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.
Gross
Johann
J
Molecular Biological Research Laboratory, Department of Otorhinolaryngology, Humboldt University, Charité Hospital, Spandauer Damm 130, 14050 Berlin, Germany. johann.gross@charite.de
Machulik
Astrid
A
Amarjargal
Nyamaa
N
Fuchs
Julia
J
Mazurek
Birgit
B
D000818:Animals; D000831:Animals, Newborn; D027321:Anion Transport Proteins; D015415:Biomarkers; D003051:Cochlea / Q000098:blood supply / Q000254:growth & development / Q000378:metabolism* / Q000473:pathology; D015870:Gene Expression; D018072:Hair Cells, Auditory, Outer / Q000098:blood supply / Q000254:growth & development / Q000378:metabolism / Q000473:pathology; D000860:Hypoxia / Q000473:pathology / Q000503:physiopathology; D007511:Ischemia / Q000473:pathology / Q000503:physiopathology; D009924:Organ Culture Techniques; D009925:Organ of Corti / Q000098:blood supply / Q000254:growth & development / Q000378:metabolism / Q000473:pathology; D011506:Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000076988:Sulfate Transporters
D027321:Anion Transport Proteins; D015415:Biomarkers; D011506:Proteins; D012333:RNA, Messenger; C409207:Slc26a5 protein, rat; D000076988:Sulfate Transporters