Julia Fuchs

Argon protects hypoxia-, cisplatin- and gentamycin-exposed hair cells in the newborn rat's organ of Corti

During the last few years, an important protective effect of the noble gas xenon against neuronal hypoxic damage was observed. However, argon (Ar), a gas from the same chemical group, but less expensive and without anesthetic effect at normobaric pressure, has not been studied in terms of possible biological effects on cell protection. Ar was tested for its ability to protect organotypic cultures of the organ of Corti from 3-5 day old rats against hypoxia, cisplatin, and gentamycin toxicity. Cultures were exposed to nitrogen hypoxia (5% CO2, 95% N2), Ar hypoxia (5% CO2, 95% Ar) or normoxia for 30 h. Ar protected the hair cells from hypoxia-induced damage by about 25%. Ar-oxygen (O2) mixtures (21% O2, 5% CO2, 74% Ar) had no effect on the hair cell survival. Cisplatin (7.5-25 microM) and gentamycin (5-40 microM) exposed in medium under air damaged the hair cells in a dose-dependent manner. The exposure of cisplatin- and gentamycin-treated cultures to the Ar-O2 atmosphere significantly reduced the hair cell damage by up to 25%. This protective effect of Ar might provide a new protective approach against ototoxic processes.

Expression of hypoxia-inducible factor-1 in the cochlea of newborn rats.

Hypoxia/ischemia is a major pathogenetic factor in the development of hearing loss. An important transcription factor involved in the signaling and adaptation to hypoxia/ischemia is the hypoxia-inducible factor-1 (HIF-1). To study HIF-1 expression we used an in vitro hypoxia model of explant and dissociated cultures of the stria vascularis, the organ of Corti with limbus and the modiolus from the cochlea of 3-5-day-old Wistar rats. Hypoxia differentially increased HIF-1 activity as measured by a reporter gene. Twenty-four hour hypoxia increased HIF-1 activity 14.1+/-3.5-fold in the modiolus, 9.4+/-3.0-fold in the organ of Corti with limbus, and 6.4+/-1.5-fold in the stria vascularis. The HIF-1alpha mRNA level was measured by quantitative reverse transcription polymerase chain reaction and showed a lower expression in the modiolus (1.3+/-0.2 pg/microg RNA) than in both the organ of Corti with limbus and the stria vascularis (2.7-3.2+/-1.3, P<0.01). Hypoxia had no effect on the HIF-1alpha mRNA levels. The region-specific regulation of HIF-1 expression on the transcriptional and posttranslational levels may expand the possibilities for adaptation of the cochlea to hypoxia.

Expression of apoptosis-related genes in the organ of Corti, modiolus and stria vascularis of newborn rats

Cell death in the inner ear tissues is an important mechanism leading to hearing impairment. Here, using microarrays and real-time RT-PCR we analyzed expression of selected apoptosis-related genes in rats inner ear. We determined the gene expression in tissues freshly isolated from neonatal rats (3-5 days old) and compared it to that of explants cultured for 24 h under normoxic or hypoxic conditions. For the analyses, we used pooled samples of the organ of Corti (OC), modiolus (MOD) and stria vascularis (SV), respectively. We observed region-specific changes in gene expression between the fresh tissues and the normoxic culture. In the OC, expression of the proapoptotic genes caspase-2, caspase-3, caspase-6 and calpain-1 was downregulated. In the MOD, the antioxidative defense SOD-2 and SOD-3 were upregulated. In the SV, caspase-2, caspase-6, calpain-1 and SOD-3 were downregulated and SOD-2 upregulated. We speculate that these changes could reflect survival shift in transcriptome of inner ear explants tissues under in vitro conditions. With the exception of SOD-2, hypoxic culture conditions induced the same changes in gene expression as the normoxic conditions indicating that culture preparation is likely the dominating factor, which modifies the gene expression pattern. We conclude that various culture conditions induce different expression pattern of apoptosis-related genes in the organotypic cochlear cultures, as compared to fresh tissues. This transcriptional pattern may reflect the survival ability of specific tissues and could become a tempting target for a pharmacological intervention in inner ear diseases.

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 24 h 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 transporter1 (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).

Role of mitochondrial uncoupling protein 4 in rat inner ear

The uncoupling protein 4 (UCP4) belongs to the mitochondrial anion transporter family. Protein tissue distribution and functions are still a matter of debate. Using an antibody we have previously shown that UCP4 appears in neurons and to a lesser extent in astrocytes of murine neuronal tissue as early as days 12-14 of embryonic development (Smorodchenko et al., 2009). Here we demonstrated for the first time that neurosensory cells such as hair cells of the inner ear and mechanosensitive Merkel cells in skin also express a significant amount of UCP4. We tested the hypothesis about whether UCP4 contributes to the regulation of oxidative stress using the model of oxygen deprivation. For this we compared the protein expression level in freshly isolated explants of organ of Corti, modiolus and stria vascularis from neonatal rats with explants cultured under hypoxia. Western blot analysis revealed that the UCP4 level was not increased under hypoxic conditions, when compared to the mitochondrial outer membrane protein VDAC or to the anti-oxidative enzyme SOD2. We moreover demonstrated that UCP4 expression is differently regulated during postnatal stages and is region-specific. We hypothesized that UCP4 may play an important role in functional maturation of the rat inner ear.

Effects of retinoic acid and butyric acid on the expression of prestin and gata-3 in organotypic cultures of the organ of corti of newborn rats

Prestin is the motor protein of the outer hair cells of the organ of Corti and a key factor in ensuring a high level of sensitivity of mammalian hearing. The factors that influence prestin expression are still largely unknown. We studied the effects of the application of retinoic acid, a ligand of a nuclear receptor, and of butyric acid, an inhibitor of histone deacetylase activity, on the expression of mRNA of prestin and Gata‐3 in the organotypic culture of the organ of Corti of newborn rats using RT‐PCR. Application of retinoic acid at concentrations of 1–50 μM results in a dose‐dependent expression decrease after two days in culture. Treatment with sodium butyrate (0.5–2 mM) elevated the expression of prestin and Gata‐3. Statistically significant correlations between Gata‐3 and prestin mRNA levels were observed under all conditions. The data indicate that retinoid nuclear transcription factors, GATA‐3 and histone acetylation/deacetylation processes may have a regulatory role to play in prestin expression.

Expression of prestin and Gata-3, -2, -1 mRNA in the rat organ of Corti during the postnatal period and in culture

Based on observations that mutations of GATA-3 are responsible for the HDR-syndrome (hypoparathyroidism, deafness, renal defects) and that GATA-transcription factors have an important role to play in inner ear development, we hypothesized that these transcription factors may be involved in regulatory changes of prestin transcription. To prove this, we examined in parallel the expression of mRNA of prestin and Gata-3,-2 and Gata-1 in the organ of Corti during early postnatal development of rats and in organotypic cultures. Remarkable relations are observed between prestin and Gata-3,-2 expression in organ of Corti preparations in vivo and in vitro: (i) Gata-3,-2 expression display similar apical-basal gradients as prestin mRNA levels. (ii) The prestin expression increases between postnatal day two and postnatal day eight by a factor of about four in the apical and middle segments and by a factor of two in the basal part. Highly significant Pearson correlation coefficients were observed between Gata-3,-2 mRNA and prestin levels when the data were evaluated by regression analyses. (iii) Parallel changes of prestin mRNA and Gata-3,-2 mRNA levels were observed in response to thyroid hormone and to gemfibrozil application. These observations suggest a regulatory role played by the Gata-3,-2 transcription factors in prestin expression.

Decrease of prestin expression by increased potassium concentration in organotypic cultures of the organ of Corti of newborn rats.

Prestin is the motor protein of the outer hair cells of the organ of Corti and a key factor in ensuring a high sensitivity level of mammalian hearing. In the present study, we examined the effects of increased extracellular potassium (K(+)) concentration on the expression of prestin mRNA and the transcription factors Gata-3 and Carf in the organotypic culture of the organ of Corti of newborn rats. Mannitol and NaCl were used to analyze possible effects of hyperosmotic stress or ion-specific changes, respectively. An increase in prestin expression by a factor of 1.5-2.0 was seen in cultures grown in the presence of 5mM K(+). Potassium concentration of 35 and 55 mM induced a parallel decrease in prestin and Carf expression, but Gata-3 expression increased. Mannitol had no effect on gene expression whereas increased NaCl concentrations decreased prestin, but not Carf expression. The data suggest that chronic depolarization might decrease the prestin expression and possibly contribute to hearing loss and tinnitus.