Short Interfering RNA against Bax Attenuates TNFα-Induced Ototoxicity in Rat Organ of Corti Explants

Abstract

Objective

Evaluate the effectiveness of short interfering RNA against Bax (Bax siRNA) as a treatment for tumor necrosis factor α (TNFα)–induced auditory hair cell (HC) loss in rat organ of Corti (OC) explants.

Study Design

Basic science.

Setting

Basic science laboratory, University of Miami Ear Institute.

Methods

Organ of Corti explants dissected from 3-day-old rats were cultured in control media, TNFα, and TNFα + Bax siRNA at 0, 48, and 72 hours in vitro. Real-time polymerase chain reaction, enzyme-linked immunosorbent assay, HC viability studies, immunofluorescence, and confocal microscopy were performed. Analysis of variance with post hoc testing was used with P < .05 considered significant.

Results

The TNFα-damaged explants demonstrated significant decreases in viable HCs in the basal turn with corresponding increased levels of Bax gene and protein expression, compared with control explant levels. The levels of viable HCs and Bax gene and protein expression in TNFα + Bax siRNA-treated explants approached levels measured in control explants. Immunolocalization studies showed increased Bax protein expression in basal turn HCs in TNFα-treated explants, whereas control explants and TNFα + Bax siRNA–treated explants had low levels of Bax expression.

Conclusion

TNFα initiates the programmed cell death of auditory HCs in OC explants through upregulation of proapoptotic Bax gene and protein expression. Bax siRNA blocks TNFα-induced apoptosis of HCs by decreasing the TNFα-induced levels of Bax mRNA and protein expression in treated explants. Bax siRNA is an effective treatment for TNFα-induced ototoxicity in OC explants in vitro and has great potential to be a therapeutic agent against trauma/inflammation-induced hearing loss.

Keywords

apoptosis auditory hair cell tumor necrosis factor Bax programmed cell death inner ear

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References

Aminpour

Tinling

Brodie

. Role of tumor necrosis factor–alpha in sensorineural hearing loss after bacterial meningitis. Otol Neurotol. 2005;26:602-609.

Van Wijk

Staecker

Keithley

Lefebvre

. Local perfusion of tumor necrosis factor alpha blocker infliximab to the inner ear improves autoimmune neurosensory hearing loss. Audiol Neurootol. 2006;11:357-365.

Zou

Pyykkö

Sutinen

Toppila

. Vibration induced hearing loss in guinea pig cochlea: expression of TNF-alpha and VEGF. Hear Res. 2005;202:13-20.

Fujioka

Kanzaki

Okano

Masuda

Ogawa

Okano

. Proinflammatory cytokines expression in noise-induced damaged cochlea. J Neurosci Res. 2006;83:575-583.

Street

Jobanputra

Proops

. Etanercept, a tumour necrosis factor alpha receptor antagonist, and methotrexate in acute sensorineural hearing loss. J Laryngol Otol. 2006;120:1064-1066.

Kim

Lee

. Cisplatin cytotoxicity of auditory cells requires secretions of proinflammatory cytokines via activation of ERK and NF-κB. J Assoc Res Otolaryngol. 2007;8:338-355.

Sutinen

Zou

Hunter

Toppila

Pyykkö

. Vibration-induced hearing loss: mechanical and physiological aspects. Otol Neurotol. 2007;28:171-177.

Khan

Szczepek

Haupt

Olze

Mazurek

. Expression of proinflammatory cytokines in cochlear explant cultures: influence of normoxia and hypoxia. Neurosci Lett. 2010;479:249-252.

Van De Water

Lallemend

Eshraghi

. Caspases, the enemy within, and their role in oxidative stress–induced apoptosis of inner ear sensory cells. Otol Neurotol. 2004;25:627-632.

10.

Wang

Ruel

Ladrech

Bonny

Van De Water

Puel

. Inhibition of the c-Jun-N-terminal kinase–mediated mitochondrial cell death pathway restores auditory function in sound exposed animals. Mol Pharmacol. 2007;71:654-666.

11.

Dinh

Haake

Chen

. 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. Neuroscience. 2008;157:405-413.

12.

Dinh

Hoang

Haake

. Biopolymer-released dexamethasone prevents tumor necrosis factor alpha–induced loss of auditory hair cells in vitro: implications toward the development of a drug-eluting cochlear implant electrode array. Otol Neurotol. 2008;29:1012-1019.

13.

Haake

Dinh

Chen

Eshraghi

Van De Water

. Dexamethasone protects auditory hair cells against TNFalpha-initiated apoptosis via activation of PI3K/Akt and NFkappaB signaling. Hear Res. 2009;255:22-32.

14.

Dinh

Bas

Chan

. Dexamethasone treatment of tumor necrosis factor–alpha challenged organ of Corti explants activates nuclear factor kappa B signaling that induces changes in gene expression that favor hair cell survival. Neuroscience. 2011;188:157-167.

15.

Maeda

Sheffield

Smith

RJH

. The therapeutic regulation of gene expression in the inner ear using RNA interference. Adv Otorhinolaryngol. 2009;66:13-36.

16.

Hannon

. RNA interference. Nature. 2002;418:244-251.

17.

Maeda

Fukushima

Nishizaki

Smith

. In vitro and in vivo suppression of GJB2 expression by RNA interference. Hum Mol Genet. 2005;14:1641-1650.

18.

Mukherjea

Jajoo

Kaur

Sheehan

Ramkumar

Rybak

. Transtympanic administration of short interfering (si)RNA for the NOX3 isoform of NADPH oxidase protects against cisplatin-induced hearing loss in the rat. Antioxid Redox Signal. 2011;13:589-598.

19.

Mukherjea

Jajoo

Whitworth

. Short interfering RNA against transient receptor potential vanilloid 1 attenuates cisplatin-induced hearing loss in the rat. J Neurosci. 2008;28:13056-13065.

20.

Kaur

Mukherjea

Sheehan

Jajoo

Rybak

Ramkumar

. Short interfering RNA against STAT1 attenuates cisplatin-induced ototoxicity in the rat by suppressing inflammation. Cell Death Dis. 2011;2:e180.

21.

Mukherjea

Jajoo, Sheehan

. NOX3 NAPDH oxidase couples transient receptor potential vanilloid 1 to signal transducer and activator of transcription 1–mediated inflammation and hearing loss. Antioxid Redox Signal. 2011;14:999-1010.

22.

Livak

Schmittgen

. Analysis of relative gene expression data using real-time quantitative PCR and the 2(–Delta Delta C(T)) method. Methods. 2001;25:402-408.

23.

Wolter

Hsu

Smith

Nechushtan

Youle

. Movement of Bax from the cytosol to mitochondria during apoptosis. J Cell Biol. 1997;139:1281-1292.