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Glaucoma is a degenerative disease that causes irreversible loss of vision and is characterized by retinal ganglion cell (RGC) loss. Others and we have demonstrated that chronic neuroinflammation mediated by reactive microglial cells plays a role in glaucomatous pathology. Exosomes are extracellular vesicles released by most cells, including microglia, that mediate intercellular communication. The role of microglial exosomes in glaucomatous degeneration remains unknown. Taking the prominent role of microglial exosomes in brain neurodegenerative diseases, we studied the contribution of microglial-derived exosomes to the inflammatory response in experimental glaucoma. Microglial cells were exposed to elevated hydrostatic pressure (EHP), to mimic elevated intraocular pressure, the main risk factor for glaucoma. Naïve microglia (BV-2 cells or retinal microglia) were exposed to exosomes derived from BV-2 cells under EHP conditions (BV-Exo-EHP) or cultured in control pressure (BV-Exo-Control). We found that BV-Exo-EHP increased the production of pro-inflammatory cytokines, promoted retinal microglia motility, phagocytic efficiency, and proliferation. Furthermore, the incubation of primary retinal neural cell cultures with BV-Exo-EHP increased cell death and the production of reactive oxygen species. Exosomes derived from retinal microglia (MG-Exo-Control or MG-Exo-EHP) were injected in the vitreous of C57BL/6J mice. MG-Exo-EHP sustained activation of retinal microglia, mediated cell death, and impacted RGC number. Herein, we show that exosomes derived from retinal microglia have an autocrine function and propagate the inflammatory signal in conditions of elevated pressure, contributing to retinal degeneration in glaucomatous conditions.

Glaucoma is a progressive chronic retinal degenerative disease and a leading cause of global irreversible blindness.This disease is characterized by optic nerve damage and retinal ganglion cell (RGC) death. The current treatmentsavailable target the lowering of intraocular pressure (IOP), the main risk factor for disease onset and development.However, in some patients, vision loss progresses despite successful IOP control, indicating that new and effectivetreatments are needed, such as those targeting the neuroprotection of RGCs. Adenosine A3receptor (A3R) activationconfers protection to RGCs following an excitotoxic stimulus. In this work, we investigated whether the activation ofA3R could also afford protection to RGCs in the laser-induced ocular hypertension (OHT) model, a well-characterizedanimal model of glaucoma. The intravitreal injection of 2-Cl-IB-MECA, a selective A3R agonist, abolished the alterationsinduced by OHT in the negative and positive components of scotopic threshold response (STR) without changing a-and b-wave amplitudes both in scotopic and photopic conditions. Moreover, the treatment of OHT eyes with the A3Ragonist promoted the survival of RGCs, attenuated the impairment in retrograde axonal transport, and improved thestructure of the optic nerve. Taking into consideration the beneficial effects afforded by 2-Cl-IB-MECA, we can envisagethat A3R activation can be considered a good therapeutic strategy to protect RGCs from glaucomatous damage. This work was supported by Foundation for Science and Technology (FCT), Portugal (Fellowships PD/BD/114115/2015 and PD/BD/127821/2016, Grant PTDC/NEU-OSD/3123/2014; Strategic Projects UID/NEU/04539/2013, UID/NEU/04539/2019 and UIDB/04539/2020; and UIDP/04539/2020 (CIBB)), FEDER-COMPETE (FCOMP-01-0124-FEDER-028417 and POCI-01-0145-FEDER-007440), and Centro 2020 Regional Operational Programme (CENTRO-01-0145-FEDER-000008: BrainHealth 2020). Spanish Ministry of Economy and Competitiveness, Instituto de Salud Carlos III, Fondo Europeo de Desarrollo Regional “Una manera de hacer Europa” (PI16/00031, SAF2015-67643-P, RD16/0008/0026, and RD16/0008/0016) and by the Fundación Séneca, Agencia de Ciencia y Tecnología Región de Murcia (19881/GERM/15).

Diabetes mellitus (DM) is a metabolic disease that affects 9% of the adult population, promoting an increase in glucose concentration that affects the corneal structure, namely, its thickness, as well as the constituents and flow of the aqueous humor. In this study, high-frequency transducers (20-MHz and 50-MHz) were used to measure and characterize changes in the corneal and aqueous humor in streptozotocin-induced type 1 diabetic rats followed over 8 weeks. Increases of 24.6 and 15.4 μm in central corneal thickness were measured with the 20-MHz and 50-MHz probes, respectively, in DM rats (p < 0.001). The increases in thickness of the different corneal layers ranged from 7% to 17%. Structural alterations of the aqueous humor were also studied by relating the amplitudes of the anterior lens and posterior cornea boundary signals, the result of which was denominated by pseudo-attenuation. The results revealed an increase of 49% at week 8 compared with the baseline values (p < 0.020, with the 50-MHz probe). This study illustrated that high-frequency ultrasound can be used to measure corneal layer thickness and study the alterations promoted by diabetes in the eye's anterior segment. Those assessments may allow early detection of DM, improving the monitoring of diabetic patients.

Glaucoma is a retinal degenerative disease characterized by the loss of retinal ganglion cells and damage of the optic nerve. Recently, we demonstrated that antagonists of adenosine A2A receptor (A2A R) control retinal inflammation and afford protection to rat retinal cells in glaucoma models. However, the precise contribution of microglia to retinal injury was not addressed, as well as the effect of A2A R blockade directly in microglia. Here we show that blocking microglial A2A R prevents microglial cell response to elevated pressure and it is sufficient to protect retinal cells from elevated pressure-induced death. The A2A R antagonist SCH 58261 or the knockdown of A2A R expression with siRNA in microglial cells prevented the increase in microglia response to elevated hydrostatic pressure. Furthermore, in retinal neural cell cultures, the A2A R antagonist decreased microglia proliferation, as well as the expression and release of pro-inflammatory mediators. Microglia ablation prevented neural cell death triggered by elevated pressure. The A2A R blockade recapitulated the effects of microglia depletion, suggesting that blocking A2A R in microglia is able to control neurodegeneration in glaucoma-like conditions. Importantly, in human organotypic retinal cultures, A2A R blockade prevented the increase in reactive oxygen species and the morphological alterations in microglia triggered by elevated pressure. These findings place microglia as the main contributors for retinal cell death during elevated pressure and identify microglial A2A R as a therapeutic target to control retinal neuroinflammation and prevent neural apoptosis elicited by elevated pressure.

Adenosine is an endogenous purine nucleoside ubiquitously distributed throughout the body that interacts with G protein-coupled receptors, classified in four subtypes: A1R, A2AR, A2BR and A3R. Among the plethora of functions of adenosine, it has been increasingly recognized as a key mediator of the immune response. Neuroinflammation is a feature of chronic neurodegenerative diseases and contributes to the pathophysiology of several retinal degenerative diseases. Animal models of retinal diseases are helping to elucidate the regulatory roles of adenosine receptors in the development and progression of those diseases. Mounting evidence demonstrates that the adenosinergic system is altered in the retina during pathological conditions, compromising retinal physiology. This review focuses on the roles played by adenosine and the elements of the adenosinergic system (receptors, enzymes, transporters) in the neuroinflammatory processes occurring in the retina. An improved understanding of the molecular and cellular mechanisms of the signalling pathways mediated by adenosine underlying the onset and progression of retinal diseases will pave the way towards the identification of new therapeutic approaches.

This work reports the development of porous poly (ε-caprolactone) (PCL)-based intraocular implants, prepared by green supercritical carbon dioxide (scCO2) foaming/mixing method (SFM), to produce implants that degrade faster than typical slow-degrading PCL-based implants. The higher porosities and surface areas of these implants led to faster degradation rates at in vitro accelerated alkaline conditions than low porosity/surface area implants prepared by hot melting processing. These porous implants also presented distinct (faster) release rates of a test-drug (dexamethasone). Additionally, these porous devices did not cause cell death and did not reduce the number of neurons, indicating that are not toxic to retinal cells. We further explored the impact of PCL-based implant to the retina by in vivo evaluation and histological analysis. Implants were surgically inserted in the vitreous of Wistar rats, and their presence did not change the function, structure and anatomy of the retina. These devices demonstrated a good intraocular tolerance, further confirming their viability for prolonged drug delivery applications. Further comprehensive studies based on this promising preliminary assessment and proof-of-concept could enable its future translation to clinical protective strategies for retinal diseases. This work wasfinancially supported by FCT, Portugal (FellowshipsPD/BD/114115/2015, PD/BD/127821/2016, and SFRH/BPD/101048/2014, Grant PTDC/NEU-OSD/3123/2014 and Strategic Projects UID/NEU/04539/2013, PEst-C/EQB/UI0102/2013, PEst-C/EQB/UI0102/2018, and PEst-C/EQB/UI0102/2019), FEDER-COMPETE (FCOMP-01-0124-FEDER-028417 and POCI-01-0145-FEDER-007440) and Centro2020 Regional Operational Programme (CENTRO-01-0145-FEDER-000008: Brain Health 2020)

Glaucoma is a progressive chronic retinal degenerative disease and a leading cause of global irreversible blindness, characterized by optic nerve damage and retinal ganglion cell (RGC) death. Elevated intraocular pressure (IOP) is a main risk factor of glaucoma. Neuroinflammation plays an important role in glaucoma. We have been demonstrating that elevated pressure triggers microglia reactivity that contribute to the loss of RGCs. Adenosine, acting on adenosine receptors, is a crucial modulator of microglia phenotype. Microglia express all adenosine receptors. Previously, we demonstrated that the activation of adenosine A3 receptor (A3R) affords protection to the retina, including RGCs, unveiling the possibility for a new strategy for glaucoma treatment. Since microglial cells express A3R, we now studied the ability of a selective A3R agonist (2-Cl-IB-MECA) in controlling microglia reactivity induced by elevated hydrostatic pressure (EHP), used to mimic elevated IOP. The activation of A3R reduced EHP-induced inducible nitric oxide synthase (iNOS) expression, microglia migration and phagocytosis in BV-2 cells. In retinal microglia, proliferation and phagocytosis elicited by EHP were also decreased by A3R activation. This work demonstrates that 2-Cl-IB-MECA, the selective agonist of A3R, is able to hinder microglia reactivity, suggesting that A3R agonists could afford protection against glaucomatous degeneration through the control of neuroinflammation.

The retinal ganglion cells (RGCs) are the output cells of the retina into the brain. In mammals, these cells are not able to regenerate their axons after optic nerve injury, leaving the patients with optic neuropathies with permanent visual loss. An effective RGCs-directed therapy could provide a beneficial effect to prevent the progression of the disease. Axonal injury leads to the functional loss of RGCs and subsequently induces neuronal death, and axonal regeneration would be essential to restore the neuronal connectivity, and to reestablish the function of the visual system. The manipulation of several intrinsic and extrinsic factors has been proposed in order to stimulate axonal regeneration and functional repairing of axonal connections in the visual pathway. However, there is a missing point in the process since, until now, there is no therapeutic strategy directed to promote axonal regeneration of RGCs as a therapeutic approach for optic neuropathies. This work was supported by Foundation for Science and Technology (FCT), Portugal (Fellowships PD/BD/114115/2015 and PD/BD/127821/2016, Grant PTDC/NEU-OSD/3123/2014 and Strategic Projects UID/NEU/04539/2013, UID/NEU/04539/2019, UIDP/04539/2020 and UIDB/04539/2020), FEDER-COMPETE POCI-01-0145-FEDER-016849, FCOMP-01-0124-FEDER-028417, POCI-01-0145-FEDER-007440) and Centro 2020 Regional Operational Programme (CENTRO-01-0145-FEDER-000008: BrainHealth 2020). Grupos UPV/EHU GIU18/50, PUE 2018-04 and ELKARTEK KK-2019/00086 to E.V.

Glaucoma is a retinal degenerative disease characterized by the loss of retinal ganglion cells and damage of the optic nerve. Recently, we demonstrated that antagonists of adenosine A2A receptor (A2AR) control retinal inflammation and afford protection to rat retinal cells in glaucoma models. However, the precise contribution of microglia to retinal injury was not addressed, as well as the effect of A2AR blockade directly in microglia. Here we show that blocking microglial A2AR prevents microglial cell response to elevated pressure and it is sufficient to protect retinal cells from elevated pressure‐induced death. The A2AR antagonist SCH 58261 or the knockdown of A2AR expression with siRNA in microglial cells prevented the increase in microglia response to elevated hydrostatic pressure. Furthermore, in retinal neural cell cultures, the A2AR antagonist decreased microglia proliferation, as well as the expression and release of pro‐inflammatory mediators. Microglia ablation prevented neural cell death triggered by elevated pressure. The A2AR blockade recapitulated the effects of microglia depletion, suggesting that blocking A2AR in microglia is able to control neurodegeneration in glaucoma‐like conditions. Importantly, in human organotypic retinal cultures, A2AR blockade prevented the increase in reactive oxygen species and the morphological alterations in microglia triggered by elevated pressure. These findings place microglia as the main contributors for retinal cell death during elevated pressure and identify microglial A2AR as a therapeutic target to control retinal neuroinflammation and prevent neural apoptosis elicited by elevated pressure.

Diabetes mellitus (DM) is a group of diseases characterized by chronic hyperglycemia caused by inadequate insulin secretion or impaired insulin action, or both. Diabetic corneal neuropathy can result in chronic, sight-threatening corneal pathology. In vivo non-invasive techniques to determine the changes in corneal thickness are of extreme importance to adequately monitor disease progression. In this work, we evaluated the effects of hyperglycemia in the corneal structure of Wistar rats using optical coherence tomography (OCT) and ultrasounds. This study showed that the central cornea thickness increased in diabetic rats, and that stroma was the corneal layer that most contributed for the central cornea thickness increase in DM. It has also been shown that the OCT technique can be used to study the alterations in the substructure of the cornea promoted by hyperglycemia in DM.

Glaucoma, a leading cause of blindness worldwide, is a degenerative disease characterized by retinal ganglion cell (RGC) loss and optic nerve atrophy. Elevated intraocular pressure (IOP) is a main risk factor for onset and progression of the disease. Since increased IOP is the only modifiable risk factor, relevant models for glaucoma would comprise RGC and optic nerve damage triggered by ocular hypertension. Animal models of glaucoma have greatly contributed to the understanding of the molecular mechanisms of this pathology, and they have also facilitated the development of new pharmacological interventions. Although animal models of glaucoma have provided valuable information about the disease, there is still no ideal model for studying glaucoma due to its complexity. There is a recognized demand for in vitro models that can replace or reduce the need for animal experiments. Several in vitro models have emerged as a great opportunity in the field of glaucoma research, helping to clarify the mechanisms involved in disease progression. Several types of equipment have been developed to expose cells and tissue cultures to elevated pressures. Herein, we discuss the methodology used to increase pressure, the main findings, and the relevance of in vitro models for the study of the pathophysiology of glaucoma.

Transient retinal ischemia is a major complication of retinal degenerative diseases and contributes to visual impairment and blindness. Evidences indicate that microglia-mediated neuroinflammation has a key role in the neurodegenerative process, prompting the hypothesis that the control of microglia reactivity may afford neuroprotection to the retina against the damage induced by ischemia–reperfusion (I–R). The available therapeutic strategies for retinal degenerative diseases have limited potential, but the blockade of adenosine A2A receptor (A2AR) emerges as candidate strategy. Therefore, we evaluated the therapeutic potential of a selective A2AR antagonist (KW6002) against the damage elicited by I–R. The administration of KW6002 after I–R injury reduced microglia reactivity and inflammatory response and afforded protection to the retina. Moreover, we tested the ability of caffeine, an adenosine receptor antagonist, in mediating protection to the retina in the I–R injury model. We demonstrated that caffeine administration dually regulated microglia reactivity and cell death in the transient retinal ischemic model, depending on the reperfusion time. At 24 h of reperfusion, caffeine increased microglial reactivity, inflammatory response and cell death elicited by I–R. However, at 7 days of reperfusion, caffeine administration decreased microglia reactivity and reduced the levels of proinflammatory cytokines and cell death. Together, these results provide a novel evidence for the use of adenosine A2AR antagonists as potential therapy for retinal ischemic diseases and demonstrate the effect of caffeine on the regulation of microglia-mediated neuroinflammation in the transient ischemic model.