1. Srdjan Vlajkovic
Cochlear Homeostasis in
Stress and Injury

2. Abnormal Cochlear Homeostasis
• Noise-induced hearing loss
• Age-related hearing loss (presbyacusis)
• Ototoxicity
• Meniere’s disease

3. 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

4. Problem?
• People working in construction or military
• Accidental exposure
Cellular bases of NIHL: prophylactic and
therapeutic drugs

5. Effects of noise on the cochlea
SL
S
L
OHC
SV
SGN
SL
IHC noise
Pillar cells

6. 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

7. 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.

8. Inflammation
CD45+ Inflammatory Cells
Hirose et al.
(2005)

9. Damage to sensory hair cells by noise
Acute trauma
http://www.iurc.montp.inserm.fr/cric/audition/english/ear/fear.htm

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

11. OHC are the most prominent target for noise

12. 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.

13. Free radical formation

14. How are ROS/free radicals formed as a result of
noise?
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.

15. 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).

16. 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.

17. Damage to the cochlea by ROS
Green fluorescence: dichlorofluorescein (DCF)

18. Apoptosis and necrosis in the noise-exposed
cochlea

19. The mechanisms of NIHL
Noise
Overdriving the Excitotoxicity Ischemia/reperfusion Inflammation
mitochondria
Free radicals
Lipid peroxidation DNA Protein
damage damage
Apoptotic and necrotic cell death
Hearing loss Adapted from Henderson et al., 2006

20. 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

21. 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

22. Sound conditioning

23. Sound conditioning and antioxidants
glutathione reductase -glutamyl cysteine synthetase catalase

24. 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.

25. Prevention of NIHL by antioxidants

26. 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.

27. Post-noise treatments
L-NAC and salicylate (Kopke et al. 2000)
Trolox and salicylate (Yamashita et al, 2005)
D-methionine (Campbell et al. 2007)

28. 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

29. Post-exposure (24 h) treatment of NIHL with a
selective A1 adenosine receptor agonist ADAC
Vlajkovic et al., 2010
• 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

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

31. HAIR CELL SURVIVAL
Multiple ADAC injections
Multiple vehicle
injections

32. 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

33. 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.

34. Cochlear blood flow and NIHL
A third point of intervention against NIHL may be
prevention of the cochlear ischemia/reperfusion
associated 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

35. 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

36. Inhibition of Src activity by KX1-004

37. Inhibition of ROS formation by KX1-004

38. 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.

39. 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.

40. 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.