Christine T. Dinh

Dexamethasone protects auditory hair cells against TNFα-initiated apoptosis via activation of PI3K/Akt and NFκB signaling

BACKGROUND Tumor necrosis factor alpha (TNFalpha) is associated with trauma-induced hearing loss. Local treatment of cochleae of trauma-exposed animals with a glucocorticoid is effective in reducing the level of hearing loss that occurs post-trauma (e.g., electrode insertion trauma-induced hearing loss/dexamethasone treatment). HYPOTHESIS Dexamethasone (Dex) protects auditory hair cells (AHCs) from trauma-induced loss by activating cellular signal pathways that promote cell survival. MATERIALS AND METHODS Organ of Corti explants challenged with an ototoxic level of TNFalpha was the trauma model with Dex the otoprotective drug. A series of inhibitors were used in combination with the Dex treatment of TNFalpha-exposed explants to investigate the signal molecules that participate in Dex-mediated otoprotection. The otoprotective capacity of Dex against TNFalpha ototoxicity was determined by hair cell counts obtained from fixed explants stained with FITC-phalloidin labeling with investigators blinded to specimen identity. RESULTS The general caspase inhibitor Boc-d-fmk prevented TNFalpha-induced AHC death. There was a significant reduction (p<0.05) in the efficacy of Dex otoprotection against TNFalpha ototoxicity when the following cellular events were blocked: (1) glucocorticoid receptors (Mif); (2) PI3K (LY294002); (3) Akt/PKB (SH-6); and (4) NFkappaB (NFkappaB-I). CONCLUSION Dex treatment protects hair cells against TNFalpha apoptosis in vitro by activation of PI3K/Akt and NFkappaB signaling.

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 TNFalphas 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.

Mechanisms of hearing loss from trauma and inflammation: otoprotective therapies from the laboratory to the clinic.

This article reviews a series of in vitro and in vivo studies that examined the otoprotective efficacy of locally delivered dexamethasone and explored the mechanisms by which dexamethasone protects auditory hair cells. These studies used auditory threshold testing in response to pure tone stimuli, organ of Corti explant cultures, FITC-phalloidin-stained explants, and surface preparations to determine hair cell density, osmotic pump delivery of dexamethasone into the scala tympani, an animal model of electrode insertion trauma (EIT)-induced hearing loss, and real-time RT-PCR studies of gene expression levels. Local delivery of two different formulations of dexamethasone conserved hearing and protected hair cells in an animal model of cochlear implantation. Dexamethasone treatment protected hair cells in organ of Corti explants exposed to an ototoxic level of an inflammatory cytokine, and gene expression studies showed that this protection was accomplished by increased expression levels of anti-apoptosis genes (e.g. Bcl-2) and decreased levels of pro-apoptosis genes (e.g. Bax). We conclude that dexamethasone is an effective otoprotective drug for both the conservation of hearing and preservation of hair cells against trauma-induced losses. Locally delivered dexamethasone is a promising therapeutic approach for the conservation of hearing during the process of cochlear implantation.

Biopolymer-released dexamethasone prevents tumor necrosis factor α-induced loss of auditory hair cells in vitro: Implications toward the development of a drug-eluting cochlear implant electrode array

Hypothesis: Polymer-eluted dexamethasone (DXM) will retain its ability to protect against tumor necrosis factor &agr; (TNF&agr;)-induced hair cell (HC) loss. Background: TNF&agr; has been shown to be associated with trauma-induced hearing loss. DXM has been demonstrated to protect the cochlea against trauma-induced hearing loss. DXM is currently administered either systemically or locally to treat patients with sudden hearing loss of unknown cause. Methods: P-3 organ of Corti explants challenged with an ototoxic level of TNF&agr; was the experimental system, and the base form of DXM (DXMb) incorporated into a biorelease polymer (i.e., SIBS) was the otoprotection molecule tested. The efficacy of otoprotection was determined by counts of fluorescein isothiocyanate-phalloidin-stained HCs and changes in gene expression. Results: HC counts show 1) SIBS alone did not protect HCs from TNF&agr; ototoxicity (SIBS versus SIBS + TNF&agr;; p < 0.001), and 2) SIBS with DXMb provides a significant level of protection against TNF&agr;-induced loss of HCs (TNF&agr; + SIBS versus TNF&agr; + SIBS/DXMb, 299 &mgr;g; p < 0.001). Gene expression results show that polymer-eluted DXMb 1) upregulates antiapoptotic genes (i.e., Bcl-2, Bcl-xl) and downregulates a proapoptotic gene (i.e., Bax) in TNF&agr;-challenged explants and 2) downregulates TNFR1 in these explants. Conclusion: Polymer-eluted DXMb retains its otoprotection capabilities in our in vitro test system of TNF&agr;-challenged organ of Corti explants by altering the pattern of gene expression to favor survival of TNF&agr;-exposed HCs. These results, although in vitro, support the application of polymer containing DXMb to electrode arrays for the conservation of hearing during cochlear implantation.

Blocking Pro-Cell-Death Signal Pathways to Conserve Hearing

The programmed cell death of stress-damaged auditory hair cells can occur through a variety of signal pathways, and therapeutic modalities that block pro-cell-death pathways are being developed and evaluated for hearing preservation. Because of their ability to have both anti-inflammatory and anti-apoptotic actions, corticosteroids have long been used to protect against several types of acute sensorineural hearing loss. Other anti-apoptotic drugs that target the mitogen-activated protein kinase (MAPK)/c-Jun-N terminal kinase (JNK) signal cascade, such as D-JNKI-1 (AM-111) and SP600125, have produced promising results both in vitro and in laboratory animal studies, with AM-111 showing promise in preliminary clinical trials. Antioxidant drugs, e.g. sodium thiosulfate, N-acetylcysteine, and D-methionine, have been shown in animal studies to attenuate permanent threshold shifts in hearing by reducing oxidative stress. In addition to reviewing selected therapeutic trends for the conservation of hearing, we review our experiences with dexamethasone and D-JNKI-1 and report results from our current research.

Conservation of hearing and protection of hair cells in cochlear implant patients' with residual hearing

This review covers the molecular mechanisms involved in hair cell and hearing losses which can result from trauma generated during the process of cochlear implantation and the contributions of both the intrinsic and extrinsic cell death signaling pathways in producing these trauma/inflammation induced losses. Application of soft surgical techniques to conserve hearing and protect auditory sensory cells during the process of cochlear implantation surgery and insertion of the electrode array during the process of cochlear implantation are reviewed and discussed. The role of drug therapy and mode of drug delivery for the conservation of a cochlear implant patients residual hearing is presented and discussed. Anat Rec, 2012.

Conservation of hearing and protection of auditory hair cells against trauma-induced losses by local dexamethasone therapy: molecular and genetic mechanisms.

Abstract Hypothesis: Dexamethasone (DXM) protects hearing against trauma-induced loss. Materials: in vivo: A guinea pig model of electrode induced trauma (EIT)-induced hearing loss was used to locally deliver dexamethasone. In vitro: TNF-α-challenged organ of Corti explants treated with DXM or polymer-eluted DXM +/− PI3K/Akt/PkB/NFkB inhibitors were used for hair cells count and gene expression studies. Results: in vivo: local DXM treatment of EIT-animals prevents trauma-induced loss of ABR thresholds that occurs in EIT-animals and EIT-animals treated with the carrier solution (i.e., AP), and prevented loss of auditory hair cells. In vitro: DXM and polymer-eluted DXM were equally effective in protecting hair cells from ototoxic levels of TNF-α Inhibitor treated explants demonstrated that DXM treatment requires both Akt/PKB and NFkB signalling for otoprotection. DXM treatment of explants showed up regulation of anti-apoptosis related genes (i.e., Bcl-2, Bcl-xl) and down regulation of pro-apoptosis related genes (i.e., Bax, TNFR-1). Conclusions: DXM exert its otoprotective action by activation of cell signal molecules (e.g., NFkB) that alter the expression of anti- and pro-apoptosis genes.

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.

The objective was to determine the role of nuclear factor kappa B (NFκB) in dexamethasone base (DXMb) protection of auditory hair cells from tumor necrosis factor-alpha (TNFα)-induced loss on gene expression and cell signaling levels. Organ of Corti (OC) explants from 3-day-old rats were cultured under one of the following conditions: (1) media only--no treatment; (2) media+TNFα; (3) media+TNFα+DXMb; (4) media+TNFα+DXMb+NFκB-Inhibitor (NFκB-I); or (5) media+TNFα+DXMb+NFκBI-Scrambled control (NFκBI-C). A total of 60 organ of Corti explants (OC) were stained with FITC-Phalloidin after 96 h in culture (conditions 1-5) for hair cell counts and imaging of surface characteristics. A total of 108 OC were used for gene expression studies (i.e. B-actin, Bax, Bcl-2, Bcl-xl, and TNFR1) after 0, 24, or 48 h in vitro (conditions 1-4). A total of 86 OC were cultured (conditions 1-3) for 48 h, 36 of which were used for phosphorylated NFκB (p-NFκB) ELISA studies and 50 for whole mount anti-p-NFκB immunostain experiments. TNFα+DXMb exposed cultures demonstrated significant upregulation in anti-apoptotic Bcl-2 and Bcl-xl genes and downregulation in pro-apoptotic Bax gene expression; DXMb treatment of TNFα explants also lowered the Bax/Bcl-2 ratio and inhibited TNFR1 upregulation. After inhibiting NFκB activity with NFκB-I, the gene expression profile following TNFα+DXMb treatment now mimics that of TNFα-challenged OC explants. The levels of p-NFκB and the degree of nuclear translocation are significantly greater in TNFα+DXMb exposed OC explants than observed in the TNFα and control groups in the middle+basal turns of OC explants. These findings were supported by the results of the hair cell counts and the imaging results obtained from the whole mount OC specimens. DXMb protects against TNFα-induced apoptosis of auditory hair cells in vitro via activation of NFκB signaling in hair cell nuclei, and regulation of the expression levels of anti- and pro-apoptotic genes and a pro-inflammatory gene.

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.

Molecular regulation of auditory hair cell death and approaches to protect sensory receptor cells and/or stimulate repair following acoustic trauma

Loss of auditory sensory hair cells (HCs) is the most common cause of hearing loss. This review addresses the signaling pathways that are involved in the programmed and necrotic cell death of auditory HCs that occur in response to ototoxic and traumatic stressor events. The roles of inflammatory processes, oxidative stress, mitochondrial damage, cell death receptors, members of the mitogen-activated protein kinase (MAPK) signal pathway and pro- and anti-cell death members of the Bcl-2 family are explored. The molecular interaction of these signal pathways that initiates the loss of auditory HCs following acoustic trauma is covered and possible therapeutic interventions that may protect these sensory HCs from loss via apoptotic or non-apoptotic cell death are explored.