Facultad de Ciencias de la Salud

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.

(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.

Connexins (Cx) are a family of transmembrane proteins that form gap junction intercellular channels that connect neighboring cells. These channels allow the passage of ions and other biomolecules smaller than 1 kDa, thereby synchronizing the cells both electrically and metabolically. Cxs are expressed in all retinal cell types and the diversity of Cx isoforms involved in the assembly of the channels provides a functional syncytium required for visual transduction. In this chapter, we summarize the status of current knowledge regarding Cx biology in retinal tissues and discuss how Cx dysfunction is associated with retinal disease pathophysiology. Although the contribution of Cx deficiency to retinal degeneration is not well understood, recent findings present Cx as a potential therapeutic target. Therefore, we will briefly discuss pharmacological approaches and gene therapies that are being explored to modulate Cx function and fight sight-threatening eye diseases.

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.