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

Abstract

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).

Gene Expression Patterns in Functionally Different Cochlear Compartments of the Newborn Rat

“…In freshly prepared tissue, about 2-10 % of all nuclei were found to be stained by propidium iodide, indicating cell damage during preparation of the cochlear tissue. After 24h in culture, the number of necrotic cells in the MOD region increased from 8 % to 25-35 %), whereas the number of such cells remained unchanged in the OC and SV regions (Gross et al, 2008). Gene expression markers indicate that two basic pathogenetic mechanisms are involved in this experimental model: mechanically induced inflammation and hypoxia.…”

“…Fragments of one ear were kept in culture under normoxic conditions, fragments of the second ear were exposed to moderate hypoxia (oxygen partial pressure inside the culture medium was 10-20 mm Hg) for 5 h, starting three hours after plating. The number of dead cells was determined in freshly prepared tissue (controls) and after 24 h in culture using the live/dead viability test by propidium iodide (PI) and calcein AM staining (Gross et al, 2008).…”

Expression of nestin-associated genes in the inner ear of newborn rats following injury and hypoxia

“…B: Propidium iodide (PI)-stained nuclei. Data from Gross et al (2008) [5] ; data indicate mean and SEM (% PI-cells, n = 12 for MOD, n = 4 for SV and OC). *p<0.01, **p>0.001.…”

“…Analysis of gene expression showed that two basic pathogenetic mechanisms are involved in this experimental model: mechanically induced inflammation and hypoxia [4] . To compare the expression changes with the cell death rate, we in parallel determined the proportion of dead cells immediately after tissue preparation and after 24 h in culture [5] . After 24 h, the cell death rate increased to about 25-30 % in the MOD, but did not change significantly in the OC and SV regions (Fig.…”

“…Briefly, the membranous cochleae from 3-5 day-old Wistar rats were dissected into the OC, MOD and SV regions and exposed to normoxic and hypoxic conditions for 24 h. For culture, the cochlear parts were incubated in 4-well tissue culture dishes. Three hours after plating, the cultures were exposed to hypoxia in an incubation chamber for 5 h. The number of dead cells was determined in freshly prepared tissue (controls) and after 24 h in culture using the live/dead viability test by propidium iodide (PI) and calcein AM staining [5] . The preparation mode is based on the assumption that the MOD region contains the cell bodies of the SGNs, the Schwann cells and parts of the peripheral and central processes, whereas the OC region contains hair cells, supporting cells and parts of peripheral processes of the SGNs.…”