1. Investigación

(1) Background: Retinitis pigmentosa (RP) is characterized by progressive photoreceptor death. A Prph2Rd2 or an rds mouse is an RP model that closely reflects human RP. The objective of this study was to investigate the relationship of rod and cone death with oxidative stress and inflammation in rds mice. (2) Methods: The retinas of control and rds mice on postnatal days (PN) 11, 17, 21, 28, 35, and 42 were used. Oxidative damage to macromolecules, glutathione (GSH and GSSG), GSH synthesis enzymes, glial fibrillar acidic protein (GFAP), ionized calcium-binding adapter molecule 1 (Iba1), and cluster of differentiation 68 (CD68) was studied. (3) Results: The time sequence of oxidative stress and inflammation changes in rds mice occurs as follows: (i) At PN11, there is a small increase in photoreceptor death and in the microglial cells; (ii) at PN17, damage to the macromolecules is observed; (iii) at PN21, the maximum photoreceptor death rate is detected and there is an increase in GSH-GSSG and GFAP; (iv) at PN21, the microglial cells are activated; and(v) at PN28, there is a decrease in GSH synthesis enzymes. (4) Conclusions: These findings contribute to the understanding of RP physiopathology and help us to understand whether oxidative stress and inflammation are therapeutic targets. These findings contribute to our understanding that, in RP, oxidative stress and inflammation evolution and their relationship are time-dependent. In this sense, it is important to highlight that both processes are potential therapeutic targets in this disease.

PURPOSE. To assess the changes in retinal morphology in a rat model of chronic glaucoma induced by ocular hypertension. METHODS. Intraocular pressure (IOP) was surgically increased through weekly injections of sodium hyaluronate (HYA) in the anterior eye chamber of the left eye of male Wistar rats, whereas the right eyes were sham operated (salt solution). During the 10-week experimental period, IOP was measured weekly with a rebound tonometer. Retinal cryosections were prepared for histological/immunohistochemical analysis and morphometry. RESULTS. IOP was higher in HYA-treated eyes than in sham-operated eyes along the 10- week period, which was significant from the fourth to the nineth week. Ocular hypertension in HYA-treated eyes was associated with morphologic and morphometric changes in bipolar cells, ON-OFF direction-selective ganglion cells, ON/OFF starburst amacrine cells, and inner plexiform layer sublamina. CONCLUSIONS. Serial HYA treatment in the rat anterior eye chamber results in mild-tomoderate elevated and sustained IOP and ganglion cell death, which mimics most human open-angle glaucoma hallmarks. The reduced number of direction-selective ganglion cells and starburst amacrine cells accompanied by a deteriorated ON/OFF plexus in this glaucoma model could lend insight to the abnormalities in motion perception observed in patients with glaucoma.

The term retinitis pigmentosa (RP) describes a large group of hereditary retinopathies. From a cellular view, retinal degeneration is prompted by an initial death of rods, followed later by cone degeneration. This cellular progressive degeneration is translated clinically in tunnel vision, which evolves to complete blindness. The mechanism underlying the photoreceptor degeneration is unknown, but several mechanisms have been pointed out as main co-stars, inflammation being one of the most relevant. Retinal inflammation is characterized by proliferation, migration, and morphological changes in glial cells, in both microglia and Müller cells, as well as the increase in the expression of inflammatory mediators. Retinal inflammation has been reported in several animal models and clinical cases of RP, but the specific role that inflammation plays in the pathology evolution remains uncertain. Sulforaphane (SFN) is an antioxidant natural compound that has shown antiinflammatory properties, including the modulation of glial cells activation. The present work explores the effects of SFN on retinal degeneration and inflammation, analyzing the modulation of glial cells in the RP rd10 mice model. A daily dose of 20 mg/kg of sulforaphane was administered intraperitoneally to control (C57BL/6J wild type) and rd10 (Pde6brd10) mice, from postnatal day 14 to day 20. On postnatal day 21, euthanasia was performed. Histological retina samples were used to assess cellular degeneration, Müller cells, and microglia activation. SFN administration delayed the loss of photoreceptors. It also ameliorated the characteristic reactive gliosis, assessed by retinal GFAP expression. Moreover, sulforaphane treatment regulated the microglia activation state, inducing changes in the microglia morphology, migration, and expression through the retina. In addition, SFN modulated the expression of the interleukins 1β, 4, Ym1, and arginase inflammatory mediators. Surprisingly, M2 polarization marker expression was increased at P21 and was reduced by SFN treatment. To summarize, SFN administration reduced retinal neurodegeneration and modified the inflammatory profile of RP, which may contribute to the SFN neuroprotective effect.

Degenerative diseases and among them those that affect the visual system, represent a huge problem for the society. This problem becomes even more important with the aging of the population. The proposed project aims to improve the quality of life of the patients by knowing the pathophysiological basis of two diseases that affect the vision: retinitis pigmentosa and myopia. The visual loss is an indisputable decrease in the quality of life and the consequences are not only restricted on a personal level, but also on a social and economic level. For all these reasons, new treatments in this diseases are urgently and precisely needed and the knowledge of the mechanisms underlying. Retinitis pigmentosa (RP) is a frequent form of retinal degeneration that is the main genetic cause of blindness in developed countries. Genetic mutations are responsible for the death of the rods. However, the death of the cones seems to occur due to the metabolic changes caused by the degeneration of the rods such as hyperoxia (oxidative stress), the secretion of various factors (cytokines, chemokines) by sticks, and so on. The death of the cones causes the loss of the central vision in RP. For this, firstly, the antioxidant response was analyzed and also the presence of oxidative stress markers in the retina of the mice with RP that could be involved in the progression or in the delay of the disease. Secondly, the expression of the glia and various growth factors were analyzed by immunohistochemistry and western blot. Finally, thioredoxin (TRX), a potent antioxidant with anti-inflammatory power, was intraperitoneally administered. We evaluated the effect of TRX on the progression of retinal degeneration in the RP model, the rd1 mouse. The results obtained in this study show that the mice with RP have a deficient antioxidant response in the retina. The mice with the treatment showed better antioxidant response, with high levels of GSH, suggesting a role of oxidative stress in the progression of the disease. Otherwise, no modification of the expression of VEGF and a tendency to increase in HGF levels was observed in the retina, with and without treatment. In the retina of these mice, a reduction in some parameters of glial activation is observed. In rd1 mice, treatment with thioredoxin, antioxidant, antiinflammatory and antiapoptotic, reduces the reactive gliosis to postnatal day 11 and 17. In short, these results confirm the hypothesis that alterations of the antioxidant system and inflammatory processes in RP are involved in the pathogenesis of the disease. Treatments of endogenous antioxidants such as TRX can be useful for the search of therapeutic targets that prevent or delay the death of photoreceptors. Our results suggest that in RP, oxidative stress, active gliosis and apoptosis, among others, play an important role in the death of retinal cells. The design of strategies that promote their blockade can be promising therapies in PR. Otherwise, myopia is an optical problem of defocusing which is due to a mismatch between the axial length of the eyeball and the lenses that comprise it. Myopia is also a medical problem that predisposes people who bear it, to suffer more frequently other ocular pathologies: retinal detachments, glaucoma, cataracts among others. There are basically two types of myopia with a very different prognosis, although the limits between both are not clearly defined. The parameter that most correlates with them is the axial length, being recognized as more appropriate to fix in 26mm the limit between both. Above these values we speak of high myopia (AM). This type of myopia is characterized by a progressive elongation of the eyeball uninterrupted during life. It is accompanied by progressive atrophy of ocular tissues and leads to blindness over the years in a large percentage of the affected population. The genetic has linked several growth factors with AM. For it, some growth factors, VEGF and HGF, were determined in patient eye samples. Thus, these factors can be related to their clinic and structural changes in the retina of these patients. The knowledge of the biochemical and molecular aspects that accompany these clinical stages will allow us to understand in depth the process and be able to find points of therapeutic approach. The results obtained in this study show that patients with AM have a poor antioxidant response in the aqueous humor. Patients with high myopia had higher levels of HGF as the degree of retinal degeneration increased, quite the opposite of what happened with VEGF, a growth factor that significantly decreases in patients with high myopia. / Las enfermedades degenerativas, y entre ellas las que afectan al sistema visual, representan un enorme problema para la sociedad, problema que será aún más importante con el envejecimiento de la población. El proyecto propuesto pretende mejorar la calidad de vida de los pacientes conociendo las bases fisiopatológicas de dos enfermedades que afectan a la visión: la Retinosis Pigmentaria (RP) y la miopía. La pérdida visual constituye una indiscutible disminución de la calidad de vida y las consecuencias no están sólo restringidas a nivel personal sino también a nivel social y económico. Por todo ello, son necesarios nuevos tratamientos en este tipo de enfermedades de forma urgente y precisa, y el conocimiento de los mecanismos subyacentes a estas enfermedades nos ayudará a encontrar estos tratamientos. La RP es una forma frecuente de degeneración retiniana que constituye la principal causa genética de ceguera en los países desarrollados. Las mutaciones genéticas son las responsables de la muerte de los bastones, los fotorreceptores situados mayoritariamente en la zona de la periferia. Sin embargo, la muerte de los conos parece que se produce debida a los cambios metabólicos que provoca la degeneración de los bastones como la hiperoxia (estrés oxidativo), la secreción de diversos factores (citoquinas, quimioquinas) por parte de los bastones, etc. La muerte de los conos provoca la pérdida de la visión central en RP. Para ello, en primer lugar, se analizó la respuesta antioxidante, la presencia de marcadores de estrés oxidativo en retina de ratones con RP que pudiesen estar implicados en la progresión o en el retraso de la enfermedad. En segundo lugar, se analizó la expresión de la glía y de diversos factores de crecimiento mediante inmunohistoquímica y western blot. Por último, se administró intraperitonealmente tiorredoxina (TRX), un potente antioxidante con poder antiinflamatorio. Se evaluó́ el efecto de la TRX sobre la progresión de la degeneración retiniana en un modelo de RP, el ratón rd1. Los resultados obtenidos en este estudio muestran que los ratones con RP presentan una deficiente respuesta antioxidante en la retina. Los ratones con el tratamiento mostraban mejor respuesta antioxidante, con niveles elevados de GSH, sugiriendo un papel del estrés oxidativo en la progresión de la enfermedad. Por otro lado, no se observó ninguna modificación de la expresión de VEGF y se observó una tendencia al aumento en los niveles de HGF en la retina de los ratones rd1, con y sin tratamiento. Con el tratamiento de TRX, en la retina de estos ratones se observa una reducción en algunos parámetros de la activación glial. En el ratón rd1, el tratamiento con TRX, reduce la gliosis reactiva a día postnatal 11 y 17. En definitiva, estos resultados confirman la hipótesis de que alteraciones del sistema antioxidante y los procesos inflamatorios en la RP están implicados en la patogénesis de la enfermedad. Tratamientos de antioxidantes endógenos como la TRX pueden ser útiles para la búsqueda de nuevas dianas terapéuticas que retrasen la muerte de los fotorreceptores. Nuestros resultados sugieren que en la RP el estrés oxidativo, la gliosis activa y la muerte celular entre otros, desempeñan un papel importante en la muerte de los fotorreceptores. Por otra parte, la miopía es un problema óptico de desenfoque que se debe a un desajuste entre la Longitud Axial (LA) del globo ocular y las lentes que lo componen. También es un problema médico que predispone a las personas que la padecen a sufrir con mayor frecuencia otras patologías oculares, tales como desprendimientos de retina, glaucoma y cataratas entre otras. Existen fundamentalmente dos tipos de miopía con pronóstico bien distinto, aunque los límites entre ambas no están netamente definidos. El parámetro que más se correlaciona con ellos es la LA, reconociéndose como más adecuado fijar en 26mm el límite entre ambas. Por encima de estos valores hablamos de Alta Miopía (AM). Este tipo de miopía se caracteriza por un alargamiento progresivo del globo ocular ininterrumpido durante la vida. Se acompaña de atrofia progresiva de los tejidos oculares y lleva a la ceguera con los años en un gran porcentaje de la población afectada. La genética ha relacionado varios factores de crecimiento con la AM. Por eso, se determinaron algunos factores de crecimiento, el factor de crecimiento endotelial vascular (VEGF) y el factor de crecimiento de hepatocitos (HGF), en muestras de humor acuoso de los pacientes. Así, se pueden relacionar estos factores con su clínica y los cambios estructurales que suceden en la retina de estos pacientes. El conocimiento de los aspectos bioquímicos y moleculares que acompañan estos estadios clínicos nos permitirá entender en profundidad el proceso de la enfermedad y poder encontrar puntos de abordaje terapéutico. Los resultados obtenidos en este estudio muestran que los pacientes con AM presentan una deficiente respuesta antioxidante en el humor acuoso. Los pacientes con alta miopía presentaban unos niveles mayores de HGF a medida que aumentaba el grado de degeneración de la retina, todo lo contrario que sucedía con el VEGF, factor de crecimiento que disminuye significativamente en los pacientes con alta miopía.

The retina may suffer neurodegenerative damages, as other tissues of the central nervous system do, and serious eye diseases may develop. One of them is age-related macular degeneration, which causes progressive loss of vision due to retina degeneration. Treatment of age-related macular degeneration focuses on antioxidant agents and anti-vascular endothelial growth factor compounds, among others, that prevent/ diminish oxidative stress and reduce neovascularisation respectively. The phytochemicals, medicinal plants and/or plant-diet supplements might be a useful adjunct in prevention or treatment of age-related macular degeneration owing to their antioxidant and anti-vascular endothelial growth factor properties. This review article presents the most investigated plants and natural products in relation to age-related macular degeneration, such as saffron, ginkgo, bilberry and blueberry, curcuma or turmeric, carotenoids, polyphenols, and vitamins C and E. This study provides up-to-date information on the effects, treatments, safety and efficiency of these phytotherapy products.

Retinitis pigmentosa (RP) is an inherited ocular disorder with no effective treatment. RP onset and progression trigger a cascade of retinal disorders that lead to the death of photoreceptors. After photoreceptors death, neuronal, glial and vascular remodeling can be observed in the retina. The purpose of this study was to study if thioredoxin (TRX) administration is able to decrease photoreceptor death in an animal model of RP (rd1 mouse), but also if it is able to modulate the retinal oxidative stress, glial and vascular changes that can be observed as the disease progresses. Wild type and rd1 mice received several doses of TRX. After treatment, animals were euthanized at postnatals days 11, 17, or 28. Glutathione (GSH) and other thiol compounds were determined by high performance liquid chromatography (HPLC). Glial fibrilary acidic protein (GFAP) and anti-ionized calcium binding adaptor molecule 1 (Iba1) were studied by immunohistochemistry. Vascular endothelial growth factor (VEGF) and hepatic growth factor (HGF) expression were determined by western blot. TRX administration significantly diminished cell death in rd1 mouse retinas and increased GSH retinal concentrations at postnatal day 11 (PN11). TRX was also able to reverse glial alterations at PN11 and PN17. No alterations were observed in retinal VEGF and HGF expression in rd1 mice. In conclusion, TRX treatment decreases photoreceptor death in the first stages of RP and this protective effect may be due in part to the GSH system activation and to a partially decrease in inflammation.