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Activation of Caspase-2 Following Noise Exposure and the Effect of Treatment with Dietary Agents on Noise-Induced Oxidat...

Permanent Link: http://ufdc.ufl.edu/UFE0042206/00001

Material Information

Title: Activation of Caspase-2 Following Noise Exposure and the Effect of Treatment with Dietary Agents on Noise-Induced Oxidative Stress and Activation of Caspases-2 and -8
Physical Description: 1 online resource (87 p.)
Language: english
Creator: Lang, Dustin
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: antioxidant, caspase, cochlea, ear, hearing, loss, loud, noise, oxidative, stress
Medicine -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Noise-induced hearing loss (NIHL) is a growing problem due to increased noise exposure in military, occupational, and recreational settings. Noise induces the formation of free radicals in the cochlea due to increased metabolic demand and reduction of cochlear blood flow. This ultimately damages and/or destroys sensory cells causing permanent hearing loss. One consequence of oxidative stress is the initiation of apoptotic signaling pathways via activation of caspases. Caspases -3, -8, and -9 are known to be up-regulated in the inner ear following noise exposure, and caspase-2 activation has been described as an initiator and/or executioner of apoptosis in other systems. Treatment with dietary antioxidants (beta-carotene, and vitamins C and E) delivered in combination with the mineral magnesium (Mg) was previously shown to be effective for the reduction of NIHL. Due to the fact that beta-carotene is metabolized to vitamin A, this treatment will subsequently be referred to as ACEMg. The purpose of this study was two-fold: 1) to investigate the activation of caspase-2 following noise exposure, and 2) to determine the effect of ACEMg treatment on noise-induced production of reactive nitrogen species (RNS) and activation of caspases-2 and -8. Caspase-2 immunolabeling was initially observed in the supporting cells of the guinea pig organ of Corti 2 hours following noise exposure, and moved transiently to the outer hair cells at 4 hours post-noise. Nitrotyrosine (3-NT), a biomarker for production of RNS, and activation of caspase-8 were assessed in order to determine the effect of ACEMg treatment on noise-induced oxidative stress and initiation of the extrinsic (death receptor mediated) apoptotic pathway respectively. The ACEMg treatment effect which was observed, while not statistically reliable under the current study design, supports further studies at later post-noise time points, when oxidative stress is at a maximum. Taken together, these immunohistochemical data support the possibility that caspase-2 plays a role in NIHL, and that the protective effect observed with ACEMg treatment involves attenuation of production of RNS and activation of caspases-2 and -8.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Dustin Lang.
Thesis: Thesis (M.S.)--University of Florida, 2010.
Local: Adviser: Leprell, Colleen G.

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Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0042206:00001

Permanent Link: http://ufdc.ufl.edu/UFE0042206/00001

Material Information

Title: Activation of Caspase-2 Following Noise Exposure and the Effect of Treatment with Dietary Agents on Noise-Induced Oxidative Stress and Activation of Caspases-2 and -8
Physical Description: 1 online resource (87 p.)
Language: english
Creator: Lang, Dustin
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: antioxidant, caspase, cochlea, ear, hearing, loss, loud, noise, oxidative, stress
Medicine -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Noise-induced hearing loss (NIHL) is a growing problem due to increased noise exposure in military, occupational, and recreational settings. Noise induces the formation of free radicals in the cochlea due to increased metabolic demand and reduction of cochlear blood flow. This ultimately damages and/or destroys sensory cells causing permanent hearing loss. One consequence of oxidative stress is the initiation of apoptotic signaling pathways via activation of caspases. Caspases -3, -8, and -9 are known to be up-regulated in the inner ear following noise exposure, and caspase-2 activation has been described as an initiator and/or executioner of apoptosis in other systems. Treatment with dietary antioxidants (beta-carotene, and vitamins C and E) delivered in combination with the mineral magnesium (Mg) was previously shown to be effective for the reduction of NIHL. Due to the fact that beta-carotene is metabolized to vitamin A, this treatment will subsequently be referred to as ACEMg. The purpose of this study was two-fold: 1) to investigate the activation of caspase-2 following noise exposure, and 2) to determine the effect of ACEMg treatment on noise-induced production of reactive nitrogen species (RNS) and activation of caspases-2 and -8. Caspase-2 immunolabeling was initially observed in the supporting cells of the guinea pig organ of Corti 2 hours following noise exposure, and moved transiently to the outer hair cells at 4 hours post-noise. Nitrotyrosine (3-NT), a biomarker for production of RNS, and activation of caspase-8 were assessed in order to determine the effect of ACEMg treatment on noise-induced oxidative stress and initiation of the extrinsic (death receptor mediated) apoptotic pathway respectively. The ACEMg treatment effect which was observed, while not statistically reliable under the current study design, supports further studies at later post-noise time points, when oxidative stress is at a maximum. Taken together, these immunohistochemical data support the possibility that caspase-2 plays a role in NIHL, and that the protective effect observed with ACEMg treatment involves attenuation of production of RNS and activation of caspases-2 and -8.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Dustin Lang.
Thesis: Thesis (M.S.)--University of Florida, 2010.
Local: Adviser: Leprell, Colleen G.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0042206:00001


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ACTIVATION OF CASPASE-2 FOLLOWING NOISE EXPOSURE AND THE EFFECT
OF TREATMENT WITH DIETARY AGENTS ON NOISE-INDUCED OXIDATIVE
STRESS AND ACTIVATION OF CASPASES-2 AND -8


















By

DUSTIN MATTHEW LANG


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2010

































2010 Dustin Matthew Lang


































To those in hearing research who may benefit from my modest contribution









ACKNOWLEDGMENTS

First, I would like to thank Dr. Colleen Le Prell for allowing me to complete my

thesis work in her laboratory, as well as for all of the training and advice she has given.

I also express my deepest gratitude to Dr. Patrick Antonelli for the opportunity to gain

invaluable experience as an undergraduate, and young graduate student. I appreciate

Dr. Gregory Schultz for his contagious enthusiasm, and willingness to offer insight and

direction. The common thread for each of these graduate committee members is that

they have made themselves available to give guidance and direction despite the

endless demands on their time. Also, I cannot overlook the contributions of Edith

"Angel" Sampson for her selfless investment in the development of my science and

character. Finally, I thank my wonderful wife Michelle for her support despite my long

hours and late nights in the lab, and my family for their unconditional support.









TABLE OF CONTENTS

page

ACKNOW LEDGM ENTS ....................... ..... .. ........... ......................................... 4

LIST OF TABLES .......... ..... .. ...................... ............. ...... ............... 7

LIS T O F F IG U R E S .................................................................. 8

LIS T O F F IG U R E S .................................................................. 8

ABSTRACT ............... .. ............................... ......... 10

CHAPTER

1 INTRODUCTION ................ .......... .......... ......... 12

An Overview of Noise-Induced Hearing Loss .......... ............................ 12
The Cochlea ............... ....................... ............. .. .......................... 17
Anatomy and Physiology ...................................... 17
Cellular Mechanisms of Noise-Induced Cochlear Pathology .......................... 20
Mechanical damage and glutamate excitotoxicity ............................... 21
Oxidative stress ................................ .. ................. .. ............. 22
Experimental Treatment for Noise-Induced Hearing Loss ............................... 27
Inhibitors of C cellular Stress Pathw ays .............................................................. 28
Neurotrophic Factors/! Neurotransmission Blockers ................ ....... ......... 29
Inhibitors of Oxidative Stress ............... ...... ................................. ......... 30
Beta-Carotene, Vitamins C and E, and Magnesium .............................. 33
Study Design ........................................ ........... ............... 35

2 MATERIALS AND METHODS ...... ............. ......................... 41

Subjects ............... ......................................... ............. ............... 41
Noise Exposure .......................... ......... .... ................... 41
E lectrophysiological Tests ...................... ....... ......... .. ............................ 41
Antioxidant Treatment...................... ..................... ............... 42
Immunohistochemistry ...... .................. .................. 43
Nitrotyrosine Immunolabeling ............................ ........... 43
C aspase-8 Im m unolabeling.......................................................... ............... 44
Caspase-2 Immunolabeling ............. .......... ................. ... .......... 44
Statistical Analysis .......................................... ............... 45

3 N IT R O T Y R O S IN E .......... ................................... ...... ............... 4 7

Results................l ................. 47.....47
RDiscussion .... ........................ ........................ 4
D is c u s s io n .............................................................................................................. 4 8









4 CASPASE-8........................... ............ ............... 54

R results ............................................................................... ............ 54
D discussion .............. ......... ..................................................................... 55

5 CASPASE-2........................... ............ ............... 60

R results ............................................................................... ............ 60
D discussion .............. ......... ..................................................................... 61

6 CONCLUSIONS AND FUTURE DIRECTIONS ................................................ 67

APPENDIX: COMMENTS ON METHODS AND STATISTICAL ANALYSIS ............... 69

LIST OF REFERENCES .............. ............................... 74

B IO G RA P H IC A L S K ET C H ................................. ......... ............................................. 87



































6









LIST OF TABLES

Table page

3-1 Difference in distribution of ACEMg treated and saline control 3-NT image
rank scores .............. ...... ......... ........ .... .......... ........... 52

4-1 Difference in distribution of ACEMg treated and saline control caspase-8
im age rank scores ................ ......... ............ .............................. 59









LIST OF FIGURES


Figure page

1-1 Typical audiogram exhibiting early NIHL....... .................... ......... 37

1-2 Light micrograph of a cross-section of the guinea pig cochlea............. ........... 37

1-3 Cross-section of the organ of Corti .................................... ........ ............... 38

1-4 Diagram showing efferent and afferent innervation of the IHC............. ........... 38

1-5 Diagram showing afferent and efferent innervation of the OHC ...................... 39

1-6 Apoptotic pathways ........ .......... .......................... ....... ........ 39

1-7 Effect of A C EM g treatm ent on N IH L............................................. ... .................. 40

1-8 Effect of ACEMg treatment on IHC and OHC loss........................ ............... 40

3-1 Epifluorescence micrographs of ACEMg treated and saline control sections
of the organ of Corti labeled with anti-3-nitrotyrosine antibody showing the
greatest observed treatment effect.................................. .... ........... 50

3-2 Epifluorescence micrographs of ACEMg treated and saline control sections
of the organ of Corti labeled with anti-3-nitrotyrosine antibody showing
median treated and control images .......... ............. ........... ................ 51

3-4 Immunostaining for NT shifts from supporting cells (Hensen, Claudius) to
OHCs, including Deiters, with a maximum at 7-10 days ............. .............. 53

4-1 Epifluorescence micrographs of ACEMg treated and saline control sections
of the organ of Corti labeled with anti-caspase-8 antibody showing the
greatest observed treatment effect...... ..................... ............... 56

4-2 Epifluorescence micrographs of ACEMg treated and saline control sections
of the organ of Corti labeled with anti-caspase-8 antibody showing median
treated and control im ages ...................................................... ............... 57

4-3 Negative control in which the primary anti-caspase-8 antibody incubation was
o m itted a nd no no ise co ntro l.................................................... .... ................. 58

5-1 Epifluorescence micrographs of the organ of Corti from the 1st turn of the
cochlea labeled with anti-caspase-2L antibody showing temporal difference in
post-noise expression........................... ...................... 63

5-2 Epifluorescence micrographs of ACEMg treated and saline control sections
of the organ of Corti labeled with anti-caspase-2L antibody showing diffuse
labeling in treated and control tissues ...... ................. ...... .......... 65









5-3 Epifluorescence micrographs of ACEMg treated and saline control sections
of the organ of Corti labeled with anti-caspase-2 antibody showing
immunolabeling concentrated in the supporting cells in 1st and 2n turns, with
labeling become ing more diffuse in the 3rd turn ................................................. 66

A-1 Auditory brainstem response (ABR) threshold shift at various post-noise time
p o in ts .................. ................................. ....... ...... ...... 7 3

A-2 Plot of statistical power vs. the odds parameter gamma ............... ............... 73









Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

ACTIVATION OF CASPASE-2 FOLLOWING NOISE EXPOSURE AND THE EFFECT
OF TREATMENT WITH DIETARY AGENTS ON NOISE-INDUCED OXIDATIVE
STRESS AND ACTIVATION OF CASPASES-2 AND -8

By

Dustin Matthew Lang

August 2010

Chair: Colleen Le Prell
Major: Medical Sciences

Noise-induced hearing loss (NIHL) is a growing problem due to increased noise

exposure in military, occupational, and recreational settings. Noise induces the

formation of free radicals in the cochlea due to increased metabolic demand and

reduction of cochlear blood flow. This ultimately damages and/or destroys sensory cells

causing permanent hearing loss. One consequence of oxidative stress is the initiation

of apoptotic signaling pathways via activation of caspases. Caspases -3, -8, and -9 are

known to be up-regulated in the inner ear following noise exposure, and caspase-2

activation has been described as an initiator and/or executioner of apoptosis in other

systems. Treatment with dietary antioxidants (3-carotene, and vitamins C and E)

delivered in combination with the mineral magnesium (Mg) was previously shown to be

effective for the reduction of NIHL. Due to the fact that 3-carotene is metabolized to

vitamin A, this treatment will subsequently be referred to as ACEMg.

The purpose of this study was two-fold: 1) to investigate the activation of caspase-

2 following noise exposure, and 2) to determine the effect of ACEMg treatment on

noise-induced production of reactive nitrogen species (RNS) and activation of









caspases-2 and -8. Caspase-2 immunolabeling was initially observed in the supporting

cells of the guinea pig organ of Corti 2 hours following noise exposure, and moved

transiently to the outer hair cells at 4 hours post-noise. Nitrotyrosine (3-NT), a

biomarker for production of RNS, and activation of caspase-8 were assessed in order to

determine the effect of ACEMg treatment on noise-induced oxidative stress and

initiation of the extrinsic (death receptor mediated) apoptotic pathway respectively. The

ACEMg treatment effect which was observed, while not statistically reliable under the

current study design, supports further studies at later post-noise time points, when

oxidative stress is at a maximum. Taken together, these immunohistochemical data

support the possibility that caspase-2 plays a role in NIHL, and that the protective effect

observed with ACEMg treatment involves attenuation of production of RNS and

activation of caspases-2 and -8.









CHAPTER 1
INTRODUCTION

An Overview of Noise-Induced Hearing Loss

Noise-induced hearing loss (NIHL) represents a growing medical problem with far-

reaching economic and social impacts. Due to the expansion of technology, we are

now exposed to higher levels of noise than ever before, both in occupational as well as

recreational settings. Education regarding the potentially harmful effects of noise,

occupational hearing preservation programs, and the technology of hearing protection

devices (HPDs) have not kept up with the increase in noise exposure. This disparity is

made evident by numerous studies citing widespread detrimental effects of noise in

various settings[1-24].

It is estimated that 5-10% of the hearing loss burden in the U.S. is caused by noise

exposure in the workplace [1], and that number rises to approximately 16% on a global

scale [2]. According to the National Institute for Occupational Safety and Health

(NIOSH), approximately 30 million people are exposed to hazardous levels of noise on

the job [25]. NIHL was also found to increase the risk of work-related accidents [3, 4].

In a Michigan study, 29.9% of those with hearing loss reported work-related noise as its

cause [5]. Those with highest risk of NIHL include construction workers, miners,

musicians, disk jockeys, law enforcement officers, and military personnel. Across all

trades, 59.7% of construction workers were found to have at least moderate NIHL [6].

Hearing loss among symphony musicians was double the rate that would be expected

for corresponding age [7, 8]. Among disc jockeys studied, 70% reported temporary

threshold shift, and 74% reported frequent tinnitus (ringing in the ears) after spending

time in the dance club [9]. A ten-year longitudinal study of police officers reveals the









detrimental effects of impulse noise from gunfire despite the use of dual protection

(earplugs and earmuffs) [10].

Diagnosis of NIHL is based upon a history of noise exposure combined with the

presence of a "noise notch" on the patient's audiogram. While varying definitions exist,

a "noise notch" is generally an increase in hearing threshold in the 4000 Hz range

(Figure 1-1), which expands to include progressively higher and lower frequencies as

the exposure to noise progresses. Coles et al. defines a noise notch as a high-

frequency notch where the hearing threshold at 3, 4, and/ or 6 kHz is at least 10 dB

greater than at 1 or 2 kHz and at least 10 dB greater than at 6 or 8 kHz [26].

Additionally, Niskar et al. gives criteria which require threshold values at 0.5 and 1 kHz

to be 5 15 dB, the greatest threshold value at 3, 4, or 6 kHz to be at least 15 dB higher

than the worst (highest) threshold value at either 0.5 or 1 kHz, and a threshold at 8 kHz

at least 10 dB better than the worst threshold at 3, 4, or 6 kHz [20]. Age-related hearing

loss (ARHL), or presbycusis also causes a threshold increase at the higher frequencies.

However, the audiogram of a person with purely ARHL is downward-sloping with

progressively higher thresholds at higher frequencies, and lacks the characteristic notch

indicating the contribution of noise. Different notch metrics can be used for diagnosis,

and one study found that these metrics can agree with expert clinical judgments [27].

Perhaps the greatest proportion of individuals with NIHL is found in the military.

Studies have shown that members of all branches of military personnel are at greater

risk for NIHL than the general population [11-14]. In 1970, 20% of all Army veterans

were entering claims for hearing loss, and the Veterans Administration (VA) paid over

$52 million in compensation. This figure does not include compensation for hearing









loss with a concurrent disability, cost of hearing aids, batteries, or repairs. Medical

evacuations for complaints related to hearing loss were conducted at an average rate of

one soldier per day during the first year of the war in Iraq. In 2004, the VA spent $108

million in disability payments to former Navy personnel, which represents a $65 million

increase from 1999. In 2006, the combined total of disability payments for hearing loss

and tinnitus were over $1 billion. This is a 319% increase since the beginning of the

war in Afghanistan in 2001 [13, 14].

According to the Occupational Safety and Health Administration (OSHA) standard

for noise exposure, a workplace hearing conservation program is required by law if

average noise levels are at or above 90 dBA as an 8-hour time-weighted average using

a 5 dB exchange rate [28]. This means that for every 5 dB increase in exposure above

90 dB, the permissible exposure time is cut in half. In 1972, NIOSH recommended a

more conservative occupational noise exposure limit of 85 dBA as an 8-hour time-

weighted average using a 3 dB exchange rate [25]. Humans have the greatest

sensitivity to sound in the range of 1000 5000 Hz, and sound measurements can be

"A weighted" which attenuates noise outside of this range. The sound level is then

reported in terms of dBA [15].

Although attempts have been made to implement noise exposure guidelines and

hearing conservation programs, there are many barriers which prevent these efforts

from being fully effective. One survey of 29 foundry companies found that all were out

of compliance with hearing conservation regulations. Furthermore, the noise exposures

that workers received during their average shift routinely exceeded 85 dBA as a time-

weighted average. Members of the management, as well as employees from each









company were interviewed, and a positive correlation was found between management

and employee knowledge of hearing conservation. In other words, as management

education with regards to hearing conservation increased, employee knowledge

increased as well [16]. These data imply that lack of knowledge has the potential to

increase the risk of occupational NIHL. There are also compliance issues such as the

failure of workers to wear hearing protection devices (HPDs) properly. One example of

this could include removing HPDs in order to communicate. This repeated removal

makes it more likely that the HPD could become dirty or be inserted poorly, both of

which would compromise the seal within or around the ear thus decreasing the level of

sound attenuation. Another factor affecting compliance is that people do not notice an

immediate hearing loss, and it is therefore difficult to convince them that they are indeed

at risk [1]. Finally, many fear that wearing HPDs may affect their ability to perform, as is

the case with symphony musicians [17], to communicate, or to hear warning signals.

Although occupational hearing loss has received much attention, this is not the

only source of harmful noise. Recreational sources of noise can include hunting, skeet

shooting, personal music players, fireworks, nightclubs, and concerts. One survey

found that most adolescents routinely listen to music at maximum volume, and feel that

they are not vulnerable to the damaging effects of noise [19]. It was estimated that

12.5% (approximately 5.2 million) of individuals age 6 19 years old and 40% of

students age 16 25 years old exhibit noise-induced threshold shifts [20]. Furthermore,

the average sound levels for a concert are between 120 140 dBA, while bars and

taverns can reach 95 dBA on a busy night [21-23]. In a web-based survey, 61% of

concert attendees reported experiencing tinnitus or temporary hearing loss, and 59%









said that they would be more likely to use hearing protection if it were recommended by

a doctor or nurse [24]. It is also noteworthy that while occupational and recreational

noise exposure are often considered separately in research studies and for the

definition of occupational noise exposure limits, one must bear in mind that the same

person who receives their full legal noise dose at work may then proceed to come home

and mow the lawn while listening to a personal music player.

Limiting exposure and appropriate use of HPDs are considered the most effective

methods of hearing loss prevention. However, even when worn according to the

manufacturer's standards, there are many instances in which HPDs cannot reduce the

level of exposure below recommended limits. The noise reduction rating (NRR), which

represents the level of sound attenuation measured in a laboratory setting, must be

stated by the manufacturer for all types of HPDs. To date, the most sophisticated

hearing protection technology provides an NRR of approximately 30dB. Additionally,

many studies have shown that real-life attenuation is nowhere near the stated NRR. In

fact, NIOSH recommends the following subtractions from the manufacturer's labeled

NRR: for earmuffs subtract 25%, for formable ear plugs subtract 50%, and for all other

ear plugs subtract 70%. Using dual protection (ear plugs and earmuffs) only adds 5-

10dB of protection.[25].

It is evident that NIHL is a growing problem which must be addressed. Efforts

should include increased education, occupational hearing conservation programs,

improving HPDs, and further research into pharmacological preventative or even

hearing rescue methods. It has been said that, "prevention of NIHL would probably do









more to reduce the societal burden of hearing loss than medical and surgical treatment

of all other ear diseases combined" [1].

The Cochlea

Anatomy and Physiology

The cochlea is the organ which is responsible for the transduction of mechanical

energy in the form of a sound stimulus into electrical energy in the form of nerve

impulses which are sent to the brain via the auditory nerve (cranial nerve VIII). The

structure and function of the cochlea has been extensively studied in many species. A

brief overview of cochlear function is provided here, however for detailed review of

current understanding the reader is referred to the following references [29-31].

In mammals, the cochlea is spiral shaped and resides within a bony encasing

(the otic capsule) in the temporal bone. The lumen of the cochlea is divided into three

fluid filled chambers called the scala vestibuli, scala media, and scala tympani. The

basilar membrane separates the scala media from the scala tympani, while Reissner's

membrane forms the partition between the scala media and the scala vestibuli. A layer

of sensory epithelium known as the Organ of Corti houses the main sensory cells called

hair cells, and lies within the scala media atop the basilar membrane. The hair cells, so

named because of tiny hair-like projections called stereocilia at the apical end of each

cell, are divided into two types: inner hair cells (IHCs) and outer hair cells (OHCs).

There are three times as many OHCs as IHCs, the two types differ in location as well as

function. The tectorial membrane is composed of acellular connective tissue, and forms

a covering over the Organ of Corti. The fluid within the scala vestibuli and scala

tympani (perilymph) has a different ion concentration than the fluid within the scala

media (endolymph). The resulting difference in current potential, called the









endocochlear potential is important for sensory cell signal transduction. This

endocochlear potential is generated and maintained by a rich vascular bed in the lateral

wall of the scala media called the stria vascularis. (Pertinent structures of the cochlea

are depicted in figures 1-2 and 1-3)

The range of frequencies that can be heard by young humans with normal hearing

is approximately 40 Hz 20 kHz, and a sound stimulus entering the ear can contain

many frequencies which must be decoded by the cochlea. In 1862, Helmholtz

suggested that the basilar membrane is composed of fibers arranged radially which

each resonate at a different frequency analogous to the strings of a harp [32]. It was

later discovered that there are physical gradations in many structures of the cochlea.

For instance, the tectorial membrane and basilar membrane both become gradually

wider and thicker from the basal to the apical end of the cochlea. These gradations do

indeed confer a tonotopic organization allowing different areas of the basilar membrane

to resonate at specific frequencies. This led Bekesy to formulate his traveling wave

model, which earned him the Nobel Prize in 1961 [33]. Additionally, the hair cell

stereocilia increase in length from base to apex, and stereociliary stiffness in inversely

correlated with length [34]. This "tuning" of the tectorial membrane, basilar membrane,

and Organ of Corti allows for transduction of high frequency sound waves at the base,

and low frequency sound waves at the apex.

The hair cells are classified as either inner hair cells (IHCs) or outer hair cells

(OHCs). In the human cochlea, there are approximately 3,500 IHCs arranged in a

single row, and 11,000 OHCs arranged in three rows. Afferent signal transduction to

the auditory nerve is the main function of IHCs. These cells receive afferent innervation









from peripheral processes of the auditory nerve called spiral ganglion cells, and

glutamate is the neurotransmitter of the IHC synapse. A spiral ganglion cell contacts

only one IHC, while each IHC is connected to multiple spiral ganglion cells. Movement

of the endolymph causes deflection of stereocilia at the apical end of the IHC, which in

turn causes ion channels to open and close according to the frequency of the sound

stimulus. Entry of K+ and Ca2+ generates a transduction current which activates voltage

sensitive Ca2+ channels and Ca2+ activated K+ channels leading to the release of

neurotransmitter into the afferent synapse at the basal end of the cell [30]. The IHCs

also receive efferent innervation from the lateral olivocochlear complex. Originating in

the auditory brainstem, these neurons synapse on the peripheral processes of the spiral

ganglion cells as opposed to the IHC body itself (Figure 1-4) [30]. The efferent synapse

contains both excitatory acetylcholinee, dynorphin, and CGRP) and inhibitory

(dopamine, enkephalin, and GABA) neurotransmitters which modulate the afferent

sensitivity to glutamate. This may be a protective mechanism to prevent overstimulation

[35].

The role of the OHC is more complex. After studying the fluid dynamics of the

cochlea, it was recognized that a model relying on passive resonance such as that

proposed by Helmholtz and Bekesy was not sufficient. The viscosity of the endolymph

would dampen the resonance of the basilar membrane leading to a lower sensitivity

than that which is actually observed. Thus, it was proposed that the cochlea must

perform some active amplification of the incoming sound stimulus [36, 37].

Accumulating evidence suggested that the OHCs provide mechanical amplification by

vibrating at the same frequency as the sound stimulus in order to prevent damping of









the traveling wave. Thus OHCs can be thought of as the mechanical effectors of the

cochlear amplifier [31]. Afferent innervation of OHCs accounts for only 5% of auditory

nerve dendrites, while they receive rich efferent innervation from the olivocochlear

bundle. Unlike the IHCs, the OHC efferents synapse on the cell body itself which

provides support for an efferent feedback mechanism (Figure 1-5). Additionally, the

concept of an afferent signal leading to efferent stimulus and further amplification lends

itself to the analogy of "feedback" in a modern sound system. This idea eventually led

to the discovery of otoacoustic emissions, or sounds generated by vibration of the inner

ear which can be recorded in the external auditory canal [38]. Later, OHC motility was

clearly shown, as hyperpolarization causes the cells to lengthen, and depolarization

leads to cell shortening [39]. This movement of OHCs was captured by video

microscopy in 1986 [40]. The next puzzle to be solved was the mechanism of OHC

motility. In 2000, a new kind of motor protein was discovered in the OHC membrane.

This protein was named prestin, and is a member of the SLC26 family of anion-

bicarbonate transporters. The name prestin is derived from "presto" which means fast

in Italian, and was given due to its ability to operate on a microsecond timescale [40].

Overall, OHC motility serves to amplify the resonance caused by the sound stimulus,

which enhances the selectivity and specificity of cochlear tuning.

Cellular Mechanisms of Noise-Induced Cochlear Pathology

Exposure to high intensity noise has the potential to damage the cochlea, and to

impede its function in various ways. This damage can occur in response to impulse

noise such as gunfire, or continuous noise exposure, as is potentially experienced with

personal music players. Overexposure can cause both mechanical damage and

metabolic damage of the hair cells and surrounding structures. The OHCs are more









susceptible to damage than IHCs, and spiral ganglion neuron degeneration occurs

following IHC loss [41]. Furthermore, OHCs at the basal end of the cochlea are more

susceptible than those at the apex [42]. Hair cell death by both necrosis and apoptosis

simultaneously was shown one hour post-noise in chinchillas exposed to a continuous

noise insult of 110 dB SPL centered at 4 kHz for a duration of one hour [43]. Necrosis

is a passive form of cell death which is characterized by nuclear swelling, rupture of the

plasma membrane, and spilling of cell contents. This causes damage to surrounding

tissue, and initiates an inflammatory response. On the other hand, apoptosis is a

programmed pathway to cell death characterized by nuclear condensation and

fragmentation, which is essential in normal growth and development for the elimination

of unwanted or damaged cells [44]. Unlike necrosis, apoptosis does not cause damage

to the surrounding tissues. Apoptosis can also be triggered inappropriately causing the

death of necessary cells [45]. One study found that 30-50% of hair cells can be lost

before any measureable hearing loss can be detected [46].

Mechanical damage and glutamate excitotoxicity

Following acoustic overexposure there is an immediate loss of hearing sensitivity.

Depending upon the intensity and type of exposure, this loss of sensitivity can recover

fully or partially with time. This immediate hearing loss which recovers with time is

commonly known as temporary threshold shift (TTS), and any persistent loss is termed

permanent threshold shift (PTS). Less intense noise exposure is usually associated

with TTS alone, and mechanical damage to hair cell stereocilia [47], as well as

glutamate excitotoxicity are mechanisms which contribute to TTS. Glutamate

excitotoxicity refers to the release of large amounts of glutamate into the IHC afferent

synapse. This over stimulates the nerve causing swelling and damage which can









usually recover with time [48]. However, recent data indicate that even when hearing

sensitivity and hair cell function fully recover with time, neural degeneration without

concurrent loss of hair cells (primary neural degeneration) may still occur[49, 50]. As

the intensity of exposure increases above approximately 125 dB, cochlear injury is

primarily caused by mechanical rather than biochemical mechanisms. Additionally,

research suggests that impulse noise is more harmful than continuous noise of the

same intensity [51]. Any damage which causes significant hair cell death induces a

PTS because mammalian hair cells cannot be regenerated.

Oxidative stress

The production of reactive oxygen and reactive nitrogen species (ROS/RNS) in the

cochlea following noise exposure has been well documented and reviewed [52-56].

ROS and RNS include free radicals such as superoxide, peroxynitrite, and hydroxyl

radicals. Excess production of free radicals can have detrimental effects because an

unpaired electron makes these molecules extremely reactive, and capable of damaging

many cellular components. For a review of free radical mechanisms see [57]. Free

radical damage has also been implicated in a number of human neurodegenerative

diseases [58].

Following noise exposure in chinchillas, there was a marked increase in ROS seen

in the OHCs [59], and administration of paraquat (which produces superoxide) to the

round window membrane resulted in PTS and hair cell loss [60]. The cochlea contains

many endogenous antioxidants such as superoxide dismutase (SOD), catalase, and

glutathione (GSH) [61-64]. Glutathione directly scavenges free-radicals. The function

of SOD is to convert the harmful superoxide anion to molecular oxygen and hydrogen

peroxide, while catalase converts hydrogen peroxide to molecular oxygen and water.









Exposure to intense sound, especially at high frequencies, exerts a greater metabolic

demand on the outer hair cells, which by nature have a high energy requirement under

normal circumstances. Superoxide, which is a byproduct of mitochondrial respiration, is

produced in greater quantities under high metabolic demand and can react with nitric

oxide to generate the highly destructive peroxynitrite radical. Additionally, the

generation of hydrogen peroxide by SOD can participate in the Haber Weiss and

Fenton reactions to produce hydroxyl radicals [55]. Cochlear damage occurs when

large scale production of free radicals overwhelms endogenous antioxidant defenses.

Glutathione peroxidase prevents oxidative damage by reducing lipid peroxides and

catalyzes endogenous GSH production. One study showed that noise-exposed

glutathione peroxidase (Gpxl) knockout mice had a higher PTS, and greater IHC and

OHC loss than wild-type controls [65]. A four-fold increase in hydroxyl radicals was also

seen within 1-2 hours after noise exposure [66]. Finally, ROS and RNS production was

shown to peak 7-10 days following noise exposure, but hair cell loss progressed for

approximately 2 weeks. This suggests that there is a window of time following exposure

during which treatment with exogenous antioxidant or endogenous antioxidant

bolstering agents may be effective [67]. The link between ROS production and cell

death is not fully understood. However, there is widespread evidence that ROS play a

major role in the following cell death mechanisms [68].

Ischemia/ reperfusion injury: Due to their motility, the OHCs have a high energy

requirement and thus a high rate of aerobic respiration. As energy demand increases

with noise exposure, mitochondrial efficiency decreases, and superoxide is released as

an unwanted byproduct of oxidative phosphorylation. The decrease in mitochondrial









efficiency can be compounded when noise-induced damage of the lateral wall

vasculature causes a decrease in cochlear blood flow [69]. This state of ischemia,

when demand for oxygen is at its highest, further increases the production of ROS.

Furthermore, as the vasculature is repaired, and the site is reperfused, the sudden

increase in oxygen once again fuels the production of ROS [52]. This mechanism can

run in a vicious cycle, as ROS production can cause further damage to the stria

vasularis and decrease cochlear blood flow [70].

Lipid peroxidation: Lipid peroxidation is another self-perpetuating process in

which free radicals catalyze the breakdown of lipid molecules within cellular

membranes. A byproduct of lipid catalysis is 8-isoprostaglandin-F2a (8-iso-PGF2a)

which causes vasoconstriction, and again leads to decreased cochlear blood flow. In

guinea pigs, there was a 30 fold increase in 8-iso-PGF2a following noise exposure, and

the extent of hair cell loss corresponded to the level of 8-iso-PGF2a production [70].

Extrinsic vs. intrinsic pathways to apoptosis: The extrinsic and intrinsic

pathways are two primary signaling cascades leading to apoptosis. The extrinsic

pathway is mediated by cell surface death receptors, while the intrinsic pathway is

mediated by the release of pro-apoptotic factors from the mitochondria (Figure 1-6) [71].

There is evidence for the utilization of both pathways in noise-induced cell death in the

inner ear. Additionally, apoptotic hair cell death can take place through either caspase-

dependent, or caspase-independent pathways. Caspases are aspartate specific

cysteine proteases which can propagate a cell death signaling cascade. Caspases -8

and -9 are classified as initiators of the apoptotic signaling pathway, and caspases -3, -









6, and -7 are apoptotic effectors [72]. Caspases -8, -9, and -3 were shown to be

activated in chinchilla OHCs following noise exposure [59].

The extrinsic pathway is initiated when the death receptor (Fas) and its

associated adaptor protein, Fas-associated death domain (FADD) dimerize with Fas

ligand, which is a member of the TNF family, to form the death inducing signaling

complex (DISC). This in turn activates the initiator caspase-8 which can either directly

activate the effector caspase-3, or cleave BID which facilitates the release of

cytochrome C mediated by the insertion of Bax or Bak into the mitochondrial

membrane. Both of these circumstances result in cell death [72]. There is evidence

that many inflammatory cytokines, including those of the TNF family which can act as a

death receptor ligand, are upregulated following noise exposure [73]. Active caspase-8

was also shown to be upregulated in hair cells following noise exposure [59].

The intrinsic pathway can be initiated by a variety of mechanisms following noise-

induced ROS production and cell damage. Damage to membrane transport proteins

leads to the influx of Ca2+ [74, 75] which in turn causes phospholipase A2 (PLA2)

activation and calpain dependent cleavage of calcineurin. Subsequently, PLA2

hydrolyzes phospholipids to pro-inflammatory mediators which lead to caspase

activation and cell death [76]. Cleavage of calcineurin allows for phosphorylation of

nuclear factor of activated T-lymphocytes (NFAT), which is a transcription factor

controlling many genes involved in regulation of cell death [77]. Calcineurin can also

dephosphorylate the pro-apoptotic regulator Bcl-2-associated death promoter (BAD),

which translocates to the mitochondria, downregulates anti-apoptotic members of the









Bcl-2 family and activates Bcl-2-associated X protein (Bax). This causes mitochondrial

membrane permeabilization, release of cytochrome C, and ultimately cell death [78].

It is clear that many of these pathways converge at the point of mitochondrial

membrane permeabilization. It is well known that the Bcl-2 family of proteins regulate

mitochondrial membrane permeability, and ultimately cell death. There are pro-

apoptotic (Bax, Bak, Bcl-Xs, Bid, Bad, and Bim), and anti-apoptotic (Bcl-2 and Bcl-XL)

Bcl-2 proteins. When the ratio shifts in favor of pro-apoptotic proteins, Bax translocates

from the cytoplasm to the mitochondria, causes membrane permeabilization, and

release of cytochrome C into the cytoplasm. Cytochrome C can then associate with

apoptotic protease-activating factor-1 (APAF-1), dATP, and procaspase-9 to form the

apoptosome. This causes activation of caspase-9, which activates effector caspases

leading to cell death [79]. Evidence of the importance of the Bcl-XL/Bak ratio in cell

survival was shown in the inner ear using two noise exposure groups. One group was

exposed to noise of a lower intensity designed to induce only TTS, while the other group

was exposed to intense noise causing PTS. Bcl-XL was upregulated in hair cells of the

TTS group, while Bak was expressed in the PTS group [80].

Other factors thought to be involved in noise-induced cell death are p53 and c-jun

NH2-terminal kinases (JNKs). The tumor suppressor protein p53 is activated by DNA

damage, and also causes loss of mitochondrial membrane integrity [81]. JNKs are

thought to function upstream of caspase activation or cytochrome C release. MAP

kinases are activated by cellular insult causing phosphorylation of the transcription

factor c-jun, which controls many genes regulating cell death [82-84].









The intrinsic pathway to cell death also consists of caspase-independent

mechanisms. For review of these mechanisms see [85]. Apoptosis inducing factor

(AIF) and endonuclease G (endo G) are two mitochondrial proteins which, like

cytochrome C, can be released upon loss of mitochondrial membrane integrity. Endo G

is a sequence non-specific DNase which normally functions in mitochondrial DNA

replication and repair [86]. AIF was identified in 1999, and is thought to have an

essential role in development [87]. Following their release, AIF and endo G translocate

to the nucleus and cause DNA fragmentation leading to cell death [88-92].

Ironically, these "caspase-independent" pathways may actually activate a unique

pathway involving caspase-2. Caspsase-2 is different than its other family members

because it has characteristics of both an initiator, and an effector caspase. It is thought

that activated caspase-2 acts upstream causing mitochondrial membrane

permeabilization and cytochrome C release. To date, caspase-2 activity in response to

noise has not been characterized in the inner ear. However, caspase-2 is known to

play a role in cell death subsequent to DNA damage [93-95]. Due to the DNA damage

caused by translocation of AIF and endo G, it is reasonable to think that caspase-2 may

also be involved in hair cell death secondary to noise exposure. Furthermore, one

study found that ROS formation led to activation of caspase-2 in a human leukemic T

cell line [96].

Experimental Treatment for Noise-Induced Hearing Loss

Although its prevalence continues to increase despite hearing conservation

programs, there are currently no FDA approved pharmacological agents for the

prevention or treatment of NIHL. However, there has been a great deal of preliminary

translational research driven by the recent increase in understanding of the cellular and









molecular pathways underlying NIHL. The complexity of these pathways implies many

possible therapeutic targets and points of intervention. Numerous drugs have entered,

or have been proposed for, clinical trials based on demonstration of protective efficacy

or even rescue of hearing loss across various animal species (For thorough review see

[97]). Most of these drugs fall into one of three categories: direct inhibitors of cellular

stress pathways, neurotrophic factors/ neurotransmission blockers, or direct inhibitors of

oxidative stress [98]. A significant barrier to clinical applicability is that many of these

treatments have only been shown to be effective when applied locally in the inner ear.

This is obviously not practical for humans anticipating, or having recently been exposed

to harmful noise levels. Thus, the ideal treatment would be one that can be given

systemically (preferably orally), and still achieve a therapeutic level in the inner ear.

Inhibitors of Cellular Stress Pathways

The noise-induced cellular stress response can lead to inflammation, disruption of

calcium homeostasis, and activation of kinase signaling cascades leading ultimately to

cell death. Glucocorticoids have long been used as nonspecific inhibitors of the stress-

induced inflammatory response. Dexamethasone, a synthetic glucocorticoid, was

infused via implanted osmotic mini-pumps directly into the scala tympani of guinea pigs,

and a significant reduction of noise-induced PTS and hair cell loss was observed.

Dexamethasone binds to glucocorticoid receptors in the inner ear and regulates

transcription of inflammatory mediators, thus reducing ischemia and ROS production

[99]. However, systemic administration of glucocorticoids is known to be associated

with many untoward side effects, making it an impractical treatment for NIHL.

As mentioned previously, noise-induced calcium influx causes neuronal damage

and phospholipase A2 activation leading to lipid peroxidation and ROS production.









Calcium dysregulation also leads to calpain dependent cleavage of calcineurin and

activation of the transcription factor NFAT leading to apoptosis. Oral administration of

the calcium channel blockers trimethadione and ethosuximide [100], or diltiazem [101]

was carried out in mice and guinea pigs respectively. Trimethadione and ethosuximide

exhibited a protective effect when given after noise exposure, and trimethadione

reduced PTS when given prior to noise exposure. Diltiazem protected OHCs from

impulse noise only when applied both before and after exposure. Inhibition of calpain or

calcineurin using leupeptin [82, 102] or cyclosporin A and FK506 [103, 104] respectively

also achieved a protective effect.

Finally, inhibition of two kinase signaling pathways was explored. These signaling

cascades regulate many genes involved in cell death, and include the Src protein

tyrosine kinase (PTK), and c-Jun N-terminal kinase (JNK) pathways. One of the

detrimental effects of Src-PTK signaling is the activation of NADPH oxidase which

causes increased superoxide production. Reduction of PTS and hair cell loss was

achieved following application of Src-PTK inhibitor KX1-004 to the round window

membrane of chinchillas [105]. Similarly, the JNK inhibitors D-JNK-1 (aka: AM-111)

[106, 107] and CEP-1347 [84] exhibited a protective effect following local administration

in various species.

Neurotrophic Factors/ Neurotransmission Blockers

Neurotrophic factors are secreted proteins which promote the survival, growth, and

differentiation of neurons. Several neurotrophic factors such as neurotrophin-3 (NT-3)

and brain-derived neurotrophic factor (BDNF) are expressed in hair cells [108]. Qiang

et al., 2004 cultured mouse spiral ganglion neurons and exposed them to high

concentrations of glutamate in order to simulate glutamate excitotoxicity. Less neuronal









cell death was seen when basic fibroblast growth factor (bFGF) was added to the

media. Next, they showed that systemic injection of bFGF in guinea pigs decreased

PTS and hair cell loss following noise exposure. The interaction of bFGF with growth

factor receptors on spiral ganglion cells and hair cells is proposed to increase

expression of oxidase, decrease NO production, modulate intracellular calcium, and

interfere with expression of pro-apoptotic proteins [109]. Similarly, regrowth of spiral

ganglion neurons following deafferentiation with aminoglycoside antibiotics was

observed with BDNF and bFGF treatment in guinea pigs.

Another approach aimed at neuroprotection involves the attenuation of glutamate

excitotoxicity using glutamate receptor antagonists, or glutamate neurotransmission

blockers. Carbamathione and caroverine are glutamate receptor antagonists which

reduced PTS following noise with systemic injection in chinchillas and local

administration in guinea pigs respectively [110, 111]. Carbamathione downregulates N-

methyl-D-aspartate (NMDA) receptor activity, while caroverine acts on both NMDA and

a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. Another

agent which exhibits NMDA receptor antagonistic properties is the mineral magnesium

[35]. Riluzole (2-amino-6-trifluoromethoxy benzothiazole) is a glutamate

neurotransmission blocker which exhibited a protective effect following local and

systemic administration in guinea pigs [112].

Inhibitors of Oxidative Stress

Acoustic overstimulation decreases mitochondrial efficiency, stimulates excess

glutamate release into the afferent IHC synapse, and induces a state of cochlear

ischemia, all of which increase production of ROS. Many approaches are being

explored which enhance endogenous cochlear antioxidant defenses, inhibit production









of ROS, or directly scavenge free radicals. Antioxidant treatments are potentially

appealing due to the fact that many of these compounds have been shown to be safe

when given orally.

Early attempts to augment endogenous antioxidant defenses used R-

phenylisopropyladenosine (R-PIA) and glutathione monoethyl ester (GSS) [113] applied

locally to the round window membrane of chinchillas. R-PIA, which increases GSH and

superoxide dismutase levels, decreased PTS and OHC loss. The cell permeable

enzyme glutathione synthetase (GSS), which is involved in GSH production also

provided a significant reduction in PTS. Other compounds which increase endogenous

GSH, including n-acetyl-cysteine (NAC) and D-methionine (D-met), reduce NIHL as

well. In addition to its ability to directly scavenge free radicals, NAC is also one of the

amino acids composing GSH, and is thus able to replenish this endogenous antioxidant

when depleted following intense noise exposure (For review see [114]). NAC has been

shown effective for the reduction [110, 115, 116] and rescue [54, 117, 118] of NIHL in

various species. It was also effective when given orally, but its efficacy was greatly

decreased [119]. D-Met also acts primarily as an indirect antioxidant by increasing

intracellular GSH levels. This is possible because methionine is used in the synthesis

of cysteine, a component of GSH. D-Met also attenuates the increase in SOD levels

which is regularly observed in the cochlea following noise exposure [120]. SOD is

responsible for the conversion of superoxide to hydrogen peroxide, which is

subsequently removed by catalase. The increase in SOD following noise is not

accompanied by an increase in catalase, therefore the excess hydrogen peroxide

creates favorable conditions for ROS production. Administration of D-Met was found to









prevent decreases in Na/K ATPase and Calcium ATPase activity, decrease intracellular

NO concentration, and prevent lipid peroxidation following noise [79].

Agents recognized for their ability to inhibit the production of ROS include

allopurinol, acetyl-L carnitine (ALCAR), and 2-phenyl-1,2-benzisoselenazol-3(2H)-one

(ebselen). Allopurinol, also used for its ability to inhibit uric acid synthesis in chronic

conditions such as gout, attenuated PTS in systemically injected rats [121]. The

mitochondrial membrane component ALCAR serves as a precursor for acetyl-CoA and

L-carnitine which carry lipids into mitochondria for p-oxidation and enhance ATP

production. Therefore, this compound improves mitochondrial efficiency, and

decreases ROS production. ALCAR was effective for reduction of hair cell loss, and for

prevention [110, 115] and rescue [118] of PTS. Ebselen is a glutathione peroxidase

mimetic which decreases hydroperoxide formation, scavenges peroxynitrite, inhibits NO

synthase, and prevents lipid peroxidation and cytochrome C release. Ebselen is

effective for the reduction of TTS, PTS, and hair cell loss [122-124]. It also reduced

noise-induced swelling of the stria vascularis in rats [125].

Finally, multiple antioxidant compounds which directly scavenge free radicals were

found to attenuate PTS and hair cell loss to varying degrees. Among these are

mannitol, salicylate, resveratrol, coenzyme Qio, and 4-hydroxy phenyl N-tert-

butylnitrone (4-OHPBN). Mannitol, a scavenger of hydroxyl radicals, attenuated PTS

when injected systemically [126]. In addition to scavenging hydroxyl radicals, salicylate

forms the iron chelator dihydrobenzoate, which prevents ROS formation by inhibiting the

iron catalyzed Fenton reaction [117]. Resveratrol, which is naturally present in grapes

and red wine, acts as an anti-inflammatory, vasodilator, and neuroprotectant in addition









to its antioxidant properties [121, 127]. Coenzyme Qlo participates in oxidative

phosphorylation as an integral member of the electron transport chain, and also has

potent antioxidant activity. Efficacy of oral administration was increased when given as

coenzyme Qlo Terclatrate (Q-Ter), a form which is highly water soluble [128][129].

Finally, 4-OHPBN, scavenges hydroxyl radicals and superoxide anions, showed a dose

dependent reduction in PTS and OHC loss in chinchillas [130]. This compound has

entered phase III clinical trials as a treatment for stroke [129].

To date, no treatment has been identified which is completely effective for the

prevention or rescue of NIHL. Significant advancements have been made in identifying

agents which are effective when given systemically, and are thus more clinically

applicable. Due to their different mechanisms of action, and the fact that no single

agent has provided complete protection, it may be beneficial to explore various

combinations of these agents.

Beta-Carotene, Vitamins C and E, and Magnesium

In 2007, Le Prell et al. administered either a combination of the antioxidants 3-

carotene and vitamins C and E, magnesium alone (Mg), or 3-carotene and vitamins C,

and E plus magnesium in guinea pigs exposed to 5 hours of 120dB SPL octave band

noise centered at 4 kHz. Given the fact that 3-carotene is metabolized to vitamin A, this

treatment will be subsequently referred to as ACEMg. Treatments were given once

daily beginning 1 hour prior to noise exposure, and continuing until day 5 post-noise.

Auditory brainstem response (ABR) thresholds were measured at day 10 following

noise exposure. Threshold shifts for the ACEMg group were significantly decreased

compared to saline control animals at 4, 8, and 16 kHz. There was no significant

difference in ABR threshold between control animals and those treated with either ACE









or Mg (Figure 1-6). Hair cell loss was also significantly reduced in the ACEMg group

compared to control and ACE or Mg alone (Figure 1-7) [131]. These data suggest a

synergistic protective effect between agents.

Each compound in this combination treatment has a distinct site and mechanism

of action for the reduction of cellular damage secondary to oxidative stress. Beta-

carotene is a fat-soluble nutrient which effectively scavenges singlet oxygen. As

previously mentioned, 3-carotene is metabolized to vitamin A. Once adequate reserves

of vitamin A have been established, excess 3-carotene in the blood is free to directly

scavenge free radicals. Singlet oxygen reacts with membrane lipids to form lipid

hydroperoxides, thus 3-carotene prevents lipid peroxidation [132]. Vitamin C (ascorbic

acid) is a water soluble essential nutrient which can directly reduce free-radicals in the

aqueous phase [133]. As well as scavenging superoxide, hydroxyl radicals, and singlet

oxygen, ascorbic acid can regenerate a-tocopherol (vitamin E) from the a-tocopheroxy

radical which results from the reduction of ROS by vitamin E [134]. Dietary or

systemically injected vitamin C reduced PTS, prevented hair cell loss, increased

endogenous antioxidants in guinea pigs and rabbits [135, 136]. Ascorbic acid also

reduced NO production by 56% in the lateral wall and 37% in the organ of Corti of

guinea pigs [137]. Vitamin E (a-tocopherol) is another lipophilic nutrient which reduces

peroxyl radicals, peroxynitrite radicals, and inhibits the propagation of lipid peroxidation

[132]. Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), a water soluble

and cell permeable derivative of a-tocopherol is often used for treatment. Pretreatment

with vitamin E significantly reduced PTS and hair cell loss, and post-treatment had a

protective effect when initiated up to 3 days post-noise [138]. Dietary vitamin E showed









a dose-dependent reduction in PTS in fathead minnows (Pimephales promelas) [139].

The protective role of magnesium in NIHL is most likely due to its role as a vasodilator.

Magnesium inhibits calcium influx into vascular smooth muscle cells by blocking calcium

channels, and also activates adenylate cyclase which forms adenosine 3',5'-

monophosphate within vascular smooth muscle cells leading to dilation [140].

Magnesium may also reduce glutamate excitotoxicity due to its NMDA-antagonistic

effect on afferent dendrites [35, 141]. The noise-induced reduction of cochlear blood

flow, as well as the associated decrease in perilymph oxygenation, was attenuated in

guinea pigs maintained on a high magnesium diet [142]. Also in guinea pigs, PTS and

susceptibility of hair cell stereocilia to noise damage showed a negative correlation with

perilymph magnesium concentration, and there was a marked reduction in TTS as

well[143, 144]. Human trials with magnesium for NIHL have also been shown to reduce

PTS as well as TTS with no side effects [145, 146].

Preliminary data from Le Prell et al. showed that type II fibrocytes, strial cell

density, and threshold sensitivity were preserved in noise-exposed CBA/J mice

maintained on a high ACEMg diet[147]. Thus, this micronutrient combination has been

shown to be effective for reduction of NIHL as a dietary supplement, and each of its

components has a very high safety profile based on AREDS seven year ACE trials in

humans.

Study Design

The aim of this study was: 1) to evaluate activation of caspase-2 following noise

exposure, and 2) to further characterize the effect of ACEMg treatment on free radical

production, and caspases -2, and -8 activation subsequent to noise exposure. The

difference in expression pattern of these target molecules known to be upregulated









following intense noise were assessed in antioxidant treated and control animals using

immunohistochemistry. There are two splice variants of caspase-2 which play opposite

roles in initiation of apoptosis. Caspase-2L induces cell death, while caspase-2s

suppresses cell death [148]. Caspase-2L was assessed in this study; there are no

selective antibodies for caspase-2s. Caspase-2 expression has been described in the

inner ear of newborn rats, where it plays a role in apoptotic cell death during

development [149]. However, whether it is activated secondary to noise exposure

remains to be determined. Production of 3-nitrotyrosine (3-NT) was assessed as an

indicator of oxidative stress in antioxidant treated and control animals. 3-NT is known to

accumulate in conditions involving oxidative stress such as Huntington's disease and

ischemic brain injury. It is also used as a reliable marker of RNS activity [67, 138, 150].

Caspase-8 expression was evaluated to determine the effect of antioxidant treatment on

the extrinsic (death receptor mediated) apoptotic pathway. We hypothesized that free

radical production (as assessed by production of 3-NT) would be reduced in antioxidant

treated ears, caspase-2 would be activated in noise exposed animals confirming its role

in noise-induced cell death, and noise-induced caspase-8 activation would not be

effected by ACEMg treatment because antioxidant treatment presumably should not

interfere with the ligation of death receptors.
















Hearing
Level
in dB


0
10
20
30
40
50
60
70
80
90
100
1


I I%"


Frequency in Hertz












_-___ -_
-r



4--- -
-


250 500 1000 2000 3000 4000 6000 8000

Right Left
I- _X


Figure 1-1. Typical audiogram exhibiting early NIHL. The bilateral decrease in hearing
sensitivity at 4000 Hz forms the characteristic "noise notch" [15].

i" ~eisoner's
Scala Membrane
estiuli Tetral Lateral Wall
9cala Memorane IApteal)





Bal ilar












Figure 1-2. Light micrograph of a cross-section of the guinea pig cochlea. Major
structures are labeled, and anatomical directions are noted in parenthesis
[30].










SCALA MEDIA
(Endolymph)


25.0 ipm
(Medhliul


(Lateral)


Stereocilia


Tectorial Membrane



S-*-


Figure 1-3. Cross-section of the organ of Corti with tectorial membrane covering the hair
cells [30].


Figure 1-4. Diagram showing efferent (E) and afferent (A) innervation of the IHC.
Excitatory (+) and inhibitory (-) neurotransmitters are shown along with their
receptors. NPR denotes neuropeptide receptors with various functions, and
IPC represents the inner phalangeal cells which surround the IHC [30].














Outer Hair Cell


ssC


-- MOC MediaI
S- Efferents
Afferent from
type 1I SGO

Figure 1-5. Diagram showing afferent (A) and efferent (E) innervation of the OHC.
Excitatory (+) and inhibitory (-) neurotransmitters and receptors are shown.
SSC denotes sub-synaptic cisternae. Outer phalangeal cells/ Deiters cells
are represented laterally (OPC/DC) [30].

Oxidative Stress
Death a

/. i, Calpain eoo 0
0NFAT 0 Glutamate
SNFAT0s of NTF
a Bad
(Casp-n~r Bid -- / ,
cvt c BakJ
4 ATPO-+ O
'!,s, X'Casp1 A
EpC yl c AIF
EndoG
*,JC~p^ ,., *f *f cl ^


Cell
Death

Casp-2.)



Figure 1-6. Apoptotic pathways. Initiation of apoptosis can occur through either the
extrinsic (death-receptor) pathway or the intrinsic (mitochondria mediated)
pathway[56].


OPC/G;


OPULCD















(n



-10
03


4kHz


8 kHz
Frequency


16 kHz


Figure 1-7. Effect of ACEMg treatment on NIHL. Auditory brainstem response (ABR)
threshold shift before and 10 days after noise exposure were significantly
reduced with ACEMg treatment, but not with either ACE, or Mg alone [131].


B
I Saline
mACE
SACEM g
ACEMg


-I bJ


10-14,99


15-20


Distance from Apex (mm)


Figure 1-8. Effect of ACEMg treatment on IHC and OHC loss. Outer hair cell loss was
significantly reduced with ACEMg treatment, but not with either ACE, or Mg
alone [131].


Saline -
PWWi ACE
Mg
Ly-0- ACEN~g


8


o









CHAPTER 2
MATERIALS AND METHODS

Subjects

Albino male guinea pigs (250-350 grams) from an approved laboratory animal

supplier (Charles River, Wilmington, MA) were used. Guinea pigs were housed in the

Animal Care Facility at the University of Florida, and were identified using ear clips.

Following arrival, all animals were given at least 48 hours to acclimate to the

environment and recover from transportation-related stress. All experimental protocols

regarding the use and care of animals were reviewed and approved by the University of

Florida Institutional Animal Care and Use Committee.

Noise Exposure

At the onset of the study, speakers were calibrated by placing microphones at the

level of the animals' heads (while the cages were unoccupied). Animals were exposed

four at a time, each in separate cages. The cages were arranged in the sound booth so

that the exposure each animal received was 114 4 dB SPL octave band noise

centered at 4 kHz, depending on location within cage. Noise exposure lasted exactly

four hours. For exposure of treated and control animals, two antioxidant treated and

two saline control animals were included in each noise exposure group. The cage

positioning of treated and control animals was alternated with each noise exposure.

Animals were unrestrained and were not anesthetized during the noise exposure period.

Electrophysiological Tests

All subjects were screened for normal hearing sensitivity at 2, 4, 8, 16, and 24 kHz

in the right and the left ear using the sound-evoked auditory brainstem response (ABR);

left ear ABR tests were repeated on one treated and one control animal per exposure









group post-noise, prior to euthanasia to verify hearing loss obtained using this

exposure. During ABR tests, animals were anesthetized with ketamine (40 mg/kg, s.c.)

and xylazine (10 mg/kg, s.c.) and neural activity in response to brief, tone pips was

measured using sterile, 27-gauge electrodes inserted subcutaneously posterior to each

pinna and at the vertex of the skull. Tone levels were decreased from 90 dB SPL to 0

dB SPL in 10-dB increments. Each pip was 10 milliseconds in duration and tones were

repeated at a rate of 17/second until 1026 responses were acquired. Threshold was

independently determined using a 25-pV Wave III response criterion. Animals were

placed on a water-circulating heating pad to maintain body temperature and lubrication

was applied to the eyes to prevent dryness during ABR procedures. The depth of the

anesthesia was measured using the pedal withdrawal reflex and additional anesthetics

administered as needed. All animals received an overdose of sodium pentobarbital

following ABR tests, and were euthanized for immunohistochemical assays.

Antioxidant Treatment

All antioxidant treated animals received a total of two treatments. The first

treatment was administered 24 hours prior to noise exposure, and the second treatment

was given 1 hour prior to noise. Control animals received saline injections equivalent to

the dose of micronutrient cocktail given in treated animals. The micronutrient cocktail of

3-carotene, vitamins C and E, plus magnesium was given as follows: vitamin A (2.1

mg/kg 3-carotene, po), vitamin C (71.4 mg/kg L-threoascorbic acid, sc), vitamin E (26

mg/kg ()-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid, "Trolox," sc),

magnesium (343 mg/ kg MgSO4, sc). Trolox is a cell-permeable, water-soluble

derivative of vitamin E. All test substances were purchased from Sigma-Aldrich (St.

Louis, MO, USA) (3-carotene, C9750, CAS 7235-40-7; L-threoascorbic acid, A5960,









CAS 50-81-7; Trolox, Fluka Chemika, 56510,CAS 53188-07-1; magnesium sulfate,

M7506, CAS 7487-88-9).

Immunohistochemistry

Following anesthesia with ketamine (40 mg/kg, s.c.) and xylazine (10 mg/kg, s.c.),

animals were euthanized at various time points via sodium pentobarbital overdose and

were decapitated. Cochlear tissues were immediately harvested and perfused with 4%

methanol-free formaldehyde. Tissues remained in fixative for 3 hours before being

rinsed and stored in phosphate buffered saline (PBS) until immunolabeling began. In

general, tissues were blocked with normal serum, permeabilized with Triton-X-100,

incubated with primary antibody, and labeled with secondary antibody. After

immunolabeling was complete, tissues were rinsed (in PBS), dissected for surface

preparations, and mounted on glass slides using VectaShield mounting medium. To

assure an accurate representation of labeling, two images were taken from each turn of

the cochlea. Images were collected using a Leica DM5500B epifluoresence

microscope, and processed with ImagePro 6.3 software. Images were acquired as a Z-

stack, deconvolved using a nearest neighbor algorithm, and tinted after they were

acquired in monochrome.

Nitrotyrosine Immunolabeling

Antioxidant treated (N=5) and saline control (N=5) tissues were harvested 2 hours

after noise exposure. All tissues were fixed as mentioned above, rinsed with PBS,

permeabilized with 0.5% Triton-X-100 for 1 hour, and rinsed again. Subsequently,

tissues were blocked with Power Block for 5 minutes. After blocking, tissues were

rinsed with PBS and incubated with anti-3-nitrotirosine mouse monoclonal antibody

(clone 39B6; Alexis Biochemicals, 1:500 for 24 hours at 4C). The primary antibody









was omitted in negative control tissues. Following PBS rinse, tissues were incubated in

the secondary antibody (1:100 AlexaFluor 488 goat anti-mouse IgG) for 1 hour at room

temperature.

Caspase-8 Immunolabeling

Antioxidant treated (N=5) and saline control (N=5) ears were processed in the

same manner as described for nitrotyrosine immunolabeling. Tissues were incubated in

mouse anti-caspase-8 monoclonal antibody (Santa Cruz Biotechnology; sc-5263, 1:500

for 48 hours at 4C). The primary antibody was omitted in negative control tissues.

Following PBS rinse, tissues were incubated in the secondary antibody (1:100

AlexaFluor 488 goat anti-mouse IgG) for 1 hour at room temperature.

Caspase-2 Immunolabeling

The purpose of caspase-2 immunolabeling was two-fold: 1) to determine if

caspase-2 immunolabeling would be observed post-noise since this has not been

described previously, and 2) to gather preliminary data concerning the effect of ACEMg

treatment on caspase-2 expression post-noise (ACEMg, N=2; saline control N=2). All

tissues were labeled for caspase-2L (long variant). Animals used for the initial

characterization of caspase-2 expression were euthanized at 2 (N=4), 4 (N=4), and 24

(N=3) hour post-noise time points. Animals euthanized with no noise-exposure served

as controls (N=4). All antioxidant treated and saline control animals were euthanized at

2 hours post-noise.

Fixed and rinsed tissues were permeabilized with 0.5% to 1% Triton-X-100 for 30

to 60 minutes. Subsequently or simultaneously, tissues were blocked with 10% Normal

Goat Serum (with or without 1% BSA) for 30 to 60 minutes, or with Power Block for 5

minutes. After blocking, tissues were rinsed in PBS, and then incubated for 24 hours in









mouse anti-caspase-2 antibody (BD Transduction Laboratories #611022, ICH-1 L) at

4C. Preliminary tests with 1-10 pg/ml initial concentrations revealed the 2.5pg/ml

concentration to produce the most specific labeling. After incubating in the primary anti-

caspase-2 antibody, tissues were rinsed in PBS. All tissues were then incubated in the

secondary antibody (1:100 Alexaflour 488 goat anti-mouse IgG) for 1 hour at room

temperature (20C).

Statistical Analysis

In order to determine antioxidant treated versus saline control group differences in

3-NT and caspase-8 expression, seven observers who were blind to study conditions

were asked to rank image sets in order from least immunolabeling within the hair cells

to most. These sets were generated by pooling images from treated and control

animals according to the section of the cochlea from which they were taken. Thus,

there were three image sets from each target molecule (3-NT and caspase-8)

corresponding to the first, second, and third turns of the cochlea. Each image set

consisted of twenty images, as there were ten animals (ACEMg treated n=5; saline

control n=5), and two images were taken from each turn of the cochlea. Each observer

was given uniform training and instruction on how to judge the images. Additionally, a

significantly positive (p<0.01) Spearman's rank correlation coefficient was observed

between each of the observers' rankings. The rank numbers assigned to each image

were averaged across all observers to give average image scores. Subsequently, the

mean of the two average image scores from each animal was taken to give average

section scores. Finally, the means of the average section scores were taken to give

average ear scores for each of the ten animals. These data were then employed to

detect group differences using the Wilcoxon-Mann-Whitney two-sample rank-sum test,









which tests the null hypothesis that the probability distributions associated with the two

populations (ACEMg treated and saline control) are equivalent. The data were

analyzed for treatment effect within each section, as well as within the ear as a whole.









CHAPTER 3
NITROTYROSINE

Production of 3-nitrotyrosine (3-NT) was assessed as an indicator of oxidative

stress in antioxidant treated (N=5) and saline control (N=5) animals. The increased

metabolic demand on hair cells due to noise-exposure causes an increase in the

production of superoxide. Superoxide then reacts with nitric oxide to generate the

peroxynitrite anion, which modifies cellular proteins to form 3-nitrotyrosine. We

hypothesized that antioxidant treatment would decrease the production of RNS, and

thus decreased labeling would be observed within the hair cells of treated ears upon

comparison with those of saline control ears.

Results

Upon initial observation, the results appeared encouraging due to the apparent

difference in immunolabeling in the hair cells between the ACEMg and saline control

groups (Figure 3-1). However, this difference was not consistently observed (Figure 3-

2), and did not prove to be statistically significant with the current methods of analysis

and sample size. Negative control tissues in which the primary anti-3-NT antibody was

omitted did not show significant non-specific labeling (Figure 3-3). The results of the

Mann-Whitney U test which was conducted for the detection of differences in the

probability distributions between the treated and control groups were as follows: median

section scores in the first turn for the ACEMg and saline control groups were 11.43 and

12.71 respectively (Mann-Whitney U = 10.0, p = 0.345 one-tailed); median section

scores in the second turn for ACEMg and saline control groups were 11.29 and 8.79

respectively (Mann-Whitney U = 12.0, p = 0.50 one-tailed); median section scores in the

third turn for the ACEMg and saline control groups were 7.00 and 14.29 respectively









(Mann-Whitney U = 11.0, p = 0.421); finally, the median average ear scores for the

ACEMg and saline control groups were 9.90 and 10.83 respectively (Mann-Whitney U =

10.0, p = 0.345). These results are summarized in Table 3-1.

Discussion

Nitrotyrosine is known to accumulate in conditions involving oxidative stress such

as Huntington's disease and ischemic brain injury [151]. It has also been used as a

reliable biomarker of RNS activity [67, 138]. The reaction of superoxide with nitric oxide

(NO) generates the highly reactive peroxynitrite anion, which modifies cellular proteins

to generate nitrotyrosine [150]. Treatment with this antioxidant cocktail was expected to

reduce the production of RNS, and thus 3-NT. This hypothesis is based on the ability of

ascorbic acid to scavenge superoxide and to reduce noise-induced NO production

[137], and also the overall efficacy of ACEMg treatment for the reduction of noise-

induced PTS as was demonstrated previously [131].

However, as was previously mentioned, we did not observe a statistically reliable

difference in 3-NT production between the treated and control groups using the current

study design. The lack of statistical significance seen in our results may be caused by

temporal and spatial variation in 3-NT production following noise. Evidence for this

theory is based on a varying pattern of 3-NT production at different post-noise time

points. A previous study showed that immunostaining for 3-NT following noise

exposure was initially low, and localized to the supporting (Hensen and Claudius) cells.

Additionally, significant immunostaining did not appear in the hair cells until later time

points (day 7-10), when 3-NT production reached a maximum (Figure 3-4) [67]. All

animals in this study were sacrificed two hours following noise exposure, which

corresponds to the time at which Yamashita et al. described 3-NT production only in the









supporting cells. Our images were analyzed with respect to immunolabeling within the

outer hair cells alone, and this may be the reason for our failure to detect a significant

treatment effect. Given this information, it may be that an appreciable treatment effect

would be more readily detectable in the 7-10 day post-noise range during the time of

peak 3-NT production. These studies are in progress.
























B














Figure 3-1. Epifluorescence micrographs of ACEMg treated (A) and saline control (B) sections of the organ of Corti
labeled with anti-3-nitrotyrosine antibody showing the greatest observed treatment effect. Sections in the left,
middle, and right columns were taken from the 1st, 2nd, and 3rd, turns of the cochlea respectively. These images
were selected from ears with the lowest treated and the highest saline average rank score.






































Figure 3-2. Epifluorescence micrographs of ACEMg treated (A) and saline control (B) sections of the organ of Corti
labeled with anti-3-nitrotyrosine antibody showing median treated and control images. Sections in the left,
middle, and right columns were taken from the 1st, 2nd, and 3rd, turns of the cochlea respectively. These images
were selected from ears with the median treated and saline rank score.


























Figure 3-3. Negative control ear in which the primary 3-NT antibody incubation step was omitted.


Table 3-1. Difference in distribution of ACEMg treated and saline control 3-NT image rank scores
1st Turn 2nd Turn 3rd Turn Ear Average
Median ACEMg 11.43 11.29 7.00 9.90
Score
Median Saline Score 12.71 8.79 14.29 10.83
Mann-Whitney U 10.0 12.0 11.0 10.0
P-value (one-tailed) 0.345 0.500 0.421 0.345












































Figure 3-4. Immunostaining for NT shifts from supporting cells (Hensen, Claudius) to
OHCs, including Deiters, with a maximum at 7- 10 days. Sections are from
an area approximately one half turn apical of the main lesion (A: control, B:
immediate, C: Day 3. D: Day 7 E: Day 10, F: Day 14). All guinea pigs were
exposed to octave band noise centered at 4 kHz and 120 dB SPL for 5 hours
[67].









CHAPTER 4
CASPASE-8

Activation of caspase-8 following noise exposure was assessed in ACEMg treated

(N=5) and saline control (N=5) ears to determine the effect of antioxidant treatment on

the extrinsic (death receptor mediated) apoptotic pathway which is characterized by

activation of caspase-8 following ligation of cell surface death receptors. We

hypothesized that ACEMg treatment would not have a considerable effect on post-noise

expression of caspase-8 due to the fact that antioxidant treatment should not prevent

the ligation of death receptors.

Results

Surprisingly, although still not statistically reliable, we observed a greater

treatment effect on caspase-8 expression than on 3-NT production (Figures 4-1 and 4-

2). Control tissues in which the primary anti-caspase-8 antibody was omitted did not

show significant non-specific labeling (Figure 4-3, A). Similarly, no caspase-8

expression was detected in control ears which were not exposed to noise (Figure 4-3,

B). The results of the Mann-Whitney U test which was conducted for the detection of

differences in the probability distributions between the treated and control groups were

as follows: median section scores in the first turn for the ACEMg and saline control

groups were 6.75 and 14.69 respectively (Mann-Whitney U = 7.0, p = 0.155 one-tailed);

median section scores in the second turn for ACEMg and saline control groups were

7.94 and 14.31 respectively (Mann-Whitney U = 10.0, p = 0.345 one-tailed); median

section scores in the third turn for the ACEMg and saline control groups were 12.56 and

6.69 respectively (Mann-Whitney U = 11.0, p = 0.579); finally, the median average ear

scores for the ACEMg and saline control groups were 9.60 and 11.98 respectively









(Mann-Whitney U = 5.0, p = 0.076). These results are summarized in Table 4-1. The

treatment effect was greatest in the first turn, and decreased though the second and

third turns. When the cochleae were analyzed as a whole, the difference in distributions

between the treated and control groups approached statistical significance.

Discussion

The apoptotic initiator, caspase-8, can either directly activate the effector caspase-

3, or cleave BID which facilitates the release of cytochrome C mediated by the insertion

of Bax or Bak into the mitochondrial membrane. Both of these circumstances result in

cell death [72]. There is evidence that many inflammatory cytokines, including those of

the TNF family which can act as a death receptor ligand, are upregulated following

noise exposure [73]. Caspase-8 was also shown to be activated in hair cells following

noise exposure [59]. Our observations confirm this finding, and suggest the possibility

that ACEMg treatment may attenuate caspase-8 activation. This protective effect could

be mediated in part by the vasodilatory properties of magnesium. Magnesium prevents

noise-induced reduction of cochlear blood flow by dilation of the vasculature within the

stria vascularis [142]. This in turn prevents ischemic injury and production of

inflammatory mediators such as those of the TNF family which, as previously

mentioned, can act as death receptor ligands [73].







































Figure 4-1. Epifluorescence micrographs of ACEMg treated (A) and saline control (B) sections of the organ of Corti
labeled with anti-caspase-8 antibody showing the greatest observed treatment effect. Sections in the left,
middle, and right columns were taken from the 1st, 2nd, and 3rd, turns of the cochlea respectively. These images
were selected from ears with the lowest treated and the highest saline average rank score.







































Figure 4-2. Epifluorescence micrographs of ACEMg treated (A) and saline control (B) sections of the organ of Corti
labeled with anti-caspase-8 antibody showing median treated and control images. Sections in the left, middle,
and right columns were taken from the 1st, 2 and 3rd, turns of the cochlea respectively. These images were
selected from ears with the median treated and saline rank score.






































Figure 4-3. Negative control in which the primary anti-caspase-8 antibody incubation was omitted (A) and no noise control
(B).









Table 4-1. Difference in distribution of ACEMg treated and saline control caspase-8 image rank scores
1st Turn 2nd Turn 3rd Turn Ear Average
Median ACEMg 6.75 7.94 12.56 9.60
Score
Median Saline Score 14.69 14.31 6.69 11.98
Mann-Whitney U 7.0 10.0 11.0 5.0
P-value (one-tailed) 0.155 0.345 0.579 0.076









CHAPTER 5
CASPASE-2

Given the fact that caspases -3, -8, and -9 have an established role in noise-

induced damage to the inner ear [59], and that caspase-2 activation has been described

in response to oxidative stress [96, 152], we sought to determine the extent of caspase-

2 activation in the cochlea following noise exposure. Caspsase-2 differs from its other

family members because it has characteristics of both an initiator, and an effector

caspase. To date, caspase-2 activity in response to noise has not been characterized

in the inner ear. Evidence which shows caspase-2 activation following ROS formation

[96] led us to hypothesize that similar activation would be observed in response to noise

insult.

Results

These data provide the first evidence that caspase-2 is activated in response to

acoustic overexposure, and imply a possible role for caspase-2 in NIHL. There was no

significant non-specific labeling of negative control tissues in which the primary anti-

caspase-2 antibody was omitted (Figure 5-1, A and B). Unexpectedly, at the 2 hour

post-noise time point, caspase-2 labeling appeared to be localized within the supporting

cells (phalangeal process of outer pillar cells and Deiters cells). This was the case in 6

of 7 ears (Figure 5-1, C and D). By the four hour post-noise time point, caspase-2

expression was observed in supporting cells (2 of 7 ears, Figure 5-1, E), but more often

in the OHCs (5 of 7 ears, Figure 5-1, F). Finally, 24 hours following noise exposure

caspase-2 immunolabeling was again observed most commonly in supporting cells (4 of

5 ears, Figure 5-1, G and H). Labeling in all control tissues not exposed to noise was









difficult to detect, was diffuse, and was not localized to a particular cell type (Figure 5-1,

I and J).

Due to the fact that the data show noise-induced activation of caspase-2, we

chose to conduct a preliminary experiment testing the effect of ACEMg treatment on this

activation. Preliminary observations of ears treated with ACEMg (N=2) versus saline

control (N=2) did not eliminate the possibility that this treatment reduces activation of

caspase-2 following noise. One of the ACEMg treated ears showed diffuse caspase-2

expression which was minimal throughout each turn of the cochlea (Figure 5-2, A).

However, the other exhibited the same characteristic pattern of labeling in the

supporting cells which was found in untreated ears (Figure 5-3, A). Further

investigation is necessary due to the small sample size. Finally, in 2 of 2 ACEMg

treated, and 2 of 2 saline control ears, caspase-2 expression decreased with distance

from the base of the cochlea.

Discussion

These data show, for the first time, the expression of caspase-2 in the inner ear

following noise exposure, and suggest a possible role for caspase-2 in noise-induced

cell death. Previously, evidence for caspase-2 in the inner ear was limited to apoptotic

cell death during development of neo-natal rats [149]. Caspase-2 activation occurs very

early after cellular insult (for review see [94, 95]), and blocking or down-regulating

caspase-2 activity inhibits the release of cytochrome c and Smac from mitochondria,

prevents translocation of Bax from the cytosol to mitochondria, and prevents

translocation of apoptosis inducing factor (AIF) from mitochondria to the nucleus [93,

94, 153]. Smac increases caspase activity by inhibiting the inhibitor of apoptosis protein

(IAP).









While much of the focus is usually on the susceptibility of hair cells to noise

damage, there is considerable evidence that supporting cells are susceptible as well.

Supporting cells, such as Hensen's cells and the outer space of Nuel was shown to

collapse at 24 hours post-noise [112]. Additionally, free-radicals were detected in

Hensen's and Claudius cells in the guinea pig after noise, and labeling spread to hair

cells as time progressed [67]. Our observation that caspase-2 expression is initially

localized to the supporting cells and progresses to OHCs, coupled with the knowledge

that caspase-2 is activated by oxidative stress [96, 152] suggests the possibility that

caspase-2 contributes to noise-induced cell death in the inner ear, and that treatment

with ACEMg may inhibit this cell death pathway. Additionally, the decreasing gradient

of caspase-2 expression from base to apex which we observed is in line with the

common knowledge that the structures at the base are more susceptible to noise-

induced damage than those at the apex.









































Figure 5-1. Epifluorescence micrographs of the organ of Corti from the 1st turn of the
cochlea labeled with anti-caspase-2L antibody showing temporal difference in
post-noise expression. Negative control in which the primary antibody was
omitted (A and B). Tissues harvested: 2 hours post-noise (C-D) showed
distinct labeling in the supporting cells in 6 of 7 ears, 4 hours post-noise
exhibited expression in supporting cells in 2 of 7 ears (E) and in the OHCs in
5 of 7 ears (F), and 24 hours post noise (G-H) found caspase-2 expression in
the supporting cells in 4 of 5 ears and only one ear with expression in the hair
cells. There were 4 no-noise control ears. The samples shown here (I-J) had
the most labeling of any no-noise control tissues.


























































Figure 5-1. Continued

























B














Figure 5-2. Epifluorescence micrographs of ACEMg treated (A) and saline control (B) sections of the organ of Corti
labeled with anti-caspase-2L antibody showing diffuse labeling in treated and control tissues. Sections in the
left, middle, and right columns were taken from the 1st, 2nd, and 3rd, turns of the cochlea respectively. Both
treated and control tissues show diffuse immunolabeling, which decreases with distance from the base.





















B
PUMP! CYUW. M


Figure 5-3. Epifluorescence micrographs of ACEMg treated (A) and saline control (B) sections of the organ of Corti
labeled with anti-caspase-2 antibody showing immunolabeling concentrated in the supporting cells in 1st and 2nd
turns, with labeling becoming more diffuse in the 3rd turn.. Sections in the left, middle, and right columns were
taken from the 1st, 2nd, and 3rd, turns of the cochlea respectively.









CHAPTER 6
CONCLUSIONS AND FUTURE DIRECTIONS

Despite efforts to educate the public about the harmful effects of noise, NIHL

continues to be a growing problem which causes significant decrease in the individual's

quality of life, and a collective economic burden. A great deal of progress has been

made in understanding the cellular and molecular mechanisms underlying this disease.

Greater understanding has sparked the development of experimental therapeutics

which intervene at various points of cell death pathways, with the ultimate goal of

prevention of hair cell loss. Despite varying degrees of success in animal models, as

well as clinical trials, there is still no FDA approved pharmacological agent for the

prevention or treatment of NIHL.

This study has taken another step in elucidating the pathways which lead to NIHL

by providing the first evidence that caspase-2 is up-regulated following intense noise

exposure. Caspase-2 expression was observed in the supporting cells of the organ of

Corti 2 hours post-noise, with the amount of expression decreasing from base to apex.

At 4 hours post-noise, expression appears to move transiently to the OHCs, with

expression once again in the supporting cells at later time points.

Noise damage is usually mediated, at least in part, by oxidative stress resulting

from high metabolic demand and noise-induced reduction of cochlear blood flow. We

also investigated the effect of the micronutrient treatment combination of 3-carotene,

vitamins C and E, plus magnesium on the activation of caspases -2 and -8, as well as

the production of 3-nitrotyrosine, a marker of RNS activity. These data support the

possibility that ACEMg treatment may attenuate the noise-induced activation of

caspases -2 and -8 as well as RNS production. Given the fact that ROS and RNS









production are known to peak at 7-10 days following noise exposure, further studies are

warranted which explore this treatment effect at later post-noise time points.

Future research will seek to characterize the effect of ACEMg treatment on other

mediators of apoptotic signaling pathways such as caspases -3 and -9 as well as

caspase-independent mediators such as endonuclease G and apoptosis inducing factor

(AIF). Given the fact that free-radical production has been implicated in hearing loss

caused by aminoglycoside antibiotics, chemotherapeutics, and age-related hearing loss,

ACEMg treatment should be studied for application in these areas as well.









APPENDIX: COMMENTS ON METHODS AND STATISTICAL ANALYSIS

In this study, individual images of noise exposed, sectioned organ of Corti from

treated and control groups were pooled and ranked in order from least immunolabeling

within the hair cells to most by a series of observers who were blind to study conditions.

A nonparametric statistical test was then used to analyze the difference in ranks

between the treated and control groups. The following discussion will correlate

previously obtained hearing data from noise exposed animals (114 dB SPL centered at

4 kHz for 4 hours) with what we would expect to see in terms of ROS production/

caspase activation, and what we actually observed. Limitations associated with the

ranking design and statistical analysis will also be commented upon.

The effect of the noise exposure used in this study on hearing sensitivity in

guinea pigs has been well characterized in our lab by measurement of auditory

brainstem response (ABR) thresholds. Figure A-1 shows average noise-induced

threshold shifts at various post-noise time points. The fact that there is a large

decrease in hearing sensitivity across all frequencies at the early post-noise time points

justifies our analysis of the entire ear taken as a whole. All of the treated and control

animals in this study were euthanized at the two hour post-noise time point. Due to the

threshold elevation at this time across all frequencies, we would expect there to be

increased free-radical production and possibly caspase activation throughout the

cochlea. Our data support this in that there is increased labeling for all targets in each

section when compared to control tissues which have not been exposed to noise. For

future studies which will examine labeling at the later post noise time points, we would

expect to find the most labeling in the high frequency (basal) region of the cochlea.









Turning now to the statistics, the nonparametric statistical test was chosen

because we are analyzing ordinal data, or qualitative data that can be ranked in order of

magnitude. Parametric statistical tests rely on certain assumptions such as that the

data are sampled from a normally distributed population. Nonparametric tests on the

other hand do not depend on the distribution of the sampled population, and are thus

referred to as distribution-free tests. Additionally, nonparametric methods are

concerned with the location of the probability distribution of the population rather than

on specific parameters of the population, such as the mean.

Specifically, the Wilcoxon-Mann-Whitney two-sample rank sum test was used to

analyze the difference in image ranks between the treated and control groups. This is a

nonparametric method which tests the null hypothesis that the probability distributions

associated with the treated and control populations are equivalent. The conditions

required for a valid rank sum test are as follows: 1) the two samples are random and

independent, and 2) the two probability distributions from which the samples were

drawn are continuous so that there are no ties. If the treated and control populations

were identical, we would expect the ranks to be randomly mixed between the two

samples. On the other hand, if the treated population tends to have less labeling for a

particular target (as hypothesized) we would expect the smaller ranks to be mostly in

the treated sample and the larger ranks to be mostly in the control sample. This

experiment defined the one-tailed alternative hypothesis to be that the distribution of

treated ranks would be less than (shifted to the left of) the distribution of control ranks.

The test statistic is calculated based on the totals of ranks (rank sums) for each of the

two samples. The greater the difference between rank sums, the greater the evidence









indicating a difference between the probability distributions of the two populations.

Once the test statistic has been calculated for a particular trial, the observed

significance level, or p-value, can be calculated based upon the sampling distribution of

the test statistic under the null hypothesis. The p-value is the probability (assuming that

the null hypothesis is true) of observing a value of the test statistic that is at least as

contradictory to the null hypothesis, and supportive of the alternative hypothesis, as the

actual one computed from the sample data. More specifically, in the case of the

Wilcoxon-Mann-Whitney test, the p-value answers this question: if the treated and

control populations really have the same median image rank score, what is the chance

that random sampling would result in a sum of ranks as far apart or more so as

observed in this experiment? We have made the assumption that the saline control

images would be ranked higher and therefore, 1-tailed p-values were reported. If the p-

value is small (<0.05), one could conclude that the treated and control populations have

different medians. If the p-value is large, there is not sufficient evidence to reject the

null hypothesis and conclude that the medians differ. This does not necessarily mean

that the medians are the same; it just means that under the current experimental

conditions there is not enough evidence to say that they differ.

One limitation was the small sample size used in this experiment. With small

sample sizes, rank tests often have little statistical power. The power of a statistical test

is defined as the probability of correctly rejecting the null hypothesis when in fact the

alternative hypothesis is true. The probability of type II error (3) is defined as the

probability of incorrectly accepting the null hypothesis when the alternative is true.

Therefore the statistical power of a test can be calculated as 1 3. For the rank sum









test, power calculations can be performed in the absence of a priori knowledge of

population variance using an odds parameter (y) to show the relationship between the

two distributions. For example, when y = 1, the distributions of ranks are identical for

the two groups, and when y = 4, the odds are 4:1 that the control animals had higher

ranks than treated [154]. In our case, the ear average data for 3-NT had a y of 1.5 and

the ear average data for caspase-8 had a y of 4. Figure A-2 plots the odds parameter

(y) against power for our two sample case with five observations per group. From this,

we can clearly see that we were not adequately powered to detect even our greatest

difference in primary outcome measure seen in the caspase-8 ear average data where

y = 4. Studies are ongoing which will both increase the sample size at the early post

noise time points, and assess the treatment effect at later post noise times.

Another possible limitation is that working with ordinal data sets in which the

images were ranked from least to most labeling may mask large differences between

individual images. For instance, the difference between images ranked 1 and 2 may be

much greater or much smaller than that between images ranked 3 and 4. This may be

alleviated by having observers score each image on a pre-determined numerical scale.

Also, an alternative to image rank analysis could be explored for future studies in which

the image collection protocols are rigorously standardized to allow for exact

measurements of either area or intensity of labeling within the hair cells to be

determined using quantitative image analysis software.












CD


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-o
0
ac
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05
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100

80


60

40


20

0


-20


Figure A-1. Auditory brainstem response (ABR)
time points.


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*1'




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threshold shift at various post-noise


gamma


Figure A-2. Plot of statistical power vs. the odds parameter gamma


I I 1111111


2 4 8 16 24


Frequency (kHz)


--- <30 min (N=4)
-v-- 31-90 min iN-9)
--- 91-240 min (N=15)
^ O 1 day (N=8)
--- 3 days (N=8)
--*- 7 days (N=8)
0 11 days (N=8)
V 15 days (N-8)
--- 21 days (N=8)
--- 35 days (N=8)


U
r;
_A-

0
0
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a
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BIOGRAPHICAL SKETCH

Dustin Matthew Lang was born and raised in Florida, where he attended

Rockledge High School. From there, he went on to receive a Bachelor of Science

degree in chemistry from the University of Florida in December of 2007. In August 2009

Dustin was married to his lovely wife, Michelle. In August of 2010, he was awarded a

Master of Science degree in medical sciences from the University of Florida.





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1 ACTIVATION OF CAS PASE2 FOLLOWING NOISE EXPOSURE AND THE EFFECT OF TREATMENT WITH DIETARY AGENTS ON NOISE INDUCED OXIDATIVE STRESS AND ACTIVATION OF CASPASES2 AND 8 By DUSTIN MATTHEW LANG A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2010

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2 2010 Dustin Matthew Lang

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3 To those in hearing research who may benefit from my modest contribution

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4 ACKNOWLEDGMENTS First, I would like to thank Dr. Colleen Le Prell for allowing me to complete my thesis work in her laboratory as well as for all of the training and advice she has given. I also express my deepest gratitude to Dr. Patrick Antonelli for the opportunity to gain invaluable experience as an undergraduate, and young graduate student. I appreciate Dr. Gregory Schultz for his contagious enthusiasm and willingness to offer insi ght and direction. The common thread for each of these graduate committee members is that they have made themselves available to give guidance and direction despite the endless demands on their time. Also, I cannot overlook the contributions of Edith An gel Sampson for her selfless investment in the development of my science and character. Finally, I thank my wonderful wife Michelle for her support despite my long hours and late nights in the lab and my family for their unconditional support

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 7 LIST OF FIGURES .......................................................................................................... 8 LIST OF FIGURES .......................................................................................................... 8 ABSTRACT ................................................................................................................... 10 CHAPTER 1 INTRODUCTION .................................................................................................... 12 An Overview of NoiseInduced Hearing Loss ......................................................... 12 The Cochlea ........................................................................................................... 17 Anatomy and Physiology .................................................................................. 17 Cellular Mechanisms of NoiseInduced Cochlear Pathology ............................ 20 Mechanical damage and glutamate excitotoxicity ...................................... 21 Oxidative stress ......................................................................................... 22 Experimental Treatment for NoiseInduced Hearing Loss ...................................... 27 Inhibitors of Cellular Stress Pathways .............................................................. 28 Neurotrophic Factors/ Neurotransmission Blockers ......................................... 29 Inhibitors of Oxidative Stress ............................................................................ 30 Beta Carotene, Vitamins C and E, and Magnesium ......................................... 33 Study Design .......................................................................................................... 35 2 MATERIALS AND METHODS ................................................................................ 41 Subjects .................................................................................................................. 41 Noise Exposure ...................................................................................................... 41 Electrophysiological Tests ...................................................................................... 41 Antioxidant Treatment ............................................................................................. 42 Immunohist ochemistry ............................................................................................ 43 Nitrotyrosine Immunolabeling ........................................................................... 43 Caspase8 Immunolabeling .............................................................................. 44 Caspase2 Immunolabeling .............................................................................. 44 Statistical Analysis ............................................................................................ 45 3 NITROTYROS INE .................................................................................................. 47 Results .................................................................................................................... 47 Discussion .............................................................................................................. 48

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6 4 CASPASE8 ............................................................................................................ 54 Results .................................................................................................................... 54 Discussion .............................................................................................................. 55 5 CASPASE2 ............................................................................................................ 60 Results .................................................................................................................... 60 Discussion .............................................................................................................. 61 6 CONCLUSIONS AND FUTURE DIRECTIONS ...................................................... 67 APPENDIX: COMMENTS ON METHODS AND STATISTICAL ANALYSIS ................. 69 LIST OF REFERENCES ............................................................................................... 74 BIOGRAPHICAL SKETCH ............................................................................................ 87

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7 LIST OF TABLES Table page 3 1 Difference in distribution of ACEMg treated and saline control 3NT image rank scores ......................................................................................................... 52 4 1 Difference in distribution of ACEMg treated and saline control caspase8 image rank scores .............................................................................................. 59

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8 LIST OF FIGURES Figure page 1 1 Typical audiogram exhibiting early NIHL.. .......................................................... 37 1 2 Light micr ograph of a cross section of the guinea pig cochlea ........................... 37 1 3 Cross section of the organ of Corti ..................................................................... 38 1 4 Diagram showing efferent and afferent innervation of the IHC ........................... 38 1 5 Diagram showing afferent and efferent innervation of the OHC ......................... 39 1 6 Apoptotic pathways ............................................................................................ 39 1 7 Effect of ACEMg treatment on NIHL ................................................................... 40 1 8 Effect of ACEMg treatment on IHC and OHC loss .............................................. 40 3 1 Epifluorescence m icrographs of ACEMg treated and saline control sections of the organ of Corti labeled with anti 3 nitrotyrosine antibody showing the greatest observed treatment effect ..................................................................... 50 3 2 Ep ifluorescence micrographs of ACEMg treated and saline control sections of the organ of Corti labeled with anti 3 nitrotyrosine antibody showing median treated and control images .................................................................... 51 3 4 Immunostaining for NT shifts from supporting cells (Hensen, Claudius) to OHCs, includin g Deiters, with a maximum at 7 10 days .................................... 53 4 1 Epifluorescence micrographs of ACEMg treated and saline control sections of the organ of Corti labeled with anti caspase8 antibody showing the greatest observed treatment effect ..................................................................... 56 4 2 Epifluorescence micrographs of ACEMg treated and saline control sections of the organ of Corti labeled with anti caspase8 antibody showing median treated and control images ................................................................................. 57 4 3 Negative control in which the primary anti caspase 8 ant ibody incubation w as omitted and no noise control .............................................................................. 58 5 1 Epifluorescence micrographs of the organ of Corti from the 1st turn of the cochlea labeled with anti caspase2L antibody showing temporal difference in post noise expression ......................................................................................... 63 5 2 Epifluorescence micrographs of ACEMg treated and saline c ontrol sections of the organ of Corti labeled with anti caspase2L antibody showing diffuse labeling in treated and control tissues ................................................................ 65

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9 5 3 Epifluorescence micrographs of ACEMg treated and saline control sections of the organ of Corti labeled with anti caspase2 antibody showing immunolabeling concentrated in the supporting cells in 1st and 2nd turns, with labeling becoming more diffuse in the 3rd turn. ................................................... 66 A 1 Auditory brainstem response (ABR) threshold shift at various post noise time points. ................................................................................................................. 73 A 2 Plot of statistical power vs. the odds parameter gamma .................................... 73

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10 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science ACTIVATION OF CASPASE2 FOLLOWING NOISE EXPOSURE AND THE EFFECT OF TREATMENT WITH DIETARY AGENTS ON NOISE INDUCED O XIDATIVE STRESS AND ACTIVATION OF CASPASES2 AND 8 By Dustin Matthew Lang August 2010 Chair: Colleen Le Prell Major: Medical Sciences Noise induced hearing loss (NIHL) is a growing problem due to increase d noise exposure in military, occupational and recreational settings N oise induces the formation of free radicals in the cochlea due to increased metabolic demand and reduction of cochlear blood flow. This ultimately damages and /or destr oys sensory cells causing permanent hearing loss One con sequence of oxidative stress is the initiation of apoptotic signaling pathways via activation of caspases. Caspases 3, 8, and 9 are known to be upregulated in the inner ear following noise exposure, and caspase2 activation has been described as an initiator and/or executioner of apoptosis in other systems. Treatment with dietary antioxidants ( carotene, and vitamins C and E ) delivered in combination with the mineral magnesium (Mg) was previously show n to be effective for the reduction of NIHL. Due to the fact that carotene is metabolized to vitamin A, this treatment will subsequently be referred to as ACEMg. The purpose of this study was twofold: 1) to investigate the activation of caspase 2 following noise exposure, and 2) to determine the effec t of ACEMg treatment on noiseinduced production of reactive nitrogen species (RNS) and activation of

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11 caspases 2 and 8 Caspase2 immunolabeling was initially observed in the supporting cells of the guinea pig organ of Corti 2 hours following noise expos ure and moved transiently to the outer hair cells at 4 hours post noise. Nitrotyrosine (3 NT), a biomarker for production of RNS, and activation of caspase8 were assessed in order to determine the effect of ACEMg treatment on noiseinduced oxidative str ess and initiation of the extrinsic (death receptor mediated) apoptotic pathway respectively. The ACEMg treatment effect which was observed, while not statistically reliable under the current study design, supports further studies at later post noise time points, when oxidative stress is at a maximum. Taken together, these immunohistochemical data support the possibility that caspase2 plays a role in NIHL, and that the protective effect observed with ACEMg treatment involves attenuation of production of RNS and activation of caspases2 and 8.

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12 CHAPTER 1 I NTRODUCTION An Overview of Noise Induced Hearing Loss Noise induced hearing loss (NIHL) represents a growing medical problem with far reaching economic and social impacts. Due to the expansion of technology, we are now exposed to higher levels of noise than ever before, both in occupational as well as recreational settings. Education regarding the potentially harmful effects of noise, occupational hearing preservation programs, and the technology of hearing protection devices (HPDs) have not kept u p with the increase in noise exposure. This disparity is made evident by numerous studies citi ng wi despread detrimental effects of noise in various settings [1 24] It is estimated that 510% of the hearing loss burden in the U.S. is caused by noise exposure in the workplace [1] and that number rises to approximately 16% on a global scale [2] According to the National Institute for Occupational Safety and Health (NIOSH), approximately 30 million people are exposed to hazardous levels of noise on the job [25] NIHL was also found to increase the risk of work related accidents [3, 4]. In a Michigan study, 29.9% of those with hearing loss reported work related noise as its cause [5]. Those with highest risk of NIHL include construction workers, miners, musicians, disk jockeys, law enforcement officers, and military personnel. Across all trades, 59.7% of construction wor kers were found to have at least moderate NIHL [6] Hearing loss among symphony musicians was double the rate that would be expected for corresponding age [7, 8]. Among disc jockeys stud ied, 70% reported temporary threshold shift and 74% reported frequent tinnitus (ringing in the ears) after spending time in the dance club [9]. A ten year longitudinal study of police officers reveals the

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13 detrimental ef fects of impulse noise from gunfire despite the use of dual protection (earplugs and earmuffs) [10] Diagnosis of NIHL is based upon a history of noise exposure combined with the presence of a noise notch on the pati ents audiogram. While varying definitions exist, a noise notch is generally an increase in hearing threshold in the 4000 Hz range ( Fig ure 11 ), which expands to include progressively higher and lower frequencies as the exposure to noise progresses. Coles et al. defines a noise notch as a highfrequency notch where the hearing threshold at 3, 4, and/ or 6 kHz is at least 10 dB greater than at 1 or 2 kHz and at least 10 dB greater than at 6 or 8 kHz [26] Additio nally, Niskar et al. gives criteria which require threshold values at 0.5 and 1 kHz to be than the worst (highest) threshold value at either 0.5 or 1 kHz, and a threshold a t 8 kHz at least 10 dB better than the worst threshold at 3, 4, or 6 kHz [20] Age related hearing loss (ARHL) or presbycusis also causes a threshold increase at the higher frequencies However, the audiogram of a person with purely ARHL is downward sloping with progressively higher thresholds at higher frequencies, and lacks the characteristic notch indicating the contribution of noise. Different notch metrics can be used for diagnosis, and one study found that these metrics can agree with expert clinical judgments [27] Perhaps the greatest proportion of individuals with NIHL is found in the military. Studies have shown that members of all branches of military personnel are at greater risk for NIHL than the general population [11 14] In 1970, 20% of all Army veterans were entering claims for hearing loss, and the Veterans Administration (VA) paid over $52 million in compensation. This figure does not include compensation for hearing

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14 loss with a concurrent disability, cost of hearing aids, batteries, or repairs. Medical evacuations for complaints related to hearing loss were conducted at an average rate of one soldier per day during the first year of the war in Iraq. In 2004, the VA spent $108 million in disability payments to former Navy personnel, which represents a $65 million incr ease from 1999. In 2006, the combine d total of disability payments for hearing loss and tinnitus were over $1 billion. This is a 319% increase since the beginning of the war in Afghanistan in 2001 [13, 14] According to the Occupational Safety and Health Administration (OSHA) standard for noise exposure, a workplace hearing conservation program is required by law if average noise levels are at or above 90 dBA as an 8hour time weighted average using a 5 dB exchange rate [28] This means that for every 5 dB increase in exposure above 90 dB the permissible exposure time is cut in half. In 1972, N IOSH recommended a more conservative occupational noise exposure limit of 85 dBA as an 8hour time weighted average using a 3 dB exchange rate [25] Humans have the greatest sensitivity to sound i n the range of 1000 5000 Hz, and sou nd measurements can be A weighted which attenuates noise outside of this range The sound level is then reported in terms of dBA [15] Although attempts have been made to implement noise exposure guidelines and hearing conservation programs, there are many barriers which prevent these efforts from being fully effective. One survey of 29 foundry companies found that all were out of compliance with hearing conservation regulations Furthermore, the noise exposures t hat workers received during their average shift routinely exceeded 85 dBA as a timeweighted average. Members of the management, as well as employees from each

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15 company were interviewed, and a positive correlation was found between management and employee knowledge of hearing conservation. In other words, as management education with regards to hearing conservation increased, employee knowledge increased as well [16] These data imply that lack of knowledge has the potential to increase the risk of occupational NIHL. There are also compliance issues such as the failure of workers to wear hearing protection devices ( HPDs) properly. One example of this could include removing HPDs in order to communicate. This repeat ed removal makes it more likely that the HPD could become dirty or be inserted poorly, both of which would compromise the seal within or around the ear thus decreasing the level of sound attenuation. Another factor affecting compliance is that people do not notice an immediate hearing loss, and it is therefore difficult to convince them that they are indeed at risk [1] Finally many fe ar that wearing HPDs may affect their ability to perform as is the case with symphony musicians [17] to communicate, or to hear warning signals. Although occupational hearing loss has received much attention, this is not the only source of harmful noise. Recreational sources of noise can inc lude hunting, skeet shooting, personal music players, fireworks, nightclubs, and concerts. One survey found that most adolescents routinely listen to music at maximum volume, and feel that they are not vulnerable to the damaging effects of noise [19] It was estimated that 12.5% (approximately 5.2 million) of individuals age 6 19 years old and 40% of students age 16 25 years old exhibit noiseinduced threshold shifts [20] Fur thermore, the average sound levels for a concert are between 120 140 dBA, while bars and taverns can reach 95 dBA on a busy night [2123] In a webbased survey, 61% of concert attendees reported experiencing tinnitus or temporary hearing loss, and 59%

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16 said that they would be more likely to use hearing protection if it were recommended by a doctor or nurse [24] It is also noteworth y that while occupational and recreational noise exposure are often considered separately in research studies and for the definition of occupational noise exposure limits, one must bear in mind that the same person who receives their full legal noise dose at work may then proceed to come home and mow the lawn while listeni ng to a personal music player. Limiting exposure and appropriate use of HPDs are considered the most effective methods of hearing loss prevention. However, even when worn according to the manufacturers standards, there are many instances in which HPDs cannot reduce the level of exposure below recommended limits. The noise reduction rating (NRR), which represents the level of sound attenuation measured in a laboratory setting, must be sta ted by the manufacturer for all types of HPDs. To date, the most sophisticated hearing protection technology provides an NRR of approximately 30dB. Additionally, many studies have shown that real life attenuation is nowhere near the stated NRR. In fact, NIOSH recommends the following subtractions from the manufacturers labeled NRR: for earmuffs subtract 25%, for formable ear plugs subtract 50%, and for all other ear plugs subtract 70%. Using dual protection (ear plugs and earmuffs) only adds 510dB of protection. [25] It is evident that NIHL is a growing problem which must be addressed. Efforts should include increased education, occupational hearing conservation programs, improving HPDs, and further research into phar macological preventative or even hearing rescue methods. It has been said that, prevention of NIHL would probably do

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17 more to reduce the societal burden of hearing loss than medical and surgical treatment of all other ear diseases combined [1] The Cochlea Anatomy and Physiology The cochlea is the organ which is responsible for the transduction of mechanical energy in the form of a sound stimulus into electrical energy in the form of nerve impulses which are sent to the brain via the auditory nerve (cranial nerve VIII). T he structure and function of the cochlea has been extensively studied in many species. A brief overview of cochlear function is provided here, however for detailed review of current understanding the reader is referred to the following references [29 31] In mammals, the cochlea is spiral shaped and resides within a bony encasing (the otic capsule) in the temporal bone. The lumen of the cochlea is divided into three fluid filled chambers called the scala vestibuli, scala media, and scala tympani. The basilar membrane separates the scala media from the scala tympani, while Reissners membrane forms the partition between the scala media and the scala vestibuli. A layer of sensory epithelium known as the O rgan of Corti houses the main sensory cells called hair cells, and lies within the scala media atop the basilar membrane. The hair cells, so named because of tiny hair like projections called stereocilia at the apical end of each cell, are divided into tw o types: inner hair cells (IHCs) and outer hair cells (OHCs) There are three times as many OHCs as IHCs, the two types differ in location as well as function. The tectorial membrane is composed of acellular connective tissue, and forms a covering over t he Organ of Corti The fluid within the scala vestibuli and scala tympani (perilymph) has a different ion concentration than the fluid within the scala media (endolymph). The resulting difference in current potential, called the

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18 endocochlear potential is important for sensory cell signal transduction. This endocochlear potential is generated and maintained by a rich vascular bed in the lateral wall of the scala media called the stria vascularis (Pertinent structures of the cochlea are depicted in figur es 1 2 and 13) The range of frequencies that can be heard by young humans with normal hearing is approximately 40 Hz 20 kHz, and a sound stimulus entering the ear can contain many frequencies which must be decoded by the cochlea. In 1862, Helmholtz sug gested that the basilar membrane is composed of fibers arranged radially which each resonate at a different frequency analogous to the strings of a harp [32] It was later discovered that there are physical gradations in many structures of the cochlea. For instance, the tectorial membrane and basilar membrane both become gradually wider and thicker from the basal to the apical end of the cochlea. These gradations do indeed confer a tonotopic organization allow ing different areas of the basilar membrane to resonate at specific frequencies This led Bekesy to formulate his traveling wave model, which earned him the Nobel Prize in 1961 [33] Additionally, the hair cell ste reocilia increase in length from base to apex, and stereociliary stiffness in inversely correlated with length [34] This tuning of the tectorial membrane, basilar membrane, and Organ of Corti allows for trans duction of high frequency sound waves at the base, and low frequency sound waves at the apex. The hair cells are classified as either inner hair cells (IHCs) or outer hair cells (OHCs). In the human cochlea, there are approximately 3,500 IHCs arranged in a single row, and 11,000 OHCs arranged in three rows. Afferent signal transduction to the auditory nerve is the main function of IHCs. These cells receive afferent innervation

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19 from peripheral processes of the auditory nerve called spiral ganglion cells, and glutamate is the neurotransmitter of the IHC synapse. A spiral ganglion cell contacts only one IHC, while each IHC is connected to multiple spiral ganglion cells. Movement of the endolymph causes deflection of stereocilia at the apical end of the IHC which in turn causes ion channels to open and close according to the frequency of the sound stimulus. Entry of K+ and Ca2+ generates a transduction current which activates voltage sensitive Ca2+ channels and Ca2+ activated K+ channels leading to the rel ease of neurotransmitter into the afferent synapse at the basal end of the cell [30] The IHCs also receive efferent innervation from the lateral olivocochlear complex. Originating in the auditory brainstem, these neurons synapse on the peripheral processes of the spiral ganglion cells as opposed to the IHC body itself (Figure 1 4 ) [30] The efferent synapse contains both excitatory (acetylcholine, dynorphin, and CGRP) and inhibi tory (dopamine, enkephalin, and GABA) neurotransmitters which modulate the afferent sensitivity to glutamate. This may be a protective mechanism to prevent overstimulation [35] The role of the OHC is more complex. After studying the fluid dynamics of the coch lea, it was recognized that a model relying on passive resonance such as that proposed by Helmholtz and Bekesy was not sufficient. The viscosity of the endolymph would dampen the resonance of the basilar membra ne leading to a lower sensitivity than that which is actually observed. Thus, it was proposed that the cochlea must perform some active amplification of the incoming sound stimulus [36, 37] Accumula ting evidence suggested that the OHCs provide mechanical amplification by vi brating at the same frequency as the sound stimulus in order to prevent damping of

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20 the traveling wave. Thus OHCs can be thought of as the mechanical effectors of the cochlear ampl ifier [31] Afferent innervation of OHCs accounts for only 5% of auditory nerve dendrites, while they receive rich efferent innervation from the olivocochlear bundle. Unlike the IHCs, the OHC efferents synapse on the cell body itself which provides support for an efferent feedback mechanism (Figure 1 5) Additionally, the concept of an afferent signal leading to efferent st imulus and further amplification lends itself to the analogy of feedback in a modern sound sys tem. This idea eventually led to the discovery of otoacoustic emissions, or sounds generated by vibration of the inner ear which can be recorded in the external auditory canal [38] Later, OHC motility was clearly sh own, as hyperpolarization causes the cells to lengthen, and depolarization leads to cell shortening [39] This movement of OHCs was captured by video microscopy in 1986 [40] The next puzzle to be solved was the mechanism of OHC motility. In 2000, a new kind of motor protein was discovered in the OHC membrane. This protein was named prestin, and is a member of the SLC26 family of anionbicarbonate transporters. The name prestin is derived from presto which means fast in Italian, and was given due to its ability to operate on a microsecond timescale [40] Overall, OHC motility serves to amplify the resonance caused by the sound stimulus, which enhances the selectivity and s pecificity of cochlear tuning. Cellular Mechanisms of NoiseInduced Cochlear Pathology Exposure to high intensity noise has the potential to damage the cochlea, and to impede its function in various ways. This damage can occur in response to impulse noise such as gunfire, or continuous noise exposure, as is potentially experienced with personal music players. Overexposure can cause both mechanical damage and metabolic damage of the hair cells and surroundi ng structures The OHCs are more

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21 susceptible to damage than IHCs, and spiral ganglion neuron degeneration occurs following IHC loss [41] Furthermore, OHCs at the basal end of the cochlea are more susceptible than thos e at the apex [42] Hair cell death by both necrosis and apoptosis simultaneously was shown one hour post noise in chinchillas exposed to a continuous noise insult of 110 dB SPL centered at 4 kHz for a duration of one hour [43] Necrosis is a passive form of cell death which is characterized by nuclear swelling, rupture of the plasma membrane, and spilling of cell contents. This causes damage to surrounding tissue, and initiates an inflammatory response. On the other hand, apoptosis is a programmed pathway to cell death characterized by nuclear condensation and fragmentation, which is essential in normal growth and development for the elimination of unwanted or damaged cells [44] Unlike necrosis, apoptosis does not cause damage to the surrounding tissues. Apoptosis can also be triggered inappropriately causing the death of necessary cells [45] One stu dy found that 30 50% of hair cells can be lost before any measureable hearing loss can be detected [46] Mechanical damage and glutamate excitotoxicity Following acoustic overexposure there is an immediate loss of hear ing sensitivity. Depending upon the intensity and type of exposure, this loss of sensitivity can recover fully or partially with time. This immediate hearing loss which recovers with time is commonly known as temporary threshold shift (TTS) and any pers istent loss is termed permanent threshold shift (PTS). Less intense noise exposure is usually associated with TTS alone, and m echanical damage to hair cell stereocilia [47] as well as glutamate excitotoxicity are mechanisms which contribute to TTS. Glutamate excitotoxicity refers to the release of large amounts of glutamate into t he IHC afferent synapse. This over stimulates the nerve causing swelling and damage which can

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22 usually recover with time [48] However, recent data indicate that even when hearing sensitivity and hair cell function fully recover with time, neural degeneration without concurrent loss of hair cells (primary neural degeneration) may still oc cur [49, 50] As the intensity of exposure increases above approximately 125 dB, cochlear injury is primarily caused by mechanical rather than biochemical mechanisms. Additionally, research sug gests that impulse noise is more harmful than continuous noise of the same intensity [51] A ny damage which causes significant hair cell death induces a PTS because mammalian hai r cells cannot be regenerated. Oxid ative stress The production of reactive oxygen and reactive nitrogen species (ROS/RNS) in the cochlea following noise exposure has been well documented and reviewed [52 56] ROS and RNS include free radicals such as superoxide, peroxynitrite, and hydroxyl radicals. Excess production of free radicals can have detrimental effects because an unpaired electron makes these molecules extremely reactive, and capable of damaging many cellular components. For a review of free radical mechanisms see [57] Free radical damage has also been implicated in a number of human neurodegenerative diseases [58] Following noise exposure in chinchillas, there was a marked increase in ROS seen in the OHCs [59] and administration of paraquat (which produces superoxide) to the round window membrane resulted in PTS and hair cell loss [60] The cochlea contains many endogenous antioxidants such as superoxide dismutase (SOD), catalase, and glutathione (GSH) [61 64] Glutathione directly scavenges freeradicals. The function of SOD is to convert the harmful superoxide anion to molecular oxygen and hydrogen peroxide, while catalase converts hydrogen peroxide to molecular oxygen and water.

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23 Exposure to intense sound, especially at high frequencies, exerts a greater metabolic demand on the outer hair cells, which by nature have a high energy requirement under normal circumstances. Superoxide, which is a byproduct of mitochondrial respiration, is produced in greater quantities under high metabolic demand and can react with nitric oxide to generate the highly destructive peroxynitrite radical. Additionally, the generation of hydrogen peroxide by SOD can participate in the Haber Weiss and Fenton reactions to produce hydroxyl radicals [55] Cochlear damage occurs when large scale production of free radicals overwhelms endogenous antioxidant defens es. Glutathione peroxidase prevents oxidative damage by reducing lipid peroxides and catalyzes endogenous GSH production. One study showed that noiseexposed glutathione peroxidase (Gpx1) knockout mice had a higher PTS, and greater IHC and OHC loss than w ild type controls [65] A four fold increase in hydroxyl radicals was also seen within 12 hours after noise exposure [66] Finally, ROS and RNS production was shown to peak 710 days following noise exposur e, but hair cell loss progressed for approximately 2 weeks. This suggests that there is a window of time following exposure during which treatment with exogenous antioxidant or endogenous antioxidant bolstering agents may be effective [67] The link between ROS production and cell death is not fully understood. However, there is widespread evidence that ROS play a major role in the following cell death mechanisms [68] Ischemia/ reperfusion injury: Due to their motility, the OHCs have a high energy requirement and thus a high rate of aerobic respiration. As energy demand increases with noise exposure, mitochondrial efficiency decreas es and superoxide is released as an unwanted byproduct of oxidative phosphorylation. The decrease in mitochondrial

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24 efficiency can be compounded when noiseinduced damage of the lateral wall vasculature causes a decrease in cochlear blood flow [69] This state of ischemia, when demand for oxygen is at its highest, further increases the production of ROS. Furthermore, as the vasculature is repaired, and the site is reperfused, the sudden increase in oxygen once again fuels the production of ROS [52] This mechanism can run in a vicious cycle, as ROS production can cause further damage to the stria vasularis and decrease cochlear blood flow [70] Lipid peroxidation: Lipid peroxidation is another self perpetuating process in which free radicals catalyze the breakdown of lipid molecules within cellular membranes. A byproduct of l ipid catalysis is 8 isoprostaglandinF2 (8 iso PGF2 ) which causes vasoconstriction and again leads to decreased cochlear blood flow. In guinea pigs, there was a 30 fold increase in 8 iso owing noise exposure, and the extent of hair cell loss corresponded to the level of 8iso production [70] Extrinsic vs. intrinsic pathways to apoptosis : The extrinsic and intrinsic pathways are two primary signaling cascades leading to apoptosis. The extrinsic pathway is mediated by cell surface death receptors, while the intrins ic pathway is mediated by the release of proapoptotic factors from the mitochondria (Figure 1 6) [71] There is evidence for the utilization of both pathways in noiseinduced cell death in the inner ear. Additionall y, apoptotic hair cell death can take place through either caspasedependent, or caspase independent pathways. Caspases are aspartate specific cysteine proteases which can propagate a cell death signaling cascade. Caspases 8 and 9 are classified as ini tiators of the apoptotic signaling pathway, and caspases 3, -

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25 6, and 7 are apoptotic effectors [72] Caspases 8, 9, and 3 were shown to be activated in chinchilla OHCs following noise exposure [59] The extrinsic pathway is initiated when the death receptor (Fas) and i ts associated adaptor protein, Fas associated death domain (FADD) dimerize with Fas ligand, which is a member of the TNF family to form the death inducing signaling complex (DISC) This in turn activates the initiator caspase8 which can either directly activate the effector caspase3, or cleave BID which facilitates the release of cytochrome C mediated by the insertion of Bax or Bak into the mi tochondrial membrane. Both of these circumstances result in cell death [72] There is evidence that many inflammatory cytokines, including those of the TNF family which can act as a death receptor ligand are upregulated following noise exposure [73] Active c aspase 8 was also shown to be upregulated in hair cells following noise exposure [59] The intrinsic pathway can be initiated by a variety of mechanisms following noiseinduced ROS production and cell damage. Damage to membrane transport proteins leads to the influx of Ca2+ [74, 75] which in turn causes phospholipas e A2 (PLA2) activation and calpain dependent cleavage of calcineurin. Subsequently, PLA2 hydrolyzes phospholipids to proinflammatory mediators which lead to caspase activation and cell death [76] Cleavage of calcineurin allows for phosphorylation of nuclear factor of activated T lymphocytes (NFAT), which is a transcription factor controlling many genes involved in regulation of cell death [77] Calcineurin can also dephosphor ylate the proapoptotic regulator Bcl 2 associated death promoter (BAD), which translocates to the mitochondria, downregulates anti apoptotic members of the

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26 Bcl 2 family and activates Bcl 2 associated X protein (Bax) This causes mitochondrial membrane permeabilization, release of cytochrome C, and ultimately cell death [78] It is clear that many of these pathways converge at the point of mitochondrial membrane permeabilization. It is well known that the Bcl 2 family of proteins regulate mitochondrial membrane permeability, and ultimately cell death. There are proapoptotic (Bax, Bak, Bcl XS, Bid, Bad, and Bim), and anti apoptotic (Bcl 2 and Bcl XL) Bcl 2 proteins. When the ratio shifts in favor of proa poptotic proteins, Bax translocates from the cytoplasm to the mitochondria, causes membrane permeabilization, and release of cytochrome C into the cytoplasm. C ytochrom e C can then associate with apoptotic proteaseactivating factor 1 (APAF 1), dATP, and procaspase 9 to form the apoptosome. This causes activation of caspase9, which activates effector caspases leading to cell death [79] Evidence of the importance of the Bcl XL/Bak ratio in cell survival was shown in the inner ear using two noise exposure groups. One group was exposed to noise of a lower intensity designed to induce only TTS, while the other group was exposed to intense noise causing PTS. Bcl XL was upregulated in hair cells of the TTS group, while B ak was expressed in the PTS group [80] Other factors thought to be involved in noiseinduced cell death are p53 and c jun NH2terminal kinases (JNKs). The tumor suppressor protein p53 is activated by DNA damage, and also causes loss of mitochondrial membrane integrity [81] JNKs are thought to function upstream of caspase activation or cytochrome C release. MAP kinases are activated by cellular insult causing phosphorylat ion of the transcription factor c jun, which controls many genes regulating cell death [82 84]

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27 The intrinsic pathway to cell death also consists of caspaseindependent mechanisms. For review of these mechani sms see [85] Apoptosis inducing factor (AIF) and endonuclease G (endo G) are two mitochondrial proteins which, like cytochrome C, can be released upon loss of mitochondrial m embrane integrity. Endo G is a sequence nonspecific DNase which normally functions in mitochondrial DNA replication and r epair [86] AIF was identified in 1999, and is thought to have an e ssential role in dev elopment [87] Following their release, AIF and endo G translocate to the nucleus and cause DNA fragmentation leading to cell death [88 92] Ironically, these caspaseindependent pathways may actually activate a unique pathway involving caspase2. Caspsase2 is different than its other family members because it has characteristics of both an initiator and an effector caspase. It is thought that activated caspase 2 acts upstream causing mitochondrial membrane permeabilization and cytochrome C release. To date, caspase2 activity in response to noise has not been characterized in the inner ear. Howev er, caspase2 is known to play a role in cell death subsequent to DNA damage [93 95] Due to the DNA damage caused by translocation of AIF and endo G, it is reason able to think that caspase2 may also be involved in hair cell death secondary to noise exposure. Furthermore, one study found that ROS formation led to activation of caspase2 in a human leukemic T cell line [96] Exp erimental Treatment for NoiseInduced Hearing Loss Although its prevalence continues to increase despite hearing conservation programs, there are currently no FDA approved pharmacological agents for the prevention or treatment of NIHL. However, there has been a great deal of preliminary translational research driven by the recent increase in understanding of the cellular and

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28 molecular pathways underlying NIHL. The complexity of these pathways implies many possible therapeutic targets and points of intervention. Numerous drugs have entered, or have been proposed for clinical trials based on demonstration of protective efficac y or even rescue of hearing loss across various animal species (For thorough review see [97] ) Most of these drugs fall into one of three categories: direct inhibitors of cellular stress pathways, neurotrophic factors/ neurotransmission blockers, or direct inhibitors of oxidative stress [98] A significant b arrier to clinical applicability is that many of these treatments have only been shown to be effective when applied locally in the inner ear. This is obviously not practical for humans anticipating, or having recently been exposed to harmful noise levels. Thus, the ideal treatment would be one that can be given systemically (preferably orally), and still achieve a therapeutic level in the inner ear. Inhibitors of Cellular Stress Pathways The noise induced cellular stress response can lead to inflammation, disruption of calcium homeostasis, and activation of kinase signaling cascades leading ultimately to cell death. Glucoc orticoids have long been used as nonspecific inhibit ors of the stressinduced inflammatory response. Dexamethasone, a synthetic glucoc orticoid, was infused via implanted osmotic mini pumps directly into the scala tympani of guinea pigs, and a significant reduction of noise induced PTS and hair cell loss was observed. Dexamethasone binds to glucocorticoid receptors in the inner ear and regulates transcription of inflammatory mediators, thus reducing ischemia and ROS production [99] However, systemic administration of glucocorticoids is known to be associated with many untoward side effects, making it an impractical treatment for NIHL. As mentioned previously, noiseinduced calcium influx causes neuronal damage and phospholipase A2 activation leading to lipid peroxidation and ROS production.

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29 Calcium dysregulation also leads to calpain dependent cl eavage of calcineurin and activation of the transcription factor NFAT leading to apoptosis. O ral administration of the calcium channel blockers trimethadione and ethosuximide [100] or diltiazem [101] was carried out in mice and guinea pigs respectively Trimethadione and ethosuximide exhibited a protective effect when given after noise exposure, and trimethadione reduced PTS when given prior to noise exposure. Diltiazem protected OHCs from impulse noise only when applied both before and after exposure. Inhibition of calpain or calcineurin using leupeptin [82, 102] or cyclosporin A and FK506 [103, 104] respectively also achieved a protective effect Finally, inhibition of two kinase signaling pathways was explored. These signaling cascades regulate many genes involved in cell death, and inc lude the Src protein tyrosine kinase (PTK), and c Jun N terminal kinase (JNK) pathways. One of the detrimental effects of Src PTK signaling is the activation of NADPH oxidase which causes increased superoxide production. Reduction of PTS and hair cell loss was achieved following application of Src PTK inhibitor KX1 004 to the round window membrane of chinchillas [105] Similarly, the JNK inhibitor s D JNK 1 (aka: AM 111) [106, 107] and CEP 1347 [84] exhibited a protective effect following local admi nistration in various species. Neurotrophic Factors/ Neurotransmission Blockers Neurotrophic factors are secreted proteins which promote the survival, growth, and differentiation of neurons. Several neurotrophic factors such as neurotrophin3 (NT 3) and brainderived neurotrophic factor (BDNF) are expressed in hair cells [108] Qiang et al., 2004 cultured mouse spiral ganglion neurons and exposed them to high concentrations of glutamate in order to simulate glutamate excitotoxicity. Less neuronal

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30 cell death was seen when basic fibroblast growth factor ( bFGF) was added to the media. Next, they showed that systemic injection of bFGF in guinea pigs decreased PTS and hair cell loss following noise exposure. The interaction of bFGF with growth factor receptors on spiral ganglion cells and hair cells is proposed to increase expression of oxidase, decrease NO production, modulate intracellular calcium, and interfere with expression of proapoptotic proteins [109] Similarly, regrowth of spiral ganglion neurons following deafferentiation with aminogly coside antibiotics was observed with BDNF and bFGF treatment in guinea pigs Another approach aimed at neuroprotection involves the attenuation of glutamate excitotoxicity using glutamate receptor antagonists, or glutamate neurotransmission blockers. Carb ama thione and caroverine are glutamate receptor antagonists which reduced PTS following noise with systemic injection in chinchillas and local administration in guinea pigs respectively [110, 111] C arbamathione downregulates N methyl D aspartate (NMDA) receptor activity, while caroverine act s on both NMDA and amino3 hydroxy 5 methyl 4 isoxazolepropionic acid (AMPA) receptors. Another agent which exhibits NMDA receptor antagonistic properties is t he mineral magnesium [35] Riluzole (2 amino 6 trifluoromethoxy benzothiazole) is a glutamate neurotransmission blocker which exhibited a protective effect following local and systemic administration in guinea pigs [112] Inhibitors of Oxidative Stress Acoustic overstimulation decreases mitochondrial efficiency, stimula tes excess glutamate release in to the afferent IHC synapse, and induces a state of cochlear ischemia, all of which increase production of ROS. Many approaches are being explored which enhance endogenous cochlear antioxidant defenses, inhibit production

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31 of ROS, or directly scavenge free radicals. Antioxidant treatments are potentially appealing due to the fact that man y of these compounds have been show n to be safe when given orally. Early attempts to augment endogenous antioxidant defenses used R phenylisopropyladenosine (R PIA) and glutathione monoethyl ester (GSS) [113] applied locally to the round window membrane of chinchillas. R PIA, which increases GSH and superoxide dismutase levels, decreased PTS and OHC loss. The cell permeable enzyme glutathione synthetase ( GSS ), which is involved in GSH production also provided a significant reduction in PTS. Other compounds which increase endogenous GSH including nacetyl cystei ne (NAC) and D methionine (D met ) reduce NIHL as well In addition to its ability to directly scavenge free radicals, NAC is also one of the amino acids composing GSH, and is thus able to replenish this endogenous antioxidant when depleted following intense noise exposure (For review see [114] ) NAC has been shown effective for the reduc tion [110, 115, 116] and rescue [54, 117, 118] of NIHL in various species. It was also effective when given orally but its efficacy was greatly decreased [119] D Met also acts primarily as an indirect antioxidant by increasing intracellular GSH levels This is possible because methionine is used in the synthesis of cysteine a component of GSH. D Met also attenuat es the increase in SOD levels which is regularly observed in the cochlea following noise exposure [120] SOD is responsible for the conversion of superoxide to hydrogen peroxide, which is subsequently removed by catalase. The increase in SOD following noise is not accompanied by an increase in catalase, therefore the excess hydrogen peroxide creates favorable conditions for ROS production. Administration of D Met was found to

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32 prevent decreases in Na/K ATPase and Calcium ATPase activity, decrease intracellula r NO concentration, and prevent lipid peroxidation following noise [79] Agents recognized for their ability to inhibit the production of ROS include allopurinol, acetyl L carnitine (ALCAR), and 2 phenyl 1,2 benzisoselenazol 3(2H) one (ebselen). Allopurinol, also used for its ability to inhibit uric acid synthesis i n chronic conditions such as gout, attenuated PTS in systemically injected rats [121] The mitochondrial membrane component ALCAR serves as a precursor for acetyl CoA and L carnitine which carry lipids into mitochondria for oxidation and enhance ATP production. Therefore, this compound improves mitochondrial efficiency, and decreases ROS production. ALCAR was effective for reduction of hair cell loss and for prevention [110, 115] and rescue [118] of PTS. Ebselen is a glutathione peroxidase mimetic which decreases hydroperoxide formation, scavenges peroxynitrite, inhibits NO synthase, and prevents lipid peroxidation a nd cytochrome C release. Ebselen is effective for the reduction of TTS, PTS, and hair cell loss [122 124] It also reduced noiseinduced swelling of the stria vascularis in rats [125] Finally, m ultiple antioxidant compounds which directly scavenge fr ee radicals were found to attenuate PTS and hair cell loss to varying degrees Among these are mannitol, salicylate, resveratrol, coenzyme Q10, and 4hydroxy phenyl N tert butylnitrone (4OHPBN). Mannitol, a scavenger of hydroxyl radicals, attenuated PTS when injected systemically [126] In addition to scavenging hydroxyl radicals, salicylate forms the iron chelator dihydrobenzoate, which prevents ROS formation by inhibiting the iron catalyzed Fenton reaction [117] Resveratrol, which is naturally present in grapes and red wine, acts as an anti inflammat ory, vasodilator, and neuroprotectant in addition

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33 to its antioxidant properties [121, 127] Coenzyme Q10 participates in oxidative phosphorylation as an integral member of the electron transport chain, and also has potent antioxidant activity. Efficacy of oral administration was increased when given as coenzyme Q10 Terclatrate (Q Ter), a form which is highly water soluble [128] [129] Finally, 4 OHPBN, scavenges hydroxyl radicals and superoxide anions, showed a dose dependent reduction in PTS and OHC loss in chinchillas [130] This compound has entered phase III clinical trials as a treatment for stroke [129] To date, no treatment has been identified which is completely effective for the prevention or rescue of NIHL. Significant advancements have been made in identifying agents which are effective when given systemically, and are thus more clinically applicable. Due to their different mechanisms of action, and the fact that no single agent has provided complete protection, it may be beneficial to explore various combinations of these agents. Beta Carotene, Vitamins C and E, and Magnesium In 2007, Le Prell et al. administered either a combination of the antioxidant s carotene and vitamins C and E magnesium alone (Mg) carotene and vitamins C, and E plus magnesium in guinea pigs exposed to 5 hours of 120dB SPL octave band noise centered at 4 kHz. carotene is metabolized to vitamin A, this treatment will be subsequently referred to as ACEMg. Treatments were given once daily beginning 1 hour prior to noise exposure, and continuing until day 5 post noise. Auditory brainstem response (ABR) thresholds were measured at day 10 following noise exposure. Threshold shifts for the ACEMg group were significantly decreased compared to saline control animals at 4, 8, a nd 16 kHz. There was no significant difference in ABR threshold between control animals and those treated with either ACE

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34 or Mg (Figure 1 6 ). Hair cell loss was also significantly reduced in the ACEMg group compared to control and ACE or Mg alone (Figure 1 7 ) [131] These data suggest a synergistic protective effect between agents Each compound in this combination treatment has a distinct site and mechanism of action for the reduction of cellular damage secondary to oxidative stress. Beta carotene is a fat soluble nutrient which effectively scavenges singlet oxygen. As carotene is metabolized to vitamin A. Once adequate reserves carotene in the blood is free to directly scavenge free radicals. Singlet oxygen reacts with membrane lipids to form lipid hydroperoxides, thus carotene prevents lipid peroxidation [132] Vitamin C (ascorbic acid) is a water soluble essential nutrient which can directly reduce freeradicals in the aqueous phase [133] As well as scavenging superoxide, hydroxyl radicals, and singlet oxygen tocopherol (vita tocopheroxy radical which results from the reduction of ROS by vitamin E [134] Dietary or systemically injected vitamin C reduced PTS, prevented hair cell loss, increased endogenous antioxidant s in guinea pigs and rabbits [135, 136] Ascorbic acid also reduced NO production by 56% in the lateral wall and 37% in the organ of Corti of guinea pigs [137] Vitamin E ( tocopherol) is another lipophilic nutrient which reduces peroxyl radicals peroxynitrite radicals, and inhibits the propagation of lipid peroxidation [132] Trolox ( 6 hydroxy 2,5,7,8 tetramethylchroman2 carboxylic acid), a water soluble and cell permeable derivative of tocopherol is often used for treatment. Pretreatment with vitamin E significantly reduced PTS and hair cell loss and post treatment had a protective effect when initiated up to 3 days post noise [138] Dietary vitamin E showed

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35 a dosedependent reduction in PTS in fathead minnows ( Pimephales promelas ) [139] The protective role of magnesium in NIHL is most likely due to its role as a vasodilator. Magnesium inhibits calcium influx into vascular smooth muscle cells by blocking calcium channels, and also activates adenylate cyclase which forms adenosine 3,5 monophosphate within vascular smooth muscle cells leading to dilation [140] Magnesium may also reduce glutamate excitotoxicity due to its NMDA antagonistic effect on afferent dendrites [35, 141] The noiseinduced reduction of cochlear blood flow as well as the associated decrease in perilymph oxygenation, was attenuated in guinea pig s maintained on a high magnesium diet [142] Also in guinea pigs, PTS and susceptibility of hair cell stereocilia to noise damage showed a negative correlation with perilymph magnesium concentration, and there was a marked reduction in TTS as well [143, 144] Human trials with magnesium for NIHL have also been shown to reduce PTS as well as TTS with no side effects [145, 146] Preliminary data from Le Prell et al. showed that type II fibrocytes, strial cell densit y, and threshold sensitivity were preserved in noiseexposed CBA/J mice maintained on a high ACEMg diet [147] T hus, t his micronutrient combination has been shown to be effective for reduc tion of NIHL as a dietary supplement, and each of its components has a very high safety profile based on AREDS seven year ACE trials in humans Study Design Th e aim of this study was: 1) to evaluate activation of caspase2 following noise exposure, and 2) to further characterize the effect of ACEMg treatment on free radical pr oduction, and caspases 2, and 8 activation subsequent to noise exposure. The difference in expression pattern of these target molecules known to be upregulated

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36 following intense noise were assessed in antioxidant treated and control animals using immunohistochemistry. There are two splice variants of caspase2 which play opposite roles in initiation of apoptosis. C aspase2L induces cell death, while caspase2S suppresses cell deat h [148] C aspase2L was assessed in this study ; there are no selective antibodies for caspase2s. Caspase 2 expression has been described in the inner ear of newborn rats, where it plays a role in apoptotic cell death during development [149] However, whether it is activated secondary to noise exposure remains to be determined. Production of 3 nitrotyrosine ( 3 NT) was assessed as an indicator of oxidative stress in antioxidant treated and control animals 3 NT is known to accumulate in conditions involving oxidative stres s such as Huntingtons disease and ischemic brain injury. It is also used as a reliable marker of RNS activity [67, 138, 150] Caspase8 expression was evaluated to determine the effect of antioxidant treatment on t he extrinsic (death receptor mediated) apoptotic pathway. We hypothesized that free radical producti on (as assessed by production of 3NT ) would be reduced in antioxidant treated ears, caspase2 would be activated in noise exposed animals confirming its r ole in noiseinduced cell death, and noiseinduced caspase 8 activation would not be effected by ACEMg treatment because antioxidant treatment presumably should not interfere with the ligation of death receptors.

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37 Figure 11. Typical audiogram exhibiting early NIHL. The bilateral decrease in hearing sensitivity at 4000 Hz forms the characteristic noise notch [15] Figure 12. Light micrograph of a cross section of the guinea pig cochlea. Major structures are labeled, and anatomical directions are noted in parenthesis [30]

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38 Figure 13. Cross section of the organ of Corti with tectorial m embrane covering the hair cells [30] Figure 14. Diagram showing efferent (E) and afferent (A) innervation of the IHC. Excitatory (+) and inhibitory ( ) neurotransmitters are shown along with their receptors. NPR denotes neuropeptide receptors with various functions and IPC represents the inner phalangeal cells which surround the IHC [30]

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39 Figure 15. Diagram showing afferent (A) and efferent (E) innervation of the OHC. Exc itatory (+) and inhibitory ( ) neurotransmitters and receptors are shown. SSC denotes subsynaptic cisternae. Outer phalangeal cells/ Deiters cells are represented laterally (OPC/DC) [30] Figure 16. Apoptotic pathways. Initiation of apoptosis can occur through either the extrinsic (deathreceptor) pathway or the intrinsic (mitochondria mediated) pathway [56]

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40 Figure 17 Effect of ACEMg treatment on NIHL. A uditory brai nstem response (ABR) threshold shift before and 10 days after noise exposure were significantly reduced with ACEMg treatment, but not with either ACE, or Mg alone [131] Figure 18 Effect of ACEMg treatment on IHC and OHC loss. Outer hair cell loss was significantly reduced with ACEMg treatment, but not with either ACE, or Mg alone [131]

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41 CHAPTER 2 MATERIALS AND METHOD S Subjects A lbino male guinea pig s (250350 grams) from an approved laboratory animal supplier (Charles River, Wilmington, MA) were used. Guinea pigs were housed in the Animal Care Facility at the University of Florida, and were identified using ear clips. Following arrival, all animals were given at least 48 hours to acclimate to the environment and recover from transportationrelated stress. All experimental protocols regarding the use and care of animals were reviewed and approved by the University of Florida Institutional Animal Car e and Use Committee. Noise Exposure At the onset of the study, speakers were calibrated by placing microphones at the level of the animals heads (while the cages were unoccupied). Animals were exposed four at a time, each in separate cages. The cages were arranged in the sound booth so that the exposure each animal received was 114 4 dB SPL octave band noise centered at 4 kHz depending on location within cage. Noise exposure lasted exactly four hours. For exposure of treated and control animals, two antioxidant treated and two saline control animals were included in each noise exposure group. The cage positioning of treated and control animals was alternated with each noise exposure. Animals were unrestrained and were not anesthetized during the noise exposure period. Electrophysiological Tests All s ubjects were screened for normal hearing sensitivity at 2, 4, 8, 16, and 24 kHz in the right and the left ear using the soundevoked auditory brainstem response (ABR); left ear ABR tests were repeated on one treated and one control animal per exposure

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42 group post noise prior to euthanasia to verify hearing loss obtained using this exposure. During ABR tests, animals were anesthetized with ketamine (40 mg/kg, s.c.) and xylazine (10 mg/kg, s.c.) and neural activity in response to brief, tone pips was measured using sterile, 27gauge electrodes inserted subcutaneously posterior to each pinna and at the vertex of the skull. Tone levels were decreased from 90 dB SPL to 0 dB SPL in 10dB increments. Each pip was 10 milliseconds in duration and tones were repeated at a rate of 17/second until 1026 responses were acquired. Threshold was independently determined using a 25V Wave III response criterion. Animals were placed on a water circulating heating pad to maintain body temperature and lubrication was applied to the eyes to prevent dryness during ABR procedures. The depth of the anesthesia was measured using t he pedal withdrawal reflex and additional anesthetics administered as needed. All animals received an overdose of sodium pentobarbital following ABR tests, an d were euthanized for immunohistochemical assays. Antioxidant Treatment All antioxidant treated animals received a total of two treatments. The first treatment was administered 24 hours prior to noise exposure, and the second treatment was given 1 hour prior to noise. Control animals received saline injections equivalent to the dose of micronutrient cocktail given in treated animals. The micronutrient cocktail of carotene, vitamins C and E, plus magnesium was given as follows: vitamin A (2.1 carotene, po), vitamin C (71.4 mg/kg Lthreoascorbic acid, sc), vitamin E (26 mg/kg () 6 hydroxy 2,5,7,8tetramethylchromane2 carboxylic acid, Trolox, sc), magnesium (343 mg/ kg MgSO4, sc). Tr olox is a cell permeable, water soluble derivative of vitamin E. All test substances were purchased from Sigma Aldrich (St. carotene, C9750, CAS 723540 7; L threoascorbic acid, A5960,

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43 CAS 50 81 7; Trolox, Fluka Chemika, 56510,CAS 53188071; magnesium sulfate, M7506, CAS 7487889). Immunohistochemistry Following anesthesia with ketamine (40 mg/kg, s.c.) and xylazine (10 mg/kg, s.c.) animals were euthanized at various time points via sodium pentobarbital overdose and were decapitated. Cochlear tiss ues were immediately harvested and perfused with 4% methanol free formaldehyde. Tissues remained in fixative for 3 hours before being rinsed and stored in phosphate buffered saline (PBS) until immunolabeling began. In general, tissues were blocked with normal serum, permeabilized with TritonX 100, incubated with primary antibody, and labeled with secondary antibody. After immunolabeling was complete, tissues were rinsed (in PBS), dissected for surface preparations and mounted on glass slides using VectaShield mounting medium To assure an accurate representation of labeling, two images were taken from each turn of the cochlea. Images were collected using a Leica DM 5500B epifluoresence microscope, and processed with ImagePro 6 .3 software. Images were acquired as a Z stack, deconvolved using a nearest neighbor algorithm, and tinted after they were acquired in monochrome. Nitrotyrosine Immunolabeling Antioxidant treated (N=5) and saline control (N=5) tissues were harvested 2 hours after noise exposure. All tissues were fixed as mentioned above, rinsed with PBS, permeabilized with 0.5% TritonX 100 for 1 hour, and rinsed again. Subsequently, tissues were blocked with Power Block for 5 minutes. After blocking, tissues were rinsed with PBS and incubated with anti 3 nitrotirosine mouse monoclonal antibody ( clone 39B6; Alexis Biochemicals, 1:500 for 24 hours at 4 C ) The primary antibody

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44 was omitted in negative control tissues. Following PBS rinse, tissues were incubated in t he secondary antibody ( 1:100 AlexaFluor 488 goat anti mouse IgG) f or 1 hour at room temperature. Caspase8 Immunolabeling Antioxidant treated (N=5) and saline control (N=5) ears were processed in the same manner as described for nitrotyrosine immunolabeling. Tissues were incubated in mouse anti caspase 8 monoclonal antibody ( Santa Cruz Biotechnology; sc 5263, 1:500 for 48 hours at 4C). The primary antibody was omitted in negative control tissues. Following PBS rinse, tissues were incubated in the second ary antibody ( 1:100 AlexaFluor 488 goat anti mouse IgG) for 1 hour at room temperature. Caspase2 Immunolabeling The purpose of caspase2 immunolabeling was twofold: 1) to determine if caspase2 immunolabeling would be observed post noise since this has not been described previously, and 2) to gather preliminary data concerning the effect of ACEMg treatment on caspase2 expression post noise (ACEMg, N=2; saline control N=2). All tissues were labeled for caspase2L (long variant). Animals used for the i nitial characterization of caspase2 expression were euthanized at 2 (N=4), 4 (N=4), and 24 (N=3) hour post noise time points. Animals euthanized with no noiseexposure served as controls (N=4). All antioxidant treated and saline control animals were eut hanized at 2 hours post noise. Fixed and rinsed tissues were permeabilized with 0.5% to 1% TritonX 100 for 30 to 60 minutes. Subsequently or simultaneously, tissues were blocked with 10% Normal Goat Serum (with or without 1% BSA) for 30 to 60 minutes or with Power Block for 5 minutes After blocking, tissues were rinsed in PBS, and then incubated for 2 4 hours in

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45 mouse anti caspase 2 antibody (BD Transduction Laboratories #611022, ICH 1L) at 4C. P reliminary tests with 1concentration to produce the most specific labeling. After incubating in the primary anti caspase2 antibody, tissues were rinsed in PBS. All tissues were then incubated in the secondary antibody ( 1:100 Alexaflour 488 goat anti mouse IgG) for 1 hour at room temperature (20C). Statistical Analysis In order to determine antioxidant treated versus saline control group differences in 3 NT and caspase8 expression, seven observers who were blind to study conditions were asked to rank image sets in order from least immunolabeling with in the hair cells to most. These sets were generated by pooling images from treated and control animals according to the section of the cochlea from which they were taken. Thus, ther e were three image sets from each target molecule (3NT and caspase8) corresponding to the first, second, and third turns of the cochlea. Each image set consisted of twenty images, as there were ten animals (ACEMg treated n=5; saline control n=5), and tw o images were taken from each turn of the cochlea. Each observer was given uniform training and instruction on how to judge the images. Additionally, a significantly positive (p<0.01) Spearmans rank correlation coefficient was observed between each of t he observers rankings. The rank numbers assigned to each image were averaged across all observers to give average image scores. Subsequently the mean of the two average image scores from each animal was taken to give average section scores. Finally, th e means of the average section scores were taken to give average ear scores for each of the ten animals These data were then employed to detect group differences using the WilcoxonMannWhitney twosample rank sum test,

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46 which tests the null hypothesis th at the probability distributions associated with the two populations (ACEMg treated and saline control) are equivalent. The data were analyzed for treatment effect within each section, as well as within the ear as a whole.

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47 CHAPTER 3 NITROTYROSINE Product ion of 3nitrotyrosine (3NT) was assessed as an indicator of oxidative stress in antioxidant treated (N=5) and saline control (N=5) animals. The increased metabolic demand on hair cells due to noiseexposure causes an increase in the production of superoxide. Superoxide then reacts with nitric oxide to generate the peroxynitrite anion, which modifies cellular proteins to form 3nitrotyrosine. We hypothesized that antioxidant treatment would decrease the production of RNS, and thus decreas ed labeling would be observed within the hair cells of treated ears upon comparison with those of saline control ears. Results Upon initial observation, the results appeared encouraging due to the apparent difference in immunolabeling in the hair cells between the ACEMg and saline control groups (Figure 31). However, this difference was not consistently observed (Figure 32), and did not prove to be statistically significant with the current methods of analysis and sample size. Negative control tissues in which the primary anti 3 NT antibody was omitted did not show significant non specific labeling (Figure 3 3) The results of the MannWhitney U test which was conducted for the detection of differences in the probability distri butions between the treated and control groups were as follows: median section scores in the first turn for the ACEMg and saline control groups were 11.43 and 12.71 respectively (Mann Whitney U = 10.0, p = 0.345 onetailed); median section scores in the second turn for ACEMg and saline control groups were 11.29 and 8.79 respectively (MannWhitney U = 12.0, p = 0.50 onetailed); median section scores in the third turn for the ACEMg and saline control groups were 7.00 and 14.29 respectively

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48 (Mann Whitney U = 11.0, p = 0.421) ; finally, the median average ear scores for the ACEMg and saline control groups were 9.90 and 10.83 respectively (MannWhitney U = 10.0, p = 0.345). These r esults are summarized in Table 31. Discussion Nitrotyrosine is known to accumulat e in conditions involving oxidative stress such as Huntingtons disease and ischemic brain injury [151] It has also been used as a reliable biomarker of RNS activity [67, 138] The reaction of superoxide with nitric oxide (NO) generates the highly reactive peroxynitrite anion, which modifies cellular proteins to generate nitrotyrosine [150] T reatment with this ant ioxidant cocktail was expected to reduce the production of RNS, and thus 3 NT This hypothesis is based on the ability of ascorbic acid to scavenge superoxide and to reduce noiseinduced NO production [137] and als o the overall efficacy of ACEMg treatment for the reduction of noiseinduced PTS as was demonstrated previously [131] However, as was previously mentioned, we did not observe a statistically reliable difference in 3NT production between the treated and control groups using the current study design. The lack of statistical significance seen in our results may be caused by temporal and spatial variation in 3NT production following noise. Evidence for this theory is based on a varying pattern of 3NT production at different post noise time points A previous study showed that immunostaining for 3NT following noise expos ure was initially low, and localized to the supporting (Hensen and Claudius) cells. Additionally, significant immunostaining did not appear in the hair cells until later time points (day 710), when 3NT produc tion reached a maximum (Figure 34) [67] All animals in this study were sacrificed two hours following noise exposure, which corresponds to the time at which Yamashita et al. described 3NT production only in the

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49 supporting cells. Our images were analyzed w ith respect to immunolabeling within the outer hair cells alone, and this may be the reason for our failure to detect a significant treatment effect. Given this information, it may be that an appreciable treatment effect would be more readily detectable i n the 710 day post noise range during the time of peak 3NT production. These studies are in progress.

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50 A B Figure 31. Epifluorescence micrographs of ACEMg treated (A) and saline control (B) sections of the organ of Corti labeled with anti 3 nitrotyrosine antibody showing the greatest observed treatment effect Sections in the left, middle, and right columns were taken from the 1st, 2nd, and 3rd, turns of th e cochlea respectively. These images were selected from ears with the lowest treated and the highest saline average rank score.

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51 A B Figure 32. Epifluorescence micrographs of ACEMg treated (A) and saline control (B) sections of the organ of Corti labeled with anti 3 nitrotyrosine antibody showing median treated and control images Sections in the left, middle, and right columns were taken from the 1st, 2nd, and 3rd, turns of the cochlea respectively. These images were selected from ears with the median treated and saline rank score

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52 Figure 33. Negative control ear in which the primary 3NT antibody incubation step was omitted. Table 31. Difference in distribution of ACEMg treated and saline control 3NT image rank scores 1 st Turn 2 nd Turn 3 rd Turn Ear Average Median ACEMg Score 11.43 11.29 7.00 9.90 Median Saline Score 12.71 8.79 14.29 10.83 Mann Whitney U 10.0 12.0 11.0 10.0 P value (one tailed) 0.345 0.500 0.421 0.345

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53 Figure 34. Immunostaining for NT shifts from supporting cells (Hensen, Claudius) to OHCs, including Deiters, with a maximum at 7 10 days. Sections are from an area approximately one half turn apical of the main lesion (A: control, B: immediate, C: Day 3. D: Day 7 E: Day 10, F: Day 14). All guinea pigs were exposed to octave band noise centered at 4 kHz and 120 dB SP L for 5 hours [67]

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54 CHAPTER 4 CASPASE8 Activat ion of caspase8 following noise exposure was assessed in ACEMg treated (N=5) and saline control (N=5) ears to determine the effect of antioxidant treatment on the extrinsic (death receptor mediated) apoptotic pathway which is characterized by activation of caspase8 following ligation of cell surface death receptors. We hypothesized that ACEMg treatment would not have a considerable effect on post noise expression of caspase 8 due to the fact that antioxidant treatment should not prevent th e ligation of death receptors. Results Surprisingly, although still not statistically reliable, we observed a greater treatment effect on caspase8 expression than on 3NT production (Figures 4 1 and 42) Control tissues in which the primary anti caspase8 antibody was omitted did not show significant nonspecific labeling (Figure 43, A). Similarly, no caspase8 expression was detected in control ears which were not exposed to noise (Figure 43, B). The results of the MannWhitney U test w hich was conducted for the detection of differences in the probability distributions between the treated and control groups were as follows: median section scores in the first turn for the ACEMg and saline control groups were 6.75 and 14.69 respectively (M annWhitney U = 7.0, p = 0.155 onetailed); median section scores in the second turn for ACEMg and saline control groups were 7.94 and 14.31 respectively (MannWhitney U = 10.0, p = 0.345 onetailed); median section scores in the third turn for the ACEMg and saline control groups were 12.56 and 6.69 respectively (MannWhitney U = 11.0, p = 0.579); finally, the median average ear scores for the ACEMg and saline control groups were 9.60 and 11.98 respectively

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55 (Mann Whitney U = 5.0, p = 0.076). These r esults are summarized in Table 41. The treatment effect was greatest in the first turn, and decreased though the second and third turns. When the cochleae were analyzed as a whole, the difference in distributions between the treated and control groups approached statistical significance. Discussion The apoptotic initiator, caspase8, can either directly activate the effector caspase3, or cleave BID which facilitates the release of cytochrome C mediated by the insertion of Bax or Bak into the mitochondrial membrane. Both of these circumstances result in cell death [72] There is evidence that many inflammatory cytokines, including those of the TNF family which can act as a death receptor ligand, are upregulated foll owing noise exposure [73] C aspase8 was also shown to be activated in hair cells following noise exposure [59] Our observations confirm this finding, and suggest the possibility t hat ACEMg treatment may attenuate caspase 8 activation. This protective effect could be mediated in part by the vasodilatory properties of magnesium. Magnesium prevents noiseinduced reduction of cochlear blood flow by dilation of the vasculature within the stria vascularis [142] This in turn prevents ischemic injury and production of inflammatory mediator s such as those of the TNF family which as previously mentioned, can act as death receptor ligands [73]

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56 A B Figure 41. Epifluorescence micrographs of ACEMg treated (A) and saline control (B) sections of the organ of Corti labeled with anti caspase 8 antibody showing the greatest observed treatment effect Sections in the left, middle, and right columns were taken from the 1st, 2nd, and 3rd, turns of the cochlea respectively. These images were selected from ears with the lowest treated and the highest saline average rank score.

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57 A B Figure 42. Epifluorescence micrographs of ACEMg treated (A) and saline control (B) sections of the organ of Corti labeled with anti caspase 8 antibody showing median treated and control images Sections in the left, middle, and right columns were taken from the 1st, 2nd, and 3rd, turns of the cochlea respectively. These images were selected from ears with the median treated and saline rank score.

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58 A B Figure 43. Negative control in which the primary anti caspase 8 antibody incubation was omitted (A) and no noise control (B).

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59 Table 41. Difference in distribution of ACEMg treated and saline control caspase8 image rank scores 1 st Turn 2 nd Turn 3 rd Turn Ear Average Median ACEMg Score 6.75 7.94 12.56 9.60 Median Saline Score 14.69 14.31 6.69 11.98 Mann Whitney U 7.0 10.0 11.0 5.0 P value (one tailed) 0.155 0.345 0.579 0.076

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60 CHAPTER 5 C ASPASE2 Given the fact that caspases 3, 8, and 9 have an established role in noiseinduced damage to the inner ear [59] and that caspase 2 activation has been described in response to oxidative stress [96, 152] we sought to determine the extent of caspase2 activation in the cochlea following noise exposure. Caspsase2 differs from its other family members because it has characteristics of both an initiator, and an effector caspase. To date, caspase2 activity in response to noise has not been characterized in the inner ear. Evidence which shows caspase2 activation following ROS formation [96] led us to hypothesize that similar activation would be obser ved in response to noise insult. Results These data provide the first evidence that caspase2 is activated in response to acoustic overexposure, and imply a possible role for caspase2 in NIHL. There was no significant nonspecific labeling of negative co ntrol tissues in which the primary anti caspase2 antibody was omitted (Figure 51, A and B). Unexpectedly, at the 2 hour post noise time point, caspase2 labeling appeared to be localized within the supporting cells (phalangeal process of outer pillar cells and Deiters cells). This was t he case in 6 of 7 ears (Figure 51, C and D). By the four hour post noise time point, caspase2 expression was observed in support ing cells (2 of 7 ears, Figure 51, E), but more often in the OHCs (5 of 7 ears, Fig ure 51, F). Finally, 24 hours following noise exposure caspase2 immunolabeling was again observed most commonly in support ing cells (4 of 5 ears, Figure 51, G and H). Labeling in all control tissues not exposed to noise was

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61 difficult to detect, was diffuse, and was not localized to a particular cell type (Figure 51, I and J). Due to the fact that the data show noiseinduced activation of caspase2, we chose to conduct a preliminary experiment testing the effect of ACEMg treatment on this activation. Prelim inary observations of ears treated with ACEMg (N=2) versus saline control (N=2) did not eliminate the possibility that this treatment reduces activation of caspase2 following noise. One of the ACEMg treated ears showed diffuse caspase2 expression which was minimal throughout ea ch turn of the cochlea (Figure 52, A). However, the other exhibited the same characteristic pattern of labeling in the supporting cells which was f ound in untreated ears (Figure 53, A). Further investigation is necessary due to the small sample size. Finally, in 2 of 2 ACEMg treated, and 2 of 2 saline control ears, caspase2 expression decreased with distance from the base of the cochlea. Discussion These data show, for the first time, the expression of caspase2 in the inner e ar following noise exposure, and suggest a possible role for caspase2 in noiseinduced cell death. Previously, evidence for caspase2 in the inner ear was limited to apoptotic cell death during development of neonatal rats [149] Caspase 2 activation occurs very early after cellular insult (for review see [94, 95] ), and blocking or downregulating caspase2 activity inhibits the release of cytochrome c and Smac from mitochondria, prevents translocation of Bax from the cytosol to mitochondria, and prevents translocation of apoptosis inducing factor (AIF) from mitochondria to the nucleus [93, 94, 153] Smac increases caspase activity by inhibiting the inhibitor of apoptosis protein (IAP).

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62 While much of the focus is usually on the susceptibility of hair cells to noise damage, there is considerable evidence that supporti ng cells are susceptible as well. Supporting cells, such as Hensens cells and the outer space of Nuel was shown to collapse at 24 hours post noise [112] Additionally, free radicals were detected in Hensens and Claudius cells in the guinea pig after noise, and labeling spread to hair cells as time progressed [67] Our observation that caspase2 expression is initially localized to the supporting cells and progresses to OHCs, c oupled with the knowledge that caspase2 is activated by oxidative stress [96, 152] suggests the possibility that caspase2 contributes to noiseinduced cell death in the inner ear, and that treatment with ACEMg may inhibit this cell death pathway. Additionally, the decreasing gradient of caspase2 expression from base to apex which we observed is in line with the common knowledge that the structures at the base are more susceptible to noiseinduced d amage than those at the apex.

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63 Figure 51. Epifluorescence micrographs of the organ of Corti from the 1st turn of the cochlea labeled with anti caspase2L antibody showing temporal difference in post noise expression. Negative control in which the primary antibody was omitted (A and B). Tissues harvested: 2 hours post noise (C D) showed distinct labeling in the supporting cells in 6 of 7 ears, 4 hours post noise exhibited expression in supporting cells in 2 of 7 ears (E) and in the OHCs in 5 of 7 ea rs (F), and 24 hours post noise (G H) found caspase2 expression in the supporting cells in 4 of 5 ears and only one ear with expression in the hair cells. There were 4 no noise control ears. The samples shown here (I J) had the most labeling of any nonoise control tissues.

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64 Figure 51. Continued

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65 A B Figure 52. Epifluorescence micrographs of ACEMg treated (A) and saline control (B) sections of the organ of Corti labeled with anti caspase 2L antibody showing diffuse labeling in treated and control tissues. Sections in the left, middle, and right columns were taken from the 1st, 2nd, and 3rd, turns of the cochlea respectively. Both treated and control tissues show diffuse immunolabeling, which decreases with distance from the base.

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66 A B Figure 53. Epifluorescence micrographs of ACEMg treated (A) and saline control (B) sections of the organ of Corti labeled with anti caspase 2 antibody showing immunolabeling concentrated in the supporting cells in 1st and 2nd turns, with labeling becoming more diffuse in the 3rd turn.. Sections in the left, middle, and right columns were taken from the 1st, 2nd, and 3rd, turns of the cochlea respectively.

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67 CHAPTER 6 CONCLUSIONS AND FUTURE DIRECTIONS Despite efforts to educate the public about the harm f ul effects of noise, NIHL continues to be a growing problem which causes significant decrease in the individuals quality of life, and a collective economic burden. A great deal of progress has been made in understanding the cellular and molecular mechanisms underlying this disease. Greater understanding has sparked the development of experimental therapeutics which intervene at various points of cell death pathways with the ultimate goal of prevention of hair cell loss. Despite varying degrees of success in animal models, as well as clinical trials, there is still no FDA approved pharmacological agent for the pr evention or treatment of NIHL. This study has taken another step in elucidating the pathways which lead to NIHL by providing the first evidence that caspase2 is upregulated following intense noise exposure. Caspase2 expression was observed in the supporting cells of the organ of Corti 2 hours post noise, with the amount of expression decreasing from base to apex. At 4 hours post noise expression appears to move transiently to the OHCs, with expression once again in the support ing cells at later time points. Noise damage is usually mediated, at least in part, by oxidative stress resulting from high metabolic demand and noiseinduced reduction of cochlear blood flow We also investigated the effect of the micronutrient treatment combination of carotene, vitamins C and E, plus magnesium on the activation of caspases 2 and 8, as well as the production of 3nitrotyrosine, a marker of RNS activity. These data support the possibility that ACEMg treatment may attenuate the noiseinduced activation of caspases 2 and 8 as well as RNS production. Given the f act that ROS and RNS

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68 production are known to peak at 710 days following noise exposure, f urther studies are warranted which explore this treatment effect at later post noise time points Future research will seek to characterize the effect of ACEMg treatm ent on other mediators of apoptotic signaling pathways such as caspases 3 and 9 as well as caspaseindependent mediators such as endonuclease G and apoptosis inducing factor (AIF). Given the fact that freeradical production has been implicated in heari ng loss caused by aminoglycoside antibiotics, chemotherapeutics, and agerelated hearing loss, ACEMg treatment should be studied for application in these areas as well.

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69 APPENDIX : COMMENTS ON METHODS AND STATISTICAL ANAL YSIS In this study, individual images of noise exposed, sectioned organ of Corti from treated and control groups were pooled and ranked in order from least immunolabeling within the hair cells to most by a series of observers who were blind to study conditions. A nonparametric statistical test was then used to analyze the difference in ranks between the treated and control groups. The following discussion will co rrelate previously obtained hearing data from noise exposed animals (114 dB SPL centered at 4 kHz for 4 hours) with what we would expect to see in terms of ROS production/ caspase activation, and what we actually observed. Limitations associated with the ranking design and statistical analysis will also be commented upon. The effect of the noise exposure used in this study on hearing sensitivity in guinea pigs has been well characterized in our lab by measurement of auditory brainstem response (ABR) thresholds Figure A 1 shows average noiseinduced threshold shifts at various post noise time points. The fact that there is a large decrease in hearing sensitivity across all frequencies at the early post noise time points justifies our analysis of the entire ear t aken as a whole. All of the treated and control animals in this study were euthanized at the two hour post noise time point. Due to the threshol d elevation at this time across all frequencies, we would expect there to be increased freeradical production and possibly caspase activation throughout the cochlea. Our data support this in that there is increased labeling for all targets in each section when compared to control tissues which have not been exposed to noise. For future studies which will examine labeling at the later post noise time points we would expect to find the most labeling in the high frequency (basal) region of the cochlea.

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70 Turning now to the statistics, the nonparametric statistical test was chosen because we are analyzing ordinal dat a, or qualitative data that can be ranked in order of magnitude. Parametric statistical tests rely on certain assumptions such as that the data are sampled from a normally distributed population. Nonparametric tests on the other hand do not depend on the distribution of the sampled population, and are thus referred to as distributionfree tests. Additionally, nonparametric methods are concerned with the location of the probability distribution of the population rather than on specific parameters of the population, such as the mean. Specifically, t he WilcoxonMannWhitney two sample rank sum test was used to analyze the difference in image ranks between the treated and control groups This is a nonparametric method which tests the null hypothesis that the probability distributions associated with the treated and control populations are equivalent. The conditions required for a valid rank sum test are as follows: 1) the two samples are random and independent, and 2) the two probability distributions from w hich the samples were drawn are continuous so that there are no ties. If the treated and control populations were identical, we would expect the ranks to be randomly mixed between the two samples. On the other hand, if the treated population tends to hav e less labeling for a particular target (as hypothesized) we would expect the smaller ranks to be mostly in the treated sample and the larger ranks to be mostly in the control sample. This experiment defined the onetailed alternative hypothesis to be that the distribution of treated ranks would be less than (shifted to the left of) the distribution of control ranks The test statistic is calculated based on the totals of ranks (rank sums) for each of the two samples. The greater the difference between r ank sums, the greater the evidence

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71 indicating a difference between the probability distributions of the two populations. Once the test statistic has been calculated for a particular trial, the observed significance level, or pvalue, can be calculated bas ed upon the sampling distribution of the test statistic under the null hypothesis. The pvalue is the probability (assuming that the null hypothesis is true) of observing a value of the test statistic that is at least as contradictory to the null hypothes is, and supportive of the alternative hypothesis, as the actual one computed from the sample data. More specifically, in the case of the Wilcoxon MannWhitney test, the p value answers this question: if the treated and control populations really have the same median image rank score, what is the chance that random sampling would result in a sum of ranks as far apart or more so as observed in this experiment? We have made the assumption that the saline control images would be ranked higher and therefore, 1tailed p values were reported. If the pvalue is small (<0.05), one could conclude that the treated and control populations have different medians. If the pvalue is large, there is not sufficient evidence to reject the null hypothesis and conclude that the medians differ. This does not necessarily mean that the medians are the same; it just means that under the current experimental conditions there is not enough evi dence to say that they differ. One limitation was the small sample size used in this experiment. With small sample sizes, rank tests often have little statistical power. The power of a statistical test is defined as the probability of correctly rejecting the null hypothesis when in fact the alternative hypothesis is true. The probability o f type II error ( ) is defined as the probability of incorrectly accepting the null hypothesis when the alternative is true. Therefore the statistical power of a test can be calculated as 1 For the rank sum

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72 test, power calculations can be performed in the absence of a priori knowledge of population variance using an odds parameter ( are 4:1 that the control animals had higher ranks than treated [154] In our case, the ear average data for 3the ear average data for caspaseFigure A 2 plots the odds parameter ( ) against power for our two sample case with five observations per group. From this, we can clearly see that we were not adequately powered to detect even our greatest difference in primar y outcome measure seen in the c aspase 8 ear average data where Studies are ongoing which will both increase the sample size at the early post noise time points, and assess the treatment effect at later post noise times. Another possible limitation is that working with ordinal data sets in which the images were ranked from least to most labeling may mask large differences between individual images. For instance, the difference between images ranked 1 and 2 may be much greater or much smaller than that between images ranked 3 and 4. This may be alleviated by having observers score each image on a pr e determined numerical scale. A lso, a n alternative to image rank analysis could be explored for future studies in which the image collection protocols are rigorously standardized to allow for exact measurements of either area or intensity of labeling within the hair cells to be determined using quantit ative image analysis software.

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73 Figure A 1. Auditory brainstem response (ABR) threshold shift at various post noise time points. Figure A 2. Plot of statistical power vs. the odds parameter gamma

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87 BIOGRAPHICAL SKETCH Dustin Matthew Lang was born and raised in Florida, where he attended Rockledge High School. From there, he went on to receive a Bachelor of Science degree in c hemistry from the University of Florida in D ecember of 2007. In August 2009 Dustin was married to his lovely wife, Michelle. In August of 2010, he was awarded a Master of Science degree in medical s ciences from the University of Florida.