Cochlear Homeostasis in

Stress and Injury

Srdjan Vlajkovic

Abnormal Cochlear Homeostasis

• Noise-induced hearing loss

• Age-related hearing loss (presbyacusis)

• Ototoxicity

• Meniere’s disease

Noise-induced hearing loss (NIHL)

A significant source of hearing loss in industrial societies.

Focus on prevention:

- hearing conservation programs

- use of protection devices

- frequent screening

- education on the causes and ways to prevent it

Problem?

• People working in construction or military

• Accidental exposure

Cellular bases of NIHL: prophylactic and

therapeutic drugs

Effects of noise on the cochlea

IHC

OHC

S

L

Pillar cells

noise

SGN

SV

SL

SL

The effect of noise on the stria vascularis and

blood vessels

• High level noise - acute swelling of the stria vascularis

• Loss of intermedate cells (permanent)

• Stria shrinks as a long-term result

• Reduction in cochlear blood flow (CBF) heavily influenced

by the length and intensity of the noise exposure

• Consequence: elevated auditory thresholds and damage to

the vital cochlear tissues

Disturbances of ionic balance in the cochlea due to the

loss of Type II and Type IV fibrocytes in the spiral

ligament. This can disrupt K+ cycling.

Inflammation

Hirose et al.

(2005)

CD45+ Inflammatory Cells

Damage to sensory hair cells by noise

Acute trauma

http://www.iurc.montp.inserm.fr/cric/audition/english/ear/fear.htm

Glutamate excitotoxicity

http://www.iurc.montp.inserm.fr/cric/audition/english/ear/fear.htm

OHC are the most prominent target for noise

Oxidative stress and hair cell death

What active mechanisms at the cellular level are triggering

hair cell death?

• A number of studies emerged showing increased

reactive oxygen species (ROS) and free radicals during

and after noise exposure.

• Free radicals are molecules with an unpaired electron

capable of altering the electron arrangements in stable

molecules.

Free radical formation

During noise exposure, the electron transport chain of the mitochondria

uses large amounts of oxygen, which can then create large amounts of

superoxide as an unwanted byproduct.

The increased superoxide can then react with other molecules to

generate higher levels of other ROS in the cochlea.

How are ROS/free radicals formed as a result of

noise?

Reactive oxygen species (ROS)

• Oxygen-based molecules that act as free radicals:

- superoxide (O2-)

- hydroxyl radical (OH-)

- peroxynitrite radical (ONOO ·1-)

• Readily capable of generating free radicals:

- hydrogen peroxide (H2O2)

- ozone (O3).

What are the mechanisms of ROS-induced loss

of sensory cells?

• ROS and free radicals are capable of damaging DNA, breaking

down lipid and protein molecules, and triggering cell death, all

of which can contribute to the loss of function seen after noise

• Lipid peroxidation: a series of reactions through which free

radicals and ROS can break down lipid molecules.

Damage to the cochlea by ROS

Green fluorescence: dichlorofluorescein (DCF)

Apoptosis and necrosis in the noise-exposed cochlea

The mechanisms of NIHL

Noise

Overdriving the

mitochondriaExcitotoxicity Ischemia/reperfusion Inflammation

Free radicals

Lipid peroxidation DNA

damage

Protein

damage

Apoptotic and necrotic cell death

Hearing loss Adapted from Henderson et al., 2006

Pharmacological interventions to reduce

hearing loss

(1) restoring the normal balance of free radicals with antioxidants

(2) reducing glutamate excitotoxicity with NMDA receptor antagonists

(3) maintaining adequate cochlear blood flow during and after noise

(4) reducing inflammation

(5) inhibiting pathways to apoptotic cell death to preserve hair cells

ROS/Antioxidant Balance

Antioxidants are molecules that scavenge ROS and convert them to less dangerous molecules.

Increasing cochlear antioxidant supplies can substantially prevent HC damage and hearing loss.

Antioxidant levels can be increased in two ways:

• application of exogenous antioxidant molecules

directly into the cochlea or systemically into the body;

• endogenously by using sound conditioning

Sound conditioning

Sound conditioning and antioxidants

glutathione reductase -glutamyl cysteine synthetase catalase

Antioxidants in prevention of NIHL

Local application (RWM):

• glutathione monoethyl ester (GSS): a precursor molecule to glutathione

Systemically injected:

• Allopurinol (an inhibitor of ROS production)

• Superoxide dismutase (ROS scavenger)

• Mannitol (a scavenger of the hydroxyl (OH-) radical)

• GSS

• LNAC (n-l-acetylcysteine): antioxidant properties and increases levels of glutathione.

• Salicylate (can scavenge the hydroxyl radical)

• Acetyl-l carnitine (ALCAR) improves mitochondrial respiration efficiency, leading to decreased ROS production during noise.

• DMET (d-methionine) increases the levels of available cochlear glutathione. ALCAR and DMET provided nearly 100% protection against noise-induced PTS, OHC and IHC loss.

Prevention of NIHL by antioxidants

Treatment after noise exposure

(rescue phenomenon)

• Prophylactic agents: administered before and

usually during and after noise exposure

• Rescue agents: first administered after noise

exposure but before permanent NIHL has occurred

• Regeneration of hair cells for permanent NIHL is a

different research area

• Rescue Phenomenon: Continued free radical formation

in the cochlea for 7-10 days after noise exposure

• First 24 hrs after noise exposure could be a critical

period for antioxidant intervention.

Post-noise treatments

D-methionine (Campbell et al. 2007)

L-NAC and salicylate (Kopke et al. 2000)

Trolox and salicylate (Yamashita et al, 2005)

Adenosine in Tissue Protection and Regeneration

• Boost antioxidant defences

• Improve blood flow and oxygen supply

• Inhibit the release of neurotransmitters

• Stabilise cells by stimulating K+ and inhibiting Ca2+

channels

• Suppress inflammation

• Promote anti-apoptotic pathways

• Promote angiogenesis

Post-exposure (24 h) treatment of NIHL with a

selective A1 adenosine receptor agonist ADAC

• Noise exposure: 110 dB SPL (8-12 kHz) for 24 hours

• ADAC administration 6 or 24 hrs after noise

• Single or multiple i.p. injections

Vlajkovic et al., 2010

Post-exposure (6 h) treatment of NIHL with ADAC

****** *** *** *** *** ***

HAIR CELL SURVIVAL

Multiple ADAC injections

Multiple vehicle

injections

Lipid peroxidation

A process through which ROS and free radicals break down lipid molecules.

It is a self-perpetuating process that may be contributing to the expansion

of the HC death lesion after noise.

0 days 2 days 4 days

Inhibition of lipid peroxidation

• Pharmacological inhibition of lipid peroxidation may be a method for rescue of hearing after noise exposure.

• A series of drugs that reduce lipid peroxidation effects in the organ of Corti (e.g. Lazaroid) were also found to limit noise-induced threshold shift.

Cochlear blood flow and NIHL

A third point of intervention against NIHL may be prevention of the cochlear ischemia/reperfusionassociated with noise exposure.

• Drugs that promote blood flow:

- Cardiac output can be increased,

- Cochlear blood vessels can be dilated,

- Blood can be thinned by expanding the plasma content.

• Inhibition of angiotensin II receptors by Sarthran leads to maintenance of normal blood vessel diameter during noise, and reduction of TTS

• Inhibition of the receptors for norepinephrine, increase CBF and reduce noise-induced TTS

Apoptotic cell death

The final point of intervention is at the level of the cellular signals responsible for apoptotic cell death.

• CEP-1347, a selective c-Jun-N-terminal (JNK) inhibitor

• KX1-004, a potent inhibitor of Src activity

• Riluzole, a neuroprotective agent that restricts

excitotoxicity and apoptotic and necrotic cell death

Inhibition of Src activity by KX1-004

Inhibition of ROS formation by KX1-004

Apoptotic pathways

In addition to the Src and JNK signaling pathways,

numerous other pathways are involved in the induction

of apoptosis.

• The caspases enzyme cascade plays a key role in the

execution of apoptotic cell death in the HC.

• The calpain enzyme pathway, a series of calcium-

dependent enzymes involved in breaking down cells

during apoptosis, has also been targeted. Leupeptin, a

calpain inhibitor, protected chinchilla HC and reduced

TTS.

Oxidative stress and acquired hearing loss

There is growing evidence that oxidative stress in the

cochlea may be a common factor for hearing loss

from aminoglycoside antibiotics, ototoxic anticancer

drugs and aging.

Recommended reading:

• Henderson et al. (2006) The role of oxidative stress in

noise-induced hearing loss. Ear & Hearing 27:1-19.

• Le Prell et al. (2007) Mechanisms of noise-induced

hearing loss indicate multiple methods of prevention

Hearing Research 226:22-43.