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University of Veterinary Medicine Hannover
Intravitreal injections in mice as a technique to induce unilateral
photoreceptor degeneration in comparison to the genetic model of
the rd10 mouse
Thesis
Submitted in partial fulfilment of the requirements for the degree
- Doctor of Veterinary Medicine -
Doctor medicinae veterinariae
(Dr. med. vet.)
by
Carina Sarah Rösch
Aachen
Hannover 2013
Academic supervisors: 1. Univ.-Prof. Dr. med. vet. Christiane Pfarrer
Department of Anatomy
University of Veterinary Medicine Hannover
2. Univ.-Prof. Dr. med. Peter Walter
Department of Ophthalmology
University Hospital RWTH Aachen
1. Referee: Univ.-Prof. Dr. med. vet. Christiane Pfarrer
Department of Anatomy, University of Veterinary Medicine Hannover
Univ.-Prof. Dr. med. Peter Walter
Department of Ophthalmology, University Hospital RWTH Aachen
2. Referee: PD Dr. Veronika M. Stein, PhD, Dipl. ECVN
University of Veterinary Medicine Hannover
Day of oral examination: 12.11.2013
This work was supported by the DFG grants WA 1472/6-1 to PW and MU 3036/3-1 to FM.
DEDICATED TO MY PARENTS
Parts of this work were submitted for publication in the following scientific journals:
Graefe´s Archive for Clinical and Experimental Ophthalmology, Springer-Verlag GmbH, Heidelberg,
Germany.
Investigative Ophthalmology & Visual Science (IOVS), ARVO JOURNAL, Rockville, Maryland,
USA.
Journal of Ophthalmology, Hindawi Publishing Corporation, 410 Park Avenue, 15th Floor, #287 pmb,
New York, NY 10022, USA.
Parts of the results of this doctoral thesis were presented as scientific posters (P) or as lecture
(L) at the following symposia and conferences:
Annual Meeting 2013 of the Association for Research in Vision and Ophthalmology (ARVO),
May 05-09, 2013 Seattle, Washington (P):
Effect of Intravitreal Injection of Iodoacetic Acid in Mice as a Model of Pharmacological Induced
Monolateral Photoreceptor Degeneration
Sarah Rösch1,2
, Stephan Hesse1, Christine Haselier
1, Babac A. Mazinani
1, Gernot Roessler
1, Christiane
Pfarrer2, Peter Walter
1
1Department of Ophthalmology, RWTH Aachen University, Germany,
2Department of Anatomy,
University of Veterinary Medicine Hannover, Germany
European Retina Meeting 2013, October 03-05, 2013 Alicante, Spain (P):
Characterisation of selective photoreceptor degeneration in mice induced by intravitreal
injections of N-methyl-N-nitrosourea in comparison to the systemic treatment and a genetic
model.
Sarah Rösch1,2
, Frank Müller3, Christiane Pfarrer
2, Peter Walter
1
1Department of Ophthalmology, RWTH Aachen University, Germany,
2Department of Anatomy,
University of Veterinary Medicine Hannover, Germany, 3Institute of Complex Systems, Cellular
Biophysics, ICS-4, Forschungszentrum Jülich GmbH, Germany
Artificial Vision, November 08-09, 2013 Aachen, Germany (L):
Effects of intravitreal injection of iodoacetic acid and N-methyl-N-nitrosourea on photoreceptor
survival in mice
Sarah Rösch1,2
, Sandra Johnen1, Frank Müller
3, Christiane Pfarrer
2, Peter Walter
1
1Department of Ophthalmology, RWTH Aachen University, Germany,
2Department of Anatomy,
University of Veterinary Medicine Hannover, Germany, 3Institute of Complex Systems, Cellular
Biophysics, ICS-4, Forschungszentrum Jülich GmbH, Germany
TABLE OF CONTENTS
LIST OF ABBREVIATIONS
1 INTRODUCTION .............................................................................................................. 9
2 LITERATURE SURVEY ................................................................................................ 11
2.1 The eye ..................................................................................................................... 11
2.2 Retinitis pigmentosa ................................................................................................. 11
2.3 Implantable visual prostheses................................................................................... 13
2.4 Genetic animal models of retinal degeneration ........................................................ 14
2.4.1 rd10 mouse ....................................................................................................... 14
2.4.2 Genetic models in larger animals than rodents ................................................ 15
2.5 Induction of photoreceptor degeneration ................................................................. 16
2.5.1 Iodoacetic acid .................................................................................................. 17
2.5.2 N-methyl-N-nitrosourea ................................................................................... 18
2.6 The intravitreal injection .......................................................................................... 18
3 AIM OF THIS THESIS .................................................................................................... 19
4 MATERIALS & METHODS ........................................................................................... 20
4.1 Animals .................................................................................................................... 20
4.2 Intravitreal injections in mice ................................................................................... 22
4.3 Electroretinography .................................................................................................. 23
4.4 Spectral domain Optical Coherence Tomography ................................................... 24
4.5 Fluorescein angiography (FA) ................................................................................. 25
4.6 Histology .................................................................................................................. 25
4.7 Urine test .................................................................................................................. 26
4.8 Statistics ................................................................................................................... 26
5 RESULTS ......................................................................................................................... 27
5.1 STUDY I ......................................................................................................................
Correlations between ERG, OCT and anatomical findings in the rd10 mouse ....... 27
5.1.1 ABSTRACT ..................................................................................................... 28
5.1.2 INTRODUCTION ............................................................................................ 29
5.1.3 MATERIALS AND METHODS ..................................................................... 30
5.1.4 RESULTS ......................................................................................................... 34
5.1.5 DISCUSSION .................................................................................................. 36
5.1.6 CONCLUSIONS .............................................................................................. 37
5.1.7 REFERENCES ................................................................................................. 38
5.1.8 FIGURES ......................................................................................................... 40
5.2 STUDY II ....................................................................................................................
The effects of iodoacetic acid on the mouse retina .................................................. 45
5.2.1 ABSTRACT ..................................................................................................... 46
5.2.2 INTRODUCTION ............................................................................................ 47
5.2.3 MATERIALS & METHODS ........................................................................... 49
5.2.4 RESULTS ......................................................................................................... 54
5.2.5 DISCUSSION .................................................................................................. 57
5.2.6 ACKNOWLEDGEMENT ............................................................................... 59
5.2.7 REFERENCES ................................................................................................. 60
5.2.8 FIGURES ......................................................................................................... 64
5.3 STUDY III ...................................................................................................................
Selective photoreceptor degeneration by intravitreal injection of MNU ................. 70
5.3.1 ABSTRACT ..................................................................................................... 71
5.3.2 INTRODUCTION ............................................................................................ 72
5.3.3 MATERIALS AND METHODS ..................................................................... 72
5.3.4 RESULTS ......................................................................................................... 80
5.3.5 DISCUSSION .................................................................................................. 84
5.3.6 ACKNOWLEDGEMENT ............................................................................... 87
5.3.7 REFERENCES ................................................................................................. 88
5.3.8 FIGURES ......................................................................................................... 91
6 GENERAL DISCUSSION ............................................................................................. 104
7 SUMMARY ................................................................................................................... 110
8 ZUSAMMENFASSUNG ............................................................................................... 112
9 REFERENCES ............................................................................................................... 114
10 ACKNOWLEDGEMENTS ........................................................................................... 123
LIST OF ABBREVIATIONS
IVI intravitreal injection
rd10 retinal degeneration; genetic blind mouse strain
RP Retinitis pigmentosa
IAA iodoacetic acid
MNU N-methyl-N-nitrosourea
ERG electroretinogram
sd-OCT spectral domain Optical Coherence Tomography
FA fluorescein angiography
RPE retinal pigment epithelial cells
PRA Progressive retinal atrophy
XLPRA X-linked progressive retinal atrophy
cGMP cyclic guanosine monophosphate
ß-PDE ß-subunit of the rod cGMP phosphodiesterase
P Postnatal days; days after birth
i.v. intravenous
i.p. intraperitoneal
BW body weight
Fig. figure
BAC Bacterial Artificial Chromosome
TG-rabbit transgenic rabbit
GAPDH glyceraldehyd-3-phosphate dehydrogenase
TUNEL terminal deoxynucleotidyl transferase dUTP nick-end labeling staining
AMD age-related macular degeneration
PBS phosphate buffered saline
NaOH sodium hydroxide
DMSO dimethylsulfoxide
PA paraformaldehyde
PB phosphate buffer
GFAP glial fibrillary acidic protein
9
INTRODUCTION
1 INTRODUCTION
In certain diseases of the neural system, the loss of function can be treated by means of
electrical stimulation provided by electronic implants. Concerning the visual system, inherited
retinal degenerations may lead to blindness and are currently not treatable. However, visual
function of impaired patients may be restored by retinal implants (WALTER 2005). Retinitis
pigmentosa (RP) is one example of an inherited human disease with selective photoreceptor
degeneration, in which distinct gene mutations lead to the degeneration of first rods and then
consecutively cones with a peripheral to central gradient, whereas the thickness of the inner
retinal layers remains unaffected (TSUBURA et al. 2010). Animal models for comparable
retinal degenerations are important tools to explore experimental therapies for currently
untreatable conditions. In rodents, genetic models are available resembling photoreceptor
degenerations, such as observed in RP. In mice, for example the retinal degeneration (rd) 10
mutant, carrying a mutation in the gene encoding the ß-subunit of rod cGMP
phosphodiesterase, was described and widely used (CHANG et al. 2007). In these mice,
comparable to RP, a progressive loss of photoreceptors - first rods, then cones - was observed
with increasing age, concomitant with a pronounced reduction in the thickness of the outer
nuclear layer (ONL), whereas the thickness of the inner retinal layers remained unaffected
(CHANG et al. 2007).
However, genetic models of animal species with larger eyes are desirable in order to evaluate
experimental surgical approaches for the treatment of retinal degenerations, but they are
intrinsically expensive and show a very slow disease progression (SCOTT et al. 2011). As
photoreceptor degeneration can also be induced pharmacologically, the aim of this project
was to establish a pharmacological model that avoids systemic side effects together with the
advantage of having an intraindividual control eye. To this end, the - in this context “new” -
technique of intravitreal injections was introduced. The local administration of this injection
technique was hypothesized to induce photoreceptor degeneration similar to the effects
obtained with systemic application, but without affecting the contralateral eye as well as the
general health status of the animal.
10
INTRODUCTION
In this work, all experiments were performed in mice. The mouse model was used in these
experiments in order to compare the results to the available genetic rd10 mouse model of
retinal degeneration.
After characterisation of wild type as well as rd10 mice using different clinical tools as for
example full-field electroretinogram (ERG), Optical Coherence Tomography (OCT) and
fluorescein angiography in vivo and final histological analysis in vitro, wild type mice were
injected systemically and intravitreally with iodoacetic acid (IAA) as well as N-methyl-N-
nitrosourea (MNU). The effects of these two chemical substances were evaluated by the same
methods (see Figure labelled “Project overview”).
11
LITERATURE SURVEY
2 LITERATURE SURVEY
2.1 The eye
The eyeball (bulbus oculi) consists of three layers:
Tunica fibrosa bulbi (including sclera and cornea)
Tunica vasculosa bulbi (including choroidea, corpora ciliares and iris)
Tunica nervosa bulbi (retina) (NICKEL et al. 2004)
This hollow sphere, the bulbus oculi, is completed by the lens, the humor aquosus and the
corpus vitreum. Together with the optic nerve, the central visual pathways and the area optica
(visual cortex), they form the sight organ (organum visum) (NICKEL et al. 2004). The tunica
nervosa bulbi consists of the stratum nervosum - with the photoreceptors (rods and cones,
stratum neuroepitheliale), the I. neuron (stratum ganglionare retinae) and the II. neuron
(stratum ganglionare n. optici) - and the stratum pigmentosum retinae (NICKEL et al. 2004).
In humans, the stratum nervosum counts up to 150 million rods and about 6 million cones
(NICKEL et al. 2004). Retinitis pigmentosa (RP) is an inherited retinal disease leading to the
degeneration of photoreceptors and consecutively leading to blindness in humans (TSUBURA
et al. 2010).
The eye of the mouse, the animal species used in these experiments, is organised in the same
way as in humans. The retina is rod dominated with only a few per cent of cones (WANG et
al. 2011). However, the cones are not organized into a central fovea, as in the human eye
(WANG et al. 2011).
2.2 Retinitis pigmentosa
Retinitis pigmentosa (RP) is an inherited, bilaterally progressive, human retinal disease -
while the name “Retinitis pigmentosa”, given by Frans Donders in 1857, indicates
misleadingly an inflammatory disease (TSUBURA et al. 2010). More than 100 different
forms of mutations, mostly missense mutations, which are associated with mutations of the
rod-specific opsin rhodopsin, are leading to the same result of phenotypic Retinitis
pigmentosa (TSO et al. 1997; HARTONG et al. 2006; WANG et al. 2011). RP shows genetic
heterogeneity with autosomal recessive, autosomal dominant and X chromosome-linked traits
(TUNTIVANICH et al. 2009).
12
LITERATURE SURVEY
During the progress of the disease, first rods and then cones degenerate, because rods seem to
secrete diffusible factors, which are essential for the surviving of the cones (TSO et al. 1997;
HICKS a. SABEL 1999). The interneurons and the ganglion cells of the inner retina remain
relatively intact (SCOTT et al. 2011). A characteristic sign of RP is the intraretinal
pigmentation in bone spicule deposits in the fundus, because the retinal pigment epithelial
cells (RPE) migrate near to the retinal blood vessels, which attenuate (TSUBURA et al.
2010). The prevalence of RP in humans is 1/4000 (TSUBURA et al. 2010). The age of onset
and rate of retinal degeneration varies because of the different forms and the inconsistent
progress; however, symptoms usually begin with loss of night vision in adolescence
(nyctalopia; due to degeneration of first rods), loss of side vision in young adulthood (tunnel
vision; begin of degeneration in peripheral visual field) and loss of central vision later in life
(HARTONG et al. 2006, TSUBURA et al. 2010).
A well-known entity in veterinary medicine is the progressive retinal atrophy (PRA) in dogs,
the canine equivalent of human RP. Rarely, PRA is also seen in the Abyssinian cat (WALDE
et al. 2008). PRA is leading to significant visual impairment, also with different age of onset
(about two years of age) (WALDE et al. 2008). In contrast to RP, in nearly all breeds PRA is
an autosomal recessive form, except the X chromosome linked trait (XLPRA) in the Siberian
Husky and the autosomal dominant form in the Bullmastiff (WALDE et al. 2008). Signs are
hyperreflexitivity of the tapetum lucidum, because of the atrophy of the outer retinal layers, a
thinning of the retinal vessels in the fundus and a brighter optic disc than normal (WALDE et
al. 2008). Hypo- and hyperpigmentation in the tapetum free areas of the fundus lead to a grey
to brown radial striation in later stages (WALDE et al. 2008). Symptoms are firstly nyctalopia
and consecutively blindness as in RP, but in most cases PRA is diagnosed after observation of
a consecutive cataract formation (WALDE et al. 2008).
In humans, no forms of treatment or effective therapies are available for progressive retinal
dystrophies (WALTER 2005). The substitution of vitamin A palmitate and omega-3-rich fish,
retards disease progression (HARTONG et al. 2006). Different research approaches exist to
find possible and successful ways of treatment and to restore sight (SCOTT et al. 2011).
Retinal transplants
Gene therapy
13
LITERATURE SURVEY
Stem cells
Implantable visual prostheses
2.3 Implantable visual prostheses
Retinal implants are electrical devices in form of microelectrode arrays, which can be fixed
epiretinally (onto the inner layers of the retina),
subretinally (between the inner layers of the retina and the pigment epithelium, at the
place of the destroyed photoreceptors),
around the optic nerve as optic nerve stimulators
or in the region of the visual cortex as cortical prostheses (WALTER 2005).
Implants of all concepts have been implanted in animal experiments as well as in pilot trials in
humans (WALTER 2005). The research group of the University Hospital RWTH Aachen is
working on the epiretinal concept. The epiretinal implant is fixed on the inner surface of the
retina using micro tacks (WALTER 2005). Basically a camera takes pictures of the
environment (FEUCHT 2005). These pictures are processed into spatial and temporal patterns
of stimulation signals and transferred to the chip by telemetry. The chip stimulates the
remaining nerval structures or cells of the retina with an electrical signal. This electrical signal
reaches the visual cortex via the optic nerve and in this manner the chip restores visual
function (FEUCHT 2005).
As one example of an epiretinal implant the ARGUS implant, which was developed in the US
(NATIONAL INSTITUTE FOR HEALTH RESEARCH 2011), has already been transplanted
in human eyes. After the development of the ARGUS I in the year 2002 (16 electrodes; tested
in six patients), now the new ARGUS II (approval 2013; Second Sight medical products,
Sylmar, California, USA) with 60 electrodes is available and can be implanted. This system is
in use in Europe and in the US for treatment of RP. Because it is the only way of RP
treatment, the charges are paid by the health insurance (NATIONAL INSTITUTE FOR
HEALTH RESEARCH 2011).
Another epiretinal implant, the EPIRET-type, was developed in cooperation with the
Department of Ophthalmology of the University Hospital Aachen (ROESSLER et al. 2009;
MENZEL-SEVERING et al. 2012). The microelectrode array of the EPIRET3, as a wireless
device entirely implanted within the eye, consists of 25 electrodes. It was implanted in a
14
LITERATURE SURVEY
prospective clinical trial into 6 blind RP- patients (ROESSLER et al. 2009; MENZEL-
SEVERING et al. 2012).
Further implants are under development. However, all kinds of implants have to be tested
before they can be implanted in humans. The experimental animals for these trials have to
show the same conditions of selective photoreceptor degeneration, as seen in human RP.
2.4 Genetic animal models of retinal degeneration
In small animals, especially in rodents, genetic models are easily commercially available.
For our study, we used the rd10 mouse to characterize the effects of the selective
photoreceptor degeneration by clinical tools, in comparison to the wild type mouse.
The animal suffering of mice, to get on both eyes completely blind, is maybe not as wearing
as for a larger animal (e.g., rabbit or cat), considering the animal species-specific ability to
suffer (TIERSCHUTZGESETZ 2013). As nocturnal animals, mice have to orientate
predominantly in the dark (ENGELHARDT a. BREVES 2004). Their most important sensory
organs for intraspecies communication and environmental monitoring are the vibrissae or
tactile hairs (AHL 1986), and their smell sense.
The strain and stress of diurnal (active during the day and sleeping at the night) or crepuscular
(active primarily during twilight) animals after development of a complete blindness seems to
be higher, although these animals have other very well developed sensory organs, too
(SMITH a. TAYLOR 1995). In these animal species, a complete blindness would be a higher
load of welfare, than to get blind on only one eye.
2.4.1 rd10 mouse
Rd is the abbreviation of “retinal degeneration” (CHANG et al. 2007). This mutant strain
occurs naturally (HICKS a. SABEL 1999). A missense point mutation in exon 13 of the ß-
subunit of the rod cGMP phosphodiesterase type 6 (ß-PDE) gene leads to the degeneration of
first rods and then consecutively cones with an, in contrast to RP, central to peripheral
gradient (CHANG et al. 2007). The degradation starts postnatally after 16 days (P 16) and the
peak of photoreceptor degeneration is reached at P 25 after birth (CHANG et al. 2007). Chang
et al. (2007) observed the complete loss of photoreceptors on P 60, while the inner retinal
layers survive and the thickness of ganglion cell layer and inner nuclear layer does not
15
LITERATURE SURVEY
decrease (LIANG et al. 2008; CHANG et al. 2007). The cells of the inner retinal layers, e.g.,
horizontal cells and rod and cone bipolar cells, morphologically change and undergo
remodelling (PHILLIPS et al. 2010).
Beside the rd10 mouse, there are many comparable genetic retinal degeneration models in
mice, e.g., the rd1 mouse. But because of the later onset and milder retinal degeneration in
contrast to the rd1 mouse (degeneration begins at P 8; with 4 weeks of age no photoreceptors
are left), the rd10 mouse provides a better genetic mouse model for human RP (CHANG et al.
2007).
2.4.2 Genetic models in larger animals than rodents
For surgical approaches, only larger animal species such as the pig, the cat, and the rabbit
come into consideration. In all three species genetic engineering and maintaining a transgenic
colony is intrinsically costly and the disease progression is slow (SCOTT et al. 2011).
While the pig is a diurnal animal, the cat and the rabbit are crepuscular animals (SMITH a.
TAYLOR 1995; ENGELHARDT a. BREVES 2004). In housing as laboratory animals and
also as pet, the cat and the rabbit had to change into diurnal as consequence of the
domestication and lighting conditions (SMITH a. TAYLOR 1995).
In general, the pig is an expensive animal and needs a lot of space (RICHTLINIE 2010/63/EU
DES EUROPÄISCHEN PARLAMENTS UND DES RATES 2010). However, the eye is
anatomically remarkably similar to the human eye (SCOTT et al. 2011). The swine retina
contains a visual streak (area centralis), which is cone-dominant, with rod enrichment in the
periphery, reminding of the human retina (WANG et al. 2011). In contrast to humans, this
region is not rod-free (SCOTT et al. 2011). A genetic RP-model of a pig was developed by
TSO et al. (1997). Although the degeneration shows features of human RP, most of the rods
are completely degenerated by nine months (TSO et al. 1997). The long period of time until
the animals express a complete disease pattern in combination with the size of the animals
leads to a difficult handling. In addition, it would be very cost intensive to pursue such a
transgenic colony (WANG et al. 2011).
The cat is used for retinal implant research in Australia. There are high demands regarding the
way these animals are kept (SMITH a. TAYLOR 1995). An important model of human
Retinitis pigmentosa in cats has been maintained for more than 28 years by Kristina
16
LITERATURE SURVEY
Narfström (MENOTTI-RAYMOND et al. 2007). But also as seen in the pig, the complete
photoreceptor degeneration and blindness is observed after a longer time period of disease
progression, in this case with the age of three to five years (MENOTTI-RAYMOND et al.
2007).
The husbandry conditions for rabbits are more easily to fulfil (SMITH a. TAYLOR 1995). In
rabbits one genetic strain was generated in Nagoya, Japan, by bacterial artificial chromosome
(BAC) transgenesis in the year 2008. This first genetic model of photoreceptor degeneration -
the transgenic (TG) rabbit - exhibited rod-dominant, progressive photoreceptor degeneration
(KONDO et al. 2009). But also in this genetic animal model, it would be extremely expensive
to get a colony of genetic rabbits to work on.
It would be useful to have the possibility to induce in reproducible manner photoreceptor
degeneration in any wanted animal species.
2.5 Induction of photoreceptor degeneration
Different physical and chemical methods are described to induce photoreceptor degeneration.
In comparison to the genetic models, they are relatively rapid and cost-effective alternatives
and can be performed in any wanted animal species (WANG et al. 2011). But only in a very
few reports describing the induction of outer retinal degenerations, the well-being, or the
effect on the health status of the experimental animals, was under consideration. In most
cases, the experiments only took a few days, and the long term effects were not seen or
evaluated.
Light exposure is a physical method and is mainly successful in albino animals. The melanin
in the pigment epithelium of pigmented eyes seems to protect against this kind of damage
(RAPP a. WILLIAMS 1980). Even if the eyes of pigmented animals are dilated, the
destruction of the photoreceptors takes longer compared to albinos, due to the absorption of
light by the pigment epithelium (RAPP a. WILLIAMS 1980). Continuous light, also with a
low light intensity, can be a toxic agent (RAPP a. WILLIAMS 1980). Animals have to be
kept in husbandry conditions since birth with very low LUX values (brightness value) or dim
cyclic light (RAPP a. WILLIAMS 1980) - so an own breeding not with the commercial high
17
LITERATURE SURVEY
LUX values is assumed. The prolonged light exposure in animals led to weight loss of 20%,
possibly caused by stress (DUREAU et al. 1996).
Outer retinal degeneration can also be induced chemically. An example is the implantation of
iron particles in the vitreous cavity (WANG et al. 1998). The cause for the iron-induced
retinopathy is an apoptotic mechanism limited to the outer nuclear layer (WANG et al. 1998).
This disease pattern is known as “siderosis bulbi” in human medicine (TERAI et al. 2011).
Beside the effects on the retina, also side effects on the iris (heterochromia, pupilloplegia) and
a shift of the lens were observed (TERAI et al. 2011).
Further the systemic application of special pharmaceuticals leads to a selective photoreceptor
degeneration. These drugs are used since about 50 years in nearly all kinds of animals. Given
systemically they have considerable systemic toxic side effects, e.g., inhibition of glycolysis
(LIANG et al. 2008) and the induction of tumours (SCHREIBER et al. 1969). Some
pharmaceuticals, e.g., sodium iodate, which leads to photoreceptor degeneration by
destruction of the retinal pigment epithelium (WANG et al. 2011), are already lethal in larger
animals such as the pig in concentrations, which have not yet a toxic effect on the retina
(NOEL et al. 2012).
2.5.1 Iodoacetic acid
Iodoacetic acid (IAA) is a photoreceptor toxin, which leads to selective photoreceptor
degeneration when administered systemically (ORZALESI et al. 1970). The inner retinal
neurons remain intact (WANG et al. 2011). The effect is described in many animal species.
IAA reacts with thiol (SH) groups, imidazole and amino groups of proteins, so that toxic side
effects after systemic application are likely. Beside the irreversible inhibition of the thiol (SH)
group of enzymes, as e.g., in the glutathione reductase (MATSUMOTO et al. 2002), IAA
inhibits the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an enzyme, which is very
important for the metabolic energy production in the glycolysis (LIANG et al. 2008, WANG
et al. 2011). Photoreceptors have a high metabolic rate and therefore seem to be particularly
sensitive for IAA (WANG et al. 2011).
However, after systemic intravenous treatment, not only the retinal enzymes are affected. The
toxic systemic side effects of IAA are reported to lead to a highly reduced animal welfare and
18
LITERATURE SURVEY
to mortality, e.g., 20% in the experiments with rabbits by ORZALESI et al. (1970) and 50%
in experiments with rats by GRAYMORE et al. (1959).
In other reports or animal species the systemic side effects on the well-being, e.g. weight loss,
were not mentioned.
2.5.2 N-methyl-N-nitrosourea
N-methyl-N-nitrosourea (MNU) is an alkylating agent, belonging to the group of the
nitrosoureas (TSUBURA et al. 2003). Like the other chemical agents of this group, it is toxic,
carcinogenic, teratogenic, embryotoxic and mutagenic (SIGMA-ALDRICH 2013). After
systemic application (i.v.) to rabbits, it leads in 70% to brain tumours and tumours of the
medulla (SCHREIBER et al. 1969).
Herrold (1967) first described that the systemic application in a variety of animals induces
photoreceptor degeneration, while inner neurons remain intact (HERROLD 1967, TSUBURA
et al. 2010). MNU leads to these retinal changes seven days after a single injection by an
apoptotic mechanism as verified by terminal deoxynucleotidyl transferase dUTP nick-end
labelling staining (TUNEL), which is a marker for apoptotic cells (TSURUMA et al. 2012).
The degradation of photoreceptors was due to oxidative stress (TSURUMA et al. 2012).
However, comparable to the systemic administration of IAA, there are also systemic side
effects.
Because of its carcinogenic properties, the user has to follow many safety measures
(protective clothing, etc.), comparable to the use of other cytostatic drugs, as also seen in the
veterinary medicine (HEINEMANN a. MEICHNER 2011).
2.6 The intravitreal injection
The intravitreal injection is a technique to bring drugs for the therapy of eye disease very
close to the area, where they are supposed to act. Under local anaesthesia the drug is placed
with an injection cannula directly into the vitreous humour of the eye (BERUFSVERBAND
DER AUGENÄRZTE DEUTSCHLANDS E.V., DEUTSCHE OPHTHALMOLOGISCHE
GESELLSCHAFT (DOG) 2010). Whereas a decade ago, intravitreal injections were only
used in sporadic, mainly urgent cases, nowadays this technique is very commonly used due to
the introduction of new drugs for the treatment of age-related macular degeneration (AMD)
19
AIM OF THIS THESIS
and other macular diseases (BERUFSVERBAND DER AUGENÄRZTE DEUTSCHLANDS
E.V., DEUTSCHE OPHTHALMOLOGISCHE GESELLSCHAFT (DOG) 2010).
3 AIM OF THIS THESIS
The aim of this project was to establish a pharmacological model of selective photoreceptor
degeneration in mice by intravitreal injection that avoids systemic side effects combined with
the advantage of having an intraindividual control eye.
20
MATERIALS & METHODS
4 MATERIALS & METHODS
4.1 Animals
The materials and methods were used and performed as described in detail in the referring
manuscripts (Study I: ”Correlations between ERG, OCT and anatomical findings in the rd10
mouse”; Study II: “The effects of iodoacetic acid on the mouse retina”, Study III: “Selective
photoreceptor degeneration by intravitreal injection of N-methyl-N-nitrosourea”).
All experiments were performed in accordance with the ARVO Statement for the use of
animals in ophthalmic and vision research and in accordance with the German Law for the
Protection of Animals and after approval was obtained by the regulatory authorities. The
proposal of the animal experiments (Study I, II, III) was approved by the LANUV
(Landesamt für Natur, Umwelt und Verbraucherschutz Nordrhein Westfalen, Recklinghausen,
Germany; file reference: 84-02.04.2011.A386).
All pigmented wild type mice (C57BL/6J) and rd10 mice were housed in typical cages with
food and water accessible ad libitum. The room was kept under a 12 h: 12 h light-dark cycle.
C57BL/6J wild type mice and rd10 mice were examined and compared at the age of 2, 3, 5, 7,
9, 12, 24, and 48 weeks (each age group n=3) using full-field electroretinography (ERG),
spectral domain Optical Coherence Tomography (sd-OCT), fluorescein angiography (FA),
Hematoxylin & Eosin histology (HE) and immunohistochemistry (IH).
Wild type mice (C57BL/6J) were treated with iodoacetic acid (IAA) or N-methyl-N-
nitrosourea (MNU). The animals were examined using full-field electroretinogram (ERG) and
spectral domain Optical Coherence Tomography (sd-OCT) five days before, as well as 7 days
and 14 days after injection of IAA and MNU, to monitor photoreceptor function and effects of
the injection. For the duration of experiments, body weight was daily recorded. Finally, two
weeks after treatment eyes were examined in histology (HE and IH).
21
MATERIALS & METHODS
A total of 20 wild type mice (C57BL/6J; 6 to 8 weeks) received systemically
(intraperitoneally and intravenously) sterile IAA (Sigma Aldrich, Germany). IAA was
dissolved just before use in sterile phosphate buffered saline (PBS) and the pH was adjusted
with NaOH to the physiological pH of 7.4.
Eight animals were treated with an intraperitoneal injection of IAA: 40 mg/kg BW (n=1), 50
mg/kg BW (n=1), 60 mg/kg BW (n=2), 65 mg/kg BW (n=1), 70 mg/kg BW (n=1), 75 mg/kg
BW (n=1) and 2 x 30 mg/kg BW in a time interval of 3.5 hours (n=1).
Twelve animals received intravenous injections of IAA: 55 mg/kg BW (n=2), 60 mg/kg BW
(n=5), 65 mg/kg BW (n=1) and 2 x 30 mg/kg BW in a time interval of 3.5 hours (n=4).
(Study II: “The effects of iodoacetic acid on the mouse retina”)
Three wild type mice (C57BL/6J; 6 to 8 weeks) received intraperitoneally sterile MNU
(Sigma Aldrich, Germany; 60 mg/kg BW). The stability of MNU is reported to be poor in
aqueous solutions and the duration of the procedure of injection is comparably long.
Therefore, MNU was dissolved in dimethylsulfoxide (DMSO; Serva, Heidelberg, Germany)
immediately before use and further diluted (sterile phosphate buffered saline, PBS). Injections
were slowly performed. Temperature of the solution was room temperature.
Animals were kept in one-way cages under a fume hood after the application of MNU (PET;
Tecniplast Deutschland GmbH; Hohenpreißenberg; Germany) under the same housing
conditions as before, because of the possible environmental hazard due to the carcinogenic
and teratogenic potential of N-methyl-N-nitrosourea. To evaluate possible systemic effects of
changing the cage conditions from normal cages to the one-way system, two untreated adult
pigmented wild type mice were kept under the same husbandry conditions (Study III:
“Selective photoreceptor degeneration by intravitreal injection of N-methyl-N-nitrosourea”).
Control animals received systemic sham injections (PBS intraperitoneal (n=3) and
intravenous (n=3) or DMSO + PBS intraperitoneal (n=3)). (Study II: “The effects of
iodoacetic acid on the mouse retina”, Study III: “Selective photoreceptor degeneration by
intravitreal injection of N-methyl-N-nitrosourea”)
22
MATERIALS & METHODS
For all experimental procedures and examinations (excluding the intraperitoneal application
of MNU and IAA, and the intravenous application of IAA), the animals were anaesthetised
with an intraperitoneal injection of ketamine (70 mg/kg Ketamin® 10%, CEVA, Germany)
and xylazine (10 mg/kg Xylazin 2% Bernburg®, Medistar, Ascheberg, Germany) in 0.1 mL
of saline. (Study I: ”Correlations between ERG, OCT and anatomical findings in the rd10
mouse”; Study II: “The effects of iodoacetic acid on the mouse retina”, Study III: “Selective
photoreceptor degeneration by intravitreal injection of N-methyl-N-nitrosourea”)
4.2 Intravitreal injections in mice
After anaesthesia by intraperitoneal injection of a mixture of ketamine (70 mg/kg Ketamin®
10%, CEVA, Germany) and xylazine (10 mg/kg Xylazin 2% Bernburg®, Medistar,
Ascheberg, Germany) in 0.1 mL of saline, the left eye was locally anaesthetised with
proxymetacainhydrochloride 0.5% eye drops (Proparakain-POS®, Ursapharm, Saarbrücken,
Germany).
A nanoliter injection system and a 3D micromanipulator (Nanoliter 2000; World Precision
Instruments, Inc. Sarasota, FL, USA) were used to conduct intravitreal injections of a
standard volume of 2 µL. After puncture in the dorso-nasal area of the limbus by a 27 gauge
steel needle (BD Microlance TM
3, Heidelberg, Germany) the volume was injected with a
glass capillary in an angle of 30°. Preliminary experiments with control animals (n=9) showed
that the injection technique worked and a volume of 2 µL could be placed safely in the
vitreous cavity of mice. As control intervention 2 µL PBS (n=3) and 2 µL PBS + DMSO
(n=3) were injected each into three eyes intravitreally. Intravitreal injections of fluorescein
(different concentrations dissolved in PBS, n=3) were performed to confirm the safety of the
injection technique. Therefore, images were taken directly after injection of fluorescein by a
confocal laser scanning microscope (Heidelberg Retina Angiograph-1, Heidelberg
Engineering, Germany). (Study II: “The effects of iodoacetic acid on the mouse retina”, Study
III: “Selective photoreceptor degeneration by intravitreal injection of N-methyl-N-
nitrosourea”)
A standard volume of 2µL with different IAA (dissolved in PBS) and MNU (dissolved in
DMSO + PBS) concentrations was injected intravitreally in the left eye of a total of 49 mice.
23
MATERIALS & METHODS
25 mice were injected with IAA: 0.1 mg/kg BW (n=3), 0.15 mg/kg BW (n=5), 0.2 mg/kg BW
(n=6), 0.25 mg/kg BW (n=4), 0.5 mg/kg BW (n=4), 1 mg/kg BW (n=3). (Study II: “The
effects of iodoacetic acid on the mouse retina”)
24 mice were injected intravitreally with MNU: 0.15 mg/kg BW (n=3), 0.2 mg/kg BW (n=3),
0.25 mg/kg BW (n=3), 0.3 mg/kg BW (n=3), 0.5 mg/kg BW (n=3), 1.0 mg/kg BW (n=3), 2.0
mg/kg BW (n=3), 3.0 mg/kg BW (n=3). (Study III: “Selective photoreceptor degeneration by
intravitreal injection of N-methyl-N-nitrosourea”)
After intravitreal injections antibiotic eye ointment (Polyspectran, Alcon Pharma GmbH,
Freiburg, Germany) was used for three days after application. All intravitreally injected
animals were examined using the same methods (ERG, OCT, Histology) and time schedule as
systemically injected animals. (Study II: “The effects of iodoacetic acid on the mouse retina”,
Study III: “Selective photoreceptor degeneration by intravitreal injection of N-methyl-N-
nitrosourea”)
4.3 Electroretinography
The ERG reveals the electrical activity of the different cell types of the retina, as the answer
to a light stimulus. The ERG wave, as a summarized potential, consists of a first negative
deflection or a-wave (mainly build by rod and cone activity) and a following positive b-wave
(formed by Müller cells and bipolar cells) (KRISHNA et al. 2002). As a non-invasive clinical
tool, it is very useful to monitor the severity and progression of retinal degeneration
(KRISHNA et al. 2002).
Before the electroretinogram procedure, mice were dark adapted (1 hour) and anaesthetised
under red dim light. Pupils were dilated using Tropicamide 2.5% eye drops (Pharmacy of the
University Hospital Aachen, Germany). Proxymetacainhydrochloride 0.5% eye drops
(Proparakain-POS®, Ursapharm, Saarbrücken, Germany) were used for local anaesthesia, and
methylcellulose (Methocel® 2%, Omni Vision, Puchheim, Germany) served for a better
contact and protected the eye against drying out. As active electrodes goldring electrodes
(animal electrode 0.5mm ø 3mm Roland Consult, Brandenburg, Germany) were placed on the
24
MATERIALS & METHODS
left and right eye. As reference, a goldring electrode was placed in direct contact to the mouth
mucosa. A subcutaneous needle electrode in the lumbar region served as ground electrode.
Ganzfeld (full-field) electroretinogram recordings (ERG) were performed according to the
ISCEV standard protocol (MARMOR et al. 2008) with the Reti System designed for rodents
(Roland Consult Electrophysiological Diagnostic Systems, Brandenburg, Germany). A- and
b-wave amplitudes and implicit times of rod and cone responses were analyzed after
averaging five responses to light stimulation. (Study I: ”Correlations between ERG, OCT and
anatomical findings in the rd10 mouse”; Study II: “The effects of iodoacetic acid on the
mouse retina”, Study III: “Selective photoreceptor degeneration by intravitreal injection of
N-methyl-N-nitrosourea”)
4.4 Spectral domain Optical Coherence Tomography
Optical coherence tomography (OCT) is a non-invasive high-resolution technique to perform
cross-sectional images of the retina in vivo. The technique is comparable to ultrasound, but
instead of using sound the OCT is an interferometric technique using light (YAMAUCHI et
al. 2012).
Spectral domain Optical Coherence Tomography (sd-OCT) scans were performed with the
Spectralis® OCT system (Heidelberg Engineering, Heidelberg, Germany) and a special lens
(+25 D lens) in front of the scanning system, to correct for rodent optics in mice. Artificial
tears (methylcellulose, Methocel® 2%, Omni Vision, Puchheim, Germany) were used
throughout the whole procedure to maintain corneal clarity. The retina was evaluated by cross
sectional images centered on the optic disk, as the main landmark and the confocal image of
the fundus was recorded and examined (IR reflection image, wavelength 715nm). Retinal
thickness was measured at six positions across the retina. For each eye these thickness values
were averaged. The thickness of the different layers of the retina was determined in the same
way. A Nikon D80 camera was used for macroscopic pictures (Nikon AF Micro Nikkor 105
mm, 2896 Pixel x 1944 Pixel x 24 Bit). (Study I: ”Correlations between ERG, OCT and
anatomical findings in the rd10 mouse”; Study II: “The effects of iodoacetic acid on the
mouse retina”, Study III: “Selective photoreceptor degeneration by intravitreal injection of
N-methyl-N-nitrosourea”)
25
MATERIALS & METHODS
4.5 Fluorescein angiography (FA)
Retinal vasculature was evaluated in anaesthesia and after the dilatation of the pupils by
Tropicamide 2.5% eye drops (Pharmacy of the University Hospital Aachen, Germany) by the
intraperitoneal application of fluorescein (10-20 µL, Fluorescein Alcon® 10%, Alcon Pharma
GMBH, Freiburg im Breisgau, Germany). Images were taken by a confocal laser scanning
microscope (Heidelberg Retina Angiograph-1, Heidelberg Engineering, Germany) in the
fluorescein angiography mode. (Study I: ”Correlations between ERG, OCT and anatomical
findings in the rd10 mouse”)
4.6 Histology
Finally, mice were euthanized by isoflurane overdosing (Forene 100% (V/V)®, Abott GmBH,
Wiesbaden, Germany), decapitated and enucleated. Histology was performed as described
earlier by Mataruga et al. (2007) and in the following three studies.
In brief, after fixation, slices were incubated in blocking solution followed by overnight
incubation in primary antibodies diluted in the same blocking solution. Primary antibodies
used were raised against glutamine synthetase (raised in mouse, 1:4000, BD Biosciences,
Germany); against GFAP (anti glial fibrillary acidic protein, raised in chicken, 1:2000, Novus,
Germany); against CabP (anti calbindin 28K, raised in mouse, 1:1000, Sigma; Germany);
against HCN1 (RTQ-7C3, raised in rat, 1:10, F. Müller, Forschungszentrum Jülich,
Germany); against PKC (anti protein kinase C α, raised in rabbit, 1:4000, Santa Cruz, USA);
against rhodopsin (1D4, 1:500, R.S. Molday, British Columbia, Canada); against calretinin
(AB1550, raised in goat, 1:3000, Millipore, Germany); against recoverin (AB5585, raised in
rabbit, 1:2000, Millipore, Germany). Sections were washed in PB and incubated with
secondary antibodies. Secondary antibodies included: donkey anti-mouse Cy3 (1:100,
Dianova, Germany); donkey anti-rat Cy3 (1:500, Dianova, Germany); donkey anti-chicken
Cy2 (1:400, Dianova, Germany); donkey anti-rabbit Cy2 (1:400, Dianova, Germany); donkey
anti-goat Alexa647 (1:200, Invitrogen, Germany); donkey anti-mouse Dy649 (1:500,
Dianova, Germany) and finally the lectin Peanut agglutinin (PNA, biotinylated, 1:1600,
Sigma Aldrich, Germany) was visualized using StreptavidinAlexa647 (1:100, Invitrogen,
Germany). Imaging was performed with a Leica TCS confocal laser scanning microscope
(Leica Microsystems, Heidelberg, Germany).
26
MATERIALS & METHODS
For Hematoxylin-Eosin staining, eyes were punctured three times at the limbus and fixated.
After dehydration and incubation in a series of increasing ethanol concentrations, followed by
xylene and paraffin, eyes were embedded in paraffin. Eyes were cut with a microtome (R.
Jung, Heidelberg, Germany), collected on slides and deparaffinised. After rehydration, slices
were stained with Hematoxylin and Eosin and images were performed with a Leica DMRX
microscope (Leica Microsystems, Heidelberg, Germany).
For thickness evaluation comparable to the OCT the papilla was used as landmark. Thickness
was determined in six positions close to the papilla and values were averaged.
(Study I: ”Correlations between ERG, OCT and anatomical findings in the rd10 mouse”;
Study II: “The effects of iodoacetic acid on the mouse retina”, Study III: “Selective
photoreceptor degeneration by intravitreal injection of N-methyl-N-nitrosourea”)
4.7 Urine test
In the first experiments with MNU injections in mice, before, two and three hours, one and
two days after systemic and intravitreal application of MNU urinalyses by UPLC were
performed (Ultra performance liquid chromatography (UPLC), Aquity, Waters). The
excretion of MNU was tested for safety reasons. (Study III: “Selective photoreceptor
degeneration by intravitreal injection of N-methyl-N-nitrosourea”)
4.8 Statistics
Retinal thickness and the thickness of different retinal layers were determined in cross
sectional images of the OCT and in histology by six vertical cuts through the retina in the
height of the papilla. The values were averaged. In the ERG a- and b-wave amplitudes and
implicit times of rod and cone responses were determined.
Statistical analysis of all experiments was performed using one-way ANOVA with
Bonferroni’s post hoc test (GraphPad Prism 5; GraphPad Software, La Jolla, CA). Values
represent mean + standard deviation.
27
RESULTS
5 RESULTS
5.1 STUDY I
Correlations between ERG, OCT and anatomical
findings in the rd10 mouse
Sarah Rösch1,2
, Sandra Johnen1, Frank Müller
3, Christiane Pfarrer
2, Peter Walter
1
SUBMITTED/ UNDER REVIEW
1. Department of Ophthalmology, RWTH Aachen University, Germany
2. Department of Anatomy, University of Veterinary Medicine Hannover, Germany
3. Institute of Complex Systems, Cellular Biophysics, ICS-4, Forschungszentrum Jülich
GmbH, Germany
Corresponding Author: Peter Walter, Department of Ophthalmology, RWTH Aachen
University, Pauwelsstr. 30, 52074 Aachen, Tel +49-241-8088191, fax +49-241-8082408,
mailto [email protected]
This work is part of a doctoral thesis at the University of Veterinary medicine Hannover
(October 2013).
Keywords: retinal degeneration, rd10 mouse
Word count: 2884
This work was supported by the DFG grants WA 1472/6-1 to PW and MU 3036/3-1 to FM.
28
RESULTS
5.1.1 ABSTRACT
Background To evaluate the correlation between ERG, OCT, and microscopic findings in the
rd10 mouse.
Methods C57BL/6J wild type mice and rd10 mice were compared at the age of 2, 3, 5, 7, 9,
12, 24, and 48 weeks (each age group n=3) using full-field electroretinography (ERG),
spectral domain Optical Coherence Tomography (sd-OCT), fluorescein angiography (FA),
Hematoxylin & Eosin histology (HE), and immunohistology (IH).
Results While in wild type mice the amplitude of a- and b-wave increased with light intensity
and with the age of the animals, the rd10 mice showed an extinction of the ERG beginning
with the age of 5 weeks. In OCT recordings the thickness of the retina decreased up to 9
weeks of age, mainly based on the degradation of the outer nuclear layer (ONL). Afterwards,
the ONL was no longer visible in the OCT. HE staining and immunohistological findings
confirmed the in vivo data.
Conclusion ERG and OCT are useful methods to evaluate the retinal function and structure in
vivo. The retinal changes seen in the OCT closely match those observed in histological
stainings.
29
RESULTS
5.1.2 INTRODUCTION
Hereditary dystrophies of the retina, such as Retinitis pigmentosa (RP), are considerable
causes of blindness in humans.[1] Research efforts concerning these currently not treatable
diseases are focused on the genetic background, the mechanisms of degeneration and on
possible treatment strategies.[2] Transgenic animal models in rodents, e.g., the retinal
degeneration (rd) 1 and rd10 mice are well characterized and described.[3-9] In rd1 and rd10
mice, missense point mutations in the gene encoding for the ß-subunit of rod cGMP
phosphodiesterase type 6 (ßPDE) result in the described disease pattern.[3-9] The gene
mutation leads to the degeneration of first rods and then consecutively cones with a central to
peripheral gradient.[9] The fact that in both animal models firstly rods and secondly cones
degenerate, which is comparable to RP,[3-9] seems to be caused by missing diffusible factors
normally secreted by the rods.[10] This kind of retinal degeneration is concomitant with a
pronounced reduction in the thickness of the outer nuclear layer (ONL).[3-9] The thickness of
the inner retinal layers remains nearly unaffected.[3-9] The retinal development in the rd1 and
rd10 mice is comparable to normal mice at 8 postnatal days (P).[2] However, while in the rd1
mice the degeneration becomes apparent with P11, in rd10 mice the degeneration starts at P16
and the peak of photoreceptor degeneration is reached at P25.[9] By P60, no photoreceptors
are left.[9]
Because of the later onset and milder retinal degeneration, the rd10 mice seem to be more
suitable to study slow disease mechanisms in RP.[8] Retinal vessels become sclerotic at 4
weeks of age, the a-wave and b-wave in the electroretinogram are visible with the age of 3
weeks and no longer detectable with the age of 2 months.[8]
To characterize retinal degeneration, monitoring of the time course of the disease is
indispensible. Using histology to observe retinal changes requires a large number of animals
to examine. Therefore it is of interest, if data from functional examinations and in vivo
imaging correlate well with histological data. We examined eyes of wild type and rd10 mice
in different age groups to determine retinal function by means of the full-field
electroretinogram (ERG) and retinal thickness by means of spectral domain Optical
Coherence Tomography (sd-OCT) scanning. The ERG examinations were performed in
general anaesthesia with ketamine and xylazine, because anaesthesia was reported to have no
influence, neither on the oscillatory potentials nor on the ERG waveforms.[11]
30
RESULTS
5.1.3 MATERIALS AND METHODS
The materials and methods were performed as also described in Study II: “The effects of
iodoacetic acid on the mouse retina” and Study III: “Selective photoreceptor degeneration by
intravitreal injection of N-methyl-N-nitrosourea”.
All experiments were performed in accordance with the ARVO Statement for the use of
animals in ophthalmic and vision research and in accordance with the German Law for the
Protection of Animals and after approval was obtained by the regulatory authorities. All
possible steps were taken to avoid animal suffering at each stage of the experiment. The
proposal of the animal experiments was approved by the LANUV (Landesamt für Natur,
Umwelt und Verbraucherschutz Nordrhein Westfalen, Recklinghausen, Germany; file
reference: 84-02.04.2011.A386).
2.1. Animals
Adult pigmented wild type mice (C57BL/6J) and rd10 mice were maintained under controlled
light conditions (12:12 hours light/dark cycle) with food and water available ad libitum.
Animals (n=3 in each group) were examined at the age of 2, 3, 5, 7, 9, 12, 24, and 48 weeks.
Animals of the same age were sacrificed for histological examinations.
For ERG, OCT, and fluorescein angiography, animals were anaesthetized with an
intraperitoneal injection of a mixture of ketamine (70 mg/kg Ketamin® 10%, CEVA,
Germany) and xylazine (10 mg/kg Xylazin 2% Bernburg®, Medistar, Ascheberg, Germany)
in 0.1 mL of saline.
2.2. Electroretinogram recordings
Electroretinogram recordings (ERG) were performed with the Reti System designed for
rodents (Roland Consult Electrophysiological Diagnostic Systems, Brandenburg, Germany).
Mice were dark adapted for one hour and the pupils were dilated using 2.5% phenylephrine
hydrochloride ophthalmic solution (Tropicamide 2.5% eye drops, Pharmacy of the University
Hospital Aachen, Germany). After local anaesthesia with proxymetacainhydrochloride 0.5%
eye drops (Proparakain- POS®, Ursapharm, Saarbrücken, Germany), a custom-made goldring
31
RESULTS
electrode (animal electrode 0.5mm ø 3mm Roland Consult) was placed on the corneal surface
of each eye. Methylcellulose (Methocel ® 2%, Omni Vision, Puchheim, Germany) served for
a good contact and to maintain corneal moisture. The reference goldring electrode was placed
on the mouth mucosa and a subcutaneous silver needle electrode in the lumbar region served
as ground electrode. The ERG was recorded as full-field ERGs according to the ISCEV
standard protocol.[12] Five responses to light stimulation were averaged. A- and b-wave
amplitudes and implicit times of rod and cone responses were analyzed.
2.3. Spectral domain Optical Coherence Tomography
Spectral domain Optical Coherence Tomography (sd-OCT) scans were done using the
Spectralis® OCT system (Heidelberg Engineering, Heidelberg, Germany). To correct for
rodent optics, the system was modified according to recommendations of the manufacturer
with a +25 D lens in front of the scanning system. Scans were obtained from the retina
centered on the optic disk as the main landmark. Retinal thickness was measured at six
positions across the retina and averaged for each eye. The thickness of the different retinal
layers was determined using the same technique. The confocal image (IR reflection image,
wavelength 715nm) of the fundus recorded by the OCT system was used for documentation.
2.4. Fluorescein Angiography
Fluorescein angiography was performed to evaluate the retinal vasculature. After dilatation of
the pupils by Tropicamide 2.5% eye drops (Pharmacy of the University Hospital Aachen,
Germany), images were taken by a confocal laser scanning microscope (Heidelberg Retina
Angiograph-1, Heidelberg Engineering, Germany) in the fluorescein angiography mode.
Directly before the measurements, mice were intraperitoneally injected with 10-20 µl
fluorescein (Fluorescein Alcon® 10%, Alcon Pharma GMBH, Freiburg im Breisgau,
Germany).
2.5. Histology
At the end of the follow-up the animals were euthanized by isoflurane (Forene 100% (V/V)®,
Abott GmBH, Wiesbaden, Germany) overdosing and decapitated. Immunohistochemistry was
performed as described earlier by Mataruga et al. (2007).16
In brief, eyes were enucleated and
32
RESULTS
opened by an encircling cut at the limbus. The retinae in the eyecup were immersion-fixed for
30 minutes in 4% paraformaldehyde (PA) in 0.1 M phosphate buffer (PB) at room
temperature and washed in PB several times. Tissue was incubated in 10% sucrose in PB for
1 hour, followed by 30% sucrose overnight. The retina was flat embedded and frozen in
optimal cutting temperature compound (NEG-50, Richard Allen Scientific, Thermo Fisher
Scientific, Germany). Vertical sections (20 μm thickness) were cut on a cryostat (HM 560
CryoStar, MICROM, Walldorf, Germany), and collected on Superfrost Plus slides (Menzel,
Braunschweig). Sections were pre-treated with blocking solution (5% chemiblocker
(Chemicon, Hofheim, Germany), 0.5% Triton-X100 in PB, and 0.05% NaN3) for 1 hour,
followed by incubation with primary antibodies over night, diluted in the same solution.
Sections were washed in PB and incubated in secondary antibodies diluted in 5%
Chemiblocker, 0.5% Triton-X100 in PB for 1 hour, washed in PB and coverslipped with
Aqua Polymount (Polysciences, Eppelheim, Germany). Sections were examined with a Leica
TCS confocal laser scanning microscope (Leica Microsystems, Heidelberg, Germany) with
63x/1.4 oil immersion lenses. Following primary antibodies were used: against GFAP (anti
glial fibrillary acidic protein; raised in chicken, 1:2000; Novus, Germany); against glutamine
synthetase (raised in mouse, 1:4000; BD Biosciences, Germany); against CabP (anti calbindin
28K; raised in mouse, 1:1000; Sigma); against PKC (anti protein kinase Cα; raised in rabbit,
1:4000; Santa Cruz); against Calretinin (AB1550, raised in goat, 1:3000; Millipore); against
HCN1 (RTQ-7C3, raised in rat, 1:10; F. Müller, Forschungszentrum Jülich); against recoverin
(AB5585, raised in rabbit, 1:2000; Millipore); against rhodopsin (1D4, 1:500; R.S. Molday,
Univ. of British Columbia). Secondary antibodies included: donkey anti-chicken Cy2 (1:400),
donkey anti-mouse Cy3 (1:100), donkey anti-rabbit Cy2 (1:400), donkey anti-rat Cy3 (1:500),
donkey anti-mouse Dy649 (1:500; all Dianova, Germany); donkey anti-goat Alexa647 (1:200;
Invitrogen, Germany). Peanut agglutinin (PNA, biotinylated, 1:1600; Sigma Aldrich;
Germany) was visualized using StreptavidinAlexa647 (1:100; Invitrogen, Germany).
For Hematoxylin-Eosin staining, eyes were enucleated, punctured and fixed by immersion as
described above. Eyes were dehydrated in a tissue dehydration automat (mtm, Fa. Slee,
Mainz, Germany), by incubation in a series of increasing ethanol concentrations (2 times x
70%, 2x 96%, 3x 100% for 1 hour), followed by xylene (3x 1 hour) and paraffin (4x 1 hour),
and embedded in paraffin. Sections of 5 μm thickness were cut with a microtome (R. Jung,
33
RESULTS
Heidelberg, Germany), collected on slides, deparaffinized, rehydrated, and stained with
Hematoxylin and Eosin. Images were performed by a Leica DMRX microscope.
The thickness of the entire retina and of the individual retinal layers was determined in both
Hematoxylin-Eosin stainings and in immunohistochemical at six positions close to the papilla.
For each retina, thickness values obtained from these positions were averaged.
34
RESULTS
5.1.4 RESULTS
ERG measurements were performed in wild type versus rd10 mice, anaesthetized with
ketamine and xylazine using goldring electrodes as active electrodes instead of contact lenses
(Figure 1A), in front of a Ganzfeld stimulator following a standardized protocol. The light-
induced electrical activity of both eyes was recorded as full-field flash ERGs as described in
the ISCEV standard protocol.[12]
Typically, the ERG waveforms consist of an early negative wave (a-wave; primary light
response in photoreceptors), followed by a large positive deflection (b-wave; dominated by
the activity of ON-bipolar cells and Müller cells). Riding on top of the b-wave are the
oscillatory potentials that probably involve inner retinal circuitry.
In wild type the amplitudes of a- and b-wave increased with light intensity and with age,
reaching their maximal levels at the age of 12 weeks (Figure 1B, 1C). In rd10 mice,
amplitudes increased only to the age of 3 weeks. In elder rd10 mice the waves of the ERG
were completely abolished (Figure 1B, 1C).
Retinal thickness was measured and the fundus of the eye was observed in vivo by Optical
Coherence Tomography (OCT) (Figure 2). For each retina, thickness was measured at six
locations close to the optic disc (examples marked by red arrows in Figure 2B, column 1).
While in wild type mice a decrease of thickness between the second and third week of age,
followed by a relatively constant retinal thickness (Figure 2, column 3) was observed, in rd10
mice a significant loss of retinal thickness was obvious (Figure 2, column 1 and 2). The outer
nuclear layer (ONL, marked by red bars in Figure 2C-F, column 1) was visible as a thin line
up to 9 weeks. Retinal separation was only observed in one 9 weeks old mouse - locally
determined directly above the optic disc - and in one 24 weeks old mouse near to the papilla
(Figure 2G, first and second column). In all other animals of each age group (n=3), no
separation was found throughout the whole retina. In Figure 2I the averaged thickness values
of the retina of wild type (Figure 2I1) and rd10 mouse (Figure 2I2) were plotted against the
age in weeks. The loss of thickness in the rd10 mice is mainly due to the thinning of the ONL
(Figure 2I3), while the inner retinal layers stayed nearly constant (Figure 2I4). The ONL
reduced its thickness up to the age of 9 weeks to 10.2% (thickness at 3 weeks: mean of 102.6
35
RESULTS
µm; thickness at 9 weeks: mean of 10.44 µm; n=3 for each age group). The ONL is
completely deleted at the age of 12 weeks.
The retinae of the two strains were also examined using fluorescein angiography (Figure 3).
No significant differences were observed.
Results from the above reported non-invasive tests were compared to data obtained with
histological techniques. For this evaluation retinal areas directly near to the optic disc were
chosen, comparable to the region used for thickness measurements in the OCT (Figure 2). In
contrast to the wild type mice, in most of the rd10 mice elder than 3 weeks we observed
artificial retinal separation, probably based on the combination of photoreceptor degeneration
and the preparation technique (Figure 4).
In the OCT measurements, the wild type retina was approximately 200 ± 10 µm thick at the
regions measured, which agreed well with measurements taken in histology with a thickness
of approximately 180 to 200 µm at the corresponding position. The preparation and
histological workup of the eyes may account for the thickness difference of about 10%.
As described with the OCT, the retinal thickness of the rd10 mice decreased with the age in
HE stains (Figure 4). In 5, 7 and 9 weeks old rd10 mice, the ONL was reduced to one row of
photoreceptor somata. To further investigate degenerative changes in the rd10 mouse retina
and to identify cellular changes, immunohistochemical techniques were employed using
specific antibodies and confocal laser scanning microscopy (Figure 5). In 7 weeks old wild
type mice 10 to 12 layers of somata were visible in the ONL (Figure 5A). In accordance with
our findings in the HE staining, in the retina of 7 weeks old rd10 mice the ONL was reduced
to one row of somata (Figure 5B staining 1, green), while the inner retina seemed to be
unaffected (Figure 5B).
In Figure 5, different cell populations in the degenerated retina are visualized using three
stainings with different combinations of antibodies. The results are consistent with those
obtained by other methods: photoreceptor somata and outer segments degenerated (staining 1)
while inner retinal cells like bipolar cells, horizontal cells and amacrine cells survived
(staining 2). In wild type retina, GFAP expression was only found in astrocytes while Müller
cells were only positive for glutamine synthetase. In rd10 mice Müller cells were also positive
for GFAP, indicating that they had become reactive (Figure 5B, staining 3, green) during the
retinal degeneration process.[13]
36
RESULTS
5.1.5 DISCUSSION
Animal models of retinal degenerations and dystrophies, like the rd10 mouse, are important to
study the underlying disease mechanisms and also to establish possible treatment
approaches.[3-9] No reduction of welfare of these animals over weeks was observed, although
a loss of eyesight developed after the age of three weeks. Many studies on retinal
degeneration rely on the combination of data obtained from large populations of experimental
animals, sacrificed at different stages of the degeneration process. Recently, non-invasive
tools like ERG, sd-OCT and fluorescein angiography have become promising methods that
might allow following retinal degeneration in individual animals. In the present study, we
show that the combination of these methods allows to precisely describe the degeneration of
the retina in vivo during the course of the degenerative process.
The data, we present here obtained by non-invasive methods, are in accordance with findings
of other groups.[8,11] The values of a- and b-wave in the ERG were comparable to the results
of other research groups.[8,9,11] Angiographic findings in the vasculature revealed no
significant differences between the wild type and rd10 mice.
Most importantly, our OCT scans proved most useful for quantitative analysis. The thickness
of the ONL or of the entire retina measured in vivo by OCT scans was very well comparable
to the thickness values obtained from HE staining or immunohistochemical analysis.
Pennesi et al. [14] found a separation between the outer retina and the pigment epithelium
(neurosensory detachment) in the retina of rd10 mice at the age of 64 days. In our series of
OCT scans, we were not able to confirm this finding. In vivo, we found neurosensory
detachment only locally in two animals. However, after OCT, when the eyes were prepared
for histology, separation was commonly observed probably induced during the histological
work-up. The difference between our results and those of Pennesi et al. [14] might be
explained by differences in the type of OCT machines. Alternatively, our results may indicate
that, although separation is not always manifest in vivo, it can be more easily induced in the
rd10 mouse eye than in wild type eyes.
We employed two histological methods that complemented one another. For the HE-staining
the whole eye was sectioned, which enabled us to also evaluate other parts than the neural
retina. As a disadvantage, sectioning of the entire eye is more prone to artifacts like retinal
separation that we observed especially in elder rd10 mice. Immunohistochemistry provides
37
RESULTS
the advantage that the major neuronal and glial cell classes of the retina can be visualized in
much detail using cell type specific antibodies. Our immunohistochemical results confirmed
the early degeneration of rod and cone photoreceptors, the reactivity of Müller cells, as well
as neuronal remodeling processes in certain types of bipolar and horizontal cells.[15]
5.1.6 CONCLUSIONS
In summary, our data indicate that non-invasive techniques such as sd-OCT scanning of the
retina in rodents are powerful tools to monitor the course of retinal degeneration with respect
to morphometric analyses and at the same time reduce the number of animals sacrificed for
histological techniques. Quantitative anatomic parameters can be extracted from sd-OCT
scans and are in good accordance with morphometric data from histological work-up.
However, single cells and their involvement in the degenerative mechanisms can only be
identified by immunohistochemical techniques using confocal fluorescence imaging
technology.
38
RESULTS
5.1.7 REFERENCES
1. E.L. Berson, “Retinitis pigmentosa: The Friedenwald Lecture”, Invest Ophthalmol Vis Sci.,
vol. 34, pp.1659-1676, 1993.
2. A. Tsubura, K. Yoshizawa, M. Kuwata, N. Uehara, “Animal models for retinitis
pigmentosa induced by MNU; disease progression, mechanisms and therapeutic trials”,
Histol Histopathol, vol. 25, pp. 933-944, 2010.
3. M.N. Delyfer, V. Forster, N. Neveux, S. Picaud, T. Léveillard, J.A. Sahel, “Evidence for
glutamate-mediated excitotoxic mechanisms during photoreceptor degeneration in the
rd1 mouse retina”, Mol Vis, vol. 11, pp. 688-696, 2005.
4. A.W. Hart, L. McKie, J.E. Morgan et al., “Genotype-phenotype correlation of mouse pde6b
mutations”, Invest Ophthalmol Vis Sci, vol. 46, pp. 3443-3450, 2005.
5. R. Gibson, E.L. Fletcher, A.J. Vingrys, Y. Zhu, K.A. Vessey, M. Kalloniatis, “Functional
and neurochemical development in the normal and degenerating mouse retina”, J Comp
Neurol, vol. 521, pp. 1251-1267, 2013.
6. C. Gargini, E. Terzibasi, F. Mazzoni, E. Strettoi, “Retinal organization in the retinal
degeneration 10 (rd10) mutant mouse: a morphological and ERG study”, J Comp
Neurol, vol. 500, pp. 222-238, 2007.
7. M.A. Chrenek, N. Dalal, C. Gardner et al., “Analysis of the RPE sheet in the rd10 retinal
degeneration model”, Adv Exp Med Biol, vol. 723, pp. 641-647, 2012.
8. B. Chang, N.L. Hawes, R.E. Hurd, M.T. Davisson, S. Nusinowitz, J.R. Heckenlively,
“Retinal degeneration mutants in the mouse”, Vision Res, vol. 42, pp. 517-525, 2002.
9. B. Chang, N.L. Hawes, M.T. Pardue et al., “Two mouse retinal degenerations caused by
missence mutations in the beta-subunit of rod cGMP phosphodiesterase gene”, Vision
Res, vol. 47, pp. 624-633, 2007.
10. D. Hicks, J. Sahel, “The Implications of Rod-Dependent Cone Survival for Basic and
Clinical Research”, Invest Ophthalmol Vis Sci, vol. 40, pp. 3071-3074, 1999.
11. S. Sugimoto, M. Imawaka, R. Imai, K. Kanamaru, T. Ito, S. Sasaki, T. Ando, T. Saijo, T.
Sato, “A procedure for electroretinogram (ERG) recording in mice - effect of
monoiodoacetic acid on the ERG in pigmented mice”, J Toxicol Sci, vol. 2, pp. 315-
325, 1997.
12. M.F. Marmor, A.B. Fulton, G.E. Holder, Y. Miyake, M. Brigell, M. Bach, “ISCEV
Standard for full-field clinical electroretinography (2008 update)”, Doc Ophthalmol,
vol. 118, pp. 69-77, 2009.
39
RESULTS
13. A.J. Eisenfeld, A.H. Bund-Milam, P.V. Sarthy, “Müller cell expression of glial fibrillary
acidic protein after genetic and experimental photoreceptor degeneration in the rat
retina”, Invest Ophthalmol Vis Sci; vol. 25, pp. 1321-1328, 1984.
14. M.E. Pennesi, K.V. Michaels, S.S. Magee, A. Maricle, S.P. Davin, A.G. Garg, M.J. Gale,
D.C. Tu, Y. Wen, L.R. Erker, P.J. Francis, “Long-term characterization of retinal
degeneration in rd1 and rd10 mice using spectral domain optical coherence
tomography”, Invest Ophthalmol Vis Sci; vol. 53, pp. 4644-4656, 2012.
15. M.J. Phillips, D.C. Otteson, D.M. Sherry, “Progression of neuronal and synaptic
remodeling in the rd10 mouse model of retinitis pigmentosa”, J Comp Neurol, vol.
518, pp. 2071-2089, 2010.
16. A. Mataruga, E. Kremmer, F. Müller, “Type 3a and type 3b OFF cone bipolar cells
provide for the alternative rod pathway in the mouse retina”, J Comp Neurol, vol. 502,
pp. 1123-1137, 2007.
40
RESULTS
5.1.8 FIGURES
Figure 1 Electroretinogram recordings in wild type and rd10 mice. (A) Left: Mice were
fixed in front of a Ganzfeld stimulator in a standardized position. Right: Goldring electrodes
were used on both eyes as active electrodes. A goldring electrode in the mouth served as
reference and a subcutaneous needle electrode in the lumbar region as ground electrode. (B)
Examples of ERGs of the left eye of a wild type (left) and rd10 mice (right) at different ages
(2, 3, 5 and 9 weeks). While in wild type the amplitudes of a- and b-waves increased with age,
in rd10 mice an ERG was only recordable untill the age of 3 weeks. (C) Amplitudes of the
ERG’s a- (left panel) and b-wave (right panel) in wild type mice (black bars) and rd10 mice
(grey bars) plotted with respect to the stimulus intensity (first row: 0.3 cds/m2, second row: 1
cds/m2 and third row: 3 cds/m
2) and to age given on the abscissa. Vertical bars indicate the
mean + standard deviation (n=3 per age group).
41
RESULTS
Figure 2 Sd-OCT of wild type and rd10 mice. (A) Set-up. The mouse is positioned in front
of the magnifying lens of the OCT on the chin rest of an equipment usually used for
application in humans. (B) - (H) OCT images of wild type and rd10 mice at different ages.
All images are taken at the height of the papilla and displayed with the pigment epithelium to
the top. Images of rd10 mice are illustrated in two ways the first (black on white) and second
column (white on black). Red arrows in (B) (first column) demonstrate the evaluation of
thickness in the height of the papilla by six averaged cuts through the retina. Red bars in (C) -
(F) (first column) mark the outer nuclear layer (ONL). In (G) (first and second column) red
stars indicate a separation of the retina from the pigment epithelium. Separation was only
observed in vivo in one animal of nine weeks and one of 24 weeks of age (G), but not at the
age of 48 weeks. (I) Evaluation of retinal thickness in wild type and rd10 mouse in the OCT.
Values obtained from six cuts through the retina at the height of the papilla were averaged and
42
RESULTS
plotted against the age (each age group: n=3). Thickness of the whole retina is illustrated in
(I1) (= wild type) and (I2) (= rd10 mouse), thickness of ONL (outer nuclear layer, somata) +
inner and outer segments of the photoreceptors (IS+OS) in (I3). In (I4) retinal thickness
except photoreceptor layers are illustrated (GC=ganglion cell layer, IPL=inner plexiform
layer, INL=inner nuclear layer and OPL=outer plexiform layer). Values represent mean ± SD
((I1) (wild type) for animals of 2 weeks: ****p<0.0001 vs. animals elder than 5 weeks; (I2)
(rd10) for animals of 2 and 3 weeks ****p<0.0001 vs. animals elder than 5 weeks; (I3) (rd10)
for animals of 2 weeks ****p<0.0001 vs. animals elder than 5 weeks; in all Figures: one-way
ANOVA with Bonferroni’s post hoc test).
Figure 3 Fluorescein angiography in wild type versus rd10 mice. In 7 and 9 weeks old
mice no significant differences in vasculature could be observed after intraperitoneal
application of fluorescein.
43
RESULTS
Figure 4 Histological work up. HE stained retina. Pigment epithelium and photoreceptors
are displayed to the top. (A) Area of evaluation. The papilla is cut directly through the
median. (B) Rd10 mouse retina at the age of five weeks (left) in contrast to wild type mouse
(right). In rd10 thickness of the ONL (yellow bars) is reduced to two layers of somata and the
retina separates from the pigment epithelium (probably induced during to the preparation
process). In wild type outer segments of the photoreceptors are in contact to the pigment
epithelium (same age; ONL= outer nuclear layer, INL= inner nuclear layer, IPL= inner
plexiform layer) (C) HE-Staining of the retina of wild type (first row) and rd10 mice (second
row) in the age of 2, 5, 9, 24 and 48 weeks. While in wild type no thickness differences are
visible, in rd10 the thickness decreased with age.
44
RESULTS
Figure 5 Immunohistochemical staining wild type (A) and rd10 retina (B). Staining 1:
Recoverin (all photoreceptors and type 2 bipolar cells) in green; rhodopsin (rod outer
segments, blue); HCN1 (processes in IPL, red). Staining 2: Horizontal cells (anti 28kDa
calcium-binding protein, red), rod bipolar cells (PKC, green), amacrine cells (calretinin, blue).
In rd10 retina, horizontal cells and bipolar cells lose their dendritic processes. Staining 3:
Cone outer and inner segment (PEA, blue), Müller cells (anti glutamine synthetase, red) and
astrocytes (anti GFAP, green). In rd10, Müller cell processes are also GFAP-positive. The
ONL in the rd10 mouse (B) is considerably reduced to only one row of somata.
45
RESULTS
5.2 STUDY II
THE EFFECTS OF IODOACETIC ACID
ON THE MOUSE RETINA
Sarah Rösch1,2
, Sandra Johnen1, Babac Mazinani
1, Frank Müller
3, Christiane Pfarrer
2,
Peter Walter1
4. Department of Ophthalmology, RWTH Aachen University, Pauwelsstr. 30, 52074
Aachen, Germany
5. Department of Anatomy, University of Veterinary Medicine, Bischofsholer Damm 15,
30173 Hannover, Germany
6. Institute of Complex Systems, Cellular Biophysics, ICS-4, Forschungszentrum Jülich
GmbH, 52428 Jülich, Germany
SUBMITTED/ UNDER REVIEW
This work is part of a doctoral thesis at the University of Veterinary medicine Hannover
(October 2013).
Corresponding Author: Peter Walter, Department of Ophthalmology, RWTH Aachen
University, Pauwelsstr. 30, 52074 Aachen, Tel +49-241-8088191, fax +49-241-8082408, mail
This work was supported by the DFG grants WA 1472/6-1 to PW and MU 3036/3-1 to FM.
46
RESULTS
5.2.1 ABSTRACT
Background: To characterize the effects of intravitreal injections of iodoacetic acid (IAA) in
comparison to its systemic application as a measure to induce unilateral photoreceptor
degeneration.
Methods: Seven weeks old C57BL/6J mice received either intravitreal injections of IAA or
systemic treatment (intraperitoneal vs. intravenous) and were observed in the following five
weeks using ERG, OCT, and histology.
Results: Systemic treatment with IAA showed high systemic toxic effects and a high
mortality in contrast to the intravitreal injection. Intraperitoneal application had no effect on
the retina. Intravenous application of 2 x 30 mg/kg BW IAA (time distance 3.5 hours)
resulted in an extinction of the ERG and a thinning of the retina, in particular of the outer
nuclear layer (ONL) indicating a photoreceptor degeneration. Animals receiving intravitreal
injections developed cataracts already in low concentrations up to 100% at 0.25 mg/kg BW.
Higher intravitreal IAA doses lead to extinguished ERGs. In histology a thinning of the entire
retina was observed that was most prominent in the inner part of the retina.
Conclusions: In contrast to intraperitoneal, intravenous application of IAA leads to a
selective photoreceptor degeneration. After intravitreal injection dense cataracts are already
observed at concentrations lower than those needed to induce changes in the ERG. ERG
results must be interpreted carefully. A thinning of all retinal layers rather than a specific
outer retinal degeneration was observed upon intravitreal injection. IAA is not a useful model
to study outer retinal degeneration in mice.
47
RESULTS
5.2.2 INTRODUCTION
Animal models for retinal degenerations are important tools to explore experimental therapies
for currently untreatable conditions. In rodents, genetic models are available resembling
photoreceptor degenerations such as observed in Retinitis pigmentosa (RP). In mice, for
example the rd1 and the rd10 mutants, carrying mutations in the gene encoding the ß-subunit
of rod cGMP phosphodiesterase, were described and widely used.1-9
In these mice,
comparable to RP, a progressive loss of photoreceptors - first rods, then cones - with age was
observed, concomitant with a pronounced reduction in the thickness of the outer nuclear layer
(ONL), while the thickness of the inner retinal layers remained unaffected.10,11
As a rat model,
the RCS (Royal College of Surgeons) rat was described and used.12-14
Experimental
treatments, e.g. the application of growth factors, were evaluated in such animal models
already years ago.15
To evaluate experimental surgical approaches for the treatment of
inherited retinal degeneration, models of animal species with larger eyes are desirable.
Nishida et al. used photodynamic therapy to induce an outer retinal degeneration in rabbits to
test the efficacy of suprachoroidal electrical stimulation.16
Recently, a genetic model was
described in rabbits17-19
, however, maintaining such a transgenic colony may be very cost
intensive and will also require other resources, possibly not easily available. Beside genetic
models, several induced models for retinal degeneration have been described. Among them
are degenerations induced by light20
and by systemically applied pharmaceuticals such as
iodoacetic acid (IAA)22-37
or N-methyl-N-nitrosourea (MNU)21
. It was described that IAA
leads to an elimination of photoreceptors while neurons of the inner retinal layers remain
unaffected.22-24
IAA covalently modifies and inhibits the glyceraldehyde 3-phosphate
dehydrogenase (GAPDH), an essential enzyme for glycolysis, which is halted.22,25
Because of
their very high metabolic rate, photoreceptors seem to be particularly sensitive for IAA, but
also other cells are influenced in their energy balance.23
Iodoacetic acid is not only reported to
irreversibly modify thiol (SH) groups as in GAPDH, it also reacts with imidazole and amino
groups of proteins,26
so that toxic side effects after systemic application are likely. The
intravenous application resulted in a degeneration of photoreceptors in mouse24
, rat26-28
,
ground squirrel29
, rabbit30-34
, pig35,36
, and monkey37
. Intravenous treatment with IAA also
causes cataracts after a latent period of one to three months30
hypothetically caused by a
blockage of the energy metabolism in the lens and inhibition of mitosis in the lens epithelium,
48
RESULTS
as a result of damage to the SH-dependent enzymatic systems.33
Further, a breakdown of the
blood-aqueous barrier resulting in an plasmoid and cellular exudation into the aqueous humor
after two to five days was reported.30,33
Intraperitoneal application is rarely described but also
leads to cataract formation twenty weeks after injection.26
However, both ways of systemic
administration are affecting the general well-being of the animal.
To avoid systemic side effects combined with the advantage of having an intraindividual
control eye, we investigated the effects of intravitreal injections of iodoacetic acid in the
retina. We hypothesized that local administration of IAA by intravitreal injection will induce
photoreceptor degeneration similar to the effects obtained with systemic application, but
without affecting the fellow eye and without affecting the general health status of the animal.
49
RESULTS
5.2.3 MATERIALS & METHODS
The materials and methods were performed as also described in Study I: ”Correlations
between ERG, OCT and anatomical findings in the rd10 mouse” and Study III: “Selective
photoreceptor degeneration by intravitreal injection of N-methyl-N-nitrosourea”.
All animal experiments adhered to the “Principles of laboratory animal care” (NIH
publication No. 85-23, revised 1985), the OPRR Public Health Service Policy on the Humane
Care and Use of Laboratory Animals (revised 1986) and the U.S. Animal Welfare Act, as
amended, as well as the current version of the German law on the protection of animals and
the local commission for animal welfare. The proposal of the animal experiments was
approved by the LANUV (Landesamt für Natur, Umwelt und Verbraucherschutz Nordrhein
Westfalen, Recklinghausen, Germany; file reference: 84-02.04.2011.A386).
Animals
Adult pigmented female wild type mice (C57BL/6J; 6 - 8 weeks) were maintained in an air-
conditioned environment under controlled light conditions (12:12 hours light/dark cycle) with
free access to food and water. Animals received sterile iodoacetic acid (IAA; Sigma Aldrich,
Seelze, Germany) in different concentrations systemically either as intraperitoneal injections
(8 animals, 40 - 70 mg/kg bodyweight (BW)) or as intravenous injections (12 animals, 55 - 65
mg/kg BW). A third group of animals received IAA as intravitreal injections (25 animals,
between 0.1 - 1 mg/kg BW). The sodium salt of IAA was dissolved in phosphate buffered
saline and neutralized to pH 7.4 with dilute NaOH immediately before use. The animals were
examined 5 days before, 7 days, 14 days and single animals up to five weeks after injection of
IAA to monitor the photoreceptor function and effects of the injection. Histological
examinations were made at two or five weeks after treatment.
For all experimental procedures and examinations except for intraperitoneal or intravenous
injections of IAA the animals were anaesthetized with an intraperitoneal injection of a
mixture of ketamine (70 mg/kg Ketamin® 10%, CEVA, Germany) and xylazine (10 mg/kg
Xylazin 2% Bernburg®, Medistar, Ascheberg, Germany) in 0.1 mL of saline.
50
RESULTS
Systemic IAA-application
For systemic application, the neutralized IAA-solution was administered intraperitoneally or
intravenously via one of the tail veins at different concentrations. The IAA-solution was at
room temperature. Very rapid injections were avoided. The concentrations were chosen
around the value of 60 mg/kg BW - a concentration reported to effectively cause
photoreceptor degeneration in mice.24
Eight animals were treated with an intraperitoneal injection of IAA: 40 mg/kg BW (n=1), 50
mg/kg BW (n=1), 60 mg/kg BW (n=2), 65 mg/kg BW (n=1), 70 mg/kg BW (n=1), 75 mg/kg
BW (n=1), and 2 x 30 mg/kg BW in a time interval of 3.5 hours (n=1).
Twelve animals received intravenous injections of IAA: 55 mg/kg BW (n=2), 60 mg/kg BW
(n=5), 65 mg/kg BW (n=1), and 2 x 30 mg/kg BW in a time interval of 3.5 hours (n=4).
Intravitreal IAA-injection
Animals were anesthetized as described before and eyes were locally anesthetized by
proxymetacainhydrochloride 0.5% eye drops (Proparakain- POS®, Ursapharm, Saarbrücken,
Germany). Eyes were punctured by a 27 gauge steel needle (BD Microlance TM
3, Heidelberg,
Germany). Intravitreal injections were performed using a nanoliter injection system with a 3D
micromanipulator (Nanoliter 2000; World Precision Instruments, Inc. Sarasota, FL, USA).
Injection volume was fixed to 2 µL. Preliminary experiments showed that this volume could
be injected safely into the vitreous cavity without touching the lens or the retina. A standard
volume of 2 µL with different IAA-concentrations was injected intravitreally in 25 mice: 0.1
mg/kg BW (n=3), 0.15 mg/kg BW (n=5), 0.2 mg/kg BW (n=6), 0.25 mg/kg BW (n=4), 0.5
mg/kg BW (n=4), 1 mg/kg BW (n=3).
After intravitreal injections, the animals were treated with antibiotic eye ointment
(Polyspectran, Alcon Pharma GmbH, Freiburg, Germany) up to three days after application.
Control interventions
Saline was injected as a control substance in the tail vein of one mouse, in the peritoneum of
three mice, and in the vitreous cavity of one eye in three mice. Control mice received the
equivalent volume of saline as the IAA treated animals. Control animals were examined five
51
RESULTS
days before, after one week, and two weeks up to four weeks after injection by recording
ERG and OCT and were finally sacrificed for histology.
In three control animals, 2 µL fluorescein solution was injected intravitreally to confirm the
correct placement of the injection cannula and the absence of leakage from the injection area.
Directly after injection images were taken by a confocal laser scanning microscope
(Heidelberg Retina Angiograph-1, Heidelberg Engineering, Germany) in the fluorescein
angiography mode to exclude side effects possibly resulting from the application technique.
Electroretinogram recordings
Electroretinogram recordings (ERG) were performed with the Reti System especially
designed for mice (Roland Consult Electrophysiological Diagnostic Systems, Brandenburg,
Germany). Animals were dark adapted for one hour. The pupils were dilated using
Tropicamide 2.5% eye drops (Pharmacy of the University Hospital Aachen, Germany) and
subsequently anaesthetized with proxymetacainhydrochloride 0.5% eye drops (Proparakain-
POS®, Ursapharm, Saarbrücken, Germany). Goldring electrodes (animal electrode 0.5mm ø
3mm Roland Consult) were placed as active electrodes on the corneal surface of both eyes.
Methylcellulose (Methocel® 2%, Omni Vision, Puchheim, Germany) was applied to keep the
mouse eye hydrated and maintain a good ionic conduction. The reference goldring electrode
was placed on the mouth mucosa and a subcutaneous silver needle electrode in the lumbar
region served as ground electrode. The fullfield-ERG was performed according to the ISCEV
standard protocol.38
Five scans to light stimulation were performed averaged at different light
intensities. A- and b-wave amplitudes and implicit times of rod and cone responses were
determined.
Spectral domain Optical Coherence Tomography
Spectral domain Optical Coherence Tomography (sd-OCT) images were obtained using the
Spectralis® OCT system (Heidelberg Engineering, Heidelberg, Germany). Retinal images
were centered on the optic disk as the main landmark and retinal thickness was determined at
six positions across the retina (Fig. 3b red arrows). For each eye these thickness values were
averaged. The thickness of the different layers of the retina was determined in the same way.
52
RESULTS
The fundus of each eye was recorded by the sd-OCT system (IR reflection image, wavelength
715nm).
Macroscopic pictures of the eye appearance were taken with a Nikon D80 camera (Objective:
Nikon AF Micro Nikkor 105 mm, 2896 Pixel x 1944 Pixel x 24 Bit).
Histology
At the end of the follow-up the animals were euthanized by isoflurane (Forene 100% (V/V),
Abott GmBH, Wiesbaden, Germany) overdosing, decapitated and enucleated.
Immunohistochemistry was performed as described earlier by Mataruga et al. (2007).40
In
brief, eyes were opened by an encircling cut at the limbus. The retinae in the eyecup were
immersion-fixed for 30 min in 4% paraformaldehyde (PA) in 0.1 M phosphate buffer (PB) at
room temperature, washed in PB several times and incubated in 10% sucrose in PB for 1 h,
followed by 30% sucrose overnight. The retina was flat embedded and frozen in optimal
cutting temperature (OCT) compound (NEG-50, Richard Allen Scientific, Thermo Fisher
Scientific, Germany). Vertical sections (20 µm thickness) were cut on a cryostat (HM 560
CryoStar; MICROM; Walldorf; Germany) and collected on Superfrost Plus slides (Menzel,
Braunschweig). Sections were pre-treated with blocking solution (5% chemiblocker
(Chemicon, Hofheim, Germany), 0.5% Triton-X100 in PB, and 0.05% NaN3) for 1 hour,
followed by incubation with primary antibodies over night, diluted in the same solution.
Sections were washed in PB and incubated in secondary antibodies diluted in 5%
Chemiblocker, 0.5% Triton-X100 in PB for 1 hour, washed in PB and coverslipped with
Aqua Polymount (Polysciences, Eppelheim, Germany). Sections were examined with a Leica
TCS confocal laser scanning microscope (Leica Microsystems, Heidelberg, Germany) with
63x/1.4 oil immersion lenses. Images were processed and printed with Adobe Photoshop. For
double or triple labelling, primary antibodies were mixed and applied simultaneously. All
secondary antibodies were highly cross-absorbed and were carefully tested to exclude
reactions with the wrong primary antibody. Concentration of the antibodies, laser intensity,
and filter settings were carefully controlled and the sequential scanning mode was employed
to completely rule out cross-talk between the fluorescence detection channels. Band pass
filters of 500–530 nm for green fluorescence (Cy2), 580–650 nm for red fluorescence (Cy3),
and 680–750 nm for infrared fluorescence (Alexa647) were used. Primary antibodies were
53
RESULTS
directed against following epitopes: GFAP (anti glial fibrillary acidic protein; raised in
chicken, 1:2000; Novus, Germany) and glutamine synthetase (raised in mouse, 1:4000; BD
Biosciences, Germany). Secondary antibodies included donkey anti-chicken Cy2 (1:400,
Dianova, Germany) and donkey anti-mouse Cy3 (1:100, Dianova, Germany). The lectin
Peanut agglutinin (PNA, biotinylated, 1:1600; Sigma Aldrich; Germany) was visualized using
StreptavidinAlexa647 (1:100, In vitrogen, Germany).
For Hematoxylin-Eosin staining, eyes were enucleated, punctured three times at the limbus
and fixed by immersion as described above. Eyes were dehydrated (dehydration automat,
mtm, Slee, Mainz, Germany) by incubation in a series of increasing ethanol concentrations (2
times x 70%, 2 x 96%, 3 x 100% for one hour), followed by xylene (3 times 1 hour) and
paraffin (4 times one hour), and embedded in Paraffin. Sections of 5 µm thickness were cut
with a microtome (R. Jung, Heidelberg, Germany), collected on slides, deparaffinized,
rehydrated, and stained with Hematoxylin and Eosin. Pictures were taken using a Leica
DMRX microscope.
The thickness of the entire retina and of the individual retinal layers was determined in both
immunohistochemical and in Hematoxylin-Eosin stainings at six positions close to the papilla.
For each retina, thickness values obtained from these positions were averaged.
54
RESULTS
5.2.4 RESULTS
Systemic application of IAA. Intraperitoneal and intravenous application of iodoacetic acid
resulted in considerable systemic side effects in mice. Intraperitoneal application of IAA
induced mortality of 25% and weight loss within the first two days of about 10% unrelated to
the dosage given. When IAA was injected intravenously, a mortality of 60%, but only little
effect on body weight was observed. Mortality after injection of IAA in the vein was related
to dosage and death occurred within the first four hours after injection. In contrast, when IAA
was administered intravitreally, no systemic side effects or even lethal outcomes were
observed. The results are summarized in Figure 1.
To monitor the functionality of the retina, light-induced electrical activity of both eyes was
recorded as full-field flash ERGs as described in the ISCEV standard protocol.38
Typically,
the ERG waveforms consist of an early negative wave (a-wave), followed by a large positive
deflection (b-wave). Whereas at least the initial portion of the a-wave reflects the primary
light response in photoreceptors, the b-wave is dominated by the activity of ON-bipolar cells
and Müller cells. Riding on top of the b-wave are the oscillatory potentials, small wavelets
that probably involve inner retinal circuitry. Systemically administered IAA did not affect the
amplitudes of either a-wave or b-wave (Fig. 2b, 2d), except when 2 x 30 mg/kg BW IAA
were applied intravenously (Fig. 2e). In these cases a reduction of the amplitudes of the a- and
b-waves could be seen as well as a loss of the oscillatory potentials.
Following the ERG recordings, retinal thickness was measured and the fundus of the eye was
observed in vivo by optical coherence tomography (OCT). Intraperitoneal application of IAA
did not induce any quantitative changes in retinal thickness as determined from OCT scans.
For each retina, thickness was measured at six locations close to the papilla (examples marked
by red arrows in Figure 3b). IAA concentrations below 60 mg/kg BW given as intravenous
injections were also not effective in reducing retinal thickness within the follow-up. Only
when 30 mg/kg BW was injected twice within 3.5 hours, a significant reduction of retinal
thickness was seen as demonstrated in Figure 3e (red bars) and Figure 3h.
OCT displayed no effect of systemic IAA administration on the retinal thickness, except for
the animal treated with 2 x 30 mg/kg BW IAA injected in the tail vein (Figure 3). This finding
was corroborated by immunohistochemical analysis of vertical sections through the retinae of
all experimental animals stained with antibodies against glutamine synthetase to visualize
55
RESULTS
Müller cells, PNA to label cone outer segments and endfeet, and anti-GFAP antibodies. Under
normal conditions, GFAP is only expressed in astrocytes. However, during retinal
degenerations, GFAP may also be observed in Müller cells, that have become reactive.39
Two
representative examples are shown in Figure 4. Only in the animal treated with the double
injection of IAA, the retinal thickness was considerably reduced due to the thinning of the
outer nuclear layer (ONL) to only 3-5 rows of photoreceptor somata (Fig. 4c), while the inner
retina seemed to be unaffected. Müller cells became reactive, as indicated by GFAP
expression (Fig. 4d). The thinning of the outer retina was reminiscent of - albeit not as
pronounced as - the photoreceptor loss observed in the retina of the rd10 mouse (Fig. 4e,
Biswas et al., in preparation).6-14
Intravitreal injection of IAA. Animals that received iodoacetic acid as intravitreal injections
showed no remarkable weight loss or other systemic side effects (see Figure 1a and 1b).
However, ocular side effects occurred in relation to the dose given. Cataract and inflammatory
reactions were most prominent. Even at the low concentration of 0.1 mg/kg BW, 20% of the
injected eyes developed cataract. With concentrations of 0.25 mg/kg BW or higher, phthisis
bulbi was frequently observed and the ERG was not recordable (Fig. 5). Due to the frequent
manifestation of cataracts and inflammatory infiltrates in the aqueous humor as verified by
HE-staining (data not shown), OCT scanning could only be performed in eyes that had
received low dosages of IAA. In those eyes where pictures could be taken, OCT recordings
were blurry and the layering of the retina seemed less clear two weeks after the injection (Fig.
6). This finding was inconsistent and in the immunohistochemical analysis the retinae showed
a normal structure and thickness (Fig. 8b). In control eyes injected with PBS, no changes in
retinal layering were observed (see Fig. 3f and 3i).
With increasing concentrations of intravitreal IAA considerable changes in the ERG were
found. ERG a- and b-wave were reduced or extinguished at concentrations of 0.25 mg/kg BW
or higher as shown in Figure 7.
In experiments with higher doses of intravitreal iodoacetic acid, histology revealed a general
thinning of the retina after two weeks (Figure 8). A specific effect of IAA on photoreceptor
survival should lead to a thinning of the outer nuclear layer, while the inner retina should
remain intact. In contrast to this, thinning was more pronounced in the inner retina (reduction
56
RESULTS
up to 44% measured in immunohistochemical sections) while only small changes were
observed in the outer nuclear layer (reduction up to 16%). Glial reaction was present as
indicated by GFAP immunoreactivity in Müller cells (Fig. 8e). Concentrations of 0.5 mg/kg
BW IAA and higher led to a gross disorganization of the retina, partially with a total
destruction of the retinal layers with interindividual differences so that morphometric
measurements did not reveal any concise results (data not shown). In all experiments, the
untreated control eyes of the animals were not affected and showed neither changes in ERG
nor in OCT measurements.
57
RESULTS
5.2.5 DISCUSSION
Animal models of retinal degenerations and dystrophies are important to study the underlying
disease mechanisms and also to establish possible treatment approaches. For retinal
dystrophies such as Retinitis pigmentosa (RP) several rodent models are established. Beside
these genetic models photoreceptor degeneration may also be induced pharmacologically. The
non-genetic models may have the advantage that they can be induced in a variety of animals
with larger eyes possibly more suitable to work on surgical methods for the treatment of
retinal dystrophies. A number of publications demonstrated that iodoacetic acid (IAA) may
lead to a blindness causing photoreceptor degeneration when administered intravenously. The
resulting histological picture is very similar to that presented in human RP.23
However, the
systemic application of IAA not only leads to bilateral disease but also to considerable
systemic side effects. Therefore, we hypothesized that IAA given intravitreally may induce
unilateral photoreceptor degeneration in mice without causing systemic side effects. The
systemic application of IAA either given as an intraperitoneal or as an intravenous injection
served as positive control.
In animals receiving systemic application, significant side effects occurred. The well-being of
the animals was strongly reduced for some hours after intravenous administration and for
some days after intraperitoneal application of IAA. Upon intravenous application, the
mortality rate in mouse was 60%, which is higher than in rat with 50%28
or in rabbit with
20%32
. Of the 12 intravenously injected animals of our study, only five survived- two with 55
mg/kg BW, two with 60 mg/kg BW and one animal which was treated by two shots of 30
mg/kg BW separated by 3.5 hours. The concentrations being used in our experiments were
derived from work of Sugimoto et al.24
Splitting the 60 mg/kg BW into two dosages given
within 3.5 hours as described in rat by Graymore et al.28
did not lower the mortality in the
mouse. But similar to their finding, also in mice 60 mg/kg BW IAA was found to more
effectively reduce photoreceptor density when administered as a divided dose. Probably the
two injection paradigm increased the pharmaceutical effect by maintaining a critical
concentration of IAA for a sufficient length of time28
. In our experiments, animals were
sacrificed two or five weeks after the injection. By that time in the two shot paradigm, the
ONL was reduced to 3 – 5 layers of somata. Detailed analysis using markers for cones (e.g.
Fig. 4c, other markers not shown) and rods (data not shown) indicate that the surviving
58
RESULTS
photoreceptor population comprised both rods and cones and that outer segments were
present. As our systemic application of IAA only served as positive controls, we did not
attempt to either monitor the time course of degeneration or to quantify whether the two cone
photoreceptor types are affected to different extent by IAA. The incomplete loss of
photoreceptors after two weeks is in agreement with histological examinations in IAA treated
rabbits (some photoreceptors survived up to four months)34
and pigs (up to 12 weeks with a
progressive loss of rods in time)35
. Interestingly, the expected effects of IAA on the retina
were not seen after intraperitoneal injection suggesting a first pass effect in the liver. The
retina remained unaffected. We did not observe cataractous changes in mice receiving
systemic application of IAA, probably because under these experimental conditions
development of cataracts was reported to be slow.23,28
Intravitreal injections in mice are difficult because the lens occupies most of the lumen of the
eye. The vitreous space is small and injections are difficult to place without touching the lens
or the retina. Therefore, we used a 3D micromanipulator and nanoliter injection system for the
injections. Injections of PBS, fluorescein and other substances carried out as negative controls
demonstrated that the injection procedure was safe and did not induce any changes in the lens
or the retina. In contrast to the systemic application the welfare of the animals was only
minimally affected upon intravitreal injections of IAA. We only noticed a slight weight loss
similar to that observed normally after an anesthetic intervention. However, the intravitreal
injection of IAA induced severe side effects, like cataract and inflammatory responses, on the
eye similar to those described after systemic application in longer experimental trials. Most
likely, the cataractous changes were not the result of a lens injury during the injection because
they appeared at about five to seven days after injection and they were not observed in control
animals receiving injections other than iodoacetic acid. Inflammatory responses with
plasmoid, fibrinous, and cellular exudation into the aqueous humor were also described by
Cibis et al. after intravenous application of IAA.23
All the described effects of iodoacetic acid
occurred with interindividual differences. This variable effect of the toxin was already
reported after intravenous application in the rabbit.23
Finally and most importantly, in contrast to the expected effect, no selective thinning of the
outer retina was observed upon intravitreal injection of IAA. Rather an overall thinning of the
retina was detected, with a tendency towards a more pronounced reduction in the inner retina.
59
RESULTS
Our results clearly indicate that IAA given intravitreally is not suitable to induce
photoreceptor degeneration to model disease processes as seen in RP or similar conditions.
Because of the cataract formation, retinal changes could not be monitored by OCT recordings
and also ERG recordings may be affected. Based on these results, we would not recommend
using IAA injections in the vitreous of mice or larger animals as a pharmaceutical model to
specifically induce degeneration of photoreceptors.
5.2.6 ACKNOWLEDGEMENT
The authors thank Christoph Aretzweiler for the excellent support in preparation and staining
of the eyes. This work was supported by the DFG grants WA 1472/6-1 to PW and MU
3036/3-1 to FM.
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RESULTS
5.2.7 REFERENCES
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3. Ahuja S, Ahuja-Jensen P, Johnson LE, Caffé AR, Abrahamson M, Ekström PA, van Veen
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4. Gibson R, Fletcher EL, Vingrys AJ, Zhu Y, Vessey KA, Kalloniatis M. (2013) Functional
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5. Gargini C, Terzibasi E, Mazzoni F, Strettoi E. (2007) Retinal organization in the retinal
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6. Chen M, Wang K, Lin B. (2012) Development and degeneration of cone bipolar cells are
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8. Chrenek MA, Dalal N, Gardner C, Grossniklaus H, Jiang Y, Boatright JH, Nickerson JM.
(2012) Analysis of the RPE sheet in the rd10 retinal degeneration model. Adv Exp Med
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9. Cang B, Hawes NL, Hurd RE, Davisson MT, Nusinowitz S, Heckenlively JR. (2002)
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10. Chang B, Hawes NL, Pardue MT, German AM, Hurd RE, Davisson MT, Nusinowitz S,
Rengarajan K, Boyd AP, Sidney SS, Phillips MJ, Stewart RE, Chaudhury R, Nickerson
JM, Heckenlively JR, Boatright JH. (2007) Two mouse retinal degenerations caused by
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11. Pang JJ, Boye SL, Kumar A, Dinculescu A, Deng W, Li J, Li Q, Rani A, Foster TC,
Chang B, Hawes NL, Boatright JH, Hauswirth WW. (2008) AAV-mediated gene
therapy for retinal degeneration in the rd10 mouse containing a recessive PDEbeta
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12. Hess HH, Newsome DA, Knapka JJ, Westney GE. (1982) Slitlamp assessment of age of
onset and incidence of cataracts in pink-eyed, tan-hooded retinal dystrophic rats. Curr
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13. Matuk Y. (1984) Retinitis pigmentosa and retinal degeneration in animals: a review. Can
J Biochem Cell Biol;62:535-546.
14. Fletcher EL, Kalloniatis M. (1997) Neurochemical development of the degenerating rat
retina. J Comp Neurol;388:1-22.
15. Baid R, Upadhyay AK, Shinohara T, Kompella UB. (2013) Biosynthesis,
Characterization, and Efficacy in Retinal Degenerative Diseases of Lens Epithelium-
derived Growth Factor Fragment (LEDGF1-326), a Novel Therapeutic Protein. J Biol
Chem;288:17372-17383.
16. Nishida K, Kamei M, Kondo M, Sakaguchi H, Suzuki M, Fujikado T, Tano Y. (2010)
Efficacy of suprachoroidal-transretinal stimulation in a rabbit model of retinal
degeneration. Invest Ophthalmol Vis Sci;51:2263-2268.
17. Jones BW, Kondo M, Terasaki H, Watt CB, Rapp K, Anderson J, Lin Y, Shaw MV, Yang
JH, Marc RE. (2011) Retinal remodeling in the Tg P347L rabbit, a large-eye model of
retinal degeneration. J Comp Neurol;519:2713-2733.
18. Muraoka Y, Ikeda HO, Nakano N, Hangai M, Toda Y, Okamoto-Furuta K, Kohda H,
Kondo M, Terasaki H, Kakizuka A, Yoshimura N. (2012) Real-time imaging of rabbit
retina with retinal degeneration by using spectral-domain optical coherence
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19. Hirota R, Kondo M, Ueno S, Sakai T, Koyasu T, Terasaki H. (2012) Photoreceptor and
post-photoreceptoral contributions to photopic ERG a-wave in rhodopsin P347L
transgenic rabbits. Invest Ophthalmol Vis Sci;53:1467-1472.
20. Harada T, Harada C, Sekiguchi M, Wada K. Light-induced retinal degeneration
suppresses developmental progression of flip-to-flop alternative splicing in GluR1. J
Neurosci. 1998;18:3336-3343.
21. Tsubura A, Yoshizawa K, Kuwata M, Uehara N. (2010) Animal models for retinitis
pigmentosa induced by MNU; disease progression, mechanisms and therapeutic trials.
Histol Histopathol;25:933-944.
22. Liang L, Katagiri Y, Franco LM, Yamauchi Y, Enzmann V, Kaplan HJ, Sandell JH.
(2008) Long-term cellular and regional specificity of the photoreceptor toxin,
iodoacetic acid (IAA), in the rabbit retina. Vis Neurosci;25:167-177.
23. Noell WK. (1953) Experimentally induced toxic effects on structure and function of
visual cells and pigment epithelium. Am J Ophthalmol;36:103-116.
24. Sugimoto S, Imawaka M, Imai R, Kanamaru K, Ito T, Sasaki S, Ando T, Saijo T, Sato T.
(1997) A procedure for electroretinogram (ERG) recording in mice – effect of
monoiodoacetic acid on the ERG in pigmented mice. J Toxicol Sci;2:315-325.
25. Winkler BS, Sauer MW, Starnes CA. (2003) Modulation of the Pasteur effect in retinal
cells: implications for understanding compensatory metabolic mechanisms. Exp Eye
Res;76:715-723.
26. Matsumoto M, Morita T, Hirayama S, Uga S, Shimizu K. (2002) Morphological analysis
of Iodoacetic acid-induced cataract in the rat. Ophthalmic Res;34:119-127.
27. Graymore C, Tansley K. (1959) Iodoacetate poisoning of the rat retina. I. Production of
retinal degeneration. Br J Ophthalmol;43,177-185.
28. Graymore C, Tansley K. (1959) Iodoacetate poisoning of the rat retina. II. Glycolysis in
the poisoned retina. Br J Ophthalmol;43:486-493.
29. Farber DB, Souza DW, Chase DG. (1983) Cone visual cell degeneration in ground
squirrel retina: disruption of morphology and cyclic nucleotide metabolism by
Iodoacetic acid. Invest Ophthalmol Vis Sci;24:1236-1249.
30. Cibis PA, Constant M, Pribyl A, Becker B. (1957) Ocular lesions produced by
Iodoacetate. AMA Arch Ophthalmol;57:508-519.
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31. Lasansky A, De Robertis E. (1959) Submicroscopic changes in visual cells of the rabbit
induced by iodoacetate. J Biophys Biochem Cytol;5:245-250.
32. Orzalesi N, Calabria GA, Grignolo A. (1970) Experimental degeneration of the rabbit
retina induced by iodoacetic acid. A study of the ultrastructure, the rhodopsin cycle and
the uptake of 14C-labelled iodoacetic acid. Exp Eye Res;9:246-253.
33. Tieri O, Vecchione L. (1963) Monolateral cataract provoked with iodoacetic acid. Acta
Ophthalmol (Copenh);41:205-212.
34. Yamauchi Y, Agawa T, Tsukahara R, Kimura K, Yamakawa N, Miura M, Goto H. (2011)
Correlation between high-resolution optical coherence tomography (OCT) images and
histopathology in an iodoacetic acid-induced model of retinal degeneration in rabbits.
Br J Ophthalmol;95:1157-1160.
35. Wang W, Fernandez de Castro J, Vukmanic E, Zhou L, Emery D, Demarco PJ, Kaplan
HJ, Dean DC. (2011) Selective rod degeneration and partial cone inactivation
characterize an iodoacetic acid model of Swine retinal degeneration. Invest Ophthalmol
Vis Sci;52:7917-7923.
36. Scott PA, Kaplan HJ, Sandell JH. (2011) Anatomical evidence of photoreceptor
degeneration induced by iodoacetic acid in the porcine eye. Exp Eye Res;93:513-527.
37. Noell WK. (1952) The impairment of visual cell structure by iodoacetate. J Cell
Physiol;40:25-55.
38. Marmor MF, Fulton AB, Holder GE, Miyake Y, Brigell M, Bach M. (2009) ISCEV
Standard for full-field clinical electroretinography (2008 update). Doc
Ophthalmol;118:69-77.
39. Eisenfeld AJ, Bund-Milam AH, Sarthy PV. (1984) Müller cell expression of glial
fibrillary acidic protein after genetic and experimental photoreceptor degeneration in
the rat retina. Invest Ophthalmol Vis Sci;25(11):1321-1328.
40. Mataruga A, Kremmer E, Müller F. (2007) Type 3a and type 3b OFF cone bipolar cells
provide for the alternative rod pathway in the mouse retina. J Comp Neurol;502:
1123-1137.
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RESULTS
5.2.8 FIGURES
Figure 1 Systemic side effects of IAA application. a Loss of body weight as an indicator of
general well-being after application of IAA given in different routes. ****p<0.0001 vs.
intraperitoneal (i.p.) application, one-way ANOVA with Bonferroni’s post hoc test.
(intravenous (i.v.): n=12; i.p.: n=8; intravitreal (IVI): n=25) b Body weight reduction after i.p.
IAA injection. For each concentration n=1; control animals (n=3 i.p.) were injected with the
same volume of PBS. c Mortality within the first two days after IAA injection. (i.v.: n=12;
i.p.: n=8; IVI: n=25). d Mortality after i.v. injection of IAA and concentration of IAA given
(control: n=1; 55 mg/kg BW: n=2; 60 mg/kg BW: n=5; 2 x 30 mg/kg BW: n=4; 65 mg/kg
BW: n=1).
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RESULTS
Figure 2 ERG after systemic IAA application. a – e Examples of full-field ERG recordings
from left eyes of mice before (black line) and one week after (grey line) systemic application
of IAA or PBS [Scotopic 3 cds/m2, 0.067 Hz]. Only in e an extinction of the ERG curve one
week after IAA application could be observed. a PBS i.p. b IAA 65 mg/kg BW i.p. c PBS i.v.
d IAA 60 mg/kg BW i.v. e IAA 2 x 30 mg/kg BW i.v. f, g Amplitude of a-wave (f) and b-
wave (g) before and two weeks after IAA application at a light intensity of 3 cds/m2 in the
scotopic ERG. Mean + SD. PBS injections served as controls.
66
RESULTS
Figure 3 Thickness evaluation in the OCT. Retinal images and thickness obtained by sd-
OCT measurements. Left panel. All images are taken at the height of the papilla and
displayed with pigment epithelium and photoreceptors to the top. Examples of sd-OCT
recordings before (left) and one week after (right) IAA (b, d, e) or PBS (a, c, f) application. a
PBS i.p. b IAA 65 mg/kg BW i.p.; red arrows indicate examples for the thickness
measurement in one retina. c PBS i.v. d IAA 60 mg/kg BW i.v. e IAA 2 x 30 mg/kg BW i.v.;
the red bars mark the outer nuclear layer, which is significantly thinner one week after
injection. f intravitreal injection of PBS. g, h Retinal thickness measurements based on sd-
OCT data 4 weeks after i.p. injection (g) and 2 weeks after i.v. injection (h) of IAA and PBS.
Values represent mean ± SD (for 2x30 mg/kg BW IAA: ****p<0.0001 vs. 0, 55, and 60
mg/kg BW, one-way ANOVA with Bonferroni’s post hoc test). i Retinal thickness
measurements under control conditions before and one week after i.p., i.v. and intravitreal
(IVI) injection of PBS.
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RESULTS
Figure 4 Confocal images of immunolabelled vertical sections obtained from retinae
prepared two weeks after systemic application. The white bars in a and c mark the
boundaries of the outer nuclear layer. Red: glutamine synthetase, Müller cells spanning nearly
the entire thickness of the retina; blue: PNA, cone outer segments and endfeet; green: GFAP.
a Control condition. b IAA i.v. 60 mg/kg BW. c IAA i.v 2x30 mg/kg BW (note that ONL
thickness and number of cones are reduced). d Glial reaction is indicated by GFAP expression
in Müller cell processes (same section as in C). e Rd10 mouse retina at the age of 50 days
displaying total loss of photoreceptors. OR: outer part of the retina; IR: inner part of the
retina; OS: outer segments; IS: inner segments; ONL: outer nuclear layer; OPL: outer
plexiform layer; INL: inner nuclear layer; IPL: inner plexiform layer; GCL: ganglion cell
layer.
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RESULTS
Figure 5 Effects of intravitreal injection of IAA. a Overview of effects. b Example of a
dense cataract seen one week after the injection of 0.5 mg/kg BW. c Phthitic eye two weeks
after injection of 0.5 mg/kg BW.
Figure 6 Retinal changes after intravitreal injection. OCT scans one and two weeks after
injection of 0.1 (a) and 0.15 mg/kg BW IAA (b). The scans were taken at the height of the
papilla (a) or at a position superior to the papilla (b). The scanning position for the cross
sectional image is indicated by the green line in the respective IR reflection image of the
fundus (b). Note that OCT scan appears blurry after two weeks making it difficult to
determine the retinal thickness accurately (position for thickness measurements indicated by
bars and double arrows).
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RESULTS
Figure 7 ERG after intravitreal IAA application. Examples of full-field ERGs recorded
before and one week after intravitreal (IVI) injection. a PBS. b 0.5 mg/kg IAA IVI. c, d
Changes in the ERG after IVI IAA application. Amplitude of a-waves (c) and b-waves (d)
corresponding to the concentration of IAA given.
Figure 8 Confocal images of immunohistochemically stained vertical sections through
the retina two weeks after injection of IAA into the vitreous. The borders of the ONL are
labelled by dashed lines. Stainings and abbreviations as in Fig. 4. a Untreated control. b 0.15
mg/kg BW IAA. c 0.2 mg/kg BW. d - f 0.25 mg/kg BW. Overall retinal thickness is reduced
in a variable way, but ONL thickness and cone density are only mildly affected. e Müller cells
express GFAP (same section as in d).
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RESULTS
5.3 STUDY III
SELECTIVE PHOTORECEPTOR DEGENERATION BY INTRAVITREAL INJECTION
OF N-METHYL-N-NITROSOUREA
Sarah Rösch1,2
, Sandra Johnen1, Anja Mataruga
3, Frank Müller
3, Christiane Pfarrer
2, and Peter
Walter1
1. Department of Ophthalmology, RWTH Aachen University, Germany
2. Department of Anatomy, University of Veterinary Medicine, Hannover Germany
3. Institute of Complex Systems, Cellular Biophysics, ICS-4, Forschungszentrum Jülich
GmbH, Germany
SUBMITTED/ UNDER REVIEW
Corresponding Author: Peter Walter, Department of Ophthalmology, RWTH Aachen
University, Pauwelsstr. 30, 52074 Aachen, Tel +49-241-8088191, fax +49-241-8082408, mail
This manuscript is part of a doctoral thesis at the University of Veterinary Medicine,
Hannover, Germany (01.10.2013).
Word count: 4417
This work was supported by the DFG grants WA 1472/6-1 to PW and MU 3036/3-1 to FM.
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5.3.1 ABSTRACT
Purpose: To characterize the effects of intravitreal injections of N-Methyl-N-nitrosourea
(MNU) in comparison to its systemic application as a measure to induce unilateral
photoreceptor degeneration.
Methods: Eight weeks old male C57BL/6J mice received either intraperitoneal injections (3
animals) or intravitreal injections (24 animals) of MNU in different concentrations and were
observed in the following two weeks using electroretinography (ERG), spectral domain
optical coherence tomography (sd-OCT), and immunohistochemistry.
Results: The intraperitoneal application of MNU showed moderate systemic toxic effects,
indicated by a loss of body weight of 12% within the first two days. In both eyes the ERG
became extinguished and sd-OCT scans showed a thinning of the retina predominantly in the
outer nuclear layer (ONL). Immunohistochemistry demonstrated the selective loss of rods and
cones. Mice receiving intravitreal injections showed nearly no weight loss after the injections
and no reduction of their general welfare. After two weeks, in the injected eye, but not in the
control eye, ERG, sd-OCT, and immunohistochemistry revealed changes identical to those
seen after systemic application.
Conclusion: The intraperitoneal application of MNU led to moderate systemic side effects in
mice and to selective photoreceptor degeneration. Intravitreal injections of MNU also induced
photoreceptor degeneration, but without systemic side effects. This tool may be helpful in
larger species where genetic models of receptor degenerations are not applicable but where
the size of the eye is more suitable to study surgical or other approaches to treat blindness
causing receptor degeneration.
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RESULTS
5.3.2 INTRODUCTION
Inherited retinal dystrophies, such as Retinitis pigmentosa (RP), are important and frequent
causes for blindness and observed in approximately 1 of 4000 persons.1,2
Research efforts are
focused on the genetic background of these diseases, on the mechanisms of degeneration, but
also on possible treatment strategies. Genetic models in rodents expressing retinal
degeneration, e.g. the retinal degeneration (rd) 1 mouse, rd10 mouse3-13
, and the RCS rat
(Royal College of Surgeons)14-17
, are indispensable tools to explore experimental therapies for
currently untreatable conditions. In rd1 and rd10 mice, mutations in the gene encoding for the
ß-subunit of rod cGMP phosphodiesterase result in the described disease pattern with an in
contrast to human RP central to peripheral gradient.3-13
Because of the later onset and milder
retinal degeneration in contrast to the rd1 mouse, the rd10 mouse provides a better genetic
mouse model for human RP.12
In the RCS rat, a deletion in the gene encoding the receptor
tyrosine kinase (Mertk) expressed in retinal pigment epithelium (RPE) cells, leads to a
disturbed phagocytosis of photoreceptor outer segments and consequently to photoreceptor
degeneration by apoptosis.17
Regardless of the cause of outer retinal degeneration in the rd1
mouse, rd10 mouse, and RCS rat, a progressive loss of photoreceptors with age comparable to
RP is observed, which is due to apoptosis.18
The thickness of inner nuclear layers and of the
ganglion cell layer remains unaffected.12-17
However, cellular and organizational and
functional changes in the inner retina were also observed.7,19
Beside retinal transplants, stem
cell therapy, and gene therapy, retinal implants have been developed and are currently used in
humans. Further development and improvement of such implants require experimental
surgical approaches performed in larger animal species than in mice or rats, e.g., in pigs, cats
or rabbits exhibiting a specific loss of photoreceptors. Genetic models in larger animals are
difficult to utilize because of costs and duration of disease manifestation.20
Photoreceptor degeneration may also be induced by extrinsic physical measures such as light
exposure or ionizing radiation on the developing retina.22,23
Retinal degenerations are also
observed after systemically applied pharmaceuticals such as iodoacetic acid (IAA)19,24,25
or N-
methyl-N-nitrosourea (MNU).2,18,26-30
After systemic treatment MNU leads dose dependent to
extensive oxidative stress resulting in retinal photoreceptor cell death due to apoptosis, what
was confirmed by TUNEL staining.26-28
Photoreceptors can be rescued by several inhibitors
of apoptosis.27,28
The toxic effects of MNU on photoreceptors in a 7-day period after systemic
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application have been described in different animals.26-30
In mice and rats, a concentration of
60 mg/kg bodyweight (BW) MNU was effective when applied intraperitoneally, while in
rabbits the intravenous application of 40 mg/kg BW effectively caused photoreceptor
degeneration.26-30
Likewise to the genetic models in mice, the loss of photoreceptors
proceeded with a central to peripheral gradient, but instead of affection of first rods and
consecutively cones, after MNU treatment both rods and cones were lost simultaneously and
to the same extent.26-30
The number of cells within the inner nuclear layer and within the
ganglion cell layer did not decrease and the inner retinal neurons showed features of
morphological remodeling comparable to the genetic model.26-30
A side effect of systemic
MNU application is the induction of neoplasms.31-33
In rabbits after systemic intravenous
injection, 68.8% of the animals developed brain tumors.31
Furthermore, tumors frequently
occurred as small intestine carcinomas and as multiple vessel wall sarcomas in different
organs.31
In rats, mammary tumors or prostate tumors can be induced by a single
intraperitoneal or intravenous injection of 50 mg/kg BW MNU.32,33
Tumors grew 12-16
weeks after injection.31-33
We were motivated to identify a safe and reproducible procedure for inducing unilateral outer
retinal degeneration by intravitreal application of pharmaceuticals, avoiding systemic side
effects and keeping the contralateral eye as an intraindividual control eye. Therefore, we
investigated the effects of intravitreal injections of MNU on the mouse retina.
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5.3.3 MATERIALS AND METHODS
The materials and methods were performed as also described in Study I: ”Correlations
between ERG, OCT and anatomical findings in the rd10 mouse” and Study II: “The effects of
iodoacetic acid on the mouse retina”.
All experiments were performed in accordance with the ARVO declaration for the use of
animals in ophthalmic and vision research and in accordance with the German Law for the
Protection of Animals and after approval was obtained by the regulatory authorities. The
proposal of the animal experiments was approved by the LANUV (Landesamt für Natur,
Umwelt und Verbraucherschutz Nordrhein Westfalen, Recklinghausen, Germany; file
reference: 84-02.04.2011.A386).
Animals
Adult pigmented male wild type mice (C57BL/6J; 8 weeks) were housed in standard
conditions under 12/12-hour light-dark cycle with food and water available ad libitum.
Because of the possible environmental hazard due to the carcinogenic and teratogenic
potential of N-methyl-N-nitrosourea (MNU), the animals were kept in special one-way cages
(PET; Tecniplast Deutschland GmbH; Hohenpreißenberg; Germany) after each MNU
application under fume hoods with the same light and food conditions as before. The mice
received sterile N-methyl-N-nitrosourea (MNU; Sigma Aldrich, Germany) in different
concentrations intraperitoneally (3 animals, 60 mg/kg BW) or intravitreally (24 animals, 0.15
- 3 mg/kg BW). Because of its poor stability in aqueous solutions and the duration of the
procedure of intravitreal injection, MNU was dissolved in dimethylsulfoxide (DMSO; Serva,
Heidelberg, Germany) immediately before use and further diluted with sterile phosphate
buffered saline (PBS). Animals were examined five days before as well as 7 days and 14 days
after injection of MNU to monitor the effects of the injection on retinal morphology and
photoreceptor function. Body weight was recorded on a daily basis during the entire
experiment. Animals were sacrificed for histological examinations two weeks after treatment.
Mice were anesthetized with ketamine (70 mg/kg Ketamin® 10%, CEVA, Germany) and
xylazine (10 mg/kg Xylazin 2% Bernburg®, Medistar, Ascheberg, Germany) in 0.1 mL of
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saline for in vivo measurements, examinations and intravitreal injections. For the systemic
treatment with MNU no anesthesia was given.
Systemic MNU-application
MNU solution was administered intraperitoneally in three mice with a concentration of 60
mg/kg BW and pre-warmed at room temperature. This concentration was described to cause
photoreceptor degeneration in mice.23
Very rapid injections were avoided.
Intravitreal MNU-injection
Intravitreal injections were performed using a 27 gauge steel needle (BD Microlance TM
3,
Heidelberg, Germany) and a nanoliter injection system with a 3D micromanipulator
(Nanoliter 2000; World Precision Instruments, Inc. Sarasota, FL, USA). After puncture in the
dorso-nasal area of the limbus, 2 µL were injected with a glass capillary in an angle of 30°
(Fig. 8, yellow arrow demonstrates direction of injection). Preliminary control experiments
showed that the injection technique was safe and that the volume (defined volume of 2 µL)
could be placed in the vitreous cavity without touching the lens or even the retina. Different
concentrations of MNU were injected intravitreally diluted with DMSO and PBS. MNU was
injected intravitreally in the left eye of 24 mice (0.15, 0.2, 0.25, 0.3, 0.5, 1.0, 2.0, 3.0 mg/kg
BW; n=3 for each concentration). We used DMSO as first solvent to guarantee better
solubility of MNU and to minimize degradation of MNU during the time consuming
intravitreal injection.
All intravitreally injected mice were treated by antibiotic eye ointment (Polyspectran, Alcon
Pharma GmbH, Freiburg, Germany) to avoid inflammatory reactions up to three days after
injection.
Control Interventions
Two wild type mice were kept under the same husbandry condition to evaluate systemic
effects of changing the cage conditions from normal cages to the one-way system. Further
control animals received intraperitoneal sham injections (PBS (n = 3) or DMSO + PBS
(n = 3)).
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Intravitreal control injections of PBS (2 µL), DMSO (maximal volume of 1 µL diluted to 2
µL with PBS) and fluorescein (different concentrations dissolved in 2 µL of PBS) were
performed to confirm the safety of the injection technique. Absence of leakage from the
injection area directly after injection of fluorescein was confirmed. Therefore, images were
taken by a confocal laser scanning microscope (Heidelberg Retina Angiograph-1, Heidelberg
Engineering, Heidelberg, Germany) in the fluorescein angiography mode.
All control animals (PBS; DMSO + PBS) were examined using the same methods and time
schedule as the experimental animals.
Electroretinogram recordings
After a dark adaption of one hour, pupils were dilated with one drop of Tropicamide 2.5% eye
drops (Pharmacy of the University Hospital Aachen, Germany). Mice were placed in front of
a full-field dome stimulator. The eyes were locally anesthetized with
proxymetacainhydrochloride 0.5% eye drops (Proparakain- POS®, Ursapharm, Saarbrücken,
Germany), and a goldring electrode (animal electrode 0.5mm ø 3mm Roland Consult,
Brandenburg an der Havel, Germany) was placed on the corneal surface of each eye. Non-
irritating artificial tears were used serving for corneal clarity and for a better ionic conduction
(Methylcellulose, Methocel® 2%, Omni Vision, Puchheim, Germany). A goldring electrode
in the mouth (contact with the mouth mucosa) served as reference electrode and a
subcutaneous silver needle electrode in the lumbar region as ground electrode.
Electroretinography was performed using the Reti System designed for rodents (Roland
Consult Electrophysiological Diagnostic Systems, Brandenburg, Germany). Mice were
examined by full-field ERGs according to the ISCEV standard protocol with five light stimuli
per recording.35
Amplitudes of the major ERG components, e.g., a- and b-wave, and implicit
times of rod and cone responses per recording were determined by averaging the responses of
the five light stimuli.
Spectral domain Optical Coherence Tomography
After anesthesia mice were positioned in front of the Spectralis® OCT system (Heidelberg
Engineering, Heidelberg, Germany) to perform the spectral domain Optical Coherence
Tomography. The scanning system was prepared additionally by a +25 D lens to correct for
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rodent optics. The optic disc was located as main landmark and retinal thickness
measurements were conducted using the cross sectional image centered on the optic disc. The
total retinal thickness as well as the thickness of the different layers was measured at six
positions across the retina (Fig. 3B, red arrows). Thickness values of each eye were averaged.
For documentation purposes, confocal images of the fundus of the evaluated eyes were
recorded (IR reflection image, wavelength 715nm). To further document possible
macroscopic changes of the treated or non-injected contralateral eyes, pictures of the mice
were taken by a Nikon D80 camera (Nikon AF Micro Nikkor 105 mm, 2896 Pixel x 1944
Pixel x 24 Bit).
Histology
After a duration of in vivo experiments of two weeks, mice were euthanized by isoflurane
(Forene 100% (V/V)®, Abott GmBH, Wiesbaden, Germany) overdosing and decapitated.
Eyes were processed in immunohistochemistry as well as in Hematoxylin and Eosin stainings.
Immunohistochemistry was performed as described earlier by Mataruga et al. (2007).37
In
brief, eyes were enucleated and opened by an encircling cut at the limbus. The retinae in the
eyecup were immersion-fixed for 30 minutes in 4% paraformaldehyde (PA) in 0.1 M
phosphate buffer (PB) at room temperature and washed in PB several times. Tissue was
incubated in 10% sucrose/PB for 1 hour, followed by 30% sucrose overnight. The retina was
flat embedded and frozen in optimal cutting temperature (OCT) compound (NEG-50, Richard
Allen Scientific, Thermo Fisher Scientific, Germany). Vertical sections (20 μm thickness)
were cut on a cryostat (HM 560 CryoStar; MICROM; Walldorf; Germany) and collected on
Superfrost Plus slides (Menzel, Braunschweig, Germany). Sections were pre-treated with
blocking solution (5% chemiblocker (Chemicon, Hofheim, Germany), 0.5% Triton-X100 in
PB, and 0.05% NaN3) for 1 hour, followed by incubation with primary antibodies over night,
diluted in the same solution. Following primary antibodies were used: anti-GFAP (anti glial
fibrillary acidic protein; raised in chicken, 1:2000; Novus, Germany), antibodies against
glutamine synthetase (raised in mouse, 1:4000; BD Biosciences, Germany), anti-CabP (anti
calbindin 28K; raised in mouse, 1:1000; Sigma, Germany), anti-PKC (anti protein kinase C α;
raised in rabbit, 1:4000; Santa Cruz, Heidelberg Germany), against Calretinin (AB1550,
raised in goat, 1:3000; Millipore, Germany), against HCN1 (RTQ-7C3, raised in rat, 1:10; F.
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Müller, Forschungszentrum Jülich, Germany), against recoverin (AB5585, raised in rabbit,
1:2000; Millipore, Germany), and against rhodopsin (1D4, 1:500; R.S. Molday, British
Columbia, Canada). Sections were washed in PB and incubated in secondary antibodies
diluted in 5% Chemiblocker, 0.5% Triton-X100 in PB for 1 hour, washed in PB and
coverslipped with Aqua Polymount (Polysciences, Eppelheim, Germany). Secondary
antibodies included: donkey anti-chicken Cy2 (1:400; Dianova, Germany), donkey anti-
mouse Cy3 (1:100; Dianova, Germany), donkey anti-rabbit Cy2 (1:400; Dianova, Germany),
donkey anti-goat Alexa647 (1:200; Invitrogen, Germany), donkey anti-rat Cy3 (1:500;
Dianova, Germany), and donkey anti-mouse Dy649 (1:500; Dianova, Germany). The lectin
Peanut agglutinin (PNA, biotinylated, 1:1600; Sigma Aldrich; Germany) was visualized using
StreptavidinAlexa647 (1:100; Invitrogen, Germany).
Sections were examined with a Leica TCS confocal laser scanning microscope (Leica
Microsystems, Heidelberg, Germany) with 63x/1.4 oil immersion lenses. Images were
processed and printed with Adobe Photoshop. For triple labeling, primary antibodies were
mixed and applied simultaneously. All secondary antibodies were highly cross-absorbed and
were carefully tested to exclude reactions with the wrong primary antibody. Concentration of
the antibodies, laser intensity, and filter settings were carefully controlled and the sequential
scanning mode was employed to completely rule out cross-talk between the fluorescence
detection channels. Band pass filters of 500 - 530 nm for green fluorescence (Cy2), 580 - 650
nm for red fluorescence (Cy3), and 680 - 750 nm for infrared fluorescence (Alexa647) were
used.
For Hematoxylin-Eosin staining, eyes were enucleated, punctured three times at the limbus
and fixed by immersion as described above. Eyes were dehydrated in a tissue dehydration
automat (mtm, Slee, Mainz, Germany) by incubation in a series of increasing ethanol
concentrations (2 times x 70%, 2 x 96%, 3 x 100% for one hour), followed by xylene (3 times
1 hour) and paraffin (4 times one hour), and embedded in paraffin. Sections of 5 μm thickness
were cut with a microtome (R. Jung, Heidelberg, Germany), collected on slides,
deparaffinized, rehydrated, and stained with Hematoxylin and Eosin. Pictures were taken
using a Leica DMRX microscope (Leica Microsystems, Heidelberg, Germany).
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The thickness of the entire retina and the individual retinal layers was determined in both
immunohistochemical and in Hematoxylin-Eosin stainings at six positions close to the papilla.
For each retina, thickness values obtained from these positions were averaged.
Urine test
Before, two and three hours, one and two days after systemic and intravitreal application of
MNU urine tests (Ultra performance liquid chromatography (UPLC); Aquity; Waters,
Eschborn, Germany) were made to test the excretion of MNU for safety reasons.
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5.3.4 RESULTS
Systemic application of MNU. Weight Loss. Intraperitoneal application of MNU resulted in
systemic side effects in mice with a weight loss of about 12% within the first two days. In
Figure 1 the weight loss after different forms of treatment with MNU is demonstrated to
reflect the well-being under changed husbandry conditions in one way cages.
Electroretinography. Systemically administered MNU led to an extinction of the scotopic
(Figure 2C - E) and photopic (Figure 3C) ERG responses after one and two weeks.
Optical Coherence Tomography. One week after intraperitoneal MNU application, a
reduction in total retinal thickness was observed, while the space between the retina and the
RPE seemed to be filled with cell debris (Figure 4C). After two weeks the debris had
disappeared completely, while the reduction of retinal thickness was more pronounced as
demonstrated in Figure 4C. Two weeks after injection, the inner nuclear layer became
adjacent to the pigment epithelium (Figure 4C).
Microscopy. Histological analysis confirmed the reduction of retinal thickness as seen in sd-
OCT recordings in-vivo with the pronounced loss of photoreceptors and the thinning of the
outer nuclear layer (ONL) after MNU treatment. Müller cells labeled with anti-GFAP (Figure
5D) had become reactive as seen also during other causes of retinal degeneration.36
Under
normal conditions, GFAP is only expressed in astrocytes. Representative sections are shown
in Figure 5. In animals that had received vehicle injection only (Figure 5B), the retina
remained unaffected. In the animals treated intraperitoneally with MNU, the ONL was
reduced to no or only one row of photoreceptor somata (Figure 5C), while the thickness of the
inner retina was statistically not different from the thickness in control eyes (Figure 8C). The
thinning of the outer retina was reminiscent of the photoreceptor loss observed in Retinitis
pigmentosa (RP) or in late stages of rodent models of RP such as in the rd10 mouse (Figure
5E).6-14
Intravitreal injection of MNU. Weight loss. Animals that received PBS, DMSO + PBS or
MNU as intravitreal injections showed no remarkable weight loss or other systemic side
effects (see Figure 1).
Electroretinography. The control interventions – injection of PBS or DMSO + PBS – as well
as the intravitreal injection of 0.15 up to 0.5 mg/kg BW MNU had no effect on the ERG. In
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animals treated with 1 and 2 mg/kg BW, scotopic (Figure 6) and photopic (Figure 3) ERGs
were affected. For both concentrations, in two of three animals the amplitudes of the a-wave
and the b-wave in the scotopic ERG were diminished and in one animal the ERG was
completely abolished. In all animals injected intravitreally with 3 mg/kg BW the scotopic
(Figure 6) and photopic (Figure 3) ERG was completely extinguished.
Optical Coherence Tomography. Sd-OCT measurements one week after the injection of 1, 2,
and 3 mg/kg BW revealed cell debris between the inner retina and the pigment epithelium,
similar to the findings observed after intraperitoneal application (compare Figure 7D - F to
Figure 4C). In animals that had received 3 mg/kg BW MNU, the somata of the photoreceptors
(outer nuclear layer, ONL) had disappeared throughout the entire retina. Upon injection of 1
or 2 mg/kg BW MNU, the cell death in the ONL was not homogeneous. In the superior
peripheral retina, i.e. close to the injection site, most or all of the cell layers in the ONL had
vanished, while in the inferior retina the reduction of ONL thickness was less than 50%.
Microscopy. The reduction of retinal thickness upon intravitreal injection of 1, 2, and 3 mg/kg
BW MNU observed in the OCT was verified by histology (Figure 8). MNU treatment led to a
thinning of the ONL, while the thickness of the inner retina was not significantly affected
(Figure 8). In animals that had received 1 or 2 mg/kg BW MNU, immunohistochemical data
(Figure 8, 9) confirmed that the degree of ONL thinning depended on the distance to the
injection site. In contrast to these findings, intravitreal injection of 3 mg/kg BW MNU
resulted in a complete loss of the ONL throughout the entire retina. All images shown in
Figure 8 are taken close to the papilla.
In Figure 9 superior parts (close to injection side) and inferior parts of the retina are illustrated
(Figure 9B, D, F and G) with a corresponding staining for rods and cones in each area (C, E,
G, I). The retinal areas with the more pronounced thickness loss in the ONL after treatment
with 1 and 2 mg/kg BW MNU (9B, D), are more close to the injection side (superior retina),
what is documented by the staining of red/green opsin (green). Red/green opsin is located in
the superior retina of mice. In the areas, where more layers of the photoreceptor somata
(ONL) remained, blue opsin staining (red) is represented, indicating an inferior retinal area.
The stainings in Figure 9 allowed the evaluation of the morphology of the photoreceptors
which remained after application of 1 or 2 mg/kg BW MNU. In surviving rods and cones, we
could observe following signs of degeneration (Figure 9). In Figure 9 B, F, D and H
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antibodies against rhodopsin and recoverin were used. Rhodopsin is presented in rod outer
segments is visualized in blue, while recoverin that is found throughout the entire
photoreceptors is shown in green. Rod outer segments, therefore, appear cyan. They are
shorter than normal in all sections shown, but are longest in regions most remote from the
injection site (Figure 9F, 1 mg/kg BW MNU, inferior part of the retina).
Cones also show features of degeneration. In Figure 9G (1 mg/kg BW MNU, inferior retina)
cones survived best, showing long outer segments stained in red for blue opsin. Closer to the
injection site (Figure 9C, 1 mg/kg BW MNU, superior retina) two abnormal features can be
observed. First, red/green opsin staining (green) is found throughout all cone compartments,
while normally only small amounts of opsin can be observed outside the outer segment.
Second, the outer segments are short and round. Close to the injection side after 2 mg/kg BW
MNU (Figure 9E), cones are completely gone (green staining represents background in inner
retinal cells), and in the referring inferior part of the retina (Figure 9I), outer segments (red)
are extremely shortened. Additionally to the photopic ERGs in Figure 3, in the same figure
immunohistochemically stained sections of intravitreally injected animals are illustrated to
underline the simultaneous death of rods and cones. In the photopic ERG cone responses are
comparably lost as well as rod driven responses (Figure 3).
In animals treated with 1 and 2 mg/kg BW MNU the stainings of HCN1 channels (red) and
recoverin in type 2 bipolar cells (green) do not reveal changes in the inner retinal layers
(Figure 9B, D, F, and H). To determine in detail the effects of MNU on inner retinal cells
additional stainings were performed. The first column of Figure 10 (A, F, K) shows
immunohistochemical stainings in wild type retina: Calbindin 28K (Figure 10A) is strongly
expressed in horizontal cell somata and their dendrites (upper cells, arrow) and weakly in
certain amacrine cells (lower part of the section). Rod bipolar cells (Figure 10F) can be
stained with antibodies against PKCα. Rod bipolar somata are usually elongated, axons are
clearly visible and axon terminals are compact. Certain amacrine cell types and three typical
bands formed by their dendrites in the IPL can be stained with antibodies against calretinin
(Figure 10K).
Upon intraperitoneal application of MNU (Figure 10B, G, L), fine horizontal cell dendrites
were lost as the presynaptic photoreceptors had disappeared, but horizontal cell somata were
mostly unchanged (Figure 10B). Some rod bipolar somata (Figure 10G, arrow) were found
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that were more roundish than usual, but most rod bipolar cells were unaffected. Amacrine
cells appeared unchanged (Figure 10L).
Upon intravitreal injection of 1 mg/kg BW (Figure 10C, H, M), 2 mg/kg BW (Figure 10D, I,
N) or 3 mg/kg BW MNU (Figure 10E, J, O), the following results were obtained. In those
regions of the retina where photoreceptors survived (1 and 2 mg/kg BW MNU), horizontal
cells (Figure 10C, D), rod bipolar cells (Figure 10H, I), and amacrine cells (Figure 10M, N)
were basically unaffected. The situation was different in those retinal regions where all
photoreceptors had disappeared, i.e. throughout the entire retina upon injection of 3 mg/kg
BW MNU (Figure 10E, J, O) and close to the injection site upon injection of 1 or 2 mg/kg
BW MNU (not shown). Here, horizontal cells were missing (Figure 10E), rod bipolar cell
somata became roundish (Figure 10J, arrow), their axons and axon terminal systems were
disorganized and more amacrine cells than usual expressed PKC (Figure 10J, arrowhead). The
calretinin positive bands in the IPL were slightly broader (Figure 10O).
In no case, the intravitreal injection affected the non-injected eye of the animal.
In none of the urine test samples MNU-molecules were found (data not shown).
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5.3.5 DISCUSSION
Genetic animal models of retinal degenerations and dystrophies, such as Retinitis pigmentosa
(RP) revealing selective photoreceptor degeneration, are important to study the underlying
disease mechanisms and also to establish possible treatment approaches. Several rodent
models are well established.3-17
In larger animals which would be more suitable to work on
surgical methods, genetic models are intrinsically expensive and disease progression is
slow.20
However, photoreceptor degeneration may also be induced pharmacologically and
may have the advantage that it can be induced in any wanted animal species.24-30
As one
possible pharmacological agent N-methyl-N-nitrosourea (MNU) was reported to lead to a
blindness causing selective photoreceptor cell apoptosis via oxidative stress when
administered intraperitoneally or intravenously.26-30
The resulting histological picture is well
analyzed in detail by different researchers26-30
and very similar to that presented in human
RP.26
However, retinal degeneration induced by systemic application of MNU not only leads
to bilateral disease, but also to a reduction of the general health status of the experimental
animals. Beside short-term effects caused by the toxicity of the substance, the induction of
tumors was described as long-term effect in rabbits and rats after systemic treatment with
MNU due to its DNA alkylating mode of action.31-33
Therefore, we proved to induce
photoreceptor degeneration in only one eye of mice by local administration of MNU and
avoiding systemic side effects.
Puthussery and Fletcher36
and Notomi et al.37
demonstrated, that intravitreally injected
adenosine triphosphate (ATP) induces photoreceptor degeneration by apoptosis, too.
However, ATP also leads to TUNEL-positive apoptotic events in the ganglion cell layer after
intravitreal application. 36
ATP was described to kill ganglion cells by excessive calcium
influx by stimulation of the P2RX7 receptor.37
In contrast to the effects of ATP on ganglion
cells, Tsuruma et al. reported, that MNU leads to radical production only in retinal cell
cultures of murine 661W photoreceptor-derived cells, but not in RGC-5, a mouse ganglion
cell line, and hence induces cell death specifically in retinal photoreceptor cells.28
The reason
why MNU toxicity is specific for photoreceptors and maybe also for horizontal cells, remains
unclear. MNU given intravitreally needs to penetrate through the retina or needs to be
transported through it to affect the outer retina. It is likely that photoreceptors are particularly
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susceptible due to its extreme high metabolic turnover in the photoreceptor outer segment /
RPE unit. Further experiments are needed to clarify the mode of MNU action.
Beside the intravitreal injections of MNU, the systemic application given as an intraperitoneal
injection served as our positive control. In these animals the well-being of the animals was
reduced for some hours after administration. They showed a reduction of weight with the
highest loss two days after injection. However, all animals survived. Regarding the outcome
of systemic MNU application, a rapid and nearly complete denudation of the ONL was
observed. Cells of the inner retina like horizontal, bipolar or amacrine cells were only slightly
affected. As the presynaptic photoreceptors were lost, horizontal cells and rod bipolar cells
lost most of their dendrites similar to findings described earlier.30
Because of the small vitreous space of the mouse eye and the proportionally large lens,
intravitreal injections in mice are difficult. To optimize the injection technique, we used a 3D
micromanipulator and nanoliter injection system. Injections of PBS, fluorescein, and
DMSO+PBS mixture, were used as negative controls, and could point out that the injection
procedure was safe. Examination of control animals succeeded without any changes in
functionality in vivo and showed no histological abnormalities. In contrast to the systemic
application, the well-being of the animals was only minimally affected upon intravitreal
injections of MNU, comparable to the welfare after an anesthetic intervention.
One week after intravitreal injection of 1, 2 or 3 mg/kg BW MNU, the OCT showed similar
changes as observed one week after intraperitoneal application. However, two weeks after 1
and 2 mg/kg BW MNU, not all somata of photoreceptors in the outer nuclear layer had
disappeared completely. The degree of photoreceptor degeneration depended on the distance
to the injection site. While at the point of injection in the superior retina no row of somata in
the ONL was left, five to ten rows of photoreceptor somata were detectable in the inferior
retina (Figure 9). Detailed analysis using photoreceptor markers (Figure 9) indicated that the
surviving photoreceptor population comprised both rods and cones, but that outer segments
were shorter than usual. Standard full-field ERG waves in animals injected with 1 and 2
mg/kg BW MNU under scotopic and photopic conditions were either diminished (2 animals
in each group) or even extinguished (one animal in each group, n=3; Figure 5D, E).
Upon injection of 3 mg/kg BW MNU, the photoreceptors (ONL) had disappeared throughout
the entire eye, while the inner retinal layers remained. Inner retinal layers of intravitreally
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injected animals (1, 2, or 3 mg/kg BW) showed no significant thickness differences to
uninjected eyes. The histological picture was reminiscent of the results obtained after
systemic treatment with MNU and results obtained with the genetic model (rd10) at postnatal
day 50 (Figure 8). Immunohistochemistry performed two weeks after injection revealed no
surviving photoreceptor population. In agreement with this finding, no responses could be
observed in the scotopic and photopic ERG two weeks after injection of MNU.
However, in those regions of intravitreally injected eyes where photoreceptors had vanished
entirely, also the horizontal and rod bipolar cells seemed to be affected. Two weeks after
injection, we noticed a complete loss of horizontal cell somata and processes in the retinal
regions where all photoreceptors had disappeared. While after systemic MNU treatment a loss
of horizontal cell processes in a study over three month has been described,30
a complete cell
loss has not been observed. In comparison, in rd10 mouse at the age of 9 months around 29%
of horizontal cell somata were lost.7
The rod bipolar cells seemed to be disorganized after an intravitreal injection of 3 mg/kg BW
MNU. For rd10 mice up to 45 days of age, no changes in the morphology, size, and
complexity of the axonal terminal systems of rod bipolar cells were found, but between 1.5
and 3.5 months of age the number or rod bipolar cells decreased by 20%.7
There are two ways how the effect of MNU on horizontal and rod bipolar cells can be
explained. First, at higher concentrations of MNU as they are reached at the site of injections
when using 1 or 2 mg/kg BW MNU or throughout the eye when using 3 mg/kg BW, MNU
might become toxic to horizontal cells and rod bipolar cells. Tsuruma et al. reported about a
decrease in thickness of the INL after intraperitoneal treatment with 75 mg/kg BW instead of
60 mg/kg BW, what may be based on a destruction of horizontal and rod bipolar cells, too.28
Second, the rapid loss of all photoreceptors at a retinal location within two weeks might have
a more pronounced effect on the survival of postsynaptic cells than the degeneration
occurring at a slower pace in the genetic model. After intravitreal injection of ATP also
degeneration of photoreceptors in variable degree is described, ranging from the destruction
of photoreceptor segments to a complete loss of retinal pigment epithelium (RPE) and in
regions of rapid and strong destruction also to highly disturbed synapses of the outer
plexiform layer.36
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In Summary, our results clearly indicate that MNU given intravitreally is a per se working
model for the induction of photoreceptor degeneration to simulate disease processes as seen in
RP or similar conditions. The mouse model has the shortcomings of a small eye. It was
difficult to achieve a homogeneous degeneration of photoreceptors throughout the retina upon
one intravitreal application. Photoreceptor loss was more pronounced close to the injection
site. This problem may not be encountered in larger eyes, such as in the rabbit, as injections of
MNU (1 - 2 mg/kg BW) at multiple positions in the large vitreal volume might result in a
more homogeneous elimination of photoreceptors. Based on these results, we would suggest
the use of MNU injections into the vitreous of mice or larger animals as a pharmaceutical
model to specifically induce degeneration of photoreceptors while maintaining an
intraindividual control eye.
5.3.6 ACKNOWLEDGEMENT
The authors thank Prof. Dr. R. Tolba and the institute of laboratory animal science of the
University Hospital RWTH Aachen for the great opportunity, for the access to one of their
laboratories, and the possibility to perform our experiments under special safety guards. We
further thank Christoph Aretzweiler for the excellent support in preparation and staining of
the eyes.
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36. Puthussery T, Fletcher E. Extracellular ATP Induces Retinal Photoreceptor Apoptosis
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5.3.8 FIGURES
Figure 1. Side effects of MNU application. Loss of body weight as an indicator of general
well-being after application of MNU given intraperitoneally (i.p.) and intravitreally (IVI) in
comparison to untreated animals and control animals in the same changed husbandry
conditions. ***p<0.003 vs. i.p. MNU application, one-way ANOVA with Bonferroni’s post
hoc test. (MNU i.p.: n=3; Control DMSO/PBS i.p.: n=3; untreated: n=2; intravitreal (IVI)
each concentration: n=3).
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Figure 2. Scotopic ERG after systemic MNU application. (A) - (C) Examples of scotopic
full-field ERG recordings from mice before (black line) and one week after (grey line)
systemic intraperitoneal application of PBS (A), DMSO + PBS (B), and 60 mg/kg BW MNU
(C) under the same husbandry conditions. Scotopic 3 cds/m2, 0.067 Hz. In (C) an extinction
of the ERG curve one week after application could be observed. (D), (E) Amplitudes of a-
wave (D) and b-wave (E) before and two weeks after i.p. MNU application (n=3) at a light
intensity of 3 cds/m2 in the scotopic ERG. Mean + SD. DMSO + PBS (n=3) and PBS (n=3)
injections served as controls (both eyes per n).
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RESULTS
Figure 3. Photopic ERG after MNU application and confocal images of
immunohistochemically stained vertical retinal sections. The a-wave and b-wave of
animals in the photopic ERG (3 cds/m2; 0,625 Hz) was diminished one week after
intraperitoneal (60 mg/kg BW, (B)) and intravitreal (C) - (E) treatment with MNU. The
contralateral eye (CL3) of the animal treated with 3 mg/kg BW revealed a normal photopic
ERG (E). The immunohistochemically stained sections were used to demonstrate the
simultaneous degeneration of rods and cones after intraperitoneal and intravitreal injection of
MNU in different concentrations (F). Stainings against red/green opsin (green, cone outer
segments in the superior retina), blue opsin (red, cone outer segments in the inferior retina)
and PEA (cones, blue) were used. The involvement of cones in the degenerative process is
seen in the photopic ERG and in histology by missing outer segments and an unusual
morphology.
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RESULTS
Figure 4. Thickness evaluation in the OCT. Retinal images and thickness obtained by sd-
OCT measurements. Left panel. All images are taken in the height of the optic disk and
displayed with pigment epithelium and photoreceptors to the top. Examples of sd-OCT
recordings before (left), one week after (middle), and two weeks after (right) intraperitoneal
PBS (A), DMSO + PBS (B), or MNU (C) application. Red arrows (B) indicate examples for
the thickness measurement in one retina. (C) MNU 60 mg/kg BW i.p.; the red bars mark the
outer nuclear layer (ONL), which is significantly thinner one and two weeks after injection;
enlargement of the area next to the optic disk shows the destruction of the ONL and the debris
in the area of the ONL. (D) Retinal thickness measurements based on sd-OCT data before,
one week, and two weeks after i.p. injection of PBS, DMSO + PBS and MNU (n=3). Values
represent mean ± SD (for 60 mg/kg BW one and two weeks after application: ****p<0.0001
vs. the controls, one-way ANOVA with Bonferroni’s post hoc test).
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RESULTS
Figure 5. Confocal images of immunolabelled vertical sections obtained from retinae
prepared two weeks after systemic application. The white bars in (A) and (B) indicate the
thickness of the outer nuclear layer. Red: glutamine synthetase, Müller cells spanning nearly
the entire thickness of the retina; blue: PNA, cone outer segments and endfeet; green: GFAP.
(A) untreated (B) DMSO i.p. (C) MNU i.p. 60 mg/kg BW (note that the ONL has nearly
vanished) (D) Glial reaction is indicated by GFAP expression in Müller cell processes (same
section as in (C)). (E) Rd10 mouse retina at the age of 50 days displaying total loss of
photoreceptors. OR: outer part of the retina; IR: inner part of the retina; OS: outer segments;
IS: inner segments; ONL: outer nuclear layer; OPL: outer plexiform layer; INL: inner nuclear
layer; IPL: inner plexiform layer; GCL: ganglion cell layer; NFL: nerve fiber layer.
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RESULTS
Figure 6. ERG after intravitreal MNU application. Examples of full-field ERGs recorded
before and one week after intravitreal (IVI) injection. (A) PBS (B) DMSO + PBS (C) 0.5
mg/kg BW MNU (D) 1 mg/kg BW MNU (E) 2 mg/kg BW MNU (F) 3 mg/kg BW MNU. (D)
– (F) A reduction of the amplitude of the ERG or a complete extinction is seen after
intravitreal injection of 1, 2, and 3 mg/kg BW MNU. In the right corner of (F) the ERG of the
contralateral, untreated right eye before and one week after treatment of the left eye
intravitreally with 3 mg/kg BW is exemplified illustrated to demonstrate that the treatment of
the left eye by intravitreal injection had no influence on photoreceptor function of the right
one. The Amplitudes of a-waves (G) and b-waves (H) are illustrated before and two weeks
after MNU application in different concentrations at a light intensity of 3 cds/m2 in the
scotopic ERG (n=3 for each concentration). In each diagram the bar designated with CL3
illustrates the values of the contralateral, right eyes of animals treated intravitreally with 3
mg/kg BW (left eye). There is no effect of intravitreal injection on the amplitude of a- and b-
wave in the ERG of the contralateral eye. Values represent mean ± SD.
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RESULTS
Figure 7. Retinal thickness evaluation using OCT upon intravitreal injection of MNU.
Retinal images and thickness obtained by sd-OCT measurements. Left panel. All images were
taken at the height of the papilla and displayed with pigment epithelium and photoreceptors to
the top. Examples of sd-OCT recordings before (left), one week after (middle), and two
weeks (right) after injection of PBS (A), DMSO + PBS (B), MNU 0.5 mg/kg BW (C), MNU
1 mg/kg BW (D), MNU 2 mg/kg BW (E), and MNU 3 mg/kg BW (F). (D) - (F) the red bars
mark the outer nuclear layer, which is significantly thinner one week after injection; the
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RESULTS
destruction of the ONL one week after application is reminiscent of the effects observed after
intraperitoneal application (compare to Figure 3C). (G) Retinal thickness measurements in the
height of the papilla based on sd-OCT data before, and two weeks after IVI injection of PBS,
DMSO/PBS and MNU. As already seen in Figure 5 CL3 illustrates the averaged retinal
thickness in the OCT of the contralateral, untreated eyes of animals injected intravitreally in
the left eye with 3 mg/kg BW MNU (highest concentration). Values represent mean ± SD (for
3 mg/kg BW MNU two weeks after application: ****p<0.0001 vs. the controls and the MNU-
treated animals before injection, one-way ANOVA with Bonferroni’s post hoc test). In (H)
the contralateral, corresponding untreated eye of the animal treated on the left eye
intravitreally with 3 mg/kg (F) two weeks after injection is exemplified illustrated. No
thickness differences or morphological changes in contrast to the eyes of control animals
could be observed (H).
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RESULTS
Figure 8. Confocal images of immunohistochemically stained vertical sections through
the retina two weeks after injection of MNU into the vitreous. The thickness of the ONL is
indicated by white bars. Stainings and abbreviations as in Figure 4. (A) Control injection of
PBS (B) Control injections of DMSO + PBS (C) 1 mg/kg BW MNU (D) 2 mg/kg BW MNU
(E) 3 mg/kg BW MNU. (C) - (E) Thickness of the ONL is progressively reduced. No
photoreceptors are left after injection of 3 mg/kg BW MNU (E). The bar diagrams in (B) and
(C) show the retinal thickness after different forms of treatment and in comparison to the
genetic model of the rd10 mouse at P50 (n=3). While in (B) the entire retinal thickness is
evaluated, in (C) only the thickness of the inner retinal layers is demonstrated, to evaluate the
possible effects of intravitreal MNU injection on these layers. (B) Retinal thickness decreases
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RESULTS
with rising intravitreal concentrations of MNU with the highest decrease after treatment with
3 mg/kg BW. The contralateral, untreated eye (CL3) is not affected regarding thickness. At
this concentration retinal thickness is comparable to the thickness after intraperitoneal (i.p.)
treatment with 60 mg/kg BW MNU or to the genetic model of the rd10 mouse. In (C) the
thickness measurements of the inner retinal layers demonstrate that intravitreal injections of
MNU have no influence on the thickness of these layers. The contralateral eyes of animals
treated by 3 mg/kg BW were not affected regarding thickness.
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RESULTS
Figure 9. Confocal images of immunohistochemically stained vertical sections through
the retina two weeks after injection of MNU into the vitreous. (A) Cartoon depicts the
injection site in the superior part of the eye. (B), (D), (F), (H): Stainings against recoverin
(green, entire photoreceptor), rhodopsin (blue, rod outer segment), and ion channel HCN1
(red, photoreceptor somata and inner segments, as well as processes in the inner retina). (C),
(E), (G), (I): Stainings against red/green opsin (green, cone outer segments in the superior
retina (C), (E)), blue opsin (red, cone outer segments in the inferior retina (G), (I)), PEA
(cones, blue). OR: outer half of the retina; IR: inner half of the retina. Photoreceptor
degeneration is more pronounced at the injection side. However, both, rods and cones, show
features of degeneration.
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RESULTS
Figure 10. Confocal images of immunohistochemically stained vertical sections through
the retina two weeks after injection of MNU. (A), (F), (K) wild type control. (B), (G), (L)
60 mg/kg BW MNU intraperitoneal. (C), (H), (M) 1 mg/kg BW MNU intravitreal. (D), (I),
(N) 2 mg/kg BW MNU intravitreal. (E), (J), (O) 3 mg/kg BW MNU intravitreal. Upper row:
antibodies against calbindin 28K yield strong label in horizontal cells (arrow) and weaker
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RESULTS
staining in amacrine cells. Middle row: PKCα is found in rod bipolar cells. Lower row:
calretinin is found in certain populations of amacrine cells. OPL: outer plexiform layer, IPL:
inner plexiform layer.
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GENERAL DISCUSSION
6 GENERAL DISCUSSION
It is important to study animal models with selective photoreceptor degeneration, which
resemble the human inherited disease Retinitis pigmentosa (RP); making reference to the
progress of degeneration, the different cell reactions and the work on possible treatment
strategies like surgical approaches. In rodents, many genetic models are available indicating
these conditions, e.g., the rd10 mouse. However, equivalent genetic models in larger animals
are intrinsically expensive and the duration until photoreceptor degeneration is completed
(TSO et al. 1997) is comparably long. Therefore, it would be very useful to have the
possibility to induce comparable outer retinal effects by administration of pharmaceuticals.
Photoreceptor degeneration could be induced in any wanted animal species over a faster
period of time. The general welfare of the experimental animals should not be affected by the
way of application. Iodoacetic acid (IAA) and N-methyl-N-nitrosourea (MNU) were used,
which are both known to lead to an outer retinal degeneration after systemic application
(GRAYMORE a. TANSLEY 1959, HERROLD 1967). However, after systemic treatment
toxic systemic side effects do occur. To this end, the - in this context “new” - technique of
intravitreal injections was introduced. The local administration of this injection technique was
hypothesized to induce photoreceptor degeneration similar to the effects obtained with
systemic application, but without affecting the contralateral eye as well as the general health
status of the animal.
Experiments were performed on wild type mice, in order to compare retinal effects to the
genetic model of the rd10 mouse, an important animal model revealing the requested and
offered conditions of retinal degeneration caused by a defect in the phosphodiesterase gene
(CHANG et al. 2007).
In the first study, the disease progression in the genetic model of the rd10 mouse was
characterised in comparison to the wild type mouse, which was examined as control.
Different in vivo and in vitro methods were used, and we demonstrated that the combination
of in vivo measurements by non-invasive tools like ERG, sd-OCT and fluorescein
angiography allow the precise description of the status of the retina during the degenerative
process. Our results were comparable to the findings of other researchers, e.g., the values of
latency (data not shown) and amplitudes of a-and b-wave in the ERG (CHANG et al. 2007,
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GENERAL DISCUSSION
SUGIMOTO et al 1997). In the fluorescein angiography, we could illustrate that there are no
significant differences between wild type and rd10 mice, although attenuated retinal vessels
were expected (BERSON 1993). The clinically used OCT was also proofed to be beneficial,
as we could clearly demonstrate that the thickness values obtained by OCT were comparable
to those obtained after histological analysis. However, retinal separation found by PENNESI
et al. (2012) at the age of 64 days, were only found in OCT measurements in vivo locally
determined in two animals (9 and 24 weeks of age). The differences in the results could be
probably explained by the use of different types of OCT devices (PENNESI et al. 2012).
However, in the histology the image of retinal separation was often observed, possibly
induced during the histological work-up. This may indicate that, although separation is not
always manifest in vivo, it can be more easily induced in the rd10 mouse in the histological
work-up, in contrast to the wild type (Study I).
Two protocols of histological work-up were chosen to enlarge the amount of data obtained.
HE-staining required sectioning of the whole eye into single slices. The advantage of this
method is the possibility to evaluate additional parts or tissues of the eye rather than the
neural retina, e.g., the lens. However, the disadvantage of this method is the frequent
observation of artifacts, like separation of the retina as seen in the rd10 mouse. In the
immunohistochemistry, only the retina was separated and cut into single slices. This method
allowed for the specific characterization of different cell classes of the retina, which can be
visualized in more detail using cell-type specific antibodies. The resulting data confirmed the
selective photoreceptor degeneration followed by retinal remodeling (CHANG et al. 2007,
PHILIPPS et al. 2010) (Study I).
In summary, the in vivo methods are very suitable to describe retinal changes and reduce the
number of animals sacrificed for histological techniques. The data obtained by these
measurements are in good accordance with the in vitro data (Study I).
After characterization of the rd10 mouse model, wild type mice were injected with two
different pharmaceuticals (iodoacetic acid (IAA; Study II) or N-methyl-N-nitrosourea (MNU,
Study III)) by different ways of application (systemic or intravitreal) in order to induce a
comparable outer retinal degeneration. Many publications, mainly in other species than mice,
demonstrate that, when both substances are systemically injected it leads to blindness causing
photoreceptor degeneration (LIANG et al. 2008, TSUBURA et al. 2010). The histologically
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GENERAL DISCUSSION
observed effect of both substances on photoreceptor survival is very similar to that presented
in the human disease of RP, when photoreceptors are completely degenerated (LIANG et al.
2008, TSUBURA et al. 2010). The systemic application of IAA, either given as an
intraperitoneal or as an intravenous injection, and the intraperitoneal application of MNU
consecutively served as positive controls in our experiments. However, both substances did
not only affect the retina after systemic treatment, they also led to a high reduction of the
welfare (Study II).
After systemic application of IAA (Study II), the welfare of the animals was sustainably
reduced for a couple of days, especially after intraperitoneal treatment. After intravenous
treatment, the highest level of mortality was observed. While the mortality rate after
intravenous IAA injections was reported to be 50% in rats (GRAYMORE et al. 1959) and
20% in rabbits (ORZALESI et al. 1970), the observed mortality in mice was 60%. IAA was
used and published in mice only once by SUGIMOTO et al. (1997), and the concentrations
(approximately 60 mg/kg BW) used in our experiments were chosen according to this
publication. We divided the 60 mg/kg BW into two dosages given within 3.5 hours
(GRAYMORE et al. 1959). This way of application did not lower the mortality, but IAA was
found to be more effective on the photoreceptor layer. The outer nuclear layer (ONL) was
reduced to 3-5 layers of somata. This effect was probably due to the maintaining of a critical
concentration for a longer and more sufficient period of time (GRAYMORE et al. 1959).
After systemic intraperitoneal application of IAA, no effects on the retina were seen, which
might be due to a minimally longer duration until passing the vasculature of the eye, in
contrast to intravenous application or because of a first pass effect in the liver (Study II).
In comparison to the systemic injection of IAA, animals receiving intraperitoneal application
of MNU (Study III) also showed a reduction of welfare for some hours after administration,
with the highest weight loss two days after injection. Nevertheless, all animals survived. The
ONL was nearly completely destroyed, comparable to the histological images of rd10 mice at
the age of P50. Inner retinal layers did not decrease in thickness. Horizontal cells and rod
bipolar cells were only slightly affected; this could be evaluated by the loss of most of their
dendrites; similar to findings described earlier (NAGAR et al. 2009) (Study III).
After systemic treatment with both of the substances, as well as our positive controls and
observation of their effects on the retinal structures and on the general welfare of the
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GENERAL DISCUSSION
experimental animals, we tried to induce a comparable retinal degeneration by intravitreal
injections of first IAA (Study II) and second MNU (Study III). Because of the small eye, the
proportionally large lens and the small vitreous space, the injection technique was difficult to
perform without affecting other structures of the eye. Therefore, the injections were
conducted using a 3D micromanipulator and a special nanoliter injection system. A volume of
2 µL was injected in the left eye of wild type mice. Previously, the method had been tested by
many test-injections (PBS, DMSO and PBS, fluorescein diluted with PBS), which were
carried out as negative controls and confirmed that the injection procedure was safe and
reproducible (Study II and Study III).
In contrast to the systemic treatment, which was conducted without anaesthesia, only a slight
weight loss was noticed after intravitreal injection, similar to that observed normally after an
anaesthetic intervention (Study II and Study III).
Intravitreally injected IAA (Study II) led to many side effects: dense cataracts, which
appeared five to seven days after injection, inflammation of different cell types in the eye
(e.g., iridocyclitis) and an exudation of blood cells into the aqueous humour. Because of the
later onset, the cataractous changes were not supposed to be the result of a lens injury during
the injection. In addition, they were not observed in control animals receiving injections other
than IAA. Due to the frequent cataract formation, retinal changes could not be evaluated in
the sd-OCT; the observed decrease of the waves in the ERG had to also be interpreted
carefully in consideration of the cataracts. After an experimental duration of two weeks, the
effects on the retina could be evaluated in immunohistochemistry. In contrast to the expected
effect of outer retinal degeneration, no selective photoreceptor degeneration was observed
upon intravitreal injection of IAA, but rather an overall thinning of the retina with a tendency
towards a more pronounced reduction of the inner retinal layers (Study II).
In summary, intravitreally injected IAA does not lead to a selective photoreceptor
degeneration. The injection led to cataract formation, inflammation and a thinning of the
retina, mainly based on a thinning of the inner retinal layers. In summary, the intravitreal
injection of IAA is no useful method to induce unilateral photoreceptor degeneration (Study
II).
One week after injection, animals treated intravitreally with MNU (1, 2 or 3 mg/kg BW
MNU; Study III) showed, correlated to the concentration, similar changes and cell debris in
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GENERAL DISCUSSION
the outer nuclear layer in the OCT as animals which were examined one week after
intraperitoneal treatment. In the groups of animals treated with 1 and 2 mg/kg BW MNU the
values of a- and b-wave in the ERG were either diminished or even extinguished. Histology
revealed that the ONL had not completely disappeared. Additionally, we pointed out that the
degree of photoreceptor degeneration depended on the distance to the injection site. Retinal
areas of treated eyes, which were not near to the injection side and were thus less affected,
comprised both rods and cones. In the group of animals treated intravitreally with 3 mg/kg
BW we observed a degeneration of all photoreceptors and somata throughout the entire eye in
the OCT. No response could be observed in the ERG. In the immunohistochemistry, we
verified the OCT findings of no surviving photoreceptors (Study III). The thickness of inner
retinal layers remained unaffected. This described histological picture was similar to those
obtained after systemic MNU treatment or to those retinal histological pictures of the rd10
mouse at P50. However, horizontal cells and rod bipolar cells were obviously more affected
than after intraperitoneal application. In those regions, where photoreceptors were completely
lost, horizontal cell somata and processes had also disappeared (Study III). In contrast, three
months after systemic treatment, only a loss of dendritic processes of horizontal cells is
described (NAGAR et al. 2009). In the genetic model of the rd10 mouse at the age of 9
months, a loss of horizontal cell somata of approximately 29% was described (GARGINI et
al. 2007).
Apart from horizontal cells, the rod bipolar cells also seemed to be affected and to be
disorganized after an intravitreal injection of 3 mg/kg BW MNU (Study III). While in rd10
mice up to 45 days of age, no changes in the morphology of these cells were found, the
number of rod bipolar cells decreased by 20% between 1.5 and 3.5 months of age (GARGINI
et al. 2007).
One explanation, as to why intravitreally injected MNU affected the horizontal and rod
bipolar cells would be that MNU, after the death of photoreceptors, might become toxic to
horizontal cells and rod bipolar cells in higher concentrations (Study III). Secondly, the more
rapid loss of photoreceptors after intravitreal injection, in contrast to the genetic model or the
systemic treatment might have a more pronounced effect on the survival of postsynaptic cells
(Study III).
109
GENERAL DISCUSSION
However, in all intravitreally treated animals the thickness of the inner retinal layers did not
significantly decrease. And most importantly, ganglion cells morphologically seemed not to
be affected after intravitreal MNU treatment.
In summary, intravitreal application of MNU led to unilateral selective photoreceptor
degeneration comparable to a complete loss of photoreceptors after systemic treatment or in
the later stages of the genetic model of the rd10 mouse. The well-being of the animal was not
more affected than after a normal anaesthetic intervention. Additionally, the contralateral eye
was not affected. We suggested the use of MNU injections into the vitreous of animals to
induce unilateral photoreceptor degeneration, while maintaining an intraindividual control eye
(Study III).
110
SUMMARY
7 SUMMARY
Sarah Rösch
Intravitreal injections in mice as a technique to induce unilateral photoreceptor
degeneration in comparison to the genetic model of the rd10 mouse
Inherited blindness causing diseases in humans are frequent and often issue of new
therapeutic strategies in research. To test these strategies, but also for a better understanding
of the pathological mechanisms, animal models that mimic pathohistological changes in the
human retina are indispensable.
For Retinitis pigmentosa, a disease with selective photoreceptor degeneration and remaining
inner retinal layers, genetic animal models in rodents allow the characterisation of the process
of degeneration. However, for the testing of visual prostheses to restore visual function or
related surgical methods, the eyes of these genetic animal models are too small. For these
experiments, animal species such as the rabbit, the cat or the pig are required, but such genetic
models for larger animals are intrinsically expensive and have a slow disease progression.
However, photoreceptor degeneration can also be induced by systemic treatment with
iodoacetic acid (IAA) and N-methyl-N-nitrosourea (MNU), both leading to substantial side
effects. To induce the same retinal changes as seen in the genetic model without affecting the
welfare of the animals, experiments were performed using the - in this context “new” -
technique of intravitreal injections. The mouse was used as animal species in these
experiments in order to compare the results to the available genetic model of the retinally
degenerated rd10 mouse.
Firstly, the process of photoreceptor degeneration and its characteristics in the genetic model
of the rd10 mouse was observed and evaluated by clinical tools (OCT, ERG, FA, histology)
in contrast to the healthy wild type mouse. Secondly, effects of systemic and intravitreal
treatment using either IAA or MNU were compared. Systemic treatment with IAA or MNU
led to outer retinal degeneration; however, the systemic application of either substance
111
SUMMARY
resulted in a high reduction of animal welfare or even in mortality (in the case of IAA) by
systemic side effects.
Upon the third approach, utilizing unilateral intravitreal injection of MNU or IAA under
anaesthesia, no reduction of animal welfare was observed and no effects were observed in the
uninjected eye. The intravitreal injection of IAA led to cataract formation and inflammatory
reactions of the whole eye. Retinal thickness was reduced mainly due to thinning of the inner
retinal layers, rather than the photoreceptor layer. Intravitreal application of MNU led to
photoreceptor degeneration similar to the effects seen after systemic treatment and resembling
the degeneration in the genetic model.
To conclude, the intravitreal injection of MNU is a suitable model to induce outer retinal
degeneration, without affecting the general health status of the animal and with preservation
of an intraindividual control eye avoiding sudden complete blindness.
112
ZUSAMMENFASSUNG
8 ZUSAMMENFASSUNG
Sarah Rösch
Technik der intravitrealen Injektion bei der Maus zur Induktion einer einseitigen
Photorezeptordegeneration im Vergleich zum genetischen Modell der rd10 Maus
Genetisch bedingte, unheilbare Netzhauterkrankungen wie Retinitis Pigmentosa (RP) führen
zu einer Degeneration der Photorezeptoren und somit final zur Erblindung. Aufgrund ihres
häufigen Vorkommens sind sie zunehmend Thema neuer Forschungsansätze auf der Suche
nach effektiven Therapiemöglichkeiten. Um sowohl diese neuen therapeutischen Ansätze
testen zu können, aber auch um die pathohistologischen Vorgänge besser verstehen zu
können, sind Tiermodelle unverzichtbar, die vergleichbare Veränderungen aufweisen.
Genetisch veränderte Tiermodelle in Nagern, die die Verhältnisse wie bei RP widerspiegeln,
sind verfügbar und erlauben eine Charakterisierung der selektiven Photorezeptordegeneration
und die Evaluierung der Auswirkungen auf die verbleibenden, intakten inneren Zellen der
Netzhaut. Für das Austesten von Sehprothesen, die eine visuelle Funktion ansatzweise
wiederherstellen sollen, oder aber für vergleichbare chirurgische therapeutische Ansätze, sind
die Augen dieser Tierspezies zu klein. Man würde hierzu Tierarten benötigen, wie z.B. das
Kaninchen, die Katze oder das Schwein, die größere Augen besitzen. Genetisch veränderte
Tiermodelle dieser Tierarten mit einer gleichartigen Erblindung sind jedoch sehr schwer zu
beziehen, sehr teuer und zeigen eine nur sehr langsame Photorezeptordegeneration.
Allerdings kann eine Photorezeptordegeneration auch durch eine systemische Verabreichung
von pharmakologischen Wirkstoffen erreicht werden, wie z.B. mit Jodessigsäure (Iodoacetic
Acid, IAA) oder n-Methyl-n-Nitrosoharnstoff (N-methyl-N-nitrosourea, MNU). Die
systemische Gabe beider Substanzen führt jedoch zu starken Beeinträchtigungen der
Tiergesundheit und des Wohlbefindens. Um die gleichen netzhautspezifischen
Veränderungen zu induzieren, ohne jedoch das Allgemeinwohl des Tieres zu beeinflussen,
wurde in den dargestellten Experimenten IAA und MNU mit der in diesem Zusammenhang
„neuen“ Methode der intravitrealen Injektion verabreicht. Als Tierspezies wurde die Maus
113
ZUSAMMENFASSUNG
gewählt, da hier das genetische Modell der rd10 Maus mit der erwünschten Form der
Photorezeptordegeneration als Vergleich herangezogen werden konnte.
Zuerst wurde die Netzhautdegeneration in der rd10 Maus mittels ERG, OCT und die
Histologie (Immunhistochemie und HE-Färbungen) im Vergleich zu der gesunden Wildtyp
Maus beobachtet und dargestellt. Anschließend wurden die Effekte durch die systemische und
intravitreale Applikation beider Substanzen analysiert. Zwar führte die systemische
Applikation als Positiv-Kontrolle wie erwartet zu einer äußeren Netzhautdegeneration, war
jedoch mit einer starken Beeinflussung des Tierwohls oder sogar Mortalität (im Fall von IAA)
verbunden.
Die unter Anästhesie durchgeführte unilaterale intravitreale Injektion beider Substanzen
führte zu keiner Reduktion des Tierwohls und zu keinerlei Effekten im zweiten,
unbehandelten Auge. Intravitreal verabreichtes IAA führte in erster Linie zu einer Katarakt
und zu Entzündungsreaktionen im Auge. Zwar war nach der Injektion die Netzhautdicke
reduziert, dies basierte jedoch vor allem auf einer Reduktion der inneren Netzhautschichten.
Die intravitreale Injektion von MNU führte zu einer Photorezeptordegeneration vergleichbar
mit der systemischen Applikation oder dem genetischen Modell.
Damit lässt sich schlussfolgern, dass die intravitreale Injektion von MNU ein gutes und
notwendiges Modell der induzierbaren Photorezeptordegeneration darstellt, in dem das
allgemeine Wohl der Versuchstiere nicht negativ beeinflusst und das kontralaterale Auge
erhalten wird und somit als intraindividuelles Kontrollauge dienen kann. Eine plötzliche,
komplette Erblindung nach Applikation wird somit vermieden.
114
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ACKNOWLEDGEMENTS
10 ACKNOWLEDGEMENTS
I would like to thank my supervisors Prof. Dr. Christiane Pfarrer and Prof. Dr. Peter Walter
for the great opportunity to work on this topic and project, and for the possibility to learn so
much about urgent themes in the complex “research”. I express my gratitude to Prof. Dr. Peter
Walter for giving me an idea about how important the connection between “research and
results” is for sufferers of different diseases.
I would like to thank the second referee PD PhD Veronika Stein for reviewing this thesis.
I would also like to thank very much the people working at the department of ophthalmology
in Aachen for their support and the very pleasant working atmosphere.
Furthermore, I would like to thank Prof. Dr. Frank Müller very much for the great support and
inspiring discussions about the project and concerning questions, and for the possibility to
prepare the immunohistochemistry in his laboratory and the use of his equipment. Also I
would like to thank Christoph Aretzweiler very much, working at the same institute for his
support in the preparation of the retinae and in immunohistological stainings of the eyes and
the time he took to explain working protocols. I learned very much.
I would especially like to thank Prof. Dr. R. Tolba and Dr. K. Scherer of the Institute of
Laboratory Animal Science in Aachen, because without their cooperativeness, their support
and their encouragement, the experiments, especially with the substance MNU, would not
have taken place. I really appreciated the opportunity to perform the experiments in an
isolated laboratory of the Institute of Laboratory Animal Science. Moreover, I would like to
thank Dr. K. Scherer very much for the enlightening talks about different themes of the
laboratory animal science.
Last but not least, I would like to thank my parents and most especially my dog Toto, for
showing an inexhaustible amount of understanding. Also, I want to thank my friends and
former working colleagues in Jülich, Aachen, Mönchengladbach and Hannover (“D”) for their
support, encouragement, helping words and/ or just for being there…
Without you, this academic journey would have been much more difficult…