Hannover Medical University Clinic of Laryngology, Rhinology and

Hannover Medical University Clinic of Laryngology, Rhinology and

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)
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
14
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)
15
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)
16
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)
17
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 fluidfilled 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 haircell/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
18
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
19
(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).
20
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).
21
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
22
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
23
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 cJun 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
24
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
25
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).
Intrinsic pathway
Extrinsic pathway
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).
26
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 linederived 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
27
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
28
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
29
(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 GFRα1 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
30
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
31
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 (TGF1/2) 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.
32
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 (TGF1/2), 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.
33
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
34
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.
Animal
group
Number
of
animals Day 0
Time course of deafening
Group 7
N = 30
AABR,
neomycin
injection
AABR
Sacrifice
Group 14
N = 30
AABR,
neomycin
injection
Group 28
N = 30
AABR,
neomycin
injection
NH
N = 15
AABR
followed by
sacrifice
Day 7
Day 14
Day 28
AABR
Sacrifice
AABR
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;
35
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 applicationspecific 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
36
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
37
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).
38
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.
39
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 nonspecific 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.
40
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
41
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
Description
Modiolus tissue pools
Tissue pools
from Tissue
contralateral ears
Group 7 (n = 40)
Group 14 (n = 40)
Group 28 (n = 40)
pools
from Both ears
deafened ears
MoC7.1=7,
MoC7.2=7, MoD7.1=7,
MoC7.3=6
MoD7.3=6
MoC14.1=7,MoC14.2=7,
MoD14.1=7,MoD14.2=7,
MoC14.3=6
MoD14.3=6
MoC28.1=7,MoC28.2=7,
MoD28.1=7,MoD28.2=7,
MoC28.3=6
MoD28.3=6
NH (n = 20)
MoD7.2=7,
NH1=7,
NH2=7,
NH3=6
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 ND1000 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.
42
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.
43
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
44
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 sampleloading 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
45
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-TechSupport/microrna-studies/tech-notes/global-mirna-profiling-higher-sensitivity-lesssample.html)
46
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
B2m
β-2-microglobulin
Rn00560865_m1
Ribosomal biogenesis and
assembly
Beta-chain of major
histocompatibility complex class I
molecules
18S
Ribosomal RNA 18S
Hs99999901_s1
Ribosome subunit
Gapdh
Glyceraldehyde-3phosphate 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
Ywhaz
Phospholipase A2
Rn00755072_m1
Heme synthesis, porphyrin
metabolism
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
47
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 (2ΔΔCт
), 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.
(Rn01402432_m1),
GFRα-1-(Rn01640513_m1),
(Rn01441745_m1),
p75NTR
–(Rn01309635_m1),
The assay IDs are: GDNF-
BDNF-(Rn01484924_m1),
TrkB-
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.
48
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.
49
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
Frequencies 1 kHz
4 kHz
dB SPL
62.5 ±8.87 40 ± 5.48
73.15±7.29
8 kHz
16 kHz
32 kHz
40 kHz
61 ± 8.31
58 ± 7.48
67± 8.43
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
50
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).
51
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).
52
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.
53
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
54
is shown in Figure 15, demonstrating a significant decrease in SGC number at all time
period of deafening.
SGC
NH
Contra day 7
Deaf day 7
Contra day 14
Deaf day 14
Contra day 28
Deaf day 28
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
55
Table 5: Density of SGC loss following neomycin treatment
Days of
Groups
deafening
NH
7
Contralateral
Deafened
14
Contralateral
Deafened
28
Contralateral
Deafened
SGC density of cells/1000 µm²
2.17 ± 0.2
2.07 ± 0.1
1.55 ± 0.23
1.88 ± 0.19
1.01 ± 0.12
1.69 ± 0.07
0.8 ± 0.04
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
56
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.
57
Table 6: RNA integrity values for rat modiolus
Description
Tissue pools from RIN
Tissue pools from
contralateral ears
deafened ears
RIN
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
MoC14.1=7
7.5
MoD14.1=7
7.9
(n = 40)
MoC14.2=7
8
MoD14.2=7
8.4
MoC14.3=6
8.6
MoD14.3=6
7.8
Group 28
MoC28.1=7
7.7
MoD28.1=7
7.7
(n = 40)
MoC28.2=7
9.4
MoD28.2=7
8.7
MoC28.3=6
8
MoD28.3=6
7.9
NH1=7
8.3
NH2=7
7.8
NH3=6
8.1
Group 7
(n = 40)
NH (n = 20)
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.
58
Deafened group 7
Contralateral group 7
RNA Integrity number (RIN):
8.9
RNA Integrity number (RIN):
Contralateral group 14
RNA Integrity number (RIN):
Deafened group 14
7.5
RNA Integrity number (RIN):
Contralateral group 28
RNA Integrity number (RIN):
8.7
7.9
Deafened group 28
7.7
RNA Integrity number (RIN):
Normal hearing
RNA Integrity number (RIN):
59
8.3
7.7
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
60
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.
NH
NH
contralateral d7
deafened d7
contralateral
d14
contralateral
deafened d14
deafened d7
A
deafened d28
deafened d14
30
A
20
15
10
5
25
15
10
20
15
10
5
5
0
0
Actb
Actb
0
Arbp
Arbp
B2m
Actb
B2m
18S
Gapdh
Arbp
Gapdh
B2m
18s
D
C
30
30
25
25
Ct average
Ct average
deafened d28
30
20
Ct average
Ct average
25
contralateral d28
B
25
30
Ct average
d7contralateral d28
contralateral d14
20
15
10
Gusb
Ppia
Gusb18S Ppia
Ppib
Ppib
20
15
10
5
5
0
0
Hprt
Tbp
Hprt
Tbp
Hm bs
Yw haz
Hmbs
Ywhaz
Pgk1
Pgk1
Rplp2
Rplp2
Tfrc
Tfrc
Ubc
Ubc
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.
61
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 neomycininduced 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
62
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.
63
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
64
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.
65
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).
66
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 semiquantitative 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 antitrkB 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.
67
Overall, no significant changes in the
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
G7
G14
Deafened cochlea
G28
G7
G14
Negative
control
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
1
1
1
1
1
2
0
-casp 3
-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.
68
7C
7D
14C
14D
28C
28D
NC
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
69
7C
7D
14C
14D
28C
28D
7D
NC
Fig. 21: Representative photomicrographs of paraffin-embedded sections showing the
distribution of GFRα-1 in the SGC following deafening. Positive immunostaining was
observed in the contra and deafened sections for all the groups. Arrows indicate SGC
positive for GFRα-1. 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
70
7C
7D
14C
14D
28C
28D
NC
Fig. 22: Representative photomicrographs of paraffin-embedded sections showing the
distribution of BDNF in the SGC following deafening. Positive immunostaining was
observed in the contra and deafened sections for all the groups. Arrows indicate SGC
positive for BDNF. 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
71
7C
7D
14C
14D
28C
28D
NC
Fig. 23: Representative photomicrographs of paraffin-embedded sections showing the
distribution of p75NTR in the SGC following deafening. Positive immunostaining was
observed in the contra and deafened sections for all the groups. Arrows indicate SGC
positive for p75NTR. 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
72
Fig. 24: Representative photomicrographs of paraffin-embedded sections showing the
distribution of trkB in the SGC following deafening. Few positive trkB positive SGC was
observed in the contra and deafened sections for all the groups. Arrows indicate SGC
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
73
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.
74
7C
7D
14C
14D
28C
28D
NC
Fig. 25: Representative photomicrographs of paraffin-embedded sections showing the
distribution of bcl2 in the SGC following deafening. Few positive bcl2 positive SGC was
observed in the contra and deafened sections for all the groups. Arrows indicate SGC
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
75
7C
7D
14C
14D
28C
28D
NC
Fig. 26: Representative photomicrographs of paraffin-embedded sections showing the
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
76
vii
7C
7D
14C
14D
28C
28D
NC
Fig. 27: Representative photomicrographs of paraffin-embedded sections showing the
distribution of caspase 9 in the SGC following deafening. Caspase 9 positive SGC was
observed in the contra and deafened sections for all the groups. Arrows indicate SGC
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
77
7C
7D
14C
14D
28C
28D
NC
Fig. 28: Representative photomicrographs of paraffin-embedded sections showing the
distribution of caspase 3 in the SGC following deafening. Caspase 3 positive SGC was
observed in the contra and deafened sections for all the groups. Arrows indicate SGC
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
78
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
79
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,
80
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
81
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
82
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
83
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 stressinduced 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
84
(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
85
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
86
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
87
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.
88
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
89
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.
90
Determination of apoptosis and cell survival signaling following
neomycin induced deafness in the rat cochlea
Souvik Kar
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 (TGF1/2) 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
GFR1, 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.
91
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 neomycininduced 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
92
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.
93
Untersuchung
der
Zellüberlebens
nach
Signalwege
Ertaubung
der
Apoptose
durch
und
des
Applikation
von
Neomycin in die Rattencochlea
Souvik Kar
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 GDNFFamilie 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
94
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 SGNDegeneration 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 mRNALevel 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
95
p75NTR sowie der Herunterregulierung der TrkB-Genexpression ist ein reziproker „crosstalk“ 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.
96
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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.
116
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
Trypsin/EDTA solution (0.05%/0.02%)
Invitrogen, Molecular Probes, Eugene,
Oregan, USA
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
117
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
118
Single-use sterile filters, 0.2 µm,
“Ministart®”
Sonicator (Ionomix)
Sartorius, Göttingen
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”
Multifuge 1s, Hereaus
IKA-Werke GmbH & Co. KG, Staufen
Tweezers “Dumont No.5”
Carl Roth GmbH & Co. KG, Karlsruhe
Pipette adjustable “Pipetman” (1-10 µl, 20200 µl, 101-1000 µl)
Precision balance “CP64’’
Gilson, Villers Le Bel/Frankreich
Ultrapure Milli-Q Biocel System Gard 2
Millipore GmbH, Schwalbach
Ionos GmbH, germany
Thermo Fischer Scientific GmbH, Bremen
Sartorius, Göttingen
Laboratory supplies and consumables II
Disposable needles BD Microlance™ 3,
23G x 11/4’’, 0.6 mm x 30 mm
Disposable needles Sterican®, 27G x
11/2’’, 0.4 mm x 12 mm
Syringes Omnifix® -F, Luer, steril,
1 ml, 2 ml, 5 ml, 10 ml
Disposable scalpel blade Nr.11 and 21
Aesculap
Latex gloves, SAFESKIN SATIN PLUS
Becton-Dickinson, Fraga (Huesca)/Spanien
Laboratory glassware (Flasks, beakers,
bottles)
Gauze, 5 cm x 5cm
Omnilab, Gehrden/VWR, Darmstadt
Braun, Melsungen
Braun, Melsungen
Product for the medicine AG, pfm, Koeln
Kimberly-Clark, Zaventem, Belgium
Lohmann & Rauscher International GmbH
& Co. KG., Rengsdorf
119
Non-absorbable suture, Seralon®, USP 3/0, Serag Weissner, Naila
DSS-15
Absorbable suture, Vicryl® rapide, USP
Ethicon GmbH, Norderstedt
3/0, RB-1
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®”
Cotton swab, 15 cm
Sartorius, Göttingen
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
Soundproof chamber, custom-made
Hico-Aquatherm heat mat 660 with
polyurethane water mat 50 cm x 30 cm
Medtronic Xomed, Jacksonville, FL, USA
Fa. Industrial acoustic company GmbH,
Niederkrüchten
Fa. Hirtz, Köln
Computer program and software
Analysis® Software
Soft Imaging Software GmbH, Münster
GraphPad Prism 3
GraphPad Software, Inc., CA, USA
120
Cell^P imaging
Olympus, Hamburg
QWin V3 Software
StepOne Plus® program
Leica Microsystems Imaging Ltd,
Cambridge, UK
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
121
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
122