Maria H. Madeira

Contribution of microglia-mediated neuroinflammation to retinal degenerative diseases.

Retinal degenerative diseases are major causes of vision loss and blindness worldwide and are characterized by chronic and progressive neuronal loss. One common feature of retinal degenerative diseases and brain neurodegenerative diseases is chronic neuroinflammation. There is growing evidence that retinal microglia, as in the brain, become activated in the course of retinal degenerative diseases, having a pivotal role in the initiation and propagation of the neurodegenerative process. A better understanding of the events elicited and mediated by retinal microglia will contribute to the clarification of disease etiology and might open new avenues for potential therapeutic interventions. This review aims at giving an overview of the roles of microglia-mediated neuroinflammation in major retinal degenerative diseases like glaucoma, age-related macular degeneration, and diabetic retinopathy.

Adenosine A2AR blockade prevents neuroinflammation-induced death of retinal ganglion cells caused by elevated pressure

BackgroundElevated intraocular pressure (IOP) is a major risk factor for glaucoma, a degenerative disease characterized by the loss of retinal ganglion cells (RGCs). There is clinical and experimental evidence that neuroinflammation is involved in the pathogenesis of glaucoma. Since the blockade of adenosine A2A receptor (A2AR) confers robust neuroprotection and controls microglia reactivity in the brain, we now investigated the ability of A2AR blockade to control the reactivity of microglia and neuroinflammation as well as RGC loss in retinal organotypic cultures exposed to elevated hydrostatic pressure (EHP) or lipopolysaccharide (LPS).MethodsRetinal organotypic cultures were either incubated with LPS (3 μg/mL), to elicit a pro-inflammatory response, or exposed to EHP (+70 mmHg), to mimic increased IOP, for 4 or 24 h, in the presence or absence of the A2AR antagonist SCH 58261 (50 nM). A2AR expression, microglial reactivity and neuroinflammatory response were evaluated by immunohistochemistry, quantitative PCR (qPCR) and enzyme-linked immunosorbent assay (ELISA). RGC loss was assessed by immunohistochemistry. In order to investigate the contribution of pro-inflammatory mediators to RGC loss, the organotypic retinal cultures were incubated with rabbit anti-tumour necrosis factor (TNF) (2 μg/mL) and goat anti-interleukin-1β (IL-1β) (1 μg/mL) antibodies.ResultsWe report that the A2AR antagonist (SCH 58261) prevented microglia reactivity, increase in pro-inflammatory mediators as well as RGC loss upon exposure to either LPS or EHP. Additionally, neutralization of TNF and IL-1β prevented RGC loss induced by LPS or EHP.ConclusionsThis work demonstrates that A2AR blockade confers neuroprotection to RGCs by controlling microglia-mediated retinal neuroinflammation and prompts the hypothesis that A2AR antagonists may be a novel therapeutic option to manage glaucomatous disorders.

Selective A2A receptor antagonist prevents microglia-mediated neuroinflammation and protects retinal ganglion cells from high intraocular pressure–induced transient ischemic injury

Glaucoma is a leading cause of vision loss and blindness worldwide, characterized by chronic and progressive neuronal loss. Reactive microglial cells have been recognized as a neuropathologic feature, contributing to local inflammation and retinal neurodegeneration. In a recent in vitro work (organotypic cultures), we demonstrated that blockade of adenosine A2A receptor (A2AR) prevents the neuroinflammatory response and affords protection to retinal ganglion cells (RGCs) against exposure to elevated hydrostatic pressure (EHP), to mimic elevated intraocular pressure (IOP), the main risk factor for glaucoma development. Herein, we investigated whether a selective A2AR antagonist (SCH 58261) could modulate retinal microglia reactivity and their inflammatory response. Furthermore, we took advantage of the high IOP-induced transient ischemia (ischemia-reperfusion, I-R) animal model to evaluate the protective role of A2AR blockade in the control of retinal neuroinflammation and neurodegeneration. Primary microglial cell cultures were challenged either with lipopolysaccharide or with EHP, in the presence or absence of A2AR antagonist SCH 58261 (50 nM). In addition, I-R injury was induced in adult Wistar rats after intravitreal administration of SCH 58261 (100 nM, 5 μL). Our results showed that SCH 58261 attenuated microglia reactivity and the increased expression and release of proinflammatory cytokines. Moreover, intravitreal administration of SCH 58261 prevented I-R-induced cell death and RGC loss, by controlling microglial-mediated neuroinflammatory response. These results prompt the proposal that A2AR blockade may have great potential in the management of retinal neurodegenerative diseases characterized by microglia reactivity and RGC death, such as glaucoma and ischemic diseases.

Transient Downregulation of Melanopsin Expression After Retrograde Tracing or Optic Nerve Injury in Adult Rats.

PURPOSE To investigate the effect of retrograde tracing or axotomy on melanopsin mRNA expression and immunodetection in albino and pigmented rat retinas. METHODS Groups were (1) intact-naïve retinas; (2) optic nerve crush (ONC) analyzed at 7 days (7d) or 2 months (2m); (3) Fluorogold (FG) tracing from the superior colliculi (SCi) analyzed at 7d or 2m; (4) tracing from the intact optic nerve (ON) with FG or hydroxystilbamidine methanesulfonate (OHSt), analyzed 3d later; and (5) sham tracing from the ON or sham surgery. Brn3a and melanopsin were double stained in whole mounts to quantify and assess the distribution of orthotopic and displaced Brn3a(+) retinal ganglion cells (Brn3a(+)RGCs) and melanopsin(+)RGCs (m(+)RGCs). Freshly dissected retinas were used for melanopsin mRNA quantitative PCR. RESULTS Tracing from the SCi did not affect the number of Brn3a(+)RGCs or m(+)RGCs counted in pigmented rats. However, only 55% of m(+)RGCs were immunodetected in albinos at 7d, although by 2m the m(+)RGCs counts returned to normal. Optic nerve tracing had a more dramatic effect (38% or 77% of m(+)RGCs were immunodetected in albino or pigmented rats) that occurred irrespectively of the tracer (OHSt or FG). This effect was not observed in the sham groups. After ONC, Brn3a(+)RGCs decreased to 37% and 8% by 7d and 2m, respectively. Melanopsin (+)RGC counts diminished to 30% at 7d, but recovered to 49% of controls by 2m. Melanopsin mRNA was downregulated after ON tracing or 7d after ONC, but did not differ from intact values 2m after ONC. CONCLUSIONS Following ON injury or retrograde tracing there is a transient melanopsin downregulation that should be taken into account when assessing m(+)RGC survival.

Caffeine administration prevents retinal neuroinflammation and loss of retinal ganglion cells in an animal model of glaucoma

Glaucoma is the second leading cause of blindness worldwide, being characterized by progressive optic nerve damage and loss of retinal ganglion cells (RGCs), accompanied by increased inflammatory response involving retinal microglial cells. The etiology of glaucoma is still unknown, and despite elevated intraocular pressure (IOP) being a major risk factor, the exact mechanisms responsible for RGC degeneration remain unknown. Caffeine, which is an antagonist of adenosine receptors, is the most widely consumed psychoactive drug in the world. Several evidences suggest that caffeine can attenuate the neuroinflammatory responses and afford protection upon central nervous system (CNS) injury. We took advantage of a well characterized animal model of glaucoma to investigate whether caffeine administration controls neuroinflammation and elicits neuroprotection. Caffeine or water were administered ad libitum and ocular hypertension (OHT) was induced by laser photocoagulation of the limbal veins in Sprague Dawley rats. Herein, we show that caffeine is able to partially decrease the IOP in ocular hypertensive animals. More importantly, we found that drinking caffeine prevented retinal microglia-mediated neuroinflammatory response and attenuated the loss of RGCs in animals with ocular hypertension (OHT). This study opens the possibility that caffeine or adenosine receptor antagonists might be a therapeutic option to manage RGC loss in glaucoma.

Having a Coffee Break: The Impact of Caffeine Consumption on Microglia-Mediated Inflammation in Neurodegenerative Diseases

Caffeine is the major component of coffee and the most consumed psychostimulant in the world and at nontoxic doses acts as a nonselective adenosine receptor antagonist. Epidemiological evidence suggests that caffeine consumption reduces the risk of several neurological and neurodegenerative diseases. However, despite the beneficial effects of caffeine consumption in human health and behaviour, the mechanisms by which it impacts the pathophysiology of neurodegenerative diseases still remain to be clarified. A promising hypothesis is that caffeine controls microglia-mediated neuroinflammatory response associated with the majority of neurodegenerative conditions. Accordingly, it has been already described that the modulation of adenosine receptors, namely, the A2A receptor, affords neuroprotection through the control of microglia reactivity and neuroinflammation. In this review, we will summarize the main effects of caffeine in the modulation of neuroinflammation in neurodegenerative diseases.

Treatment with A2A receptor antagonist KW6002 and caffeine intake regulate microglia reactivity and protect retina against transient ischemic damage

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.

Melanopsin expression is an indicator of the well-being of melanopsin-expressing retinal ganglion cells but not of their viability

Light is an electromagnetic stimulus that in mammals is sensed by specialized neurons in the retina. The physiological response to light encompasses two fundamental and different functional outputs: image-forming and non-image forming. The image-forming response is classically known as vision, while the non-image forming responses include the circadian photo-entrainment and the pupillary reflex. Each one is processed by different neurons.

Glia-Mediated Retinal Neuroinflammation as a Biomarker in Alzheimer's Disease.

Alzheimers disease (AD) is the most common type of dementia worldwide; it is characterized by a progressive decline in cognitive functions and memory, resulting from synaptic and cell loss, and accompanied by a strong neuroinflammatory response. Besides the vast progress in the understanding of the pathophysiology of AD in the past decades, there is still no effective treatment. Moreover, the diagnosis occurs usually at an advanced stage of the disease, where the neurological damage has already occurred. The identification of biomarkers that would allow an early diagnosis of this disease is a major goal that would also help managing AD progression. Due to its cellular and physiological resemblances with the brain, the retina has long been regarded as a window to the brain. Several brain manifestations have been associated with retinal alterations. In AD patients, some structural and functional alterations in the retina can be associated with disease onset. However, only a few studies have focused on the alterations in retinal glial cells associated with AD. This review aims at giving an overview of the AD-associated retinal alterations, particularly in glial cells. The documented alterations in retinal glia will be discussed concerning their potential to predict the brain alterations occurring in AD.

Elevated Pressure Changes the Purinergic System of Microglial Cells

Glaucoma is the second cause of blindness worldwide and is characterized by the degeneration of retinal ganglion cells (RGCs) and optic nerve atrophy. Increased microglia reactivity is an early event in glaucoma that may precede the loss of RGCs, suggesting that microglia and neuroinflammation are involved in the pathophysiology of this disease. Although global changes of the purinergic system have been reported in experimental and human glaucoma, it is not known if this is due to alterations of the purinergic system of microglial cells, the resident immune cells of the central nervous system. We now studied if elevated hydrostatic pressure (EHP), mimicking ocular hypertension, changed the extracellular levels of ATP and adenosine and the expression, density and activity of enzymes, transporters and receptors defining the purinergic system. The exposure of the murine microglial BV-2 cell line to EHP increased the extracellular levels of ATP and adenosine, increased the density of ecto-nucleoside triphosphate diphosphohydrolase 1 (E-NTPDase1, CD39) and decreased the density of the equilibrative nucleotide transporter 2 as well as the activity of adenosine deaminase. The expression of adenosine A1 receptor also decreased, but the adenosine A3 receptor was not affected. Notably, ATP and adenosine selectively control migration rather than phagocytosis, both bolstered by EHP. The results show that the purinergic system is altered in microglia in conditions of elevated pressure. Understanding the impact of elevated pressure on the purinergic system will help to unravel the mechanisms underlying inflammation and neurodegeneration associated with glaucoma.