Dexamethasone treatment of naïve organ of Corti explants alters the expression pattern of apoptosis-related genes.
BACKGROUND
Dexamethasone treatment of organ of Corti explants challenged with an ototoxic level of an inflammatory cytokine modulates NFkappaB signaling and the expression levels of both pro-and anti-apoptosis-related genes. It is not known if naïve organ of Corti explants will respond in a similar manner to treatment with a corticosteroid. This study examines the response of naïve organ of Corti explants to treatment with dexamethasone.


METHODS
Three-day-old rat organ of Corti explants were cultured for 1, 2, or 4 days. Four-day in vitro cultures were fixed, stained with FITC-phalloidin and hair cells were counted. ELISA was performed on 2-day cultures to determine the levels of phosphorylated nuclear factor kappa B protein. One- and 2-day cultures were studied with real-time RT-PCR for expression levels of beta-actin, Bax, Bcl-xl, Bcl-2 and TNFR1 genes with mean fold changes determined with the 2(-)(DeltaDeltaCt) method. All mean fold changes in gene and protein expression were analyzed by the Kruskal-Wallis non-parametric test.


RESULTS
There were no significant differences in hair cell counts between naïve explants and explants treated with dexamethasone. Dexamethasone treatment of naïve explants resulted in a significant increase (p<0.01) in the level of phosphorylated-nuclear factor kappa B protein. Bax expression was significantly decreased (p<0.01) in the dexamethasone-treated explants compared to untreated-naïve explants at 1 and 2 days. TNFR1 expression was significantly reduced in dexamethasone-treated explants at 1 (p<0.01) and 2 days (p=0.001). Both Bcl-2 and Bcl-xl expression levels were significantly increased in dexamethasone-treated cultures compared to naïve-cultures at 2 days in vitro (p<0.001). Dexamethasone-treated explants showed a significant decrease in the Bax/Bcl-2 ratio at both 1 (p=0.004) and 2 days (p<0.001) in vitro.
Hoang
Kimberly N
KN
University of Miami Ear Institute, Department of Otolaryngology, University of Miami, FL 33136-1015, USA.
Dinh
Christine T
CT
Bas
Esperanza
E
Chen
Shibing
S
Eshraghi
Adrien A
AA
Van De Water
Thomas R
TR
D000704:Analysis of Variance; D000818:Animals; D000831:Animals, Newborn; D000893:Anti-Inflammatory Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects* / Q000235:genetics; D002452:Cell Count; D003907:Dexamethasone / Q000494:pharmacology*; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression / Q000187:drug effects*; D016328:NF-kappa B / Q000378:metabolism; D009924:Organ Culture Techniques; D009925:Organ of Corti / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D010766:Phosphorylation; 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; D020133:Reverse Transcriptase Polymerase Chain Reaction; D018709:Statistics, Nonparametric; D013997:Time Factors; D051028:bcl-2-Associated X Protein / Q000235:genetics / Q000378:metabolism; D051020:bcl-X Protein / Q000235:genetics / Q000378:metabolism
D000893:Anti-Inflammatory Agents; D016328:NF-kappa B; D019253:Proto-Oncogene Proteins c-bcl-2; D047888:Receptors, Tumor Necrosis Factor, Type I; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein; D003907:Dexamethasone