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Received 16 October 2013 / Accepted in revised form 25 February 2014 / Available online 12 March 2014, The DBA/2J mouse is a model of ocular hypertension and retinal ganglion cell (RGC) degeneration, the main features of which are iris pigment dispersion (IPD) and iris stromal atrophy (ISA). These animals also experience glaucomatous changes, including an increase in intraocular pressure (IOP) beginning at about 9-12 months of age and sectorial RGC death in the retina. The aim of this study was to determine the onset of functional changes exhibited by DBA/2J mice in the inner retina. This was performed by means of electroretinographic recordings (scotopic threshold response, STR) and their correlation with morphological changes (loss of RGCs). To this end, we recorded the scotopic threshold response in control C57BL/6J and in DBA/2J mice at different ages. The RGCs, in both DBA/2J and C57BL/6J animals, were identified at 15 months of age by retrograde tracing with an analogue of fluorogold, hydroxystilbamidine methanesulfonate (OHSt), applied on the superior colliculi. Whole mount retinas were processed to quantify the population of RGCs identified by fluorogold tracing and Brn3a immunodetection, and were counted using image analysis software; an isodensity contour plot was generated for each retina. DBA/2J mice showed a significant reduction in the positive STR (pSTR) amplitudes at 12 months of age, as compared to control C57BL/6J mice of the same age. The pSTR mean amplitude decreased to approximately 27.82% of the values recorded in control mice (p=0.0058). STR responses decreased in both strains as a result of the natural process of aging, but the decrease was more pronounced in DBA/2J mice. Furthermore, quantification of the total number of RGCs identified by OHSt and Brn3a expression showed a reduced population of RGCs in DBA/2J mice as compared to control mice. Regression analysis revealed significant correlations between the decrease in pSTR and a non-homogeneous reduction in the number of RGCs throughout the retina. Our results indicate the existence of a co, Ministerio de Ciencia e Innovación (MICINN), Ministerio de Economía y Competitividad (MINECO), Instituto de Salud Carlos III, Pharmacological Biochemistry of the Eye Research Group (OCUPHARM), Unidad Docente de Bioquímica y Biología Molecular, Fac. de Óptica y Optometría, TRUE, pub

© 2013 de Hoz et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited., In the mouse model of unilateral laser-induced ocular hypertension (OHT) the microglia in both the treated and the normotensive untreated contralateral eye have morphological signs of activation and up-regulation of MHC-II expression in comparison with naive. In the brain, rod-like microglia align to less-injured neurons in an effort to limit damage. We investigate whether: i) microglial activation is secondary to laser injury or to a higher IOP and; ii) the presence of rod-like microglia is related to OHT. Three groups of mice were used: age-matched control (naive, n=15); and two lasered: limbal (OHT, n=15); and non-draining portion of the sclera (scleral, n=3). In the lasered animals, treated eyes as well as contralateral eyes were analysed. Retinal whole-mounts were immunostained with antibodies against, Iba-1, NF-200, MHC-II, CD86, CD68 and Ym1. In the scleral group (normal ocular pressure) no microglial signs of activation were found. Similarly to naive eyes, OHT-eyes and their contralateral eyes had ramified microglia in the nerve-fibre layer related to the blood vessel. However, only eyes with OHT had rod-like microglia that aligned end-to-end, coupling to form trains of multiple cells running parallel to axons in the retinal surface. Rod-like microglia were CD68+ and were related to retinal ganglion cells (RGCs) showing signs of degeneration (NF-200+ RGCs). Although MHC-II expression was up-regulated in the microglia of the NFL both in OHT-eyes and their contralateral eyes, no expression of CD86 and Ym1 was detected in ramified or in rod-like microglia. After 15 days of unilateral lasering of the limbal and the non-draining portion of the sclera, activated microglia was restricted to OHT-eyes and their contralateral eyes. However, rod-like microglia were restricted to eyes with OHT and degenerated NF-200+ RGCs and were absent from their contralateral eyes. Thus, rod-like microglia seem be related to the neurodegeneration associated with HTO., ISCIII, Ministerio Español de Ciencia e Innovación, Ministerio Español de Economía e Innovación, Gobierno Regional de Murcia, Fundación Séneca, Unidad Docente de Inmunología, Oftalmología y ORL, Fac. de Óptica y Optometría, TRUE, pub

Received: 24 October 2018; Accepted: 03 March 2020; Published: 17 March 2020, Microglial activation is associated with glaucoma. In the model of unilateral laser-induced ocular hypertension (OHT), the time point at which the inflammatory process peaks remains unknown. Different time points (1, 3, 5, 8, and 15 d) were compared to analyze signs of microglial activation both in OHT and contralateral eyes. In both eyes, microglial activation was detected in all retinal layers at all time points analyzed, including: i) increase in the cell number in the outer segment photoreceptor layer and plexiform layers (only in OHT eyes) from 3 d onward; ii) increase in soma size from 1 d onward; iii) retraction of the processes from 1 d in OHT eyes and 3 d in contralateral eyes; iv) increase in the area of the retina occupied by Iba-1+ cells in the nerve fiber layer/ganglion cell layer from 1 d onward; v) increase in the number of vertical processes from 1 d in contralateral eyes and 3 d in OHT eyes. In OHT eyes at 24 h and 15 d, most Iba-1+ cells were P2RY12+ and were down-regulated at 3 and 5 d. In both eyes, microglial activation was stronger at 3 and 5 d (inflammation peaked in this model). These time points could be useful to identify factors implicated in the inflammatory process., Ministerio de Economía y Competitividad (España), Ministerio de Ciencia e Inovación (España), Ministerio de Educación, Cultura y Deporte (España), Universidad Complutense de Madrid, Depto. de Inmunología, Oftalmología y ORL, Unidad Docente de Inmunología, Oftalmología y ORL, Fac. de Óptica y Optometría, Instituto de Investigaciones Oftalmológicas Ramón Castroviejo, Fac. de Medicina, TRUE, pub, Pagado por el autor

Glaucoma is an age-related neurodegenerative disease that begins at the onset of aging. In this disease, there is an involvement of the immune system and therefore of the microglia. The purpose of this study is to evaluate the microglial activation using a mouse model of ocular hypertension (OHT) at the onset of aging. For this purpose, we used both naive and ocular hypertensives of 15-month-old mice (early stage of aging). In the latter, we analyzed the OHT eyes and the eyes contralateral to them to compare them with their aged controls. In the eyes of aged naive, aged OHT and aged contralateral eyes, microglial changes were observed compared to the young mice, including: (i) aged naive vs young naive: An increased soma size and vertical processes; (ii) aged OHT eyes vs young OHT eyes: A decrease in the area of the retina occupied by Iba-1 cells and in vertical processes; and (iii) aged contralateral vs young contralateral: A decrease in the soma size and arbor area and an increase in the number of microglia in the outer segment layer. Aged OHT eyes and the eyes contralateral to them showed an up-regulation of the CD68 expression in the branched microglia and a down-regulation in the MHCII and P2RY12 expression with respect to the eyes of young OHT mice. Conclusion: in the early phase of aging, morphological microglial changes along with changes in the expression of MHCII, CD68 and P2RY12, in both naive and OHT mice. These changes appear in aged OHT eyes and the eyes contralateral to them eyes.

Received 28 August 2018, Revised 23 April 2019, Accepted 10 May 2019, Available online 16 May 2019. This article is dedicated to Prof. Jesús Pintor, who died at the time this manuscript was under revision., Glaucoma is a common cause of visual impairment and blindness, characterized by retinal ganglion cell (RGC) death. The mechanisms that trigger the development of glaucoma remain unknown and have gained significant relevance in the study of this neurodegenerative disease. P2X7 purinergic receptors (P2X7R) could be involved in the regulation of the synaptic transmission and neuronal death in the retina through different pathways. The aim of this study was to characterize the molecular signals underlying glaucomatous retinal injury. The time-course of functional, morphological, and molecular changes in the glaucomatous retina of the DBA/2J mice were investigated. The expression and localization of P2X7R was analysed in relation with retinal markers. Caspase-3, JNK, and p38 were evaluated in control and glaucomatous mice by immunohistochemical and western-blot analysis. Furthermore, electroretinogram recordings (ERG) were performed to assess inner retina dysfunction. Glaucomatous mice exhibited changes in P2X7R expression as long as the pathology progressed. There was P2X7R overexpression in RGCs, the primary injured neurons, which correlated with the loss of function through ERG measurements. All analyzed MAPK and caspase-3 proteins were upregulated in the DBA/2J retinas suggesting a pro-apoptotic cell death. The increase in P2X7Rs presence may contribute, together with other factors, to the changes in retinal functionality and the concomitant death of RGCs. These findings provide evidence of possible intracellular pathways responsible for apoptosis regulation during glaucomatous degeneration., Ministerio de Economía y Competitividad de España (MINECO), Ministerio de Salud, Servicios Sociales e Igualdad, Fundación Séneca, Agencia de Ciencia y Tecnología de la Región de Murcia (España), Unidad Docente de Bioquímica y Biología Molecular, Fac. de Óptica y Optometría, TRUE, pub

Received 4 October 2017, Revised 23 February 2018, Accepted 7 March 2018, Available online 9 March 2018. Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.exer.2018.03.006., The immune system plays an important role in glaucomatous neurodegeneration. Retinal microglial reactivation associated with ganglion cell loss could reportedly contribute to the glaucoma progression. Recently we have described signs of microglia activation both in contralateral and ocular hypertension (OHT) eyes involving all retinal layers 15 days after OHT laser induction in mice. However, no works available have analyzed the microglial activation at earliest time points after OHT induction (24 h) in this experimental model. Thus, we seek to describe and quantify signs of microglia activation and differences depending on the retinal layer, 24 h after unilateral laser-induced OHT. Two groups of adult Swiss mice were used: age-matched control (naïve) and lasered. In the lasered animals, OHT eyes as well as contralateral eyes were analyzed. Retinal whole-mounts were immunostained with antibodies against Iba-1 and MHC-II. We quantified the number of microglial cells in the photoreceptor layer (OS), outer plexiform layer (OPL), and inner plexiform layer (IPL); the number of microglial vertical processes connecting the OPL and OS; the area of the retina occupied by Iba-1+ cells (Iba1-RA) in the nerve fiber layer-ganglion cell layer (NFL-GCL), the total arbor area of microglial cells in the OPL and IPL and; Iba-1+ cell body area in the OPL, IPL and NFL-GCL. In contralateral and OHT eyes the morphological features of Iba-1+ cell activation were: migration, enlargement of the cell body, higher degree of branching and reorientation of the processes, radial disposition of the soma and processes toward adjacent microglial plexuses, and presence of amoeboid cells acting as macrophages. These signs were more pronounced in OHT eyes. Most of Iba-1+ cells did not express MHC-II; rather, only dendritic and rounded cells expressed it. In comparison with naïve eyes, in OHT eyes and contralateral eyes no significant differences were found in the microglial cell number; but there was, Oftared, Instituto de Salud Carlos III (Madrid), Ministerio de Economía y Competitividad (MINECO), ISCIII-Subdirección General de Redes y Centros de Investigación Cooperativa, Programa Europeo FEDER, Fundación Séneca, Agencia de Ciencia y Tecnología Región de Murcia, Ministerio de Educación, Cultura y Deporte de España, Unidad Docente de Inmunología, Oftalmología y ORL, Fac. de Óptica y Optometría, TRUE, pub

The development of novel non-viral delivery vehicles is essential in the search of more efficient strategies for retina and brain diseases. Herein, optimized niosome formulations prepared by oil-in water (o/w) and film-hydration techniques were characterized in terms of size, PDI, zeta potential, morphology and stability. Three ionizable glycerol-based cationic lipids containing a primary amine group (lipid 1), a triglycine group (lipid 2) and a dimethylamino ethyl pendent group (lipid 3) as polar head-groups were part of such niosomes. Upon the addition of pCMS-EGFP plasmid, nioplexes were obtained at different cationic lipid/DNA ratios (w/w). The resultant nioplexes were further physicochemically characterized and evaluated to condense, release and protect the DNA against enzymatic digestion. In vitro experiments were performed to evaluate transfection efficiency and cell viability in HEK-293, ARPE-19 and PECC cells. Interestingly, niosome formulations based on lipid 3 showed better transfection efficiencies in ARPE-19 and PECC cells than the rest of cationic lipids showed in this study. In vivo experiments in rat retina after intravitreal and subretinal injections together with in rat brain after cerebral cortex administration showed promising transfection efficiencies when niosome formulations based on lipid 3 were used. These results provide new insights for the development of non-viral vectors based on cationic lipids and their applications for efficient delivery of genetic material to the retina and brain. © 2015 Elsevier Ltd.

The present study aimed to evaluate the incorporation of protamine into niosome/DNA vectors to analyze the potential application of this novel ternary formulation to deliver the pCMS-EGFP plasmid into the rat retina. Binary vectors based on niosome/DNA and ternary vectors based on protamine/DNA/niosomes were prepared and physicochemically characterized. In vitro experiments were performed in ARPE-19 cells. At 1:1:5 protamine/DNA/niosome mass ratio, the resulted ternary vectors had 150 nm size, positive charge, spherical morphology, and condensed, released, and protected the DNA against enzymatic digestion. The presence of protamine in the ternary vectors improved transfection efficiency, cell viability, and DNA condensation. After ocular administration, the EGFP expression was detected in different cell layers of the retina depending on the administration route without any sign of toxicity associated with the formulations. While subretinal administration transfected mainly photoreceptors and retinal pigment epithelial cells at the site of injection, intravitreal administration produced a more uniform distribution of the protein expression through the inner layers of the retina. The protein expression in the retina persisted for at least one month after both administrations. Our study highlights the flattering properties of protamine/DNA/niosome ternary vectors for efficient and safe gene delivery to the rat retina.