Expression of genes implicated in oxidative stress in the cochlea of newborn rats.

Oxidative stress is an important mechanism inducing ototoxicity-, age- and noise-induced hearing loss. To better understand this phenomenon, we examined cochlear tissues for the expression of following genes involved directly or indirectly in the oxidative stress response: glyceraldehyde-3-phosphate dehydrogenase (Gapdh); solute carrier family-2 (facilitated glucose transporter), member-1 (Slc2a1); heme oxygenase-1 (Hmox1); heme oxygenase-2 (Hmox2); inducible nitric oxide synthase-2 (Nos2); transferrin (Tf); transferrin receptor (Tfrc); glutathione S-transferase A3 (Gsta3) and metallothionein-1a (Mt1a). Cochlear tissues were dissected from the p3-p5 Wistar rats, divided into the organ of Corti (OC), modiolus (MOD) and stria vascularis together with spiral ligament (SV + SL) and processed immediately or cultured under normoxic conditions or a short-term, mild hypoxia followed by re-oxygenation. After 24 h, explants were collected and total RNA isolated, transcribed and amplified in the real time RT-PCR. We found all genes listed above expressed in the freshly isolated cochlear tissues. In the OC and MOD, Slc2a1, Tf, and Mt1a were expressed on a lower level than in the SV + SL. In the OC, Hmox1 was expressed on a lower level than in the MOD and SV + SL. Hypoxic and normoxic cultures increased the transcript number of Gapdh, Slc2a1 and Hmox1 in all cochlear tissues. The expression of Nos2, Tf, Gsta3 and Mt1a increased in a tissue-specific manner. In the SV + SL, Mt1a expression decreased after normoxic and hypoxic conditions. Taken together, using real time RT-PCR, our results imply that oxidative stress may be an important component of cochlear injury during the developing period. In spite of the immaturity of the tissue, a differential response of antioxidant enzymes/proteins with respect to the pathway, the expression levels and regions was observed.

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).

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 rat's 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.

Up-regulation of prestin mRNA expression in the organs of Corti of guinea pigs and rats following unilateral impulse noise exposure.

Prestin is the motor protein of the outer hair cells (OHCs) and is required for both their electromotility and for cochlear amplification. We investigated the prestin mRNA expression in guinea pigs and rats in relation to the degree of noise-induced hearing loss (NIHL) induced by unilateral impulse noise exposure (167dB peak SPL) for 2.5-5 min. Distortion product otoacoustic emissions (DPOAE) and auditory brainstem responses were recorded before and one week post exposure. Prestin mRNA was examined by quantitative reverse transcription-polymerase chain reaction. Either the whole organs of Corti or the apical, middle and basal parts were examined separately. The specimens were pooled and grouped according to the degree of NIHL measured in the exposed ears. In rats, the number of hair cells was counted. A clear base-to-apex gradient in the prestin mRNA expression was found to exist in guinea pig and rat controls. In both species, there was an increase in the number of prestin RNA transcripts at a mean NIHL of about 15-25 dB indicating an up-regulation in the remaining intact cells. In rats, this degree of NIHL corresponded to an OHC loss of about 40%. Interestingly, the contralateral ears also revealed an up-regulation of prestin mRNA accompanied by significant DPOAE improvements.

Apoptosis, necrosis and hypoxia inducible factor-1 in human head and neck squamous cell carcinoma cultures.

The objective of this study was to examine the mode of cell death and the hypoxia inducible factor-1 (HIF-1) expression of human head and neck squamous cell carcinoma (HNSCC) exposed to hypoxia in vitro. Apoptosis and necrosis rates were examined using flow cytometry. The findings suggest that HNSCC cells show a considerable heterogeneity in cell size and in response to hypoxia. A small-cell population showed a high spontaneous apoptosis and necrosis rate which was in-sensitive to hypoxia. A large-cell population responded to hypoxia by increase of apoptosis rate in parallel to recruitment of HIF-1. Hypoxia led to increased HIF-1alpha protein levels in nuclear extract using ELISA-binding activity. In all cells, accumulation of HIF-1 in the nuclei during hypoxia and a rapid degradation of HIF-1 in the post-hypoxic period were observed immunocytochemically. The HIF-1alpha mRNA level showed an expression of 10-40 pg/microg total RNA and remained unchanged in one cell line, while slightly decreasing in the other. Remarkably, no increased luciferase activity response was found on the reporter gene level using pGL3 reporter gene with three erythropoietin hypoxia responsive elements, either by hypoxia or by application of lactacystin, desferrioxamine or CoCl2. These findings suggest that, in HNSCC cells, hypoxia induces HIF-1alpha to stabilize and accumulate in the cell nuclei but have a cell-specific transcriptional complex.

Expression of prestin mRNA in the organotypic culture of rat cochlea.

To quantitate in absolute terms the prestin mRNA levels in the explant culture of rat cochlea, we used competitive RT-PCR with a synthetic internal cRNA standard. Prestin gene expression was found at levels of 100 fg specific mRNA/microg total RNA on postnatal day 3, which corresponds to about 300 copies per outer hair cell (OHC) and is indicative of an intermediate level of expression. Two days of culturing resulted in an increase of prestin mRNA levels and in the formation of an apical-basal gradient (p<0.001). To elucidate the variations the prestin mRNA levels undergo as a result of damage to the organ of Corti, we exposed the explant cultures to ischemia and hypoxia. While total RNA was observed to remain unchanged, the numbers of OHCs and the prestin mRNA levels were found to decrease by about 20% and 35%, respectively, compared to normoxia. In conclusion, we showed that the prestin mRNA levels during in vitro development increase and form an apical-basal gradient within 2 days in culture, similar to the postnatal in vivo development. Hypoxia and ischemia result in a decrease of the prestin mRNA level in parallel with OHC loss. The prestin mRNA level can therefore be used as marker of damage to or loss of OHCs.

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.