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Dentro de la estructura del ojo de los vertebrados, la retina es tal vez el tejido que más capta la atención de los científicos por ser el primer actor en la recepción y transmisión del estímulo visual. La retina si bien forma parte de un sentido periférico, posee gran cantidad de homologías con el Sistema Nervioso Central. Esto provoca que los eventos y patologías asociadas a la retina puedan, hasta cierto punto, relacionarse con las enfermedades neurodegenerativas del cerebro más difíciles de estudiar como son el mal de Parkinson y la enfermedad de Alzheimer. En el caso particular de la retina, existen dos enfermedades aún hoy incurables como son la Retinitis Pigmentosa (RP) y la Degeneración Macular asociada a la Edad (AMD, por sus siglas en ingles). La muerte de las neuronas fotorreceptoras cumple un rol fundamental en el desarrollo de estas enfermedades, que se basan en la pérdida gradual y prácticamente irreversible de la visión. En la retina, las células gliales de Müller proveen el sostén necesario para la estructura de la misma. Se ha reportado que estas células expresan marcadores de células madre por lo cual están involucradas en la regeneración de la retina ante un eventual daño. Existen ya terapias destinadas a estimular el desarrollo de dichas células madre. Sin embargo, aún es necesario un mayor avance en la búsqueda de tratamientos destinados al mantenimiento y posterior diferenciación de las células regeneradas, evitando el proceso de muerte al que están expuestas. En otras palabras, evitar que las neuronas fotorreceptoras sean sometidas al proceso de suicidio celular conocido en líneas generales como Apoptosis. Dentro del enorme abanico de causas que pueden generar apoptosis en una neurona, la falta de factores tróficos o de crecimiento es una de ellas. Este trabajo de tesis buscó revertir la muerte provocada por la falta de factores tróficos en un modelo de cultivo neuronal puro compuesto en su mayor proporción por neuronas fotorreceptoras. En la primera parte nos enfocamos en estudiar el rol de la Melatonina como agente neuroprotector. Los resultados apuntan a que la Melatonina resulta eficaz para disminuir el avance de la apoptosis a días avanzados en el desarrollo. Pudimos demostrar que el efecto que la Melatonina ejerce específicamente en neuronas fotorreceptoras, está dado por la activación de la vía de señalización de ERK/MAPK. Respecto a la capacidad de la Melatonina de mantener la población de neuronas fotorreceptoras, no se vio un aumento en la expresión de proteínas del ciclo visual. Por el contrario, se observó un aumento de la expresión génica del factor de transcripción CRX (Cone Rod Homeobox) que ayudaría a mantener el fenotipo de neuronas fotorreceptoras a aquellas que hayan iniciado el proceso de diferenciación. Estos resultados sugieren que la Melatonina, podría ejercer su acción protectora directamente en las neuronas fotorreceptoras, e indirectamente, a través del estímulo de la población de neuronas que acompaña a los fotorreceptores en el ciclo visual, como ser las células amacrinas. La segunda parte de este trabajo se enfocó en la caracterización de la vía de muerte por falta de factores tróficos. En ese sentido, la vía de NO-GAPDH-SIAH consiste en la translocación a núcleo de la enzima GAPDH y el posterior desencadenamiento de la muerte celular. Comprobamos que efectivamente en nuestro modelo se verifica la translocación nuclear; y que al pre-tratar los cultivos con un inhibidor selectivo de dicha translocación se recupera significativamente la perdida de la viabilidad en los cultivos neuronales. Los resultados de esta tesis por un lado enriquecen el conocimiento sobre el rol de la Melatonina como agente neuroprotector en la retina. Por otro lado contribuyen a esclarecer los mecanismos de muerte en las células de la retina ante la falta de factores tróficos para poder, en un futuro, unificar su tratamiento. / The retina is probably the most interesting tissue within the structure of vertebrate eye, given its role as the first one in charge of receiving and transmitting the visual stimuli. Even though the retina is part of a peripheral sensory system, it shares many homologies with the Central Nervous System. This allows correlating to some extent the pathologic events associated with the retina with neurodegenerative illness more difficult to study such as Alzheimer’s and Parkinson’s disease. There are two main disorders of the retina named Retinitis Pigmentosa and Age-related Macular Degeneration. Those two pathologies share a common trait which is the death of photoreceptor cells as the main cause of gradual and irreversible loss of vision. In the retina, Müller glial cells are in charge among other functions, to support and maintain the structure of the retina. It has been reported for this cells the expression of stem cell markers and the involvement on retina regeneration after damage. Even though there are treatments nowadays that aim to stimulate Müller glia development of their stem potential, treatments aiming to the maintenance and ulterior differentiation of newly regenerated cells are just as important. The general idea is to prevent newly generated photoreceptor cells to undergo cell suicide, also known as Apoptosis. Within the large range of causes that can trigger apoptosis on a neuron, the lack of growth factors is one of them. In this thesis we aim to revert photoreceptor cell death induced by lack of growth factors, on a model of pure neuronal cultures. On the first chapter we focused on assessing the role of Melatonin as a neurotrophic factor. Results showed that Melatonin is effective on slowing down the apoptotic pathway after 4 days of development. We proved the involvement of Melatonin on the activation of the ERK/MAPK pathway directly on photoreceptor cells. Regarding Melatonin as a factor promoting neuronal differentiation, we could not observe an overexpression of visual cycle proteins. On the other hand, we observed the genetic overexpression of a transcription factor (Cone Rod Homeobox) which will help maintain the phenotype of those neurons that started developing as photoreceptors. Our results suggest that Melatonin could accomplish neuroprotection by either acting directly on photoreceptor cells, or indirectly through engaging the neurons that aid said cells on the visual cycle. The second part of this thesis focuses on the characterization on the cell death pathway caused by lack of trophic factors. On that matter, the NO-GAPDH-SIAH pathway consist in nuclear translocation of the glycolytic enzyme GAPDH followed by onset of neuronal death. We found that nuclear translocation of GAPDH occurs in our model, and that by pre-treating cultures with R-(-)-Deprenyl (a selective inhibitor for such translocation), we recovered neuronal cultures from loss of cell viability. The results shown on this thesis on one hand contribute to deepen the knowledge of Melatonin as a neuroprotective agent in the retina. On the other hand they shed light into the mechanisms involving neuronal death on lack of trophic factors so we may, in the future, unify their treatment.

Las enfermedades neurodegenerativas se caracterizan por la pérdida progresiva e irreversible de neuronas, lo cual perjudica seriamente las funciones neurológicas, por lo que es indispen-sable encontrar una solución efectiva a esta problemática. El sistema nervioso es de difícil acceso y de una enorme complejidad. Utilizar modelos que permitan analizar la génesis y la muerte neuronal, resulta clave para comprender la evolu-ción y establecer potenciales tratamientos para las enferme-dades neurodegenerativas. La retina es un excelente sistema para indagar en estos procesos, dado que forma parte del sis-tema nervioso central y presenta una estructura laminar sen-cilla. En la retina, las enfermedades neurodegenerativas que afectan a las neuronas fotorreceptoras - como la Retinitis Pigmentaria o la Degeneración Macular - culminan con la ce-guera de los pacientes afectados. Actualmente se presentan dos posibles estrategias de tratamiento para estas enfermeda-des: la primera busca conocer los mecanismos que conducen a la apoptosis neuronal, de manera de evitarla mediante el uso de factores que promuevan la supervivencia de los fotorreceptores. La segunda propone regenerar las neuronas perdidas durante la enfermedad mediante el uso de stem cells o células madre, las cuales se caracterizan por la auto-reno-vación y multipotencialidad. Las células gliales de Müller(CGM) serían stem cells en la retina. Numerosos trabajos indican que las CGM son capaces de de-diferenciarse y proliferar luego de un daño en la retina, para luego expresar marcadores de fo-torreceptores. Este comportamiento es ampliamente recono-cido en peces y anfibios, sin embargo resulta más restringido en aves y mamíferos. Para poder utilizarlas en el tratamiento de patologías degenerativas, es clave conocer varios aspec-tos de la biología de las CGM como stem cells en animales superiores. Por otra parte, la rápida muerte por apoptosis de las células regeneradas dificulta notablemente el éxito de las terapias de reemplazo. Por lo tanto, es importante dilucidar cómo evitar dicha muerte. Estudios previos de nuestro labora-torio indicaron que las CGM expresaron marcadores de multipo-tencialidad por largos períodos in vitro, y que esta expresión era regulada por la interacción con neuronas de retina o el agregado de factores tróficos. El objetivo del presente trabajo de Tesis fue evaluar el potencial comportamiento como célu-las madre de las CGM de roedores, determinando si eran capa-ces de generar neuronas fotorreceptoras in vitro. Para ello di-señamos tres sistemas de cultivos celulares de retina (neuro-nales puros, gliales puros y cultivos mixtos neuro-gliales) en los cuales analizamos la presencia, origen y evolución de pro-genitores multipotentes, determinando si retenían las caracte-rísticas de multipotencialidad a través de los pasajes. A conti-nuación evaluamos la capacidad de diferenciación de dichos progenitores en fotorreceptores maduros y funcionales; y si el agregado de factores lipídicos podía prevenir la apoptosis de los fotorreceptores regenerados. En el Primer Capítulo evalua-mos si las CGM presentaban dos características de las células madre: la capacidad de expresar marcadores de multipoten-cialidad a través de los pasajes, y de generar progenitores neuronales. Determinamos que tras el repique de cultivos enri-quecidos en CGM, las CGM retuvieron la expresión de Nestina, a la vez que co-existieron con una población de progenitores que bajo condiciones adecuadas se diferenciaron en neuronas La existencia de progenitores en estos cultivos secundarios planteó cuatro preguntas: De dónde provienen los progenito-res? Expresan otros marcadores de multipotencialidad? Es posible diferenciarlos en fotorreceptores? Existen factores que promuevan su supervivencia y/o diferenciación? Antes de avanzar con ellas evaluamos si las dos poblaciones mayorita-rias en cultivos primarios de retina (CGM y neuronas), influían en el destino que los progenitores adoptaban in vitro. Deter-minamos que sólo la interacción de CGM con neuronas de reti-na permitía la generación y/o conservación de progenitores multipotentes por largos períodos en cultivo. En el Segundo Capítulo analizamos el origen y la expresión de marcadores de multipotencialidad en los progenitores observados tras el repi-que. Determinamos que los progenitores expresaron -además de Nestina- Sox-2 y Pax-6 a la vez que conservaron su capa-cidad proliferativa. Estas características se observaron sólo en los pasajes de cultivos mixtos neuro-gliales: los cultivos puros neuronales no sobrevivieron al repique, mientras que los cultivos puros de CGM no generaron progenitores de retina. Los progenitores retuvieron la expresión de Pax-6 y Sox-2 y continuaron proliferando aún luego de cuatro pasajes sucesi-vos de cultivos mixtos. Algunos de ellos, inclusive, expresaron Crx, hecho que indicaba su capacidad de diferenciación en neuronas fotorreceptoras. Para establecer el origen de los pro-genitores de retina en los repiques, utilizamos una sonda fluorescente que nos permitió identificar la progenie tanto de los progenitores como de las CGM. Este análisis sugirió que los progenitores multipotentes observados en los sucesivos repi-ques se originarían en progenitores pre-existentes preserva-dos por la interacción con las CGM y no en las CGM. En el tercer capítulo investigamos la capacidad de diferenciación de los progenitores multipotentes en neuronas fotorreceptoras. Determinamos que en los cultivos mixtos neuro-gliales, bajo condiciones adecuadas de cultivo los progenitores se diferen-ciaron en fotorreceptores maduros y funcionales, que expre-saron marcadores característicos de fotorreceptores (como Crx, Opsina, Periferina) a la vez que fueron capaces de res-ponder a la luz y de capturar glutamato por mecanismos de alta afinidad. Por último, investigamos si era posible bloquear o retrasar el desarrollo de la apoptosis en las células regene-radas. Determinamos que la administración de DHA y S1P - dos moléculas lipídicas con efectos anti-apoptóticos en fo-torreceptores-disminuyeron significativamente la apoptosis neuronal en los repiques. En conclusión, en este trabajo de Tesis establecimos una nueva función para las células gliales de Müller: su capacidad de transformar a las células progeni-toras de retina en células multipotentes, y de promover su diferenciación en neuronas fotorreceptoras. Esta novedosa función podría ser relevante al momento de diseñar nuevas estrategias para el tratamiento de enfermedades neurode-generativas de la retina. / Neurodegenerative diseases are characterized by progressi-ve and irreparable neuronal death, which ends up in major neurological dysfunctions. This scenario has prompted re-searchers to find an effective cure for these diseases; however, few results have been achieved so far. Studying the nervous system is very complicated, because of its difficult access and the vital functions that rely upon it. Thus, having an appropriate model to study neuronal genesis and degeneration is very important in order to find a valid treatment for neurodegenerative diseases. The retina is an excellent model for this type of studies: it is part of the central nervous system, has a very simple structure and is readily accessible. Retinal neurodegene-rative diseases, like Retinitis Pigmentosa (RP) or age- related macular degeneration (AMD), are characterized by extensive photoreceptor loss, which results in visual impairment and/or complete blindness. Two possible strate-gies are now being considered to find a cure for these pa-thologies: one strategy is to avoid neuronal degeneration, by using trophic factors that promote neuronal survival. The second strategy proposes using stem cells to replace the neurons lost during neurodegenerative diseases. Müller glial cells (MGC) are possible candidate to behave as stem cells in the retina. Several studies indicate that, after retinal injury, MGC are capable of de-differentiating and proliferating, and later expressing photoreceptor markers. This regenerative capacity is very robust in lower verte-brates, but much more restricted in birds and mammals.In addition, for stem cells to be useful in the treatment of retinal neurodegenerative diseases, an important issue needs to be solved: the rapid and extensive apoptosis of newly generated neurons. In this regard, it is essential to find molecules able to promote neuronal survival.Pre-vious studies from our laboratory indicate that MGC ex-press stem cell markers for several days in vitro. Moreo-ver, this expression can be regulated by both interactions with retinal neurons and supplementation with trophic factors. The present Doctoral Thesis studied the behavior of MGC as stem cells in rodent retinas, evaluating the ability of MGC to originate photoreceptors in vitro through the generation of multipotent retinal progenitors. We used three different types of retinal cultures (pure neuronal, pure glial and mixed neuro-glial cultures), in which we analyzed the presence, origin and evolution of multipotent retinal progenitors. We evaluated their multi-potentiality in passages of the diverse culture systems and their later ability to differentiate into functional photoreceptors. Finally, we investigated if supplementa-tion with docosahexaenoico acid and sphingosine-1-phospate (two lipid molecules with anti-apoptotic effects in photo-receptors in vitro) could promote the survival of newly regenerated neurons. In the First Chapter, we evaluated if MGC presented at least two of the main stem cell features: multipotentiality preserved through successive re-seedings and the ability to give rise to neuronal progenitors. Our results indicated that MGC retained the expression of stem cell marker Nestin after being re-seeded; in addition,they co-existed with a population of progenitors that also ex-pressed Nestin and, eventually, differentiated into neurons. The presence of neuronal progenitors in these se-condary cultures raised four important questions: Which cells originate these progenitors? Do they express other stem cell markers, besides Nestin? Is it possible to indu-ce their differentiation into photoreceptors? Is there any trophic factor that may promote their survival and/or indu-ce their differentiation into neurons? Before looking for answers to these questions, we evaluated if the two most abundant cells in retinal cultures (MGC and neurons) could influence photoreceptor fate in culture. We determined that progenitors were only able to retain the expression of stem cell marker Nestin for several days in vitro in mixed neuron-glial cultures. In the Second Chapter we ana-lyzed the source and the expression of other stem cell mar-kers in multipotent progenitors present in secondary cultu-res. Our results indicated that progenitors expressed Sox-2 and Pax-6, and preserved the ability to proliferate. The-se characteristics were only found in secondary mixed neuro-glial cultures; re-seeding of pure neuronal cultures led to generalized cell death, while re-seeding of pure glial cultures only generated glial cells, since no proge-nitors where found in this condition. To our surprise, pro-genitors from mixed cultures could be consecutively re-seeded until the fourth passage still preserving the ex-pression of stem cell features while some of them began to express Crx (an early transcription factor for photorecep-tors). To address the question about the source of the pro-genitors in the re-seedings, we used a fluorescent probe that separately labeled the progeny of both MGC and proge-nitors. We determined that multipotent progenitors did not originate from MGC; instead, they derived from pre-exis-tent progenitors present in the donor retina, which were preserved after passages due to their interactions with MGC. In the Third Chapter we investigated the ability of multipotent progenitors to differentiate into photorecep-tors. We determined that in secondary mixed cultures, pro-genitors differentiated into mature photoreceptors that expressed Opsin, Crx and peripherin. Moreover, they displa-yed functional features: they responded to light stimuli and showed high affinity-glutamate uptake, characteristics found in mature photoreceptors in the retina in vivo. Fi-nally, we evaluated if docosahexaenoic acid (DHA) and sphingosine-1-phosphate (S1P) promoted the survival of newly generated neurons. Our results indicated that supple-mentation of secondary mixed cultures with both DHA and S1P significantly reduced the number of apoptotic neurons, suggesting they might be useful in preventing neuronal apoptosis after their regeneration is achieved.We conclude that MGC may have an alternative function in retina regene-ration: besides giving birth to neurons -as many others researchers suggest-, we believe that MGC may help to res-tore neuronal populations by preserving a pool of progeni-tors in a multipotent state, and by later inducing their differentiation into mature and functional photoreceptors.

O sistema circadiano das aves é composto pela retina, a região homóloga aos núcleos supraquiasmáticos de mamíferos (NSQ) e a glândula pineal. A retina apresenta muitos eventos fisiológicos rítmicos, como por exemplo os movimentos das células fotorreceptoras em vertebrados não mamíferos, a expressão de opsinas, regeneração do cromóforo visual e produção e liberação de melatonina e dopamina. Todos estes eventos rítmicos são coordenados para prever alterações nas condições luminosas que ocorrem durante o dia, otimizando a função retiniana. Neste trabalho foi investigada a expressão de componentes chave de um sistema circadiano, incluindo os dois genes de melanopsina, Opn4x e Opn4m, os genes de relógio Clock e Per2, e os genes das enzimas chave da síntese de melatonina, N-Acetiltransferase, e de dopamina, Tirosina Hidroxilase, em células da retina de embriões de galinha. Culturas primárias de retina de embriões de galinha com 8 dias foram preparadas no ZT0 (quando as luz é acesa) e semeadas na densidade de 107 células por frasco de 25 cm2 . As células foram mantidas em ambiente úmido, com 5% CO2, a 40o C, em escuro constante, fotoperíodo 12C:12E, fotoperíodo 12C:12E seguido de escuro constante, ou em escuro constante na presença e na ausência de glutamato 100 μM por 12 h. A extração de RNA total foi feita ao longo de 24 horas com intervalo de três horas tendo início no ZT0 do sexto dia. As amostras foram submetidas a RT-PCR seguido de PCR quantitativo para a quantificação de RNAm. Para confirmar a expressão da proteína OPN4x foi realizado ensaio imunohistoquímico com anticorpos anti-melanopsina de galinha desenvolvidos em coelho. Também foi feita a quantificação da concentração das proteínas OPN4x, CLOCK e TIROSINA HIDROXILASE através da técnica de Western Blot. A quantificação do RNAm em escuro constante não apresentou ritmos de transcrição para nenhum gene. Já as células mantidas em fotoperíodo 12C:12E apresentaram padrões rítmicos de transcrição para Clock, Per2, Opn4m, N-Acetiltransferase e Tirosina Hidroxilase. Glutamato 100 μM foi eficaz em induzir ritmo em Clock, e inibiu drasticamente a expressão de Tirosina Hidroxilase e, apenas mais pontualmente, de Opn4x e Opn4m. Ensaios de viabilidade celular e fragmentação de DNA por citometria de fluxo demonstraram que essa inibição não foi resultante de ação tóxica ou apoptótica do glutamato. O neurotransmissor não teve qualquer efeito sobre a transcrição de Per2 e de N-Acetiltransferase. A quantificação protéica não indicou a presença de ritmo para CLOCK, OPN4x ou TIROSINA HIDROXILASE. A grande variabilidade inter-ensaios nos resultados de quantificação protéica sugere uma menor sensibilidade e precisão para esse método, quando comparado a PCR quantitativo. Nossos resultados indicam que as células de retina de embrião de 8 dias de galinha em cultura já contêm um relógio funcional, porém, este necessita do ciclo claro-escuro ou glutamato para sua sincronização. / The avian circadian system is composed by the retina, the mammalian homolog region of the supra-chiasmatic nucleus (SNC) and the pineal gland. The retina itself shows many rhythmic physiological events, such as movements of photoreceptor cells, opsin expression, retinaldehyde re-isomerization, melatonin and dopamine production and release. Altogether these rhythmic events are coordinated to predict environmental changes in light conditions during the day, optimizing retina function. In this work we investigated the expression of key components of a circadian system, including the two melanopsin genes, Opn4x, Opn4m, as well as the Clock, Per2, N-Acetyltransferase and Tyrosine Hidroxylase genes in chick embryo retinal cells. Primary cultures of chicken retina from 8-day-old embryos were prepared at ZT0 (lights on) and seeded at the density of 107 cells per 25 cm2 culture flask. The cells were kept in a humidified incubator in a 5% CO2 atmosphere at 40o C in constant dark, in 12L:12D, in 12L:12D followed by constant dark, or in constant dark in the absence or presence of 100 μM glutamate for 12 h starting at ZT0 of the fifth day in vitro. Total RNA extraction was performed along 24 hours every three hours starting at ZT0 of the sixth day. The samples were submitted to RT-PCR followed by quantitative PCR for mRNA quantification. To analyze the Opn4x expression in these cells we performed an immunocytochemistry analysis with antibodies anti-chicken melanopsin developed in rabbit. We also quantified the protein levels of OPN4x, CLOCK AND TYROSINE HYDROXYLASE by Western Blot. The mRNA quantification showed no rhythm of transcription for any gene in cells kept in constant dark. However under a light-dark cycle, Clock, Per2, Opn4m, N-Acetyltransferase and Tyrosine Hydroxylase presented rhythm patterns of transcription. 100 μM glutamate was able to induce rhythmic expression of Clock, and strongly inhibited the expression of Tyrosine Hydroxylase and, just punctually, of Opn4x and Opn4m. Assays of cell viability and DNA fragmentation using flow cytometry demonstrated that the inhibition did not result of glutamate toxic or apoptotic actions. The neurotransmitter had no effect on Per2 and N-Acetyltransferase transcription. Protein quantification by Western Blot showed no rhythmic oscillation of CLOCK, OPN4x or TYROSINE HYDROXYLASE. The great variability inter-assays seen in the results of protein quantification suggests that this method is less precise and sensitive than quantitative PCR. The present data show evidences that chicken embryonic retinal cells contain a functional circadian Clock. However light-dark cycle or glutamate stimuli are needed to its synchronization.

Orientador: Renée Laufer Amorim / Coorientador: José Luiz Laus / Banca: Juliany Quitzan Gomes / Banca: Alvio Isao Shiguematsu / Resumo: Não disponível / Abstract: Purpose: The aims of this study were to better understand the mechanism of cell death by apoptosis in a glaucoma model (ischemia / reperfusion) and evaluate the role of apoptosis in this model and if treatment with Sildenafil helps prevent apoptosis. Methods: 36 rats, from 4 to 6 months, males, Lewis and weighing ± 350g were divided in 5 groups: control group (6 animals) and for groups with ischemia / reperfusion (7 and 21 days), two groups consisting of ten animals treated with sildenafil and two groups of Five animals treated with placebo. Paracentesis of the anterior chamber with needle 30G coupled to saline (0.9%) was made and maintained for 60 minutes. Intraocular pressure was measured by rebound tonometer (Tonovet®). There was histological, morphometric by hematoxylin and eosin and, immunohistochemical staining and qRT-PCR analysis by Caspase-7, Caspase-6, Caspase-9, Tnf-r2, Fas-l, Bcl-2 and Bax. For statistic analysis we used ANOVA and t-test for morphometric analysis and, for immunohistochemistry and qRT-PCR, Fisher exact test was employed with a statistical significance level of p <0.05 Results: Histology and morphometric analysis, proved more changes in the untreated group compared to the treatment and control group. Analysis of immunohistochemistry and qRT-PCR observed the more significant expression in untreated eyes. Conclusion: Sildenafil apperead to be protective to ganglion cell apoptosis. Cell survival was evident in histology and morphometry. For immunohistochemistry and RT-PCR was observed protective effect in the apoptosis pathways with similar or below expression compared to the control / Mestre

A síntese de melatonina na retina ocorre de maneira circadiana na camada fotorreceptora, e age localmente promovendo a adaptação fisiológica da retina ao escoto-período. Sabendo-se que o estresse oxidativo contribui com o desenvolvimento da retinopatia diabética e que a melatonina é um poderoso antioxidante natural, o objetivo deste trabalho foi avaliar na retina de animais diabéticos o conteúdo de melatonina e as enzimas envolvidas em sua via de síntese. Ratos Wistar (250-280g, 12h/12h claro/escuro) tiveram o quadro diabético induzido por STZ e foram tratados ou não com insulina. Os animais foram sacrificados 3 dias pós-indução. Os resultados mostraram que o diabetes causa a diminuição no conteúdo de melatonina retiniana devido à redução da atividade da AANAT causada pela queda no conteúdo de AMPc. O tratamento com insulina restabeleceu tanto o perfil de atividade da AANAT quanto à síntese de melatonina. Supõe-se que as retinas dos animais diabéticos estão mais vulneráveis aos danos decorrentes do quadro diabético, já que a síntese da melatonina está diminuída. / Melatonin controls several retinal physiologic rhythmic phenomena besides being the most powerful natural free radical scavenger. The aim of this work is evaluating the retinal melatonin diurnal profile of diabetic rats. Male Wistar rats were diabetic-induced by an injection of STZ and some of them were treated with insulin (6U/day). Retinas were obtained throughout the 24h light-dark cycle for retinal melatonin content, enzymes activity, cAMP content and gene expression analysis. Control animals showed a retinal melatonin daily rhythm with high levels at night while diabetic rats presented a reduction. The AANAT activity showed a daily rhythm with high levels at night in the control group. This daily fluctuation was vanished in STZ-induced diabetic rats and insulin-treatment restored both parameters. We suppose the AANAT activity reduction observed is due to the reduced cAMP content. As melatonin plays an important role modulating circadian functions, this impairment could compromise the retinal physiological homeostasis.

Die vorliegende kumulative Habilitationsschrift von Dr. rer. nat. Antje Grosche umfasst neben einer Einführung in die Physiologie der dominierenden Gliazellen der Netzhaut – der Müllerschen Gliazellen - zehn Originalarbeiten zur Bedeutung von Gliotransmittern für die Volumenregulation der Müllerzellen. Eine effiziente Volumenregulation gilt als Voraussetzung dafür, dass Müllerzellen ihrer Rolle als stabilisierendes Element hinsichtlich des Ionen- und Wasserhaushaltes in der Netzhaut gerecht werden können. Aus Vorarbeiten war bekannt, dass Müllerzellen typische Eigenschaften, wie die hohe Kaliumleitfähigkeit ihrer Membranen und damit einhergehend eine ausgeprägte Fähigkeit zur Volumenregulation, in der pathologisch veränderten Netzhaut verlieren (Pannicke et al., 2004; Wurm et al., 2006b). Diese Reaktion der Müllerzellen auf Netzhautschädigungen wird als gliotische Aktivierung bezeichnet und konnte in der soeben beschriebenen Ausprägung im ersten Abschnitt der vorliegenden Arbeit an Netzhäuten von Patienten mit uvealem Melanom bestätigt werden (Grosche et al., 2012). Weiterhin wurde die Relevanz einer in früheren Arbeiten identifizierten glutamaterg-purinergen Signalkaskade für die Funktion von gliotischen Müllerzellen detailliert untersucht. Im ersten Abschnitt dieser Habilitationsschrift wird nachgewiesen, dass sowohl im Modell für eine akute Schädigung der Netzhaut (Netzhautablösung im Schwein, Wurm et al., 2011), als auch bei einer chronischen Erkrankung (Streptozotocin-induzierter Diabetes in der Ratte, Wurm et al., 2008c) durch Aktivierung besagter Kaskade trotz verminderter Kaliumströme die Volumenregulation der Zellen wieder hergestellt werden kann. Ein zweiter Schwerpunkt der Untersuchungen war die umfassende Analyse der Expressions-muster und Funktion der an der Volumenregulation beteiligten Rezeptoren (insbesondere P2Y-Rezeptoren) in juvenilen und adulten Müllerzellen. Daten über die Differenzierung von Müllerzellen der Ratte während der ersten drei Postnatalwochen unterstreichen die herausragende Bedeutung von P2Y-Rezeptoren bzw. belegen deren Beteiligung an verschiedenen Müllerzellfunktionen (Volumenregulation, kalziumabhängige Signalwege). Interessanterweise wurden die meisten Effekte von ATP über die Aktivierung von P2Y1-Rezeptoren vermittelt (Wurm et al., 2009a, 2010). Zudem konnte durch Einsatz von P2Y1-Rezeptor-, IP3 Rezeptorsubtyp 2- und A1-Rezeptor-defizienten Mäusen belegt werden, dass ein störungsfreies Wirken der glutamaterg-purinergen Signalkaskade (neben der hohen Kaliumleitfähigkeit der Membranen) auch in unbehandelten Müllerzellen essentiell für deren Volumenregulation ist (Wurm et al., 2009b, 2010; Lipp et al., 2009). Im dritten Themenkomplex wurde der Frage nachgegangen, welche Mechanismen bei der Freisetzung der in die Volumenregulation involvierten Gliotransmitter eine Rolle spielen. So konnte eine exozytotische Ausschüttung von Glutamat bestätigt werden, während die Freisetzung von ATP primär durch Hemichannels (Linnertz et al., 2011; Brückner et al., 2012; Slezak et al., 2012) und die von Adenosin durch Nukleosidtransporter sowie extrazelluläre Degradation von ATP/ADP vermittelt wird (Wurm et al., 2010). Abschließend ordnet das Kapitel „Zusammenfassung und Ausblick“ die neu gewonnenen Erkenntnisse dieser Habilitationsschrift über die Rolle von Gliotransmittern für die Funktionen von Müllerzellen (einschließlich unveröffentlichter Daten aus weiterführenden Experimenten) für zukünftige Forschungsvorhaben ein.