1. Investigación

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.

Progesterone (PG) may provide protection to the retina during retinitis pigmentosa, but its topical ocular supply is hampered by PG poor aqueous solubility and low ocular bioavailability. The development of e cient topical ocular forms must face up to two relevant challenges: Protective barriers of the eyes and lack of validated ex vivo tests to predict drug permeability. The aims of this study were: (i) To design micelles using Pluronic F68 and Soluplus copolymers to overcome PG solubility and permeability; and (ii) to compare drug di usion through the cornea and sclera of three animal species (rabbit, porcine, and bovine) to investigate interspecies di erences. Micelles of Pluronic F68 (3–4 nm) and Soluplus (52–59 nm) increased PG solubility by one and two orders of magnitude, respectively and exhibited nearly a 100% encapsulation e ciency. Soluplus systems showed in situ gelling capability in contrast to the low viscosity Pluronic F68 micelles. The formulations successfully passed the hen’s egg-chorioallantoic membrane test (HET-CAM) test. PG penetration through rabbit cornea and sclera was faster than through porcine or bovine cornea, although the di erences were also formulation-dependent. Porcine tissues showed intermediate permeability between rabbit and bovine. Soluplus micelles allowed greater PG accumulation in cornea and sclera whereas Pluronic F68 promoted a faster penetration of lower PG doses.

Exposure to sunlight and contact with atmospheric oxygen makes the eye particularly susceptible to oxidative stress, which can potentially produce cellular damage. In physiological conditions, there are several antioxidant defense mechanisms within the eye. Glutathione (GSH) is the most important antioxidant in the eye; GSH deficit has been linked to several ocular pathologies. The aim of this study was to explore the potential for newly developed formulations allowing controlled delivery of antioxidants such as GSH and vitamin C (Vit C) directly to the eye. We have investigated the stability of antioxidants in aqueous solution and assessed ex-vivo the di usion of GSH through two ocular membranes, namely cornea and sclera, either in solution or included in a semisolid insert. We have also carried out the hen’s egg-chlorioallantoic membrane test (HET-CAM) to evaluate the ocular irritancy of the di erent antioxidant solutions. Our results showed that GSH is stable for up to 30 days at 4 C in darkness and it is not an irritant to the eye. The di usion studies revealed that the manufactured formulation, a semisolid insert containing GSH, could deliver this tripeptide directly to the eye in a sustained manner.

The interactions between steroid gonadal hormones and the retina (a part of the visual system and the central nervous system (CNS)) have received limited attention and beneficial effects of these hormones in retinal diseases is controversial. Retinitis pigmentosa (RP) is the most common cause of retinal hereditary blindness and to date no treatment is available. However, results regarding the effects of progesterone on the progression of RP are promising. With the idea of demonstrating if the progesterone retinal protection in RP is related to its possible anti-inflammatory properties, we have administered orally progesterone to rd10 mice, an animal model of RP. We observed that progesterone decreased photoreceptors cell death, reactive gliosis and the increase in microglial cells caused by RP. We also examined the expression of neuronal and inducible nitric oxide synthase (nNOS and iNOS), the enzyme responsible for NO production. The results demonstrated a decrease in nNOS expression only in control mice treated with progesterone. Inflammation has been related with an increase in lipid peroxidation. Noticeably progesterone administration was able to diminish retinal malondialdehyde (MDA, a lipid peroxidation product) concentrations in rd10 mice. Altogether, we can conclude that progesterone could be a good therapeutic option not only in RP but also for other retinal diseases that have been associated with inflammation and lipid peroxidation.