Age-related macular degeneration (AMD) is the leading cause of blindness worldwide, affecting an estimated 50 million individuals aged over 65 years. Environmental and genetic risk-factors contribute to the development of AMD. An AMD-risk locus on chromosome 10q26 spans two genes, ARMS2 and HTRA1, and controversy exists as to which variants are responsible for increased risk of disease. Recent work suggests that HTRA1 expression levels are significantly increased in carriers of the risk haplotype associated with AMD. However, relatively little is known about the interactions, substrate specificity and roles in disease played by this secreted serine protease. This thesis aims to elucidate the potential role played by HTRA1 in AMD pathogenesis. A combination of tandem affinity purification (TAP) and yeast two-hybrid techniques was used to identify interacting partners of HTRA1. A number of proteins, with diverse roles in the alternative complement pathway, cell signaling, cell-matrix interactions, inflammation, angiogenesis and fibrosis, were identified. These are attractive candidates for further study as such processes are disturbed in AMD, implicating HTRA1 and its binding partners in disease development. One interacting partner, Complement Factor D (CFD), is a key activator in the alternative complement pathway. CFD, a 24 kDa serine protease, is expressed as an inactive zymogen, from which a signal peptide and activation peptide are cleaved before release of the mature, active protein into the circulation. In vitro studies show that CFD interacts with, and can be a substrate for, HTRA1. The interacting domain between the two proteins is localised to a region of 30 amino acids at the N-terminal end of proCFD. The 5 amino acid pro-peptide of CFD appears to be both necessary and sufficient for proteolysis of CFD by HTRA1. Investigation of the functional relevance of the interaction between HTRA1 and CFD shows that proCFD is cleaved by HTRA1, whilst mature CFD is not subjected to proteolysis. HTRA1-mediated cleavage of CFD forms an active protease, leading to activation of factor B in the alternative complement pathway in in vitro assays. Furthermore, a normal complement response is restored to CFD-depleted serum by addition of proCFD activated by HTRA1. Thus, an HTRA1- mediated increase in alternative complement pathway activity may explain a proportion of the AMD-risk attributed to the chr10q26 locus. Genetic and protein-based approaches were used to study the potential role of CFD in AMD pathogenesis, independent of an interaction with HTRA1. An intronic SNP, rs3826945, was significantly associated with increased risk of AMD in two British case-control cohorts, and in a combined meta-analysis with 4 additional cohorts from North America and Europe (p-value = 0.032, Odds Ratio = 1.112 in 4765 cases and 2693 controls). Assessment of copy number variation and sequencing of CFD did not identify any functional variants which may explain the association with disease. However, plasma levels of CFD were measured by ELISA in 751 AMD cases and 474 controls, and were found to be significantly elevated in AMD cases compared to controls (p-value = 0.00025). This further implicates complement activation in AMD pathogenesis, and makes CFD an attractive candidate for therapeutic intervention. An alteration in the level of activated CFD, possibly mediated via an interaction with HTRA1, either at the systemic or local tissue level, may play a role in disease development and progression.

Yes / Microperimetry, also referred to as fundus perimetry or fundus-driven perimetry, enables simultaneous acquisition of visual sensitivity and eye movement data. We present sensitivity data collected from 60 participants with normal vision using gaze-contingent perimetry. A custom designed spatially dense test grid was used to collect data across the visual field within 13° of fixation. These data are supplemental to a study in which we demonstrated a spatial interpolation method that facilitates comparison of acquired data from any set of spatial locations to normative data and thus screening of individuals with both normal and non-foveal fixation (Denniss and Astle, 2016)[1].

Yes / Purpose: To demonstrate methods that enable visual field sensitivities to be compared with normative data without restriction to a fixed test pattern. Methods: Healthy participants (n = 60, age 19–50) undertook microperimetry (MAIA-2) using 237 spatially dense locations up to 13° eccentricity. Surfaces were fit to the mean, variance, and 5th percentile sensitivities. Goodness-of-fit was assessed by refitting the surfaces 1000 times to the dataset and comparing estimated and measured sensitivities at 50 randomly excluded locations. A leave-one-out method was used to compare individual data with the 5th percentile surface. We also considered cases with unknown fovea location by adding error sampled from the distribution of relative fovea–optic disc positions to the test locations and comparing shifted data to the fixed surface. Results: Root mean square (RMS) difference between estimated and measured sensitivities were less than 0.5 dB and less than 1.0 dB for the mean and 5th percentile surfaces, respectively. Root mean square differences were greater for the variance surface, median 1.4 dB, range 0.8 to 2.7 dB. Across all participants 3.9% (interquartile range, 1.8–8.9%) of sensitivities fell beneath the 5th percentile surface, close to the expected 5%. Positional error added to the test grid altered the number of locations falling beneath the 5th percentile surface by less than 1.3% in 95% of participants. Conclusions: Spatial interpolation of normative data enables comparison of sensitivity measurements from varied visual field locations. Conventional indices and probability maps familiar from standard automated perimetry can be produced. These methods may enhance the clinical use of microperimetry, especially in cases of nonfoveal fixation.

Age related macular degeneration (AMD) is a public health concern in an aging society. The retinal pigment epithelium (RPE) layer of the eye is a principal site of pathogenesis for AMD. Morphological characteristics of the cells in the RPE layer can be used to discriminate age and disease status of individuals. In this thesis three genotypes of mice of various ages are used to study the predictive abilities of these characteristics. The disease state is represented by two mutant genotypes and the healthy state by the wild-type. Classification analysis is applied to the RPE morphology from the different spatial regions of the RPE layer. Variable reduction is accomplished by principal component analysis (PCA) and classification analysis by the k-nearest neighbor (k-NN) algorithm. In this way the differential ability of the spatial regions to predict age and disease status by cellular variables is explored.

This research was designed to address important issues for the effective prescription of, and training in the use of, magnifiers for reading patients with visual impairment. The emphasis was on the development of simple methods of assessment and training that could be easily implemented, at no great cost, by low vision practitioners in clinical practice. To ensure that the results would be widely applicable, the research focused on subjects with age-related macular degeneration (AMD) using stand magnifiers (being the most common cause of low vision and the most commonly prescribed magnifiers respectively). From this research, modifications to the current methods of reading rehabilitation are suggested to more effectively improve low vision reading for the millions of people with low vision around the world. The magnification and reading performance achieved with the magnifier determined by the fixed acuity reserve method was as valid as that achieved with the magnifier determined by the individual acuity reserve method. The fixed acuity reserve is a simpler method to calculate the required magnification, as it requires only near visual acuity and the patient's goal reading task. This method was primarily used to select the appropriate illuminated stand magnifiers for the subjects participating in the subsequent studies and is recommended for use as the starting point in clinical low vision practice. The main study of this thesis was a longitudinal investigation of the benefit of large print reading practice on reading performance with stand magnifiers. Instead of the intensive training programs on magnifier use which have been suggested by previous studies, this study aimed to investigate the effect of simple large print reading practice, under either full or restricted field of view (the latter simulated by a practice stand), on reading rate with stand magnifiers for subjects with AMD. The experimental hypothesis was that reading practice prior to the prescription of stand magnifiers would improve reading performance with the stand magnifiers for subjects with AMD. As previous studies have shown, reading rate reduced when a stand magnifier was first introduced. One week of reading practice on large print, with or without a reduced field of view, gave an improvement in reading rate with the stand magnifier for passages of text (such that the reading rates with and without magnifiers were not significantly different). There was a suggestion that this practice may give a more rapid improvement in reading rate than that achieved by the control subjects who did not do any large print reading practice, but this did not reach statistical significance. Even very brief reading with the stand magnifiers by the control subjects gave some improvement in reading rate. Therefore, home or in-office reading practice on large print or with magnifiers is recommended for patients with AMD before magnifiers are prescribed. Subjects who had neither reading practice nor exposure to the magnifier prior to its prescription required two weeks practice using their stand magnifiers to achieve their maximum reading rate. This suggests that home practice in using stand magnifiers is beneficial and a follow up visit is recommended two weeks after the provision of a magnifier to assess any change in reading rate. If no improvement in the magnifier reading rate is found or the rate is less than the reading rate on large print without a magnifier, further investigations of the patients' vision and/or their magnifier manipulation strategy are necessary. In the last study, a simple method aimed at alleviating difficulties with magnifier manipulation and navigation, the attachment of a line guide to the base of the stand magnifier, was investigated using both objective methods (recording magnifier movements and reading rate measures) and subjective methods (simple questionnaire). Although there was no improvement in the objective measures of reading or navigation performance with the line guide, more than half of the subjects with low vision preferred to have the line guide on their stand magnifiers. This suggests that the objective measures might not be sensitive enough to predict the subjective response, or that other factors that were not measured in this study influenced subjects' preferences in selecting the line guide (e.g., psychological support provided by the line guide in reading orientation). Clinically, the subjective response of patients to the use of low vision aids as well as their motivation are important criteria for success in low vision rehabilitation. There was a tendency for less experienced users to prefer the line guide to assist their use of the stand magnifier for reading. Therefore, a line guide could be offered as a preliminary training aid when stand magnifiers are first prescribed for AMD patients. Possible improvements to the design of the line guide were identified. Further research is required to assess the benefits of this or similar devices for new magnifier users and to understand the difficulties that people with visual impairment have with page navigation in order to determine improved methods of training navigation strategies. The unique contribution of this study to the field of low vision rehabilitation is that the benefit of short-term reading practice, on large print or with magnifiers, as simple, cheap methods of enhancing reading performance with stand magnifiers was demonstrated. The results of this study have led to the development of recommendations for assessing and training AMD patients who are prescribed stand magnifiers.

La Dégénérescence Maculaire Liée à l’Age (DMLA) est la première cause de cécité légale dans les pays industrialisés chez les personnes âgées. L’atrophie géographique – l’une des formes tardives de la DMLA - est caractérisée par la perte de l’épithélium pigmentaire et la dégénérescence des photorécepteurs. Nous groupe a montré précédemment que dans l’atrophie géographique, les phagocytes mononucléés (PMs) s’accumulent dans l’espace sous-rétinien, et induisent la dégénérescence rétinienne via la production d’IL-1β. Dans un premier temps, nous montrons que la présence de PMs sous-rétiniens est associée à la perte des bâtonnets et la dégénérescence des segments de cônes dans la zone de transition de patients atrophiques. Nous montrons ensuite dans différents modèles in vivo et ex vivo que les macrophages récapitulent ces effets, et qu’IL-1β est nécessaire à la perte des segments externes des cônes induite par les PMs. Dans un deuxième temps, nos résultats montrent qu’IL-1β induit indirectement la mort des bâtonnets, en perturbant l’homéostasie rétinienne du glutamate. L’inhibition des récepteurs glutamatergiques pour prévenir l’excitotoxicité du glutamate, ou la supplémentation en cystine favorisant la restauration de la machinerie neuronale antioxydante, permettent de protéger les bâtonnets de la toxicité induite par IL-1β. L’ensemble de nos résultats démontre le rôle joué par IL-1β dans la dégénérescence des segments de cônes et la perte des bâtonnets dans l’inflammation sous-rétinienne. Cette étude permettra la mise au point de thérapies innovantes, afin de lutter contre la forme atrophique de la DMLA, pour laquelle il n’existe actuellement aucun traitement. / In geographic atrophy (GA), one of the late forms of Age-related Macular Degeneration (AMD), an extending atrophic zone forms, characterized by the loss of retinal pigment epithelium and photoreceptor degeneration. Subretinal mononuclear phagocytes (MPs) accumulate in GA, and are associated with IL-1β-dependent retinal degeneration. First, we confirmed that subretinal accumulation of MPs is associated with rod degeneration and cone segment loss in the transitional zone in GA human samples. Using ex vivo and in vivo models, we then demonstrated that MPs-derived IL-1β leads to severe cone segment degeneration. Therefore, inhibiting subretinal MP accumulation or IL-1β might protect the cone segment, and help preserve high acuity daytime vision in conditions characterized by subretinal inflammation. Second, we showed that IL-1β effect on rod degeneration is indirect, and mediated by glutamate. Our results indicate that IL-1β impairs Müller glial cells glutamate recycling, and subsequently leads to the extracellular increase in glutamate content. Inhibiting glutamate receptors to prevent excitotoxicity, or exogenous cystine supplementation to supply antioxidant metabolism, are sufficient to protect rods from IL-1β-induced neurotoxicity. Our results provide new perspectives to treat pathologies associated with subretinal inflammation such as late AMD. Our results collectively demonstrated that MP-derived IL-1β induces cone segment loss, and glutamate homeostasis disruption associated with rod degeneration. This study will help with the development of new therapeutic strategies in dealing with inflammatory retinal pathologies as geographic atrophy.

Les rétinopathies ischémiques et la forme humide de la dégénérescence maculaire liée à l’âge (DMLA) sont la principale cause de malvoyance respectivement chez les personnes en âge de travailler et les personnes agées. Les néovascularisations choroïdiennes (NVC) et rétiniennes et l’œdème maculaire associés à ces pathologies sont traités à l’aide de biomolécules qui ciblent uniquement la voie de signalisation des VEGF. Le développement de thérapies indépendantes de cette voie permettrait d’offrir aux patients résistants aux anti-VEGF une alternative thérapeutique pour préserver leur vision. Les intégrines αvβ3 et αvβ5, impliquées dans la néovascularisation oculaire, apparaissent ainsi comme une cible alternative intéressante. La Lébécetine (LCT), une lectine de type-C, de 30 kDa et de structure hétérodimèrique issue de venin de serpent Macrovipera lebetina interagit spécifiquement avec les intégrines α5β1 et αvβ3, αvβ5. La LCT a une activité anti-angiogénique in vitro sur des cellules endothéliales microvasculaire du cerveau humain (HBMEC) et in vivo sur le modèle de la membrane chorioallantoïde du poulet (CAM). Afin d’étudier son effet sur la néovascularisation oculaire, nous avons eu recours aux modèles d’angiogenèse ex vivo utilisant des explants aortique ou choroïdien cultivés en présence de LCT, puis son effet a été évalué in vivo dans un modèle de NVC chez la souris et également sur la néovascularisation rétinienne dans le modèle de rétinopathie induite par l’oxygène (RIO). Nos données démontrent qu’une injection unique de LCT est capable de réduire la NVC et rétinienne dans ces modèles sans affecter les vaisseaux quiescents matures indiquant un bon profil d’innocuité. / Ischemic retinopathies and the wet form of age-related macular degeneration (AMD) are characterized by devastating angiogenesis responsible for the majority of irreversible blindness. Current therapies include use of anti-VEGF agents to reduce choroidal neovascularization and edema. These treatments are effective in most cases, but spontaneous or acquired resistance to anti-VEGF highlight a need for additional alternative therapies. In recent years, pharmacological inhibition of αvβ3 and αvβ5, which regulate endothelial cell proliferation and stabilization, have emerged as new therapeutic tools for the treatment of these diseases. Lebecetin (LCT), a 30-kDa heterodimeric C-type lectin that is isolated from Macrovipera lebetina venom, interacts with α5β1 and αv-containing integrins (αvβ3, αvβ5). We previously showed that LCT has an anti-angiogenic effect in vitro on human brain microvascular endothelial cells (HBMEC) and in vivo in a chick chorioallontoic membrane assay (CAM). To evaluate the inhibitory effect of LCT on ocular angiogenesis, we cultured aortic and choroidal explants in the presence of LCT and analyzed the effect of LCT on choroidal neovascularization in the mouse CNV model and on retinal neovascularization in the oxygen induced retinopathy (OIR) model. Our data demonstrated that a single injection of LCT efficiently reduced choroidal and retinal neovascularization in these models with no significant effect on mature blood vessels predicting a good safety profile.

Immunosenescence is considered a possible factor in the development of age-related macular degeneration and choroidal neovascularization (CNV). However, age-related changes of myeloid cells (MCs), such as microglia and macrophages, in the healthy retina or during CNV formation are illdefined. In this study, Cx3cr1-positive MCs were isolated by fluorescence-activated cell sorting from six-week (young) and two-year-old (old) Cx3cr1GFP/+ mice, both during physiological aging and laser-induced CNV development. High-throughput RNA-sequencing was performed to define the age-dependent transcriptional differences in MCs during physiological aging and CNV development, complemented by immunohistochemical characterization and the quantification of MCs, as well as CNV size measurements. These analyses revealed that myeloid cells change their transcriptional profile during both aging and CNV development. In the steady state, senescent MCs demonstrated an upregulation of factors contributing to cell proliferation and chemotaxis, such as Cxcl13 and Cxcl14, as well as the downregulation of microglial signature genes. During CNV formation, aged myeloid cells revealed a significant upregulation of angiogenic factors such as Arg1 and Lrg1 concomitant with significantly enlarged CNV and an increased accumulation of MCs in aged mice in comparison to young mice. Future studies need to clarify whether this observation is an epiphenomenon or a causal relationship to determine the role of immunosenescence in CNV formation.

Vision impairment and blindness are in industrialized countries primarily caused by the degeneration of the retina, the light sensing tissue inside the eye. The degeneration, occurring in diseases like age-related macular degeneration (AMD) or retinitis pigmentosa (RP), can be caused by environmental factors as well as genetic defects and thus shows diverse pathologies. In all conditions, the light detecting photoreceptors (rods and/or cones) are dying caused by either direct photoreceptor damage or as a secondary effect following degeneration of supporting cells. Although promising treatment approaches are currently under investigation, up to date it is not possible to cure these diseases. Amongst these therapeutic strategies, pre-clinical studies evaluating the replacement of degenerated cells by transplantation of new photoreceptors demonstrated promising results. First studies conducted the specific enrichment and transplantation of primary photoreceptors derived from postnatal mice and their sufficient integration and differentiation into mature photoreceptors in wild-type as well as degenerated mouse retinae. Recent experiments additionally proved the recovery of some dim-light vision after transplantation in mice lacking night sight. The in vitro differentiation of whole eye cups containing photoreceptors, out of human or mouse ES or iPS cells, peaked in the transplantation of ES-derived photoreceptors into wild-type as well as degenerated mice and the integration and maturation of these cells. These observations are encouraging, but prior to a save implementation of this strategy into a clinical routine, several further hurdles need to be challenged. Collection of photoreceptors out of whole retinal tissues prior to transplantation was shown to be an important step to reach high integration rates. Additionally, transplantation of photoreceptors derived from stem cells comprises the risk of tumor formation after transplantation and thus also requires depletion of inadvertent cells. Therefore, we established the enrichment of photoreceptors using the cell surface marker dependent method magnetic-activated cell sorting (MACS). For identification of suitable target-specific surface markers, we characterized young transplantable mouse photoreceptors using microarray analysis and screened their transcriptome. Amongst others, ecto-5´nucleotidase (Nt5e, termed CD73) was identified being a rod photoreceptor specific cell surface protein. Thus, we enriched young photoreceptors with CD73-dependent MACS with sufficient purity and transplanted these cells into the subretinal space of wild-type mice. In contrast to unsorted retinal cells, enriched photoreceptors integrated in significantly higher number into the host retina, proving that MACS is a suitable alternative for specific photoreceptor enrichment. Testing other proteins, identified as photoreceptor specific, for MACS suitability and the translation of this approach to photoreceptors, derived from mouse as well as human iPS or ES cells, should be the focus of consecutive investigations. The integration of grafted cells into the retina is a complex process dependent on a variety of influencing factors. Transplantation experiments in aging wild-type mice and a rod-depleted mouse model, containing a retina composed of cone and cone-like photoreceptors, indicated that the activation of Müller glia cells facilitates integration of transplanted photoreceptors. Besides that, reduced outer limiting membrane (OLM) integrity, increased subretinal graft distribution or reduced retinal cell density are further suggested as potential cell engraftment enhancers. These factors might open up important possibilities of host retina manipulation to increase cell integration rates. Although retinal transplantation experiments were in addition to mice also performed using pigs or rats as hosts, the transplantation of enriched single photoreceptors, following the protocols successfully established in mice, has not been performed in other species. Nevertheless, transferring this technique is important and would allow better predictions for future application in human patients. Therefore, we transferred our protocol, using CD73 based MACS, to the rat and successfully enriched rat photoreceptors with sufficient purity. We subsequently transplanted these cells into the subretinal space of rats as well as mice and observed limited integration capacity of grafted cells. Only few transplanted rat photoreceptors were localized in the rat retina, lacking proper photoreceptor morphology. Especially regarding a perspective clinical application in humans, these data are remarkable. They imply the question, whether low integration in rat represents a general problem and might thus also be relevant for treatment in humans, or whether the rat retina forms just an exception. Thus, further detailed analysis of the cellular and molecular mechanisms underlying the integration process of transplanted photoreceptors represent an essential prerequisite for the development of a safe and efficient therapy, aiming to treat retinal degenerative diseases characterized by photoreceptor loss. / Degenerationserkrankungen der Netzhaut (Retina) sind in Industrieländern die Hauptursache für verminderte Sehfähigkeit und Blindheit. Sowohl Umweltfaktoren als auch vererbte Mutationen können Defekte wie altersbedingte Makuladegeneration (AMD) oder Retinitis pigmentosa (RP) auslösen und führen zu einem sehr variablen Krankheitsbild. Eine Gemeinsamkeit aller Formen ist das Absterben der lichtdetektierenden Fotorezeptoren (Stäbchen und/oder Zapfen). Dieses kann entweder durch direkte Schädigung, oder als Sekundäreffekt nach Degeneration der unterstützenden Zellen erfolgen. Obwohl im Moment vielversprechende Behandlungsansätze untersucht werden, ist es zurzeit nicht möglich, retinale Degenerationserkrankungen dieser Art zu heilen. Ein erfolgversprechender Ansatz könnte jedoch der Ersatz der degenerierten Zellen durch transplantierte Fotorezeptoren sein. Erste Studien demonstrierten die spezifische Anreicherung von primären Fotorezeptoren aus der Netzhaut neugeborener Mäuse und deren subretinale Transplantation in Wildtyp-Mäuse und Mausmodelle mit retinaler Degeneration. Die transplantierten Zellen integrierten in die Empfängernetzhaut und entwickelten sich in ausgereifte Fotorezeptoren und konnten unter anderem bei nachtblinden Mäusen die Sehfähigkeit bei Dunkelheit verbessern. Die Differenzierung von humanen oder murinen ES- und iPS-Zellen in vitro in vollständige Retinae und die Transplantation daraus gewonnener Fotorezeptoren in Mäuse, bilden vorläufig den Höhepunkt dieser Entwicklung. Obwohl die Fortschritte der jüngsten Vergangenheit beeindruckend sind, sollten vor der sicheren und effektiven Anwendung einer retinalen Zellersatztherapie als therapeutische Maßnahme beim Menschen noch einige wissenschaftliche Fragestellungen beantwortet werden. Studien zeigen, dass Zellpopulationen, die direkt aus der Spendernetzhaut entnommen und transplantiert wurden, auf Grund ihrer Heterogenität in geringeren Zahlen in die Empfängerretina einwandern als angereicherte Fotorezeptoren. Zusätzlich besteht bei unsortierten Zellen, die aus Stammzellpopulationen gewonnen wurden, das Risiko einer Tumorbildung. Daher haben wir die magnetisch-aktivierte Zellsortierung (MACS) zur Anreicherung junger Fotorezeptoren etabliert. Die dabei benötigten, für Fotorezeptoren spezifischen, Oberflächenproteine wurden mit Hilfe von Microarray-Analysen des Transkriptoms junger Stäbchen von Mäusen identifiziert. Dabei wurde unter anderem die 5\'-Nukleotidase (Nt5e, CD73) entdeckt, die uns die erfolgreiche Anreicherung junger Mausfotorezeptoren mit Hilfe von CD73-vermitteltem MACS erlaubte. Die Transplantation dieser angereicherten Zellpopulation in die Netzhaut von Empfängertieren resultierte in einer signifikant erhöhten Integrationsrate im Vergleich zu nicht-angereicherten retinalen Zellen. Die Überprüfung der Nutzbarkeit weiterer identifizierter Oberflächenproteine zur Zellanreicherung bzw. die Übertragung der etablierten Protokolle zur Zellsortierung und Transplantation auf Fotorezeptoren aus ES- und iPS-Zellkulturen, sollten im Fokus nachfolgender Experimente stehen. Die Integration transplantierter Zellen in die Empfängernetzhaut ist ein komplexer Prozess und von unterschiedlichen Einflussfaktoren abhängig. Durch Transplantationsexperimente in alternden Wildtyp-Mäusen und einem Mausmodell, dessen Fotorezeptorschicht keine Stäbchen und stattdessen nur Zapfen und zapfenähnlichen Fotorezeptoren aufweist, konnte gezeigt werden, dass vor allem die Aktivierung von Müllerzellen die Integrationsrate der Fotorezeptoren erhöht. Neben dieser sogenannten Gliose werden weitere Faktoren, wie die reduzierte Stabilität der äußeren Grenzmembran, die flächenmäßig größere Verteilung der transplantierten Zellen im subretinalen Raum oder die reduzierte Dichte der Zellen in der äußeren Körnerschicht, als potentielle integrationsfördernde Komponenten in Betracht gezogen. Diese bilden interessante Schwerpunkte für weitere Forschungen, um eine ausreichende Zellintegration durch Manipulation der Empfängernetzhaut, auch in der klinischen Anwendung, zu erreichen. Obwohl Transplantationsexperimente zusätzlich zur Maus auch in anderen Empfängerspezies, wie Ratten und Schweinen, durchgeführt wurden, liegen bis jetzt keine Studien vor, die die in der Maus erfolgreich etablierten Protokolle der Zellanreicherung und Transplantation von Fotorezeptor-Suspensionen in diesen Spezies reproduzierte. Der Transfer dieser Technik und eine Generalisierung der Anwendbarkeit eines Fotorezeptorersatzes durch Transplantation in verschiedenen Säugetierarten geben jedoch wichtige Hinweise für eine mögliche Translation dieser Technologie für klinische Anwendungen. Deshalb haben wir unser bereits an der Maus getestetes Protokoll auf die Ratte übertragen und erfolgreich Fotorezeptoren der Ratte mit Hilfe von CD73-vermitteltem MACS angereichert. Nach deren Transplantation in die Netzhaut von Ratten und Mäusen zeigten die Rattenfotorezeptoren aber eine stark verminderte Integrationsfähigkeit und das Fehlen einer reifen Fotorezeptormorphologie. Speziell in Hinsicht auf eine zukünftige klinische Anwendung sind diese Ergebnisse relevant, da sie die Frage aufwerfen, ob die mangelnde Integration in der Ratte ein generelles Problem darstellt und daher auch beim Menschen zu erwarten ist, oder ob sie nur eine Ausnahme im Rattenmodell bildet. Aus diesem Grund bildet die weitere Erforschung der zellulären und molekularen Mechanismen der Integration transplantierter Fotorezeptoren eine wichtige Grundlage für die Entwicklung einer sicheren und effizienten Therapie mit dem Ziel, degenerative Netzhauterkrankungen zu heilen.

Las enfermedades neurodegenerativas cursan con la degeneración y muerte de las neuronas las cuales son incapaces de suplir su propia muerte. Debido a esta característica enfermedades como el Alzheimer, el Parkinson o el Glaucoma no tienen cura en la actualidad. La retina es una proyección del sistema nervioso central (SNC) encapsulada en el globo ocular y aislada del resto del SNC, lo que la hace fácilmente accesible a la manipulación experimental. En este trabajo estudiamos las alteraciones funcionales de la retina con el electrorretinograma de campo completo (ERG), técnica basada en el registro de la respuesta eléctrica retiniana tras la presentación de un estímulo de luz homogéneo. Así hemos estudiado los efectos de la sección del nervio, del aumento de presión intraocular y de la fototoxicidad, dichas lesiones afectan selectivamente a determinadas poblaciones neuronales retinianas e imitan enfermedades neurodegenerativas como el Glaucoma o la Degeneración Macular Asociada a la Edad. / Neurodegenerative diseases are characterized by degeneration and death of neurons which are unable to recover after a given insult, thus impairing functional recuperation. Because of this, there is no cure for diseases such as Alzheimer, Parkinson or Glaucoma. The retina is a projection of the central nervous system (CNS) located in the eye and therefore, isolated from the rest of the CNS. This makes the retina a very good model for experimental manipulation. In this work we have studied the functional changes in the retina by full field electroretinograme (ERG). This technique records the electric response of the retina after presentation of homogeneous light stimuli. We have studied the effects that optic nerve sections, increase of the intraocular pressure and phototoxicity have on ERG recordings. These lesions impair selectively certain retinal neuronal populations and imitate neurodegenerative diseases as Glaucoma or Age-related Macular Degeneration.