Cellular and molecular changes in animal
models of retinal degeneration
Graduate School for Cellular and Biomedical Sciences
University of Bern
PhD Thesis
Submitted by
Rahel Zulliger
from Madiswil/BE
Thesis advisor
PD Dr. Volker Enzmann
Department of Ophthalmology
Medical Faculty of the University of Bern
2
3
Accepted by the Faculty of Medicine, the Faculty of Science and
the Vetsuisse Faculty of the University of Bern at the request of the
Graduate School for Cellular and Biomedical Sciences
Bern,
Dean of the Faculty of Medicine
Bern,
Dean of the Faculty of Science
Bern,
Dean of the Vetsuisse Faculty Bern
4
5
Declaration of Originality
Last name, first name:
Zulliger, Rahel
Matriculation number:
03-129-681
I hereby declare that this thesis represents my original work and that I have used no
other sources except as noted by citations.
All data, tables, figures and text citations which have been reproduced from any other
source, including the internet, have been explicitly acknowledged as such.
I am aware that in case of non-compliance, the Senate is entitled to divest me of the
doctorate degree awarded to me on the basis of the present thesis, in accordance
with the “Statut der Universität Bern (Universitätsstatut; UniSt)”, Art. 20, of
17 December 1997.
Place, date
Signature
………………………………………
…………………………………………………
6
Abstract
Age-related macular degeneration (AMD) and retinitis pigmentosa (RP) are two of the
leading causes for blindness in the developed countries. For both diseases
successful, vision-restoring treatment has yet to be found. For the development of
new therapeutic approaches animal models are highly useful. Besides frequently
used genetic mouse models, it is as well possible to induce retinal degeneration
pharmacologically by the application of retinotoxic compounds. In this study, two such
compounds have been used: Sodium iodate (NaIO3), which induces cell death in the
retinal pigment epithelium (RPE) and causing the degeneration of the photoreceptor
cells as a secondary effect and N-methyl-N-nitrosourea (MNU), which directly induces
degeneration in photoreceptors without affecting the RPE cells.
To investigate the course of degeneration induced by NaIO3 or MNU, mice treated
with the compounds underwent visual testing with the cued water maze and the
optokinetic reflex measurement (OKR) for a prolonged period of time as well as
individual measurements of the photoreceptor function with electroretinography
(ERG). To assess the extent of the damage, histological sections of the mouse eyes
were prepared and analyzed using morphometry and optical coherence tomography
(OCT). In addition, autofluorescence measurements on whole eye flat mounts to
monitor RPE cell integrity were recorded. Deeper insights into the pathology were
gained by immunohistochemistry, qRT-PCR and Western blot for molecules involved
in major cell death pathways. Electron microscopy rendered more precise information
on ultrastructural changes after the treatment with the retinotoxins.
Both compounds are feasible to induce a time- and concentration-dependent retinal
degeneration, even though with different severity and progression. The degeneration
can be easily quantified with tests of visual function and by measuring retinal
thickness in tissue sections. Whereas the RPE cell layer is completely disrupted by
the application of NaIO3, MNU only induces an initial swelling that disappears after
two
weeks,
a
difference
clearly
visible
in
electron
microscopic
pictures.
Autofluorescence analysis on whole eye flat mounts showed also patchy loss of RPE
cells due to NaIO3-application but not after the application of MNU. On a molecular
basis, NaIO3 induces a caspase 3-dependent cell death in photoreceptors, while MNU
does not lead to the activation of caspase 3, a key player of apoptosis.
7
The described pharmacological models are feasible to mimic features of AMD and
RP. Differences between the two diseases are reflected in the animal models
especially concerning their impact on RPE cells. The exact mechanism of action in
cell death execution is only partially revealed up to now, but both models have been
characterized well enough to test for instance experimental regenerative therapies.
8
Table of contents
Abstract ................................................................................................................................. 7
Table of contents ................................................................................................................... 9
1.
Abbreviations ................................................................................................................12
2.
Overall introduction .......................................................................................................15
2.1 Structure of the eye .................................................................................................15
2.2 Pathologies of the eye .............................................................................................18
3. Chapter I – Retinal degeneration induced by NaIO3 .........................................................20
3.1 Introduction .................................................................................................................20
3.1.1 Age-related macular degeneration........................................................................20
3.1.2 Pathophysiology of dry AMD ................................................................................22
3.1.3 Animal models of AMD .........................................................................................23
3.1.4 RPE degeneration induced by NaIO3....................................................................24
3.1.5 Outlook on future therapy approaches ..................................................................24
3.2 Results........................................................................................................................26
3.2.1 Decreased visual function after patchy loss of retinal pigment epithelium induced
by low-dose sodium iodate ............................................................................................26
3.3 Additional Results .......................................................................................................34
3.3.1 NaIO3 leads to an activation of caspase 3 ............................................................34
3.3.2 Detection of molecular PCD pathways with quantitative Polymerase Chain
Reaction (qRT-PCR) .....................................................................................................36
3.3.3 Application of NaIO3 leads to change in the protein level of caspase 1 .................39
3.3.4 NaIO3 is cytotoxic for RPE cells in vitro.................................................................40
3.3.5 Electron microscopy reveals ultrastructural changes ............................................42
3.3.6 Retinal degeneration induced by NaIO3 is visible in the OCT and the ERG ..........44
3.4 Additional material and methods .................................................................................46
3.4.1 Immunohistochemistry ..........................................................................................46
3.4.2 Quantitative Polymerase Chain Reaction (qRT-PCR) ...........................................46
9
3.4.3 XTT/M30 ..............................................................................................................48
3.4.4 Electron microscopy (EM) .....................................................................................49
3.4.5 Western Blotting ...................................................................................................49
3.4.6 Electroretinography (ERG) ...................................................................................50
3.4.7 Optical coherence tomography (OCT) ..................................................................51
3.5 Discussion ..................................................................................................................52
4. Chapter II – Retinal degeneration induced by MNU ..........................................................57
4.1 Introduction .................................................................................................................57
4.1.1 Retinitis pigmentosa .............................................................................................57
4.1.2 Pathophysiology of retinitis pigmentosa ................................................................58
4.1.3 Animal models of retinitis pigmentosa...................................................................59
4.1.4 Retinal degeneration induced by MNU .................................................................60
4.1.5 Outlook on future therapy approaches ..................................................................61
4.2 Results........................................................................................................................63
4.2.1 Caspase-3-independent photoreceptor degeneration by N-methyl-N-nitrosourea
(MNU) induces morphological and functional changes in the mouse retina ...................63
4.3 Additional Results .......................................................................................................89
4.3.1 Thickness of the ONL is decreased after the application of MNU .........................89
4.3.2 Autofluorescence is not influenced by application of MNU ....................................91
4.3.3 RPE cells are intact on the ultrastructural level .....................................................92
4.3.4 Electroretinography (ERG) shows impaired photoreceptor function after MNU
application .....................................................................................................................93
4.3.5 Retinal degeneration induced by MNU is visible in the OCT .................................95
4.3.6 Upregulation in mRNA expression does not lead to higher protein levels .............97
4.4 Additional material and methods .................................................................................98
4.4.1 Histology ..............................................................................................................98
4.4.2 Whole eye flat mounts ..........................................................................................98
4.4.3 Immunohistochemistry..........................................................................................98
10
4.4.4 Electron microscopy (EM) .....................................................................................99
4.4.5 Western Blotting ...................................................................................................99
4.4.6 Electroretinography (ERG) ...................................................................................99
4.4.7 Optical coherence tomography (OCT) ..................................................................99
4.5 Discussion ................................................................................................................100
5. Overall discussion ..........................................................................................................105
5.1 Outlook ..................................................................................................................106
6. References .....................................................................................................................107
7. List of publications ..........................................................................................................113
7.1 Original papers ......................................................................................................113
7.2 Abstracts ...............................................................................................................113
8. Acknowledgements ........................................................................................................115
11
1. Abbreviations
AMD
age-related macular degeneration
ARBP
acidic ribosomal phosphoprotein
B2M
β-2-microglobulin
bFGF
basic fibroblast growth factor
BRB
blood-retina-barrier
BSA
bovine serum albumin
BW
body weight
CNTF
ciliary neurotrophic factor
DAPI
4’,6-diamidin-2-phenylindol
DHA
docosahexaenoic acid
DMEM
Dulbecco’s modified Eagle medium
EM
electron microscopy
ER
endoplasmatic reticulum
ERG
electroretinography
GCL
ganglion cell layer
H&E
hematoxylin and eosin
HPRT1
hypoxanthine-guanine phosphoribsyltransferase
ICE
IL-1β converting enzyme
INL
inner nuclear layer
i.p.
intraperitoneal
IPL
inner plexiform layer
12
i.v.
intravenous
MNU
N-methyl-N-nitrosourea
NaIO3
Sodium iodate
NSR
neurosensory retina
OCT
optical coherence tomography
OKR
optokinetic reflex
ONL
outer nuclear layer
OPL
outer plexiform layer
PCD
programmed cell death
PEDF
pigment epithelium-derived factor
PFA
paraformaldehyde
PGK
phosphoglycerate kinase 1
PI
post injection
PPIA
peptidylpropyl isomerase A, cyclophilin A
qRT-PCR
quantitative real-time polymerase chain reaction
RP
Retinitis pigmentosa
RPE
retinal pigment epithelium
TBS
tris-buffered saline
TUNEL
TdT-mediated dUTP-biotin nick end labeling
VEGF
vascular endothelial growth factor
13
14
2. Overall introduction
2.1 Structure of the eye
Vision is probably our most
important
sense
for
interaction
with
the
environment
and
the
perception of light happens
exclusively
in
the
eye.
Specialized nerve cells, the
photoreceptors, absorb light
with a spectrum of 400-700
nm
9
and transform it into an
electrical potential that is
then transmitted over the
optic nerve into the brain,
Fig. 1: Detailed cellular structure of the retina. The description
where
the
information
of the layers with the concerning cell types is indicated on the
processed
right. The cell types marked in red are affected in diseases
Some parts of the eye are
1
marked in blue below .
and
is
analyzed.
not directly involved in the
light perception, but are rather providing the right environment. The cornea and the
sclera form together a fibrous coat enveloping the eye, giving a structure and
protecting the inner tissues. In addition, the light is refracted mainly at the cornea. The
lens is responsible for the exact focusing of the light beam on the retina by changing
its form and thereby its refractive index. Another important structure of the anterior
part of the eye is the iris, which can allot the amount of light penetrating the eye by
altering its diameter just like the diaphragm of a camera. The vitreous is located
between the anterior and the posterior part of the eye, supporting the round shape of
the eye 10 .
The retina is located at the posterior pole of the eye (figure 1). It consists of the
neurosensory retina containing neural cells and the retinal pigment epithelium (RPE).
Underneath the RPE cells is the Bruch’s membrane, modified connective tissue
separating the retina from the underlying choriocapillaris. The choriocapillaris itself
contains a dense network of capillaries supplying the outer retina with nutrients. The
retina has several regions with distinct functions
15
10
. In the central retina, positioned in
the focus of the lens, is the macula lutea containing the fovea, a spot with a high
density of photoreceptors responsible for high-resolution central vision like reading
and recognizing faces
11
. The peripheral retina towards the iris has a lower resolution
and is required for peripheral vision. Next to the macula, in the nasal direction, lies the
optic disc, where the neuronal fibers from the retina congregate into the optic nerve.
This area depicts no retinal layering and is therefore called the blind spot 10 .
The RPE is a cell monolayer
and serves several roles in the
retina. First, it is part of the
blood-retina-barrier (BRB) that
secludes the retina from the
blood stream and selectively
provides
nutrients
neurosensory
disposes
for
retina
waste
the
and
products.
Second, it absorbs light with
its melanin pigment granules,
reducing light scattering and
protecting
the
eye
from
Fig. 2: Illustration of the visual cycle indicating the recycling
cycle of 11-cis-retinal. Through absorption of photons, 11-
dangerous irradiation. Third, it
cis-retinal is isomerized into all-trans-retinal, which is then
is
the
transported into the RPE cell and re-isomerized. Recycled
regeneration of 11-cis-retinal,
11-cis-retinal will be reshipped into the photoreceptors for
a
reuse .
responsible
for
5
molecule
used
in
the
photoreceptors to transform light into an electric potential (Figure 2). But probably the
most important function of the RPE cells is the phagocytosis of the photoreceptor
outer segments. As light is inducing oxidation of many substrates, the photoreceptor
cells shed about 10% of their outer segments every day to prevent the accumulation
of toxic photo-oxidative products. After the phagocytosis, the discarded segments are
processed in the lysosomes of the RPE cells and either disposed into the blood
stream or stored in granules. These waste product granules, also known as lipofuscin
granules, can be oxidized as well and are probably involved in age-related
degenerations 12 .
16
The neurosensory retina is built in
layers, consisting of different cell
types. Coming from the RPE cell
layer, the first structure are the
segments of the photoreceptors,
divided
in
inner
segments.
The
nuclei
located
are
and
outer
photoreceptor
above
the
segments and this area is called
outer nuclear layer (ONL). There
are
two
different
types
of
photoreceptors found in the human
retina, the rods and the cones.
Rods are much more sensible for
light
than
cones
and
are
responsible for the perception in
Fig. 3: Illustration of a rod photoreceptor. The synaptic
low-light situations and for contrast
body (bottom) is in direct contact with the overlying
and movement vision (figure 3).
bipolar and horizontal cells. Next to the nucleus in the
The
inner
segment
are
the
organelles
needed
for
maintenance of the cell. The outer segment consists
cones
are
generally
less
sensible, but they can distinguish
12
only of the discs filled with chromophore and the
colors
. For color perception,
attached rhodopsin. Apical discs are shed in the
humans have three different types
subretinal space, where they are phagocytosed from
of cones with a specialized set of
2
the RPE cells (top) .
opsins. Opsin is a protein bound to
the chromophores catching the photons in the photoreceptors. Even small changes in
the opsin amino acid sequence leads to a change in the wavelength that can be
absorbed, raising the possibility to have color vision 9 . The electric potential produced
by the absorption of light is transmitted to the neurons overlying the photoreceptors in
the inner nuclear layer (INL). The two nuclear layers are divided by the outer
plexiform layer, harboring the synaptic junctions between the cells
1
. The INL
contains neurons involved in the signal transduction, processing and redistribution
like horizontal cells, amacrine cells and bipolar cells. The ganglion cell layer, the
outmost layer of the retina, is in contact with the vitreous and connected through the
inner plexiform layer, another synaptic layer, with the INL. In the ganglion cells the
last part of information processing and gathering is performed before it is sent via the
17
optic nerve to the brain. More or less found throughout the retina are astrocytes and
Müller cells, which are glia cell types providing retinal scaffolding, and the microglia, a
subpopulation of the monocyte system capable of phagocytosis when activated
10
.
2.2 Pathologies of the eye
Vision is considered the most important of our five senses since ages and the loss of
vision can lead to severe depressions
13
. Therefore and because of the obvious
economic impact of blindness, pathologies concerning the eye are a major issue
14
.
Cataract is still the leading cause of blindness in the world, even though the disease
is curable by surgery. This is mainly due to an underdeveloped health care system in
the developing countries. The same holds true for glaucoma, which is affecting about
70 million people worldwide and is treatable quite easily in the early stages by
lowering the intraocular pressure. Infectious diseases leading to blindness, like
trachoma, are as well widespread in impoverished countries and will be treated with
improved health care programs systematically targeting the source of infection
15
.
Other pathologies cannot be treated up to now, especially inherited retinal
degenerations. The most prominent one is retinitis pigmentosa, targeting directly the
photoreceptor cells, while others, like Stargardt’s disease, are primarily affecting the
RPE cells
10
. Inherited retinal degenerations mostly lead to visual impairment at
young age, while acquired degenerations need more time to develop. Age-related
macular degeneration (AMD) is the cause of irreversible central vision loss in people
over 50 years of age. The incidence of the disease is increasing together with mean
age of the population and currently, treatment is only available for a fraction of AMD
patients
7
. Another pathology with an increasing incidence due to the ageing
population and the higher rate of obesity is diabetic retinopathy, a consequence of
diabetes mellitus. Blindness occurs in up to 4% of the patients with diabetes type I
and the only effective treatment so far is the rather invasive laser coagulation
15
.
According to the WHO, 314 million people worldwide are visually impaired, 45 million
of them are legally blind. As 85% of the blind patients live in a developing country,
developmental assistance and global health care programs will probably alleviate the
issue, but they will simultaneously lead to more cases of age-related visual
impairment as they augment the mean age of the population as well. This trend is
already noticeable, as the WHO recorded an increase in cases of blindness related to
higher life expectancy
16
. Therefore, blindness is unlikely to become a minor health
issue in the future.
18
The aim of the present study was to investigate animal models of retinal
degeneration. Animal models help to understand pathologies and are very useful
tools to test new therapeutic approaches. Therefore, two different pharmacological
models were chosen, MNU and NaIO3, inducing two different forms of retinal
degeneration in mice. The treated animals were extensively studied in terms of visual
acuity and histology of the eye and molecular tests were applied to investigate
involved cell death pathways.
19
3. Chapter I – Retinal degeneration induced by NaIO3
3.1 Introduction
3.1.1 Age-related macular degeneration
Fig. 4: Age-related macular degeneration (AMD) overview. The early stages of AMD with drusen as
well as hyper- and hypopigmentation are depicted in column A and B. Column C and D show the two
final stages of the disease with either geographic atrophy in the dry form (C) or with subretinal
7
hemorrhages in the wet form of AMD (D) .
Age-related macular degeneration (AMD) accounts for about half of the cases of
vision impairment or legal blindness in developed countries. As the disease only
affects elderly people over the age of 50, the prevalence of AMD is increasing with
the demographic shift towards an ageing society
7, 17
. The area in the eye affected is
the macula, a circular area at the center of the retina, which is responsible for highresolution vision. If the function of the macula is impaired, the patients lose their
ability to read and identify small details, therefore the disease has a huge impact on
the daily life of affected people
7
. The first changes in the retina do not impact the
visual acuity and are only visible as small yellow spots called drusen and hyper- or
hypo-pigmentated changes of the macula during an ophthalmological examination
17
(figure 4). Drusen are focal deposits of debris between retinal pigment epithelium
(RPE) and Bruch’s membrane and their growth and extension are major indicators for
a progress of AMD, but their exact formation pathway is not known
7
. After the initial
phase of the disease, two late stages can be distinguished: the atrophic or dry form,
which leads to degeneration of RPE and choriocapillaris, and the exudative or wet
20
form,
which
includes
neovascularization,
hemorrhages
and vessel leakage
11
. Although the
wet form is only present in about
10% of the patients, it can lead to
sudden vision loss within weeks by
bleedings
or
fluid
leakage
and
therefore accounts for over 80% of
the
cases
of
severe
visual
impairment or legal blindness
7
(figure 5).
However, the only therapy strategies
available for AMD are all aimed to
stop
the
neovascularization
and
vessel leakage in the wet form.
Fig. 5: Illustration of the macula and the pathological
conditions during AMD. The macula is located at the
focus of the light in the central part of the eye (A,
depicted with a blue square). A normal macula
Lasers
vessels
are
used
and
the
to
coagulate
photodynamic
therapy combines light
sensitive
shows continuous morphology in all cell layers (B).
dyes with laser beams to clog new,
In the dry form of AMD, the loss of cells in the RPE
weak vessels without too much
cell layer leads to a secondary degeneration of the
thermal damage. Nevertheless, both
overlying ONL, the attached inner and outer
segments (IS/OS) and the underlying choriocapillaris
therapies
have
severe
adverse
(C). In the wet form of AMD, blood vessels penetrate
effects and cannot be used if the
the Bruch’s membrane (BM) and enter the retina.
affected area is too close to the
RPE cells migrate in the outer retina and as a
macula, because the treatment does
consequence of the bleeding into the subretinal
space the sensory retina will detach and cells in the
not only clog the vessels, but also
destroys
6
ONL degenerate (D) .
the
overlying
photoreceptors, leaving behind a
blind spot. The latest therapy for wet AMD is an intravitreal injection of anti-vascular
endothelial growth factor (VEGF) antibodies, inhibiting the induction of sprouting
vessels. This treatment is even able to improve vision of patients, something that was
never achieved by laser therapy. However, to maintain vision gain, it has to be
administered repeatedly. In addition, the intravitreal injection is objectionable and can
lead to severe adverse effects as intraocular infection 7 .
21
On the other hand, up to now no approved therapy is available for the treatment of
dry AMD, which is affecting even more patients. One of the reasons for this deficit is
the fact, that most of the underlying pathology is largely obscure.
3.1.2 Pathophysiology of dry AMD
Dry AMD is also called geographic atrophy, because in the macular region RPE
degeneration occurs in growing patches and therefore the overlying photoreceptor
layer is no longer functional. Pathological changes inside the RPE cells seem to be
crucial for the onset and progression of AMD and are associated with degenerative
changes in the supportive Bruch’s membrane underneath. The genesis of drusen is
not fully understood, but it is
probably
tightly
metabolic
processes
RPE
cells,
linked
in
which
to
the
are
responsible for the turnover
and recycling of photoreceptor
outer
segments.
During
regeneration of 11-cis-retinal
for the photoreceptors, a lot of
waste material is stored in
lysosomes
and
has
disposed
for
a
to
be
proper
lysosomal function. Proportions
of this waste material cannot be
Fig. 6: Processing of photoreceptor outer segments by
fully degraded and are stored
phagocytosis into the RPE cells. Phagosomes fuse there
as lipofuscin in granules inside
with the lysosomes containing the digestive enzymes and
the cells (figure 6). Lipofuscin is
form the secondary lysosomes. The digestion process is
accumulating
partially incomplete and therefore lipofuscin granules can
4
arise .
during
lifetime
and very sensitive to oxidation
processes, where toxic lipid
products can arise. Accumulation of the toxic waste products in concert with
additional factors can lead to RPE cell death at a certain age. However, involvement
of lipofuscin in the pathophysiology of AMD is not yet proven. It is known, that AMD
patients have higher amounts of lipofuscin in their RPE cell layer. Further evidence
for a pathogenic association is the fact that changes in RPE autofluorescence, which
is originating mainly from the lipofuscin, is a leading sign of disease progression
22
18
.
The most important external risk factor is smoking, which seems to enhance the
activation of inflammatory mediators and therefore contributing to the oxidative stress
build upon the RPE. As internal factors, several risk alleles from genes important for
the complement cascade were identified. Thereby, mutations in inhibitory
complement factors seem to enhance the inflammation cycle, adding to the oxidative
stress in the eye. Several other risk factors, like light exposure and diet were
discussed, but most of them are inconclusive. In summary, AMD is a multifactorial
disease and therefore hard to predict and even harder to prevent
19
. One of the
problems in solving these issues is a lack of animal models of AMD.
3.1.3 Animal models of AMD
Research on populations of rhesus monkeys revealed pathogenic changes in the
macula similar to early AMD with clearly visible drusen in elderly individuals, but this
does not provide an alternative to small laboratory animals out of ethical reasons
20
.
Mice are widely used model organisms for many human diseases, but the major
disadvantage for the use in AMD research is the fact that mice are lacking a macula.
Therefore, a specific AMD model will not be achievable in this species. However,
mice have still some advantages over other species. They are easily bred, have a
high genetic homology to humans and are easily genetically modified
21
. Various
genetic mouse models are available that display at least some of the characteristics
of AMD. Mutations in the Mdm1 gene lead to the formation of lipofuscin-like granules
and significant retinal hypo- and hyper-pigmentation as well as to hyperpigmented
and atrophic RPE cells. The RPE cell loss ultimately induces photoreceptor cell
death, which is displayed by decrease in ONL thickness and a progressive amplitude
loss in electroretinography (ERG)
22
. Transfection with an additional VEGF gene
linked to an RPE65 promoter leads to intrachoroidal neovascularization and shows
some features of early wet AMD
21
. Pharmacological models are established as well
and have several advantages: First, the onset of disease can be arbitrarily set by the
researcher, enabling the use of adult mice. Genetic models normally have an early
onset, sometimes even before birth, which can lead to interference with normal
postpartal reorganization of the retina. Second, transgenic models have an intrinsic
progression rate and severity of the degeneration, which cannot be changed.
Pharmacological models provide the possibility to control these features by changing
the dosage. For example, the immunization of mice with carboxyethylpyrrole
connected to mouse serum albumin leads to a degeneration of the RPE cells with
23
sub-RPE deposits and accumulation of complement component C3d
23
. Another
commonly used pharmacological compound is sodium iodate (NaIO3).
3.1.4 RPE degeneration induced by NaIO3
The retinotoxicity of iodic compounds is known since 1926, when patients treated with
the new disinfectant septojod suffered from sudden but reversible blindness.
Experiments with rabbits revealed NaIO3 as the toxic component and histological
analysis of the rabbit eyes showed a selective damage of the RPE cell layer. Low
doses of the compound are enough to induce the degeneration and do not affect
other organs, making NaIO3 a specific toxin to induce retinal damage
compound works as well in rats and mice with adjusted concentrations
25, 26
24
. The
.
The mechanism of action of NaIO3 is still unknown and its specificity for RPE cells not
fully understood. It is known, that NaIO3 inhibits several enzymes including lactate
dehydrogenase, which are existing in the RPE but are not unique to these cells. The
ability of NaIO3 to destroy the zonula occludens, the basis of the blood-retina-barrier,
could also account for the toxic effect, but not solely. The fact that non-pigmented
animals are less susceptible for the NaIO3-induced RPE degeneration is supporting
the theory of creation of toxic melanin products by the compound. However, other
pigmented cells like choroidal cells are not affected and therefore this can as well be
only part of the effect
27
. Distribution studies with I131-labeled NaIO3 revealed that
RPE cells take up much higher quantities of the toxin than any other cell in the body 28
. Together, all of these fragments could be an explanation for the unique susceptibility
of RPE cells for NaIO3.
NaIO3 induced retinal degeneration displays two features similar to AMD: First, low
doses lead to a patchy loss of the RPE cells leaving spots void of autofluorescence
as in geographic atrophy. Without the sustaining RPE cell layer, the overlying
photoreceptors rapidly degenerate leading to vision impairment
27
. Second, the RPE
loss does not only affect the photoreceptors but also the underlying choriocapillaris,
causing atrophy
29
. Of course, NaIO3 does not induce the same pathology as AMD,
but gives at least the possibility to study RPE degeneration in a time- and
concentration-dependent manner.
3.1.5 Outlook on future therapy approaches
The ultimate goal of a feasible animal model is the better understanding of pathology
and the consequential development of successful treatment. Currently, some new
24
therapeutic approaches are tested experimentally and others are already in the
clinical trial phase. As a natural anti-angiogenic factor, pigment epithelium growth
factor (PEDF) seems to be an obvious choice for medication of wet AMD and the
substance has been proven useful in first clinical applications. Together with other
anti-angiogenic
drugs,
PEDF
might
reform
the
therapy
for
choroidal
neovascularization 30 .
The bigger challenge is still the treatment of the geographic atrophy, where currently
is no medication available. An invasive approach is the relocalization of the macula
from the patch of degenerated RPE cells to a different, non-diseased part of the eye.
This might preserve the photoreceptors in the macula and their function, but the
surgery bears a high risk for blindness by retinal detachment and is entailing
extensive muscle surgery to prevent ambiopia
30
. To replace the degenerated cells
transplantation of allogeneic RPE cells has been performed but unfortunately failed to
yield any improvement of vision by inducing a graft rejection. To circumvent the
immunological problems, autologous RPE cells from the periphery of the eye or even
the iris pigment epithelium have been transplanted. The biggest concern about
autologous transplantation is the fact that the transplanted RPE cells have the same
age and the same susceptibility as the degenerated ones and are therefore prone to
undergo the same fate. Furthermore, to improve the survival of the transplanted cells,
sheets of RPE cells still attached to the underlying Bruch’s membrane have been
introduced. However, up to date none of the transplantation regimes was successful
in restoring vision 31 .
25
3.2 Results
3.2.1 Decreased visual function after patchy loss of retinal pigment epithelium
induced by low-dose sodium iodate
Luisa M. Franco, Rahel Zulliger, Ute E. K. Wolf-Schnurrbusch, Yoshiaki Katagiri,
Henry J. Kaplan, Sebastian Wolf and Volker Enzmann
The aim of this study was to investigate the effect of sodium iodate (NaIO 3) on RPE
cells and to monitor the secondarily induced photoreceptor cell death leading to visual
impairment in the mouse.
Three different concentrations were applied, 15, 25 and 35 mg/kg bodyweight (BW),
and the visual function was assessed at different time points PI with cued water maze
and optokinetic reflex measurements. After the mice were sacrificed, the enucleated
eyes were either embedded in paraffin for tissue sections or prepared for whole eye
flat mounts. In tissue sections, the thickness of the ONL was measured and the
number of photoreceptor nuclei per row was counted.
Application of NaIO3 led to a time- and concentration-dependent decrease in visual
function. Application of 15 mg/kg BW induced only small morphological changes in
the retina, whereas 35 mg/kg BW almost destroyed the whole ONL. The optokinetic
reflex emerged as more sensitive method for visual function testing than behavioral
studies in the cued water maze. Measurements of the autofluorescence on flat
mounts showed a patchy loss of RPE cells after the application of NaIO3, which led to
a decrease in thickness of the ONL in tissue sections.
26
27
28
29
30
31
32
33
3.3 Additional Results
3.3.1 NaIO3 leads to an activation of caspase 3
TUNEL-staining was applied on paraffin sections to detect cell death. Positive nuclei
were found in the ONL only with higher doses, 25 and 35 mg/kg, applied from day 3
to day 28 PI (figure 7). Paraffin sections were as well stained with an antibody against
cleaved caspase 3, one of the main actors in apoptosis. The staining clearly revealed
activation after the application of NaIO3, again only with the two higher doses applied.
The positively stained nuclei restricted to the ONL confirmed our findings in the H&Estaining that only the outer retina but not the INL is affected. Caspase 3 positive cells
were found throughout the whole retina and the induction was also concentrationdependent (figure 8). Caspase 3 activation peaked at day 3 PI, but positive nuclei
were found up to day 28 PI, demonstrating an ongoing process of degeneration.
Fig. 7: TUNEL-staining in paraffin sections of retinal tissue after treatment with NaIO3. Treatment with 15
mg/kg did not lead to any positive staining in the eye at day 7 PI (B), as in control animals (A). Cells positive
for the TUNEL-staining (green) are most prominent after treatment with 35 mg/kg at day 3 PI (D), but some
were as well visible after treatment with 25 mg/kg at day 7 PI (C). The staining was positive up to day 14 PI
(E) and day 28 PI (F) after treatment with 35 mg/kg. Nuclei are stained with DAPI. (GCL = ganglion cell
layer, IPL = inner plexiform layer, INL = inner nuclear layer, ONL = outer nuclear layer, RPE = retinal
pigment epithelium)
34
Fig. 8: Immunohistochemistry for cleaved caspase 3 in retinal tissue sections after treatment with NaIO3.
Cells positive for cleaved caspase 3 (green) are found in the ONL after treatment with 35 mg/kg at day 3 PI
(E) but as well with 25 mg/kg (day 7 PI, C). No positive cells are present at day 28 PI (D) after treatment with
25 mg/kg but with 35 mg/kg (F). No positive cells are found in sections after treatment with 15 mg/kg (day 3,
B) as well as in control sections (A).
35
3.3.2 Detection of molecular PCD pathways with quantitative Polymerase Chain
Reaction (qRT-PCR)
The reference gene analysis yielded three suitable genes for the gene analysis in
photoreceptors: Phosphoglycerate kinase 1 (PGK), peptidylpropyl isomerase A,
Cyclophilin A (PPIA) and Hypoxanthine-guanine phosphoribsyltransferase (HPRT1).
For the gene analysis in RPE cells, three candidates were found as well:
Peptidylpropyl isomerase A, Cyclophilin A (PPIA), acidic ribosomal phosphoprotein
(ARBP) and beta-2-microglobulin (B2M). Out of logistic reasons, only two were used
each, PGK and PPIA for neurosensory retina and PPIA and ARBP for RPE cells.
For the qRT-PCR experiments cells from the RPE cell layer and cells from the
neurosensory retina were used and the gene expression in both tissues was
compared between treated and control animals at day 3, 7 and 10 PI. In the
neurosensory retina, the highest change in gene expression was observed in the
caspase 1 gene with an upregulation of about 25-fold at day 3 PI. Caspase 1
expression then was diminished to control level and increased again at day 10 PI, but
only to about 10-fold. This pattern of regulation with an instant increase or decrease,
an insignificant regulation at day 7 and then again a significant change compared to
control at day 10 PI was seen as well in other PCD genes like caspase 2, 7, 8 and 12.
The only exception to this pattern was the regulation of Cathepsin S, where
upregulation increased over time to significance at day 7 and 10 PI. Downregulation
was occurring with caspase 2 and 7, indicating that some apoptotic pathways are
shut down (figure 9 and table 1). In the RPE cell layer, the gene regulation did not
follow a certain pattern. From the 12 genes tested, significant regulation occurred only
in three genes at the p-value < 0.05 level. Caspase 1 was upregulated in the RPE
cells as well, but only about 2-fold at day 7 PI. Cathepsin S was significantly
upregulated at day 10 PI and calpain downregulated at day 7 PI. This was in contrast
to neurosensory retina, where neither of these genes was regulated. Caspase 12 was
upregulated at day 10 PI, but only at a p-value < 0.1 level (figure 10 and table 2).
36
Fig. 9: qRT-PCR for expression of PCD-specific genes in the neurosensory retina after treatment with
NaIO3 (n=5 for treatment group, n=3 for control group, mean ± SD). Cathepsin S, caspase 1, 6, 8 and
12 showed an upregulation at different time points. For caspase 2 and 7, a downregulation was
measured. Significant results are depicted by an asterisks (p-value <0.05).
Table 1: qRT-PCR results from neurosensory retina after treatment with NaIO3. Statistically significant
changes in gene expression are written in bold (mean fold change of mRNA expression, standard deviation
in brackets).
37
Fig. 10: qRT-PCR for expression of PCD-specific genes in the RPE cell layer after treatment with NaIO3
(n=5 for treatment group, n=3 for control group, mean ± SD). Cathepsin S and caspase 1 showed an
upregulation at day 10 and day 7 PI, respectively. For calpain 2 downregulation was measured at day 7
PI. Significant results are depicted by an asterisks (p-value <0.05).
Table 2: qRT-PCR results from RPE cell layer after treatment with NaIO3. Statistically significant changes in
gene expression are written in bold (mean fold change of mRNA expression, standard deviation in brackets).
38
3.3.3 Application of NaIO3 leads to change in the protein level of caspase 1
Concomitantly with the mRNA expression, the protein expression of caspase 1 was
increased significantly 3 days after the application of 25 mg/kg BW NaIO3 in the
quantitative western blot (figure 11). The downregulation of caspase 6 and 7 and the
upregulation of caspase 12 on mRNA level did not lead to any change in the protein
content.
Fig. 11: Western blot detected comparison of caspase 1 protein levels in the neurosensory retina
(NSR) in mice treated with 25 mg/kg BW NaIO3 and without treatment at day 3 PI. The
integrated intensity was normalized to actin levels before statistical analysis. Protein level of
caspase 1 was significantly higher in the control animals than in the treated ones (p-value <0.05).
39
3.3.4 NaIO3 is cytotoxic for RPE cells in vitro
Application of NaIO3 on cultured human RPE cells led to a significant decrease in cell
viability measurable by XTT assay (figure 12). The decrease was concentration- and
time-dependent, as well as depending on the source of the primary cell culture.
Staining with the M30-antibody revealed no positive cells after NaIO3-treatment with
lethal doses in contrast to the treatment with staurosporine used as a positive control
(figure 13).
Fig. 12: XTT viability assay on cultured human RPE cells after treatment with NaIO3 or staurosporine (n=6,
mean ± SD). The viability was measured 3 h and 8 h after addition of the XTT reagent and the background
absorption at 650 nm was subtracted from the absorption at 450 nm. Viability decreased with increasing
concentration of either of the two compounds added and was significantly different from the untreated control
(asterisks, p-value <0.05).
40
Fig. 13: Staining for the apoptotic fragment of cytokeratin 18 (M30). Positive cells after treatment with 1 μM
staurosporine (B, C and D) are stained in green and contain a fragmented nucleus (stained with DAPI)
characteristic for apoptosis. Cells treated with 12 mM NaIO3 do not show any green staining, only nuclei
stained with DAPI (A, E and F). Counterstaining with an anti-actin antibody reveals distorted cell shape in
staurosporine-treated cells (B) as well as in NaIO3-treated cells (A), indicating ongoing cell death.
41
3.3.5 Electron microscopy reveals ultrastructural changes
In the electron microscopic pictures destruction of the RPE cell layer was visible and
remaining RPE cells had a rounded shape and retracted microvilli indicating a
stressful condition. The underlying structures, Bruch’s membrane and choriocapillaris,
appeared swollen, but remained intact. On the other side of the RPE cells, the
number of photoreceptor cells decreased and their outer segments were completely
lost (figure 14 and 15).
Fig. 14: Ultrastructural pictures of photoreceptor nuclei in mice. In untreated mice, nuclei are evenly distributed
and have a specific morphology with a bright rim and a darker center (arrows, A). After the treatment with 35
mg/kg BW NaIO3, some of the nuclei have a characteristic apoptotic appearance with chromatin condensation
(arrowheads), while others still look physiologically normal (arrows) (day 3 PI, B).
42
Fig. 15: Ultrastructural pictures of the mouse retina. Healthy RPE cells express microvilli (arrows) on their
apical surface wrapping around the outer segments of photoreceptor cells (arrowheads) and the Bruch’s
membrane beneath the RPE cells is very compact (asterisks) (A). After the treatment with 35 mg/kg BW
NaIO3 (day 3 PI, B and C) the RPE cells did not express any microvilli anymore (arrows) and the pigment
granules were localized all over the cell body instead of only apically. The photoreceptor outer segments
appeared disturbed and were not engulfed by microvilli anymore (arrowheads, B). Single RPE cells were
detached from the monolayer and lost their membrane integrity (circled in B). The Bruch’s membrane starts
to swell and becomes less compact (asterisk, C). After 14 days PI, the remaining RPE cells displayed round
shape and the epithelial monolayer was disrupted (C). The photoreceptor outer segments were missing and
the remaining photoreceptor nuclei were located close to the RPE cells (green diamonds, D).
43
3.3.6 Retinal degeneration induced by NaIO3 is visible in the OCT and the ERG
Application of NaIO3 induced dose-dependent decrease in ERG a- and b-wave
amplitudes (figure 16) and to alteration of the c-wave shape (figure 17). However, the
changes were described only qualitatively. Because of the huge variations between
animals and time points surveys with more animals have to be undertaken in the
future for a qualitative analysis.
In picture taken with the OCT, layering of the mouse retina was clearly visible with
RPE cells appeared as smooth monolayer. After the treatment with 25 mg/kg BW, the
layering gradually disappeared, the RPE monolayer disintegrated and single RPE cell
migrated into the overlying retina (figure 18).
Fig. 16: Scotopic electroretinography (ERG) measured after the application of NaIO3. Amplitude of a- and bwave were significantly smaller in the treated animal (B, 25 mg/kg BW NaIO 3) compared to control (A) at day 3
PI.
44
Fig. 17: Scotopic ERG measured after the application of NaIO3. The c–wave amplitude was distorted and
diminished after the application of 25 mg/kg BW NaIO3 (B) compared to control (A) at day 3 PI.
Fig. 18: Optical coherence tomography (OCT) pictures made from the same mouse before (day 0), 7 and 21
days after the treatment with 25 mg/kg BW NaIO3. On the left side, fundus pictures indicate the location where
the sectional pictures were taken (green lines). In the sections (middle column), layering of the retinal cells was
clearly visible before treatment but diminished gradually after treatment over time. A closer look at the RPE cell
layer (blue squares equal inserts on the right), revealed a distortion of this cell layer, shown by the dark spots
inside the outer retina and the disintegration of the dark layer at the bottom of the picture.
45
3.4 Additional material and methods
3.4.1 Immunohistochemistry
Paraffin tissue sections (7
m) were used for TUNEL-staining with the In situ Cell
Death Detection kit, Fluorescein green (Roche Applied Science, Rotkreuz,
Switzerland; excitation wavelength 450-500 nm, emission wavelength 515-565 nm).
Immunohistochemistry was performed on the sections with an antibody against
cleaved caspase 3 (rabbit, 1:200, Cell Signaling Technology, Danvers MA, USA). The
primary antibody was diluted in Tris-buffered saline (Sigma-Aldrich, Buchs,
Switzerland) with 0.1% Triton X-100 (Sigma-Aldrich), 1% BSA (Sigma-Aldrich) and
2% goat serum (Dako Schweiz AG, Baar, Switzerland) and incubated overnight at
4°C. As secondary antibody goat α-rabbit Alexa Fluor® 488 nm (LuBioScience,
Luzern, Switzerland) was applied. The secondary antibody was diluted in 1:500 in
TBS with 0.1% Triton X-100 (Sigma-Aldrich) and 1% BSA (Sigma-Aldrich) for 1h at
room temperature. Isotype control antibodies were used as negative controls (normal
rabbit IgG and mouse IgG, Santa Cruz Biotechnology, Inc., Heidelberg, Germany).
3.4.2 Quantitative Polymerase Chain Reaction (qRT-PCR)
Mice were sacrificed 3, 7 and 10 days PI (25 mg/kg bodyweight NaIO3) and
enucleated eyes were immediately immersed in RNAlater (Qiagen, Hombrechtikon,
Switzerland). Neurosensory retina tissue was prepared by an incision at the ora
serrata followed by a circumpolar cut. The anterior part of the eye as well as the lens
was discarded. The neurosensory retina was separated from the eyecup and further
processed to cDNA with the μMACS™ RNA isolation kit and the μMACS™ One-step
cDNA kit (Miltenyi Biotech GmbH, Bergisch-Gladbach, Germany) including a DNAse I
treatment as recommended by the manufacturer (DNAse I, Ambion Inc., Austin,
USA).
All primers were quality controlled by sequencing the template on a genetic analyzer
ABI PRISM® 3100 (Applied Biosystems Inc., Foster City, USA). Primer sequences are
depicted in table 3. The PCR samples were labeled with the BigDye® Terminator V1.1
cycle sequencing kit (Applied Biosystems Inc.). Reference gene analysis was
performed with a Mouse Endogenous Control Gene Panel (TATAA Biocenter AB,
Gothenburg, S).
qRT-PCR was performed with iQ™ SYBR® Green Supermix (Bio-Rad Laboratories,
Reinach, Switzerland) on a MyiQ™ Single color Real-time PCR detection system
46
(Bio-Rad Laboratories). PCR samples were preheated to 95°C for 5 min and internal
calibration was done. Fifty cycles were performed, each consisting of 30 s
denaturation at 95°C, a 30s annealing at 58°C and a 30 s extension at 72°C.
Statistical analysis was performed with an unpaired two-tailed t-Test on the GenEx
program
(MultiD
Analyses
AB,
Gothenburg,
Sweden).
Table 3: Primer of programmed cell death-specific genes used for qRT-PCR.
RPE was prepared after the neurosensory retina was separated from the eyecup.
Therefore the eyecup was placed in cold PBS and the RPE cells were scraped off
with a glass rod. The suspended cells were centrifuged and resuspended in RLT
buffer with 1% β-mercaptoethanol. The RNA preparation was executed with the
RNeasy® Micro kit (Qiagen). Quality and amount of RNA were quantified after
preparation with the NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific,
47
NanoDrop Products, Wilmington, USA) and the Experion™ RNA HighSens Analysis
kit on the Experion™ System (Bio-Rad Laboratories).
The amount of RNA yielded from RPE was not sufficient for qRT-PCR and therefore
the samples were amplified with the WT Ovation™ RNA amplification system
(NuGen® Technologies Inc., Bemmel, Netherlands) and purified with the QIAQuick®
PCR purification kit (Qiagen). After amplification, the cDNA was tested for quality and
amount again with the NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific)
and the Experion™ RNA HighSens Analysis kit on the Experion™ System (Bio-Rad
Laboratories). qRT-PCR was performed with iQ™ SYBR® Green Supermix (Bio-Rad
Laboratories) on a MyiQ™ Single color Real-time PCR detection system (Bio-Rad
Laboratories). PCR samples were preheated to 95°C for 5 min and internal calibration
was applied. Fifty cycles were performed, each consisting of 30 s denaturation at
95°C, a 30s annealing at 58°C and a 30 s extension at 72°C. Statistical analysis was
conducted with an unpaired, two-tailed t-Test on the GenEx program (MultiD
Analyses AB).
3.4.3 XTT/M30
Human RPE cells were cultured in DMEM (GIBCO, Invitrogen AG, Basel,
Switzerland) with 2% Antibiotic/Antimycotic solution (10’000 units/ml penicillin and
10’000 µg/ml streptomycin (GIBCO)) and 10% fetal calf serum (Connectorate,
Dietikon, Switzerland). For the XTT assay, 6000 RPE cells/ well were seeded in a 96well plate. A 240 mM NaIO3-solution in cell culture medium was a used as a stock
solution and the appropriate amount of this stock solution was added to each well to
achieve 3, 6 and 12 mM end concentration. As positive control for apoptosis, 1 mM
staurosporine (Sigma-Aldrich) was added to the cells. Before measurement, the
reagents of the Cell proliferation kit II (XTT; Roche Diagnostics AG) were mixed
according to the manufacturer’s instructions. Statistical analysis was performed using
Kruskal-Wallis analysis on ranks and pairwise comparison with Tukey in the
SigmaStat 3.5 Software (Systat Software GmbH, Erkrath, Germany).
For staining purposes the RPE cells were seeded on fibronectin-covered glass discs
in a 6-well plate (300’000 cells/well) and cultured in the same cell culture medium
described above with identical concentrations of NaIO3 and staurosporine. For the
staining, antibodies against M30, an apoptotic fragment of cytokeratin 18 (mouse,
1:100, Enzo Life Sciences AG, Lausen, Switzerland), and actin (rabbit, 1:200, Cell
Signaling Technology) were used. All primary antibodies were diluted in PBS
48
(Invitrogen AG, Basel, Switzerland) with 1% BSA (Sigma-Aldrich) and incubated for
30 min at room temperature. As secondary antibody, Alexa Fluor® goat anti-mouse
488 nm and goat anti-rabbit 594 nm (1:200, Invitrogen AG) were used. All secondary
antibodies were diluted in PBS (Invitrogen AG). After the staining procedure, the
glass discs were mounted on glass slides with Vectashield Fluorescent Mounting
medium with DAPI (Vector Laboratories Inc., Burlingame, USA).
3.4.4 Electron microscopy (EM)
Mice treated with 25 or 35 mg/kg BW NaIO3 were sacrificed at different time points PI
and enucleated. The eyes were fixed with Karnowksy fixation medium (1 %
paraformaldehyde (Sigma-Aldrich), 3 % glutaraldehyde (Sigma-Aldrich) and 3 %
sodium cacodylate-HCl (Science Services, Munich, Germany)) for at least 24 h before
the lens was removed through a cut in the cornea and the eyes were fixed for another
24 h. The eyecups were washed with EM-buffer (2.5 % glutaraldehyde (SigmaAldrich) and 0.1 M sodium cacodylate-HCl (Science Services)) and then postfixed in
4% osmiumtetroxide (Science Services). The tissue was dehydrated, washed with a
resin/1,2-propylene oxide (Merck KGaA, Darmstadt, Germany) mixture and mounted
in resin (ERL 4221, DER 736, NSA and DMAE, mixed according to the manufacturer
(Science Services).
After the 3 days the hardened resin-embedded tissue was trimmed with an Ultratrim
and cut with an Ultracut E (Reichert Microscope Services, Depew, NY, USA) and a
Ultra 45° diamond knife (Diatome AG, Biel, Switzerland) in 80 nm thick sections and
applied on copper grids (G100H-C3, Science Services). The sections were contrasted
with 0.1 % lead citrate and then visualized on a CM 12 electron microscope (Philips
Applied Technologies, Eindhoven, Netherlands) with tungsten cathode and a 12
Morada megapixel camera (Olympus Soft Imaging Solutions GmbH, Münster,
Germany).
3.4.5 Western Blotting
Mice treated with 25 mg/kg BW NaIO3 were sacrificed at day 3 PI. For protein
extraction, eyes were enucleated and put in PBS (Sigma-Aldrich) on ice. The
preparation was performed in wash buffer consisting of PBS (Sigma-Aldrich) and 1
mM Na3VO4 (Sigma-Aldrich). The eyes were opened by an incision at the ora serrata
followed by a circumpolar cut. The anterior part and the lens were discarded. The
neurosensory retina was separated from the eyecup and put into wash buffer. After a
centrifugation the supernatant was discarded and the retinas immersed with lysis
49
buffer consisting of RIPA buffer (Sigma-Aldrich) with 1 mM Na3VO4 (Sigma-Aldrich)
and one tablet of CompleteMini protease inhibitor (Roche Diagnostics AG). The tissue
was homogenized with scissors and after a second centrifugation, the supernatant
was frozen. The concentration of the protein was measured with the Experion™
Pro260 analysis kit (Bio-Rad Laboratories).
Protein samples were loaded on a 15% Criterion™ Tris-HCl resolving gel (Bio-Rad
Laboratories) and blotted on a PVDF membrane (Bio-Rad Laboratories) with a
Criterion™ Blotter. For detection of the protein, the following antibodies diluted in TBS
with 0.1 % Tween-20 (Sigma-Aldrich) and 5% milk (Sigma-Aldrich) were used: rabbit
anti-caspase 6, rabbit anti-caspase 7 (1:1000, Cell Signaling Technology), rabbit anticaspase 1, rabbit anti- caspase 12 (1:1000, Abcam, Cambridge, UK) and mouse antiactin (1:10’000, Cell Signaling Technology). As secondary antibodies the following
were used: IRDye® 800CW conjugated goat anti rabbit IgG, IRDye® 800CW
conjugated goat anti mouse IgG, IRDye® 680 conjugated goat anti rabbit IgG and
IRDye® 680 conjugated goat anti mouse IgG (LI-COR® Biosciences, Lincoln, NE,
USA). Pictures were taken and processed with an ODYSSEY® Infrared Imaging
System (LI-COR® Biosciences). From the questioned bands identified by size
markers the integrated intensity was calculated with the band width and the signal
intensity. The bands were normalized to the actin band, to remove the variance
introduced by unequal amount of protein loaded. Statistical analysis was done using
Kruskal-Wallis analysis on ranks and pairwise comparison with Tukey in the
SigmaStat 3.5 Software (Systat Software GmbH).
3.4.6 Electroretinography (ERG)
Mice were treated with 15, 25 or 35 mg/kg BW NaIO3. Animals were dark adapted
overnight and all procedures were carried out under dim red light. Mice with
tropicamide dilated pupils (Mydriaticum) were anesthetized with i.p. injection of
ketamine (Ketalar® 50 mg) / medetomidine (Dormitor®) (Pfizer AG, Zürich,
Switzerland) and placed on a specially designed heated ERG mouse table (Roland
Consult, Stasche & Finger GmbH, Brandenburg, Germany). Gold wires (0.2 mm)
were positioned in the mouth as reference electrodes. The ground electrode was
placed in the tail and connected to the table. The ERG responses were recorded
simultaneously from both eyes by means of gold wire electrodes, which were
positioned to touch the central cornea of each eye. Corneal hydration was maintained
throughout the examination by the application of a small drop of 2% methylcellulose
50
(Methocel, OmniVision® GmbH, Puchheim, Germany). Standardized flashes of light
were presented to the mouse in a Q400 Ganzfeld bowl and responses were analyzed
using a RetiScan-RetiPort electrophysiology unit (Roland Consult). The standard
protocol used is based on the methods approved by the International Clinical
Standards Committee for human ERG. Rod-isolated responses were recorded using
a −25 dB dim white flash (maximal flash intensity of 3 cd/s/m-2) presented under
scotopic conditions. The intensity of the flash was increased stepwise (-20 dB, -10
dB). Finally, the maximal combined rod–cone response was recorded at 0 dB
intensity. The recording time was then increased to measure the c-wave (1.5 s). For
calculating the ERG amplitudes the standard convention where a-waves are
measured from the baseline to the trough and b-waves from the a-wave trough to the
b-wave peak were followed. The c-wave was measured from the baseline to the
peak. After the measurements anesthesia was revoked by i.p. injection of atipamezol
(Antisedan®, Pfizer AG).
3.4.7 Optical coherence tomography (OCT)
Mice were treated with 15, 25 or 35 mg/kg BW NaIO3. Mice with tropicamide dilated
pupils (Mydriaticum) were anesthetized with i.p. injection of ketamine (Ketalar® 50
mg) / medetomidine (Dormitor®) (Pfizer AG). Methylcellulose (Methocel 2%,
OmniVision® GmbH) was applied to negate refractive power of the air/cornea
interface. A contact lens (power + 4) (Bausch+Lomb Swiss AG, Zug, Switzerland)
was used to reduce the risk of corneal dehydration and edema and act as collimator.
Furthermore, to adapt for the optical qualities of the mouse eye a commercially
available 78-D double aspheric fundus lens (Volk Optical Inc., Mentor, OH, USA) was
mounted directly in front of the camera unit. Animals were placed before the
instrument on a custom-made holding table and SD-OCT imaging was performed in
the same session as cSLO using Spectralis™ HRA+OCT device (Heidelberg
Engineering, Heidelberg, Germany). All measurements were taken at 1.4 mm
eccentricity from the optic nerve head to mirror the SD-OCT based thickness
measurement along a circular ring scan (r = 1.4 mm) centered on the optic nerve
head.
Respective
thickness
was
quantified by computer
assisted
manual
segmentation analysis using the proprietary Heidelberg Eye Explorer (version 1.6.2.0)
(Heidelberg Engineering, Heidelberg, Germany). After the measurements anesthesia
was revoked by i.p. injection of atipamezol (Antisedan®, Pfizer AG).
51
3.5 Discussion
Previous experiments with NaIO3 in mice were conducted with high doses, up to 70
mg/kg BW i.v., and mainly focused on histological findings
26, 27
. To be able to draw
conclusions about the progression of the degeneration, experiments with low
concentrations and a slower progression rate are necessary to monitor the ongoing
pathology. Histology data described above indicate that the application of low-dose
NaIO3 leads to a concentration- and time-dependent degeneration of the RPE cells.
Secondarily, the overlying photoreceptor cells in the ONL degenerate as well, without
harming the retinal cells in the INL. However, the degeneration is ongoing up to 3
months, where a significant decrease on ONL thickness was measured with the
lowest dose, 15 mg/kg BW, used. Although the highest dose applied, 35 mg/kg BW,
was about half the highest dose used in previous studies, it still led to total destruction
of the photoreceptor cell layer, although in a delayed manner. The same result was
found in the RPE autofluorescence, where the lower doses applied led to patchy
appearance like in geographic atrophy
32
. Of interest was the fact that the area void
of autofluorescence was significantly smaller with all doses applied at 3 months PI
compared to 28 days PI. This somehow indicates a rebound of the autofluorescence,
but up to now, there is no evidence that the RPE cells recover likewise. Other groups
described proliferation of RPE cells after small lesions in rat retinas, but these results
are rather not transferable to our model, as we induce larger lesions 33 .
To have a deeper insight into the pathology after application of NaIO 3, paraffin
sections were stained using the TUNEL method. Thereby, findings by others 2 could
be confirmed with our dose regime. Many cells in the ONL were positively stained with
a peak of positive TUNEL-staining at day 3 PI and positive cells up to day 28 PI.
However, with the lowest dose applied the retinal degeneration develops barely
detectable as the ONL thickness was significantly decreased only at 3 months PI
without detection of an increase of TUNEL-positive cells compared to untreated
animals at any time point. As the degeneration is progressing very slowly, we might
just miss the rare events of cell death caused by this low concentration. As TUNELstaining is not exclusively specific for apoptotic events, another staining against the
cleaved and therefore activated form of caspase 3, one of the hubs of apoptotic cell
death
34
, has been applied. The staining showed an almost identical pattern to the
TUNEL-staining, with peak activity at day 3 PI and persisting staining up to day 28 PI.
Again, the application of 15 mg/kg BW did not induce any cleavage of caspase 3.
52
In contrast to earlier studies, a significant decrease in visual acuity as well with a
lower dose, 25 mg/kg BW, could be shown even in the cued water maze27 . However,
the initial significant difference of the treated animals to the untreated control group
was lost during the course of the experiment due to the re-learning effect. This effect
cannot be prevented and was originally wanted as the water maze was invented to
test memory and learning ability of rodents
35
. It has not been shown in previous
studies because concentrations used in that study were much higher and the induced
degeneration therefore much more prominent
27
. To be more accurate, a more
sensitive method to measure visual acuity was used, namely the OptoMotry system to
measure the optokinetic reflex. With this method, even with a dose as low as 15
mg/kg BW, a significant difference compared to untreated animals could be measured
36
. There was also no intrinsic learning effect confounding the results, so that a
significant difference to the untreated animals was found at all time points measured.
ERG measurements showed a comparable progression of retinal function loss after
NaIO3 application. Decrease in ERG amplitude 3 days after the injection of 25 mg/kg
was significant and continued over the investigated time period. Another feature of
the NaIO3-model was the change in the c-wave originating from the RPE. C-wave
amplitude was significantly diminished and the curve shape distorted, indicating that
RPE were affected first by the compound
37
. To follow morphological changes in the
retina in the same animal OCT measurements will be a feasible method in the future.
First data showed a thinning of all retinal layers and patchy disintegration of the RPE
cell layer
38
. This degeneration of the pigment epithelium can also be visualized by
electron microscopy. Thereby, it could be visualized that RPE cells first retracted their
microvilli, then changed from a cubical into a round shape and finally disappeared.
That left easily distinguished gaps in the monolayer and was followed by
degenerating of the photoreceptors. They lost their outer segments and also vanished
completely from the retina 29 .
For a first overview of the cell death pathways switched on in the neurosensory retina
(NSR) after the application of NaIO3, arrays of quantitative RT-PCR to investigate
changes in PCD-specific gene expression were performed. Gene expression analysis
does, however, not give conclusive information about the expression of the end
product of the gene, the protein, as there are many regulation checkpoints between
mRNA transcription and protein synthesis, but it offers a good starting point for
protein measurements. Most of the investigated genes showed some common
53
pattern of regulation with an early change at day 3 PI, then a return to control levels at
day 7 PI and a late change at day 10 PI. This could be correlated to the two-part
pathology induced by NaIO3. The first changes would then be induced by the
administration of the compound itself and the second changes at day 10 PI by the
dying RPE cells. Surprisingly, the activation of caspase 3 on the protein level did not
have any significant influence on its gene expression. However, we are not able to
detect very small changes in gene expression, given that one of the disadvantages of
pharmacological models is the high variability between animals thereby reducing
sensitivity.
The most pronounced changes in gene expression were measured in the caspase 1
gene, with an upregulation of about 25-fold at day 3 PI and about 10-fold at day 10 PI.
Caspase 1 is not directly involved in cell death pathways but is a member of the socalled inflammatory caspases. Its main task is to cleave the pro-form of IL-1β into its
active form, therefore it is known as IL-1β converting enzyme (ICE)
39
. However, as
levels of activated IL-1β have not been investigated, an induction of innate immunity
cannot be proofed by the gene expression increase. On the other hand, caspase 1 is
able to induce the infiltration of the tissue by immune cells from the blood and this has
been described after the application of NaIO3 in mice
26
. Cathepsin S results can be
interpreted in association with caspase 1. The mRNA expression of the cathepsin S
gene did not follow the same pattern as most of the other genes, as it showed an
increasing upregulation significant at day 7 and 10 PI. Cathepsins are able to induce
a caspase-independent cell death or even necrosis when they are released from the
lysosome in case of damage. Cathepsins are as well responsible for the cleavage of
caspase 1 into its active form, explaining its upregulation during inflammation
40
.
Another member of the group of inflammatory caspases is caspase 12, which was
significantly upregulated at day 3 and 10 PI. Caspase 12 is a major component of the
apoptotic pathway induced by stress in the endoplasmatic reticulum (ER), but up to
now there is no evidence, that NaIO3 induces ER-stress. However, caspase 12 also
initiates a downstream cascade ultimately leading to the activation of caspase 3,
which can be seen in our model 41 .
Caspase 8 is a member of the initiator caspases, which are inducing downstream
caspase cleavage upon an apoptosis-inducing event 39 . After application of NaIO3 the
expression of the caspase 8 gene was upregulated at day 3 and 10 PI. This is
consistent with the other results, as caspase 8 activates caspase 3 and is involved in
54
the initiation of innate immunity. Caspase 8 activation is induced by several receptors
from the tumor necrosis factor (TNF) receptor superfamily 1, but again, there is no
evidence for such an activation after the application of NaIO3 up to now
42
. Another
initiator caspase is caspase 2, which was downregulated at day 3 and 10 PI in our
model. As caspase 2 is activated by DNA damage in the cell, downregulation of the
gene indicates that this PCD mode of action is not activated after NaIO 3 application
39
.
Caspase 6 belongs to the group of effector caspases, which are responsible for the
completion of cell death, cleaving nucleases and proteases to initiate the ultimate
decomposition of the cell. It reacts concomitant with caspase 3 and is mostly
activated simultaneously and therefore the upregulation at day 10 PI is
comprehensible. The same can be said for caspase 7, the third effector caspase
besides 6 and 3. However, the gene expression for caspase 7 is downregulated at
day 3 and 10 PI. This seems somewhat contradictory, but can be explained by the
fact, that caspase 7 is a feedback loop for the activation of caspase 3 by cleaving
caspase 12, which in return activates more caspase 3 39 .
Atg5 is one of the major promoters of autophagy
43
. No regulation of Atg5 gene
expression was found after NaIO3 treatment and therefore it can be suggested that
autophagy plays no important role in the pathology. Any regulation neither in calpain
1, 2 nor 10 was found. Therefore, this is most likely another cell death pathway not
involved in NaIO3-induced degeneration is apoptosis induction by calpains. Thereby,
membrane damage or calcium transport activation lead to influx of Ca2+-ions from the
outside of the cell or the inside of the mitochondrion into the cytosol, where the ions
activate calpains, which in return initiate apoptotic cell death 44 .
To repeat the qRT PCR experiments with RPE tissue an amplification step to yield
enough cDNA had to be implemented. However, through this step more variability
has been added to the samples and the possibility of losing small differences along
the procedure increased. This might be the reason that only three genes were
significantly regulated and all of them at only one time point. Caspase 1 is
upregulated in the RPE cells as well, but only with a moderate increase of about 2fold at day 7 PI. RPE cells are part of the blood-retina-barrier (BRB) and initiation of
inflammatory processes has to start with weakening of the BRB and entering of cells
of the immune system into the eye
45
. The upregulation of cathepsin S at day 10 PI is
consistent with the upregulation of the gene in the neurosensory retina. As cathepsin
55
S is involved in necrotic cell death 40 , this supports the hypothesis, that RPE cells die
by necrosis after application of NaIO3. However, the time point seems to be very late,
as it has been reported that degeneration of the RPE starts already 6h after
application
26
. The downregulation of calpain 2 would suspend Ca2+-induced
apoptosis, but could also be a sign of general downregulation of unused genes 44 .
Western blot analysis of caspase 6, 7 and 12 did not reveal any changes of the
protein expression in the NSR after the treatment with NaIO3 at day 3 PI, even though
there is a significant change on the mRNA expression level. The amount of change
may be too small to be detected as there was a significant change in the protein
amount of caspase 1, also highly upregulated at the mRNA level. However, we were
only able to check on pro-caspase 1 and are therefore unable to make assumptions
on the level of activation of the enzyme.
To get more evidence for necrosis in RPE cells an in vitro assay has been used. This
was necessary as pigmentation in RPE cells interferes in situ with staining
fluorophores. Cytokeratin 18 is a cytoskeletal protein found in epithelial cells and
specifically cleaved during apoptosis. This apoptosis-specific fragment is detected by
a monoclonal antibody M30
46
. NaIO3-treated cultured RPE cells were never
positively stained, whereas cells treated with staurosporine, known to induce
apoptosis
47
, were clearly positive for the cleaved protein. To prove, that the NaIO 3
doses used in vitro were lethal to the cells, an XTT assay was performed in parallel.
In the viability assay, a significant decrease in cell viability was detectable after NaIO 3
application and it was in the range of the staurosporine-induced cytotoxicity. In
conclusion, RPE cells die by the NaIO3 treatment, but the cytokeratin 18 is not
cleaved in an apoptotic fashion. This is of course no proof, especially as it is known
that in autophagic cell death cleavage does as well not occur
46
, but it is another
piece of evidence for a necrotic cell death in RPE cells after NaIO3 treatment.
56
4. Chapter II – Retinal degeneration induced by MNU
4.1 Introduction
4.1.1 Retinitis pigmentosa
Retinitis
(RP)
is
pigmentosa
used
as
a
general term for a wide
collection
of
retinal
degenerative
diseases
caused
inherited
by
mutations. With a rather
high prevalence of up to
1:3000,
retinitis
pigmentosa is still the
leading
cause
blindness
people
in
in
of
young
developed
Fig. 19: Posterior part of an eye removed from a patient with
countries. The pattern of
retinitis pigmentosa showing characteristic pathology. Arrows
pathology
indicate deposits of bone spicule pigment and the arrowhead points
heterogeneous and the
is
very
3
at the waxy pale optic nerve head .
age of onset reaches
from infancy to adulthood. Generally, both eyes are involved and the disease is
characterized by progression of the degeneration. Typically, the disease starts with
impairment of night vision by primarily affecting the rods and then proceeds with
deterioration of the peripheral daytime vision. That leads to the so called tunnel
vision, as the cones degenerate secondarily, and ultimately to blindness
48
. Up to
date, 45 genes were identified, where a mutation can lead to retinitis pigmentosa.
However, that covers only about 60 % of the patients. Some mutations can induce
syndromic forms of RP, also affecting hearing or kidney function, as in Usher- or
Bardet-Biedl-syndrome, respectively. Most of the mutations cause damage isolated to
the eye and one of the most prominent genes affected is RHO, responsible for the
production of the rhodopsin in rod photoreceptors. It has been found mutated in 25 %
of autosomal-dominant RP cases. Many patients have almost unique mutations and
that fact impedes the search for therapy severly
49
. Diagnosis of RP is mostly done
by the symptoms described by patients, as night blindness and tunnel vision are quite
57
characteristic. Beyond that, funduscopy can deliver some evidence for the diseases.
A typical fundus anomaly in RP patients shows pigmentation alterations, so called
bone spicules, in addition to attenuated retinal vessels and waxy pallor of the optic
nerve (figure 19). A method to detect RP very early is electroretinography (ERG),
which shows an abnormal electric response of the photoreceptors even before
symptoms are experienced 50 .
Currently, there is no treatment available for retinitis pigmentosa, but studies with
nutritional supplements showed some effect on the disease progression. A few
patients seem to benefit from the supplementation with vitamin A, but these results
are highly controversial and the treatment is not recommended as a general therapy
2
. Other anti-oxidants and nutrients like lutein or ascorbic acid are prescribed to
optimize the microenvironment of the photoreceptors to somehow slow down the
progression of the degeneration. But in contrast to the findings with the vitamin Asupplementation, none of the drugs yielded a significant benefit for the patients
50
.
4.1.2 Pathophysiology of retinitis pigmentosa
To describe the pathophysiology of retinitis pigmentosa it is imperative to know the
underlying mutations, which is unfortunately the case in only 60 % of the patients. For
mutations in the RHO gene, the processes ultimately inducing photoreceptor cell
death were studied profoundly. Mutations in this gene lead to misfolding of the
produced rhodopsin protein, which then affects the tight outer segment packaging.
This mispackaging has an effect on the turnover rate of the photoreceptor segments,
causing abnormally large shedding. Excess shedding can only be maintained for a
certain amount of time, before it induces rod photoreceptor cell death. The onset of
the degeneration depends on the amount of misfolded rhodopsin, causing high
variance in patients with different mutations. The disease shows often autosomaldominant inheritance pattern with the expression of only small amounts of mutated
rhodopsin already sufficient to induce degeneration
2
. An autosomal-recessive
inheritance pattern is caused by another well-studied mutation in the RPE65 gene.
The gene codes for an isomerase in the RPE cells, responsible for one of the steps in
recycling of 11-cis-retinal. A malfunction of the RPE65 enzyme disrupts the visual
cycle and induces cell death in photoreceptors as their function is abolished, when no
11-cis-retinal is delivered by the RPE cells
51
. However, for many of the known genes
causing RP, the mechanism of degeneration is not fully understood.
58
In some cases, an association between the mutated gene and the pathology in the
eye is not obvious from the beginning. The mutations leading to systemic forms of
RP, like Usher- or Bardet-Biedl-syndrome, were identified in genes coding for ciliary
proteins. As photoreceptor outer segments are a specialized form of sensory cilia the
pathology detected in these patients revealed deeper insights into the structure of
photoreceptors 52 .
In conclusion, the pathophysiology in RP is as diverse as the underlying genes and
their mutations.
4.1.3 Animal models of retinitis pigmentosa
Naturally
mutations
occurring
leading
to
retinal degeneration are
found in mouse strains
in
almost
the
same
manner as in humans.
As laboratory animals
are often inbred, such
mutations are detected
now and then by their
spreading
Some
phenotype.
of
Fig. 20: Analysis of different cell death mechanisms correlated to
these
time (days after birth) in the rd1 mouse. Up to four different
mutations
could
be
processes are going on at the same time, rendering detection of
correlated
with
the
mutation-caused degeneration rather complicated. Therefore,
human
providing
counterpart,
many controversial results have been published, probably because
8
of measuring the same marker at different time points .
excellent
animal models for studying the disease. For example, the rd1 mouse with mutation in
the Pde6b gene also occurring in human RP patients. However, mouse models
available show the same diversity as the different forms of RP and exhibit several
hallmarks of the disease like vessel attenuation, pigment patches in the fundus,
decrease in ERG amplitudes and a progressive degeneration of photoreceptors. The
rd5 mouse is even suffering from hearing loss, being a model for Usher syndrome
54
53,
.
In addition to these naturally occurring mouse models, researcher started to develop
transgenic mice after they identified the genes responsible for RP development. The
59
RPE65-/- mouse allows insight into the function of the protein and the process of
degeneration caused by its functional failure
55
. Various mutations were also induced
in the rhodopsin gene, reflecting the situation in humans, where over 100 distinct
mutations are found
56-58
. As already pointed out, genetic models harbor some
disadvantages, as their onset is very early and the progression quite fast (figure 20).
This is especially true for some of the retinal degeneration mutants like rd3, where
eight weeks after birth no photoreceptor cells are left in the retina
54
.
One of the most commonly used RP models is the induction of retinal degeneration
by light exposure. Time point of onset can be freely chosen and the severity of the
degeneration can be easily modulated by adjusting light intensity and exposure time
59
.
A pharmacological model for retinal degeneration, known already since the 1950’s,
uses iodoacetic acid (IAA), which has been injected into cats, rabbits, pigeons, turtles
and frogs. During ERG measurements a decrease in ERG amplitude was found and
led to the conclusion that IAA is causing photoreceptor cell death
60
. Later on,
experiments with rats were conducted, but in this species a retinal degeneration was
only inducible by the simultaneous injection of IAA and sodium malate. The ERG
findings were confirmed by histology, where the reduction of photoreceptor cells in
the ONL was easily detectable
61, 62
. Additional experiments have revealed, that the
IAA is accumulating in the retinal tissue with high affinity and is not damaging the
RPE cell layer at all 63 . However, IAA was tried for use in mice as a model for RP, but
there was no sublethal dose inducing retinal degeneration.
4.1.4 Retinal degeneration induced by MNU
In cancer research, N-Methyl-N-nitrosourea (MNU) is a commonly used compound to
induce tumors in rodents
64
. Eventually, it was discovered that it is as well inducing
retinal degeneration, acting quite specifically on photoreceptors
65
. First experiments
revealed, that the degeneration is independent of the pigmentation of animals,
working equally well in albino and pigmented animals. It has been demonstrated, that
the degeneration starts already 24 h after the application and that RPE cells are
mostly unharmed
66
. However, the fate of RPE cells after MNU application is
controversially discussed. Some groups showed detached and migrated RPE,
whereas others stated no changes in the RPE cell layer
67, 68
. MNU is thought to
induce cell death by alkylating the DNA and thereby forcing the cells into apoptosis
69
. This assumption is supported by other groups working with alkylating agents. They
60
could show that methylation of the DNA is leading to PARP activation as a DNA
repair mechanism and that this overactivation is exhausting the cells energy level,
inducing a regulated form of necrotic cell death
70
. More recent experiments could
avoid retinal degeneration by MNU by application of a PARP inhibitor71, 71 .However,
other research groups found evidence of apoptosis via the activation of caspase 3
72
.
Therefore, it is necessary to state, that the pathogenesis leading to retinal
degeneration induced by MNU is not yet fully understood.
4.1.5 Outlook on future therapy approaches
As already mentioned above, the only treatment for RP available at the moment is the
supplementation with vitamin A, which only slows the progression of the disease
down and is controversially discussed because of opposed results and risk of severe
adverse effects. Another compound tested is docosahexaenoic acid (DHA), a longchain omega-3 fatty acid that could be used by the photoreceptors to build up
membranes of their outer segments. However, studies indicate that the shown effect
is coupled to vitamin A supplementation, as both compounds cause a deceleration of
the pathology alone but do not have a cumulated effect together. Generally, treatment
of RP patients is limited to nutrient supplementation and therapy of associated ocular
manifestations as cataract or macular edema 50 .
A logical approach to prevent RP would be replacement of the mutated genes before
onset of photoreceptor cell death. Gene therapy is widely discussed for the treatment
of inherited diseases including RP and some major progresses were recently made.
Most research groups work with well-known adenoviral vectors, infecting cells
specifically and depositing the transgenic DNA. These viruses are however not
feasible for long-term transfection, as a potent T-cell-mediated immune response is
triggered by their presence. Lentiviruses are integrating permanently in the genome of
the host cell and have therefore a better long-term expression, but raise some safety
concerns, because their integration can lead to cancer
73
. To circumvent the problem
of immunogenicity, non-viral vectors have been invented. One of the more successful
approaches is the use of nanoparticles composed of DNA and lipoproteins, where in
experiments with rds+/- mice, a murine RP model, partial functional rescue of vision
has been shown
74
. Nonetheless, gene therapy for RP will not be available soon.
First of all, the many different genes affected in RP will prevent the development of a
universal treatment for the disease. The gene therapy would need to be tailored for
almost every single patient, depending on the mutation involved. In addition, the
61
therapy for dominant gene mutation is not as simple as for the recessive ones, where
just a non-mutated form of the gene has to be introduced to reestablish function.
Experiments with RNA interference and ribozymes show also positive results, but the
techniques are still a long way off the use in humans 50 .
Furthermore, gene therapy has the disadvantage that the treatment needs to start
before the damage occurred. However, many patients do not realize a degeneration
going on until the impairment of the peripheral vision is already pronounced. At this
stage of the disease, a huge part of the rod photoreceptors and some of the cone
photoreceptors are already lost and the remaining cells are under stressed
conditions. Recently, the idea of replacing the degenerated cells was brought up. In
this field, animal experiments are currently conducted with stem cells from distinct
origins. Transplantation of pre-differentiated embryonic stem cells in RPE65rd12 mice
yielded higher ERG response. However, severe adverse effects like tumor formation
or retinal detachment have been found
75
. One way to circumvent at least the
rejection of transplanted cells by the immune system is the use of autologous stem
cells. Cells obtained from the bone marrow and injected intravenously showed as well
a slowdown of disease progression in the RCS rat
76
. Stem cells secrete also certain
cytokines, known as growth or survival factors. Examples are pigment epitheliumderived factor (PEDF), ciliary neurotrophic factor (CNTF) or basic fibroblast growth
factor (bFGF). Several factors have been tested for effectiveness and showed some
effects in animal models, but none has emerged successfully from clinical trials up to
now 77-79 .
A more technical approach to restore visual function in blind RP patients is the
invention of retinal prostheses, a technical device with light-sensitive detectors linked
to the nervous system of the patient and delivering the information from the light
perception to the brain. Different methods are under development: microchips placed
epi- or subretinal, electrical stimulation of the retinal ganglion cells, the optic nerve or
even the visual cortex directly. Unfortunately, most of the devices did not live up to
their expectations, as their resolution was not sufficient or their implantation had too
many severe side effects. Up to now, this treatment is not an option, but with the
technical progress, this may be possible in the future 50, 80 .
62
4.2 Results
4.2.1 Caspase-3-independent photoreceptor degeneration by N-methyl-Nnitrosourea (MNU) induces morphological and functional changes in the mouse
retina
Rahel Zulliger, Stéphanie Lecaudé, Sylvie Eigeldinger-Berthou, Ute E.K. WolfSchnurrbusch, Volker Enzmann
N-methyl-N-nitrosourea (MNU) induces retinal degeneration in mice without affecting
the RPE cells. In contrast to genetic models of retinal degeneration the
pathomechanism of retinotoxic drugs like MNU is not fully understood. This study was
conducted to investigate influence of MNU on visual function, retinal morphology and
underlying molecular pathways of cell death.
Three different concentrations of MNU, 30, 45 and 60 mg/kg BW, were applied and
mice were sacrificed at several time points to measure thickness of the ONL in eye
tissue sections. To follow the progression visual function tests, namely cued water
maze and optokinetic reflex measurements, were conducted during the experiment.
Molecular changes were investigated by immunohistochemistry and qRT-PCR.
The highest dose of MNU applied led to instant loss of vision at day 1 PI. With the
other concentrations, a decrease in visual function over time was detected. The
thickness of the ONL declined concomitantly. TUNEL-staining revealed positive cells
in the ONL with 45 and 60 mg/kg BW, but no cleaved caspase 3-positive cells were
detected at any time point. With qRT-PCR, a gene expression change of caspase 1
and 12 was discovered.
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
4.3 Additional Results
4.3.1 Thickness of the ONL is decreased after the application of MNU
Application of MNU led to a significant decrease in thickness of the ONL at all time
points and with all doses applied (figure 21). The number of nuclei in the ONL
decreased concomitantly (figure 22), but the INL remained unaffected.
Fig. 21: Morphometric measurements of the outer nuclear layer (ONL) of mice treated with MNU (n=5,
mean ± SD). Thickness of the ONL was calculated by measuring the area of the ONL and dividing it by
the length of the area at six different locations in the retina. Mean values from healthy animals are
represented by the bar specified as control. Significant changes compared to control were found at all
time points PI after the MNU application with all concentrations (asterisk, p-value <0.05).
89
Fig. 22: Counts of number of nuclei in row of mice treated with MNU (n=5, mean ± SD) at six different
locations in the retina. Mean values from healthy animals are represented by the bar specified as control.
Significant changes compared to control were detected at all time points with all concentrations (asterisk, pvalue <0.05).
90
4.3.2 Autofluorescence is not influenced by application of MNU
Used as a marker for RPE integrity, autofluorescence measurements on whole eye
flat mounts were prepared after treatment with all concentrations at all investigated
time points. Considering the fact, that in the H&E-stained sections no degeneration in
the RPE cell layer was detected, no change in autofluorescence was expected.
Statistically no difference between the treated animals and the control animals was
found. Nevertheless, the appearance of the flat mount pictures changed with the
surface looking less smooth than in control animals, probably due to phagocytosis of
the dying photoreceptors by the RPE (figure 23).
Fig. 23: Autofluorescence pictures of retinal pigment epithelium cells (RPE) after treatment with MNU. At
day 7 PI after the application of 30 mg/kg MNU (B) no difference compared to control was found (A).
However, the injection of 45 mg/kg induced changes in the pattern of autofluorescence early at day 3 PI
(C), which are sustained up to day 21 PI (D), even though there was no statistical significant change in
autofluorescence intensity. The same was seen after the application of 60 mg/kg at day 3 PI (E) or day 21
PI (F) but more prominent than with the lower dose.
91
4.3.3 RPE cells are intact on the ultrastructural level
Pictures taken with the electron microscope confirmed the almost total destruction of
the ONL nine days after the application of 45 mg/kg. As already seen in the H&Estained paraffin sections, the RPE cell layer was mostly unaffected and appeared as
a physiologically normal monolayer with microvilli. Throughout the retina some RPE
cells displayed large vacuoles and look swollen, but with the monolayer still intact.
RPE cells with dissolving nuclei and an irregular distribution of pigment granules can
also be found, but only occasionally. Next to the RPE cell layer, some rare single
cells, with a macrophage-like appearance, have been found (figure 24).
Fig. 24: Ultrastructural pictures of the mouse retina. Healthy RPE cells express microvilli
(arrows) on their apical surface wrapping around the outer segments of photoreceptor cells
(arrowheads; A). After the treatment with 45 mg/kg BW MNU some RPE cells contain large
vacuoles (asterisk) and look swollen, but they still express microvilli (arrows) and form a
continuous monolayer (C, day 9 PI). However, most RPE appeared physiologically normal (B,
day 9 PI). At this time point, no photoreceptor outer segments or nuclei were left in the ONL.
Single macrophage-like cells were detectable next to the RPE cells after the treatment (D).
92
4.3.4 Electroretinography (ERG) shows impaired photoreceptor function after
MNU application
After the application of MNU the a- and b-wave amplitude decreased in a
concentration- and time-dependent manner (figure 25). The c-wave amplitude
decreased in concomitant manner (figure 26). Only qualitative analysis was
performed because the huge variations between animals and time points would
require higher animal numbers for statistical analysis.
Fig. 25: Scotopic electroretinography (ERG) measured after the application of MNU. Amplitudes of
a- and b-wave were significantly smaller in the treated animal (B, 60 mg/kg BW MNU) compared to
untreated control (A). This was seen already at day 1 PI, whereas one week after the application no
ERG was measurable anymore (data not shown).
93
Fig. 26: Scotopic ERG measured after the application of MNU. The amplitude of the c–wave was diminished
at day 1 PI after the application of 60 mg/kg BW MNU (B) compared to the untreated control (A). After the aand b-wave were obliterated within one week, the c-wave was also not measurable anymore.
94
4.3.5 Retinal degeneration induced by MNU is visible in the OCT
Layering of the mouse retina was clearly distinguishable in pictures taken with the
OCT. After treatment with 45 mg/kg BW, the layering gradually disappears until it was
barely visible (figure 27). Baseline fundus photographs showed an equally distributed,
weak autofluorescence. After the treatment with 30 mg/kg BW the autofluorescence is
much stronger, but concentrated in small, bright spots beginning at day 3 PI (figure
28).
Fig. 27: Optical coherence tomography (OCT) pictures of mice eyes taken in the infrared channel.
The sections indicated by the green circles in the fundus picture (left) are shown on the right. The
different layers of the retina were well distinguishable from each other before the treatment (day 0).
One day after the treatment with 45 mg/kg BW MNU the layering was already disturbed, a process
that continued until day 3 PI.
95
Fig. 28: Autofluorescence of a mouse eye before (left) and 7 days after (right) the treatment with 30
mg/kg BW MNU, The autofluorescence is weak and evenly distributed across the fundus before the
treatment. However, it became much more prominent and concentrated in spots over time.
96
4.3.6 Upregulation in mRNA expression does not lead to higher protein levels
From the cell death genes specific with changed mRNA expression after MNU
application three were tested for altered protein levels with quantitative Western blot.
Caspase 1 and 12 showed a significant upregulation of mRNA expression three days
after the application of 45 mg/kg BW MNU (figure 29). However, the protein level of
both molecules was significantly lower in the neurosensory retina of the treated
animals than in the controls. The mRNA expression of caspase 7 was significantly
downregulated at day 3 PI, but there was no change in protein level detectable at the
same time point.
Fig. 29: Comparison of Western blot-detected caspase protein levels in the neurosensory
retina (NSR) of mice treated with 45 mg/kg MNU BW and without treatment at day 3 PI. The
integrated intensity was normalized to actin levels before statistical analysis. Protein levels of
caspase 1 and 12 were both significantly higher in the control animals than in the treated ones
(asterisks, p-value <0.05).
97
4.4 Additional material and methods
4.4.1 Histology
Eyes were enucleated after euthanasia at day 3, 7, 14 and 21 and fixed for at least 18
h with 4% PFA (Sigma-Aldrich, Buchs, Switzerland) in PBS. The right eyes were
dehydrated, embedded in paraffin and 7 µm sections were cut. The sections were
then stained with Mayer’s hemalaun solution (Merck KGaA, Darmstadt, Germany) /
eosin G solution (Carl Roth GmbH, Karlsruhe, Germany; H&E) and analyzed with
Image-Pro plus© (Media Cybernetics, Bethesda MD, USA). Thickness of outer (ONL)
and inner nuclear layer (INL) were measured and rows of nuclei in the ONL were
counted at six different locations in each eye (400, 1000 and 1600 µm superior and
inferior from the edge of the optic nerve head). Statistical analysis was performed
using Kruskal-Wallis analysis on ranks and pairwise comparison with Tukey in the
SigmaStat 3.5 Software (Systat Software GmbH, Erkrath, Germany).
4.4.2 Whole eye flat mounts
For flat mount preparation an incision was made at the ora serrata and followed by a
circumpolar cut. The anterior part of the eye as well as the lens and the neurosensory
retina was discarded. In the remaining eyecup three centered cuts were executed and
the tissue was flattened with the RPE side up on a glass slide and mounted with
Fluorescent Mounting Medium (Dako Schweiz AG, Baar, Switzerland). The
appearance of RPE fluorescence in these flat mounts was studied on a confocal
microscope (SP 2; Leica Microsystems, Heerbrugg SG, Switzerland) with an argon
laser (wavelength, 488 nm), and digital images were acquired to compare control and
MNU-treated specimens. Finally, the area without autofluorescence was quantified
with imaging software Image-Pro plus© (Media Cybernetics, Bethesda MD, USA).
Statistical analysis was performed using Kruskal-Wallis analysis on ranks and
pairwise comparison with Tukey in the SigmaStat 3.5 Software (Systat Software
GmbH).
4.4.3 Immunohistochemistry
Immunohistochemistry was performed on paraffin sections with antibodies against
caspase 6 (rabbit, 1:100, Santa Cruz Biotechnology Inc., Heidelberg, Germany). The
antibody was diluted in tris-buffered saline (Sigma-Aldrich, Buchs, Switzerland) with
0.1% Triton X-100 (Sigma-Aldrich), 1% BSA (Sigma-Aldrich) and 2% goat serum
(Dako Schweiz AG, Baar, Switzerland) and incubated overnight at 4°C. As secondary
antibody, goat α-rabbit Alexa Fluor® 488 nm (LuBioScience, Luzern, Switzerland) was
98
applied. The secondary antibody was diluted in 1:500 in TBS with 0.1% Triton X-100
(Sigma-Aldrich) and 1% BSA (Sigma-Aldrich) for 1h at room temperature. Isotype
control antibodies were used as negative controls (normal rabbit IgG and mouse IgG,
Santa Cruz Biotechnology, Inc., Heidelberg, Germany).
4.4.4 Electron microscopy (EM)
Mice treated with 45 mg/kg BW MNU were sacrificed at different time points PI and
enucleated. The eyes were processed according to the same protocol used for MNUtreated eyes (See description above).
4.4.5 Western Blotting
Mice were treated with 45 mg/kg BW MNU and sacrificed 3 days PI. Eyes were
enucleated and treated according to the protocol used for NaIO3-treated mice (See
description above).
4.4.6 Electroretinography (ERG)
Animals were treated with 30, 45 or 60 mg/kg BW MNU and ERG measurements
were carried out in exactly the same manner as described above for NaIO 3-treated
mice.
4.4.7 Optical coherence tomography (OCT)
Animals were treated with 30, 45 or 60 mg/kg BW MNU and OCT pictures were taken
in exactly the same manner as described above for NaIO3-treated mice.
99
4.5 Discussion
Previous studies with MNU were mostly conducted with high concentrations only and
without any functional tests assessed. Therefore several doses have been tested to
investigate if MNU leads to a dose-dependent retinal degeneration. Regarding retinal
thickness of the eyes of treated mice no linear correlation between dose and damage
could be found. Whereas barely any effect was induced by using 30 mg/kg BW, the
whole retina was destroyed with 60 mg/kg BW. With a linear correlation an
intermediate dose would lead to an intermediate result, but with 45 mg/kg BW almost
the same outcome as with 60 mg/kg BW was found. However, the results were seen
time delayed. One explanation for this course of progression is the so-called hockey
stick model, where a compound induces damage only above a certain threshold. The
cell is able to repair the occurring damage below this threshold, but the degeneration
follows a steep and linear correlation to the dose above it
81
. In the H&E-stained
pictures of early time points, 3 and 7 days PI, the RPE cells looked stressed and
swollen when treated with 45 or 60 mg/kg BW. However, two weeks PI, the RPE
appears completely normal again as in untreated animals. This has also been found
by others, although this group described a migration of pigmented cells towards the
INL, what was not the case in our animals
66
. The autofluorescence measured on
whole eye flat mounts did not change quantitatively after the application of MNU,
another cue for the intactness of the RPE cell layer. However, chances could be
observed qualitatively. The autofluorescence pattern lost its smooth appearance and
looks more disturbed and random than in untreated controls.
The TUNEL-staining was applied to the same paraffin sections to get a first indication
on involved cell death pathways. Positive cells were detected at all time points after
the application of 45 or 60 mg/kg BW, except for late time points, where no cells in
the ONL were left to be positive. The application of 30 mg/kg BW did not induce any
TUNEL-positive cells at any time point. As TUNEL-staining is not very specific for
apoptosis the activation of caspase 3, one of the major players of apoptosis, has
been investigated
34
. In previous MNU studies caspase activity assays yielded
positive results for the involvement of caspase 3
69, 72
. In contrast to this, we could not
detect an activation of caspase 3 using immunohistochemistry. The discrepancy is
probably arising because of the different detection methods used. To test, if another
effector caspase, caspase 6, might be involved in the execution of cell death, staining
of tissue sections with an adequate antibody was accomplished. The staining for
whole length caspase 6 revealed an accumulation of the protein between the inner
100
and the outer layer of the retina in healthy mouse retinas, which has never been
described before. After the treatment with 45 or 60 mg/kg BW MNU, the protein
disappears gradually from the retina until day 3, where it is completely absent. This
could be due to protein relocalization or cleavage in the active form or just for
degradation purposes.
The application of 30 mg/kg BW MNU did not have any effect on the performance of
the mice in the cued water maze. On the other hand, the application of 60 mg/kg BW,
led to high increase in latency and path length, with significant difference to the
untreated control animals. The application of 45 mg/kg BW yielded a significant
difference at day 3 and 7 PI only, afterwards, the animals performed equally well as
the control animals. This learning effect, also clearly visible in the decreasing path
length and latency over time in the control group, is confounding smaller differences
between groups and the method can even fail to show a difference in visual acuity,
when the animals are not completely blind
35
. This has not been shown by previous
studies with other compounds, because for the first time lower doses not leading to
blindness have been used
27
. Optokinetic reflex measurements are far more
sensitive, as they are based on an automatic reflex and are therefore not influenced
by learning and memory
36
. With this method, a significant difference in visual acuity
after treatment with 30 mg/kg BW could be detected and that difference was also
significant after the treatment with 45 mg/kg BW up to day 21 PI. As already found
using the cued water maze testing, application of 60 mg/kg BW leads to an almost
immediate blindness of the mice.
ERG results measured after the application of MNU showed a comparable effect of
the drug as the optokinetic reflex measurement. The compound induced an
immediate decrease in amplitude measured at day 1 PI and led to a total obliteration
of the electronic response of the photoreceptors after one week with the highest dose,
60 mg/kg BW, applied. A similar decrease in ERG amplitude is seen as well in
genetic models of retinal degeneration
21, 37
. Future measurements have to
investigate the loss of electrophysiological function in a time-dependent manner. An
ongoing degeneration of the photoreceptor cells was shown as well by measurements
with retinal optical coherence tomography (OCT). With OCT, one is able to follow the
morphological changes in the retina in the same animal over time, replacing
measurements with tissue sections from several animals at the different time points
and thereby reducing animal numbers
38
. In pictures taken one week after the
101
application of MNU, changes in the autofluorescence comparable to the ones taken
from whole eye flat mounts were also seen. There seems to be a change in the RPE
cell layer, even though it does not lead to the destruction of the RPE cells. In electron
microscopy pictures a continuous monolayer of pigmented cells beneath the
remaining retina was visualized. In some parts, gaps are visible between two RPE
cells, but both cells were connected via cellular contacts. They were clearly originated
from RPE cells, as the cells displayed microvilli and contained melanin granules.
Another explanation for the spotted autofluorescence pattern in the OCT after MNU
application could be an irregular distribution of melanin and lipofuscin granules, as
seen under the electron microscope. How this irregular distribution arises is not
known, it could develop either during cell division or cell elongation. In general, the
RPE cells were not affected directly by the MNU. Another observation were
macrophage-like cells, which could represent either invading immune cells from the
blood stream or differentiated microglia from the overlying retina 82 .
As a starting point for more comprehensive protein studies to investigate the cell
death pathways involved, changes in mRNA expression of several PCD specific
genes have been tested. However, the mRNA expression data can be used as an
indicator only, as cell death is regulated on many levels. Enough cDNA from the
neurosensory retina to do our PCR experiments could be harvested up to day 10 PI,
even though the thickness of the ONL is already strongly decreased at this time point.
From the 12 genes tested for altered mRNA expression, exactly half showed a
significant response to the application of 45 mg/kg BW MNU. No activation of
caspase 3 on the protein level and as well no regulation on the mRNA expression
level has been found, confirming our previous data. The disappearance of full length
caspase 6 appears unconnected to the gene regulation as no regulation of its mRNA
was detected.
The most distinct change in gene regulation, about 10-fold, was found in the caspase
1 gene, as well known as IL-1β converting enzyme (ICE), for its involvement in
inflammation
39
. The mRNA level was only altered at day 3 PI, not at any other time
point. This indicates a peak of the immediate reaction at that time point. However, as
the activation of IL-1β has not been investigated, there is no proof for an active
inflammation. Caspase 1 belongs to the so-called inflammatory caspases, together
with caspase 12, which was upregulated even before caspase 1 at day 1 and 3 PI.
Therefore it is reasonable to hypothesize, that there is some kind of immune
102
response induced by MNU. Especially, as an infiltration with macrophages was
67
already stated by another group
. Caspase 12 is also an initiator of apoptosis
induced by stress in the endoplasmatic reticulum (ER) and therefore a candidate for
the apoptosis-induction by MNU
83
. Another candidate is caspase 8, which was
upregulated at day 3 PI and can be activated by calpain 2, which was upregulated at
the same time point 84 . Calpain 2 can be activated by an elevated level of Ca2+ ions in
the cell, either caused by leakage of the cell membrane or transport of the ions from
the mitochondrion to the cytosol
44
. The other calpains we tested, calpain 1 and 10,
were not regulated at all. Downstream of the initiator caspases act the effector
caspases 3, 6 and 7, ultimately leading to the destruction of the cell
39
. Caspase 3
and 6 are not regulated at all at the mRNA level and caspase 7 was downregulated at
all time points. Because caspase 3 was not activated, it is likely caspase 6 executing
the cell death if any caspase is involved. This would confirm the findings from the
immunohistochemistry, where disappearance of the inactive full length caspase 6
protein was observed.
The fact that caspase 2, the initiator caspase after DNA damage, was downregulated
at day 1 and 3 PI, indicates that the DNA methylation by MNU does not directly
induce apoptosis
85
. However, DNA damage can lead to cell death by another
pathway not involving caspase 2 but an enzyme called poly ADP-ribose polymerase
(PARP). PARP is activated by severe DNA damage and a mechanism of the cell to
repair DNA strand breaks. But overactivation of this enzyme can lead to cell death via
energy depletion, as the mending of DNA strand breaks needs a large amount of
NAD+ which is otherwise used for the production of ATP. This mechanisms has been
suggested by other groups using NAD+ precursors to inhibit cell death after MNU
application, too
71, 86
. No regulation was detected for the autophagy marker Atg5,
therefore this process does not likely play a role in the MNU-induced degeneration 43 .
The same holds true for cathepsin S, which would be involved in the induction of an
innate immune response 40 .
Western blot analysis did not reveal any significant changes of caspase 7 in the NSR
after the treatment with MNU at day 3 PI. A change in protein amount was probably
too small to be detected. In contrast to this, the amount of caspase 1 and 12 was
significantly smaller in the treated animals compared to the control animals. This is in
contrast to mRNA expression, which was significantly upregulated at the same time
point. This could be due to a negative feedback ensuing downregulation of the
103
translation of the relevant mRNA or by a very high turnover of the resulting protein,
either by activation or degradation.
104
5. Overall discussion
Both investigated compounds, MNU and NaIO3, induce retinal degeneration after
single application with a conjoint measurable decrease in visual function. Apart from
this, the two animal models are rather different from each other. NaIO 3 is primarily
affecting the RPE cell, leading to the disruption of the epithelial monolayer and
induces the degeneration of the photoreceptors secondarily. Unlike NaIO3, MNU
induces cell death in photoreceptors directly, without harming the RPE cells
irrevocably. NaIO3-induced degeneration shows time- and concentration dependent
linear development and, even with the highest doses applied, the total destruction of
the ONL takes more than a month. MNU in the contrary depicts a very fast onset,
leading to blindness with the highest dose applied already at day 1 PI. The
progression is much faster, leaving no photoreceptors in the outer retina 14 days PI.
In addition, the degeneration is indeed dependent on time, but not on dose, at least
not in a linear manner. According to our experiments, the correlation is more
threshold-based, with all doses over the threshold leading to similar outcome.
Although the two models have different method of action, there are similarities in the
execution of the degeneration. Both induce apoptosis in the photoreceptor cells:
NaIO3 induces the major apoptotic pathway involving caspase 3, while MNU leads to
caspase 3-independent cell death. Both compounds induce a massive change in
mRNA expression of the caspase 1 gene, but only NaIO3 leads also to accumulation
of the corresponding protein. Additional caspase genes are as well regulated in
similar manner in both models, indicating that they have common execution
sequences, even though the main cell death pathway activated is discriminative.
The impact on visual function appeared similar in both models except for the already
discussed kinetic differences. However, there are differences in the ERG
measurements. NaIO3 leads to disturbance of the c-wave not seen in the MNU model,
indicating the fact that MNU is not directly interfering with RPE cells. The same holds
true for the autofluorescence, where the differences are owed as well to the distinct
effect on the RPE cell layer, main source of the autofluorescence.
The models can be used to mimic two distinct forms of photoreceptor degeneration in
the human system. NaIO3 leads to a RPE-dependent degeneration of the
photoreceptor layer as seen in dry AMD, whereas MNU does affect photoreceptors
directly as observed in RP. Although some features of the human diseases are met,
105
these animal models are of course no specific models for AMD and RP. However,
they are useful to study some aspects of the diseases and can serve as tools for
testing of new retinal therapies. Pharmacological models have some advantages
compared to genetic models, especially the fact that baseline measurements of the
visual functions can be performed before the application of the compound and so
alleviate testing for potential benefits of treatment. In addition, as the measurements
can be executed on adult mice, there are no restrictions in the methods to use. Even
though pharmacological models are always strained with a larger variance between
animals it is still possible to induce retinal degeneration in a highly reliable manner.
5.1 Outlook
To receive a more detailed insight of how MNU and NaIO3 interact with the different
layers of the retina and induce cell death, more experiments will be needed. In
addition to the already tested cell types, the photoreceptors and the RPE cells, there
are more cells of interest, like the Müller cells and the ganglion cells. It has to be
considered as well that different types of photoreceptors, namely the rods and the
cones, could be affected differentially. Furthermore, to clarify if the pharmaceutically
induced degeneration is eliciting an immune response particular attention has to be
paid on possible immigration of immune cells and the secretion of pro-inflammatory
cytokines.
Nevertheless, the models can already serve as tools to investigate beneficial effects
of experimental treatments. The NaIO3-model is already implemented to the
microenvironment for the transplantation of stem cells in a approach of regenerative
restoration of damaged retina. It is also planned to use MNU-treated mice to assess
the efficacy of neuroprotective agents.
106
6. References
1. Naik R, Mukhopadhyay A, Ganguli M. Gene delivery to the retina: focus on non-viral
approaches. Drug Discov Today. 2009;14:306-315.
2. Berson EL. Retinitis pigmentosa: unfolding its mystery. Proc Natl Acad Sci U S A.
1996;93:4526-4528.
3. Milam AH, Li ZY, Fariss RN. Histopathology of the human retina in retinitis
pigmentosa. Prog Retin Eye Res. 1998;17:175-205.
4. Kennedy CJ, Rakoczy PE, Constable IJ. Lipofuscin of the retinal pigment epithelium:
a review. Eye (Lond). 1995;9 ( Pt 6):763-771.
5. McBee JK, Palczewski K, Baehr W, Pepperberg DR. Confronting complexity: the
interlink of phototransduction and retinoid metabolism in the vertebrate retina. Prog
Retin Eye Res. 2001;20:469-529.
6. Tezel TH, Bora NS, Kaplan HJ. Pathogenesis of age-related macular degeneration.
Trends Mol Med. 2004;10:417-420.
7. Jager RD, Mieler WF, Miller JW. Age-related macular degeneration. N Engl J Med.
2008;358:2606-2617.
8. Sancho-Pelluz J, Arango-Gonzalez B, Kustermann S, et al. Photoreceptor cell death
mechanisms in inherited retinal degeneration. Mol Neurobiol. 2008;38:253-269.
9. Solomon SG, Lennie P. The machinery of colour vision. Nat Rev Neurosci.
2007;8:276-286.
10. Forrester J.V., Dick A.D., McMenamin P.G., Lee W.R. The eye. Second edition ed.:
W.B. Saunders; 2002.
11. Fine SL, Berger JW, Maguire MG, Ho AC. Age-related macular degeneration. N Engl
J Med. 2000;342:483-492.
12. Kevany BM, Palczewski K. Phagocytosis of retinal rod and cone photoreceptors.
Physiology (Bethesda ). 2010;25:8-15.
13. De LD, Hickey PA, Meneghel G, Cantor CH. Blindness, fear of sight loss, and suicide.
Psychosomatics. 1999;40:339-344.
14. Frick KD, Gower EW, Kempen JH, Wolff JL. Economic impact of visual impairment
and blindness in the United States. Arch Ophthalmol. 2007;125:544-550.
15. Congdon NG, Friedman DS, Lietman T. Important causes of visual impairment in the
world today. JAMA. 2003;290:2057-2060.
16. http://www.who.int/mediacentre/factsheets/fs282/en/index.html. WHO. 2010.
17. Chakravarthy U. Age related macular degeneration. BMJ. 2006;333:869-870.
18. Sparrow JR, Boulton M. RPE lipofuscin and its role in retinal pathobiology. Exp Eye
Res. 2005;80:595-606.
107
19. de Jong PT. Age-related macular degeneration. N Engl J Med. 2006;355:1474-1485.
20. Engel HM, Dawson WW, Ulshafer RJ, Hines MW, Kessler MJ. Degenerative changes
in maculas of rhesus monkeys. Ophthalmologica. 1988;196:143-150.
21. Rakoczy PE, Yu MJT, Nusinowitz S, Chang B, Heckenlively JR. Mouse models of
age-related macular degeneration. Exp Eye Res. 2006:741-752.
22. Chang B, Mandal MN, Chavali VR, et al. Age-related retinal degeneration (arrd2) in a
novel mouse model due to a nonsense mutation in the Mdm1 gene. Hum Mol Genet.
2008;17:3929-3941.
23. Hollyfield JG, Bonilha VL, Rayborn ME, et al. Oxidative damage-induced
inflammation initiates age-related macular degeneration. Nat Med. 2008;14:194-198.
24. Sorsby A. Experimental pigmentary degeneration of the retina by sodium iodate. Br J
Ophthalmol. 1941;25:58-62.
25. Gong L, Wu Q, Song B, Lu B, Zhang Y. Differentiation of rat mesenchymal stem cells
transplanted into the subretinal space of sodium iodate-injected rats. Clin Experiment
Ophthalmol. 2008;36:666-671.
26. Kiuchi K, Yoshizawa K, Shikata N, Moriguchi K, Tsubura A. Morphologic
characteristics of retinal degeneration induced by sodium iodate in mice. Curr Eye
Res. 2002;25:373-379.
27. Enzmann V, Row BW, Yamauchi Y, et al. Behavioral and anatomical abnormalities in
a sodium iodate-induced model of retinal pigment epithelium degeneration. Exp Eye
Res. 2006;82:441-448.
28. Orzalesi N, Calabria GA. The penetration of I-131 labeled sodium iodate into the
ocular tissues and fluids. Ophthalmologica. 1967;153:229-238.
29. Korte GE, Reppucci V, Henkind P. RPE destruction causes choriocapillary atrophy.
Invest Ophthalmol Vis Sci. 1984;25:1135-1145.
30. Morris B, Imrie F, Armbrecht AM, Dhillon B. Age-related macular degeneration and
recent developments: new hope for old eyes? Postgrad Med J. 2007;83:301-307.
31. Binder S, Stanzel BV, Krebs I, Glittenberg C. Transplantation of the RPE in AMD.
Prog Retin Eye Res. 2007;26:516-554.
32. Holz FG. [Autofluorescence imaging of the macula]. Ophthalmologe. 2001;98:10-18.
33. von Leithner PL, Ciurtin C, Jeffery G. Microscopic mammalian retinal pigment
epithelium lesions induce widespread proliferation with differences in magnitude
between center and periphery. Mol Vis. 2010;16:570-581.
34. Stadelmann C, Lassmann H. Detection of apoptosis in tissue sections. Cell Tissue
Res. 2000;301:19-31.
35. Morris R. Developments of a water-maze procedure for studying spatial learning in
the rat. J Neurosci Methods. 1984;11:47-60.
108
36. Prusky GT, Alam NM, Beekman S, Douglas RM. Rapid quantification of adult and
developing mouse spatial vision using a virtual optomotor system. Invest Ophthalmol
Vis Sci. 2004;45:4611-4616.
37. Weymouth AE, Vingrys AJ. Rodent electroretinography: methods for extraction and
interpretation of rod and cone responses. Prog Retin Eye Res. 2008;27:1-44.
38. Drexler W, Fujimoto JG. State-of-the-art retinal optical coherence tomography. Prog
Retin Eye Res. 2008;27:45-88.
39. Chowdhury I, Tharakan B, Bhat GK. Caspases - an update. Comp Biochem Physiol B
Biochem Mol Biol. 2008;151:10-27.
40. Conus S, Simon HU. Cathepsins: key modulators of cell death and inflammatory
responses. Biochem Pharmacol. 2008;76:1374-1382.
41. Morishima N, Nakanishi K, Takenouchi H, Shibata T, Yasuhiko Y. An endoplasmic
reticulum stress-specific caspase cascade in apoptosis. Cytochrome c-independent
activation of caspase-9 by caspase-12. J Biol Chem. 2002;277:34287-34294.
42. Maelfait J, Beyaert R. Non-apoptotic functions of caspase-8. Biochem Pharmacol.
2008;76:1365-1373.
43. Yousefi S, Simon HU. Apoptosis regulation by autophagy gene 5. Crit Rev Oncol
Hematol. 2007;63:241-244.
44. Vosler PS, Brennan CS, Chen J. Calpain-mediated signaling mechanisms in neuronal
injury and neurodegeneration. Mol Neurobiol. 2008;38:78-100.
45. Crane IJ, Liversidge J. Mechanisms of leukocyte migration across the blood-retina
barrier. Semin Immunopathol. 2008;30:165-177.
46. Ueno T, Toi M, Linder S. Detection of epithelial cell death in the body by cytokeratin
18 measurement. Biomed Pharmacother. 2005;59 Suppl 2:S359-S362.
47. Gil J, Almeida S, Oliveira CR, Rego AC. Cytosolic and mitochondrial ROS in
staurosporine-induced retinal cell apoptosis. Free Radic Biol Med. 2003;35:15001514.
48. Kalloniatis M, Fletcher EL. Retinitis pigmentosa: understanding the clinical
presentation, mechanisms and treatment options. Clin Exp Optom. 2004;87:65-80.
49. Hartong DT, Berson EL, Dryja TP. Retinitis pigmentosa. Lancet. 2006;368:17951809.
50. Shintani K, Shechtman DL, Gurwood AS. Review and update: current treatment
trends for patients with retinitis pigmentosa. Optometry. 2009;80:384-401.
51. Cai X, Conley SM, Naash MI. RPE65: role in the visual cycle, human retinal disease,
and gene therapy. Ophthalmic Genet. 2009;30:57-62.
52. Liu Q, Zhang Q, Pierce EA. Photoreceptor sensory cilia and inherited retinal
degeneration. Adv Exp Med Biol. 2010;664:223-232.
109
53. Heckenlively JR, Chang B, Erway LC, et al. Mouse model for Usher syndrome:
linkage mapping suggests homology to Usher type I reported at human chromosome
11p15. Proc Natl Acad Sci U S A. 1995;92:11100-11104.
54. Chang B, Hawes NL, Hurd RE, Davisson MT, Nusinowitz S, Heckenlively JR. Retinal
degeneration mutants in the mouse. Vision Res. 2002;42:517-525.
55. Redmond TM, Yu S, Lee E, et al. Rpe65 is necessary for production of 11-cis-vitamin
A in the retinal visual cycle. Nat Genet. 1998;20:344-351.
56. Huang PC, Gaitan AE, Hao Y, Petters RM, Wong F. Cellular interactions implicated in
the mechanism of photoreceptor degeneration in transgenic mice expressing a
mutant rhodopsin gene. Proc Natl Acad Sci U S A. 1993;90:8484-8488.
57. Naash MI, Hollyfield JG, al-Ubaidi MR, Baehr W. Simulation of human autosomal
dominant retinitis pigmentosa in transgenic mice expressing a mutated murine opsin
gene. Proc Natl Acad Sci U S A. 1993;90:5499-5503.
58. Wilson JH, Wensel TG. The nature of dominant mutations of rhodopsin and
implications for gene therapy. Mol Neurobiol. 2003;28:149-158.
59. Marti A, Hafezi F, Lansel N, et al. Light-induced cell death of retinal photoreceptors in
the absence of p53. Invest Ophthalmol Vis Sci. 1998;39:846-849.
60. Noell WK. The effect of iodoacetate on the vertebrate retina. J Cell Physiol.
1951;37:283-307.
61. GRAYMORE C, TANSLEY K. Iodoacetate poisoning of the rat retina. II. Glycolysis in
the poisoned retina. Br J Ophthalmol. 1959;43:486-493.
62. GRAYMORE C, TANSLEY K. Iodoacetate poisoning of the rat retina. I. Production of
retinal degeneration. Br J Ophthalmol. 1959;43:177-185.
63. Orzalesi N, Calabria GA, Grignolo A. Experimental degeneration of the rabbit retina
induced by iodoacetic acid. A study of the ultrastructure, the rhodopsin cycle and the
uptake of 14C-labeled iodoacetic acid. Exp Eye Res. 1970;9:246-253.
64. Leung WK, Wu KC, Wong CY, et al. Transgenic cyclooxygenase-2 expression and
high salt enhanced susceptibility to chemical-induced gastric cancer development in
mice. Carcinogenesis. 2008;29:1648-1654.
65. Smith SB, Cooke CB, Yielding KL. Effects of the antioxidant butylated hydroxytoluene
(BHT) on retinal degeneration induced transplacentally by a single low dosage of Nmethyl-N-nitrosourea (MNU). Teratog Carcinog Mutagen. 1988;8:175-189.
66. Yuge K, Nambu H, Senzaki H, et al. N-methyl-N-nitrosourea-induced photoreceptor
apoptosis in the mouse retina. In Vivo. 1996;10:483-488.
67. Nakajima M, Nambu H, Shikata N, Senzaki H, Miki H, Tsubura A. Pigmentary
degeneration induced by N-methyl-N-nitrosourea and the fate of pigment epithelial
cells in the rat retina. Pathol Int. 1996;46:874-882.
110
68. Nambu H, Yuge K, Nakajima M, et al. Morphologic characteristics of N-methyl-Nnitrosourea-induced retinal degeneration in C57BL mice. Pathol Int. 1997;47:377-383.
69. Yoshizawa K, Nambu H, Yang J, et al. Mechanisms of photoreceptor cell apoptosis
induced by N-methyl-N-nitrosourea in Sprague-Dawley rats. Lab Invest.
1999;79:1359-1367.
70. Zong WX, Ditsworth D, Bauer DE, Wang ZQ, Thompson CB. Alkylating DNA damage
stimulates a regulated form of necrotic cell death. Genes Dev. 2004;18:1272-1282.
71. Miki K, Uehara N, Shikata N, Matsumura M, Tsubura A. Poly (ADP-ribose)
polymerase inhibitor 3-aminobenzamide rescues N-methyl-N-nitrosourea-induced
photoreceptor cell apoptosis in Sprague-Dawley rats through preservation of nuclear
factor-kappaB activity. Exp Eye Res. 2007;84:285-292.
72. Yoshizawa K, Yang J, Senzaki H, et al. Caspase-3 inhibitor rescues N -methyl- N nitrosourea-induced retinal degeneration in Sprague-Dawley rats. Exp Eye Res.
2000;71:629-635.
73. Liu MM, Tuo J, Chan CC. Gene therapy for ocular diseases. Br J Ophthalmol. 2010.
74. Cai X, Nash Z, Conley SM, Fliesler SJ, Cooper MJ, Naash MI. A partial structural and
functional rescue of a retinitis pigmentosa model with compacted DNA nanoparticles.
PLoS One. 2009;4:e5290.
75. Wang NK, Tosi J, Kasanuki JM, et al. Transplantation of reprogrammed embryonic
stem cells improves visual function in a mouse model for retinitis pigmentosa.
Transplantation. 2010;89:911-919.
76. Wang S, Lu B, Girman S, et al. Non-invasive stem cell therapy in a rat model for
retinal degeneration and vascular pathology. PLoS One. 2010;5:e9200.
77. Bok D. Ciliary neurotrophic factor therapy for inherited retinal diseases: pros and
cons. Retina. 2005;25:S27-S28.
78. Chader GJ. Surmountable challenges in translating pigment epithelium-derived factor
(PEDF) therapy from animal models to clinical trials for retinal degenerations. Retina.
2005;25:S29-S30.
79. Lavail MM. Survival factors for treatment of retinal degenerative disorders: preclinical
gains and issues for translation into clinical studies. Retina. 2005;25:S25-S26.
80. Winter JO, Cogan SF, Rizzo JF, III. Retinal prostheses: current challenges and future
outlook. J Biomater Sci Polym Ed. 2007;18:1031-1055.
81. Johnson GE, Doak SH, Griffiths SM, et al. Non-linear dose-response of DNA-reactive
genotoxins: Recommendations for data analysis. Mutat Res. 2009;678:95-100.
82. Xu H, Chen M, Manivannan A, Lois N, Forrester JV. Age-dependent accumulation of
lipofuscin in perivascular and subretinal microglia in experimental mice. Aging Cell.
2008;7:58-68.
111
83. Szegezdi E, Fitzgerald U, Samali A. Caspase-12 and ER-stress-mediated apoptosis:
the story so far. Ann N Y Acad Sci. 2003;1010:186-194.
84. Frisch SM. Caspase-8: fly or die. Cancer Res. 2008;68:4491-4493.
85. Krumschnabel G, Sohm B, Bock F, Manzl C, Villunger A. The enigma of caspase-2:
the laymen's view. Cell Death Differ. 2009;16:195-207.
86. Kiuchi K, Yoshizawa K, Shikata N, Matsumura M, Tsubura A. Nicotinamide prevents
N-methyl-N-nitrosourea-induced photoreceptor cell apoptosis in Sprague-Dawley rats
and C57BL mice. Exp Eye Res. 2002;74:383-392.
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7. List of publications
7.1 Original papers
Franco LM*, Zulliger R*, Wolf-Schnurrbusch UE, et al. Low dose sodium iodate
induces patchy loss of retinal pigment epithelium and decreases visual function in a
mouse model. Invest Ophthalmol Vis Sci. 2009. *Equal contribution
Retina: Rasagiline interferes with neurodegeneration in the prph2/rds mouse. Sylvie
Eigeldinger-Berthou, Claudia Meier, Rahel Zulliger, Stéphanie Lecaudé, Volker
Enzmann, Gian-Marco Sarra. Submitted for publication
Graefe's
Arch
Clin
Exp
Ophthalmol:
Caspase-3-independent
photoreceptor
degeneration by N-methyl-N-nitrosourea (MNU) induces morphological and functional
changes in the mouse retina. Rahel Zulliger, Stéphanie Lecaudé, Sylvie EigeldingerBerthou, Ute E.K. Wolf-Schnurrbusch, Volker Enzmann. Submitted for publication
7.2 Abstracts
Retinal degeneration: Models and experimental therapies. Rahel Zulliger, Sylvie
Eigeldinger-Berthou, Volker Enzmann. Swiss Eye Research Meeting 2008
Sodium iodate-induced changes in the mouse retina are time and concentration
dependent. Rahel Zulliger , Sylvie Eigeldinger-Berthou, Volker Enzmann. Tag der
klinischen Forschung 2008
MNU induces dose-dependent cell death in the mouse retina. Rahel Zulliger, Sylvie
Eigeldinger-Berthou, Ute E.K. Wolf-Schnurrbusch, Volker Enzmann. Swiss Eye
Research Meeting 2009
N-Methyl-N-Nitrosourea (MNU) induces concentration-dependent cell death in
photoreceptors of the mouse retina. Rahel Zulliger, Sylvie Eigeldinger-Berthou, Ute
E.K. Wolf-Schnurrbusch, Volker Enzmann. ARVO 2009 annual meeting, Fort
Lauderdale
In vitro transformation of bone marrow-derived stem cells during coculture with retinal
pigment epithelium cells. Stéphanie Lecaudé, Rahel Zulliger, Volker Enzmann. ARVO
2009 annual meeting, Fort Lauderdale
Detection of different apoptotic pathways in animal models of retinal degeneration.
Rahel Zulliger, Sylvie Eigeldinger-Berthou, Stéphanie Lecaudé, Ute E.K. Wolf113
Schnurrbusch, Volker Enzmann. Swiss Eye Research Meeting 2010, ARVO 2010
annual meeting, Fort Lauderdale (Travel Grant Award)
In vitro characterization of retina-committed bone marrow-derived progenitor cells.
Stéphanie Lecaudé, Rahel Zulliger, Sebastian Wolf, Volker Enzmann. ARVO 2010
annual meeting, Fort Lauderdale
Two pharmacological animal models of retinal degeneration: their impact on function,
morphology and induction of cell death in the retina. Rahel Zulliger, Sylvie
Eigeldinger-Berthou, Stéphanie Lecaudé, Ute E.K. Wolf-Schnurrbusch, Volker
Enzmann. Young Researcher Vision Camp 2010
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8. Acknowledgements
I would like to express my gratitude to my supervisor, PD Dr. Volker Enzmann, for
giving me the opportunity to conduct my PhD-thesis in his lab and for his excellent
supervision, especially during the final stages of my doctoral studies. I would also like
to thank Prof. Dr. Robert Rieben and Prof. Dr. Martin Frenz for their mentoring during
my thesis and their guidance in scientific and personal questions. I would also like to
thank Prof. Dr. Anne-Catherine Andres for acting as a substitute examiner at my
thesis defense.
I would like to thank Monika Kilchenmann, Silvana Müller and Sorin Ciocan for their
outstanding technical support and all members from the research lab for the warm
atmosphere and the numerous discussions. I am thankful to Dr. Sylvie EigeldingerBerthou and Dr. Stéphanie Lecaudé for their training and supervision in technical
questions. They were a huge support with all the little things that can render the life of
a PhD-student quite difficult. I would also like to thank Dr. Ute Wolf, for spending
some of her precious time to help me, whenever I needed it.
During my PhD-thesis, I met many people who inspired me with their ideas and let me
benefit from their huge knowledge. I would like to thank them all, even though I’m not
able to list them by name.
At last, I would like to thank my family, my friends and especially my mother, for
keeping me grounded and going with me through the ups and downs of life. Without
you, all the achievements of the last years would not have been possible.
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