THESIS DOCTOR OF PHILOSOPHY (Ph.D.)

Transcription

University of Veterinary Medicine Hannover
Clinics for Laryngology, Rhinology and Otology, Hannover Medical School
Center for Systems Neuroscience
CELL-BASED DRUG DELIVERY
TO OPTIMISE THE ELECTRODE-NERVE INTERFACE
THESIS
Submitted in partial fulfilment of the requirements for the degree
DOCTOR OF PHILOSOPHY
(Ph.D.)
awarded by the University of Veterinary Medicine Hannover
by
Odett Kaiser
Saalfeld / Saale
Hannover 2013
Supervisor:
Prof. Dr. Andrej Kral
Supervision Group: Prof. Dr. Sabine Kästner
Prof. Dr. Susanne Petri
Prof. Dr. Hans Gerd Nothwang
1st Evaluation:
Prof. Dr. Andrej Kral
Clinic for Laryngology, Rhinology and Otology
Hannover Medical School
Prof. Dr. Sabine Kästner
Small Animal Clinic
University of Veterinary Medicine Hannover, Foundation
Prof. Dr. Susanne Petri
Department of Neurology
Hannover Medical School
2nd Evaluation:
Dr. rer nat. habil. Hugo Murua Escobar
Hematology, Oncology and Palliative Medicine
University of Rostock
Date of final exam:
25th October 2013
Sponsorship: Georg-Christoph-Lichtenberg scholarship
Parts of the thesis have been published previously in:
Neuropharmacology (Chapter 2)
PLOS One (Chapter 3)
Results of this thesis were presented in form of presentations or posters
at the following conferences:
Stöver T., Kaiser O., Paasche G., Hoffmann A., Lenarz T., Warnecke A.
“TGF-beta Superfamilie-Mitglied Activin A verbessert das Überleben von
Spiralganglienzellen in vitro“
In: 83rd Annual Meeting of the German Society of Oto-Rhino-Laryngology,
Head and Neck Surgery (Mainz 2012); presentation
Warnecke A., Kaiser O., Paasche G., Stöver T., Kral A., Schäck L., Lenarz T.
“Steigerung des neuroprotektiven Effektes von Activin A und BDNF durch
Zugabe von Erythropoietin“
In: 84th Annual Meeting of the German Society of Oto-Rhino-Laryngology,
Head and Neck Surgery (Nürnberg 2013); presentation
Kaiser O., Wissel K., Alious P., Lenarz T., Reuter G., Warnecke A.
“Die dissoziierte Colliculus inferior Kultur als in vitro Modell für
Untersuchungen der Gewebe-Implantat-Interaktion beim AMI“
In: Annual Meeting of the German Society for Biomaterials (Hamburg, 2012);
poster
To
My family
“Nur wer seine Träume lebt, kann seine Sehnsucht stillen.”
Sergio Bambaren
Index
INDEX
INDEX .............................................................................................................................. I
LIST OF ABBREVIATIONS ................................................................................................ III
LIST OF FIGURES ........................................................................................................... VI
SUMMARY ........................................................................................................................ 1
ZUSAMMENFASSUNG ....................................................................................................... 3
1.
CHAPTER 1: GENERAL INTRODUCTION ....................................................................... 7
1.1
Basic Anatomy of the Ear and the Physiology of Hearing ......................................... 7
1.2
Auditory Pathway - From the Cochlea to the Brain ................................................13
1.2.1
The Inferior Colliculus – The Auditory Convergence Centre .................................. 15
1.3
Classification of Hearing Loss ...........................................................................16
1.4
Sensorineural Hearing Loss in the Cochlea - CI-Research....................................... 17
1.5
Neurotrophic Factors - Pharmacologically active Substances ................................. 18
1.5.1
Neurotrophins ........................................................................................19
1.5.2
Transforming Growth Factor - beta Superfamily .............................................. 20
1.5.3
Erythropoietin ....................................................................................... 22
1.5.4
Known Interactions of the different Substances ............................................... 23
24
1.6
Auditory Midbrain Implant and Inferior Colliculi: When the CI is no Implant Option
1.7
Aims for the Thesis ......................................................................................... 24
2. CHAPTER 2: TGF-BETA SUPERFAMILY MEMBER ACTIVIN A ACTS WITH BDNF AND
ERYTHROPOIETIN TO IMPROVE SURVIVAL OF SPIRAL GANGLION NEURONS IN VITRO . 27
2.1
3.
Abstract
....................................................................................................... 27
CHAPTER 3: DISSOCIATED NEURONS AND GLIAL CELLS DERIVED FROM RAT INFERIOR
COLLICULI AFTER DIGESTION WITH PAPAIN ............................................................. 29
3.1
Abstract
....................................................................................................... 29
4. CHAPTER 4: GENERAL DISCUSSION ..........................................................................31
4.1
Activin A In Combination with BDNF and EPO as potential Drug Delivered by Cells ...31
4.1.1
Short Summary of the Results obtained within the First Part ................................31
4.1.2
Interactions of the different Substances and the (possible) Cross-Talk of their Signalling
Pathways ............................................................................................. 33
4.1.3
4.2
Long-term Factor Application
.................................................................... 40
Establishment of a primary, dissociated Culture of the Rat IC ................................ 42
~I~
Index
5.
4.2.1
Short Summary of the Results obtained within the Second Part ............................ 42
4.2.2
Identification of different Cell Types ............................................................. 44
4.3
Biohybrid Neuroprostheses.............................................................................. 46
4.4
Conclusion .................................................................................................... 48
REFERENCES ........................................................................................................... 51
AFFIDAVIT .....................................................................................................................61
ACKNOWLEDGEMENTS ................................................................................................... 62
~ II ~
List of Abbreviations
LIST OF ABBREVIATIONS
µL
microlitre
µm
micrometre
ABI
auditory brainstem implant
AcvR
activin receptor
AFM
atomic force microscopy
AMI
auditory midbrain implant
BAMBI
BMP and activin membrane-bound inhibitor
BDNF
brain-derived neurotrophic factor
BMP
bone morphogenetic protein
BSA
bovine serum albumin
CaMK
calmodulin-dependent protein kinases
cAMP
cyclic adenosine monophosphate
CI
cochlear implant
CN
cochlear nucleus
CRYPTO
cryptic family protein 1B
d
days
DAPI
4',6-diamidino-2-phenylindole
DMEM
Dulbecco’s Modified Eagle Medium
DMSO
dimethyl sulfoxide
EDTA
ethylenediaminetetraacetic acid
EPO
erythropoietin
FCS
fetal calf serum
Fig.
Figure
GAP-43
growth associated protein 43
GDFs
growth and differentiation factors
GDNF
glial cell line-derived neurotrophic factor
GFAP
glial fibrillary acidic protein
h
hour
HBSS
Hank's Balanced Salt Solution
HC
hair cell
hMSC
human mesenchymal stem cell
~ III ~
IC
inferior colliculi
ICC
central nucleus of the inferior colliculus
IHC
inner hair cell
JAK2
Janus tyrosine kinase 2
JNK
c-Jun N-terminal kinases
kDa
kilo Dalton
L
litre
LLN
lateral leminiscal nuclei
MAG
myelin associated glycoprotein
MAPK/ERK
Mitogen-activated protein kinases/extracellular signal-regulated
kinases
MGB
medial geniculate body
min
minutes
mL
millilitre
mM
millimolar
MOC
medial olivocochlear bundle
n.s.
not significant
NF
neurofilament (200 kDa)
NF2
neurofibromatosis type II
NF-κB
nuclear factor kappa-light-chain-enhancer of activated B cells
ng
nanogram
NGF
nerve growth factor
nm
nanometre
NMDAR
N-methyl-D-aspartate receptor
nN
nano Newton
NT-3
neurotrophin-3
NT-4/5
neurotrophin-4/5
NTDK
Neural Tissue Dissociation Kit
NTF
neurotrophic factors
OHC
outer hair cell
P3
postnatal day 3
p75NTR
pan-neurotrophin receptor (low affinity receptor)
PABI
penetrating auditory brainstem implant
~ IV ~
PBS
phosphate buffered saline
PCD
programmed cell death
PFA
paraformaldehyde
PI3K/Akt
Phosphatidylinositide-3 kinase/Akt
PKA
protein kinase A (cAMP-dependent)
PLC
phospholipase C
rpm
rounds per minute
RT
room temperature
SEM
standard error of mean
SGN
spiral ganglion neurons
SMAD
SMAD is a contraction of SMA: Caenorhabditis elegans protein
SMA from gene sma for small body size and MAD: Mothers Against
Decapentaplegic
SNHL
sensory neural hearing loss
SOC
superior olivary complex
STAT5
signal transducer and activator of transcription 5
Tab.
Table
TGF-β
transforming growth factor-beta
Trk
tyrosin kinase receptor
TUJ1
neuronal class III β-tubulin
U
units
w/o
without
~V~
List of Figures
LIST OF FIGURES
Figure 1-1 Schematic representation of a section through one of the turns of the
cochlea (modified from Bloom and Fawcett, 1975) ............................. 10
Figure 1-2 Overview over the organ of Corti (modified from Fettiplace and
Hackney, 2006) ..................................................................................... 11
Figure 1-3 Schematic illustration of the auditory pathway (modified from Winer
and Schreiner, 2005). ............................................................................14
Figure 4-1 Possible cross-talk of the signalling pathways (figures modified by
Sabine Gebhardt from (Chang et al., 2002; Kumral et al., 2011;
Reichardt, 2006) and rearranged for this thesis) (next page) ............. 35
Figure 4-2 Intrinsic and extrinsic pathway of apoptosis (modified from Tizard,
2004) .................................................................................................... 39
~ VI ~
Summary
SUMMARY
Cell-based drug delivery to optimise the electrode-nerve interface
Odett Kaiser
Hearing loss drastically impairs the quality of life. Restoration of the hearing
sensation with auditory prostheses is emerging as one of the high end technology
therapies in the 20th and 21st century. Despite the advances in technology, several
limitations have been described that are based on biological challenges.
Sensorineural hearing loss is characterized by the loss of sensory cells followed by a
progressive degeneration of spiral ganglion neurons (SGN). The overall aim is thus
the improvement of the preconditions for electrical stimulation - of the remaining
SGN - in auditory implants. In the herein presented thesis, this was implemented by
the identification of novel factors as well as combinatorial approaches for
neuroprotection.
Neuroprotective properties of the growth factor activin A as well as its
corresponding receptors were revealed on SGN for the first time. In order to activate
the intracellular signalling cascade, activin binds to its the dimeric type II receptor.
This binding activates the dimeric type I receptor to form an active tetrameric
complex. Especially when activin A was applied in combination with brain-derived
neurotrophic factor (BDNF) and erythropoietin (EPO), a high percentage of the
seeded neurons (up to 60 %) were prevented from apoptosis with activin A.
Inhibition of the EPO pathway resulted in a neuronal survival comparable to the
double combination (activin A with BDNF). Since EPO does not enhance the
neuroprotective effects of activin A or BDNF when applied as a double combination,
~1~
Summary
we may conclude that the combinatorial code of factors act in a synergistic rather
than in an additive manner.
For electrical stimulation, the insertion of electrodes into the cochlear nucleus
is challenging according to its anatomical localization within the brain stem. By
contrast, the central nucleus of the IC is anatomically easy to distinguish and to
access making the implantation of auditory prosthesis into the midbrain a novel and
interesting alternative. Thence, a primary dissociated culture of the rat inferior
colliculus (IC) was established for the first time, revealing all relevant cell types of the
IC: neurons and glial cells. Such a culture system can serve as basis for investigations
concerning neuroprotection in the central auditory system as well as in order to
reduce gliosis.
For long-term drug delivery of neurotrophic factors, a cell-based application
system can be used. Cells providing the neuronal structures to be excited with
neurotrophic factors can be delivered via the electrode. This has been investigated for
the cochlea in some studies; however, it has not been reported for the central
auditory pathway so far. The factor combination investigated in the present thesis
seems to be a promising candidate for a drug locally delivered by cells. Such cellcoatings of the electrode surface shall disembogue in the development of biohybrid
electrodes furthering the biocompatibility by the creation of a biomimetic surface.
Translating biological and physical modifications of electrode surfaces in the
clinic in the long term, we may be able to enhance the outcome of patients with
cochlear and retrocochlear hearing loss treated with auditory neuroprostheses.
~2~
Zusammenfassung
ZUSAMMENFASSUNG
Zell-basierte
Faktorapplikation
zur
Optimierung
der
Schnittstelle
zwischen Nerv und Elektrode
Odett Kaiser
Eine Hörstörung führt zwangsläufig zu einem Verlust der Lebensqualität. Die
Wiederherstellung des Gehörs mittels auditorischer Neuroprothesen bietet eine der
Therapien mit high-end Technologie des 20. und 21. Jahrhunderts. Trotz der
technologischen Entwicklungen ergeben sich einige Limitationen, die der Klärung auf
biologischer Basis bedürfen. Hochgradiger Schwerhörigkeit liegt ein Verlust des
sensorischen Epithels zu Grunde, welcher wiederum die Degeneration der
Spiralganglienneurone (SGN) bedingt. Das Hauptziel ist daher die Verbesserung der
Voraussetzungen für die elektrische Stimulation u.a. der verbliebenen SGN mittels
auditorischer Implantate.
In dieser Studie wurden erstmals die neuroprotektiven Effekte des
Wachstumsfaktors
Aktivin A
sowie
die
Expression
seiner
korrespondieren
Rezeptoren in SGN gezeigt. Um die intrazellulären Signalwege zu aktivieren, bindet
Aktivin an den dimeren Typ II Rezeptor, welcher wiederum den dimeren Typ I
Rezeptor rekrutiert; zusammen wirken sie als aktives Tetramer. Insbesondere die
Kombination von Aktivin mit brain-derived neurotrophic factor (BDNF) und
Erythropoietin (EPO) konnte ein signifikanter Prozentsatz der eingesäten Neurone
(bis zu 60 %) vor Apoptoseprozessen geschützt werden. Die Hemmung des
Signalweges von EPO führte zu einer Reduktion der neuronalen Überlebensraten.
Diese entsprachen denen der kombinierten Therapie mit BDNF und Aktivin A. Da
~3~
Zusammenfassung
EPO in Kombination entweder mit BDNF oder mit Aktivin A keine weitere
Verstärkung der Neuroprotektion erwirkt, kann davon ausgegangen werden, dass
hier ein synergistisches und kein additives Zusammenspiel aller Faktoren vorliegt.
Die Insertion von Elektroden zur elektrischen Stimulation des cochleären
Nukleus stellt auf Grund seiner anatomischen Lokalisation im Hirnstamm eine
Herausforderung dar. Auf Grund einfacher Diskriminierung des inferioren Colliculus
(IC) vom umgebenden Gewebe ist die Lokalisation des zentralen Kerns des IC und
somit eine Elektrodeninsertion eine vielversprechende Alternative. Daher wurde in
der vorliegenden Studie erstmalig eine dissoziierte IC Kultur etabliert, in der alle
relevanten
Zelltypen
Zellkultursystem
enthalten
dient
als
sind:
Basis
Neurone
für
und
Gliazellen.
Untersuchungen
Solch
hinsichtlich
ein
der
Neuroprotektion im zentralen auditorischen System und der Reduktion der
Glianarbe.
Für die Langzeit-Applikation neurotropher Faktoren erscheinen Zell-basierte
Systeme zur Faktorbereitstellung attraktiv. Zellen, die die Zielstrukturen der
neuroprothetischen Implantate mit neurotrophen Faktoren versorgen, können
mittels Elektrode lokal eingebracht werden. Dieser Ansatz wird bereits in einigen
Studien für das Innenohr verfolgt, ist aber bislang nicht für zentrale auditorische
Implantate beschrieben. Die Dreifach-Kombination der Faktoren, die in dieser Arbeit
als besonders neuroprotektiv identifiziert wurden, stellt eine potentielle Substanz
dar, die zellvermittelt verabreicht werden kann. Dieser Ansatz der Zellbeschichtung
von Oberflächen soll in die Entwicklung sogenannter Biohybrid-Elektroden münden.
Die
so
entwickelten
biomimetischen
Oberflächen
können
eine
bessere
Biokompatibilität sowie die Reduktion der Fremdkörperreaktion gewährleisten.
~4~
Zusammenfassung
Werden solche biologischen und physikalischen Oberflächenmodifikationen in
die klinische Anwendung überführt, könnte eine deutliche Verbesserung des Hörens,
welches sowohl mit cochleären als auch mit zentral-auditorischen Neuroprothesen
möglich ist, erzielt werden.
~5~
Zusammenfassung
~6~
Chapter 1: General Introduction
1. CHAPTER 1: GENERAL INTRODUCTION
Generally, about 360 million people (328 million adults and 32 million
children) suffer from hearing loss (WHO, 2013). Treatment of choice for
sensorineural hearing loss is the implantation of an electrode array into the
respective target structure in order to gain hearing sensation. After implantation, a
fibrillary sheath around the electrode acts as isolator and thus, impairs the electrical
stimulation of neurons. An overall aim in hearing research is to enhance the clinical
outcome of implanted patients. The aim of this thesis was to improve the nerveelectrode interface and therefore the preconditions for electrical stimulation. To get
an idea of the underlying pathomechanisms and the treatment options, the anatomy
and physiology of the ear and the ascending auditory pathway as well as researchbased possibilities to improve the clinical outcome are described briefly in the
following subchapters.
1.1
BASIC ANATOMY OF THE EAR AND THE PHYSIOLOGY OF HEARING
Anatomically and physiologically, the ear can be subdivided into the outer,
middle and internal ear.
The outer ear consists of the pinna, concha and the ear canal (external
auditory meatus) and all three anatomical units together are responsible to lead the
air to the ear drum (tympanic membrane). The pinna and concha reflect sound waves
into the ear canal and provide cues for the localization of the sound source. These
cues depend on the intensity and timing differences when a sound wave reaches the
left and the right ear. The localization cues are limited when the sound source is
located behind, above or below the midline of both ears. The second major function
~7~
of the outer ear is the increase of sound pressure at the tympanic membrane
throughout the major resonance of the concha and the external ear canal. This
increase enhances the frequency dependent efficiency of the energy transfer to the
middle ear (Pickles, 2008).
The middle ear is located in the temporal bone and consists of the tympanic
membrane and the ossicular chain (malleus, incus and stapes), which is located in the
middle ear cavity. The malleus is coupled on one side to the tympanic membrane and
on the other side to the incus. Both ossicles (malleus and incus) create a rigid
connection and transfer the force coming from the ear drum to the stapes. The
footplate of the stapes is attached to the flexible wall of the oval window of the inner
ear and therefore, the ossicles connect the outer with the internal ear. The middle ear
is also connected with the pharynx via the Eustachian tube (Nickel et al., 2004). In
many small animals, there is also a bony bulge, called bulla, an enlarging cavity of the
middle ear and increasing the response to low-frequency sounds. The major function
of the middle ear is the adaptation of the impedance, which means that the sound
energy from the external auditory meatus (outer ear) is coupled to the fluid-filled
cochlea (internal ear). This impedance adaptation is based on two principles: (1)
increase of the pressure of the sound waves (by decreasing the area of action:
tympanic membrane is larger than the footplate of the stapes) and (2) the lever action
of the ossicles that increases the force of the sound and decreases the velocity of the
stapes. The transmission of the sound waves also depends on the frequency of the
stimulus (the most efficient transmission is around 1-2kHz) and can be modified by
on the middle ear muscles (M. tensor tympani and M. stapedius) whose contraction
stiffens the ossicular chain (Pickles, 2008).
~8~
The internal ear (vestibulocochlear organ; inner ear) is also called labyrinth
due to its fluid-filled sacs and tubules, which are suspended in cavities.
Physiologically, the internal ear is responsible for the perception of acoustic stimuli
(acoustic organ) as well as for the equilibrium (vestibular system). It is located in the
petrous portion of the temporal bone. The membranous labyrinth with its
corresponding, fluid-filled saccules and tubules lines the bony or osseous labyrinth.
In the bony labyrinth, two major cavities can be divided: the vestibule and the cochlea
with its organ of Corti (Bloom and Fawcett, 1975).
The cochlea is spirally coiled around its axis: the modiolus. The modiolus is a
spongy bone containing blood vessels as well as the somata and central processes of
nerve fibres of the auditory nerve. The lumen of the bony labyrinth is divided
longitudinally by membranes into three compartments (scala vestibuli, scala media
and scala tympani; cf. Fig. 1-1). The scala vestibuli and scala media (cochlear duct)
are separated by the Reissner’s membrane (vestibular membrane). The basilar
membrane (composed of the osseus and membranous spiral lamina) defines the
border between the scala tympani and scala media. The scalae vestibuli et tympani
are perilymphatic (like normal cellular fluid) spaces and both communicate at the
apex (helicotrema). The scala media is filled with endolymph, a positive charged fluid
and this positive potential is driven by active Na+/K+-and active N+/2Cl-/K+- ion
pumps in the stria vascularis (Bloom and Fawcett, 1975; Pickles, 2008).
~9~
Figure 1-1 Schematic representation of a section through one of the turns of the cochlea
(modified from Bloom and Fawcett, 1975)
The organ of Corti is located upon the basilar membrane, in the cochlear
duct. It contains the receptor cells called hair cells (HC) and various supporting
cells. There is one row of inner hair cells (IHC) and between three and (towards the
apex) five rows of outer hair cells (OHC). On the top of the HCs, V-shaped (OHC) or
straight (IHC) rows of stereocilia are projecting through the reticular membrane (see
below; cf. Fig. 1-2). Though misleading, the term stereocilia is commonly used.
However, histologically precisely stereocilia should be referred to as stereovilli since
they contain actin filaments rather than microtubuli. About three rows of stereocilia
are anchored in the cuticular plate of one OHC and the shorter cilia are connected at
~ 10 ~
their top with tip-links to the next bigger one. The tallest cilium on one HC is located
distal from the modiolus. At the basis of the HCs, nerve endings are located. These
terminals are associated with the afferent fibres of the auditory nerve. The several
types of supporting cells are tall, slender cells extending from the basilar
membrane to the free surface of the organ of' Corti forming with their endings the
reticular lamina. The supporting cells include inner and outer rod/pillar cells, inner
and outer phalangeal cells (outer phalangeal of Deiters), border cells, cells of Hensen,
Claudius cells and Boettcher cells. The organ of Corti is covered by the tectorial
membrane, a gelatinous and fibrous flap (cf. Fig. 1-1, 1-2). Only the longest hairs of
the OHCs but not the IHCs are embedded in the tectorial membrane (Bloom and
Fawcett, 1975; Pickles, 2008).
Figure 1-2 Overview over the organ of Corti (modified from Fettiplace and Hackney,
2006)
A transverse section through a middle turn of the cochlea is depicted. The lateral wall is located
on the right side. When the travelling wave occurs, the basilar membrane gets displaced from the
hinge point to the lateral wall. Caused by the displacement, the lateral part of the organ of Corti is
elevated and thus shearing occurs between the tectorial membrane and the hair bundles.
~ 11 ~
The vibration of the stapes is transmitted through the oval window opening
into the scala vestibuli. Hence, the cochlear fluids get dislocated and a progressive,
wave-like displacement (travelling wave pattern) of the basilar membrane is
produced. This displacement depends on the physical properties (stiffness and mass)
of the basilar membrane: The distance (length of the basilar membrane) from the
organ of Corti to the spiral prominence is short (near) at the base and wide at the
apex. This results in a stiffness and a mass gradient from the base (thinless mass
but high stiffness) to the apex (widehigh mass but less stiffness). Thus, high
frequencies are represented at the base and low frequency stimuli near the apex. Due
to its tonotopic organization, the maximal displacement of the basilar membrane is
reached at the characteristic frequency of the stimulus with a sharp tuning.
Throughout this displacement, shearing forces occur between the tectorial membrane
and the organ of Corti. Thus, the stereocilia of the HCs are deflected and tip links get
opened. This process couples the stimulus-induced movements to the actual
mechanotransducer channels in the membrane and the mechanical signal is
converted in a chemical and electrical signal: opening of the tip-links leads to K+influx along the potential gradient into the HCs with a negative intracellular resting
potential and the HCs depolarise. This causes the release of neurotransmitter and
therefore activation of auditory nerve fibres. Although both HC-types send afferences
to the auditory nerve fibres, the OHCs act as mechanical amplifier, whereas the IHCs
transmit the electrical signal due to their sharp tuning curves.
The afferent fibres convey auditory information to the central nervous
system along the auditory pathway. The cell bodies of the afferences are located in the
spiral ganglion (located in the modiolus, cf. Fig. 1-2). These spiral ganglion neurons
~ 12 ~
(SGN) project with one process to the HCs and with one to the cochlear nucleus.
About 90 – 95 % bipolar neurons connect directly with the IHCs and have thick,
myelinated fibres (type I or radial fibres). Approximately 20-30 type I fibres project
to one IHC. The remaining 5 – 10 % fibres (type II or outer spiral fibres) are thin and
unmyelinated with monopolar cell bodies and form the outer spiral bundle. About six
fibres innervate one OHC and each type II neuron connects up to 50 OHC (Pickles,
2008).
Efferent fibres arise in the brainstem (periolivary neuclei of the superior
olivary complex, SOC). The number of efferences innervating one HC declines from
the base to the apex and surround the base of the HC with its afferent terminal. The
olivocochlear system can be divided into the medial olivocochlear bundle (MOC;
projecting to the OHCs from the contralateral side) and the lateral olivocochlear
bundle (LOC; projecting to the ipsilateral IHCs) (Pickles, 2008).
1.2
AUDITORY PATHWAY - FROM THE COCHLEA TO THE BRAIN
After transduction of the mechanical input to an electrical signal in the organ
of Corti, the information of the auditory signal (decoded in duration, intensity and
frequency) is transmitted centrally via the spiral ganglion neurons (SGN) forming the
auditory nerve. The auditory pathway from the cochlea to the brain is shown in Fig. 13.
~ 13 ~
Figure 1-3 Schematic illustration of the auditory pathway (modified from Winer and
Schreiner, 2005).
For simplicity, only the major ascending connections are shown. Local circuit arrangements and
descending connections have been omitted. The major regions, which are investigated within this
thesis, are highlighted in pink.
In short, the first central unit is the cochlear nucleus (CN) in the
brainstem. It is responsible for the genesis of basic response patterns and emergence
of parallel pathways. There is also an array of descending projections from higher
order auditory structures providing feedback. From the CN, the information is send
to the SOC in the brainstem, where binaural pathways are constructed and the
establishment of timelines occurs. The processed information is transmitted to the
pons, where the lateral leminiscal nuclei (LLN) are located and to the midbrain,
where the inferior colliculus (IC) acts as a converging centre. The ascending input
~ 14 ~
is then send to the medial geniculate body (MGB; nucleus geniculate) in the
thalamus. There, the auditory information is modulated by the cortical and the limbic
system before it is processed in the auditory cortex that is located in the temporal
area within the lateral sulcus. Along the way upwards to the cortex, most of the
information is projected to the contralateral ear, whereas also information is
transmitted to the ipsilateral ear (Pickles, 2008; Winer and Schreiner, 2005).
The tonotopical organization of the processing structures and therefore
also the presentation of the stimuli is preserved from the cochlea up to the auditory
cortex. For extensive information about the auditory pathway the reader is referred to
Winer and Schreiner (2005) or Popper and Fay (1992).
1.2.1
The Inferior Colliculus – The Auditory Convergence Centre
The inferior colliculi (IC) are located in the midbrain and can be identified as
the two caudal prominences of the corpora quadrigemina (quadruplet bodies). The
superior colliculi form the anterior pair of the corpora quadrigemina and display a
large visual reflex centre, whereas the IC represent an auditory processing,
transmission and reflex centre: it is a convergence centre for the most ascending
auditory projections. Ventrally, beneath the IC, the tegmental brainstem (pedunculi
cerebri) is located.
Along the neuronal architecture, the IC can be divided into three parts: the
central nucleus, the external nucleus and the dorsal cortex. Those parts have
different innervation patterns and as a result have also different function.
The central nucleus (ICC) is the essential element in the converging sending
projections to the LLN. The ICC has a laminar structure formed by the afferent axons
and the dendrites of the intrinsic neurons. Disk-shaped neurons arborising their
~ 15 ~
dendrites along the laminar plane dominate the histology. The remaining cells are
represented by stellate cells with spheric or ovoid dendritic fields (cross-over of
multiple laminae), which are acting as interneurons within the nucleus. The
tonotopic organization is also consistent within the IC(C): the different laminae
correspond to different iso-frequency planes (low frequencies dorsal, high
frequencies ventral).
The external nucleus receives input from the contralateral CN and the central
IC as well as somatosensory input from the trigeminal nuclei. The dorsal cortex
receives input from the contralateral IC and descending inputs from the auditory
cortex. Although both nuclei tend to be broad tuned, they are also tonotopically
organized. The dorsal cortex is driven monaurally and the external nucleus binaurally
(Pickles, 2008; Winer and Schreiner, 2005). For more detailed information the
reader is referred to Winer and Schreiner (2005).
1.3
CLASSIFICATION OF HEARING LOSS
Hearing loss or deafness is a disease affecting one or both ears with an
increasing incidence in industrial countries. It can be classified as mild, moderate,
severe or profound. The causes are various. According to the location of occurrence,
hearing loss can be divided (in the periphery) into two major forms: conductive and
sensorineural hearing loss.
Conductive hearing loss occurs when the outer or middle ear are affected
in their normal physiology. The problem can occur in every crucial part up to the
inner ear e. g. the blockage of the outer ear canal, rupture of the tympanic membrane
or immobilized ossicles. These problems result in an impaired coupling to the inner
ear. Thus, there is a frequency-dependent attenuation of the stimulus (Pickles, 2008).
~ 16 ~
Conductive hearing loss, if not possible to be treated according to the aetiology (e. g.
removal of obstruction, ossicular chain reconstruction, stapedotomy) can be
compensated by normal hearing aids or even implantation of prosthesis recovering
the amplification of the ossicular chain.
When the inner ear or parts of the ascending auditory pathway are affected,
sensorineural hearing loss (SNHL) arises. SNHL can be further subdivided
according to the location of the impairment in cochlear or retrocochlear/central
hearing loss.
In the cochlea, SNHL is morphologically characterized by the loss of IHC
followed by a progressive degeneration of SGN. This can be caused by several
aetiologies: genetically driven (congenital), complications during pregnancy and
birth, infectious diseases, ototoxic agents, injury/trauma, noise or age-related
(presbyacusis) (WHO, 2013). Treatment of choice is the implantation of an auditory
prosthesis. If the problem is located in the cochlea (profound or severe SNHL), the
patient gets implanted with an electrical array into the cochlea (cochlear implant
device; CI) in order to stimulate remaining HC in a distinct manner (cf. 1.4).
Central prostheses are the treatment of choice for retrocochlear or central
hearing loss, when the cochlea can no longer act as an implant target (cf. 1.6).
1.4
SENSORINEURAL HEARING LOSS IN THE COCHLEA - CI-RESEARCH
The majority of the hearing impaired patients suffer from sensorineural
hearing loss (SNHL). Worldwide, as of December 2010, approximately 219,000
hearing impaired people were fitted with a CI (NIDCD, 2011).
Benefits from the CI vary among the patients and can be affected by the
number of functional SGN stimulated by the CI (Gantz et al., 1993; Nadol et al., 1989;
~ 17 ~
Xu et al., 2012). The general goal is to enhance the electrode-nerve-interface and
hence the outcome of the implantation. One way to reach this goal is to influence the
cell behaviour by modifying surface properties of the CI. These surface properties
may be altered by mechanical/physical patterning (Paasche et al., 2009; Reich et al.,
2008; 2012) and by chemical (Yue et al., 2011), biochemical or biological
modification of the surface of the CI electrode (consisting of silicone as carrier and
platinum for the contacts). Biochemical modification includes the coating of the
implant surface with pharmacologically active substances to achieve neuroprotection,
reduction of inflammation or cell adhesion (Bohl et al., 2012; Ramekers et al., 2012),
whereas biological modification aims for the sustained production of factors by cells
after cell coating of the implant surface (Rejali et al., 2007; Warnecke et al., 2012).
Furthermore, pharmacologically active substances may be administered
directly intra operationem or over a limited period via osmotic pumps (Agterberg et
al., 2008; Scheper et al., 2009). However, pump based delivery systems increase the
surgical complexity. In addition, the stability of delivered factors, if stored
subcutaneously in mini-osmotic pumps, is imperilled. Viral vectors may be also used
to transfect host cells for endogenous factor production (Kanzaki et al., 2002;
Lalwani and Mhatre, 2003; Nakaizumi et al., 2004), albeit they involve potential
toxicity and infection.
1.5
NEUROTROPHIC FACTORS - PHARMACOLOGICALLY ACTIVE SUBSTANCES
Nevertheless, a standard procedure to increase neuronal protection and to
gain neuronal regeneration is the application of neurotrophic factors (NTF).
Several families of NTF have been described: e.g. neurotrophins, fibroblast growth
factor (FGF) family and transforming growth factor-beta (TGF-β) superfamily.
~ 18 ~
The neurotrophic effect of NTF on SGN can be enhanced by combined
electrical stimulation in deafened guinea pigs (Chikar et al., 2008; Kanzaki et al.,
2002; Scheper et al., 2009; Shepherd et al., 2005; Yamagata et al., 2004), making
NTF-application together with the implantation an attractive option to enhance the
performance of the CI.
1.5.1
Neurotrophins
Neurotrophins consist
of
nerve growth factor
(NGF),
brain-derived
neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4/-5 (NT-4/5). They are ubiquitously expressed and secreted by neurons, glial cells, sensory cells
and muscle fibres (reviewed in Ramekers et al., 2012). Neurotrophins transmit their
actions through corresponding high affinity (tyrosine kinase) receptors (Trk) and one
low affinity receptor (p75NTR): NGFTrkA, BDNF (also NT-4/5, NT-3)TrkB and
NT-3TrkC (Hansen et al., 2001; Ramekers et al., 2012). To a lesser extent, binding
to TrkA and TrkB has also been observed for NT-3 (cf. Fig. 4-1) (Hansen et al., 2001;
Ramekers et al., 2012). Even though expression of the neurotrophins and their
receptors varies during development and adulthood (Gillespie and Shepherd, 2005;
Ramekers et al., 2012), BDNF and NT-3 and their high affinity receptors (TrkB and
TrkC) are highly expressed whereas NGF, NT-4/5 and their corresponding receptors
as well as the p75NTR are absent during development. By contrast, only NT-3 and all
high affinity receptors (TrkA, TrkB and TrkC) are expressed in adults, whereas NGF,
BDNF and NT-4/-5 are absent (reviewed in Ramekers et al., 2012). A plethora of
studies were performed investigating the impact of neurotrophins in the inner ear: In
particular, BDNF and NT-3 were tested in vitro and in vivo to prevent SGN from
degeneration (for review see Ramekers et al., 2012).
~ 19 ~
Exemplary for BDNF, the intracellular signalling will be described briefly: after
binding
to
its
corresponding
TrkB
receptor,
effects
are
mediated
via
phosphatidylinositide-3 kinase/Akt (PI3K/Akt) signalling, mitogen-activated kinase
pathways (MAPK/ERK) and phospholipase C (PLC; cf. Fig. 4-1) (reviewed in Gupta et
al., 2013).
1.5.2
Transforming Growth Factor - beta Superfamily
The transforming growth factor-beta (TGF-β) superfamily can be divided
based on their structural features into the TGF-β family, activins/inhibins, bone
morphogenetic proteins (BMPs)/growth and differentiation factors (GDFs) and the
glial cell line-derived neurotrophic factor (GDNF) family members (Weiskirchen et
al., 2009).
1.5.2.1
TGF-β superfamily-member: Glial Cell Line-Derived Neurotrophic Factor
Family
The GDNF family members are GDNF, neurturin, persephin and artemin
(Weiskirchen et al., 2009).
Within the inner ear, besides the neurotrophins, only GDNF family members
like GDNF (Boström et al., 2010; Fransson et al., 2010; Liu et al., 2008) and to a
lesser degree artemin (Warnecke et al., 2010; Wissel et al., 2006) have been
investigated thoroughly in vitro and in vivo so far.
1.5.2.2 TGF-β superfamily-member: Activins
The expression of several other receptors for NTF in SGN encourages the
search for new factors in order to induce more suitable or tailored effects on the
target cells. For example, in the mammalian inner ear, SGN express activin receptor
~ 20 ~
IIA (AcvR IIA), whereas the presence of the activin receptor IIB (AcvR IIB) is limited
to the inner and outer pillar cells of the organ of Corti (McCullar et al., 2010).
Although the corresponding receptors are represented in the cochlea, the role of
activins in the mammalian inner ear still remains to be elucidated (cf. 1.5.4, 1.7 and
Chapter 2).
Generally, activins are important regulators and key elements in hormone
secretion, regulation of developmental events and apoptosis, and the repair of various
tissues and organs (reviewed in Ageta and Tsuchida, 2011; Werner and Alzheimer,
2006). Besides this, it was also shown that activin A can induce neuroprotection as
well as neuroregeneration (e.g. induction of neuronal differentiation in a
neuroblastoma cell line; Suzuki et al., 2010). Shortly after treatment with kainic acid,
activin A is also upregulated in the hippocampus (Tretter et al., 1996). In addition, it
is known to mediate the neuroprotective effects of basic FGF in vivo (Tretter et al.,
2000). During embryonic development, activins are expressed in the central nervous
system and exert neuroprotective effects (Tsuchida et al., 2009).
Activins were first discovered as inducers of follicle-stimulating hormone
(FSH) release (Ling et al., 1986; Vale et al., 1986). Historically, they were named as
inhibins, which are heterodimers either composed of αβA (inhibin A) or αβB chains
(inhibin B) (Vale et al., 1990). By contrast, activins are dimeric peptides with a lack of
an α-subunit and composed of βA-, βB-, βC- or βE- chains cross-linked via disulphide
bridges. Three different forms of activin exist in various tissues: activin A (βA βA),
activin AB (βA βB), activin B (βB βB) (Tretter et al., 1996; Vale et al., 1990; Werner and
Alzheimer, 2006) whereas βC- and βE- subunits are predominantly expressed in the
liver (Tsuchida et al., 2009). The biological effect is mediated by binding to a
transmembrane serine/threonine-kinase type II-receptor (AcvR IIA or AcvR IIB).
~ 21 ~
After binding, this complex recruits the membrane-associated type I-receptor via
phosphorylation
(AcvR IB/Alk4,
AcvR IA/Alk2;
de
Caestecker,
2004).
The
intracellular signalling is mediated either via a SMAD-dependent or a SMADindependent pathway (cf. Fig. 4-1; SMAD is a contraction of SMA: Caenorhabditis
elegans protein SMA from gene sma for small body size and MAD: Mothers Against
Decapentaplegic). The SMAD-dependent intracellular signalling cascade includes
recruitment of receptor SMADs (SMAD2 and SMAD3), which translocate into the
nucleus and multimerise with Co-SMAD (SMAD4). This Co-SMAD modulates as
transcription factor complex the expression of a large variety of genes. Beside the
SMAD-dependent pathway, SMAD-independent pathways like the p38 MAPK and
JNK (c-Jun N-terminal kinases) of the MAPK/ERK can be also activated (Chen et al.,
2006; de Caestecker, 2004; Suzuki et al., 2010; Tsuchida et al., 2009; Werner and
Alzheimer, 2006; Xia and Schneyer, 2009).
1.5.3
Erythropoietin
Erythropoietin (EPO), the key modulator of erythropoiesis (Brines and
Cerami, 2005; Goldwasser et al., 1990), is a glycoprotein with a molecular weight of
30.4 kDa. Mainly synthesized and expressed in the kidney in adults. Within the fetal
system, a significant amount of this protein is also produced by the liver (Kumral et
al., 2011). Surprisingly, EPO is also expressed in the brain, suggesting its involvement
in neuronal processes including embryonic development. It has been shown that EPO
is released in the nervous system after hypoxic damage (Genc et al., 2011). Its
receptor is distributed on nearly all cell types of the brain (neurons, astrocytes,
endothelial cells and microglia). The expression of EPO receptors in the guinea pig,
our preferred animal model for the testing of neurotrophic effects in vivo, was
~ 22 ~
reported by Cayé-Thomasen (2005). Despite its negative publicity as doping
substance, EPO is one the most promising emerging neurotrophic factors (Genc et al.,
2011), since it effectively can inhibit apoptosis. Other attributes of EPO include antiinflammatory, anti-oxidative, angiogenic and anti-epileptic.
Major intracellular pathways that are activated by EPO in order to exert its
abundant effects have been identified. After binding to its receptor, EPO activates at
least three intracellular signalling cascades (cf. Fig. 4-1): (1) Janus tyrosine kinase 2
(JAK2)/signal transducer and activator of transcription 5 (STAT5), (2) PI3K/Akt, and
(3) RAS/MAPK (Marzo et al., 2008). Thus, inhibition of EPO can be achieved e.g. by
administering of JAK2 inhibitors like AG490 or JAK2 inhibitor II (Sandberg et al.,
2005). From previous experiments, we expected a selective effect upon the neurite
outgrowth of SGN mediated by EPO and even enhanced after the addition of BDNF
(Berkingali et al., 2008).
1.5.4
Known Interactions of the different Substances
Known effects of activin A upon EPO, of EPO upon BDNF as well as shared
features of activin A and BDNF are described in subchapter 2.2. To the best of our
knowledge, nothing is known about the action of the combined application of all
three factors. Thus, within the first study of this thesis, the effects of activin A, BDNF
and EPO individually and in combination were investigated (Chapter 2). Possible
underlying mechanisms are discussed the Discussion of chapter 2 as well as in
subchapter 4.1.2.
~ 23 ~
1.6
AUDITORY MIDBRAIN IMPLANT
IS NO IMPLANT OPTION
AND
INFERIOR COLLICULI: WHEN
THE
CI
Central prostheses are implanted when hearing cannot be restored via a CI.
This applies especially for people with bilateral vestibular schwannomas in
association with neurofibromatosis type 2 (NF2), severe post-inflammatory
ossification of the cochlea and modiolus or bilateral temporal bone fractures
(reviewed in Shannon, 2012). In these cases, prostheses stimulating the central
auditory pathway are necessary to gain auditory perception. The auditory brainstem
implants (ABI) and the penetrating auditory brainstem implants (PABI) are used to
stimulate the cochlear nucleus (CN) (Hendricks et al., 2008; Shannon, 2012). For the
stimulation at a higher level within the central auditory pathway proximal to a
damaged cochlear nucleus, the IC can be chosen as target for a central auditory
prosthesis: the auditory midbrain implant (AMI). Further details are given in the
Introduction of Chapter 3.
1.7
AIMS FOR THE THESIS
Within the first study (Chapter 2), the general aim was to improve the
preconditions for electrical stimulation via a CI in the cochlea by application of
factors and therewith to optimize the electrode-nerve interaction. Thus, we examined
the impact of activin A on SGN in vitro as a potential factor for (cell-based) drug
delivery. Furthermore, the application of activin A, EPO and BDNF individually or in
combination were also tested to modulate the effects achieved by activin A. This
combined treatment may increase the number of surviving neurons and decrease the
anatomical distance between neurons and CI by the induction of neurite outgrowth
towards the implant.
~ 24 ~
Within the second study (Chapter 3), the aim was to establish a dissociated
culture of the inferior colliculus, which is an implant target when the CI is no implant
option for hearing restoration. This dissociated culture can act as a fundamental test
system e.g. for identification of suitable substances (or neurotrophic factors) which
can modulate the nerve-electrode interaction as well as to test alteration of the
surface of the electrode.
~ 25 ~
~ 26 ~
6BChapter 2: TGF-Beta Superfamily Member Activin A
2. CHAPTER 2: TGF-BETA SUPERFAMILY MEMBER ACTIVIN A
AND
ACTS WITH
BDNF
ERYTHROPOIETIN TO IMPROVE SURVIVAL OF SPIRAL GANGLION NEURONS
IN VITRO
Odett Kaiser1, Gerrit Paasche1, Timo Stöver2, Stefanie Ernst3, Thomas Lenarz1,
Andrej Kral1, Athanasia Warnecke1*
1Department
2Department
of Otolaryngology, Hannover Medical School, Hannover, Germany
of Otolaryngology, University Hospital, Johann Wolfgang Goethe
University, Frankfurt am Main, Germany
3Institute
for Biometry, Hannover Medical School, Hannover, Germany
*Corresponding
author
This manuscript has been accepted for publication in Neuropharmacology and can
be found: Neuropharmacology. 2013 Aug 22;75C:416-425.
Science direct: http://dx.doi.org/10.1016/j.neuropharm.2013.08.008
2.1
ABSTRACT
Activins are regulators of embryogenesis, osteogenesis, hormones and
neuronal survival. Even though activin receptor type II has been detected in spiral
ganglion neurons (SGN), little is known about the role of activins in the inner ear. An
activin-mediated neuroprotection is of considerable clinical interest since SGN are
targets of electrical stimulation with cochlear implants in hearing impaired patients.
Thus, the presence of activin type-I and type-II receptors was demonstrated
immunocytochemically and the individual and combined effects of activin A,
~ 27 ~
6BChapter 2: TGF-Beta Superfamily Member Activin A
erythropoietin (EPO) and brain-derived neurotrophic factor (BDNF) on SGN were
examined in vitro.
SGN isolated from neonatal rats (P 3-5) were cultured in serum-free medium
supplemented with activin A, BDNF and EPO. Compared to the negative control,
survival rates of SGN were significantly improved when cultivated individually with
activin A (p < 0.001) and in combination with BDNF (p < 0.001). Neither neurite
outgrowth nor neuronal survival was influenced by the addition of EPO to activin Atreated neurons. However, when all three factors were added, a significantly
(p < 0.001) improved neuronal survival was observed (61.2 ± 3.6 %) compared to
activin A (25.4 ± 2.1 %), BDNF (22.8 ± 3.3 %) and BDNF+EPO (19.2 ± 1.5 %). Under
the influence of the EPO-inhibitors, this increase in neuronal survival was blocked.
Acting with BDNF and EPO to promote neuronal survival in vitro, activin A presents
an interesting factor for pharmacological intervention in the inner ear. The present
study demonstrates a synergetic effect of a combined therapy with several trophic
factors.
Keywords: activin, brain-derived neurotrophic factor, erythropoietin, spiral
ganglion neurons, inner ear
~ 28 ~
7BChapter 3: Dissociated Neurons and Glial Cells derived from Rat Inferior Colliculi
3. CHAPTER 3: DISSOCIATED NEURONS
AND
GLIAL CELLS
DERIVED FROM
RAT
INFERIOR COLLICULI AFTER DIGESTION WITH PAPAIN
Odett Kaiser, Pooyan Aliuos, Kirsten Wissel, Thomas Lenarz, Darja Werner, Günter
Reuter, Andrej Kral, Athanasia Warnecke*
Department of Otolaryngology, Hannover Medical School, Hannover, Germany
*Corresponding author
This manuscript has been accepted for publication in PLOS One and can be found
from the 5th December 2013 onwards on the homepage of the journal
(http://www.plosone.org/).
Science direct: http://dx.doi.org/10.1371/journal.pone.0080490
3.1
ABSTRACT
The formation of gliosis around the electrode impairs electrode-tissue-
interaction and thus, hearing sensation with central auditory prostheses like the
brainstem implant and the midbrain implant. Altering of material properties can
hinder unspecific growth of glial tissue around the electrode. In vitro screening of this
tissue-material-interaction requires an adequate cell culture system. Hitherto, an
adequate in vitro model utilizing dissociated cells has not been described for the IC
and was thus aim of this study. Therefore, IC were isolated from neonatal rats (P3-5).
In screening experiments using four dissociation methods (Neural Tissue
Dissociation Kit [NTDK] T, NTDK P; NTDK PN and a validated protocol for the
dissociation of spiral ganglion neurons [SGN]), the optimal media and seeding
~ 29 ~
7BChapter 3: Dissociated Neurons and Glial Cells derived from Rat Inferior Colliculi
densities were identified. Thereafter, a dissociation protocol containing only the
proteolytic enzymes of interest (trypsin or papain) was tested. For analysis, cells were
fixed and immunolabelled using glial- and neuron-specific antibodies. Adhesion and
survival of dissociated neurons and glial cells isolated from the IC were demonstrated
in all experimental settings. Hence, preservation of type-specific cytoarchitecture
with sufficient neuronal networks only occurred in cultures dissociated with NTDK P,
NTDK PN and fresh prepared papain solution. However, cultures obtained after
dissociation with papain, seeded at a density of 2 x 104 cells/well and cultivated with
Neuro Medium for 6 days reliably revealed the highest neuronal yield with excellent
cytoarchitecture of neurons and glial cells. In order to minimize reactive gliosis
formation around the implantation site and potentially for a more effective hearing
restoration, the herein described dissociated culture can be utilized as in vitro model
to screen interactions between cells of the IC and surface modifications of the
electrode.
Keywords: inferior colliculus, auditory midbrain implant, neuron-tissueinteraction, trypsin, papain, primary culture
~ 30 ~
Chapter 4: General Discussion
4. CHAPTER 4: GENERAL DISCUSSION
The aim of this thesis was to improve the preconditions for electrical
stimulation in auditory implants. This was implemented on the one hand by the
identification of activin A as an agent with neuroprotective properties on SGN,
especially when applied in combination with BDNF and EPO. These factors could be
used for the establishment of a local cell-based drug delivery. On the other hand, a
primary, dissociated culture of the rat IC was established serving as basis for
investigations on the cell-material-interaction such as cytotoxicity, adhesion,
proliferation and inflammation, on intercellular interactions and on neuroprotection
in order to reduce gliosis and improve the preconditions for electrical stimulation
with central auditory prosthesis.
Parts of the discussion, which are not explicitly described within the herein
presented chapter, can be found in detail in the Discussion of Chapter 2 and 3,
respectively. Especially the results from the establishment of the primary culture are
extensively discussed in the discussion of Chapter 3. Therefore, only additional
aspects that have not been included in the submitted manuscripts have been
discussed in detail.
4.1
4.1.1
ACTIVIN A IN COMBINATION
DELIVERED BY CELLS
WITH
BDNF
AND
EPO
AS POTENTIAL
DRUG
Short Summary of the Results obtained within the First Part
Within the first part of the thesis (Chapter 2), we could demonstrate the
presence of both types of the activin receptors (AcvR IIA-B and AcvR IA-C) in
postnatal rat spiral ganglion neurons for the first time (Fig. 2-1). A different
~ 31 ~
expression pattern for type II receptors (AcvR IIA in spiral ganglion cells, whereas
AcvR IIB expression is limited to inner and outer pillar cells) was reported previously
for adult murine inner ears by McCullar and colleagues (2010). Thus, differences
between neonatal and adult inner ears must be considered.
Activin A can exert its effects via binding to its receptors AcvR IIA or IIB and
the neuroprotective effect of activin A upon SGN is proven in our dissociated
cultures. This neuroprotective effect of activin A can be enhanced substantially (up to
the 2.7-fold) with the combined application of all three factors (activin A, BDNF and
EPO). This is of particular importance, since there is the possibility of
neuroprosthetic intervention through cochlear implants when the improvement of
SGN survival is of high clinical relevance.
Although application of activin A, BDNF and EPO as triple combination
providing a combinatorial code that allows for a significant enhanced neuroprotective
effect, this was not the case if EPO was applied only in a double combination with
BDNF or activin A. In order to investigate the role of EPO in the increase in neuronal
survival after treatment with all three factors and to rule out the influence of the
endogenous EPO-production, cultures were exposed to the following EPO-inhibitors:
AG490 and JAK2 inhibitor II. When applying the triple combination, inhibition of
the EPO-receptors resulted in a neuronal survival comparable to the double
combination (activin A with BDNF). Thus, it seems that the combined application of
factors acts synergistically in order to promote survival in SGN and that this increase
is EPO dependent. Interestingly, the neuronal outgrowth is unaffected by the use of
EPO-inhibitors in the cultures supplemented with EPO in combination with activin
and BDNF.
~ 32 ~
In previous studies the significant influence of EPO upon the neurite
outgrowth of SGN was demonstrated without affecting cell survival (Berkingali et al.,
2008). Contrary to what we expected from previous studies, only a tendency towards
longer neurites was observed in the present study due to EPO. Methodological
differences can be the cause for the lack of significance within the present study (cf.
2.5).
4.1.2 Interactions of the different Substances and the (possible) CrossTalk of their Signalling Pathways
4.1.2.1 Inhibition of Activins
Since activin exerts its main effects after binding to and activating the
AcvR IIA or IIB, the biological effects can be controlled by various substances:
Follistatin binds activin and acts as an inhibitor (Nakamura et al., 1990), whereas
BAMBI is a membrane-bound bone morphogenic protein (BMP) - and activin
receptor inhibitor (BMP and activin membrane-bound inhibitor) (Xia and Schneyer,
2009) since it acts as a pseudoreceptor. CRYPTO (cryptic family protein 1B) forms an
inert complex with activin and AcvR IIA and therefore prevents the activation of
type-I receptors (Kelber et al., 2008; Onichtchouk et al., 1999). Also, chimeras of the
receptors (McCullar et al., 2010) compete with the homologous endogenous receptors
and are therefore possible inhibitors of downstream signalling. Inhibin and other
TGF-β family proteins are structurally similar and can also bind to the AcvR II. SB431542 leads to a dose-dependent inhibition of Alk4 (Suzuki et al., 2010). Inhibition
of members of the intracellular signalling cascade (Kurisaki et al., 2008; Xia and
Schneyer, 2009) is also a possibility to clarify the various intracellular pathways
which can be activated after application of different substances.
~ 33 ~
4.1.2.2 Possible Cross-Talk of the “main” Pathways
Neuroprotective effects observed after application of the factors as a triple
combination seem to be synergistic rather than additive. After binding to its
receptors, the molecular pathways activated by BDNF, EPO and activin are diverse
and may be shared by all three factors: Intracellular downstream signalling of activin
comprises a SMAD-dependent as well as a SMAD-independent cascade (p38 MAPK
and JNK of the MAPK pathways, cf. 1.5.2.2, Fig. 4-1). The latter one seemed to be
mainly responsible for the mediation of neuronal differentiation and survival (Suzuki
et al., 2010) as well as for NMDAR activation (by Ca2+ influx) in hippocampal
neurons (Kurisaki et al., 2008). As described in subchapter 2.5, the MAPK pathways
can also be activated by neurotrophins. Particularly for BDNF, cellular effects can be
attributed to different MAPK/ERK downstream cascades: In neurons, neurite
development is mediated by activation of MAPK/ERK signalling pathways, whereas
synaptic plasticity is triggered by PLC downstream signalling. Activation of PI3K
promotes growth and survival of cells (reviewed in Gupta et al., 2013). Especially in
SGN, survival is mediated by the activation of both MAPK/ERK and PI3K pathways
(Hansen et al., 2001). Furthermore, the low affinity receptor of the neurotrophins
p75NTR is a member of the tumour necrosis receptor superfamily and is able to
interact (cross-talk) beside the neurotrophins with Trk family members. Thus,
activation of p75NTR by BDNF can influence the mechanism of action e.g. by leading
to downstream activation of NF-κB signalling (nuclear factor kappa-light-chainenhancer of activated B cells; Fig. 4-1) (reviewed in Gupta et al., 2013).
Moreover, activin A can modulate cell responses upon EPO (Maguer-Satta et
al., 2003) and EPO receptor binding activates the JAK2. That initiates several
intracellular signalling cascades (cf. 1.5.3, 2.5), whereby at least two are shared with
~ 34 ~
BDNF (PI3K and MAPK). This sharing of the signalling pathways could be the reason
for the fact that EPO itself induces upregulation of the BDNF expression, which was
reported previously (Hu et al., 2011; Mengozzi et al., 2012). Thus, the SMADindependent pathways of the activin signalling as well as effects of BDNF and EPO
are mediated by the MAPK, p38 and/or PI3K and probably can be shared by each
other (Fig. 4-1).
Inhibition of EPO results in a neuronal survival comparable to the double
combination (activin A with BDNF). As described in Results and Discussion section
of Chapter 2 and as well as in subchapter 4.1, the neuronal outgrowth is reduced in
the cultures supplemented with EPO individually after application of EPO-inhibitors.
Interestingly, this outgrowth is unaffected when cultures are treated with EPO in
combination with activin and BDNF. This indicates that neuronal outgrowth seems to
be not only mediated by EPO. Therefore, it seems possible that only if applied in the
triple combination, the factors augmented each other’s action with such an impact
that was not possible in any of the double combinations.
Figure 4-1 Possible cross-talk of the signalling pathways (figures modified by Sabine
Gebhardt from (Chang et al., 2002; Kumral et al., 2011; Reichardt, 2006) and rearranged
for this thesis) (next page)
From left to right, activin receptors (grey), the EPO receptor (blue), high affinity (Trk) and low
affinity neurotrophin receptors (p75NTR) are anchored in the cell membrane (black lines). The
interaction of BDNF with the signalling pathways is indicated in dark pink. The main interactions
of the neurotrophins with their high affinity receptors are depicted in black, whereas their
interactions at a lower extend are presented in light grey.
SMURF: E3 ubiquitin-protein ligase; inhibitor SMAD: SMAD 7; SARA: SMAD anchor for receptor
activation; IAP: inhibitor of apoptotic protein; BCL-2 and BCL-XL: antiapoptotic gene; PKC:
protein kinase C; DAG: diacylglycerol; IP3: inositol 1,4,5-trisphosphate (=triphosphoinositol), P:
phosphate; all other abbreviations are explained in the text.
~ 35 ~
~ 36 ~
4.1.2.3 Intracellular Potentiation of the Signalling
Neurotrophins induce multiple intracellular signalling pathways which all
together seem to contribute in the mediation of the action. For SGN (and also many
neurons of the central and peripheral nervous system), beside the PI3K and
MAPK/ERK pathway, the presence of an autocrine neurotrophic mechanism (cf.
Fig. 4-1) contributing to neuronal survival and plasticity especially in depolarized
cells is suggested (Hansen et al., 2001). In the absence of depolarization, this
mechanism seems to provide an insufficient survival-promoting stimulus (Hansen et
al., 2001).
Furthermore, the activation of cAMP signalling via elevated Ca2+ can also
contribute in neuronal survival by direct suppression of the apoptosis machinery
after depolarization. Increased calcium levels can activate calmodulin-dependent
protein kinases II and IV (CaMKII, CaMKIV) as well as cyclic AMP-dependent
protein kinase (protein kinase A; PKA) (Bok et al., 2003; Hansen et al., 2001; 2003).
Activation of both molecules initiates the phosphorylation of cAMP response element
binding protein (CREB; cf. Fig. 4-1). The cAMP pathway can be also activated by
glutamatergic input which leads to an intracellular increase of Ca2+ (Lachica et al.,
1995). Especially N-methyl-D-aspartate subtype of glutamate receptors (NMDARs)
are responsible for the Ca2+ elevation in rat hippocampal neurons and can be
influenced by BDNF as well as by activins (in a SMAD-independent manner; with
different kinetics). Furthermore, it is suggested that endogenous activin is secreted by
neurons themselves and may affect the L-glutamate-dependent Ca2+ influx of
neurons (Kurisaki et al., 2008). Thus, besides the “main” pathways which can be
shared by the different substances tested within the first part of the thesis, the action
of the substances can be modified by intracellular potentiation (autocrine
~ 37 ~
neurotrophin mechanism as well as intracellular Ca2+ levels). This could be an
explanation for the fact that neuronal outgrowth was unaffected only in factor
combinations (EPO with BDNF as well as EPO with activin A or triple combination)
after EPO-inhibition.
Further research is necessary in order to identify the exact molecular
mechanisms and their possible interactions induced in SGN after treatment with
BDNF, activin A and EPO administered as triple combination. Additionally, the
obtained in vitro results must be clarified by in vivo experiments.
All three factors are upregulated after injury in the nervous system. This
increased expression may be an autocrine-paracrine endogenous protection. Several
clinical trials have been performed already for BDNF (Ochs et al., 2000), as well as
EPO in adults (Haljan et al., 2009) and preterm infants/newborns (reviewed in Xiong
et al., 2011). Thence, these factors either individually or in combination are promising
candidates for a neuroprosthesis-related pharmacotherapy.
4.1.2.4 Inhibition of Apoptosis
Apoptosis is the best characterized form of programmed cell death (PCD). It
can be initiated by normal body processes (development, tissue remodelling,
response to pathogenic infections or irreparable cell damage) (Feng et al., 2011a) or
induced by several factors via two major pathways: the extrinsic and the intrinsic
pathway (cf. Fig. 4-2) (Hengartner, 2000; Tizard, 2004). The extrinsic pathway (also
called “death receptor” pathway) is triggered by cytokines such as tumour necrosis
factor-alpha (TNF-α) or other TNF-superfamily ligands acting throughout specific
receptors. Apoptotic stimuli lead to an activation of the intrinsic (or mitochondriadependent) pathway (Riedl and Shi, 2004; Tizard, 2004). Independently from the
~ 38 ~
stimulus, the effector caspase-3 is the key component in apoptosis and responsible
for the mediation of DNA-fragmentation. Beside these two pathways, likewise the
MAPK pathway especially JNK downstream signalling can be involved in PCD
(reviewed in Alam et al., 2007; Diaz, 2009).
Figure 4-2 Intrinsic and extrinsic pathway of apoptosis (modified from Tizard, 2004)
The constitutively homotrimeric ligands such as Fas ligand bind to death receptors and lead to the
formation of death–inducing signalling complexes (DISC) (Riedl and Shi, 2004; Tizard, 2004).
This complex recruits the initiator caspases, caspase-8 or capsase-10. These initiator caspases
then cleave and activate the effector caspase, capsase-3 (Riedl et al., 2004). Apoptotic stimuli
trigger the release of several proteins such as cytochrome c, SMAC (second mitochondria-derived
activator of caspases)/DIABLO (direct inhibitor of apoptosis (IAP) - binding protein with low pI),
AIP (apoptosis-inducing factor), EndoG (endonuclease G) and OMI/HTRA2 (high-temperaturerequirement protein A2) from the intermembrane space of mitochondria. There can be also a
cross-talk between the extrinsic and the intrinsic pathway mediated by capsase-8 mediated
cleavage of BID (a member of the BCL2 family) triggering the release of mitochondrial proteins
(Riedl and Shi, 2004; Tizard, 2004).
~ 39 ~
Within the inner ear, apoptosis plays an important role in the developing
cochlea (Fekete et al., 1997; Nishizaki et al., 1998; Zheng and Gao, 1997) and follows a
highly specific spatial and temporal pattern as published for rats (Nikolic et al.,
2000). It is also described that excessive apoptosis in the inner ear can be caused by
various ototoxic factors such as cisplatin (Zhang et al., 2003), TNF-a (Pregi et al.,
2009), aminoglycosides (Cunningham et al., 2002; Lee et al., 2004) or salicylate
(Chen et al., 2010; Feng et al., 2010; Feng et al., 2011a; 2011b; Wei et al., 2010). In
addition, other causes, e.g. microcirculation chaos (Nakashima et al., 2003; Seidman
et al., 1999; Taniguchi et al., 2002), presbyacusis (Bao and Ohlemiller, 2010; Park et
al., 2010), autoimmune dysfunction (Ma et al., 2000; Watanabe et al., 2001, 2002)
and injury (Ding et al., 2010; Zhou et al., 2009) have been described. Regardless the
injury, formation of reactive oxygen species (ROS) can occur as a consequence and
also induce apoptosis (Jeong et al., 2010; Pai et al., 1998). Thus, neuronal survival
can be achieved by the inhibition of apoptosis.
As published previously, in vitro blockage of caspases inhibits apoptotic death
induced by aminoglycosides or cisplatin in HCs (Cheng et al., 2003; Matsui et al.,
2002; Matsui et al., 2003) and (Liu et al., 1998). Such inhibitors can be used in order
to verify if the neuronal survival, which was observed after the application of activin A
and BDNF individually or in any factor combination (also with EPO), is mediated by
the inhibition of (pro-) apoptotic proteins. In this context, further experiments are
necessary.
4.1.3
Long-term Factor Application
In order to increase neuroprotection, a factor combination may be a better
choice than applying only one factor individually. Unfortunately, the stability of
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neurotrophins is limited and they are not able to penetrate the blood-brain (Poduslo
and Curran, 1996) or the blood-cochlea barrier (Kishino et al., 2001). Therefore, oral
application is not an option when the factor should act in the cochlear. In addition, an
increased number of side effects can be expected after systemic application when
compared to local single dose infusion into the inner ear. For long-term application,
those factors can be applied via pump based systems in vivo. Nonetheless, when the
reservoir in the pump is empty, it must be removed or at least exchanged if the supply
of factors should be warranted over an extended period. These measures, however,
may increase the surgical complexity and also the risk of inflammation. Also,
reported consequences of the cessation of the factor treatment (e.g. via pump based
systems combined with electrical stimulation) are controversial. Agterberg and
colleagues (2009) published that temporary treatment with BDNF prevented
degeneration of SGN even after cessation of treatment: SGN packing densities were
comparable with those obtained in cochleae of normal-hearing guinea pigs and three
times greater than in the untreated contralateral cochleae. By contrast, Gillespie and
colleagues (2003) as well as Shepherd and colleagues (2008) showed that the
number of SGN was similar to that in untreated, contralateral cochleae, already
2 weeks after withdrawal of BDNF and ongoing electrical stimulation after BDNF
treatment only partially prevented degeneration of SGCs in the basal turn (Shepherd
et al., 2008). Thus, an alternative system for long-term drug delivery is needed. In
order to prolong the factor supply and to administer the factors more locally to
reduce possible side effects, cells could be used as drug-delivery system. In order to
apply BDNF-producing cells onto electrodes, two different coating strategies were
tested in vivo as published previously (Rejali et al., 2007; Warnecke et al., 2012).
Both research groups used fibroblasts as cell source for drug-delivery. In order to
~ 41 ~
reduce possible side effects induced by the cells per se and to enhance clinical
relevance, an autologous cell source would be more suitable for long-term drug
delivery (cf. 4.3). Besides the drug delivery, also a cell replacement by these cells may
be achieved. Such cell replacement may be necessary for regeneration of structures
damaged due to the insertion process, such as walls surrounding the scala tympani.
In addition, functional regeneration may be achieved for missing HCs and
degenerated SGN in order to regain the physiological function of the inner ear.
4.2 ESTABLISHMENT OF A PRIMARY, DISSOCIATED CULTURE OF THE RAT IC
The formation of gliosis around the electrode impairs electrode-tissueinteraction and thus, hearing sensation with central auditory prostheses like the
brainstem implant and the midbrain implant. Altering of material properties can
hinder unspecific growth of glial tissue around the electrode. In vitro screening of this
tissue-material-interaction requires an adequate cell culture system. Hitherto, an
adequate in vitro model utilizing dissociated cells has not been described for the IC
and was thus aim of the second part of the thesis (Chapter 3).
4.2.1
Short Summary of the Results obtained within the Second Part
4.2.1.1 Initial Screening to identify important Parameters
In initial screening experiments, four different dissociation protocols (Neural
Tissue Dissociation Kit [NTDK] T, NTDK P, NTDK PN and SGN-protocol), two
different media (Panserin 401 and MACS® Neuro Medium) as well as different cell
seeding numbers (1 x 104, 2 x 104, 3 x 104 and 4 x 104 cells/well) were investigated (cf.
3.3). The following parameters were identified after screening: (1) Supplemented
MACS® Neuro Medium improved the maintenance of neurons when compared to
~ 42 ~
Panserin 401. In addition, a prolonged cultivation period (up to 6 days) enhanced
extension of neurites and the formation of a neuronal mesh. (2) A seeding density of
2 x 104 cells/well was identified as optimal for investigation of cell architecture. (3)
The degree of dissociation seemed to have no influence upon the confluency of the
cells and the neuronal yield. (4) The cytoskeletal marker TUJ1 (neuronal class III βtubulin) and GFAP (glial fibrillary acidic protein) were set as standard marker for
neurons and astrocytes, respectively in order to evaluate the cultures.
Of all kits used for dissociation, best cell and neuronal yield was obtained by
the NTDK P (containing papain) and NTDK PN. Digestion with NTDK T generated
insufficient results. Additionally, kits rendered only varying results.
4.2.1.2 Proteolytic Enzymes
Thus, fresh prepared solutions containing only the enzymes of interest trypsin (in another variation to the previously established SGN-protocol) and papain
(the main component of the NTDK P; for 30 and 90 min) with and without the
addition of DNase I during the trituration - were investigated (cf. Materials and
Methods of Chapter 3). The optimal seeding density as well as the optimal medium
identified in the initial screening experiments were used for cultivation. Higher cell
numbers and reproducible results were obtained after digestion with the proteolytic
enzymes trypsin and papain (30 min) than with the kits: sufficient in cell yield and
distribution as well as less variations in confluency and neuronal yield. Cell clusters
were absent. An excellent cytoarchitecture of oligodendrocytes as well as highly
branched neurons and astroglia were only obtained after dissociation with papain for
30 minutes (cf. Results of Chapter 3). The addition of DNase I for trituration of cells
seem not advantageous over the use of an enzyme-free trituration solution (HBSS).
~ 43 ~
Based on these results, digestion with papain for 30 minutes without DNase I for
trituration was determined as the most suitable method for the dissociation of the IC
tissue.
4.2.1.3 Cultivation Parameters can be adjusted to the requested Conditions
Depending on the envisaged experiments, different preconditions are
necessary: In order to investigate adhesion forces with the material surface with
AFM, single cells without neuronal networks and cell-cell-adhesions are in favour
(Aliuos et al., 2013). This can be realized by e.g. reducing the seeding number or
cultivation period. By contrast, in order to screen novel materials or material
modifications in terms of their biocompatibility, a homogenous culture with high cell
yield is necessary.
4.2.2
Identification of different Cell Types
As described in the Introduction of Chapter 3, formation of gliosis after
implantation of a central neural prosthesis may act as an isolator for electrical
stimulation and hinder a focused activation of the auditory system (McCreery et al.,
2007; 2010). Additionally, astrocytes represent the key component in reactive gliosis
in adult CNS (Hatten et al., 1991; Ridet et al., 1997). Thus, we tested the presence of
neurons and glial cells in order to be able to influence them e.g. by alteration of
surface materials.
Although several neuronal markers were tested, only TUJ1 seemed to be
appropriate for the evaluation of the cytoarchitecture (cf. Result of Chapter 3). Glial
cells are divided into two families based on their germ layer origin: microglia (small
size and mesodermal origin) and macroglia (large size and ectodermal origin).
Macroglia can be further subdivided into oligodendrocytes and astrocytes with
~ 44 ~
distinct functions. Oligodendrocytes are mainly responsible for the myelination
process in the central and peripheral nervous system. Since dissociated cultures of
the IC presented oligodendrocytes and astrocytes (after co-staining with anti-myelinassociated glycoprotein antibody [MAG] and glial fibrillary acidic protein [GFAP]; cf.
Results section of Chapter 3 and Table 3), respectively, myelination in the IC seem to
start at early postnatal stages. This was similarly described previously by other
groups based on stains of slices of the IC: at P 4 in the rat (Hafidi et al., 1996) and in
gerbils (Ridet et al., 1996) and between P 0 and P 7 in rats (Hafidi and Galifianakis,
2003).
Astrocytes ensure the maintenance of the extracellular environment and the
stabilization of cell-cell communications within the central nervous system (Bélanger
et al., 2013; Desclaux et al., 2009; Souza et al., 2013). The presence of astrocytes was
revealed by the labelling of the cultures with GFAP: the intense cytoplasmic
immunolabelling and astral-like cytoarchitecture supported their astroglial identity
as described previously for primary astrocytes (Desclaux et al., 2009; Souza et al.,
2013). After co-staining with S100 (calcium-binding protein synthesized in astrocytes
and also other glial cells) and GFAP, similar cells could be labelled. Furthermore,
cells positive for S100 showed a distribution comparable to GFAP positive cells (cf.
Supplemental Figure 3-3): located around TUJ1 positive cells within the IC culture.
Thence, S100 as well as GFAP can be used for the identification of astrocytes. By
contrast, Hafidi and Galifianakis (2003) observed a different distribution pattern for
GFAP and S100 in histological slices of the rat IC and hypothesised that two distinct
subtypes of astrocytes are present in the midbrain. GFAP positive cells are also
present in the CN (Rak et al., 2011).
~ 45 ~
After AFM scanning, cells were attributed according their morphology to
different cell types (Figure 5 of Chapter 3). Besides astrocytes (third row; star-like
phenotype of the cell) and oligodendrocytes (second row; similar to maturating
oligodendrocyte progenitor cells presented by Othman and colleagues (2011)),
probably neurons (lower row) and also vascular cells (first row; characteristic
polygonal or irregular shape; (Kakade and Mani, 2013; Vandenhaute et al., 2012))
could be delineated. Alterations in the morphology can be caused by the reduced
seeding density, whereby direct communication of cells via cell-cell interactions was
not possible. This leads to the fact that e.g. astrocytes could not pursue their
physiological function supplying neuronal metabolism (Bélanger et al., 2013). Similar
was described for primary embryonic Purkinje neurons, which died within a few days
after omitting the astrocytic layer (Brorson et al., 1991).
Thence, dissociated culture of the IC revealed neurons, astrocytes and
oligodendrocytes. Those cell types are of main interest, when investigating cellmaterial-interaction such as cytotoxicity, adhesion, proliferation and inflammation,
as well as intercellular interactions and achieving neuroprotection or reduction of
gliosis.
4.3 BIOHYBRID NEUROPROSTHESES
The idea is to coat cells onto electrodes affording less-immunogenic as well as
trophic support, e.g. via NTF resulting in the development of a biohybrid auditory
prosthesis that offers a biomimetic surface for improved biocompatibility and enables
cell-based local drug delivery for the protection of auditory neurons. This application
of such biohybrid electrodes is not restricted to the cochlea. Richter and colleagues
(2011) described such an approach for electrodes for deep brain stimulation. Using a
~ 46 ~
cell layer that provides a “native, fully and actively integrating brain-mimicking
interface” would improve nerve-electrode interaction and reduce the foreign body
reaction and thus glial formation.
Once established, such biohybrid electrodes can be transferred into the clinic
for the improved activation and preservation of auditory neurons (i.e. peripheral and
central). Thus, biointegration and compatibility of the electrode will be increased and
thereby, the immune response after electrode implantation will be reduced. Since
human mesenchymal stem cells (hMSC) exert immunomodulatory effects upon
transplantation (Seo and Cho, 2012; Stagg and Galipeau, 2013), they can be utilised
for the local delivery of trophic factors that are produced either endogenously or after
genetic modification (e.g. BDNF). Human MSC can be easily obtained from the adult
organism in an autologous or allogenic manner. They have been obtained from
various organs and tissues, e.g. bone marrow, skin, adipose tissue, liver and gastric
epithelium. One major advantage of MSC is the fact that they only express MHC class
I and not MHC class II. They can suppress ongoing immune responses and thus the
foreign body reaction can be prevented. Furthermore, they are multipotent, i.e. they
preserve the potential for differentiation along their lineage (into adipose tissue, bone
and skin) (Lanza, 2009) as well as into neurons (Bae et al., 2007; Wislet-Gendebien
et al., 2005). Human MSC secrete numerous trophic factors that modulate
inflammation and apoptosis. A variety of cytokines and growth factors that act
primarily through paracrine mechanisms in order to exert neurogenesis,
remyelinisation and synaptic connection of damaged neurites as well as the
suppression of inflammation are secreted from hMSC. Among the MSC-secreted
growth factors, BDNF, GDNF, NGF and ciliary NTF are identified as neuroprotective
agents for the primary auditory neurons (c.f. 1.5). Therefore, hMSC can be used as a
~ 47 ~
paracrine delivery system for such growth factors. Additionally, they can be
genetically modified in order to enhance the expression of these therapeutic agents.
In our laboratory, initial studies have been performed using NIH3T3
fibroblasts coated on silicone model electrodes as a delivery for BDNF (Warnecke et
al., 2012). Such systems have been investigated in terms of their neuroprotective
properties on the first auditory neurons, the SGN. Since the biohybrid-electrodebased strategies for the improvement of tissue-electrode interaction can be also used
for central auditory prosthesis, an in vitro system for screening experiments is of high
interest in basic research. Although a primary dissociated culture was described for
the cochlear nucleus using trypsin (Rak et al., 2011) or papain (Fitzakerley et al.,
1997) for digestion, it has never been described for the IC before.
4.4 CONCLUSION
An improvement of the tissue-electrode interaction in auditory prostheses can
be achieved via protection of peripheral and central auditory neurons and by
reduction of the fibrosis/gliosis formation around the electrode.
A neuroprotection has been achieved in peripheral auditory neurons via a
combinatorial code provided by three different factors (activin A, BDNF and EPO)
with neurotrophic properties in this thesis for the first time. In order to screen-test
such neurotrophic properties on central auditory neurons, dissociated cell cultures
are ideal. For the IC, the structure to be excited in the midbrain with auditory
prostheses, a dissociated culture has been established in the present study. The
adequate neuronal yield in these cultures enables the screening of novel
neuroprotective factors or their combinations. In addition, the herein described
dissociated IC culture contains astrocytes, the main component for the formation of
~ 48 ~
gliosis around electrodes inserted in the brain. Hence, the screening of electrode
properties on the adhesion and proliferation of IC-derived astrocytes is possible.
For long-term drug delivery of neurotrophic factors, a cell-based application
system can be used. This has been investigated for the cochlea in some studies;
however, it has not been reported for the central auditory pathway so far. Cells
providing the neuronal structures to be excited with neurotrophic factors can be
delivered via the electrode. Such cell-coatings of the electrode surface shall
disembogue
in
the
development
of
biohybrid
electrodes
furthering
the
biocompatibility by the creation of a biomimetic surface.
Further research shall concentrate on the in vivo testing of combinatory
applied neurotrophins as well as on initial screening of the impact of such factors and
surface modification in IC cultures.
~ 49 ~
~ 50 ~
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~ 59 ~
~ 60 ~
Affidavit
AFFIDAVIT
I herewith declare that I autonomously carried out the PhD-thesis entitled
“Cell-based drug delivery to optimise the electrode-nerve interface”.
No third party assistance has been used.
I did not receive any assistance 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: Clinics for Laryngology,
Rhinology and Otology, Hannover Medical School
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.
_____________________
21.11.2013, Odett Kaiser
~ 61 ~
Acknowledgements
ACKNOWLEDGEMENTS
This work has been financially supported by the Lower Saxony Ministry of Education
and Culture and the Hannover Medical School.
I would like to thank the “Niedersächsischen Ministerium für Wissenschaft und
Kultur” providing me with a three-year Georg-Christoph-Lichtenberg scholarship.
I thank my supervisor group Prof. Andrej Kral, Prof. Sabine Kästner, Prof. Susanne
Petri as well as my supervisor from Oldenburg Prof. Hans Gerd Nothwang for their
support during this Ph.D.
Further, I want to thank Prof. Timo Stöver for providing me the topic and Prof.
Thomas Lenarz for giving me the opportunity to perform this thesis.
Furthermore, I thank all the members of the ZSN and “Hearing” for the great time
during the courses, summer schools and meetings. The former and current members
of the coordination office answered any open question and provided help.
My special thanks go to Dr. Athanasia Warnecke for ideas to rearrange the content of
the Ph.D., for the weekend and late night calls, for critical reading and the
constructive ideas of the manuscripts as well as the thesis. But most importantly is:
You believed in me.
I would like to thank also our technical assistant Darja Werner for all the help and the
fun while working together! Kirsten Wissel, thank you for your sympathetic ear and
your advice. Also, I want to thank all the other colleagues for the great atmosphere,
the joy and their attempts to socialise me during the extensive period of preparing the
manuscripts and thesis. You are great!
~ 62 ~
Acknowledgements
Alice, we started together with the Ph.D. and we’ll finish together with the Ph.D. but
our friendship will survive.
I want to thank the three grandmas and two grandpas of Luca - you believed in me
and you supported me to do it my way. Last but not least: Patrick and Luca - my
*Sonnenscheine* and *honeicakiehorsies* - nothing I would write describes anything
you did or you are or you will be for me. Thank you!
~ 63 ~

THESIS DOCTOR OF PHILOSOPHY (Ph.D.)

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