Hannover Medical University Clinic of Laryngology ... · 1. Prof. Prof. h. c. Dr. med. Thomas...

Hannover Medical University

Clinic of Laryngology, Rhinology and Otology /

University of Veterinary Medicine Hannover

Determination of apoptosis and cell survival signaling

following neomycin induced deafness in the rat cochlea

THESIS

Submitted in the partial fulfillment of the requirements for the degree

DOCTOR OF PHILOSOPHY (Ph.D)

awarded by the University of Veterinary Medicine Hannover

by

Souvik Kar

Balasore, Orissa, India

Hannover, 2012

Supervisor

Prof. Prof. h.c. Dr. Med. Thomas Lenarz, Professor and Chairman, Department of

Otolaryngology, Hannover Medical School, Germany

Supervisor group:

1. Prof. Prof. h. c. Dr. med. Thomas Lenarz, Department of Otolaryngology, Hannover

Medical School, Germany

2. Prof. Dr. Martin Stangel, Department of Neurology, Hannover Medical School,

Germany

3. PD. Dr. Karl-Heinz Esser, Auditory Neuroetholgy and Neurobiology Laboratory,

Institute of Zoology, Hannover, Germany

External referee

Mrs. Priv-Doz. Minoo Lenarz, MD, Charité-Universitätsmedizin Berlin, CC 16:

Audiology/Phoniatrics, Ophthalmology and ENT- Medicine, Clinic for Ear, Nose and

Throat Medicine, Berlin, Germany

Date of oral exam: 30-31st March, 2012

This work has been supported by the Georg-Christoph-Lichtenberg Fellowship by the

State of Lower Saxony, Germany

“The process of scientific discovery is, in effect, a continual flight from

wonder”

Albert Einstein

dedicated to my beloved family and my love

Abbreviations

AABR Acoustic auditory brainstem response

AN Auditory nerve

Apaf Apoptosome complex

APAF-1 apoptotic protease activating factor

ATP Adenosine tri-phosphate

Bcl-2 B-cell lymphoma 2

BDNF Brain derived neurotrophic factor

BMP Bone morphogenetic protein

CNS Central nervous system

Cyt-c Cytochrome c

CREB cyclic AMP response element-binding

DAPI 4', 6-diamidino-2-phenylindole

dBSPL decibel sound pressure level

DNA deoxy ribo-nucleic acid

DIABLO Direct inhibitor of apoptosis (IAP)-binding protein

DISC Disc-inducing signaling complex

EGF Epidermal growth factor

EtOH Ethanol

EDTA Ehtylenediamine tetra-acetic acid

FRET Fluorescence resonance energy transfer

FADD Fas-associated protein with death domain

FITC Fluorescein isothiocyanate

FGF-1 Fibroblast growth factor-1

GDNF Glial cell-derived neurotrophic factor

GFRα1 GDNF-family receptor-α

GPI Glycosylphosphatidylinositol

IHC Inner hair cell

IGF Insulin-like growth factor

IgG Immunoglobulin-G

JNK Jun-NH2-terminal kinase pathway

kHz Kilohertz

mRNA messenger ribonucleic acid

MAP mitogen activated protein

NTN Neuritrin

NT-3 Neurotrophin-3

NTF Neurotrophic factor

NF-κB Nuclear factor- κB

NIHL Noise induced hearing loss

NH Normal hearing

OHC Outer hair cell

PCR Polymerase chain reaction

PBS Phosphate buffer saline

PNS Peripheral nervous system

PSP Persephin

PCD Programmed cell death

p75NTR p75 neurotrophin receptor

RIN RNA integrity number

ROS Reactive oxygen species

TNFR Tumor necrosis factor receptor

TRAIL TNF-related apoptosis-inducing ligand

TUNEL Terminal deoxynucleotidyl transferase dUTP nick end labeling

Trk-B Tyrosine kinase receptor-B

TDT Tucker Davies Technologies

tRNA Transfer ribonucleic acid

30S 30 Svedberg unit

Acknowledgements

First of all, I would like to express my deep gratitude towards my supervisor, Prof. Prof.

h. c. Dr. med. Thomas Lenarz for providing me ample opportunity, and whose guidance,

support and healthy critics motivated me to learn and carry out this research project

successfully. I would also like to thank my former supervisors, Prof. Dr. med. Timo

Stoever and Dr. med. Minoo Lenarz for their scientific support, constructive comments

and providing me a platform to carry out independent research. I am happy to extend my

heartful thanks to Dr. Kirsten Wissel, whose support and proper guidance motivated me

to complete this work. Her positive comments and regular discussions helped me to carry

out my work independently with great interest. I am delighted to thank my ex- and

present co-supervisors, Prof. Krampfl, Prof. Claudia Grothe, Prof. Dr. Martin Stangel and

PD. Dr. Karl-Heinz Esser, who were helpful and supportive in providing scientific advice

and suggessations in finishing my Ph.D work. Further, I thank Dr. Verena Scheper for her

strong support, help in animal related surgeries, which gave me confidence in carrying

out my experiments properly with animals. Many thanks for Peter Erfurt for showing me

how to work with cochlear histology and providing me with technical expertise in

paraffin and cryo embedding. In addition, I am thankful to all my former and present

colleagues in my department, Gerogios, Hubert, Meli, Sussane, Gerrit, Athanasia, Roger,

Heike, Alice, Odett, Darja, Antonina, Saied, Behrouz, Nadine, Thilo for sharing good

times during my Ph.D work. I cannot forget to deeply thank my close friends and

colleagues, Ronnie, Alex, Ugur and Pooyan, Vikram, Dharmesh and Kiran who were

always by my side during my hard and good times. I am thankful to Mrs. Gabi

Richardson and Dr. Thorsten Schweizer for their prompt help and response anything

related to official matters in our department. I would like to express my thanks to our

collaborators, Dr. Ananta Paine providing me with the opportunity to use their real time

PCR machine, Dr. Oliver Boch for helping us in running the housekeeping control panel

experiments and Dr. med. Arndt Vogel for providing the TUNEL assay kit. I wish to

acknowledge my thanks to Zentrum für Systemische Neurowissenschaften (ZSN) Ph.D

programme for providing me the financial support to carry out my Ph.D work in

hannover. I cannot miss to express my gratitude to our former ZSN coordinator, Dagmar

Esser who was very friendly and supportive from the beginning of my Ph.D career here. I

extend my thanks to our new ZSN coordinator, Prof. Beatrice Grummer and Dr. Tina

Selle who have been enough helpful in providing suggessations regarding the ZSN

curriculum. My indebted obligations always will remain towards my parents and family

without whose support and encouragement I may not have been here. I cannot conclude

this acknowledgement without the mention of one name, Arpita, my wife, for whom there

is no words to thank. She has been my source of inspiration, strength, encouragement and

love in all the hard and good times.

Contents

Abbreviations .................................................................................................................... 4

Acknowledgements ........................................................................................................... 7

Introduction ..................................................................................................................... 13

1.1 The hearing organ ................................................................................................. 13

1.1.1 Inner ear ................................................................................................. 14

1.2 Auditory physiology of the inner ear ...................................................................... 18

1.3 Auditory dysfunction .............................................................................................. 19

1.4 Aminoglycoside induced ototoxicity ...................................................................... 21

1.4.1 Mechanism of aminoglycoside ototoxicity .......................................................... 22

1.5 Neurotrophic factors and their receptors ................................................................ 27

1.5.1 The neurotrophic factor hypothesis.................................................................. 30

1.6 Aims of the study .................................................................................................... 33

Materials and Methods ................................................................................................... 34

2.1 Neomycin induced deafness ................................................................................... 34

2.1.1 Housing of animals .......................................................................................... 34

2.1.2 Animal model of deafness................................................................................ 34

2.1.3 Acoustically evoked auditory brainstem response (AABR) ................................ 35

2.1.4 Deafening procedure and surgery ........................................................................ 36

2.2 Cochlear histology and immunohistochemistry ..................................................... 37

2.2.1 Cochlea harvesting and paraffin embedding .................................................... 37

2.2.2 Hematoxylin and eosin staining ....................................................................... 38

2.3 Spiral ganglion cell (SGC) density measurement ................................................... 38

2.4 Immunohistochemistry ........................................................................................... 40

2.5 Gene expression analysis ........................................................................................ 41

2.5.1 Modioli preparation ......................................................................................... 41

2.5.2 Total RNA isolation ......................................................................................... 41

2.5.3 RNA quality assessment .................................................................................. 43

2.5.4 cDNA synthesis ............................................................................................... 44

2.5.5 Real-time PCR ................................................................................................. 44

2.5.5.1 Principle of real-time PCR ............................................................................ 44

2.5.5.2 Housekeeping genes ...................................................................................... 45

2.5.5.3 Real-time PCR assay ..................................................................................... 48

2.6 Statistical analysis ................................................................................................... 49

Results .............................................................................................................................. 50

3.1 Unilateral deafening by neomycin resulted in profound hearing loss .................... 50

3.2 Significant reduction of Spiral ganglion cell density in the deafened cochleae 7, 14

and 28 days after ototoxic deafening ............................................................................ 54

3.3.Gene expression results .......................................................................................... 57

3.3.1 Quality assessment of total RNA ..................................................................... 57

3.3.2 RPLP2 as housekeeping gene .............................................................................. 60

3.3.3 Gene expression patterns of neurotrophic and apoptosis related genes following

deafening ....................................................................................................................... 62

3.4 Immunohistochemistry ........................................................................................... 67

3.4.1 Immunohistochemical expression and distribution of neurotrophic factors in

the cochlear tissues ................................................................................................... 67

3.4.2 Immunohistochemical expression and distribution of apoptosis related

molecules in the cochlear tissues .............................................................................. 74

Discussion......................................................................................................................... 79

4.1 Effect of ototoxicity on the auditory threshold of rats ............................................ 80

4.2 Significant reduction of SGC density following deafening .................................... 81

4.3 Relative gene expression changes in the normal and deaf animals following

deafening ....................................................................................................................... 82

4.4 Methodological considerations ............................................................................... 89

Summary .......................................................................................................................... 91

Zussamenfassung ............................................................................................................ 93

References ........................................................................................................................ 97

Declaration..................................................................................................................... 122

Introduction

1.1 The hearing organ

The uniqueness of the mammalian ear is attributed to perceive sound from the

environment. The ear is broadly divided into three major parts: outer, middle and inner

ear. The outer ear constitutes the pinna and the external auditory meatus. The pinna helps

to collect sound waves from the external enviornment and directs them to the external

auditory meatus. Therefore, the outer ear contributes to the localization of the source of

sound energy. The auditory canal is filled with cerumen/ear wax which helps to protect

the middle ear from the entry of dust and airborne particles. The outer ear canal behaves

as a resonant tube, similar to an organ pipe.

Fig.1: The hearing organ. The picture displays a coronal view of a mammalian ear

illustrating the external ear (external auditory meatus), middle ear (tympanic membrane,

malleus, incus and stapes) and inner ear (the cochlea and the vestibule).

(Modified from http://www.virtualmedicalcentre.com/anatomy.asp?sid=29)

14

The tympanic membrane and the auditory ossicles: malleus (hammer), incus (anvil) and

stapes (stirrup), together constitutes the middle ear (Figure 1). The middle ear helps to

transfer and match the sound waves from low impedance of air to the high impedance of

cochlear fluid of the inner ear through the ossicular chain (Kurokawa et al., 1995). They

act as a hydraulic press in which the surface area of the tympanic membrane is 21 times

larger that of the stapes footplate. Therefore, the force caused due to the sound pressure

in the air acting on the tympanic membrane is concentrated through the small area of the

stapes footplate, resulting in a pressure increase. Moreover, the lever arm formed by the

rotating malleus about its pivot is somewhat longer than that of the incus, providing

another factor of 1.3 times increase in pressure. The tympanic membrane vibrates under

the influence of alternating sound pressure. Movement of the tympanum causes the

mallues and incus to rotate as a unit about a pivot point, and this movement causes the

stapes to rock back and forth, setting up a wave of sound pressure in the fluid of the inner

ear. Besides acting as a hydraulic press, the middle ear comprises of two muscles, the

tensor tympani and the stapedius muscle. These muscles help to stiffen-up the ossicular

chain to afford some protection and lessen the intensity of very strong stimuli, thus,

minimizing possible damages to the inner ear (Wilson, 1987).

1.1.1 Inner ear

The inner ear (Figure 2) consists of a membranous cavity encased in an osseous

labyrinth. The inner ear is innervated by the eighth cranial nerve in all vertebrates and has

the receptors for hearing and balance. The osseous labyrinth of the inner ear can be

subdivided into the spirally shaped cochlea, and the vestibule. The cochlea helps to

tranform the mechanical sound energy into electric impulses which are conveyed to the

auditory cortex via the auditory nerve (Roland et al., 1997). The vestibule plays a

dominant role in the spatial orientation of the head and adjusts the muscular activity and

body posture (Figure 3).

The cochlea is a spirally coiled structure embedded in the bony modiolus. It is conical in

form and placed almost horizontally in front of the vestibule. Its base corresponds with

the inferior part of the internal acoustic meatus and is perforated by numerous apertures.

The osseous spiral lamina (lamina spiralis ossea) accommodates a bony shelf projecting

15

from the modiolus to the interior of the spiral canal. Surrounded by the spiral canal is the

spiral ganglion of the cochlea (SGC), made up of of bipolar nerve cells which constitutes

the cells of auditory nerve. The SGC project its central process into the osseous to

synapse with the hair cells (Roland et al., 1997). Anatomically, the middle and inner ear

communicates via two small opening in the temporal bone, the oval and round window.

The cochlea is subdivided into three fluid-filled compartments. The scala vestibule and

scala tympani are filled with perilymph and scala media filled with endolymph (Roland et

al., 1997). The base of the scala media represents the basilar membrane and its upper

margin consists of vestibular membrane (Reisssner’s membrane), extending between the

upper edge of the stria vascularis and the inner margin of spiral limbus (Roland et al.,

1997). The ionic composition of the fluid in the scala media is similar to that of

intracellular fluid, rich in potassium and low in sodium and the fluid in the scala vestibuli

and scala tympani is similar to that of extracellular fluid, rich in sodium and poor in

potassium (Wangemann, 2006). Located at the opening near the apical termination of the

bony labyrinth is the helicotrema, which allows communication between the scala

vestibule and scala tympani. Situated on the basilar membrane is the organ of Corti and

comprises of outer hair cells (OHC), inner hair cells (IHC), supporting cells and the

tectorial membrane (Figure 2). The basilar membrane distinguishes sound vibration

according to the frequency and the organ of Corti associated with the hair cell transform

these vibrations of the basilar membrane into a neural code. The supporting cell

comprises of the inner and outer pillar cells, inner and outer phalangeal cells, Hensen

cells, and Claudius cells. A large number of nerve fibers run helically and radially

through the tunnel of Corti, seperating the IHC and OHC. At the apical part of the OHC,

100- 200 stereocilia are anchored into the cuticular plate representing a “V” or “W”

pattern. The stereocilia are bathed by the endolymph, while other receptor cells including

the Hensen and Claudius cells are surrounded by the cortilymph located in the

extracellular spaces of organ of Corti (Raphael et al., 2003). The IHC helps to convert the

mechanical sound signal into electrical impulses by sending the signals to the auditory

cortex via the auditory nerve (Deol et al., 1979), thus acting as mechanotransducers.

Nearly 12,000 OHC, each containing 100-200 stereocilia arranged in parallel rows

perform the function of electromotility (Brownell, 1990)

16

Fig. 2: Inner ear anatomy. The inner ear comprises of the vestibular apparatus and

cochlea. The cochlea is shown in cross section (upper left) and subsequent panels. The

cross section shows the location of scala media in between scala vestibule and tympani.

The organ of Corti illustrates the location of the hair cells between the basilar and

tectorial membrane.

(Modifiedfrom:http://www.ncbi.nlm.nih.gov/books/NBK10946/figure/A895/?report=obj

ectonly)

17

and 3500 IHC forms a single row of a letter U. Overlying the organ of Corti is the

tectorial membrane attached to the limbus lamina spiralis close to the inner edge of the

vestibular membrane.

Located medial to the tympanic cavity, behind the cochlea and infront of the semicircular

canals is the organ of balance, the vestibule (Wright, 1983). It is somewhat ovoid in

shape, but flattened transversely. The mammalian vestibular apparatus comprises of

semicircular canals, a utricle and a saccule (Figure 3). The saccule and the utricle

together are referred as otolith organs. The semicircular canals are located at right angles

to each other at the opposite side of the head and allow us to sense the direction and

speed of angular acceleration. At the base of each canal are located small swellings called

ampulla, which contain sensory receptors. The function of the vestibular apparatus is to

send symmetrical impulses to the auditory cortex in the brain for the proper coordination

of balance and movement. (O’Reilly et al., 2011)

Fig. 3: The vestibular system: Cross section through the vestibular system comprising

semicircular canals, utricle, saccule and ampullae. The semicircular canals are oriented

along three planes of movement with each plane at right angles to the other two and are

filled with endolymph.

(Modified from: http://weboflife.nasa.gov/learningResources/vestibularbrief.htm)

18

1.2 Auditory physiology of the inner ear

The physiology of hearing is associated with the transmission of sound vibrations and

generation of neural impulses. The mammalian cochlea as described earlier is divided

longitudinally into three fluid-filled scalae (the scala vestibule, the scala tympani, and the

scala media). The scala media runs throughout the length of the cochlear duct and

provides a suitable ionic evniornment to the sensory hair cells (Ashmore, 2008), while

the scala vestibuli and the scala tympani are joined by an opening at the apex of the

cochlea, the helicotrema. Acoustic signals entering via the auditory canal propagates to

the tympanic membrane, setting it into a vibratory motion. Such vibrations are

mechanically transmitted by the ear ossicles to the fluid-filled cochlea. Inside the fluid-

filled cochlea, the basilar membrane undergoes an oscillatory motion matching the

frequency of the sound signal, resulting in a waveform. A travelling waveform is

spatially confined along the length of the basilar membrane, and the location of

maximum amplitude is dependent upon the frequency of sound. That implies, the higher

the frequency the more disturbance is created at the proximal end of the basilar

membrane as illustrated in Figure 4. The sensory hair cells located within the organ of

Corti receiving maximal mechanical stimuli from the basilar membrane, transduce into

electrical signals and generate sensory outflow from the cochlea. Due to the repeated

vibration and oscillations of the basilar membrane, a shearing force is generated in the

organ of Corti forcing the stereocilia of the sensory hair cells to bend. A change in the

tension caused by the bending of the stereocilia produces a change in the OHC motility,

subsequently increasing the sensitivity and frequency selectivity of the basilar membrane.

On the other hand, the shear movement of the tectorial membrane forces the stereocilia of

the IHC to bend, resulting in the release of neurotransmitters across the hair-

cell/auditory-nerve synapse (Robertson, 2002). Such movement of stereocilia is

responsible for the opening of transduction ion channels in the IHC, allowing the entry of

K+

and calcium ions to generate transduction current (depolarization), resulting in the

release of neurotransmitter at the hair cell base (Zhang et al., 1999). Conversely,

movement of stereocilia in the opposite direction closes the ion channels and blocks the

release of the neurotransmitter, leading to hyperpolarization. Moreover, the release of

19

neurotransmitter requires a synapse where there is a rapid post-synaptic effect and

recovery. A postsynaptic potential of sufficient magnitude is propagated along the

auditory nerve to the brain, which is finally accomplished as an action potential or spike

(Hossain et al., 2005).

Fig. 4: Auditory transduction in the inner ear: Schematic representation of a cochlear

duct demonstrating sound waves travelling along the length of the basilar membrane in

response to a pure tone. The high frequency sounds are more restricted towards the

proximal end and the low frequency sounds are more confined towards the distal end of

the basilar membrane, giving rise to a topographical mapping of frequency.

(Modified from Hole’s Human Anatomy and Physiology, 7th

edition, by Shier et al.

copyright © 1996 TM Higher Education Group, Inc)

1.3 Auditory dysfunctions

Auditory dysfunction is a complete or partial decrease in the ability to detect or

discriminate sounds from one or both the ears. It is caused by a wide range of biological

and environmental factors: genetic, due to complications by birth or by the use of

ototoxic drugs, and exposure to excessive noise. Hearing loss is generally described as

mild, moderate, severe, or profound, depending on how well an individual can perceive

the frequencies or sound intensities associated with speech. Previous report suggests that

approximately 1-6 children per 1,000 new borns suffer from congenital hearing loss

20

(Cunningham et al., 2005). Depending on which part of the hearing system is affected,

auditory dysfunctions can be categorized into conductive and sensorineural neural

hearing loss (SNHL).

Conductive hearing loss is a common type of dysfunction with complications in the

conductance of sound through the outer ear canal to the tympanum and the tiny ossicles

of the middle ear. A person with such type of hearing loss has the inner ear capable of

normal function. Conductive hearing loss may result due to accumulation of ear wax

resulting in obstruction, damage to the tympanic membrane, middle ear infection and

dislocated ear ossicles (Briggs et al., 1994). For example, otitis media (inflammation of

the middle ear) is a common form of conductive hearing loss in children suffering from

chronic ear infections (Paradise et al., 1990). However, if left untreated, otitis media

might result in a build-up of fluid and a ruptured tympanum.

SNHL develop when the mechanical sound energy is properly conducted to the cochlea

via the oval window, but not appropriately converted into a neural signal to be carried by

the auditory nerve. As a result, an individual’s ability to discriminate between sounds of

different intensities and frequency is thus, affected. SNHL causes hearing loss to the

neurosensory elements in the inner ear (Kopecky et al., 2011). Generally the loss is

profound and nearly always permanent and irreversible. SNHL might be acquired or

congenital. Acquired SNHL are mostly caused by ototoxic drugs and medications

(Shepherd et al., 2004; Yamagata et al., 2004), noise exposure (Nam et al., 2000), normal

aging process (Robinson et al., 1979), infections, trauma (Kohut et al., 1996), neoplasm

(Moffat et al., 1994) and idiopathic or systemic conditions (Hughes et al., 1996), such as,

Meniere’s diseases, autoimmune diseases (Salley et al., 2001), neurological disorders,

bony abnormalities (Isaacson et al., 2003) and endocrine disorders, whereas congenital

SNHL might be due to inherited hearing loss, prematurity, German measles (rubella) or

cytomegalovirus and jaundice. A great majority of SNHL is associated with the

degeneration of the hair cells followed by a number of pathological changes in the organ

of Corti (Shepherd et. al., 2004). In few cases, SNHL may also involve the VIIIth cranial

nerve (the vestibulocochlear nerve) or the auditory portions of the brain (Lavi et al.,

2001).

21

1.4 Aminoglycoside induced ototoxicity

Ototoxic trauma induced by aminoglycosides causes hair cell loss and subsequent

degeneration of the auditory nerve resulting in permanent bilateral, high-frequency

SNHL and temporary vestibular dysfunction (Guthrie, 2008). Despite their ototoxic and

nephrotoxic nature, aminoglycosides are still used globally as antibiotics due to their low

cost and antimicrobial properties (Grohskopf et al., 2005). Ototoxicity might be also

induced clinically by loop diuretics, macrolide antibiotics and cisplatin (Arslan et al.,

1999). Ototoxicity gained its popularity with the discovery of the antibiotic, streptomycin

from Streptomyces griseus in 1944 by Selman A. Waksman. However, when this drug

was applied to patients suffering from tuberculosis, they developed irreversible cochlear

and vestibular dysfunctions (Kahlmeter et al., 1984). Aminoglycosides are known to have

variable cochleotoxicity and vestibulotoxicity (Selimoglu et al., 2003). Their

cochleotoxicity affects the high frequency from the base and then extend towards the low

frequency at the apex of the cochlea over time in a dose-dependent manner (Aran et al.,

1975). Becvarovski showed that round window membrane permeability is an important

cause for aminoglycosides ototoxicity (Becvarovski et al., 2004). The most common

forms of aminoglycosides are streptomycin, kanamycin, gentamicin, neomycin,

tobramycin, netilmicin and amikacin. Among them, gentamicin, streptomycin are

primarily vestibulotoxic, whereas neomycin, kanamycin and amikacin are cochleotoxic

(Selimoglu et al., 2003). Tobramycin is known to be vestibulotoxic and cochleotoxic

(Yorgason et al., 2006). Little is known about netilmicin ototoxicity but its ototoxic

potential is considered to be low (Selimoglu et al., 2003). The vestibulotoxic nature of

gentamicin was used advantageously to treat Meniere’s disease (Light et al., 2003).

Kanamycin being less toxic than neomycin was used against gram-negative bacteria since

early 1960s (Brewer et al., 1977). Amikacin, a semi synthetic aminoglycoside derived

from kanamycin is less ototoxic and effective against pseudomonas and gram-negative

bacteria (Schuknecht, 1993). Neomycin is considered one of the potent cochleotoxic

aminoglycoside when administered orally and in high doses compared to other ototoxic

antibiotics (Gookin et al., 1999). The ototoxicity levels resulting in apical surface damage

induced by aminoglycosides in hair cells are of the order: neomycin > gentamicin >

dihydrostreptomycin > amikacin > neamine > spectinomycin (Kotecha et al., 1994).

22

Neomycin is active against bacteria growing aerobically and is produced by Streptomyces

fradiae. It is considered as an effective inhibitor of protein synthesis (Pestka, 1971;

Weisblum et al., 1968).

Figure 5: Overview of the clinically important aminoglycosides with respect to their year

of discovery, organism in which there were isolated, chemical formula and functions.

1.4.1 Mechanism of aminoglycoside ototoxicity

Aminoglycosides are multifunctional hydrophilic amino-modified sugars used in the

treatment of gram-negative and gram-positive infections (Haasnoot et al., 1999). Being

polycations and polar, they are poorly absorbed into the gastrointestinal tract and are

easily excreted by the normal kidney. Aminoglycosides possess bactericidal properties

23

and are involved in the disruption of the plasma membrane, intracellular binding to

ribosomal subunits and drug uptake (Davis, 1987). High concentrations of

aminoglycosides in the plasma can result in transfer to the perilymph and endolymph in

the cochlea and its subsequent accumulation resulting in ototoxicity (Steyger et al.,

2008). The half-life period of aminoglycosides in the perilymph is 10-15 times longer

than present in serum (Lortholary et al., 1995). Within cells, aminoglycosides are

localized in the endosomal and lysosomal vacuoles and Golgi complex, cytosol and in the

nucleus (Yorgason et al., 2006). Aminoglycosides are selectively diffused into the

lysosome-like intracytoplasmic vesicles by IHC via endocytosis, where they are finally

accumulated. Once the accumulation exceeds the vesicle’s capacity, the vesicle

membrane is disrupted and they slowly diffuse into the cytoplasm (Hashino et al., 2000).

Previous studies have clarified, in part, the mechanism by which aminoglycosides

induces apoptosis (Forge et al., 2000; Pirvola et al., 2000; Rybak et al., 2003), and

apopnecrosis-like events in the hair cells (Formigli et al., 2000; Jaattela et al., 2002; Jiang

et al., 2005). Apoptosis involves controlled auto-digestion whereas necrosis accompanies

rapid swelling and lysis in the cell. Aminoglycosides induces ototoxicity by binding to

the 30S ribosomal subunit through an energy dependent mechanism causing misreading

of the mRNA or terminating ongoing translation of mRNA (Moazed et al., 1987; Fourmy

et al., 1996; Rybak et al., 2007). Once bound, they can remain in the hair cells for several

months, increasing the risk of ototoxicity. The mechanism of ribosomal subunit binding

has been more extensively studied in paromomycin, which is structurally related to

neomycin (Ma et al., 2002; Recht et al., 1996; Carter et al., 2000). Several molecular

models have been proposed to explain aminoglycoside ototoxicity. One of them involves

the energy dependent reversible binding of aminoglycoside to the plasma membrane and

subsequent binding to the precursor, phosphatidylinositol biphosphate (Schacht et al.,

1986, Williams et al., 1987). The widely accepted theories of aminoglycosides

ototoxicity demonstrates the generation of reactive oxygen species (ROS) (Clerici et al.,

1996; Song et al., 1996; Hirose et al., 1997; Conlon et al., 1999; Rybak et al., 2007). As

illustrated in Figure 5, ROS are the products of oxygen metabolism and are produced by

the formation of aminoglycoside-iron complex which catalyses their production from the

unsaturated fatty acids (Lesniak et al., 2005). A unique role of ROS lies in the

24

understanding that animals overexpressing superoxide dismutase (a superoxide

scavenging enzyme) generated by the membrane-bound enzyme complex, NADPH

oxidase, demonstrate less susceptibility to aminoglycoside ototoxicity compared to the

wild types (Sha et al., 2001). It has been previously reported that streptomycin stimulates

the formation of ROS which is considered to be a crucial factor for inner ear damage

(Horiike et al., 2003; Nakagawa et al., 1999; Takumida et al., 2002). Reports have shown

that iron chelators ameliorate aminoglycoside-induced cochleo- and vestibule-toxicity

(Conlon et al., 1998; Song et al., 1996). ROS mediated signaling pathways involve the c-

Jun N-terminal kinase (JNK) apoptotic pathway (Tournier, 2000). The c-Jun N-terminal

kinase (JNK) belong to the mitogen-activated protein kinase family are responsive to

stress and play a role in T-cell differentiation and apoptosis (Davis, 2000; Pearson et al.,

2001). Inhibition of the JNK signaling using cell permeable peptide protects hair cells

from apoptosis (Pirvola et al., 2000; Wang et al., 2003; Eshraghi et al., 2007). Following

aminoglycoside-induced ototoxicity, a large number of free-radical species, including

oxygen and nitrogen free-radical species have been detected in the inner ear, which is

believed to initiate the apoptotic cascade (Roland et al., 2004). Thus, there has been a

significant effort in discovering the cell signaling pathways by which aminoglycosides

induces apoptosis in the inner ear (Rybak et al., 2003; Jiang et al., 2006; Yu et al., 2010).

Apoptosis is primarily regulated by the activation of caspases through either extrinsic or

intrinsic signaling pathways (Rybak et al., 2003). The intrinsic signaling pathway

involves the activation of procaspase-9 in the mitochondria and the formation of

apoptosome, a cytosolic death signaling protein that is produced upon release of

cytochrome c from the mitochondria (Mak et al., 2002; Salvesen et al., 2002). Studies

have demonstrated that pro- and antiapoptotic Bcl-2 family members interact at the

surface of the mitochondria, competing for the regulation of cytochrome c release. The

dimerization of procaspase-9 leads to the activation of caspase-9 (Denault et al., 2002),

thus activating procaspase-3, -6 and -7 which result in the mediation and execution of cell

death (Earnshaw et al., 1999). The extrinsic apoptotic singnaling is mediated via the

activation of death receptors, which are cell surface receptors. These death receptors

involved in signaling belong to the tumor necrosis factor receptor (TNFR) gene

superfamily, including TNFR-1, Fas/CD95, and the TNF-related apoptosis-inducing

25

ligand (TRAIL) receptors DR-4 and DR-5 (Ashkenazi, 2002). The receptor Fas and

p75NTR

both comprises of death domains in their cytoplasmic regions, which are

important for inducing apoptosis (Strasser et al., 2000). Subsequent signaling is mediated

by the adapter molecules, Fas-associated protein with death domain (FADD) possessing

death domains. Such adapter molecules are further recruited to the death domain of the

activated death receptor constituting death inducing signaling complex (DISC).

Autocatalytic activation of procaspase-8 at the DISC leads to the release of active

caspase-8, which activates downstream effector caspases resulting in cell death (Figure

6).

Fig. 6: Mechanism of aminoglycoside-induced outer hair cell death: (1)

Aminoglycoside enter the outer hair cell through the mechano-electrical transducer

channels (2) formation of an aminoglycoside-iron complex which react with electron

donors forming (ROS) (3) ROS activate JNK (4) translocation to nucleus (5) further

translocation to mitochondria (6) causing release of cytochrome-c which can trigger (7)

apoptosis via caspase pathways. (Rybak et al., 2007). AG: aminoglycoside; Fe: iron;

ROS: reactive oxygen species; Cyt c: cytochrome c; JNK: c-Jun N-terminal kinase

26

Several studies have shown that the extrinsic signaling pathways also contribute to

apoptosis in the inner ear. Fas/FasL signaling has been shown to be involved in

gentamicin-induced ototoxicity (Bae et al., 2008; Jeong et al., 2010), and following

induction of labyrinthitis (Bodmer et al., 2003). It has been ealier documented that

following aminoglycoside-induced hair cell damage, a progressive loss of SGC occurs as

a result of reduced hair-cell derived neurotrophic support (Dodson et al., 2000).

Withdrawal of neurotrophic support induces ROS production in the SGC culture (Huang

et al., 2000). According to a recent study, the transcription factor, nuclear factor-κB is

believed to mediate protection against kanamycin-induced ototoxicity (Jiang et al., 2005).

Fig. 7: Schematic representation of apoptosis involing the intrinsic and extrinsic

signaling pathways resulting in caspase activation. FADD: Fas-Associated protein with

Death Domain; (Mak et al., 2002).

Extrinsic pathway

Intrinsic pathway

Extrinsic pathway

Intrinsic pathway

27

1.5 Neurotrophic Factors and their receptors

Neurotrophic factors are endogenous proteins which affect development, homeostasis,

survival and regeneration of neuronal as well as non-neuronal tissues (Sariola et al.,

2003). They are soluble homodimeric proteins with molecular weights between 13 and 24

kDa (McDonald et al., 1991; Holland et al., 1994). Neurotrophic factors play an

important role in complex behaviors like depression (Vaidya et al., 2001) and learning

(Mangina et al., 2006). In relation to previous studies, Neurotrophic factors have been

predicted to be involved in establishing neuronal connections in the developing brain

(Thoenen, 1995). Chronic intrinsic deprivation of these factors eventually leads to

apoptosis and death of specific populations of neurons in the adult brain. Neurotrophic

factors are classified into four categories: the neurotrophin super family; glial cell line-

derived neurotrophic factor (GDNF) family; neuropoietic cytokines and non-neuronal

growth factor super-family (Siegel et al., 2000).

Neurotrophin superfamily comprises the nerve growth factor (NGF), brain-derived

neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5)

(Bothwell, 1995; Reichardt et al., 2006). Neurotrophins interact via two receptors:

tropomysin-related kinases (TrkA, TrkB and TrkC) and p75NTR

(Reichardt et al., 2006).

NGF binds to TrkA, BDNF and NT-4/5 interacts through TrkB receptor, and NT-3

activates TrkC receptor (Patapoutian et al., 2001) (Figure 7). All the four neurotrophins

bind to the p75NTR

receptor (Dechant et al., 2002). NGF was the first neurotrophin to be

discovered and was characterized by Rita-Levi-Montalcini and colleagues, when they

demonstrated that NGF promoted neurite outgrowth of sympathetic neurons in chicken

(Levi-Montalcini et al., 1951). BDNF was discovered in the year 1982 through studies

performed by Barde and his colleagues (Barde et al., 1982), who showed that they are

structurally related to NGF. BDNF and NT-3 are known to be crucial for the cochlear

development and SGC survival in the inner ear. These neurotrophins are localized within

the organ of Corti, and their receptors are shown to be expressed by the SGC (Ernfors et

al., 1992; Pirvola et al., 1992; Ylikoski et al., 1993; Schecterson et al., 1994; Wheeler et

al., 1994). Studies have shown that mice lacking the gene NT-3 and BDNF, demonstrated

a significant reduction in the number of SGC (Farinas et al., 1994; Ernfors et al., 1995),

suggesting their importance as trophic factors during cochlear development. Also, in

28

animal models of deafness, neurotrophins BDNF and NT-3 provide protection against

ototoxic agents (Zheng et al., 1996). Neurotrophin-4/5 is the recently discovered

member of the neurotrophin family. They are responsible for inducing SGC survival in

early postnatal rats with the survival enhancing capacity reported to be similar to BDNF,

but stronger than NT-3 (Zheng et al., 1995).

GDNF family belongs to the transforming growth factor-β (TGF-β) and comprises of

GDNF (Lin et al., 1993), neurturin (NRTN) (Kotzbauer et al., 1996), persephin (PSPN)

(Milbrandt et al., 1998) and artemin (Baloh et al., 1998), which are functionally and

structurally related. GDNF is an important survival factor of motor neurons (Henderson

et al., 1994), neurons of the central nervous system (CNS) and peripheral nervous system

(PNS). Studies have determined the neurotrophic support of GDNF in neurite outgrowth,

cranial nerve and spinal cord motor neurons, basal forebrain cholinergic neurons,

purkinje cells and in dorsal ganglion and sympathetic neurons (Arenas et al., 1995; Huber

et al., 1995; Lapchak et al., 1996; Lin et al., 1993). In the inner ear, studies have

indicated that the IHC of neonatal and mature rat cochlea are responsible for GDNF and

its receptor synthesis (Ylikoski et al., 1998). GDNF gene therapy in combination with

electrical stimulation was shown to promote greater SGC survivility than either treatment

alone (Kanzaki et al., 2002), whereas overexpression of GDNF in the inner ear protected

the hair cells against degeneration induced by aminglycoside ototoxicty (Kawamoto et

al., 2003). Studies have indicated that NRTN, which promoted survival of sympathetic

and sensory neurons and the dorsal root ganglia, activated mitogen activated protein

(MAP) kinase signaling in cultured sympathetic neurons (Kotzbauer et al., 1996). PSPN

was first isolated and characterized by Milbrandt, and was shown to promote motor

neurons survival in-vitro and in-vivo after sciatic nerve axotomy (Milbrandt et al., 1998).

Gene expression pattern of neurturin, persephin and artemin has been previously well

documented in the inner ear tissues of normal hearing animals, suggesting their

functional importance and interactions between sensory cells and the auditory system

(Stoever et al., 2000; 2001).

The GDNF family ligands (GLFs) binds a GDNF-family receptor-α (GFRα) coupled to

the plasma membrane through glyocoslyphosphatidyl inositol (GPI) anchored protein

(Airaksinen et al., 2002). Each GFRα receptor binds specifically to one of the four

29

ligands and induces ligand specificity: GDNF-GFRα-1, NRTN-GFRα-2, ART-GFRα-3,

and PSPN-GFRα-4. GDNF signaling is mediated through a receptor complex consisting

of GFRα-1 and Ret. GFRα-1 is a high-affinity glyocoslyphosphatidyl inositol (GPI)

anchored protein, whereas Ret is an associated low-affinity transmembrane protein.

Binding of GDNF to its receptor GFRα-1 activates the Ret receptor tyrosine kinase which

triggers autophosphorylation of the tyrosine residues and initiates a number of

intracellular downstream signaling proteins (Figure 8). The phosphotyrosine residues

further activates various signaling pathways including the RAS/ERK, PI3K/AKT, MAPK

and JNK (Takahashi, 2001), responsible for a variety of cell process involving cell

survival, proliferation and neuronal differentiation.

Fig. 8: Neurotrophin receptors. Neurotrophin members bind specifically to Trk

receptors. The p75NTR

receptor binds to all members of the neurotrophins. NTN:

neurotrophin; BDNF: brain-derived neurotrophic factor; NGF: nerve growth factor.

(Siegel et al., 2000).

The neurotrophic cytokine family comprises of ciliary neurotrophic factor (CNTF),

leukemia inhibitory factor (LIF), interleukin-6 (IL-6) and cardiotrophin-1 (CT-1)

(Sleeman et al., 2000). The non-neuronal growth factor constitutes the acidic fibroblast

growth factor (a-FGF), basic fibroblast growth factor (b-FGF), epidermal growth factors

30

(EGF) and insulin-like growth factor (IGF), which are important for regulating neuronal

growth and survival of neuritis (Pincus et al., 1998).

Fig. 9: GDNF receptor signaling cascade: A. GDNF dimmer interacts with two GFRα-

1 molecules forming a complex which binds to Ret following dimerization. B.

Dimerization of Ret leads to autophosphorylation of tyrosine residues of the two subunits

and leads to activation of Ret.

Modified from: (http://ethesis.helsinki.fi/julkaisut/laa/biola/vk/wartiovaara/review.html)

1.5.1 The Neurotrophic factor hypothesis

In the developing PNS, several neurons are committed to undergo death after their axons

reach their target field. Developing neurons depend on neurotrophic factors released by

target tissues for survival and more than 50% of the neurons die during development

following programmed cell death/apoptosis (Huang et al., 2001; Ginty et al., 2002). With

the discovery of NGF in 1951, came the first molecular realization of the neurotrophic

factor concept. Earlier studies based on neuron-target interactions, naturally occurring

cell death and the discovery of NGF, led to the concept that neurons compete with each

other for trophic support supplied in limited amounts (Barde, 1988; Oppenheim, 1989).

Such limited availability of trophic support is thought to match the number of neurons to

31

the size and requirements of their target field because manipulating the target field size

before innervation affects the number of neurons that survive (Davies et al., 1996).

Earlier, a large body of evidence reported that the retrograde transport of NGF/receptor

complex was necessary for the regulation of trophic support to the cell body. One of such

evidence supports the fact that changes in the neurotrophin levels in the target during

development correlates with changes in neuronal survival (Anderson et al., 1995).

Another body of evidence demonstrated that by severing the axons connecting the

ganglions cells (Yawo, 1987) or by the administration of colchicine (Purves, 1976),

neuronal death occurred following loss of neurotrophic support. Later, further

investigation regarding neurotrophins and their trophic roles led to the hypothesis that

they specifically bind to high-affinity receptors of the tyrosine kinase family which

induces a cascade of intracellular events resulting in long-term cellular responses

(Barbacid, 1994), and the retrograde mode of transport for the expression of trophic

properties may not necessarily be involved. Such experimental manipulations and

observations resulted in the modified version of the neurotrophic factor hypothesis. As

suggested by Mattson, 1998, deafferentiation of neurons induces a loss of trophic factor

leading to the formation of free radicals and up-regulation of cell death cascades either

through apoptosis or necrosis. Consistent with this hypothesis, numerous studies have

demonstrated that neurotrophic factors are essential for neuronal survival in the inner ear.

BDNF and NT-3 are secreted by the cochlear hair cells and are important for the

development and survival of SGC (Zheng et al., 1995; Marzella et al., 1999; Farinas et

al., 2001; Rubel et al., 2002). In vivo studies performed on BDNF prevented SGC

degeneration in ototoxically deafened guinea pigs following two-weeks (Miller et al.,

1997), four-weeks (Gillespie et al., 2003), and eight-weeks of treatment (Staecker et al.,

1996). In addition, GDNF was shown to be crucial for neuronal and SGC integrity and

survival (Wissel et al., 2008; Scheper et al., 2009), and helped to prevent SGC

degeneration following deafening, via local application (Fransson et al., 2010) and

through mini-osmotic pumps (Ylikoski et al., 1998). Scheper and colleagues showed that

SGC density was increased significantly following delayed treatment of GDNF/electrical

stimulation in deafened guinea pigs. Similar studies also demonstrated that direct infusion

of BDNF or NT-3 into the scala tympani resulted in profuse outgrowth of neurites in

32

guinea pigs which also prevented SGC degeneration from aminoglycoside toxicity

(Ernfors et al., 1996; Staecker et al., 1996). Studies have also shown that gene transfer

via (adeno-associated virus) AAV-BDNF, (herpes simplex-1 vector) HSV-BDNF,

adenovirus-BDNF and adenovirus-GDNF vectors promoted survival of SGC in different

animal models of aminoglycoside ototoxicity (Staecker et al., 1998; Yagi et al., 2000;

Lalwani et al., 2002; Nakaizumi et al., 2004). Later investigations indicated that

neurotrophic factors plays an important role in regulating development, maintenance and

survival of SGC in the inner ear, further supporting the concept of neurotrophic factor

hypothesis as proposed by Mattson. Although a large number of basic and clinical

researches support the concept of neurotrophic factor hypothesis; studies have also

implicated contradictory results with respect to neurotrophic hypothesis. It was reported

that an abnormally accelerated degeneration of SGC resulted following interruption of

BDNF treatment (Gillespie et al., 2003; Shepherd et al., 2008). Gillespie showed that the

number of SGC was not significantly distinct from that in deafened, untreated cochlea

following cessation of treatment, and Shepherd determined a slow rate of SGC

degeneration following chronic electrical stimulation. It was recently shown that GDNF,

artemin, BDNF and transforming growth factor beta 1 and 2 (TGF1/2) were

significantly upregulated in the rat cochlea 26 days following aminoglycoside ototoxicity

(Wissel et al., 2006a; Wissel et al., 2006b), suggesting a deprivation-induced

upregulation of these endogenous trophic factors which might be responsible for

activating certain cellular mechanism in the cell bodies required for survival and

protection. Despite a large number of research work explaining the critical role of NTF in

regulation of neuronal survival and function exist, the diversity of functions regulated by

neurotrophic factors (including neuronal migration, neurite outgrowth, axon guidance and

synaptic plasticity) suggests that considerable revisions are required for the neurotrophic

factor hypothesis to account for the extreme versatility and beauty of neurotrophic factors

action.

33

1.6 Aim of the study

As mentioned previously, degeneration of SGC is followed by loss of neurotrophic

support which leads to cell death via apoptosis or necrosis. This hypothesis has been

supported by several studies demonstrating the role of neurotrophic factors in survival

and providing trophic support to SGC from noise and drug-induced deafness. Recently,

Wissel and colleagues have shown that mRNA transcription of the GDNF family

member artemin and GDNF, as well as BDNF and transforming growth factor beta 1

and 2 (TGF1/2), were significantly upregulated in the rat cochlea following 26 day of

neomycin-induced deafness. However, despite the progress in characterization of the

functions of neurotrophic factors in the inner ear, their specific contributions to

protection, recovery and apoptosis and how they are affected by interactions between

hair cells, supporting cells and spiral ganglion cells (SGC) are yet to be elucidated. In

the present study, we tested this hypothesis that these factors paly a significant role in

SGC degeneration in the context of apoptotic mechanism by gene expression and protein

expression analysis at different time intervals following deafening in the rat cochlea.

The aim of this study is:

1) To determine the extent of SGC degeneration induced by neomycin at 7, 14 and 28

days by morphometry.

2) To study whether the expression of mRNAs for the GDNF, BDNF, and their

receptors, GFRα-1, TrkB, p75NTR

, proapoptotic bax, anti-apoptotic bcl2, and caspase 9,

3 are changed in the normal and deafened rat cochlea at day 7, 14 and 28 following

deafening.

3) To determine the cellular localization of GDNF, BDNF, GFRα-1, TrkB, p75NTR

,

GFRα-1, bax, bcl2, caspase 9, 3 by immunohistochemsitry in the normal and deafened

cochleae.

34

Materials and Methods

A list of the solutions, regaents and chemicals, pharmaceuticals, laboratory equipments

including consumption of materials and programs and softwares used in this study is

given in a tabular form in the appendix I.

2.1 Neomycin induced deafness

2.1.1 Housing of animals

Housing conditions and all in vivo procedures were performed in accordance with the

regulations for care and use of laboratory animals at the Central Animal Laboratory,

Hannover Medical School, Hannover, Germany and was approved by the responsible

committee of the regional government, LAVES, approval number 07/1267. The animals

had free access to food and drinking water and were placed on a 12h light/dark cycle. All

animals were kept for one week to adapt to the housing conditions before treatment.

2.1.2 Animal model of deafness

Sprague-dawley rats (n = 105), irrespective of their sex weighing between 230-370g,

purchased from Charles river, Germany were included into this study. According to

table.1, animals were divided into three groups: group 7 (n = 30), 14 (n = 30), 28 (n = 30)

referring to 7, 14 and 28 days of deafness respectively. Normal hearing group (NH; n =

15) represented animals without any further treatment. At time point zero (considered as

day one), rats were anesthetized by intra-peritoneal injection of ketamine hydrochloride

(80 mg/kg) and xylazine (10 mg/kg). Subsequently, the hearing thresholds of all animals

were measured by acoustically evoked auditory brainstem response (AABR) to confirm

normal hearing. NH animal group was included to compare the effect of neomycin on the

deafened and corresponding contralateral ears. The animals to be deafened were

unilaterally injected with 10% neomycin sulfate via cochleostomy into the scala tympani.

The hearing status was monitored by AABR at 7, 14 and 28 days, respectively, to

confirm deafness (Table 1). Only those animals were considered for further experiments

35

which showed a threshold shift of 40 dB SPL or more in the deafened ears compared to

the normal hearing thresholds.

Table 1: Overview of the experimental groups and their designation in the thesis, the

number of animals used for each group and the time course of deafening and sacrifice.

NH: Normal hearing animals; AABR: acoustically evoked auditory brainstem response

2.1.3 Acoustically evoked auditory brainstem response (AABR)

For the measurement of frequency specific AABR, first described by Jewett and

Williston, 1971, Tucker-Davis Technologies (TDT) system was used. Prior to

measurement, the rats were anesthetized with intraperitoneal (i.p.) injection of a mixture

of ketamine hydrochloride (80 mg/kg) and xylazine (10 mg/kg). Analgesia was evoked

by injecting 5 mg/kg carprofen. The anesthetized animals were placed on a heating pad in

a soundproof room. The acoustic stimuli were delivered through electrostatic speakers

and calibrated earphones prepared from 200 µl pipette tips. The electric potentials were

acquired by sub-dermal electrodes placed such that the positive pole was at the vertex;

Animal

group

Number

of

animals

Time course of deafening

Day 0 Day 7 Day 14 Day 28

Group 7 N = 30 AABR,

neomycin

injection

AABR

Sacrifice


neomycin

injection

AABR

Sacrifice


neomycin

injection

AABR

Sacrifice

NH N = 15 AABR

followed by

sacrifice

36

the negative pole was on the left and right mastoid, and the ground electrode in the neck

of the rats. The signals were relayed via a preamplifier and a fiber-optic cable to the base

station. The various hardware components of the measuring units were controlled by a

computer connected to the Tucker Davies Technologies (TDT) system and application-

specific software HughPhonics (Lim and Anderson, 2006). It was necessary to perform a

preliminary test by means of broadband noise allowing the determination of the degree of

artifact suppression, which was a function of the ambient noise signals at 100-500 micro

volts depending on the surrounding background noise. Corresponding to the hearing

status of the animal, the dB levels were selected for the measurement of AABR, every 10

dB steps. The following frequencies were considered as standard for measurement: 1

kHz, 4 kHz, 8 kHz, 16 kHz, 32 kHz and 40 kHz. The determination of the cutoff

frequencies of the band pass filter was performed with a high pass of 300 Hz and a low

pass of 3000Hz, to suppress the inclusion of background frequencies. The generated

stimuli indicated duration of 10 ms with a square cosine rise and fall time of 1msec. The

recorded neurological signals from the animals were digitized and averaged at 200-250

cycles per stimulation. Created via the TDT system and HughPhonics software, the raw

data were converted and analyzed with custom written MATLAB® software. These

values were collected for all frequencies and averaged within an animal group for each

measurement day and the standard deviation was determined. This was compared for

every individual frequency from both the ears and with a shift in hearing threshold

following day 7, 14 and 28 of deafening. The differences in dB levels between the left

and right ear of each group were also evaluated.

2.1.4 Deafening procedure and surgery

Following AABR evaluation, additional half doses of ketamin hydrochloride and

xylazine were given on observation of pedal reflexes. A retroauricular incision was made

on the left ear with a fine scalpel to expose the tympanic bullae and a hole was drilled

with a fine needle to get access to the scala tympani. The round window membrane was

identified and exposed by further clipping off the lateral wall of the bullae and cautering

the blood vessel. Under microscope view, a fine lancet was used to carefully incise the

round window membrane. A blunt needle connected to a 10 µl graduated Hamilton micro

37

syringe was used to inject 5 µl of 10% neomycin solution slowly for 5 min into the scala

tympani avoiding any air bubbles. The opened tympanic bullae were sealed with bone

cement and the wound was sutured in two layers. During surgery, the animal’s body

temperature was maintained at 37ºC using water circulated heating pad. No surgery was

performed in the corresponding contralateral ears. Following deafening, the animals were

kept under infrared light until they recovered from anesthesia. All animals received water

and food ad libitum. Antibiotic prophylaxis was performed by adding 1.87ml of a

combination of trimethoprime and sulfamethoxazole (Cotrim K-rathiopharm®) to 500 ml

of drinking water.

2.2 Cochlear histology and immunohistochemistry

2.2.1 Cochlea harvesting and paraffin embedding

SGC density (n = 5) and immunohistochemical analysis of protein expression (n = 5) in

deafened and normal hearing animals was achieved by harvesting cochleae and through

histology. First, the animals were transcardially perfused under deep anesthesia, the

skulls were exposed and opened and the temporal bones were harvested. Subsequently

the bullae were opened immediately under microscopic view and the cochleae were

cleared from bony tissues and muscles with the help of fine forceps. The following

manual dissection procedure of the cochleae was carried out in 0.1M phosphate-buffered

saline PBS, pH 7.45, followed by the transfer to 4% cold paraformaldehyde and fixed

overnight at 4ºC to maintain the integrity of the subcellular structures. After fixation, the

samples were rinsed three times in 0.1 M PBS, pH 7.45 for 5 min each in a shaker at

room temperature to remove the excess paraformaldehyde. The cochlea samples were

decalcified for two weeks in 20% ethylenediamine tetra-acetic acid (EDTA) dissolved in

0.1 M PBS, pH 7.45, at 37ºC and the fresh EDTA solution was changed every alternate

day. When the cochleae were soft enough for paraffin embedding, the decalcification was

stopped by washing three times in 0.1M PBS, pH 7.45, for 5 min each. Dehydration steps

were carried out for 2 h in 50%, 70%, 90% and 100% ethanol followed by incubation in

methlybenzoate overnight. Next day, the cochleae were immersed in molten paraffin for

38

8 h at 56ºC followed by the exchange of fresh paraffin and hardening at room

temperature. The paraffin-blocks were serially cut at 5 µm in a midmodiolar plane by

using a manually operated microtome.

2.2.2 Hematoxylin and eosin staining

For examination of the structural morphology of rat cochlea and the extent of

degeneration of SGC induced by neomcyin, the cochlear sections were stained with

hematoxylin and eosin (H&E). Hematoxylin is uptaken by cell nuclei appearing blue

colored spots whereas eosin stained the cytoplasm and other cellular structures

pink/orange. With such type of routine staining, the subcellular structures in a cell are

clearly identified under the view of a microscope. The routine H&E staining was

performed as follows: by rinsing twice in xylene for 5min each for deparaffinization of

the tissue sections. The slides were then rehydrated in ethanol series: 100%-90%-70%-

50%-30% and distilled water for 3 min each. The slides were dipped in hematoxylin for

45 sec, washed twice in distilled water for 3 min each and then immersed in eosin Y

solution for 45 sec. After washing twice for 3 min, the slides were dehydrated in ethanol

series: 30%-50%-70%-90%-100% for 2 min each. The slides were then dipped twice in

xylene for 2 min each for clearing the ethanol. The sections were mounted with an

appropriate mounting media, entellan and then coverslipped. The stained slides were

further used for SGC counting.

2.3 Spiral ganglion cell (SGC) density measurements

For quantitative determination of the SGC survival, n = 3 and n = 4 animals for NH,

group 7, 14 and 28 were included. Midmodiolar sections for the quantitative analysis of

SGC densities were considered as there are four profiles of Rosenthal’s canal (R1- 4),

Figure 9. The cochlear turns in rats were specified as R1-4 from basal to apical as

previously described by Song et al., 2008. We used a systematic random sampling for

SGC counts. The first midmodiolar section were randomly selected and every fifth

proximate section was chosen for cell counts, thus analyzing five sections each separated

with a distance of 25 µm (Scheper et al., 2009).

39

Fig. 10: Mid-modiolar cochlear section: A mid-modiolar hematoxylin-eosin stained

cross-section of the rat cochlea depicting four regions of the Rosenthal’s canal (R1-4)

used for SGC counts. R1: basal; R2: mid basal; R3: mid apical; R4: apical; OC: organ of

Corti, SGC: spiral ganglion cells, SV: scala vestibuli, SM: scala media, ST: scala

tympani. Scale bar = 100 µm.

SGC numbers were assessed in each of the four (R1-4) cross-sectional profiles of the

Rosenthal’s canal on each of the five sections of the cochleae. SGC were counted and

analyzed assuming that each cell has a distinct nucleus with a minimum nuclear

membrane diameter of 12 µm. The number of SGC was treated as a whole by counting of

both type I cells (95% of the total SGC population) and type II cells (5% of the total

SGC) in a random manner. The measurement and analysis were performed

microscopically at a magnification of 200x (Olympus CKX41). Images of the cell counts

were taken using a CCD color camera and processed by image analyzing software

(Version. 3.2). The cross-sectional area of each Rosenthal’s canal was determined and the

SGC density was calculated as number of cells/1000 µm².

40

2.4 Immunohistochemsitry

N=3 animals belonging to normal and deafened individuals from groups 7, 14 and 28,

respectively were considered for protein expression analysis. The animals were deeply

anesthetized and cochleae were harvested and processed as previously described.

The paraffin sections were rehydrated and dehydrated again in ethanol series as described

in subchapter 2.2.2. Following rehydration, each section was marked using PAP pen

marker to avoid mixing of antibody solutions from other sections on the slides. The

markings were air dried and the antigen retrievel was performed by treating the tissues

with 0.05% trypsin for 15 min at 37ºC inside a humidified chamber. Washing steps were

carried out three times for 5 min each in 0.1 M PBS, pH 7.45, in a rotary shaker.

Following antigen retrievel step, the slides were incubated with 5% normal goat serum

dissolved in 0.1 M PBS, pH 7.45 for 1 h at room temperature to block the binding of non-

specific antibodies. Paraffin slides were incubated with 100 µl of the following primary

polyclonal rabbit antibodies overnight at 4ºC in a humidified chamber: anti-GDNF

(1:100), -GFRα-1 (1:200), -BDNF (1:250), -p75NTR

(1:50), -TrkB (1:100), -bcl2 (1:100),

-Bax (1:100), -caspase 9 (1:50), -caspase 3 (1:50), respectively diluted 1.5% normal goat

sera in 0.1 M PBS, pH 7.45. The slides were washed three times in 0.1 M PBS, pH 7.45,

for 5 min at room temperature in a rotary shaker. Specific antigen antibody interactions

were detected by incubating the sections with 100 µl of cy3-conjugated goat anti-rabbit

secondary antibody IgG (H+L) diluted 1:200 in 0.1 M PBS, pH 7.45, for a period of 1 h

at room temperature in darkness followed by rinsing and drying. Negative control tissue

sections were prepared following replacement of primary antibodies by 0.1 M PBS, pH

7.45. Finally, the sections were mounted with 4’, 6-diamidino-2-phenylindole (DAPI) for

nuclear counterstaining. Images were visualized by fluorescence microscope (excitation

at 649 nm and emission at 670 nm) captured digitally and analyzed using the CCD

camera, Leica DFC 320 coupled with the analysis software, Leica QWinV3.

A semi-quantification scoring method was employed to access anti-GDNF, GFRα-1,

BDNF, p75NTR

, TrkB, bcl2, Bax, caspase 9, caspase 3 immunostaining in the contra and

deaf cochlea tissues: 0 = no fluorescence; 1 = weak, but specific fluorescence; 2 =

distinct, specific fluorescence; and 3 = severe specific fluorescence, U = non-specific

fluorescence in the cochlear tissues.

41

2.5 Gene expression analysis

2.5.1 Modioli preparation

N = 20 modioli from the groups 7, 14 and 28 respectively, and n = 20 modioli from the

NH group were included for gene expression profiling. The animals were subjected to

deep anesthesia followed by sacrifice and opening of the temporal bone as described

previously in subchapter 2.2.1. The surface of the tympanic bullae was ruptured with a

fine needle to expose the cochlea. All bony materials and muscles surrounding the

cochlea were removed. Further manual dissection of the cochleae was performed under

the microscopic view. The bony cochlear capsule was carefully opened with fine forceps

and the membranous labyrinth was exposed. First, the spiral ligament was gently

removed and the organ of Corti was slowly separated from the modiolus. Finally, the

entire modiolus was dissected manually in ice-cold RNase Later® solution and

subsequently stored at -80ºC for RNA isolation.

2.5.2 Total RNA isolation

RNA isolation was carried out using RNAase free conditions. Each groups representing

contralateral and deafened modioli including those of normal hearing animals (NH) were

further subdivided into tissue pools consisting of n=7, n=7 and n=6 individuals

illustrated in Table 2. Tissue pooling was performed to consider the biological variability

for statistical assessment. Total RNA was isolated using the silica gel matrix that

selectivey binds to nucleic acids (Micro-to-Midi Total RNA Purification system). Tissues

were disrupted and homogenized in 750 µl lysis buffer containing 1% β-mercaptoethanol,

guanidinium isothiocyanate and ceramic beads for 10-15 sec with a

microdismembranator Ionomix. The lysates were then transferred to a 2 ml reaction tube

and centrifuged at 12,000 x g for 2-4 min at room temperature to separate the solid pellets

from the supernatant. One volume of 70% ethanol was added to the RNA containing

supernatant and transferred to the RNeasy spin columns, mixed carefully and placed in a

2 ml collection tube and centrifuged at 12,000 x g for 15 sec, followed by washing of the

42

spin catridge with 700 µl of Wash Buffer I and centrifugation at 12,000 x g for 15 sec.

The washing step was continued by adding 500 µl of Wash Buffer II and centrifugation at

Table 2

MoC7.1, MoC7.2, MoC7.3, MoC14.1, MoC14.2, MoC14.3, MoC28.1, MoC28.2, MoC28.3 and MoD7.1,

MoD7.2, MoD7.3, MoD14.1, MoD14.2, MoD14.3, MoD28.1, MoD28.2, MoD28.3 refers to contralateral

and deaf tissue samples dissected from rats following 7, 14 and 28 days of deafening,

respectively. NH = normal hearing animals.

12,000 x g for 15 sec. The spin cartridge was centrifuged at 12,000 x g for 1 min to dry

the membrane finally. Total RNA was eluted twice by pipetting 30 µl of RNAase-free

water to the centre of the spin cartridge and incubating for 1 min followed by

centrifugation for 2 min at 12,000 x g. The RNA yield was measured by NanoDrop ND-

1000 spectrophotometer at an absorbance of 260 nm. An absorbance of one unit at 260

nm corresponded to 40 µg of RNA per ml (A260 = 1 = 40 µg/ml). RNase free water was

used as the reference. A ratio of 1.8-2.1 between the absorbance values at 260 nm and

280 nm (A260/A280) provided an estimation of RNA purity with respect to contaminants,

such as protein. RNA integrity was further determined by the bioanalyzer (Agilent 2100)

using RNA 6000 Pico Kit.

Description Modiolus tissue pools

Tissue pools from

contralateral ears

Tissue pools from

deafened ears

Both ears

Group 7 (n = 40) MoC7.1=7, MoC7.2=7,

MoC7.3=6

MoD7.1=7, MoD7.2=7,

MoD7.3=6

Group 14 (n = 40) MoC14.1=7,MoC14.2=7,

MoC14.3=6

MoD14.1=7,MoD14.2=7,

MoD14.3=6

Group 28 (n = 40) MoC28.1=7,MoC28.2=7,

MoC28.3=6

MoD28.1=7,MoD28.2=7,

MoD28.3=6

NH (n = 20) NH1=7, NH2=7,

NH3=6

43

2.5.3 RNA quality assessment

For the successful gene expression profiling, RNA of high quality and integrity is

essential (Winnepenninckx et al., 1995). Detection of the quality and quantity of 28S and

18S ribosomal RNA (rRNA) bands on traditional agarose gels is not effective because the

appearance of RNA bands can be affected by various parameters, like electrophoresis

conditions, amount of RNA loaded and the saturation of ethidium bromide fluorescence.

In order to overcome such issues, we preferred to use Agilent bioanalyzer 2100 which

uses a combination of microfluids, capillary electrophoresis, and fluorescence to analyze

both RNA concentration and integrity. Moreover, Agilent bioanalyzer requires small

inputs, which conveniently allows assay of RNA quality in limiting samples. The

bioanalyzer uses the principle of capillary electrophoresis to move the sample through a

gel matrix. RNA samples from deaf and normal individuals were run with RNA 6000

Pico LabChip kits on Agilent 2100 bioanalyzer as per the manufacturer’s instruction.

RNA samples were denatured for 2 min at 70ºC and cooled down on ice. Charged

biomolecules were electrophoretically driven across a voltage gradient. Because of a

constant mass-to-charge ratio and the presence of a polymer matrix, the molecules are

separated by mass. The smaller fragments migrate faster than the larger ones. RNA 6000

Pico Ladder was used as a reference for data analysis. The RNA Pico Ladder displayed

six RNA fragments ranging in size from 0.2 to 6.0 kb at a total concentration of 1 ng/µl.

Integrity of RNA was assessed by visualization of the intact 18S and 28S ribosomal RNA

bands. RNA quality was determined by the RNA Integrity Number (RIN, Agilent

Technologies) ranging from 1 to 10, with 1 referring to the most degraded and 10, the

most intact RNA (Schroeder et al., 2006). A RIN value more than 7 was considered to

have a good RNA quality and RIN value less than 7 was not considered for further

downstream gene expression analysis.

44

2.5.4 cDNA synthesis

Single stranded cDNA synthesis was performed according to manufacturer

recommendation using High Capacity cDNA Reverse Transcription Kit (Applied

Biosystems). Briefly, a 20 µl reaction mixture containing 200 ng of total cellular RNA, 1

µl of multiscribe reverse transcriptase (50 U/µl), 2 µl of 10x reverse transcriptase (RT)

buffer, 0.8 µl of 25X dNTP-Mix (100 mM), 2 µl 10X Random Primers, and 1 µl RNase

inhibitor (20 U/µl) was incubated at 25ºC for 10 min. Following denaturation step, the

reverse transcription of the single strands was annealed at 37ºC for 2 h. After cDNA

synthesis, the enzyme activity was terminated at 85ºC for 5 min followed by gradual

cooling down to 4ºC. The cDNA samples were stored at - 20ºC until further use prior to

real-time PCR.

2.5.5 Real-time PCR

2.5.5.1 Principle of real-time PCR

Classical PCR relies on the linear amplification and end-point detection of the target

using a strand specific primer. The factors affecting amplification efficiencies in classical

PCR include the efficiency of primer composition and concentrations, enzyme activity,

pH, cycle number and temperature. Since polymerase chain reaction results in a million

fold amplification, variations in any of the above factors will significantly affect the final

output. However, such issues are overcome by real-time PCR which is based on the

principle of polymerase chain reaction, where the amplified DNA is detected as the

reaction progress in real time. Such process involves either non-specific detection of

product using fluorescent dyes which intercalate with the double-stranded DNA, or

through sequence-specific DNA probes (taqman) consisting of oligonucleotides labeled

with fluorescent dyes that is detected by hybridization of the probe with its

complementary DNA. TaqMan® chemistry is based on measuring increase in

fluorescence. TaqMan® probe has a fluorescent reporter dye at the 5’ end and a quencher

at the 3’ end of the template. During the extension phase of the polymerization cycle, the

reporter dye is cleaved from the probe by the 5’ nuclease activity of the taq-DNA

45

polymerase. Following removal from the probe, the dye emits its characteristic

fluorescence, and the increase in fluorescence is proportional to the amount of amplicons.

2.5.5.2 Housekeeping genes

An internal standard is necessary for normalization of gene expression values and to

compensate variations in RNA isolation, cDNA synthesis, preparation of polymerase

chain reaction (PCR) assays and efficiency of the taq polymerase. In general,

housekeeping genes are widely used as internal standards due to their stable and

consecutive RNA expression independently of the cellular conditions in the tissues to be

examined. However, numerous studies have shown that the expression levels of

housekeeping genes in normal and tumor tissues fluctuated considerably (Schmid et al.,

2003; De Kok et al., 2005). Therefore, 16 frequently used housekeeping genes were

examined in the modiolus from deafened and normal hearing animals and their suitability

as internal standard throughout the experimental conditions were tested on the rat cochlea

using real-time PCR. The genes and their cellular functions are described in table 3. The

rat endogenous control plate consisted of 384 wells preloaded with taqman primers and

probes specific for the respective housekeeping genes. The plate contained eight sample-

loading ports, each connected by a micro channel to 48 miniature reaction wells (16 x 3)

for a total of eight samples per plate (Figure 11). 70 ng cDNA each from three different

tissue pools from each group was added to 50 µl of TaqMan® Universal PCR Master

Mix (2x) in a total volume of 100 µl, which was loaded into one of the eight sample ports

according to the manufacturer’s instructions. Following centrifugation at 1200 rpm for 2

min, the plate was sealed to prevent cross-contamination. Each plate was placed in the

sample block of an ABI Prism® 7900HT sequence detection system and the PCR was

run. The thermal cyclic conditons were as follows: 2 min at 50ºC for incubation, 10 min

at 95ºC (activation), denaturation at 95ºC for 15 sec and annealing and extension at 60ºC

for 1 min. The Sequence Detection Software v2.1 (SDS 2.1) measured the fluorescence

intensity of the reporter (FAM™) and the quencher dye (TAMRA™) and calculated the

increase in normalized reporter emission intensity over the amplification phase.

Following PCR, the number of PCR cycles to reach the fluorescence threshold in each

sample was specified as the cycle threshold (Cт), which is proportional to the negative

46

logarithm of the initial amount of cDNA input. Cт values of 16 houskeeping genes in one

tissue were directly related, since the input of cDNA was equal for each PCR reaction.

For statistical verification, three PCR assays were run.

Fig. 11: TaqMan low-density array: An ideograph presenting 384 well rat endogenous

control plate predesigned with specific primers and probes (taqman assays). The left

panel of the diagram shows eight fill ports where the tissue samples are loaded for further

gene expression analysis.

(Modified from: http://www.invitrogen.com/site/us/en/home/References/Ambion-Tech-

Support/microrna-studies/tech-notes/global-mirna-profiling-higher-sensitivity-less-

sample.html)

47

Table 3: Selected housekeeping genes for gene expression analysis

Gene symbol Gene name Gene assay ID Cellular function

Actb Actin β Rn00667869_m1 Cytoskeletal structural protein

Arbp Acidicribosomal phosphoprotein Rn00821065_g1

Ribosomal biogenesis and assembly

B2m β-2-microglobulin Rn00560865_m1

Beta-chain of major histocompatibility complex class I molecules

18S Ribosomal RNA 18S Hs99999901_s1 Ribosome subunit

Gapdh Glyceraldehyde-3-phosphate dehydrogenase Rn99999916_s1 Glycolysis, transcription activation

Gusb Beta glucurodinase Rn00566655_m1 Cell division

Ppia Peptidylprolyl isomerase A Rn00690933_m1 Protein folding

Ppib Peptidylprolyl isomerase B Rn00574762_m1 Cell adhesion, cell migration

Hprt Hypoxanthine guanine phosphoribosyl transferase Rn01527840_m1 Purine metabolism

Tbp TATA-box binding protein Rn01455648_m1 Transcriptional initiation

Hmbs Hydroxymethylbilane synthase Rn00565886_m1

Heme synthesis, porphyrin metabolism

Ywhaz Phospholipase A2 Rn00755072_m1

Catalytically hydrolyses arachidonic aicd and lysophospholipids

Pgk1 Phosphoglycerate kinase 1 Rn00821429_g1 Key enzyme in glycolysis

Rplp2 Ribosomal protein, large P2 Rn01479927_g1 Protein synthesis

Tfrc Tansferrin receptor Rn01474695_m1 Cellular uptake of iron

Ubc Ubiquitin C Rn01789812_g1 Protein degradation

48

2.5.5.3 Real-time PCR assay

Inventoried TaqMan assays (custom made) consisted 20X mix of PCR primers (forward

and reverse) and TaqMan® MGB (minor groove-binder) probes (FAM™ dye labeled).

TaqMan® MGB probes consisted of a fluorescent reporter dye (FAM™) at the 5’ end

and a quencher (TAMRA™) at the 3’ end. Amplification of cDNA from contralateral,

deafened and NH groups was performed using TaqMan® Universal PCR Master Mix

(2X), 20X TaqMan Gene Expression Assay mix and 10 ng cDNA in a total volume of 20

µl per reaction well. The conditions of each PCR cycle was performed on a

StepOnePlus® real-time PCR system at 50ºC for 15 min and 95ºC for 10 min followed

by 40 cycles at 95ºC for 15 sec and 60ºC for 1 min. The amplified products were detected

by monitoring the fluorescence activity of the reporter dye throughout the PCR cycle.

The gene expression results were calculated using the relative quantification method (2-

ΔΔCт), where the cDNA achieved from NH animals was denoted as the calibrator (Livak et

al., 2001). The calibrator value derived from the equation (2-0

) was equivalent to 1, and

everyother sample was expressed relative to this. The ΔCт corresponded to threshold

cycle of the target minus that of housekeeping gene, and ΔΔCт represented the ΔCт of

each target minus the calibrator. For calibrator, i.e reference RNA representing NH

animals, the equation corresponding to relative quantification (2-0

), is equivalent to 1 and

everyother sample is expressed relative to this. The assay IDs are: GDNF-

(Rn01402432_m1), GFRα-1-(Rn01640513_m1), BDNF-(Rn01484924_m1), TrkB-

(Rn01441745_m1), p75NTR

–(Rn01309635_m1), Bax-(Rn01480158_m1), bcl2-

(Rn00437783_m1), caspase 9-(Rn00668554_m1), and caspase 3-(Rn00563902_m1),

where “m1” suffix in the above context suggests that the assay spans an exon junction,

and would not be impacted by genomic DNA contamination. For statistical verification,

three PCR assays were run.

49

2.6 Statistical Analysis

Statistical analysis of data was performed using GraphPad Prism 5. A factorial design

two-way ANOVA was estimated for correlating the differences in the SGC density,

AABR thresholds and differential gene expression results in the deaf, contralateral and

NH groups following 7, 14 and 28 days of neomycin treatment. Data from AABR

thresholds, SGC density and gene expression analysis were expressed as mean ± standard

error of mean (SEM). A paired-sample t-test was used to evaluate the statistical

significance of differences between the deaf and the contralateral cochlea for each group.

A difference was considered statistically significant at *p < 0.05; **p < 0.01; ***p <

0.001.

50

Results

3.1 Unilateral deafening by neomycin resulted in profound hearing

loss

To confirm deafness due to neomycin, hearing thresholds were evaluated in the animals

prior to and 7, 14 and 28 days following deafening. The animals were divided into four

groups as described earlier in section 2.1.2. Normal hearing (NH) animals without

undergoing any treatment demonstrated a baseline threshold level less than equal to 40

dB SPL, and deafened groups exhibited a threshold shift greater than equal to 40-60 dB

SPL from the normal hearing range. All experimental animals receiving unilateral

neomycin injections demonstrated profound unilateral hearing loss in the left (deafened)

ear, while no significant changes in the threshold level was observed in the contralateral

ears following deafening. AABR recordings from both ears of normal hearing rats (NH, n = 10) at various test

frequencies were averaged to determine the normal hearing thresholds demonstrated

below.

Table 4: Summary of the mean AABR threshold values from both ears of normal hearing

animal (NH) at 1, 4, 8, 16, 32 and 40 kHz test frequencies

As displayed from the table above, normal hearing rats reached a baseline hearing

threshold at 40 ± 5.48 dB SPL at 8 kHz cochlear frequency range. The frequency at 8

kHz were preferred for AABR analysis, since the animals maximal hearing sensitivity

was found within the range of 8-32 kHz. In addition, the test frequencies at 1, 4, 16, 32

and 40 kHz were also included for comparison.

Auditory threshold shift at test frequencies 1, 4, 8, 16, 32 and 40 kHz following 7 (n = 7),

14 (n = 10) and 28 (n = 10) days prior to and after deafening are demonstrated in Figure.

12 (A, B, C and D). As shown in Figure. 12 (A-D), y-coordinate represents sound

intensities in decibels (dB) at 10 dB intervals and x-coordinate represent different test

Frequencies 1 kHz 4 kHz 8 kHz 16 kHz 32 kHz 40 kHz

dB SPL 73.15±7.29 62.5 ±8.87 40 ± 5.48 61 ± 8.31 58 ± 7.48 67± 8.43

51

frequencies (kHz). AABR measurements in Figure 12A demonstrated a slight but not

significant increase of 5 dB, 15 dB and 10 dB hearing threshold in the contralateral ears

for group 7 [45 dB SPL (± 5.47)], 14 [55 dB SPL (± 11.78)] and 28 [50 dB SPL (± 5)]

when compared to the NH group (40 ± 5.48 dB SPL) following deafening. Following 7

days of deafening, a significant threshold increase of 57.17 dB was observed in the

deafened ears [(98.83 dB SPL (± 2.04)] when compared to the animals prior to deafening

(day 0) [41.66 dB SPL (± 17.22)] (p < 0.001), Figure 12B. In addition, a significant

threshold increase of 55 and 57.62 dB SPL was found in the deafened ears for group 14

[94 dB SPL (± 5.47)] and 28 [93.33 dB SPL (± 11.54)] when compared to their

respective day 0 ears prior to deafening (p < 0.001), Figure 12 C and D. No significant

changes were observed in the contralateral sides at test frequencies 1, 4, 16, 32 and 40

kHz following 7, 14 and 28 days of deafening. The deafened ears demonstrated profound

threshold shifts at 98.83 dB SPL (± 2.04), 94 dB SPL (± 5.47) and 93.33 dB SPL (±

11.54) for group 7, 14 and 28, respectively when compared to NH group (Figure. 12).

An additional comparative analysis was performed by plotting bar graphs indicating the

difference in the threshold values at different time periods before and after deafening at 8

kHz cochlear frequency (Figure. 13). The difference between the threshold of normal

hearing (NH) [40 dB SPL (± 5.48)] animals and rats prior to deafening (day 0), for group

7 [45 dB SPL (±5.47)] was found to be not significant, which was also the same for

group 14 and 28 day 0 animals. The deafened ears from animals in group 7 [98.83 dB

SPL (± 11.54)], group 14 [94 dB SPL (± 5.47)] and group 28 [93.33 dB SPL (± 11.54)]

demonstrated a significant threshold shift of 58.83, 54 and 53.33 dB when compared to

the rats from the NH group [40 dB SPL (± 5.48)] (p < 0.001).

52

Fig.12: Mean AABR threshold data from group A: NH and contralateral ears post

deafening, B: 7 (n = 7), C: 14 (n = 10) and D: 28 (n = 10) days following unilateral

neomycin injection at test frequencies 1, 4, 8, 16, 32, and 40 kHz. AABR are shown for

day 0 before and 7, 14 and 28 days after deafening. The NH group (n = 10) have been

included for comparison with deafened and contralateral ears. Test frequency at 8 kHz

prior to deafening showed a normal hearing threshold near the range of 40 dB SPL (±

5.48) for NH group. No significant differences were observed at test frequencies 1, 4, 16,

32 and 40 kHz. NH: normal hearing; Day 0: AABR threshold prior to neomycin

deafening. Error bars represent standard error of mean (SEM).

53

Fig. 13: Comparative mean auditory threshold shifts at frequency 8 kHz prior to and

following neomycin treatment at day 7, 14 and 28. A significant increase in the threshold

values was observed in the deafened ears for group 7 (98.83 ± 2.04 dB SPL), 14 (94 ±

5.47 dB SPL) and 28 (93.33 ± 11.54 dB SPL), indicating profound hearing loss as a result

of neomycin injection. Significant changes are indicated by black (deafened versus day 0

animals) and red (deafened versus NH animals) asterisks (*p < 0.05, **p < 0.01, ***p <

0.001). Vertical bars represent standard deviation. NH = normal hearing; G7, G14 and

G28 = group 7, 14 and 28 respectively; d0 = animals prior to deafening.

We did not observe any significant changes in the hearing threshold of the contralateral

ears for group 7, 14 and 28 throughout the deafening period. Moreover, no significant

changes were noticed in the animals prior to deafening (day 0) corresponding to all the

time periods.

54

3.2 Significant reduction of spiral ganglion cell density in the

deafened cochleae 7, 14 and 28 days after ototoxic deafening

To confirm the spiral ganglion cell (SGC) degeneration at different time period following

deafening, histological images from normal hearing (NH), contralateral and deafened

cochleae were analyzed in the rat cochlea. Light microscopic examination demonstrated a

subsequent loss of SGC numbers and peripheral process in the contralateral and deafened

cochleae post-deafening compared to the normal hearing animals. Chracteristic features

in the deaf cochleae included shrinked somata, vacuolated cytoplasm and the presence of

widened space among the neurons when compared to spherical and intact cell bodies in

the normal cochleae (Figure 14). Mid-modioar sections from deafened cochleae were

compared to the NH and contralateral sides for group 7, 14 and 28 (Figure 15). NH (n =

3) animals without any treatment were introduced considering that unilateral deafening

induced a partial deafness in the contralateral side of the cochlea via the cochlear

aqueduct (Stoever et al., 2000). Every fifth serial section was considered for SGC counts.

SGC in each Rosenthal’s canal (R1-4) were counted in an arbitrary manner without

considering basal and apical turns.

Morphometrical estimation across the time period of deafening showed a significant

decrease of SGC density in the deafened cochleae from animals of the group 7 (1.55 ±

0.23, SGC/1000 µm², p < 0.05), 14 (1.01 ± 0.12, SGC/1000 µm², p < 0.01) and 28 (0.8 ±

0.04, SGC/1000 µm², p < 0.01) when compared to the NH (2.17 ± 0.2 SGC/1000 µm²),

(Figure 14 A, C, E and G). In addition, SGC loss was observed in the contralateral

cochleae from animals of the group 7 (2.07 ± 0.1, SGC/1000 µm²), 14 (1.88 ± 0.19,

SGC/1000 µm²) and 28 (1.69 ± 0.07, SGC/1000 µm, p < 0.05) in relation to NH

following deafening (Figure 14 A, B, D and F), respectively. A significant loss of SGC

was also noticed between contralateral and deaf cochleae from animals of the group 7

(2.07 ± 0.1 versus 1.55 ± 0.23, p < 0.05), 14 (1.88 ± 0.19 versus 1.01 ± 0.12, p < 0.01)

and 28 (1.69 ± 0.07 versus 0.8 ± 0.04), respectively. The average SGC loss among NH,

contralateral and deaf cochleae from animals of the group 7, 14 and 28 are illustrated in

Table 5. A comparative SGC density loss at 7, 14 and 28 days prior to and post deafening

SGNs

55

is shown in Figure 15, demonstrating a significant decrease in SGC number at all time

period of deafening.

Fig. 14: Representative mid modiolar cross sections from Rosenthal’s canal illustrating

SGC loss following deafening. A: SGC from NH animals with intact and closely packed

nucleus. B, C: Sections from a contralateral and deafened cochlea at day 7 after

deafening. D, E: Midmodiolar sections from 14 day contra and deafened cochlea with

subsequent loss in SGC number. F, G: Cochlear sections from 28 days contralateral and

deaf animal with a significant loss of SGC somata after deafening. Black and blue arrow

represents intact and shrinked SG somata. Scale bar=100 µm

SGNs

NH Contra day 7 Deaf day 7

Contra day 14 Deaf day 14

Deaf day 28Contra day 28

SGC

56

Table 5: Density of SGC loss following neomycin treatment

SGC = spiral ganglion cell; NH = normal hearing animals

Fig. 15: Mean density of SGC loss following unilateral neomycin induced deafening. A

significant reduction in SGC density was observed between deafened day 7, 14 and 28 (p

< 0.05) compared to NH. Each bar represents (n=4) animals used for statistical analysis.

Error bars represents standard deviation and significant changes are indicated by red

[(compared to NH) and black asterisks (compared between contra and deaf cochleae and

between time periods) (*p < 0.05, **p < 0.01, ***p < 0.001)]. NH = normal hearing; C

= contralateral and D = deafened cochleae

Days of

deafening

Groups SGC density of cells/1000 µm²

NH 2.17 ± 0.2

7 Contralateral 2.07 ± 0.1

Deafened 1.55 ± 0.23





57

3.3 Gene expression results

3.3.1 Quality assessment of total RNA

Modiolus constitutes a major portion of auditory nerve and SGC. In order to the study

gene expression profiling, a RNA quality control is essential. For the estimation of RNA

quality in the modiolus, quality control from Agilent bioanalyzer was performed Quality

of total RNA extracted from normal hearing (NH), contralateral and deafened modiolus

from group 7, 14 and 28 was performed by capillary electrophoresis using Agilent 2100

Bioanalyzer, described in section 2.5.3. The RNA intergrity numbers (RIN) from one

tissue pool representing group 7 (contralateral=MoC7.1=7, deafened= MoD7.1=7), 14

(contralateral=MoC14.1=7, deafened= MoD14.1=7), 28 (contralateral= MoC28.1=7, deafened=

MoD28.1=7) and NH (NH1=7) are represented in Figure 16. The RIN of all the tissue pools

from NH, group 7, 14 and 28 is shown in Table 6. RNA concentration was measured in

picograms per microlitre (pg/µl) and the integrity was analyzed based on RIN value. A

value of 1 represented a completely degraded RNA and 10 indicated an intact RNA

(Imbeaud et al., 2005). All modiolar samples from group 7, 14 and 28 yielded relatively

intact RNA with a RIN of approximately more than 7.5, and a distinct 18S and 28S

ribosomal peak. Representative group 7 contralateral modiolus shown here yielded the

highest RIN = 8.9, while group 14 contralateral modiolus yielded the lowest RIN = 7.5.

RIN for group 7 and 14 deafened modiolus was 8.7 and 7.9, respectively. RIN for group

28 contralateral, deafened and NH were found to be 7.7, 7.7 and 8.3, respectively. Total

RNA yielding RIN less than 7.5 were discarded from further analysis.

C

E

F

58

Table 6: RNA integrity values for rat modiolus

MoC7.1, MoC7.2, MoC7.3, MoC14.1, MoC14.2, MoC14.3, MoC28.1, MoC28.2, MoC28.3 and MoD7.1,

MoD7.2, MoD7.3, MoD14.1, MoD14.2, MoD14.3, MoD28.1, MoD28.2, MoD28.3 refers to contralateral

and deaf modiolar samples dissected from rats following 7, 14 and 28 days of deafening,

respectively. RIN = RNA integrity number; NH = normal hearing animals.

Fig. 16: Representative baseline RNA quality control for NH, contralateral and deafened

modiolar samples: Modiolar tissue was dissected from the fresh frozen tissue block and

total RNA was extracted. Using the RNA 6000 Pico LabChip kit and 2100 Bioanalyzer,

RNA quality was assessed. As shown in the electrophoregram below, RNA integrity

numbers was highest for contralateral group 7 (8.9) and lowest for contralateral group 14

modiolus (7.5). The 18S and 28S peaks could be clearly marked in the

electropherograms. 18S:18S bands; 28S: 28S bands; RIN: RNA integrity number.

Description

Tissue pools from

contralateral ears

RIN Tissue pools from

deafened ears

RIN

Group 7

(n = 40)

MoC7.1=7 8.9 MoD7.1=7 8.7

MoC7.2=7 9.2 MoD7.2=7 10

MoC7.3=6 9.9 MoD7.3=6 8.1

Group14

(n = 40)

MoC14.1=7 7.5 MoD14.1=7 7.9

MoC14.2=7 8 MoD14.2=7 8.4

MoC14.3=6 8.6 MoD14.3=6 7.8

Group 28

(n = 40)

MoC28.1=7 7.7 MoD28.1=7 7.7

MoC28.2=7 9.4 MoD28.2=7 8.7

MoC28.3=6 8 MoD28.3=6 7.9

NH (n = 20) NH1=7 8.3

NH2=7 7.8

NH3=6 8.1

59

Contralateral group 7 Deafened group 7


RNA Integrity number (RIN): 8.9 RNA Integrity number (RIN): 8.7



Normal hearing


RNA Integrity number (RIN): 8.3

60

3.3.2 RPLP2 as the housekeeping gene

To compensate variations in RNA isolation, cDNA synthesis, preparation of polymerase

chain reaction (PCR) assays and efficiency of the taq polymerase, 16 commonly used

housekeeping genes were examined in the modiolus from deafened and normal hearing

animals and their suitability as internal standard throughout the experimental conditions

were tested. Total RNA was extracted from NH, deafened and contralateral modioli,

reverse transcribed and quantified using real-time PCR. The average Cт was used to

determine the standard deviation of each gene across the different tissue samples listed in

Table 7.

Table 7: Mean Cт and std.dev.for selecting the most appropriate housekeeping gene for

semi-quantitative analysis. Cт average: threshold cycle; Std.dev: standard deviation

Cт average: threshold cycle; std.dev: standard deviation

Housekeeping Genes Cт average Std.dev

Actb 20.2 0.58

Arbp 22.54 0.61

B2m 20.67 1.24

18S 9.01 0.25

Gapdh 20.62 0.32

Gusb 27.59 0.84

Ppia 21.01 0.34

Ppib 23.29 0.5

Hprt 25.87 0.23

Tbp 27.86 0.23

Hmbs 26.99 0.36

Ywhaz 22.88 0.32

Pgk1 23.15 0.18

Rplp2 22.4 0.10

Tfrc 24.62 0.44

Ubc 24.24 0.5

61

The gene expression analysis with the lowest standard deviation of Cт, therefore the

lowest variation accross tissue samples were identified as, RPLP2 (22.40 ± 0.10), Pgk1

(23.15 ± 0.18), Hprt (25.87 ± 0.231) andTbp (27.86 ± 0.23), Figure. 17. By these

findings, RPLP2 demonstrated the lowest standard deviation and the most stably

expressed throughout the deafening period. RPLP2 plays an important role in the

elongation step of protein synthesis and is a member of the ribosomal protein L12P

family. Tbp and Gusb showed higher variability across samples and thus, had higher

standard deviation values (27.86 ± 0.23, 27.59 ± 0.84) across the modiolus from group 7,

14 and 28. 18S displayed the lowest threshold values (9.01 ± 0.25) throughout the

deafening period, suggesting their high abundancy of the total RNA in the modiolus of

the rat cochlea. Gapdh (20.62 ± 0.32) and Actb (20.2 ± 0.58), which are commonly used

as endogenous controls, demonstrated higher variability across modilar tissue samples.

Fig. 17: Comparative gene expression analysis of 16 endogenous controls A, B, C and D

across NH, contralateral and deafened modiolus following deafening using two-way

ANOVA. RPLP2 (22.40 ± 0.1) shown with a red circle was identified as the suitable

housekeeping gene based upon lowest standard deviation and most stable expression

across the tissue samples. Error bars indicate standard deviation. NH=normal hearing.

Actb Arbp B2m 18S0

5

10

15

20

25

30

NH

contralateral d7

deafened d7

contralateral d14

deafened d14

contralateral d28

deafened d28 A

Ct

av

era

ge

Actb Arbp B2m 18S0

5

10

15

20

25

30

NH

contralateral d7

deafened d7

contralateral d14

deafened d14

contralateral d28

deafened d28

A

Ct

av

era

ge

Gapdh Gusb Ppia Ppib0

5

10

15

20

25

30

B

Ct

av

era

ge

Hprt Tbp Hmbs Yw haz0

5

10

15

20

25

30

C

Ct

av

era

ge

Pgk1 Rplp2 Tfrc Ubc0

5

10

15

20

25

30

D

Ct

av

era

ge

Actb Arbp B2m 18s Gapdh Gusb Ppia Ppib

Hprt Tbp Hmbs Ywhaz Pgk1 Rplp2 Tfrc Ubc

62

3.3.3 Gene expression patterns of neurotrophic and apoptosis related genes

following deafening

To understand the role of neurotrophic factors in the survival of SGC following

neomycin-induced cell death, a part of our study was devoted to analyze the gene

expression changes of these growth factors at various time intervals in the normal and

deafened animals. Gene expression analysis was carried out on GDNF, GFRα-1, BDNF,

TrkB and p75NTR

, bcl2, bax, caspase 9 and 3 at 7, 14 and 28 days following neomycin-

induced deafness. The target genes were normalized with the housekeeping gene, RPLP2

across the modiolar samples.

As illustrated in Figure 18A, GDNF mRNA expression in the 28 day deafened modiolus

and contralateral sides demonstrated a 2.69 fold increase (p < 0.001). A slight but

significant increase was observed in the deafened groups between group 7 and 28 (2.14 ±

0.37 versus 3.69 ± 0.5) and between group 14 and 28 (2.04 ± 0.16 versus 3.69 ± 0.5, p <

0.01). No significant expression changes could be seen in the contralateral modiolus

throughout the deafening period. A comparative analysis demonstrated a significant

expression of GDNF in contralateral tissue samples for group 7 (p < 0.001), and in

deafened samples for group 7 (p < 0.01), group 14 (p < 0.001) and group 28 (p < 0.001)

following deafening.

No significant change in the GFRα-1 expression was found in the deafened group 7 with

respect to contralateral side (1.59 ± 0.08 versus 1.24 ± 0.21). A significant increase in the

mRNA activity of GFRα-1 was observed between deafened and contralateral modiolus

for group 14 (1.85 ± 0.11 versus 0.88 ± 0.05, p < 0.001) and group 28 (1.96 ± 0.04 versus

1.01 ± 0.08, p < 0.001). Transcription activity of GFRα-1 was also slightly increased

between group 7 and 28 in deafened modiolus (1.59 ± 0.08 versus 1.96 ± 0.04, p < 0.01).

No statistical significant changes in mRNA expression level were noticed between

deafened group 14 and 28 and in the contralateral modiolus between all the time periods.

The deafened modiolus for group 7, 14 and 28 had a significant increase (p < 0.001) in

the GFRα-1 mRNA level compared to the NH tissue samples (Figure. 18 B).

BDNF mRNA activity was significantly increased between the contralateral and deafened

modiolus following deafening for group 14 (2.13 ± 0.19 versus 2.69 ± 0.15, p < 0.01) and

28 (3.01 ± 0.34 versus 4.27 ± 0.45, p < 0.01). A high elevated BDNF expression could be

63

observed between the deafened group 7 and 28 (1.78 ± 0.09 versus 4.27 ± 0.45, p <

0.001) and between group 14 and 28 (2.69 ± 0.15 versus 4.27 ± 0.45, p = 0.05). We did

not observe any significant changes in the contralateral modiolus between the time

periods, although BDNF transcription activity was increased. Significant elevated levels

of expression was marked in the deafened modiolus for group 7 (p < 0.05), 14 (p < 0.01),

28 (p < 0.01) and in the contralateral for group 14 (p < 0.05) compared to NH group,

Figure. 18 C.

p75NTR

transcription activity was found to be upregulated between deafened and

contralateral modiolus for group 7 (1.5 ± 0.22 versus 2.26 ± 0.11, p < 0.01) and an slight

rise in the p75NTR

mRNA activity was also observed for group 14 (2.18 ± 0.44 versus

2.01 ± 0.16) and 28 (2.82 ± 0.45 versus 2.32 ± 0.16), Figure. 18 D. No significant

transcription activity for p75NTR

was observed in deafened and contralateral modiolus

throughout the deafening period. A significant effect was noticed between deafened

group 7 and 28 (p < 0.01). p75NTR

mRNA level was increased in the contralateral tissue

samples for group 7 (p < 0.001), 14 (p < 0.001), 28 (p < 0.001) and in deafened tissue

samples for group 7 (p < 0.05), 14 (p < 0.01), 28 (p < 0.01) in relation to NH groups.

Interestingly, TrkB, one of the receptor for BDNF, was downregulated in the deafened

modiolus for group 7, 14 and 28, Figure. 18 E. Followig 7 days of deafening, a robust

downregulation of TrkB was observed in the deafened modiolus compared to

contralateral side (0.51 ± 0.23 versus 1.06 ± 0.13, p < 0.01). TrkB was also seen to be

downregulated for group 14 (0.68 ± 0.22 versus 1.01 ± 0.02, p < 0.05) and significantly

for group 28 (0.77 ± 0.06 versus 1.12 ± 0.1, p < 0.05). No significant changes in the

mRNA expression level were noticed in the deafened and contralateral modiolus at

different time periods throughout the deafening period.

64

Fig. 18: Expression of growth factor mRNA in the deafened and contralateral modiolus

of rat cochleae following deafening. A-E) Graphs showing mRNA activity fold changes

of GDNF, GFRα-1, BDNF, p75NTR,

and TrkB expression in the modiolus of rat cochleae

following deafening compared to NH animals (calibrator) and normalized to the

housekeeping gene by ΔΔCт method. Significant changes are indicated by black (deaf

versus contralateral) and red (deaf and contralateral versus NH) asterisks (*p < 0.05, **p

< 0.01, ***p < 0.001).

Bcl2 gene expression was downregulated in the deafened modiolus throughout the

deafening period. Gene expression level of Bcl2 was significantly downregulated in the

deafened modiolus compared to the contralateral for group 7 (0.52 ± 0.02 versus 1.2 ±

0.12, p < 0.001) and 14 (0.66 ± 0.03 versus 1.19 ± 0.16, p < 0.001). Decreased mRNA

activity was also observed for group 28 between deaf and contralateral modiolus (0.62 ±

0.16 versus 0.89 ± 0.04), and was statistically insignificant. No significant change at the

mRNA level was observed in the contralateral side throughout the deafening time

65

periods. Bcl2 expression level in the deafened modiolus at for group 7 14 (p = 0.0001)

was significantly decreased compared to NH groups, Figure. 19 F.

Bax mRNA activity increased following day 7 (1.28 ± 0.04, p < 0.001) until 14 days

(1.63 ± 0.35) and was dramatically reduced following day 28 (1.18 ± 0.05) in the

deafened modiolar tissues compared to the NH groups. No significant expression pattern

in Bax mRNA gene was observed in the contralateral modiolus following 7, 14 and 28

days deafening. The gene expression level insignificantly increased between contralateral

and deafened modiolus for group 7 (1.02 ± 0.2 versus 1.28 ± 0.04) and significantly for

group 14 (1.04 ± 0.08 versus 1.63 ± 0.35, p < 0.05), but no such significant correlation

was observed for group 28 (1.15 ± 0.04 versus 1.18 ± 0.05) following deafening, Figure.

19 G.

No correlation in the differential gene expression level was observed for caspase 9 in the

contralateral tissue samples for all the groups studied. A slight mRNA activity in caspase

9 was observed between contralateral and deafened modiolus for group 7 (0.96 ± 0.1

versus 1.11 ± 0.11), and group 14 (0.94 ± 0.03 versus 1.23 ± 0.18), whereas caspase 9

mRNA level was significantly elevated between contralateral and deafened modiolus for

group 28 (1.01 ± 0.06 versus 1.56 ± 0.09, p < 0.001) following deafness. The difference

in the gene expression patterns between group 7 and 28 deafened modiolus (1.11 ± 0.11

versus 1.56 ± 0.09, p < 0.01) was found to be significant, Figure. 19 H.

Caspase 3 was significantly increased in the deafened modiolus for group 7, 14 and 28

following deafening. The increased expression was significant between group 7 and 28

(1.16 ± 0.11 versus and 2.26 ± 0.22, p < 0.001) and between group 14 and 28 (1.23 ±

0.06 versus 2.26 ± 0.22, p < 0.001) in the deafened modiolus. No significant mRNA

activity in the contralateral modiolus was noticed for different time periods. A slight

insignificant elevation in the caspase 3 mRNA activity was also observed between

contralateral and deafened modiolus for group 7 (0.65 ± 0.1 versus 1.16 ± 0.11) and

group 14 (0.86 ± 0.08 versus 1.23 ± 0.06), but the elevated expression level was

significant for group 28 (0.51 ± 0.2 versus 2.26 ± 0.22, p < 0.001) following deafening.

We also observed a significant increase (p = 0.02) in caspase 3 activity in the deafened

modiolus for group 28 compared to NH groups, Figure. 19 I.

66

Fig. 19: Expression of apoptosis related molecules mRNA in the deafened and

contralateral modiolus of rat cochleae following deafening, F-I: Graphs showing mRNA

activity fold changes of bcl2, bax, caspase 9 and caspase 3 expression in the modiolus of

rat cochleae following deafening compared to NH animals (calibrator) and normalized to

housekeeping gene by the ΔΔCт method. Significant changes are indicated by black (deaf

versus contralateral) and red (deaf and contralateral versus NH) asterisks (*p < 0.05, **p

< 0.01, ***p < 0.001).

67

3.4 Immunohistochemistry

3.4.1 Immunohistochemical expression and distribution of NTF in the

cochlear tissues

Immunohistochemistry was performed to semi-quantify and localize GDNF, GFRα-1,

BDNF, trkB and p75NTR

in the SGC for group 7, 14 and 28 following deafening. GDNF,

a secretory protein was found to be localized in the SGC of both deafened and

contralateral cochleae for group 7, 14 and 28. Negative control sections incubated

without the primary antibody demonstrated no immunostaining. No specific change in

expression pattern was detected in all the groups throughout the time periods, except that

GDNF for group 7 and 28 deafened cochlea showed intense immunostaining. Although

GFRα-1 was found to be localized in the cell membrane of SGC, we could not detect any

change in the expression pattern for all the groups following deafening. A slight

immunoreactivity was detected in the neuronal fibres surrounding the SGC. No staining

was observed in the negative control section where the anti-GFRα-1 was substituted with

PBS. BDNF, a secretory protein showed a strong immunostaining in the SGC of the

Rosenthal’s canal. In relation to the gene expression data, BDNF immunoreactivity was

slightly increased with increase in time periods for all groups as demonstrated by semi-

quantitative scoring. Few SGC and fibres demonstrated immunoreactivity to BDNF in the

contralateral group 14 and 28. No staining was observed in the negative control sections

incubated only with secondary antibody. Secretory p75NTR

immunoreactivty did not

change throughtout the time periods, although few p75NTR

positive SGC could be

detected both in the contralateral and deafened cochlea. A slight increase in the p75NTR

expression was noticed in the group 28 deafened cochlea tissues, where most

immunoreactivity was confined to the degenerating SGC and its neighbouring cells. Very

faint immunostaining was detected in the tissue sections treated for anti-trkB, both in the

contralateral and deaf cochlea for all the groups. Few SGC was stained positive for anti-

trkB in the cell membrane of contralateral cochlea, while negligible staining was detected

in the group 14 and 28 deafened cochlea. Negative control tissue sections demonstrated

no immunostaining in the tissue sections. Overall, no significant changes in the

68

expression patterns of growth factors and its receptors could be detected in the normal

and deaf cochlea tissues by immunohistochemistry as detailed in Table 8.

Table 8: Indirect immunofluorescence of GDNF and its receptor GFRα-1, BDNF and its

receptor trkB, p75NTR

and apoptosis inducing molecules such as bax, bcl2, caspase 9 and

3 on SGC bodies from NH and deaf rat cochlea.

Protein Contralateral cochlea Deafened cochlea Negative

control G7 G14 G28 G7 G14 G28

-GDNF 1 1 1 3 1 3 0

-GFRα-1 1 1 1 1 1 1 0

-BDNF 2 2 3 2 2 3 0

-trkB 1 1 1 2 1 1 0

-p75NTR

2 1 1 1 1 2 0

-Bcl2 1 1 1 1 1 1 0

-Bax 2 2 1 2 1 1 0

-casp 9 1 1 1 1 1 2 0

-casp 3 1 1 1 1 1 2 0

-casp 9 = caspase 9; -casp 3 = caspase 3; G7, G14, G28 = group 7, 14 and 28; Rating: 0 =

no fluorescence; 1 = weak, but specific fluorescence; 2 = distinct, specific fluorescence;

and 3 = severe specific fluorescence, U = non-specific fluorescence.

69

Fig: 20: Representative photomicrographs of paraffin-embedded sections showing the

distribution of GDNF in the SGC following deafening. Positive immunostaining was

observed in the contra and deafened sections for all the groups. Arrows indicate SGC

positive for GDNF. 7C, 7D, 14C, 14D, 28C, 28D = contra and deaf cochlea for group 7,

14 and 28 and NC = negative control. Scale bar = 100 µm, x400

7C 7D

14C 14D

28C 28D

NC

70

Fig. 21: Representative photomicrographs of paraffin-embedded sections showing the

distribution of GFRα-1 in the SGC following deafening. Positive immunostaining was


positive for GFRα-1. 7C, 7D, 14C, 14D, 28C, 28D = contra and deaf cochlea for group 7,


7C

7D

7D

14C 14D

28C 28D

NC

71


distribution of BDNF in the SGC following deafening. Positive immunostaining was


positive for BDNF. 7C, 7D, 14C, 14D, 28C, 28D = contra and deaf cochlea for group 7,


7C 7D

14C 14D

28C 28D

NC

72


distribution of p75NTR

in the SGC following deafening. Positive immunostaining was


positive for p75NTR

. 7C, 7D, 14C, 14D, 28C, 28D = contra and deaf cochlea for group 7,


7C 7D

14C 14D

28C 28D

NC

73


distribution of trkB in the SGC following deafening. Few positive trkB positive SGC was


positive for trkB. 7C, 7D, 14C, 14D, 28C, 28D = contra and deaf cochlea for group 7, 14

and 28 and NC = negative control. Scale bar = 100 µm, x400

74

3.4.2 Immunohistochemical expression and distribution of apoptosis related

molecules in the cochlear tissues

Immunostaining for nuclear specific anti-bcl-2 did not change significantly over the

different periods in the contralateral and deafened cochlea, although few SGC were found

to be positive in the contralateral groups. No specific staining was observed in the

deafened groups despite some non-specific background staining. Negative control

sections demonstrated no immunostaining. A significant number of SGC were densely

labeled with cytoplasmic anti-bax in the contra and deafened cochlea for all the groups. A

large number of positive SGC was detected in contralateral cochlea compared to

deafened ones, where the remaining surviving SGC expressed bax protein. No

immunostaining was detected in the negative control incubated only with secondary

antibody. No significant changes in the immunostaining patterns could be detected in the

cytoplasmic caspase 9 labeling. Numerous positive SGC for caspase 9 was seen in the

contralateral side compared to deafened, although the immunoreactivity was found to be

more intense in the deafened 14 and 28 groups, suggesting cells lying close to SGC might

express caspase 9 following degeneration. Negative control tissue sections were free from

any kind of immunostaining. Cytoplasmic caspase 3 immunoreactivty was detected in the

SGC of contralateral and deafened individuals following deafening. A large number of

positive SGC for caspase 3 was seen in the contralateral side compared to the deafened.

We also noticed an intense immunostaining in the group 28 deafened cochlea compared

to its contralateral side. Even few neuronal fibres were also labeled positive for caspase 3

in the contralateral group 7 individual. No immunoreactivity was observed in the

negative control where anti-caspase 3 was omitted with PBS.

75


distribution of bcl2 in the SGC following deafening. Few positive bcl2 positive SGC was


positive for bcl2. 7C, 7D, 14C, 14D, 28C, 28D = contra and deaf cochlea for group 7, 14

and 28 and NC = negative control. Scale bar = 100 µm, x400

7C 7D

14C 14D

28C 28D

NC

76


distribution of bax in the SGC following deafening. Bax positive SGC was observed in

the contra and deafened sections for all the groups. Arrows indicate SGC positive for bax.

7C, 7D, 14C, 14D, 28C, 28D = contra and deaf cochlea for group 7, 14 and 28 and NC =

negative control. Scale bar = 100 µm, x400

7C 7D

14C 14D

28C 28D

NC

77


distribution of caspase 9 in the SGC following deafening. Caspase 9 positive SGC was


positive for caspase 9. 7C, 7D, 14C, 14D, 28C, 28D = contra and deaf cochlea for group

7, 14 and 28 and NC = negative control. Scale bar = 100 µm, x400

vii

7C 7D

14C 14D

28C 28D

NC

78


distribution of caspase 3 in the SGC following deafening. Caspase 3 positive SGC was


positive for caspase 3. 7C, 7D, 14C, 14D, 28C, 28D = contra and deaf cochlea for group

7, 14 and 28 and NC = negative control. Scale bar = 100 µm, x40

7C 7D

14C 14D

28C 28D

NC

79

Discussion

Although aminoglycosides are one of the most potent antibiotics effective against

infectious disease such as tuberculosis, their prominent side effect include cochlear,

vestibular and renal toxicity, which proves to be a limiting factor for their use globally

(Schacht, 1998). These ototoxic drugs destroy the sensory hair cells that are responsible

for hearing and balance, resulting in sensorineural hearing loss (SNHL). Several studies

have shown that it is possible to protect the spiral ganglion cells (SGC) from noise and

drug-induced apoptosis by local application of neurotrophic factors into the inner ear

(Miller et al., 1997; Ylikoski et al., 1998; Shinohara et al., 2002; Gillespie et al., 2003;

Staecker et al., 1996; Schindler et al., 1995; Yagi et.al., 1999). The loss of neurotrophic

factors followed by neuronal degeneration subsequently culminates in a change in the

oxidative state of the cell (Dugan et al., 1997; Mattson, 1998). With reference to

“neurotrophin hypothesis” suggested by Mattson (1998), the differentiation of neurons is

accompanied by a gradual reduction of neurotrophic factor which contributes to the

formation of free radicals/reactive oxygen species (ROS). The generation of ROS

destroys the cellular components and eventually leads to apoptosis or necrosis (Priuska et

al., 1995; Davies, 1996; Deshmukh et al., 1997; Dodson, 1997; Van De Water et al.,

2004). It was earlier demonstrated that chronic administration of aminoglycosides into

the cochlea resulted in multiple forms of cell death, rather than the involvement of

classical apoptosis (Jiang et al., 2006).

Recent studies have shown that GDNF, its receptor GFRα-1, and BDNF expression were

significantly upregulated in the rat auditory nerve 26 days following neomycin-induced

deafness (Wissel et al., 2006). Such upregulation is contradictory to the hypothesized

decreased neurotrophic input following cell death. In the present work, we tested the

hypothesis, if these neurotrophic factors play a significant role in SGC degeneration via

apoptosis performing gene expression analysis at different time intervals in the rat

cochlea, following neomycin administration. Briefly, the extent of SGC degeneration was

correlated to the acoustic auditory brainstem response (AABR) threshold shifts and the

differential expression of GDNF and its receptor GFRα-1, BDNF and its receptor trkB

80

and p75NTR

and components of the apoptotic machinery; bax, bcl2, caspase 9 and

caspase-3 following 7, 14 and 28 days of neomycin-induced deafening in the rat cochlea.

4.1 Effect of ototoxicity on the auditory threshold of rats

The rationale behind using frequency specific AABR was to monitor the hearing

thresholds of rats prior to and 7, 14 and 28 days post deafening. Previous studies have

reported that neomycin intervention into the rat cochleae can induce severe threshold

changes following deafening (Conlon et al., 1998). Such changes are mostly

accompanied by increase in the threshold shift evoked potentials at various test

frequencies. The frequency of 8 kHz was considered as the appropriate hearing

sensitivity for rats. The normal hearing animal groups (NH), which underwent no

treatment, demonstrated a hearing threshold of less than equal to 43 dB SPL (Tan et al.,

2006). The hearing threshold level for group 7, 14 and 28 prior to deafening (day 0) were

found to be within this normal hearing range. Minor fluctuations observed in the

threshold level could be attributed to the variations in the hearing status of each

individual animal via cochleostomy and the surrounding noise interference from the

sound proof chamber where each measurement was performed. In comparison to the NH

and d0 groups, neomycin induced severe hearing loss in the neomycin deafened ears. The

AABR threshold graphs for deaf animals demonstrated threshold shifts of more than 50

dB SPL, while a slight but not significant increase in threshold level were seen in the

contralateral side following deafening. We consider this to be due to the transfer of

neomycin into the contralateral ear via the cochlear aqueduct which is the most likely

route of functional communication between the cerebrospinal fluid and the perilymphatic

space of the inner ear (Stoever et al., 2000). Since this minimal threshold shift was found

to be insignificant, the hearing status on the contralateral side was considered in the range

of normal hearing. As illustrated in Figure 13, a threshold shift of 58.83 dB, 54 dB and

53.33 dB was noticed for deafened group 7, 14 and 28, respectively in comparison to NH

controls. The abovementioned findings were validated with histological analysis, where a

decrease in the SGC density was consistent with the shift in the threshold levels for the

deafened and contralateral ear. No significant difference was observed at test frequencies

1, 4, 16, 32 and 40 kHz prior to and after deafening for all the groups. Taken together,

81

these results indicates that neomycin induced a severe and permanent hearing loss in the

deafened ears (> 90 dB SPL), and a slight but not significant threshold shift was noticed

in the contralateral ears compared to NH controls.

4.2 Significant reduction of SGC density following deafening

Neomycin administration induces SGC degeneration in a time dependent manner

following hair cell loss (Webster et al., 1981; Bae et al., 2008; Dodson, 1997).

Characteristic features of the degenerated SGC in the deafened cochlea include

cytoplasmic vacuolation and fragmented nuclear chromatin (Bae et al., 2008). The

process of SGC death over a function of time is also species dependent. Previous studies

indicate that the rate of SGC loss ranges from weeks to months in guinea pigs (Dodson et

al., 1997). Bichler et al., 1983 determined that the time course of SGC death in rats might

extend over a period of 1 year. A significant reduction in SGC numbers (Figure 15) was

observed in the neomycin treated groups (0.8 ± 0.04, SGC/1000 µm2) as compared to NH

controls (2.17 ± 0.2 SGC/1000 µm2) in a time dependent manner (p < 0.001). This

represents a loss of approximately 63.14% of the total number of SGC at 28 days post

deafening (Bae et al., 2008). A significant SGC loss following 7 and 14 days of

deafening in deafened as well as the contralateral cochleae has also been reported

previously by other groups (Bae et al., 2008; Dodson, 1997; Dodson and Mohuiddin,

2000; Alam et al, 2007). Neuronal loss, though insignificant was also seen in the

contralateral cochleae across all the time period of deafening. The rate of SGC

degeneration induced by intracochlear neomycin administration seems to be comparable

to the systemic administration of amikacin in deafened rats (Bichler et al., 1983), which

is in lines with the fact that different aminoglycosides do not affect the rate of loss of

SGC (Alam et al., 2007). The unilateral deafening created a partial decline in SGC

density in the contralateral cochleae, which was functionally manifested as an increase in

threshold shift. And as mentioned previously, the contralateral cochleae has been shown

to be affected during the deafening procedure through the cochlear aqueduct, a route for

functional communication between the CSF (cerebro-spinal fluid) and the perilymphatic

space of the inner ear (Stoever et al., 2000; Lalwani et al., 2002). The pattern of SGC

degeneration were similar to that previously manifested in different animal models

82

subjected to ototoxic drugs (Keithley et al., 1979; Dodson, 1997; Dodson et al., 2000;

Cardinaal et al., 2000, Bae et al., 2008). The causal factors leading to the degeneration of

SGC by aminoglycosides is not yet known, although apoptosis or necrosis might

contribute to such phenomenon. Although the SGC degeneration is heterogenous in

nature, it is not evident why some cells undergo degeneration at a given time while others

may survive for weeks or months (Alam et al., 2007). Alam hypothesized that the

neurotrophic support from the supporting cells adjacent to the SGC distal processes might

be a contributing factor in explaining the heterogeneity of SGC following degeneration.

As the distal processes begin to degenerate, the individual SGC loose access to the

supporting cells at different time points, and finally die. Investigating the phenomenon

why a particular cell survives, may be instructive in the development of new therapeutic

strategies in the future and help to restore the SGC survival in profoundly deaf

individuals.

4.3 Relative gene expression changes in the normal and deaf animals

following deafening

Since neurotrophic factors play an important role in the survival and maintenance of

neurons, a part of our study was devoted to analyze the gene expression changes of these

growth factors at various time intervals in the normal and deafened animals.

Neurotrophins bind to two distinct types of receptors: Trk receptor tyrosine kinase family

and the p75 neurotrophin receptor p75NTR

, which is well known as the low affinity

receptor (Patapoutian et al., 2001; Dechant et al., 2002). Neurotrophic factors, especially,

glial cell-line derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor

(BDNF) have been shown to play an important role in the development and maintenance

of the auditory system (Gillespie et al., 2005). Previous studies have shown that the

neurotrophins BDNF and neurotrophin-3 (NT-3) influence SGC survival by binding to

their high-affinity Trk receptors (Ernfors et al., 1995). The protective and survival role of

GDNF is well documented in various studies both in vitro (Ylikoski et al., 1998; Qun et

al., 1999) and in vivo (Yagi et al., 2000; Kanzaki et al., 2002; Scheper et al., 2009). The

83

cell death observed following neomycin-induced deafness prompted us to question the

role of these neurotrophic factors at different time periods of deafening.

GDNF is expressed in the inner hair cells (Ylikoski et al., 1998) and in SGC (Stoever et

al., 2000). Studies have shown that the levels of GDNF protein was increased following 8

hours of noise overstimulation in the rat cochlea (Nam et al., 2000), and GDNF mRNA

expression was found to increase in the cochlea of P26 wild-type and homozygous

transgenic mice (Stankovic et al., 2004). The importance of GDNF has also been shown

in a variety of nerve cells, like dopaminergic neurons (Choi-Lundberg et al., 1997),

corticospinal neurons (Giehl et al., 1998) and motoneurons (Henderson et al., 1994).

Studies have demonstrated that GDNF mRNA is significantly increased following

traumatic spinal cord injury (Satake et al., 2000), in the brains of scrapie-infected mice

(Lee et al., 2006). Our gene expression results show that GDNF is significantly

upregulated at mRNA level at 28 days of neomycin-induced SGC loss in the deafened

modiolus with a slight increase also noticeable at day 7 and 14. GDNF mRNA expression

in the contralateral side was slightly downregulated following 14 and 28 days of

deafening. Immunohistologically also GDNF was found to be localized in the SGC of

both contralateral and deafened cochleae. Significant upregulation of endogenous GDNF

in the modiolus suggests a “growth factor deprived” expression of GDNF upon deafening

(Wissel et al., 2006). However, the molecular events contributing to the downregulation

of GDNF in the contralateral side are speculative and yet to be determined. In addition,

the GDNF receptor GFRα-1 demonstrated a significant increase in the transcription

activity 7, 14 and 28 days following deafening in the deafened modiolus. Our findings are

consistent with previous stress induced studies showing an upregulation of GFRα-1 in the

sensory neurons and spinal motoneurons of aged rats (Bergman et al., 1999) and in

dentate gyrus, cortex and striatum in a rat model of stroke (Sarabi et al., 2001).

Immnunohistochemical localization of GFRα-1 in the SGC was determined both in the

contralateral and deafened cochleae following deafening (Ylikoski et al., 1998). As

GFRα-1 expression is dependent on the upregulation of GDNF, an increase in the

expression pattern of GFRα-1 over a time period might enhance the responsiveness to

GDNF and reduce neuronal degeneration (Sarabi et al., 2001). The gradual increase of

GFRα-1 mRNA expression in the deafened cochleae in a time dependent manner may

84

suggest its consistent neuroprotective role following cochlear insults. Such simultaneous

increase in the expression of both GDNF and its receptor following cochlear insult is

mediated through its receptor complex composed of GFRα-1 and c-Ret proto-oncogene

(Jing et al., 1996; Trupp et al., 1995). Previous reports exhibit that extrinsic

administration of GDNF promote SGC survival in injury models (Yagi et al., 2000;

Ylikoski et al., 1998). Thus, it is unlikely that an upregulation of GDNF and its receptor

GFRα-1 mRNA in the deafened modiolus contributes to SGC death in our study. This

may indicate that the dying SGC retain their projection to the cochlear nucleus which is

the primary source of neurotrophic support and they receive trophic support from the

adjacent Schwann cells and supporting cells (Stankovic et al., 2004). The degeneration of

SGC is expected to withdraw only a fraction of the neurotrophic support available,

however, the residual trophic support is also insufficient to preserve the SGC in the long

term (Alam et al., 2007). Therefore, even though the neurons may have an increased

upregulation of survival factors following trauma, the SGC may still require higher levels

of neurotrophic factors for their survival (Wissel et al., 2006) from the Schwann cells and

satellite cells. Activation of satellite glia and Schwann cells in response to stress and

inflammation influences neighbouring neurons to proliferate and survive, although the

underlying mechanism is still not known (Hanani et al., 2005). In order to understand

how a balance between mechanism of cell survival and death is achieved in a stress-

induced cochlea, the delicate physical and molecular interactions between SGC and

Schwann cells has to be investigated in a finer detail.

Brain-derived neurotrophic factor (BDNF), a member of the nerve growth factor family

of trophic factors, promotes neuronal survival, regulates nerve fibre elongation in both

the central and peripheral nervous system, and has been implicated in synaptic plasticity

in the mammalian central nervous system (Ikeda et al., 2001; Lu, 2003; Maison et al.,

2009). BDNF mediates its biological function via two receptor systems: TrkB and

p75NTR

. It is well known that BDNF selectively binds TrkB with high affinity (Barbacid

et al., 1994; Schimmang et al., 1995; Patapoutian et al., 2001; Qian et al., 2006). Upon

binding, BDNF triggers dimerization and autophosphorylation of its tyrosine residues and

activates different signaling pathways (Vetter et al., 1991; Ohmichi et al., 1992; Stephens

et al., 1994). A number of intracellular signaling pathways such as Map-Erk Kinase

85

(MEK)-MAP-kinase are activated by binding of BDNF to TrkB, which results in the

phosphorylation of cyclic-AMP response element binding protein, CREB, a transcription

factor vital for neuronal survival and function (Patapoutian et al., 2001).). All

neurotrophins including BDNF bind with low affinity to the p75NTR

receptor, a member

of the tumor necrosis factor receptor superfamily, which is widely expressed in the

developing neurons during synaptogenesis and developmental cell death while it is

downregulated during maturation. Upon ligand binding, p75NTR

initiates several

intracellular death signaling pathways, including the Jun kinase and ceramide production.

Our gene expression results provide evidence that the degenerating SGC exhibit an

augmented BDNF and p75NTR

expression and a concomitant decrease in TrkB expression

at all time periods of deafness. A minor but statistically insignificant change in the

expression level of BDNF and aforementioned receptors was also observed in the

contralateral ears throughout the deafening period. Upon immunostaining BDNF and its

receptor p75NTR

and TrkB were found to be localized in the SGC of deafened and

contralateral cochleae. Lately, p75NTR

receptor has been found to be upregulated under

pathological conditions, inflammatory response (Lee et al., 2001; Dowling et al., 1999;

Roux et al., 1999) and following trauma or stress in the neuronal and glial populations

(Dechant et al., 2002; Cragnolini et al., 2008). The role of p75NTR

as a mediator of

apoptosis following stress or injury has also been elucidated (Kaplan et al., 2000; Zhu et

al., 2003; Tan et al., 2006; Yoon et al., 1998). In contrast, TrkB receptor has been found

to be downregulated in SGC following aminoglycoside-induced degeneration (Tan et al.,

2006), following status epilepticus in the rat hippocampus (Unsain et al., 2009) and in rat

cortical neurons (Qiao et al., 1998). Our results relating to TrkB downregulation and

p75NTR

upregulation in the deafened cochlea following deafness are consistent with the

above findings. We speculate there exists a fine balance between the survival and cell

death signal in a degenerating neuron. When a neuron becomes futile in its struggle for

adequate amounts of appropriate neurotrophic factors, the signaling pathway mediated

through p75NTR

supersede these suboptimal survival signals and ensure rapid apoptosis in

that neuron. The molecular mechanism following the stress mediated upregulation of

p75NTR

.and the detailed understanding of the antagonistic interplay between p75NTR

and

86

TrkB post aminoglycoside-induced trauma will provide an interesting platform for future

therapeutics.

Tissue homeostasis is regulated by an exquisite interplay between cell survival and

apoptosis, the cell suicidal program which is mediated by cysteinyl aspartate proteases

called caspases B. Bcl2 (B-cell lymphoma 2) family of intracellular proteins which can

exhibit both pro and anti-apoptotic activities have been studied extensively in the past

due to their contribution as central regulators of caspase activation; predominantly

caspase 9. The Bcl2 family members can be grouped classically into a class I of proteins

that inhibit apoptosis and class II that promote apoptosis. We focused our study on bcl2

and bax which are representative members of the first and second class of Bcl2 family

members respectively. While bax promotes caspase activation by inducing

permeabilisation of the outer mitochondrial membrane and subsequent release of

apoptogenic molecules such as cytochrome c which initiate the cascade of activated

caspases, bcl2 has been shown to dimerise with bax and inhibit its pro-apoptotic

activities. Members of the Bcl2 family proteins have also been implicated in the survival

(Staecker et al., 2007) or apoptosis (Rong et al., 2008) of SGC deprived of trophic

factors. Our gene expression results presented in Figure 19 suggested an upregulation of

bax, caspase 9 and 3 mRNA expression in the deafened modiolus over all the time period

of deafness.

Caspase-9 is an upstream molecule activated from the mitochondria during apoptosis.

Activation of caspase-9 involves the release of cytochrome-c from the mitochondria

(Stennicke et al., 2000). Neomycin administration induced a slight but significant

upregulation of caspase-9 following 28 days of deafening period. A slight but

insignificant upregulation of caspase-9 mRNA level was also noticed in the deafened

modiolus following day 7, 14 of deafening. Although caspase-9 was found to be localized

in the neurons by immunohistochemistry in the contralateral side, no significant changes

at the transcription level was observed throughout the deafening period. Possible

explanations for the slight but not significant upregulation of caspase-9 following

deafening might be due to the predominant contribution of the extrinsic pathway of

apoptosis mediated via the association of Fas death receptor on the target cell with its

87

cognate ligand FasL (Cheng et al., 2002; Bae et al., 2008), culminating in the activation

of upstream caspase-8 and further relaying of the signal to downstream caspase-3, 6 and

7. In addition, one might also suggest the involvement of necrosis-like programmed cell

death (Leist et al., 2001), or paraptosis (Sperandio et al., 2004) or a combination of both.

Caspase-3 known to be the common junction between the intrinsic and extrinsic cell

death pathway and is one of the key regulators of cell death. It executes apoptosis by the

cleavage of proteins necessary for cell survival, including bcl2 and cytoskeletal proteins

(Kirsch et al., 1999; Kothakota et al., 1997). We showed that caspase-3 mRNA level was

upregulated in the deafened modiolus following 28 days of deafening, while gene

expression at day 7 and 14 demonstrated a slight upregulation. No significant changes in

the expression patterns for caspase-3 were noticed in the contralateral sides following

deafening, although immunoreactivity was seen in the SGC of deafened and contralateral

cochleae. As previously reported, we also witnessed an upregulation in caspase-3

expression over a time period in the deafened modiolus, hence suggesting its role in

SGC-induced apoptosis following aminoglycoside induced deafening (Mangiardi et al.,

2004; Lee et al., 2004).

On the contrary, bcl2 mRNA level was sparingly expressed throughout various time

points in the deafened cochlea, resulting in a decreased bcl2 to bax ratio. This is in

agreement with the previous findings where bax, caspase 3 and 9 were found to be

upregulated while bcl2 mRNA was downregulated following cisplatin (Garcı´a-Berrocal

et al., 2007) and gentamicin treatment (Bae et al., 2008) in the cochlea. Although

overexpression of bcl2 might prove to be protective for neurons deprived of neurotrophic

factors in vitro and in vivo, it is yet elusive to which extent bcl2 can rescue the

degenerating neurons and support their survival (Davies, 1995). The current study

provides evidence that neomycin administration results in caspase-induced cell death in

the degenerated SGC, which is also well supported by the auditory threshold shift and

SGC cell count data post deafening. Bax mRNA levels demonstrated a slight

upregulation until day 14 in the deafened modiolus and gradually tapered at day 28,

indicating its role in apoptosis and mediating SGC death. To iterate, the decreased

mRNA expression of bcl-2 together with the antagonistic increase in expression of bax as

88

well as caspase-3 and 9 in the neomycin deafened modiolus may suggest a critical role

played by Bcl-2 family proteins in the initiation of apoptosis.

In summary, neomycin administration into the rat cochlea induced degeneration of SGC

in a time dependent manner, which is evident by significant shift in threshold levels

measured by AABR and SGC density assessment. The gene expression results

demonstrated a significant upregulation of endogenous GDNF and its receptor, GFRα-1

in the deafened modiolus, indicating a deprivation-induced upregulation following

neomycin injection. We speculate that this upregulation at the mRNA level is contributed

by the consistent neurotrophic support from the glial cells and Schwann/satellite cells

following deafening, where the degenerating SGC retain their projection into the cochlear

nucleus for survival. The degenerative changes observed in the SGC due to

aminoglycoside application mediates apoptosis, thus rendering the existing neurotrophic

support futile for the survival of existing neurons. The significant upregulation of BDNF

and its receptor p75NTR

and a concurrent downregulation of TrkB receptor in the

deafened modiolus suggested a reciprocal cross-talk between the two receptors. In view

of these results, we may speculate that BDNF interacts through an alternative pathway

with p75NTR

rather than utilizing TrkB receptor pathway to induce cell death in SGC.

Neomycin-induced degeneration of SGC resulted in upregulation of pro-apoptotic bax

and caspase-3 and downregulation of anti-apoptotic bcl2 gene in the deafened and

contralateral modiolus, which may indicate the SGC death by apoptosis. However, we

did observe a slight upregulation of caspase-9 in the deaf modious, although not

significant, may indicate the involvement of alternative death signaling pathways

following deafening as previously discussed. To conclude, our differential gene

expression patterns following deafening find consensus with the “neurotrophic factor

hypothesis” which states that cell death associated with degeneration of neurons is due to

deprivation of neurotrophic factors. Our results showed that the neurotrophic factors were

significantly upregulated following neomycin-induced deafness in the rat cochleae and it

is not the deprivation of neurotrophic factors but a delicate balance between the complex

signaling network of cell survival and cell death which mediates neuronal degeneration.

89

Fig. 29: Schematic representation of neomycin-induced degeneration of SGC in the rat

cochlea. Degeneration of SGC follows deprivation-induced upregulation of GDNF and

receptor GFRα-1, and this support is extended by the Schwann cells. BDNF upregulation

in degenerating neurons may relay different signaling pathways, while binding of BDNF

to TrkB mediates cell survival, binding to low affinity p75NTR

promotes SGC death via

caspase-dependent or independent cell death.

4.4 Methodological considerations

Although the classical method of normalization of a single housekeeping gene is assumed

to be invariable among different tissue samples, their individual expression may vary

with respect to different experimental conditions, disease state, neoplasia, age of the

animal and the tissue being analyzed, hence, influencing the correct interpretation of

results. To circumvent such issues, high density endogenous control array pre-designed

with primers and probes for 16 housekeeping genes was preferred over the normalization

against a single gene. The modiolus contains the cell bodies of the SGC and auditory

nerve (Stoever et al., 2001). The current study focused on the gene expression pattern of

GDNF, GFRα-1, BDNF, TrkB, p75NTR

, bcl2, bax, caspase-9 and -3 mRNA in the

90

modiolus using real-time PCR and localized these proteins to the SGC of the auditory

nerve in the cochlea using immunohistochemistry. For the estimation of RNA quality in

the modiolus, quality control from Agilent bioanalyzer was performed. A RNA integrity

number (RIN) of more than 7 was found in all the tissue pools constituting normal

hearing (NH), contralateral and deafened modiolus, indicating that the RNA extracted

from these tissue pools were intact and not degraded. The highly specific Taqman gene

expression assays (“m1”) used in the study spanning the exon junction may exclude gene

products with similar homologs and off-target sequences, and are not affected by

genomic DNA contamination. Lastly, the amplification efficiency regarding the potential

contamination of modiolus from the surrounding mixed tissues has to be considered.

Nevertheless, our statistical significant gene expression results shows that the mere

contaminants present in the modiolus may not influence the changes at the gene

expression levels.

91

Determination of apoptosis and cell survival signaling following

neomycin induced deafness in the rat cochlea

Summary

Aminoglycoside-induced sensorineural hearing loss (SNHL) is associated with loss of

hair cells followed by secondary degeneration of spiral ganglion cell (SGC). By the

common “neurotrophin hypothesis”, cell death associated with degeneration of neurons

may reflect deprivation of neurotrophic factors. Contrary to this, recent studies have

reported that gene expression profiling of neurotrophic factors, GDNF, BDNF, artemin,

GFRα-1 and transforming growth factor beta 1 and 2 (TGF1/2) were significantly

upregulated following 26 days of neomycin-induced deafness in the rat cochleae. The

reasons for the contradictory findings may be as follows: (i) it is well known that some

growth factors or their receptors both contribute to the cell survival and protection as well

as cell death. However, it is still unclear in which way they shift the delicate balance of

survival and cell death, (ii) The autocrine neurotrophic factor expression may be induced

at a time point the degeneration process can only be slowed down, (iii) overexpression of

the neurotrophic factors may not be able to ensure the SGC survival throughout a longer

period following the neomycin induced degeneration., thus, additional external

application of neurotrophic factors may be needed.

In consideration of the neurotrophin hypothesis and the previous findings in gene

expression analysis following 26 days deafness, this study presents the results of SGC

survival and gene expression profiling of GDNF, BDNF, their corresponding receptors

GFR1, TrkB and p75NTR

, pro- and anti-apoptotic molecules in the rat cochleae

following 7, 14 and 28 days deafening. Hereby, we suppose, that the ongoing of the SGC

degeneration despite the strong neurotrophic factor upregulation might be the result of

deprivation induced induction of the neurotrophic factor expression.

Souvik Kar

92

Hearing sensitivity was confirmed by frequency specific acoustic auditory brainstem

responses (AABR) and SGC loss density was estimated morphometrically by cell counts

to determine the extent of degeneration in the Rosenthal’s canal at different time periods

following deafening. As shown by immunohistochemical staining the expression of

GDNF, BDNF, GFRα-1, TrkB, p75NTR

, bcl-2, bax, caspase-9 and caspase-3 was positive

to SGC. Differential gene expression patterns of GDNF, GFRα-1, BDNF, p75NTR

and

caspase-3 were significantly upregulated following 28 days of deafening. We observed a

slight upregulation of pro-apoptotic molecule bax at 14 day and caspase-9 at 28 days in

the deafened modiolus, respectively. There were no statistical significant differences in

the expression level in the contralateral modioli at days 7 and 14 post deafening. In

contrast, a reciprocal expression of TrkB and p75NTR

was noticed, in which p75NTR

was

upregulated and TrkB was downregulated in the deaf modiolus. These findings

demonstrated that neomycin induced a high and low-frequency hearing loss in the

deafened and contralateral ear, respectively. SGC cell counts confirmed a significant loss

of neurons in a time dependent manner. As revealed by the data from gene expression

analysis, substantial increase of GDNF and its receptor GFRα-1 following deafening

indicated deprivation-induced upregulation in the SGC after hair cell loss. The

degenerating SGC are maintained to survive for a longer time period by the Schwann

cells, which remain a potential source of neurotrophic support. Deafened animals

exhibiting higher expression levels of BDNF and p75NTR

and reduced TrkB mRNA,

indicated a reciprocal cross-talk following deafening. We speculated that p75NTR

binds to

BDNF and triggers apoptosis in neurons resulting in the degeneratuin process. This

hypothesis may be supported by the upregulation of bax and caspase-3 in the deafened

modiolus. We assume that an alternate cell-death pathway is induced in our neomycin-

induced deafness model that may trigger such phenomenon. To summarize, the

differential gene expression changes of GDNF and its receptor GFRα-1, BDNF, p75NTR

and TrkB indicate a fine-tuned orchestration between cell survival and death in this

neomycin-deafness model. Moreover, these changes in the expression levels following

deafening are in consistence with the ‘neurotrophin hypothesis’ by which apoptosis of the

SGC death is a result of deprivation of neurotrophic factors not only supplied by the

Schwann cells. Understanding the response of Schwann cells to deafening might facilitate

93

SGC maintenance and regeneration studies. Future studies should aim to investigate

extended time period of deafness and try to understand the similarities and differences

between ototoxic mechanisms underlying SNHL, in order to develop more protective

strategies to minimize their ototoxicity.

94

Untersuchung der Signalwege der Apoptose und des

Zellüberlebens nach Ertaubung durch Applikation von

Neomycin in die Rattencochlea

Zusammenfassung

Innenohrschwerhörigkeit ist mit dem Verlust der Haarzellen durch sekundäre

Degeneration der Spiralganglion-Neuronen (SGN) nach einem Ototrauma verbunden.

Nach der „Neurotrophin-Hypothese“ erfolgt die Degeneration der Hörnerven nach

Haarzellverlust durch Unterbrechung/Entzug der trophischen Aktivität neurotropher

Faktoren (NTF) als Folge der von sensorischen Zellen induzierten Apoptose. Gemäß den

Ergebnissen der Genexpressionsanalyse der bisher untersuchten Mitglieder der GDNF-

Familie und TGF-Superfamilie, die als sog. „survival“-Faktoren vor den Folgen

ototoxischen Traumas im Innerohr von Nagetieren charakterisiert sind, läßt sich die oben

genannte Hypothese nicht aufrechterhalten: Die Transkriptionsrate von GDNF, artemin

und BDNF wurde im Hörnerven erwachsener Ratten 26 Tage nach der Ertaubung durch

Neomycin entgegen dem Postulat der „Neurotrophin-Hypothese“ erhöht. Folgende

Gründe für die erhöhte Expression könnten eine Rolle spielen: (i) Es ist bekannt, dass

einige Wachstumsfaktoren sowohl zur Zellprotektion beitragen als auch die Apoptose

fördern können. Unklar ist, wann und bei welchen externen und/oder intrazellulären

Stimuli sich die Funktionen umkehren. (ii) Die autokrine Expression der NTF wird zu

einem späteren Zeitpunkt induziert und verlangsamt den Degenerationsprozeß. (iii) Das

Überleben der SGN nach einem aminoglykosid-induzierten Degenerationsprozess für

einen längeren Zeitraum ist nicht ausreichend, so dass zusätzlich NTF von außen

appliziert werden muss.

Basierend auf der Neurotrophin-Hypothese und den Ergebnissen der Genexpressionstudie

in ertaubten Ratten nach 26 Tagen untersucht die vorliegende Studie die Expression von

NTF im Rahmen der Induktion apoptotischer Signale in den Modioli der Ratten-Cochleae

7, 14 und 28 Tage nach der Neomycin induzierten Ertaubung auf mRNA- und

Souvik Kar

95

Proteinebene. Der Hörstatus der Versuchstiere wurde elektrophysiologisch mittels

„acoustically evoked auditory brainstem response“ (AABR) vor und 7, 14 und 28 Tage

nach der Ertaubung bestimmt. Die differentielle Genexpression von GDNF und BDNF,

deren korrespondierenden Rezeptoren GFRα-1, p75NTR und Trk-B, Bcl-2

(antiapoptotisches Signalmolekül), Bax, Caspasen 3 und 9 (apoptitische Signalmoleküle)

wurden mittels real time-PCR untersucht. Zur Bestimmung der Ausdehnung der SGN-

Degeneration zu den oben angegebenen Zeitpunkten nach der Neomycin-Injektion wurde

die Dichte der SGN histologisch durch Auszählen noch intakter Soma der SGN in den

Rosenthal-Kanälen ermittelt. Die subzelluläre Lokalisation von neurotrophen und

apoptotischen Markern auf Proteinebene wurde immunhistochemisch bestimmt.

Immunhistochemisch wurde die Expression von GDNF, BDNF, GFRα-1, TrkB, p75NTR

,

bcl-2, bax, caspase-9 and caspase-3 im Spiralganglion nachgewiesen.

Die Analyse der Transkriptionsaktivität im Modiolus 28 Tage nach der Ertaubung zeigte

eine signifikante Hochregulierung von GDNF und dessen Rezeptor GFRα-1, BDNF und

dessen Rezeptor p75NTR sowie Caspase-3 im Vergleich zu den normal hörenden (NH)

Tieren. Im Gegensatz dazu wurde die Genexpression des BDNF-spezifischen Rezeptors

TrkB im Vergleich zur NH-Gruppe am Tag 28 signifikant herunterreguliert. Der mRNA-

Level des proapoptotischen Signalmoleküls Bax war nur am Tag 14 der Ertaubung

signifikant erhöht, während an Tag 28 die Transkriptionsaktivität wiederum

herunterreguliert wurde. Im Vergleich zur NH-Gruppe wurde ebenfalls nur 28 Tage nach

der Ertaubung eine signifikante Erhöhung der Caspase-3-Genexpression nachgewiesen.

Dagegen wurden keine signifikanten Änderungen der Caspase-9-Genexpression an allen

Untersuchungstagen gefunden. Für die Bcl2-Genexpression wurde eine leichte, aber

nichtsignifikante, Herunterregulierung 7, 14 und 28 Tage nach der Ertaubung ermittelt.

Keine statistisch signifikanten Änderungen im Expressionslevels wurden in den

kontralateralen Ohren der Versuchstiere gefunden, die für 7 und 14 Tage ertaubt waren.

Unsere Daten in der Bestimmung der SGN-Dichte bestätigten einen signifikanten Verlust

der SGN mit zunehmender Zeitdauer der Ertaubung. Darüber hinaus sprechen die

Aktivierung von Bax, Caspase-9 und -3 sowie die verringerte Genexpressionsaktivität

des antiapoptotischen Bcl-2 für eine caspaseinduzierte mitochondriale Apoptose in

ertaubten Ratten. Im Zusammenhang mit dem Anstieg der Genexpression von BDNF und

96

p75NTR

sowie der Herunterregulierung der TrkB-Genexpression ist ein reziproker „cross-

talk“ im Degenerationsprozeß der SGN zu vermuten: Durch Bindung von BDNF an

p75NTR

wurde möglicherweise die Apoptose in den Neuronen getriggert. Die

Hochregulierung von bax und caspase-3-mRNA unterstützt diese Annahme.

Möglicherweise stellen hauptsächlich Schwannzellen die Quelle für die erhöhten Level

der GDNF- und GFR-1-mRNA dar und die autokrine Expression der NTF als „survival

Faktoren“ in den SGN spielt selbst keine Rolle mehr. Nach dieser Schlußfolgerung kann

das Postulat der Neurotrophin-Hypothese als erfüllt betrachtet werden. Welche Rolle

Schwannzellen im Degenerationsprozeß und dem Erhalt der SGN spielt, wird weiterhin

Gegenstand der Innenohrforschung sein.

97

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Appendix I

Preparation of solutions

Phosphate buffered saline (PBS)

For preparation of phosphate buffered saline (0.14 M NaCl, 0.010 M PO4-buffer,

0.0003 M KCl; pH 7.45), one tablet of PBS is dissolved in 500 ml of distilled water on a

magnetic stirrer and then filtered sterile.

Paraformaldehyde 4% (PFA)

For the preparation of 4% paraformaldehyde in a half-liter PBS prepared above, 20 g of

PFA is added and heated on a magnetic stirrer at 60 ºC. The turbid solution is changed

into a clear solution by adding 1M NaOH solution. Finally, the solution is filtered and

stored at 4 ºC for further use.

0.1% Triton X-100

0.1% Triton X-100 is prepared by dissolving 1 ml of the viscous liquid in 1 liter of PBS

and mixed thoroughly unless the solution becomes clear.

20% Ehtylenediaminetetraacetic acid (EDTA)

20 g of ethylenediaminetetraacetic acid is dissolved in 1 liter of PBS and mixed

thoroughly on a magnetic stirrer until the solution in clear and the solution is stored in

refrigerator.

0.1% Diethylpyrocarbonate (DEPC) water

DEPC water is used to remove excess RNase from the tissue samples for gene expression

studies. We dissolved 1 ml of DEPC in 1 liter of autoclaved distilled water and mixed

thoroughly overnight at room temperature until the solution turns clear.

1% Eosin Y stock solution

To 200 ml of distilled water 800 ml of 95% ethanol is added. To this solution, 10 g of

eosin Y is added and mixed on a magnetic stirrer overnight. Finally, the solution is

filtered and stored at room temperature.

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Solutions and reagents

Acetic acid Merck, Darmstadt

Denatured ethanol, 70% Hannover Medical School, Hannover

1% Eosin Y Merck, Darmstadt

Ethanol for molecular biology Merck, Darmstadt

Formaldehyde Merck, Darmstadt

Hematoxylin Merck, Darmstadt

Sodium hydroxide 10 mol/l, high purity Merck, Darmstadt

Paraformaldehyde (PFA) Merck, Darmstadt

Phosphate buffered saline (PBS) tablets Invitrogen, GIBCO™, Karlsruhe

ProLong® Gold antifade reagent with

DAPI

Invitrogen, Molecular Probes, Eugene,

Oregan, USA

Trypsin/EDTA solution (0.05%/0.02%) Bichrom AG, Berlin

Pharmaceuticals

Neomycin tri-sulfate salt hydrate Sigma, CA, USA

Carprofen (50 mg/ml), Rimadyl®,

20 ml

Pfizer GmbH, Karlsruhe

Ketamin (100 mg/ml), Ketamin Gräub

10%, 10 ml

A. Albrecht GmbH & Co., Aulendorf

Xylazin (20 mg/ml), Rompun® 2%, 20 ml Bayer Vital GmbH, Leverkusen

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Ringer Acetat Infusionlösung, 1000 ml Fa. B. Braun Melsungen AG, Melsungen

Xylonest® 1%, 10 ml AstraZeneca GmbH, Plankstadt

Cotrim K-ratiopharm® Saft, 250 ml,

Sulfamethoxazol (200 mg) and

Trimethoprim (40 mg)

Ratiopharm GmbH, Ulm

Laboratory supplies and consumables I

Autoclave Systec, Germany

Coverslips 22 X 22 mm Menzel Glass, Braunschweig

Centrifuge tubes, 15 ml conical Biochrom AG, Berlin

Centrifuge tubes, 20 ml conical Cellstar

Cryostat JUNG CM3000 Leica

Latex gloves SAFESKIN SATIN PLUS Kimberly-Clarke, Zaventem, Belgium

Lab glassware (flasks, bottles and beakers) Omnilab, Gehrden/VWR, Darmstadt

Olympus light microscope Olympus, Hamburg

Mini spin plus centrifuge Eppendorf, germany

Nanodrop Peqlab biotechnologie, Germany

pH meter Sartorius, Germany

Pipette tips, 1-10 µl, 1-200 µl, 101-1000 µl TipOne, Starlab, Ahrensburg

Real time-PCR (StepOne)® Applied biosystem, USA

Real time-PCR (7900 HT) Applied biosystem, USA

Stereomicroscope` Leica, germany

Superfrost® slides Menzel glass, Braunschweig

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Single-use sterile filters, 0.2 µm,

“Ministart®”

Sartorius, Göttingen

Sonicator (Ionomix) Ionos GmbH, germany

Vortex Genie 2 Scientific industries, Germany

Freezing tubes ”Nalgene“, 1.8 ml Nalge Co., Rochester, USA

Reaction vessel “Safe-Lock’’, 2 ml Omnilab, Gehrden

Freezer “Typ56134”, 4º C, -20º C Liebherr, Bieberach

Magnetic stirrer with hot plate “K-RET

basic”

IKA-Werke GmbH & Co. KG, Staufen

Multifuge 1s, Hereaus Thermo Fischer Scientific GmbH, Bremen

Tweezers “Dumont No.5” Carl Roth GmbH & Co. KG, Karlsruhe

Pipette adjustable “Pipetman” (1-10 µl, 20-

200 µl, 101-1000 µl)

Gilson, Villers Le Bel/Frankreich

Precision balance “CP64’’ Sartorius, Göttingen

Ultrapure Milli-Q Biocel System Gard 2 Millipore GmbH, Schwalbach

Laboratory supplies and consumables II

Disposable needles BD Microlance™ 3,

23G x 11/4’’, 0.6 mm x 30 mm

Becton-Dickinson, Fraga (Huesca)/Spanien

Disposable needles Sterican®, 27G x

11/2’’, 0.4 mm x 12 mm

Braun, Melsungen

Syringes Omnifix® -F, Luer, steril,

1 ml, 2 ml, 5 ml, 10 ml

Braun, Melsungen

Disposable scalpel blade Nr.11 and 21

Aesculap

Product for the medicine AG, pfm, Koeln

Latex gloves, SAFESKIN SATIN PLUS Kimberly-Clark, Zaventem, Belgium

Laboratory glassware (Flasks, beakers,

bottles)

Omnilab, Gehrden/VWR, Darmstadt

Gauze, 5 cm x 5cm Lohmann & Rauscher International GmbH

& Co. KG., Rengsdorf

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Non-absorbable suture, Seralon®, USP 3/0,

DSS-15

Serag Weissner, Naila

Absorbable suture, Vicryl® rapide, USP

3/0, RB-1

Ethicon GmbH, Norderstedt

Surgical gloves, supermed® supreme, Semperit Technische Product, Wien,

Österreich

Petri dishes, 60 mm x 15 mm Corning Inc., New York, USA

Pipette tips, 1-10 µl, 1-200 µl, 101-1000 µl TipOne, Starlab, Ahrensburg

Single use sterile filters, 0.2 µm,

“Ministart®”

Sartorius, Göttingen

Cotton swab, 15 cm WDT eG, Garbsen

Equipments for AABR-measurement

ESI electrostatic speaker with base station Tucker-Davis Technologies (TDT),

Alachua, FL, USA

Microstimulator Base Station RX7

Multifunction Processor RX6

Pentusa Base Station RX5

Security LI-CONN connector,

1.5 mm

Sigma-Delta Medusa preamplifier

RA16PA, 16 channels

Needle electrodes, 12 mm Medtronic Xomed, Jacksonville, FL, USA

Soundproof chamber, custom-made Fa. Industrial acoustic company GmbH,

Niederkrüchten

Hico-Aquatherm heat mat 660 with

polyurethane water mat 50 cm x 30 cm

Fa. Hirtz, Köln

Computer program and software

Analysis® Software Soft Imaging Software GmbH, Münster

GraphPad Prism 3 GraphPad Software, Inc., CA, USA

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Cell^P imaging Olympus, Hamburg

QWin V3 Software Leica Microsystems Imaging Ltd,

Cambridge, UK

StepOne Plus® program Applied Biosystem, CA, USA

Microsoft Office® Microsoft Deutschland GmbH

Microsoft Excel® Microsoft Deutschland GmbH

MATLAB Software MathWorks, Natick, MA, USA

HughPhonics LIM u. ANDERSON 2006

TDT Software Alachua, FL, USA

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Declaration

I hereby declare that I have autonomously carried out my Ph.D thesis entitled:

“Determination of apoptosis and cell survival signaling following neomycin-induced

deafness in rat cochlea”

I did not receive any assistance from in return for payment by consulting agencies or any

other person. No one received any kind of payment for direct or indirect assistance in

correlation to the content of the submitted thesis.

I conducted the project at the following institution:

Department of Otolaryngology, Hannover Medical School, Hannover, Germany

The thesis has not been submitted elsewhere for an exam, as thesis or for evaluation in a

similar context.

I hereby affirm the above statements to be complete and true to the best of my

knowledge.

Souvik Kar, May 2012

Hannover Medical University Clinic of Laryngology ... · 1. Prof. Prof. h. c. Dr. med. Thomas...

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Transcript of Hannover Medical University Clinic of Laryngology ... · 1. Prof. Prof. h. c. Dr. med. Thomas...