Tracing the influence of prenatal risk factors on the offspring retina: Focus on development and putative long-term consequences.

BACKGROUND: Pregnancy represents a window of vulnerability to fetal development. Disruptions in the prenatal environment during this crucial period can increase the risk of the offspring developing diseases over the course of their lifetime. The central nervous system (CNS) has been shown to be particularly susceptible to changes during crucial developmental windows. To date, research focused on disruptions in the development of the CNS has predominantly centred on the brain, revealing a correlation between exposure to prenatal risk factors and the onset of neuropsychiatric disorders. Nevertheless, some studies indicate that the retina, which is part of the CNS, is also vulnerable to in utero alterations during pregnancy. Such changes may affect neuronal, glial and vascular components of the retina, compromising retinal structure and function and possibly impairing visual function. METHODS: A search in the PubMed database was performed, and any literature concerning prenatal risk factors (drugs, diabetes, unbalanced diet, infection, glucocorticoids) affecting the offspring retina were included. RESULTS: This review collects evidence on the cellular, structural and functional changes occurring in the retina triggered by maternal risk factors during pregnancy. We highlight the adverse impact on retinal development and its long-lasting effects, providing a critical analysis of the current knowledge while underlining areas for future research. CONCLUSIONS: Appropriate recognition of the prenatal risk factors that negatively impact the developing retina may provide critical clues for the design of preventive strategies and for early therapeutic intervention that could change retinal pathology in the progeny.

A longitudinal multimodal in vivo molecular imaging study of the 3xTg-AD mouse model shows progressive early hippocampal and taurine loss.

The understanding of the natural history of Alzheimer's disease (AD) and temporal trajectories of in vivo molecular mechanisms requires longitudinal approaches. A behavioral and multimodal imaging study was performed at 4/8/12 and 16 months of age in a triple transgenic mouse model of AD (3xTg-AD). Behavioral assessment included the open field and novel object recognition tests. Molecular characterization evaluated hippocampal levels of amyloid ß (Aß) and hyperphosphorylated tau. Magnetic resonance imaging (MRI) included assessment of hippocampal structural integrity, blood-brain barrier (BBB) permeability and neurospectroscopy to determine levels of the endogenous neuroprotector taurine. Longitudinal brain amyloid accumulation was assessed using 11C Pittsburgh compound B positron emission tomography (PET), and neuroinflammation/microglia activation was investigated using 11C-PK1195. We found altered locomotor activity at months 4/8 and 16 months and recognition memory impairment at all time points. Substantial early reduction of hippocampal volume started at month 4 and progressed over 8/12 and 16 months. Hippocampal taurine levels were significantly decreased in the hippocampus at months 4/8 and 16. No differences were found for amyloid and neuroinflammation with PET, and BBB was disrupted only at month 16. In summary, 3xTg-AD mice showed exploratory and recognition memory impairments, early hippocampal structural loss, increased Aß and hyperphosphorylated tau and decreased levels of taurine. In sum, the 3xTg-AD animal model mimics pathological and neurobehavioral features of AD, with early-onset recognition memory loss and MRI-documented hippocampal damage. The early-onset profile suggests temporal windows and opportunities for therapeutic intervention, targeting endogenous neuroprotectors such as taurine.

Sex-specific changes in peripheral metabolism in a model of chronic anxiety induced by prenatal stress.

BACKGROUND: Prenatal stress is associated with increased susceptibility to psychiatric and metabolic disorders later in life. Prenatal exposure to stress mediators may have sex-dependent effects on offspring brain and metabolic function, promoting a sex-specific vulnerability to psychopathology and metabolic alterations at adulthood. In this work, the impact of prenatal stress on glucose homeostasis and peripheral metabolism of male and female offspring was investigated in a chronic anxiety animal model. METHODS: Pregnant Wistar rats were injected with saline or glucocorticoid (dexamethasone: 1 mg/kg, subcutaneous) at gestational days 18 and 19. Male and female offspring weight was monitored, and anxious-like behaviour and peripheral insulin-sensitive tissues were analysed at adulthood. RESULTS: At birth, females and males prenatally exposed to stress presented decreased body weight which remained low in females. At adulthood, a morphological disorganization of the Langerhans islets was observed in both sexes prenatally exposed to stress, yet not changes in insulin levels were detected. Also, prenatal stress increased glucose transporter 4 (GLUT-4) levels in female and male adipose tissues and decreased insulin receptor levels in the liver and skeleton muscle but only in females. CONCLUSIONS: Exposure to stress mediators in critical periods of development negatively affects behaviour and metabolism. Prenatal stress programmes offspring peripheral metabolism in a sex-specific manner, emphasizing that the response to stress in critical periods of development may be sex-specific having each sex different vulnerabilities to psychiatric and metabolic disorders. Considering sex-specificities may provide critical clues for the design of preventive strategies and for early therapeutic intervention.

Microglia cytoarchitecture in the brain of adenosine A2A receptor knockout mice: Brain region and sex specificities.

Microglia cells exert a critical role in brain development, mainly supported by their immune functions, which predicts an impact on the genesis of psychiatric disorders. In fact, microglia stress during gestation is, for instance, associated with chronic anxiety and cognitive deficits accompanied by long-lasting, region- and sex-specific changes in microglia morphology. We recently reported that the pattern of microglia morphologic plasticity, which is sex-determined, impacts on anxious-like behaviour and cognition. We also reported that the pharmacologic blockade of adenosine A2A receptors (A2A R) is able to reshape microglia morphology, in a sex-specific manner and with behavioural sequelae. In order to better understand the role of A2A R in the sex differentiation of microglia, we now compared their morphology in wild-type and A2A R knockout male and female C57BL/6 mice in two cardinal brain regions implicated in anxiety-like behaviour and cognition, the prefrontal cortex (PFC) and the dorsal hippocampus (dHIP). We report interregional differences between PFC and dHIP in a sex-specific manner: while males presented more complex microglia in the dHIP, microglia from females had a more complex morphology in the PFC. Surprisingly, the genetic deletion of A2A R did not alter these sex differences, but promoted the exclusive remodelling (increase in complexity) in PFC microglia from females. These findings further support the existence of a heterogeneous microglial network, distinct between sexes and brain regions, and help characterizing the role of A2A R in the sex- and brain region-specific morphologic differentiation of microglia.

Microglial Activation in the Retina of a Triple-Transgenic Alzheimer's Disease Mouse Model (3xTg-AD).

Alzheimer's disease (AD) is the most common type of dementia in the world. The main biomarkers associated with AD are protein amyloid-ß (Aß) plaques and protein tau neurofibrillary tangles, which are responsible for brain neuroinflammation mediated by microglial cells. Increasing evidence has shown that the retina can also be affected in AD, presenting some molecular and cellular changes in the brain, such as microglia activation. However, there are only a few studies assessing such changes in the retinal microglia in animal models of AD. These studies use retinal sections, which have some limitations. In this study, we performed, for the first time in a triple-transgenic AD mouse model (3xTg-AD), a quantitative morphometric analysis of microglia activation (using the anti-Iba-1 antibody) in retinal whole-mounts, allowing visualization of the entire microglial cell, as well as its localization along the extension of the retina in different layers. Compared to age-matched animals, the retina of 3xTg-AD mice presents a higher number of microglial cells and a thicker microglial cell body area. Moreover, the microglia migrate, reorient, and retract their processes, changing their localization from a parallel to a perpendicular position relative to the retinal surface. These findings demonstrate clear microglia remodeling in the retina of 3xTg-AD mice.

Region-specific control of microglia by adenosine A2A receptors: uncoupling anxiety and associated cognitive deficits in female rats.

Epidemiologic studies have provided compelling evidence that prenatal stress, through excessive maternal glucocorticoids exposure, is associated with psychiatric disorders later in life. We have recently reported that anxiety associated with prenatal exposure to dexamethasone (DEX, a synthetic glucocorticoid) correlates with a gender-specific remodeling of microglia in the medial prefrontal cortex (mPFC), a core brain region in anxiety-related disorders. Gender differences in microglia morphology, the higher prevalence of anxiety in women and the negative impact of anxiety in cognition, led us to specifically evaluate cognitive behavior and associated circuits (namely mPFC-dorsal hippocampus, dHIP), as well as microglia morphology in female rats prenatally exposed to dexamethasone (in utero DEX, iuDEX). We report that iuDEX impaired recognition memory and deteriorated neuronal synchronization between mPFC and dHIP. These functional deficits are paralleled by microglia hyper-ramification in the dHIP and decreased ramification in the mPFC, showing a heterogeneous remodeling of microglia morphology, both postnatally and at adulthood in different brain regions, that differently affect mood and cognition. The chronic blockade of adenosine A2A receptors (A2A R), which are core regulators of microglia morphology and physiology, ameliorated the cognitive deficits, but not the anxiety-like behavior. Notably, A2A R blockade rectified both microglia morphology in the dHIP and the lack of mPFC-dHIP synchronization, further heralding their role in cognitive function.

Subtle thinning of retinal layers without overt vascular and inflammatory alterations in a rat model of prediabetes.

Purpose: Diabetic retinopathy is a neurovascular disease characterized by increased permeability of the blood-retinal barrier, changes in the neural components of the retina, and low-grade chronic inflammation. Diabetic retinopathy is a major complication of diabetes; however, the impact of a prediabetic state on the retina remains to be elucidated. The aim of this study was to assess possible early retinal changes in prediabetic rats, by evaluating changes in the integrity of the blood-retinal barrier, the retinal structure, neural markers, and inflammatory mediators. Methods: Several parameters were analyzed in the retinas of Wistar rats that drank high sucrose (HSu; 35% sucrose solution during 9 weeks, the prediabetic animal model) and were compared with those of age-matched controls. The permeability of the blood-retinal barrier was assessed with the Evans blue assay, and the content of the tight junction proteins and neural markers with western blotting. Optical coherence tomography was used to evaluate retinal thickness. Cell loss at the ganglion cell layer was assessed with terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay and by evaluating the immunoreactivity of the Brn3a transcription factor. To assess retinal neuroinflammation, the mRNA expression and protein levels of inducible nitric oxide synthase isoform (iNOS), interleukin-1 beta (IL-1ß), and tumor necrosis factor (TNF) were evaluated. Iba1 and MHC-II immunoreactivity and translocator protein (TSPO) mRNA levels were assessed to study the microglial number and activation state. Results: The thickness of the inner retinal layers of the HSu-treated animals decreased. Nevertheless, no apoptotic cells were observed, and no changes in retinal neural markers were detected in the retinas of the HSu-treated animals. No changes were detected in the permeability of the blood-retinal barrier, as well as the tight junction protein content between the HSu-treated rats and the controls. In addition, the inflammatory parameters remained unchanged in the retina despite the tendency for an increase in the number of retinal microglial cells. Conclusions: In a prediabetic rat model, the retinal structure is affected by the thinning of the inner layers, without overt vascular and inflammatory alterations. The results suggest neuronal dysfunction (thinning of the inner retina) that may precede or anticipate the vascular and inflammatory changes. Subtle structural changes might be viewed as early disturbances in an evolving disease, suggesting that preventive strategies (such as the modification of diet habits) could be applied at this stage, before the progression toward irreversible dysfunction and damage to the retina.

Impact of type 1 diabetes mellitus and sitagliptin treatment on the neuropeptide Y system of rat retina.

BACKGROUND: Neuropeptide Y (NPY) is a neuromodulator that is expressed in the retina. Increasing evidence suggests that NPY has pronounced anti-inflammatory effects, which might depend on the inhibition of dipeptidyl-peptidase-IV (DPP-IV). The aim of this study was to investigate the impact of type 1 diabetes mellitus (DM) and sitagliptin, a DPP-IV inhibitor, on the NPY system in the retina using an animal model. METHODS: Type 1 DM was induced in male Wistar rats by an intraperitoneal injection of streptozotocin. Starting 2 weeks after DM onset, animals were treated orally with sitagliptin (5 mg/kg.day) for 2 weeks. The expression of NPY and NPY receptors (Y1 , Y2 and Y5 receptors) was measured by quantitative polymerase chain reaction, Western blot and/or enzyme-linked immunosorbent assay. The immunoreactivity of NPY and NPY receptors was evaluated by immunohistochemistry, and the [35 S]GTPγS binding assay was used to assess the functional binding of NPY receptors. RESULTS: DM decreased the mRNA levels of NPY in the retina, as well as the protein levels of NPY and Y5 receptor. No changes were detected in the localization of NPY and NPY receptors in the retina and in the functional binding of NPY to all receptors. Sitagliptin alone reduced retinal NPY mRNA levels. The effects of DM on the NPY system were not affected by sitagliptin. CONCLUSION: DM modestly affects the NPY system in the retina and these effects are not prevented by sitagliptin treatment. These observations suggest that DPP-IV enzyme is not underlying the NPY changes detected in the retina induced by type 1 DM.

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.

Elevated Glucose and Interleukin-1ß Differentially Affect Retinal Microglial Cell Proliferation.

Diabetic retinopathy is considered a neurovascular disorder, hyperglycemia being considered the main risk factor for this pathology. Diabetic retinopathy also presents features of a low-grade chronic inflammatory disease, including increased levels of cytokines in the retina, such as interleukin-1 beta (IL-1ß). However, how high glucose and IL-1ß affect the different retinal cell types remains to be clarified. In retinal neural cell cultures, we found that IL-1ß and IL-1RI are present in microglia, macroglia, and neurons. Exposure of retinal neural cell cultures to high glucose upregulated both mRNA and protein levels of IL-1ß. High glucose decreased microglial and macroglial cell proliferation, whereas IL-1ß increased their proliferation. Interestingly, under high glucose condition, although the number of microglial cells decreased, they showed a less ramified morphology, suggesting a more activated state, as supported by the upregulation of the levels of ED-1, a marker of microglia activation. In conclusion, IL-1ß might play a key role in diabetic retinopathy, affecting microglial and macroglial cells and ultimately contributing to neural changes observed in diabetic patients. Particularly, since IL-1ß has an important role in retinal microglia activation and proliferation under diabetes, limiting IL-1ß-triggered inflammatory processes may provide a new therapeutic strategy to prevent the progression of diabetic retinopathy.

Disruption of a neural microcircuit in the rod pathway of the mammalian retina by diabetes mellitus.

Diabetes leads to dysfunction of the neural retina before and independent of classical microvascular diabetic retinopathy, but previous studies have failed to demonstrate which neurons and circuits are affected at the earliest stages. Here, using patch-clamp recording and two-photon Ca(2+) imaging in rat retinal slices, we investigated diabetes-evoked changes in a microcircuit consisting of rod bipolar cells and their dyad postsynaptic targets, AII and A17 amacrine cells, which play an essential role in processing scotopic visual signals. AII amacrines forward their signals to ON- and OFF-cone bipolar cells and A17 amacrines provide GABAergic feedback inhibition to rod bipolar cells. Whereas Ca(2+)-permeable AMPA receptors mediate input from rod bipolar cells to both AII and A17 amacrines, diabetes changes the synaptic receptors on A17, but not AII amacrine cells. This was expressed as a change in pharmacological properties and single-channel conductance of the synaptic receptors, consistent with an upregulation of the AMPA receptor GluA2 subunit and reduced Ca(2+) permeability. In addition, two-photon imaging revealed reduced agonist-evoked influx of Ca(2+) in dendritic varicosities of A17 amacrine cells from diabetic compared with normal animals. Because Ca(2+)-permeable receptors in A17 amacrine cells mediate synaptic release of GABA, the reduced Ca(2+) permeability of these receptors in diabetic animals leads to reduced release of GABA, followed by disinhibition and increased release of glutamate from rod bipolar cells. This perturbation of neuron and microcircuit dynamics can explain the decreased dynamic range and sensitivity of scotopic vision that has been observed in diabetes.

Dipeptidyl peptidase-IV inhibition prevents blood-retinal barrier breakdown, inflammation and neuronal cell death in the retina of type 1 diabetic rats.

Diabetic retinopathy, a leading cause of vision loss in working-age population, is often associated with inflammation and apoptosis. We have previously reported that sitagliptin, a DPP-IV inhibitor, exerts beneficial effects in the retina of type 2 diabetic animals. The present study aimed to evaluate whether sitagliptin can exert protective effects in the retina of type 1 diabetic animals by a mechanism independent of insulin secretion and glycemia normalization. Streptozotocin-induced diabetic rats were treated orally with sitagliptin (5mg/kg/day) for the last two weeks of 4 weeks of diabetes. Sitagliptin treatment did not change the weight and glucose, HbA1c or insulin levels. However, it prevented the diabetes-induced increase in DPP-IV/CD26 activity and levels in serum and retina. Sitagliptin also prevented the increase in blood-retinal barrier (BRB) permeability and inhibited the changes in immunoreactivity and endothelial subcellular distribution of occludin, claudin-5 and ZO-1 proteins induced by diabetes. Furthermore, sitagliptin decreased the retinal inflammatory state and neuronal apoptosis. Sitagliptin inhibited the BRB breakdown in a type 1 diabetic animal model, by a mechanism independent of normalization of glycemia, by preventing changes in tight junctions (TJs) organization. Sitagliptin also exerted protective effects against inflammation and pro-apoptotic state in the retina of diabetic rats. Altogether, these results suggest that sitagliptin might be envisaged to be used to prevent or delay some of the alterations associated with the development of diabetic retinopathy.

c-Src function is necessary and sufficient for triggering microglial cell activation.

Microglial cells are the resident macrophages of the central nervous system. Their function is essential for neuronal tissue homeostasis. After inflammatory stimuli, microglial cells become activated changing from a resting and highly ramified cell shape to an amoeboid-like morphology. These morphological changes are associated with the release of proinflammatory cytokines and glutamate, as well as with high phagocytic activity. The acquisition of such phenotype has been associated with activation of cytoplasmic tyrosine kinases, including those of the Src family (SFKs). In this study, using both in vivo and in vitro inflammation models coupled to FRET-based time-lapse microscopy, lentiviruses-mediated shRNA delivery and genetic gain-of-function experiments, we demonstrate that among SFKs c-Src function is necessary and sufficient for triggering microglia proinflammatory signature, glutamate release, microglia-induced neuronal loss, and phagocytosis. c-Src inhibition in retinal neuroinflammation experimental paradigms consisting of intravitreal injection of LPS or ischemia-reperfusion injury significantly reduced microglia activation changing their morphology to a more resting phenotype and prevented neuronal apoptosis. Our data demonstrate an essential role for c-Src in microglial cell activation.

Diabetic hyperglycemia reduces Ca2+ permeability of extrasynaptic AMPA receptors in AII amacrine cells.

There is increasing evidence that diabetic retinopathy is a primary neuropathological disorder that precedes the microvascular pathology associated with later stages of the disease. Recently, we found evidence for altered functional properties of synaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in A17, but not AII, amacrine cells in the mammalian retina, and the observed changes were consistent with an upregulation of the GluA2 subunit, a key determinant of functional properties of AMPA receptors, including Ca(2+) permeability and current-voltage (I-V) rectification properties. Here, we have investigated functional changes of extrasynaptic AMPA receptors in AII amacrine cells evoked by diabetes. With patch-clamp recording of nucleated patches from retinal slices, we measured Ca(2+) permeability and I-V rectification in rats with ∼3 wk of streptozotocin-induced diabetes and age-matched, noninjected controls. Under bi-ionic conditions (extracellular Ca(2+) concentration = 30 mM, intracellular Cs(+) concentration = 171 mM), the reversal potential (Erev) of AMPA-evoked currents indicated a significant reduction of Ca(2+) permeability in diabetic animals [Erev = -17.7 mV, relative permeability of Ca(2+) compared with Cs(+) (PCa/PCs) = 1.39] compared with normal animals (Erev = -7.7 mV, PCa/PCs = 2.35). Insulin treatment prevented the reduction of Ca(2+) permeability. I-V rectification was examined by calculating a rectification index (RI) as the ratio of the AMPA-evoked conductance at +40 and -60 mV. The degree of inward rectification in patches from diabetic animals (RI = 0.48) was significantly reduced compared with that in normal animals (RI = 0.30). These results suggest that diabetes evokes a change in the functional properties of extrasynaptic AMPA receptors of AII amacrine cells. These changes could be representative for extrasynaptic AMPA receptors elsewhere in AII amacrine cells and suggest that synaptic and extrasynaptic AMPA receptors are differentially regulated.

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.

Diabetes causes transient changes in the composition and phosphorylation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors and interaction with auxiliary proteins in the rat retina.

PURPOSE: The impairment of glutamatergic neurotransmission has been associated with diabetic complications in the central nervous system, such as diabetic retinopathy. Here, we investigated the effect of elevated glucose exposure and diabetes on α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor composition, subunit phosphorylation, and the association of the GluA2 subunit with accessory proteins in the retina. METHODS: The subunit composition of AMPA receptors and the association of the GluA2 subunit with modulatory proteins were evaluated with coimmunoprecipitation in retinal neural cell cultures and in the retina of experimentally induced-diabetic rats. The phosphorylation status of AMPA receptor subunits was evaluated with western blotting. RESULTS: In retinal neural cell cultures, elevated glucose did not significantly alter the composition of AMPA receptors, namely, the interactions between the GluA1, GluA2, and GluA4 subunits, but reduced GluA2 association with GRIP1. Moreover, elevated glucose did not cause changes on the level of GluA1 phosphorylated at serine residues 831 and 845. Diabetes induced early transitory changes in the interaction between AMPA receptor subunits GluA1, GluA2, and GluA4. At 8 weeks of diabetes, the content of GluA1 phosphorylated at serine 831 or serine 845 in the retina increased, compared to age-matched controls. CONCLUSIONS: Taken together, these results suggest that diabetes induces dynamic changes in AMPA receptor subunit composition, which could affect glutamatergic transmission in the rat retina.

Diabetes induces changes in KIF1A, KIF5B and dynein distribution in the rat retina: implications for axonal transport.

Diabetic retinopathy is a leading cause of vision loss and blindness. Disruption of axonal transport is associated with many neurodegenerative diseases and might also play a role in diabetes-associated disorders affecting nervous system. We investigated the impact of type 1 diabetes (2 and 8 weeks duration) on KIF1A, KIF5B and dynein motor proteins in the retina. Additionally, since hyperglycemia is considered the main trigger of diabetic complications, we investigated whether prolonged exposure to elevated glucose could affect the content and distribution of motor proteins in retinal cultures. The immunoreactivity of motor proteins was evaluated by immunohistochemistry in retinal sections and by immunoblotting in total retinal extracts from streptozotocin-induced diabetic and age-matched control animals. Primary retinal cultures were exposed to high glucose (30 mM) or mannitol (osmotic control; 24.5 mM plus 5.5 mM glucose), for seven days. Diabetes decreased the content of KIF1A at 8 weeks of diabetes as well as KIF1A immunoreactivity in the majority of retinal layers, except for the photoreceptor and outer nuclear layer. Changes in KIF5B immunoreactivity were also detected by immunohistochemistry in the retina at 8 weeks of diabetes, being increased at the photoreceptor and outer nuclear layer, and decreased in the ganglion cell layer. Regarding dynein immunoreactivity there was an increase in the ganglion cell layer after 8 weeks of diabetes. No changes were detected in retinal cultures. These alterations suggest that axonal transport may be impaired under diabetes, which might contribute to early signs of neural dysfunction in the retina of diabetic patients and animal models.

Role of microglia adenosine A(2A) receptors in retinal and brain neurodegenerative diseases.

Neuroinflammation mediated by microglial cells in the brain has been commonly associated with neurodegenerative diseases. Whether this microglia-mediated neuroinflammation is cause or consequence of neurodegeneration is still a matter of controversy. However, it is unequivocal that chronic neuroinflammation plays a role in disease progression and halting that process represents a potential therapeutic strategy. The neuromodulator adenosine emerges as a promising targeting candidate based on its ability to regulate microglial proliferation, chemotaxis, and reactivity through the activation of its G protein coupled A2A receptor (A2AR). This is in striking agreement with the ability of A2AR blockade to control several brain diseases. Retinal degenerative diseases have been also associated with microglia-mediated neuroinflammation, but the role of A2AR has been scarcely explored. This review aims to compare inflammatory features of Parkinson's and Alzheimer's diseases with glaucoma and diabetic retinopathy, discussing the therapeutic potential of A2AR in these degenerative conditions.

Calcium-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors trigger neuronal nitric-oxide synthase activation to promote nerve cell death in an Src kinase-dependent fashion.

In the retina information decoding is dependent on excitatory neurotransmission and is critically modulated by AMPA glutamate receptors. The Src-tyrosine kinase has been implicated in modulating neurotransmission in CNS. Thus, our main goal was to correlate AMPA-mediated excitatory neurotransmission with the modulation of Src activity in retinal neurons. Cultured retinal cells were used to access the effects of AMPA stimulation on nitric oxide (NO) production and Src phosphorylation. 4-Amino-5-methylamino-2',7'-difluorofluorescein diacetate fluorescence mainly determined NO production, and immunocytochemistry and Western blotting evaluated Src activation. AMPA receptors activation rapidly up-regulated Src phosphorylation at tyrosine 416 (stimulatory site) and down-regulated phosphotyrosine 527 (inhibitory site) in retinal cells, an effect mainly mediated by calcium-permeable AMPA receptors. Interestingly, experiments confirmed that neuronal NOS was activated in response to calcium-permeable AMPA receptor stimulation. Moreover, data suggest NO pathway as a key regulatory signaling in AMPA-induced Src activation in neurons but not in glial cells. The NO donor SNAP (S-nitroso-N-acetyl-DL-penicillamine) and a soluble guanylyl cyclase agonist (YC-1) mimicked AMPA effect in Src Tyr-416 phosphorylation, reinforcing that Src activation is indeed modulated by the NO pathway. Gain and loss-of-function data demonstrated that ERK is a downstream target of AMPA-induced Src activation and NO signaling. Furthermore, AMPA stimulated NO production in organotypic retinal cultures and increased Src activity in the in vivo retina. Additionally, AMPA-induced apoptotic retinal cell death was regulated by both NOS and Src activity. Because Src activity is pivotal in several CNS regions, the data presented herein highlight that Src modulation is a critical step in excitatory retinal cell death.

Differential contribution of the guanylyl cyclase-cyclic GMP-protein kinase G pathway to the proliferation of neural stem cells stimulated by nitric oxide.

Nitric oxide (NO) is an important inflammatory mediator involved in the initial boost in the proliferation of neural stem cells following brain injury. However, the mechanisms underlying the proliferative effect of NO are still unclear. The aim of this work was to investigate whether cyclic GMP (cGMP) and the cGMP-dependent kinase (PKG) are involved in the proliferative effect triggered by NO in neural stem cells. For this purpose, cultures of neural stem cells isolated from the mouse subventricular zone (SVZ) were used. We observed that long-term exposure to the NO donor (24 h), NOC-18, increased the proliferation of SVZ cells in a cGMP-dependent manner, since the guanylate cyclase inhibitor, ODQ, prevented cell proliferation. Similarly to NOC-18, the cGMP analogue, 8-Br-cGMP, also increased cell proliferation. Interestingly, shorter exposures to NO (6 h) increased cell proliferation in a cGMP-independent manner via the ERK/MAP kinase pathway. The selective inhibitor of PKG, KT5823, prevented the proliferative effect induced by NO at 24 h but not at 6 h. In conclusion, the proliferative effect of NO is initially mediated by the ERK/MAPK pathway, and at later stages by the GC/cGMP/PKG pathway. Thus, our work shows that NO induces neural stem cell proliferation by targeting these two pathways in a biphasic manner.