New Insights on Dietary Polyphenols for the Management of Oxidative Stress and Neuroinflammation in Diabetic Retinopathy.

Diabetic retinopathy (DR) is a neurodegenerative and vascular pathology that is considered one of the leading causes of blindness worldwide, resulting from complications of advanced diabetes mellitus (DM). Current therapies consist of protocols aiming to alleviate the existing clinical signs associated with microvascular alterations limited to the advanced disease stages. In response to the low resolution and limitations of the DR treatment, there is an urgent need to develop more effective alternative therapies to optimize glycemic, vascular, and neuronal parameters, including the reduction in the cellular damage promoted by inflammation and oxidative stress. Recent evidence has shown that dietary polyphenols reduce oxidative and inflammatory parameters of various diseases by modulating multiple cell signaling pathways and gene expression, contributing to the improvement of several chronic diseases, including metabolic and neurodegenerative diseases. However, despite the growing evidence for the bioactivities of phenolic compounds, there is still a lack of data, especially from human studies, on the therapeutic potential of these substances. This review aims to comprehensively describe and clarify the effects of dietary phenolic compounds on the pathophysiological mechanisms involved in DR, especially those of oxidative and inflammatory nature, through evidence from experimental studies. Finally, the review highlights the potential of dietary phenolic compounds as a prophylactic and therapeutic strategy and the need for further clinical studies approaching the efficacy of these substances in DR management.

The Role of Cannabinoids in CNS Development: Focus on Proliferation and Cell Death.

The active principles of Cannabis sativa are potential treatments for several diseases, such as pain, seizures and anorexia. With the increase in the use of cannabis for medicinal purposes, a more careful assessment of the possible impacts on embryonic development becomes necessary. Surveys indicate that approximately 3.9% of pregnant women use cannabis in a recreational and/or medicinal manner. However, although the literature has already described the presence of endocannabinoid system components since the early stages of CNS development, many of their physiological effects during this stage have not yet been established. Moreover, it is still uncertain how the endocannabinoid system can be altered in terms of cell proliferation and cell fate, neural migration, neural differentiation, synaptogenesis and particularly cell death. In relation to cell death in the CNS, knowledge about the effects of cannabinoids is scarce. Thus, the present work aims to review the role of the endocannabinoid system in different aspects of CNS development and discuss possible side effects or even opportunities for treating some conditions in the development of this tissue.

Gut microbiota: A potential therapeutic target for management of diabetic retinopathy?

Diabetic Retinopathy (DR) is one of the main complications of Diabetes Mellitus (DM), drastically impacting individuals of working age over the years, being one of the main causes of blindness in the world. The existing therapies for its treatment consist of measures that aim only to alleviate the existing clinical signs, associated with the microvasculature. These treatments are limited only to the advanced stages and not to the preclinical ones. In response to a treatment with little resolution and limited for many patients with DM, investigations of alternative therapies that make possible the improvement of the glycemic parameters and the quality of life of subjects with DR, become extremely necessary. Recent evidence has shown that deregulation of the microbiota (dysbiosis) can lead to low-grade, local and systemic inflammation, directly impacting the development of DM and its microvascular complications, including DR, in an axis called the intestine-retina. In this regard, the present review seeks to comprehensively describe the biochemical pathways involved in DR as well as the association of the modulation of these mechanisms by the intestinal microbiota, since direct changes in the microbiota can have a drastic impact on various physiological processes. Finally, emphasize the strong potential for modulation of the gut-retina axis, as therapeutic and prophylactic target for the treatment of DR.

TRPA1 mediates damage of the retina induced by ischemia and reperfusion in mice.

Oxidative stress is implicated in retinal cell injury associated with glaucoma and other retinal diseases. However, the mechanism by which oxidative stress leads to retinal damage is not completely understood. Transient receptor potential ankyrin 1 (TRPA1) is a redox-sensitive channel that, by amplifying the oxidative stress signal, promotes inflammation and tissue injury. Here, we investigated the role of TRPA1 in retinal damage evoked by ischemia (1 hour) and reperfusion (I/R) in mice. In wild-type mice, retinal cell numbers and thickness were reduced at both day-2 and day-7 after I/R. By contrast, mice with genetic deletion of TRPA1 were protected from the damage seen in their wild-type littermates. Daily instillation of eye drops containing two different TRPA1 antagonists, an oxidative stress scavenger, or a NADPH oxidase-1 inhibitor also protected the retinas of C57BL/6J mice exposed to I/R. Mice with genetic deletion of the proinflammatory TRP channels, vanilloid 1 (TRPV1) or vanilloid 4 (TRPV4), were not protected from I/R damage. Surprisingly, genetic deletion or pharmacological blockade of TRPA1 also attenuated the increase in the number of infiltrating macrophages and in the levels of the oxidative stress biomarker, 4-hydroxynonenal, and of the apoptosis biomarker, active caspase-3, evoked by I/R. These findings suggest that TRPA1 mediates the oxidative stress burden and inflammation that result in murine retinal cell death. We also found that TRPA1 (both mRNA and protein) is expressed by human retinal cells. Thus, it is possible that inhibition of a TRPA1-dependent pathway could also attenuate glaucoma-related retinal damage.

Chlorogenic acids inhibit glutamate dehydrogenase and decrease intracellular ATP levels in cultures of chick embryo retina cells.

Chlorogenic acids (CGAs) are a group of phenolic compounds found in worldwide consumed beverages such as coffee and green tea. They are synthesized from an esterification reaction between cinnamic acids, including caffeic (CFA), ferulic and p-coumaric acids with quinic acid (QA), forming several mono- and di-esterified isomers. The most prevalent and studied compounds are 3-O-caffeoylquinic acid (3-CQA), 4-O-caffeoylquinic acid (4-CQA) and 5-O-caffeoylquinic acid (5-CQA), widely described as having antioxidant and cell protection effects. CGAs can also modulate glutamate release from microglia by a mechanism involving a decrease of reactive oxygen species (ROS). Increased energy metabolism is highly associated with enhancement of ROS production and cellular damage. Glutamate can also be used as an energy source by glutamate dehydrogenase (GDH) enzyme, providing α-ketoglutarate to the tricarboxylic acid (TCA) cycle for ATP synthesis. High GDH activity is associated with some disorders, such as schizophrenia and hyperinsulinemia/hyperammonemia syndrome. In line with this, our objective was to investigate the effect of CGAs on GDH activity. We show that CGAs and CFA inhibits GDH activity in dose-dependent manner, reaching complete inhibition at high concentration with IC50 of 52 µM for 3-CQA and 158.2 µM for CFA. Using live imaging confocal microscopy and microplate reader, we observed that 3-CQA and CFA can be transported into neuronal cells by an Na+-dependent mechanism. Moreover, neuronal cells treated with CGAs presented lower intracellular ATP levels. Overall, these data suggest that CGAs have therapeutic potential for treatment of disorders associated with high GDH activity.

The (Na(+)/K (+))-ATPase activity in the developing rat retina: the role of insulin-like growth factor-I (IGF-I).

In this work, the (Na(+)/K(+))-ATPase activity was evaluated during the early stages of the postnatal development of rat retina and showed an almost three-time increase from P0 to P14. Expression of the three catalytic subunit isoforms (α1, α2, and α3) of the (Na(+)/K(+))-ATPase was also evaluated by immunoblot in the same period, but no correlation to the catalytic activity increment was observed. On the other hand, immunolocalization of these three α-catalytic isoforms in the developing retina showed an age-related pattern. Involvement of IGF-I in the stimulation of the (Na(+)/K(+))-ATPase was investigated. Our results demonstrate that the exogenous IGF-I (10 ng/mL) stimulates enzyme activity at the age of P7 only. Incubation of retinas with 10 µM I-OMe-AG 538 (inhibitor of the IGF-I receptor) indicates that the basal (Na(+)/K(+))-ATPase activity is sustained by endogenous IGF-I in P7 animals. These data were corroborated by an age-dependent decrease in the immunodetection of endogenous IGF-I as well as in the phosphorylation level of its cognate receptor in rat retina homogenates. The signaling pathway involved in IGF-I-induced modulation of the (Na(+)/K(+))-ATPase was also investigated. Our data show that the inhibitory effects induced by I-OMe-AG 538 and the PI 3-kinase inhibitor Ly 294002 on the basal (Na(+)/K(+))-ATPase activity were non-cumulative. Furthermore, IGF-I induced phosphorylation of PKB in a Ly 294002-sensitive manner. Together, these data demonstrate that the PI 3-kinase/PKB signaling pathway is involved in the IGF-I-sustained basal (Na(+)/K(+))-ATPase activity during the first 7 days of the postnatal development of rat retina.

Expression of A1 adenosine receptors in the developing avian retina: in vivo modulation by A(2A) receptors and endogenous adenosine.

Little is known about the mechanisms that regulate the expression of adenosine receptors during CNS development. We demonstrate here that retinas from chick embryos injected in ovo with selective adenosine receptor ligands show changes in A1 receptor expression after 48 h. Exposure to A1 agonist N6-cyclohexyladenosine (CHA) or antagonist 8-Cyclopentyl-1, 3-dipropylxanthine (DPCPX) reduced or increased, respectively, A1 receptor protein and [³H]DPCPX binding, but together, CHA+DPCPX had no effect. Interestingly, treatment with A(2A) agonist 3-[4-[2-[[6-amino-9-[(2R,3R,4S,5S)-5-(ethylcarbamoyl)-3,4-dihydroxy-oxolan-2-yl]purin-2-yl]amino] ethyl]phenyl] propanoic acid (CGS21680) increased A1 receptor protein and [³H]DPCPX binding, and reduced A(2A) receptors. The A(2A) antagonists 7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-trizolo[1,5-c] pyrimidine (SCH58261) and 4-(2-[7-amino-2-[2-furyl][1,2,4]triazolo[2,3-a][1,3,5]triazo-5-yl-amino]ethyl)phenol (ZM241385) had opposite effects on A1 receptor expression. Exposure to CGS21680 + CHA did not change A1 receptor levels, whereas CHA + ZM241385 or CGS21680 + DPCPX had no synergic effect. The blockade of adenosine transporter with S-(4-nitrobenzyl)-6-thioinosine (NBMPR) also reduced [³H]DPCPX binding, an effect blocked by DPCPX, but not enhanced by ZM241385. [³H]DPCPX binding kinetics showed that treatment with CHA reduced and CGS21680 increased the Bmax, but did not affect Kd values. CHA, DPCPX, CGS21680, and ZM241385 had no effect on A1 receptor mRNA. These data demonstrated an in vivo regulation of A1 receptor expression by endogenous adenosine or long-term treatment with A1 and A(2A) receptors modulators.

Neurochemical phenotype and birthdating of specific cell populations in the chick retina

The chick embryo is one of the most traditional models in developing neuroscience and its visual system has been one of the most exhaustively studied. The retina has been used as a model for studying the development of the nervous system. Here, we describe the morphological features that characterize each stage of the retina development and studies of the neurogenesis period of some specific neurochemical subpopulations of retinal cells by using a combination of immunohistochemistry and autoradiography of tritiated-thymidine. It could be concluded that the proliferation period of dopaminergic, GABAergic, cholinoceptive and GABAceptive cells does not follow a common rule of the neurogenesis. In addition, some specific neurochemical cell groups can have a restrict proliferation period when compared to the total cell population.

O embrião de galinha é um dos mais tradicionais modelosde estudos da neurociência do desenvolvimento e seu sistema visual tem sido um dos mais exaustivamente estudado. Aretina tem sido utilizada como modelo para estudar o desenvolvimento do sistema nervoso. Aqui, nós descrevemos as características morfológicas que caracterizam cada estádio da retina em desenvolvimento e os estudos do período de neurogênese de algumas subpopulações de células neuroquímicamente específicas da retina usando uma combinação de imunohistoquímica e autoradiografia de timidina-tritiada. Conclui-se que o período de proliferação das células dopaminérgicas, GABAérgicas, colinoceptivas e GABAceptivas não segue uma regra comum. Além disso, alguns grupos celulares neuroquimicamente distintos podem ter um período de proliferaçãomais restrito quando comparado ao da população total destas células.

Ethanol increases GABA release in the embryonic avian retina.

Several mechanisms underlying ethanol action in GABAergic synapses have been proposed, one of these mechanisms is on GABA release. Here, we report that in ovo exposure to ethanol induces an increase on GABA release in the embryonic chick retina. Eleven-day-old chick embryos (E11) received an injection of either phosphate buffer saline (PBS) or ethanol (10%, v/v, diluted in PBS), and were allowed to develop until E16. A single glutamate stimulus (2 mM) showed approximately a 40% increase on GABA release in E16 retinas when compared to controls. The effect was dependent on NMDA receptors and GAD65 mRNA levels, which were increased following the ethanol treatment. However, the numbers of GABA-, GAD-, and NR1-immunoreactive cells, and the expression levels of these proteins, were not affected. We conclude that ethanol treatment at a time point when synapses are being formed during development selectively increases GABA release in the retina via a NMDA receptor-dependent process.

Neurochemical phenotype and birthdating of specific cell populations in the chick retina.

The chick embryo is one of the most traditional models in developing neuroscience and its visual system has been one of the most exhaustively studied. The retina has been used as a model for studying the development of the nervous system. Here, we describe the morphological features that characterize each stage of the retina development and studies of the neurogenesis period of some specific neurochemical subpopulations of retinal cells by using a combination of immunohistochemistry and autoradiography of tritiated-thymidine. It could be concluded that the proliferation period of dopaminergic, GABAergic, cholinoceptive and GABAceptive cells does not follow a common rule of the neurogenesis. In addition, some specific neurochemical cell groups can have a restrict proliferation period when compared to the total cell population.

Glutamate receptors modulate sodium-dependent and calcium-independent vitamin C bidirectional transport in cultured avian retinal cells.

Vitamin C is transported in the brain by sodium vitamin C co-transporter 2 (SVCT-2) for ascorbate and glucose transporters for dehydroascorbate. Here we have studied the expression of SVCT-2 and the uptake and release of [(14)C] ascorbate in chick retinal cells. SVCT-2 immunoreactivity was detected in rat and chick retina, specially in amacrine cells and in cells in the ganglion cell layer. Accordingly, SVCT-2 was expressed in cultured retinal neurons, but not in glial cells. [(14)C] ascorbate uptake was saturable and inhibited by sulfinpyrazone or sodium-free medium, but not by treatments that inhibit dehydroascorbate transport. Glutamate-stimulated vitamin C release was not inhibited by the glutamate transport inhibitor l-beta-threo-benzylaspartate, indicating that vitamin C release was not mediated by glutamate uptake. Also, ascorbate had no effect on [(3)H] D-aspartate release, ruling out a glutamate/ascorbate exchange mechanism. 2-Carboxy-3-carboxymethyl-4-isopropenylpyrrolidine (Kainate) or NMDA stimulated the release, effects blocked by their respective antagonists 6,7-initroquinoxaline-2,3-dione (DNQX) or (5R,2S)-(1)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801). However, DNQX, but not MK-801 or 2-amino-5-phosphonopentanoic acid (APV), blocked the stimulation by glutamate. Interestingly, DNQX prevented the stimulation by NMDA, suggesting that the effect of NMDA was mediated by glutamate release and stimulation of non-NMDA receptors. The effect of glutamate was neither dependent on external calcium nor inhibited by 1,2-bis (2-aminophenoxy) ethane-N',N',N',N',-tetraacetic acid tetrakis (acetoxy-methyl ester) (BAPTA-AM), an internal calcium chelator, but was inhibited by sulfinpyrazone or by the absence of sodium. In conclusion, retinal cells take up and release vitamin C, probably through SVCT-2, and the release can be stimulated by NMDA or non-NMDA glutamate receptors.

Signal transduction pathways associated with ATP-induced proliferation of cell progenitors in the intact embryonic retina.

ATP and ADP induce retinal cell proliferation through activation of PKC and extracellular signal-regulated kinases (ERKs). Here, we characterized the effect of purinergic agonists on the turnover of phosphoinositides and activation of ERKs during development of the chick embryo retina. When intact retinas were incubated with ATP, ADP or UTP, a dose-dependent accumulation of [(3)H]-phosphoinositides was observed (% of control, EC(50): 548+/-20.5%, 0.18 mM; 314+/-53.8%, 0.51 mM; 704+/-139.9%, 0.018 mM, respectively). Only the response promoted by ADP was completely inhibited by the P2 receptor antagonists, PPADS and suramin. All the responses decreased with the progression of retinal development. Western blot assays revealed that ATP, ADP and UTP stimulated the phosphorylation of ERKs in the chick embryo retina very early during development (% of control: 174+/-16; 199+/-16.4 and 206+/-37, respectively). The responses to ADP and UTP were transient and dose-dependent, showing EC(50) values of 0.12 mM and 0.009 mM. The response to ADP was inhibited by the antagonists PPADS and suramin and by U73122 and chelerythrine chloride, which block PLC and PKC, respectively. Conversely, chelerythrine chloride did not block the response induced by UTP. Immunohistochemical analysis revealed that ATP and ADP induced the phosphorylation of ERKs in cells of the neuroblastic layer of retinas from embryos at E8. Our data showed that ATP, ADP and UTP stimulate the turnover of InsPs and promoted the activation of ERKs in the chick embryo retina. ADP, through activation of P2Y(1) receptors, activated ERK pathway through PLC and PKC and UTP, via P2Y(4)-like receptors, induced the phosphorylation of ERKs through a pathway that did not involve PKC.

Local differences in GABA release induced by excitatory amino acids during retina development: selective activation of NMDA receptors by aspartate in the inner retina.

Glutamate and GABA are the major excitatory and inhibitory neurotransmitters in the CNS. In the retina, it has been shown that glutamate and aspartate and their agonists kainate and NMDA promote the release of GABA. In the chick retina, at embryonic day 14 (E14), glutamate and kainate were able to induce the release of GABA from amacrine and horizontal cells as detected by GABA-immunoreactivity. NMDA also induced GABA release restricted to amacrine cell population and its projections to the inner plexiform layer (E14 and E18). Although aspartate reduced GABA immunoreactivity, specifically in amacrine cells of E18 retinas, it was not efficient to promote GABA release from retinas at E14. As observed in differentiated retinas, dopamine inhibited the GABA release promoted by NMDA and aspartate but not by kainate. Our data show that different retinal sites respond to distinct EAAs via different receptor systems.

GABA(Abeta2-3) immunoreactive cells in the developing chick retina.

Gama-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system (CNS). It has been shown that GABA is an important factor for CNS maturation and that its functions are mainly mediated by GABA(A) receptors. Thus, in order to fully comprehend the role of GABA during development, it is essential to establish the developmental features of the catalytic subunits (beta) of GABA(A) receptor. Here, we determine the ontogenesis and neurogenesis of cells expressing beta2-3 subunits of GABA(A) receptor (GABA(Abeta2-3)) in the chick retina. In the ontogenetic experiments, only the immunohistochemistry for GABA(Abeta2-3) approach was employed. For neurogenesis a double-labeling method (autoradiography and immunohistochemistry) was applied. [H(3)]-thymidine was injected into eggs (2-11 days) and the embryos were sacrificed at embryonic day 19 (E19). GABA(Abeta2-3) immunohistochemistry was processed and then autoradiography was performed. We used a cumulative counting method to quantify the autoradiographic grains. The ontogenesis study revealed that at E9, GABA(Abeta2-3) immunoreactivity was restricted to the inner plexiform layer and the first cell bodies immunoreactive to GABA(Abeta2-3) were seen at E14. Thereafter, the number of cell bodies and the intensity of GABA(Abeta2-3) immunoreactivity increased until the adult pattern was established. The neurogenesis study showed that cells that will express GABA(Abeta2-3) were generated between E6 and E9. In addition, from E7 to E9 the rate of neurogenesis of GABA(Abeta2-3) immunoreactive cells quickly increases. Therefore, the detection of GABA(Abeta2-3) occurred only after the end of generation period of this cell population.