1. Investigación

The expressions of ion channels by Müller glial cells (MGCs) may change in response to various retinal pathophysiological conditions. There remains a gap in our understanding of MGCs' responses to photoreceptor degeneration towards finding therapies. The study explores how an inhibition of store-operated Ca2+ entry (SOCE) and its major component, Orai1 channel, in MGCs protects photoreceptors from degeneration. The study revealed increased Orai1 expression in the MGCs of retinal degeneration 10 (rd10) mice. Enhanced expression of oxidative stress markers was confirmed as a crucial pathological mechanism in rd10 retina. Inducing oxidative stress in rat MGCs resulted in increasing SOCE and Ca2+ release-activated Ca2+ (CRAC) currents. SOCE inhibition by 2-Aminoethoxydiphenyl borate (2-APB) protected photoreceptors in degenerated retinas. Finally, molecular simulations proved the structural and dynamical features of 2-APB to the target structure Orai1. Our results provide new insights into the physiology of MGCs regarding retinal degeneration and shed a light on SOCE and Orai1 as new therapeutic targets.

The retina is highly susceptible to the generation of toxic reactive oxygen species (ROS) that disrupt the normal operations of retinal cells. The glutathione (GSH) antioxidant system plays an important role in mitigating ROS. To perform its protective functions, GSH depends on nicotinamide adenine dinucleotide phosphate (NADPH) produced through the pentose phosphate pathway. This work develops the first mathematical model for the GSH antioxidant system in the outer retina, capturing the most essential components for formation of ROS, GSH production, its oxidation in detoxifying ROS, and subsequent reduction by NADPH. We calibrate and validate the model using experimental measurements, at different postnatal days up to PN28, from control mice and from the rd1 mouse model for the disease retinitis pigmentosa (RP). Global sensitivity analysis is then applied to examine the model behavior and identify the pathways with the greatest impact in control compared to RP conditions. The findings underscore the importance of GSH and NADPH production in dealing with oxidative stress during retinal development, especially after peak rod degeneration occurs in RP, leading to increased oxygen tension. This suggests that stimulation of GSH and NADPH synthesis could be a potential intervention strategy in degenerative mouse retinas with RP.

(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.

Progesterone (PG) diminishes free radical damage and thus can afford protection against oxidative stress affecting the retina. The therapeutic use of PG is limited because it is a highly hydrophobic steroid hormone with very low solubility in water. This is the main drawback for the therapeutic application of PG at ocular level. The aims of this study were: (i) to analyze if PG causes ocular irritation (ii) to validate a HPLC method to determine PG in ex vivo studies and (iii) to evaluate PG permeation through cornea and sclera. A high performance liquid chromatographic method was developed and validated to detect PG incorporated to β-cyclodextrin using a Waters Sunfire C18 (150 × 4.6 mm) reverse-phase column packed with 5 μm silica particles using a mobile phase consisted of a mixture of acetonitrile (ACN) and pure water 80:20 (v/v), pH 7.4. The limit of detection and the limit of quantification for 50 μL injection of PG were found to be 0.42 and 1.26 μg/mL, respectively. The calibration curve showed excellent linearity over the concentration range (0.5 μg/mL to 100 μg/mL). As proof of concept, ex-vivo experiments to investigate PG permeation through cornea and sclera with vertical diffusion cells were carried out to quantify PG diffusion. Ex vivo experiments demonstrate its applicability to investigate permeation levels of PG from 6.57 ± 0.37 μg/cm2 at cornea and 8.13 ± 0.85 μg/cm2 sclera. In addition, at the end of diffusion studies the amount of PG retained in each tissue was also quantified, and it was 40.87 ± 9.84 μg/cm2 (mean ± SD; n = 6) in cornea and 56.11 ± 16.67 μg/cm2 (mean ± SD; n = 6) in sclera.

(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.

Retinitis pigmentosa (RP) is an inherited eye disorder which triggers a cascade of retinal disorders leading to photoreceptor cell death and for which there is currently no effective treatment. The purpose of this research was to study whether ocular administration of a solution of progesterone (PG) in -cyclodextrins (CD) could delay photoreceptor cell death and counteract the gliosis process in an animal model of RP (rds mice). The possible effect of PG reaching the contralateral eye through the circulatory system was also evaluated. Finally, this research discusses and evaluates the diffusion of the drug from possible topical formulations for ocular administration of PG. A group of rds mice received one drop of a solution of PG in CD every 12 h for 10 days to the left eye, while the right eye was left untreated. Another group of rds mice (control) received the drug vehicle (PBS) on the left eye and, again, the right eye was left untreated. Once the treatment was finished on postnatal day 21, the animals were euthanized and histological immunofluorescence studies (TUNEL, GFAP, and DAPI staining) were carried out. Our results showed that the administration of a solution of PG in CD (CD-PG) as drops significantly decreased cell death and inflammation in the retina of the PG-treated eyes of rds mice. No effect was seen in the contralateral eye from PG that may have entered systemic circulation. In conclusion, CD-PG applied topically as drops to the eye decreases photoreceptor cell death in the early stages of RP, delaying vision loss and decreasing gliosis.

Oxidative stress is associated with multiple pathologies such as cancer and can exacerbate the development of them. In this work, we have studied the antioxidant capacity of 5-Fluorouracile (5-FU) which is an antineoplastic drug that is used in the treatment of colorectal cancer. 5-FU is a compound that has a chemical structure similar to uracil and is also fluorinated. New fluorinated derivates previously obtained in our laboratory were tested to study its antioxidant activity. All the compounds analyzed were able to inhibit lipid peroxidation when used in concentrations of 10 μM.

Ochratoxin A (OTA) is a common secondary metabolite of Aspergillus ochraceus, A. carbonarius, and Penicillium verrucosum. This mycotoxin is largely present as a contaminant in several cereal crops and human foodstuffs, including grapes, corn, nuts, and figs, among others. Preclinical studies have reported the involvement of OTA in metabolic, physiologic, and immunologic disturbances as well as in carcinogenesis. More recently, it has also been suggested that OTA may impair hippocampal neurogenesis in vivo and that this might be associated with learning and memory deficits. Furthermore, aside from its widely proven toxicity in tissues other than the brain, there is reason to believe that OTA contributes to neurodegenerative disorders. Thus, in this present in vivo study, we investigated this possibility by intraperitoneally (i.p.) administering 3.5 mg OTA/kg body weight to adult male mice to assess whether chronic exposure to this mycotoxin negatively affects cell viability in the dentate gyrus of the hippocampus. Immunohistochemistry assays showed that doses of 3.5 mg/kg caused a significant and dose-dependent reduction in repetitive cell division and branching (from 12% to 62%). Moreover, the number of countable astrocytes (p < 0.001), young neurons (p < 0.001), and mature neurons (p < 0.001) negatively correlated with the number of i.p. OTA injections administered (one, two, three, or six repeated doses). Our results show that OTA induced adverse effects in the hippocampus cells of adult mice brain tissue when administered in cumulative doses.

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.