Dexamethasone protects organ of corti explants against tumor necrosis factor-alpha-induced loss of auditory hair cells and alters the expression levels of apoptosis-related genes.
OBJECTIVE
Determine the molecular mechanism(s) behind tumor necrosis factor-alpha (TNFalpha)-induced loss of auditory hair cells and the ability of dexamethasone base (DXMb) to protect against TNFalpha ototoxicity.


METHODS
Hair cell counts: Three-day-old rat organ of Corti explants were cultured under three different conditions: 1) untreated-control; 2) TNFalpha (2 mug/ml); and 3) TNFalpha (2 mug/ml)+DXMb (70 mug/ml) for 4 days, fixed, and stained with FITC-phalloidin. Hair cells were counted in the basal and middle turns. Gene expression: total RNA was extracted from the three different groups of explants at 0, 12, 24 and 48 h. Using quantitative real-time RT-PCR, mRNAs were transcribed into cDNAs and amplification was performed using primers for rat ss-actin (housekeeping gene), TNFR1, Bcl-2, Bax, and Bcl-xl.


RESULTS
DXMb protected explant hair cells from TNFalpha-induced loss. Bax gene expression was greater in TNFalpha-exposed explants compared with TNFalpha+DXMb-treated explants at 48 h (P=0.023), confirmed by the increase in the Bax/Bcl-2 ratio at 48 h (P<0.001). These results correlated with increased TNFR1 expression at 24 h (P=0.038). DXMb otoprotection in TNFalpha-exposed cultures was accompanied by an up-regulation of Bcl-xl at both the 24 (P<0.001) and 48 h time points (P=0.030) and up-regulation of Bcl-2 expression at 24 h (P=0.018). DXMb treatment also prevented increases in the expression levels of Bax, TNFR1, and the Bax/Bcl-2 ratio that occurred in untreated TNFalpha-exposed explants.


CONCLUSIONS
TNFalpha's ototoxicity may be mediated through an up-regulation of Bax and TNFR1 expression as well as an increase in the Bax/Bcl-2 ratio. DXMb protects the organ of Corti against TNFalpha ototoxicity by up-regulating Bcl-2 and Bcl-xl expression and by inhibiting TNFalpha-induced increases in Bax, TNFR1, and the Bax/Bcl-2 ratio. These results support the use of local dexamethasone treatment to conserve hearing following a trauma.
Cochlear Implant Research Program, University of Miami Ear Institute, Department of Otolaryngology, University of Miami, Miller School of Medicine, 1600 Northwest 10th Avenue, RMSB 3160, Miami, FL 33136-1015, USA.
C T
CT
Dinh
S
S
Haake
S
S
Chen
K
K
Hoang
E
E
Nong
A A
AA
Eshraghi
T J
TJ
Balkany
T R
TR
Van De Water
D000704:Analysis of Variance; D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis / Q000187:drug effects* / Q000235:genetics; D003907:Dexamethasone / Q000494:pharmacology*; D015870:Gene Expression / Q000187:drug effects*; D005938:Glucocorticoids / Q000494:pharmacology*; D006198:Hair Cells, Auditory / Q000187:drug effects*; D009924:Organ Culture Techniques; D009925:Organ of Corti / Q000166:cytology* / Q000187:drug effects; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000235:genetics / Q000378:metabolism; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000633:toxicity; D051028:bcl-2-Associated X Protein / Q000235:genetics / Q000378:metabolism; D051020:bcl-X Protein / Q000235:genetics / Q000378:metabolism
D005938:Glucocorticoids; D019253:Proto-Oncogene Proteins c-bcl-2; D047888:Receptors, Tumor Necrosis Factor, Type I; D014409:Tumor Necrosis Factor-alpha; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein; D003907:Dexamethasone