Gimeno Hernández, Roberto

(1) Background: the aim of this work was to study microglia and autophagy alterations in a one retinitis pigmentosa (RP) model at different stages of the disease (when rods are dying and later, when there are almost no rods, and cones are the cells that die. (2) Methods: rd1 mice were used and retinas obtained at postnatal days (PN) 11, 17, 28, 35, and 42. Iba1 (ionized calcium-binding adapter molecule 1) was the protein selected to study microglial changes. The macroautophagy markers Beclin-1, Atg5, Atg7, microtubule-associated protein light chain 3 (LC3), and lysosomal-associated membrane protein 2 (LAMP2) (involved in chaperone-mediated autophagy (CMA)) were determined. (3) Results: the expression of Iba1 was increased in rd1 retinas compared to the control group at PN17 (after the period of maximum rod death), PN28 (at the beginning of the period of cone death), and PN42. The number of activated (ameboid) microglial cells increased in the early ages of the retinal degeneration and the deactivated forms (branched cells) in more advanced ages. The macroautophagy markers Atg5 at PN11, Atg7 and LC3II at PN17, and Atg7 again at PN28 were decreased in rd1 retinas. At PN35 and PN42, the results reveal alterations in LAMP2A, a marker of CMA in the retina of rd1 mice. (4) Conclusions: we can conclude that during the early phases of retinal degeneration in the rd1 mouse, there is an alteration in microglia and a decrease in the macroautophagy cycle. Subsequently, the CMA is decreased and later on appears activated as a compensatory mechanism.

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.

Retinitis pigmentosa (RP) is a group of inherited retinopathies characterized by photoreceptors death. Our group has shown the positive progesterone (P4) actions on cell death progression in an experimental model of RP. In an effort to enhance the beneficial effects of P4, the aim of this study was to combine P4 treatment with an antioxidant [lipoic acid (LA)] in the rd1 mice. rd1 and control mice were treated with 100 mg/kg body weight of P4, LA, or a combination of both on postnatal day 7 (PN7), 9, and 11, and were sacrificed at PN11. The administration of LA and/or P4 diminishes cell death in rd1 retinas. The effect obtained after the combined administration of LA and P4 is higher than the one obtained with LA or P4 alone. The three treatments decreased GFAP staining, however, in the far peripheral retina, and the two treatments that offered better results were LA and LA plus P4. LA or LA plus P4 increased retinal glutathione (GSH) concentration in the rd1 mice. Although LA and P4 are able to protect photoreceptors from death in rd1 mice retinas, a better effectiveness is achieved when administering LA and P4 at the same time.

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.