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1. EIT involves oxidative stress and lipid peroxidation early on after the

implantation.

2. L-NAC, Mannitol and Dex are effective alone in protecting the

sensory cells in vitro at high doses.

3. A cocktail containing L-NAC, Mannitol and Dex at much lower doses

of each compound, is effective in protecting sensory cells.

4. The three compounds can be combined with a synergistic effect

allowing a decrease in the potential side effects of each of the

compound.

Otoprotection by a Combination of L-NAC, Mannitol and Dex in an In Vitro Model of Cochlear Implant Trauma

Adrien A Eshraghi, MD; Jeenu Mittal, MSc; Jonathan Roell, BS; Mateo Guardiola, MS; Fred F Telischi, MD;

Dustin Lang, MS; Thomas R Van De Water, PhD; Esperanza Bas, PhD; Chhavi Gupta, PhD; Helio Rodrigues, MD

University of Miami Hearing Research Laboratory, Department of Otolaryngology, University of Miami Miller School of Medicine, Miami, USA,

Adrien A Eshraghi MD, MSc, FACS

Professor of Otolaryngology and Biomedical Engineering

Director, Hearing Research Laboratory

Co-Director, University of Miami Ear Institute

University of Miami Miller School of Medicine

Email: aeshraghi@med.miami.edu, Phone::304-243-1484

1. Eshraghi AA, et al. (2005). Pattern of hearing loss in a rat model of cochlear implantation trauma. Otol Neurotol. 26: 442-7.

2. Eshraghi AA, et al. (2013). Molecular mechanisms involved in cochlear implantation trauma and the protection of hearing and auditory sensory cells by inhibition of c-Jun-N-terminal kinase signaling. Laryngoscope. 123:S1-14.

3. Bas E et al. (2012). Mannitol protects hair cells against tumor necrosis factor α-induced loss. Otol Neurotol. 33(9): 1656-1663.

4. Feghali JG et al. (2001). L-n-acetyl-cysteine protection against cisplatin-induced auditory neuronal and hair cell toxicity. Laryngoscope. 111: 1147-1155.

5. Kopke RD et al. Am J Otol (1997) Use of organotypic cultures of Corti's organ to study the protective effects of antioxidant molecules on cisplatin-induced damage of auditory hair cells. 18(5):559-71.

6. Eshraghi AA, et al. (2007) Local dexamethasone therapy conserves hearing in an animal model of electrode insertion trauma-induced hearing loss. Otol Neurotol. 28(6):842-9

7. Hamernik RP, et al. (2008). The effectiveness of N-acetyl-L-cysteine (L-NAC) in the prevention of severe noise-induced hearing loss. Hear Res 239:99–106.

Materials and Methods

1. The EIT resulted in an increased production of the total reactive

oxygen species (ROS) in both the HCs and the supporting cells

(SCs).

2. There was an increase of total hair cell (THC) loss in the EIT OC

explants when compared with control group HC counts or the tri-

therapy cochlea.

3. We defined the dosage of L-NAC, Mannitol and Dex for the

survival of 50% protection of hair cells in vitro. Their combination

provided close to 96% protection demonstrating a synergistic

effect.

4. This combination therapy may be beneficial in other type of inner

ear trauma that can result in hair cell loss.

Results/Discussion

Conclusion

Results:FITC-phalloidin staining of HCs & HC counts

The pattern of hearing loss post cochlear electrode

implant insertion trauma (EIT) is described as an acute

loss of hearing followed by the gradual loss of residual

hearing on the days following the implantation.(1) This

inner ear trauma initiates multiple molecular

mechanisms such as oxidative stress, JNK activation

and caspase-3 activation in hair cells (HCs) or support

cells (SCs) resulting in initiation of programmed cell

death within the damaged tissues of the cochlea which

leads to loss of residual hearing.(2) In earlier studies L-

N-acetylcysteine (L-NAC) (an antioxidant), Mannitol

(osmotic and diuretic effects) and dexamethasone

(Dex) (a steroid) have been shown independently to

protect the HCs loss against different types of inner ear

trauma.(3-7) These 3 molecules have different types of

otoprotective properties that might have synergistic

effects.

Molecular mechanisms involved in EIT (2)

Background

References Contact Information

Objectives

To test the otoprotective effects of the tri-therapy (L-

NAC + Mannitol + Dex) in an in vitro model of inner ear

trauma (e.g. cochlear implantation trauma).

Supported by research grant from MED-EL Corporation, Innsbruck, Austria to Dr. Adrien A Eshraghi, MD, FACS

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The organ of Corti from control explants shows three well organized

rows of outer hair cells (OHCs) and a single row of inner hair cells

(IHCs), while the organ of Corti having EIT shows areas of damaged

OHC and IHCs with missing hair cells or damaged stereocilia. The

explants exposed to EIT and treated with L-NAC (5mM) + Mannitol

(100mM) + Dex (20 μg/mL) shows the same pattern of preservation

of OHC and IHCs organization as seen in control explants. The

graph show the percentage of living OHCs in each group .

Cochlea explants were dissected from P-3 rats and

placed in serum-free media. Explants were divided into

multiple groups:

1. Control groups (6 explants per group; a total of 12

explants):

1. Control (no trauma, no drug)

2. EIT (implant trauma , no drug)

2. Ten experimental groups (6 explants per group; a total

of 60 explants):

1. EIT + L-NAC (5, 2 or 1 mM)

2. EIT + Mannitol (100, 50 or 10 mM)

3. EIT + Dex (20, 10 or 5 µg/mL)

4. EIT +L-NAC + Mannitol + Dex

In the EIT groups, a 0.28-mm diameter monofilament

fishing line was introduced through the small

cochleostomy located next to the round window area,

allowing for an insertion of between 110 and 150

degrees. Oxidative stress was studied in all explants

post this EIT.

After EIT was caused, explants were cultured in media

containing L-NAC alone, Mannitol alone or Dex alone at

decreasing concentrations.

Concentrations of L-NAC, Mannitol and Dex that showed

50 percent protection of hair cell loss individually were

used as a combination in the experimental group 4.

.

• For EIT in the 3-day-old (P-3) rat cochlea,

a 0.28-mm diameter monofilament fishing

line was introduced through the small

cochleostomy located next to the round

window area, allowing for an insertion of

between 110 and 150 degrees. (3) • After EIT was caused, explants were

cultured in media containing the single

therapeutic compound (L-NAC, Mannitol,

and Dex) at varying concentrations as well

as the cocktail containing all three.

CellROX™ labeling is seen in the middle and basal turns of the

EIT exposed specimens in both HCs and SCs, but is absent in the

control and EIT + L-NAC + Mannitol + Dex specimens. The graph

shows the mean signal intensity of CellROX™ labeling (n = 9

samples) in the HCs and SCs for each group (control, EIT, and EIT

+L-NAC + Dex + Mannitol).

Result: Immunostaining for ROS (CellROX)

EIT procedure for the 3-day-old (P-3) rat cochlea

Surface preparations of HCs and SCs stained with anti-HNE (red)

and DAPI (blue) of the middle and basal turns from each group

(control, EIT, and EIT + L-NAC + Mannitol + Dex) are

represented. Anti-HNE labeling is seen in the basal turn of the

EIT exposed specimens but is absent in the control and EIT+ L-

NAC + Mannitol + Dex specimens. The graphs shows the mean

signal intensity of anti-HNE labeling (n=9 samples) in the HCs

and SCs for each group (control, EIT, and EIT + L-NAC + Mannitol + Dex).

Result: Immunostaining for membrane damage (HNE)

Results: FITC-phalloidin staining of HCs

While the lower concentrations of L-NAC

(2mM), Mannitol (10mM) and Dex

(5µg/ml) showed only partial protection

against HCs loss, the cocktail containing

those same concentrations combined

showed a total protection against HCs

loss.