined during the application of kanamycin and ethacrynic acid, which are known to damage the hair cells in the inner ear. For the continuous recording of glutamate, the microdialysis technique combined with an enzyme-linked fluorometric assay was used. In guinea pigs receiving a loading dose of 800 mg/kg of kanamycin subcutaneously followed 3 h later by an i.v. injection of 40 mg/kg of ethacrynic acid, a marked glutamate release was clearly found about 2 h after the injection of ethacrynic acid. Injection of kanamycin or ethacrynic acid alone did not produce any change in the perilymphatic glutamate. The morphological changes induced by the administration of both drugs indicated that the collapsing hair cells might release glutamate into the perilymphatic space. The present findings provide additional evidence that glutamate acts as an aggravating factor in aminoglycoside-induced ototoxicity.

Lectin binding patterns in nonsensory regions of rat cochlea during postnatal development.

The distribution of glycoconjugates was examined in the nonsensory regions of the rat cochlea during postnatal development using biotin-conjugated lectins. Temporal bones of rats at postnatal d 1 and at wk 2, 4 and 6 were fixed in 4% paraformaldehyde and 0.1% glutaraldehyde and processed for paraffin wax embedding. The dewaxed sections were incubated with 7 biotinylated lectins, followed by avidin-biotin-peroxidase complex. A different staining pattern was observed in the stria vascularis, spiral ligament and spiral limbus in the age groups examined. The staining intensity varied between lectins and the reaction product exhibited limited disparity. The staining intensity for WGA increased with age in all the 3 nonsensory regions. The staining patterns for the other lectins differed in the various nonsensory regions examined indicating tissue specificity. The limited variations in the lectin binding patterns after 2nd wk of postnatal life also indicate that the changes in the carbohydrate moieties are established during the fetal period of cochlear development and limited changes take place during postnatal maturation of the nonsensory regions.

beta2-adrenergic but not vasopressin V2 receptor mRNAs are expressed in the stria vascularis of the rat inner ear.

Activation of beta2-adrenergic receptors (beta2-AR) and of V2-vasopressin receptors (V2-VR) has been shown to stimulate cAMP production in the stria vascularis. Expression of these receptors in this epithelial structure has never been investigated at the mRNA level. A quantitative assay based on the reverse transcriptase-polymerase chain reaction (RT-PCR) method was developed to study the expression of beta2-AR, vasopressin (V1a,V1b, V2) and oxytocin receptors in rat microdissected stria vascularis fragments. Steady-state mRNA levels were measured using mutant cRNAs as internal standards. We consistently found evidence of the expression of beta2-AR transcripts in the stria vascularis; however, no such evidence of V2-VR mRNA expression was found in studies of the same samples. As our method is sensitive enough to detect 200 mRNA molecules, V2-VR mRNA is thought to be present, if at all, at a concentration that is 40 times lower than that of the beta2-AR transcripts. V1a-VR mRNA was found to be present in trace amounts and is the only subtype of the vasopressin-oxytocin receptor family expressed in this epithelium. These data demonstrate, at the mRNA level, the expression of beta2-AR in the stria vascularis, and indicate that V2-VR transcripts are not present in this structure.

Expression of the gap-junction connexins 26 and 30 in the rat cochlea.

Gap junction channels which are responsible for direct intercellular communication are composed of connexin proteins. Different connexins are distributed in a tissue-specific manner. Up to now only connexin26 has been identified to be widely expressed in the inner ear. In order to investigate the role of additional gap junction proteins, the expression of connexin30 and 43 was investigated in the rat cochlea. Connexin26 and connexin30 were both expressed in the spiral limbus, the spiral ligament, the stria vascularis and between supporting cells of the organ of Corti. Double-labeling experiments suggest that both connexins are partly colocalized between cells. Weak staining of connexin43 could only be detected in the stria vascularis, the spiral ligament and between organ of Corti supporting cells. The corresponding transcripts for connexin26, 30 and 43 could be detected by Northern blot analysis. The expression of different gap junction channels in the cochlea suggests functional diversity. Gap junctions in the inner ear may control ion concentrations of cochlear fluids or act as conduits through which glucose and other metabolites diffuse.

Use of electron spectroscopic imaging to determine element composition of the melanin granules in the stria vascularis of the guinea pig.

Electron spectroscopic imaging (ESI) was used to analyze the element content of melanin granules in the stria vascularis seen in ultrathin sections of Spurr-embedded cochleae of the guinea pig. To determine element composition, ESI images were taken at different ionization edges, and non-specific background signals were subtracted digitally by an image processing system. The presence of calcium and nitrogen in the melanin granules could be demonstrated clearly. The calcium identified in the melanin granules was then compared with the spatial distributions of calcium binding sites after the application of an antimonate precipitation method, which was used to localize loosely bound calcium. Despite a high calcium concentration within the granules, only very small single scattered calcium precipitates could be detected between these structures as compared with the amount of calcium precipitates attached to the plasma membrane or located within the cell nuclei. The nearly complete absence of precipitates within the melanin granules after the application of antimonate suggests differences in calcium binding and mobility involved in various physiological processes of ion balance regulation within the stria vascularis.

Expression of growth factors and their receptors in the postnatal rat cochlea.

RT-PCR was used to assay for growth factors and receptors from seven different protein families in cochlea tissues of the juvenile rat. There was a broad representation of the growth factor families in all the cochlea tissues examined, though the organ of Corti and stria vascularis expressed a greater variety than the spiral ganglion. This broad expression suggests that a variety of known growth factors play significant roles in the development, maintenance, and repair of the inner ear. The results of this survey serve as a basis for the design of future in vitro experiments that will address the ability of growth factors to protect hair cells from damage and to evoke a repair-regeneration response by injured hair cells.