Microglia as therapeutic targets in retinal degeneration: role of translocator protein (18 κDa) (TSPO) and minocycline.

Microglia are the resident immune cells of the central nervous system (CNS) and play an important role in innate immune defense as well as tissue homeostasis. Chronic microglial reactivity, microgliosis, is a general hallmark of inflammatory and degenerative diseases that affect the CNS, including the retina. There is increasing evidence that chronic microgliosis is more than just a bystander effect, but rather actively contributes to progression of degeneration through processes such as toxic nitric oxide (NO) production and even phagocytosis of stressed but viable photoreceptors. Therefore immunmodulation of microglia presents a possible therapeutic strategy for retinal degenerations. Notably, the expression of the mitochondrial translocator protein 18 (κDa) (TSPO) is highly elevated in reactive microglia as seen in several neuroinflammatory diseases such as Alzheimer’s disease, Parkinson’s disease and multiple sclerosis. Therefore it is used as a gliosis biomarker in the brain. Moreover TSPO ligands show potent effects in resolving neuroinflammatory brain disorders. However, TSPO expression in the eye had not been investigated before. Further, it was unknown whether TSPO ligands’ potent immunomodulatory effects could be used to treat retinal degenerations. To fill this gap, the study aimed to analyze whether TSPO is also a potential biomarker for degenerative processes in the retina. Moreover the thesis attempted to determine whether a specific TSPO ligand, XBD173, might modulate microglial reactivity and is a potent therapeutic, to treat retinal degenerative diseases. The findings revealed that TSPO is strongly upregulated in microglial cells of retinoschisin-deficient (RS1-/y) mice, a model of inherited retinal degeneration and in a murine light damage model. A co-localization of TSPO and microglia was furthermore detectable in human retinal sections, indicating a potential role for TSPO as a biomarker for retinal degenerations. In vitro assays showed that the TSPO ligand XBD173 effectively inhibited features of microglial activation such as morphological transformation into reactive phagocytes and enhanced expression of pro-inflammatory cytokines. XBD173 also reduced microglial migration and proliferation and reduced their neurotoxic potential on photoreceptor cells. In two independent mouse models of light-induced retinal degeneration, the treatment with XBD173 reduced accumulation of amoeboid, reactive microglia in the outer retina and attenuated degenerative processes, indicated by a nearly preserved photoreceptor layer. A further question addressed in this thesis was whether minocycline, an antibiotic with additional anti-inflammatory properties is able to reduce microglial neurotoxicity and to protect the retina from degeneration. Minocycline administration dampened microglial pro-inflammatory gene expression, NO production and neurotoxicity on photoreceptors. Interestingly, in addition to its immunomodulatory effect, minocycline also increased the viability of photoreceptors in a direct manner. In the light damage model, minocycline administration counter-acted microglial activation and blocked retinal degeneration. Taken together these results identified TSPO as a biomarker for microglial reactivity and as therapeutic target in the retina. Targeting TSPO with XBD173 was able to reverse microglial reactivity and could prevent degenerative processes in the retina. In addition, the study showed that the antibiotic minocycline effectively counter-regulates microgliosis and light-induced retinal degeneration. Considering that microgliosis is a major contributing factor for retinal degenerative disorders, this thesis supports the concept of a microglia-directed therapy to treat retinal degeneration.

Numb/Numbl-Opo antagonism controls retinal epithelium morphogenesis by regulating integrin endocytosis

Polarized trafficking of adhesion receptors plays a pivotal role in controlling cellular behavior during morphogenesis. Particularly, clathrin-dependent endocytosis of integrins has long been acknowledged as essential for cell migration. However, little is known about the contribution of integrin trafficking to epithelial tissue morphogenesis. Here we show how the transmembrane protein Opo, previously described for its essential role during optic cup folding, plays a fundamental role in this process. Through interaction with the PTB domain of the clathrin adaptors Numb and Numbl via an integrin-like NPxF motif, Opo antagonizes Numb/Numbl function and acts as a negative regulator of integrin endocytosis in vivo. Accordingly, numb/numbl gain-of-function experiments in teleost embryos mimic the retinal malformations observed in opo mutants. We propose that developmental regulator Opo enables polarized integrin localization by modulating Numb/Numbl, thus directing the basal constriction that shapes the vertebrate retina epithelium.

Endocytosis Of Tight Junctions Caveolin Nitrosylation Dependent Is Improved By Cocoa Via Opioid Receptor On Rpe Cells In Diabetic Conditions.

Retinal pigment epithelium cells, along with tight junction (TJ) proteins, constitute the outer blood retinal barrier (BRB). Contradictory findings suggest a role for the outer BRB in the pathogenesis of diabetic retinopathy (DR). The aim of this study was to investigate whether the mechanisms involved in these alterations are sensitive to nitrosative stress, and if cocoa or epicatechin (EC) protects from this damage under diabetic (DM) milieu conditions. Cells of a human RPE line (ARPE-19) were exposed to high-glucose (HG) conditions for 24 hours in the presence or absence of cocoa powder containing 0.5% or 60.5% polyphenol (low-polyphenol cocoa [LPC] and high-polyphenol cocoa [HPC], respectively). Exposure to HG decreased claudin-1 and occludin TJ expressions and increased extracellular matrix accumulation (ECM), whereas levels of TNF-α and inducible nitric oxide synthase (iNOS) were upregulated, accompanied by increased nitric oxide levels. This nitrosative stress resulted in S-nitrosylation of caveolin-1 (CAV-1), which in turn increased CAV-1 traffic and its interactions with claudin-1 and occludin. This cascade was inhibited by treatment with HPC or EC through δ-opioid receptor (DOR) binding and stimulation, thereby decreasing TNF-α-induced iNOS upregulation and CAV-1 endocytosis. The TJ functions were restored, leading to prevention of paracellular permeability, restoration of resistance of the ARPE-19 monolayer, and decreased ECM accumulation. The detrimental effects on TJs in ARPE-19 cells exposed to DM milieu occur through a CAV-1 S-nitrosylation-dependent endocytosis mechanism. High-polyphenol cocoa or EC exerts protective effects through DOR stimulation.

Applications Of Visual Evoked Potentials And Fourier-domain Optical Coherence Tomography In Parkinson's Disease: A Controlled Study.

The goal of this cross-sectional observational study was to quantify the pattern-shift visual evoked potentials (VEP) and the thickness as well as the volume of retinal layers using optical coherence tomography (OCT) across a cohort of Parkinson's disease (PD) patients and age-matched controls. Forty-three PD patients and 38 controls were enrolled. All participants underwent a detailed neurological and ophthalmologic evaluation. Idiopathic PD cases were included. Cases with glaucoma or increased intra-ocular pressure were excluded. Patients were assessed by VEP and high-resolution Fourier-domain OCT, which quantified the inner and outer thicknesses of the retinal layers. VEP latencies and the thicknesses of the retinal layers were the main outcome measures. The mean age, with standard deviation (SD), of the PD patients and controls were 63.1 (7.5) and 62.4 (7.2) years, respectively. The patients were predominantly in the initial Hoehn-Yahr (HY) disease stages (34.8% in stage 1 or 1.5, and 55.8 % in stage 2). The VEP latencies and the thicknesses as well as the volumes of the retinal inner and outer layers of the groups were similar. A negative correlation between the retinal thickness and the age was noted in both groups. The thickness of the retinal nerve fibre layer (RNFL) was 102.7 μm in PD patients vs. 104.2 μm in controls. The thicknesses of retinal layers, VEP, and RNFL of PD patients were similar to those of the controls. Despite the use of a representative cohort of PD patients and high-resolution OCT in this study, further studies are required to establish the validity of using OCT and VEP measurements as the anatomic and functional biomarkers for the evaluation of retinal and visual pathways in PD patients.

Increased Circulating Pedf And Low Sicam-1 Are Associated With Sickle Cell Retinopathy.

Sickle cell retinopathy (SCR) develops in up to 30% of sickle cell disease patients (SCD) during the second decade of life. Treatment for this affection remains palliative, so studies on its pathophysiology may contribute to the future development of novel therapies. SCR is more frequently observed in hemoglobin SC disease and derives from vaso-occlusion in the microvasculature of the retina leading to neovascularization and, eventually, to blindness. Circulating inflammatory cytokines, angiogenic factors, and their interaction may contribute to the pathophysiology of this complication. Angiopoietin (Ang)-1, Ang-2, soluble vascular cell adhesion molecule-1, intercellular adhesion molecule (ICAM)-1, E-selectin, P-selectin, IL1-β, TNF-α, pigment epithelium derived factor (PEDF) and vascular endothelial growth factor plasmatic levels were determined in 37 SCD patients with retinopathy, 34 without retinopathy, and healthy controls. We observed that sICAM-1 is significantly decreased, whereas PEDF is elevated in HbSC patients with retinopathy (P=0.012 and P=0.031, respectively). Ang-1, Ang-2 and IL1-β levels were elevated in SCD patients (P=0.001, P<0.001 and P=0.001, respectively), compared to controls, and HbSS patients presented higher levels of Ang-2 compared to HbSC (P<0.001). Our study supports the possible influence of sICAM-1 and PEDF on the pathophysiology of retinal neovascularization in SCD patients.

Polyphenol-enriched Cocoa Protects The Diabetic Retina From Glial Reaction Through The Sirtuin Pathway.

Cocoa is rich in flavonoids, which are potent antioxidants with established benefits for cardiovascular health but unproven effects on neurodegeneration. Sirtuins (SIRTs), which make up a family of deacetylases, are thought to be sensitive to oxidation. In this study, the possible protective effects of cocoa in the diabetic retina were assessed. Rat Müller cells (rMCs) exposed to normal or high glucose (HG) or H2O2 were submitted to cocoa treatment in the presence or absence of SIRT-1 inhibitor and small interfering RNA The experimental animal study was conducted in streptozotocin-induced diabetic rats randomized to receive low-, intermediate-, or high-polyphenol cocoa treatments via daily gavage for 16 weeks (i.e., 0.12, 2.9 or 22.9 mg/kg/day of polyphenols). The rMCs exposed to HG or H2O2 exhibited increased glial fibrillary acidic protein (GFAP) and acetyl-RelA/p65 and decreased SIRT1 activity/expression. These effects were cancelled out by cocoa, which decreased reactive oxygen species production and PARP-1 activity, augmented the intracellular pool of NAD(+), and improved SIRT1 activity. The rat diabetic retinas displayed the early markers of retinopathy accompanied by markedly impaired electroretinogram. The presence of diabetes activated PARP-1 and lowered NAD(+) levels, resulting in SIRT1 impairment. This augmented acetyl RelA/p65 had the effect of up-regulated GFAP. Oral administration of polyphenol cocoa restored the above alterations in a dose-dependent manner. This study reveals that cocoa enriched with polyphenol improves the retinal SIRT-1 pathway, thereby protecting the retina from diabetic milieu insult.

Estudo da circulação retrobulbar e do campo visual após dose única oral de citrato de sildenafil (Viagra®)

Purpose: To analyze the effects of 100 mg of sildenafil citrate (Viagra®) on the retrobulbar circulation and visual field. Methods: A double masked, placebo controlled study was conducted in 10 males with a mean age of 27.7 + 5.68 years. The right eye of each volunteer underwent orbital color Doppler imaging and automated perimetry (Humphrey, program 30-2, Full-Threshold Strategy) at 3 occasions: baseline, 1 hour after placebo and 1 hour after 100 mg of sildenafil. The foveal threshold and the mean deviation (MD) were analyzed by automated perimetry on the three occasions. Color Doppler imaging allowed the measurement of the peak systolic velocity (PSV), end diastolic velocity (EDV) and Pourcelot index (PI) in the central retinal artery and ophthalmic artery. Results: The foveal threshold and the mean deviation did not show a significant change following the administration of sildenafil. The ophthalmic artery peak systolic velocity and end diastolic velocity significantly increased after the administration of sildenafil (p<0.001). The hemodynamic parameters in the central retinal artery and the ophthalmic artery PI did not significantly change. Conclusions: Sildefanil citrate increased the blood flow velocities in the ophthalmic artery in normal subjects, with no significant changes in the foveal threshold and mean deviation in the automated perimetry.

Efeitos da constrição pupilar na perimetria de freqüência dupla em olhos normais

Purpose: To study the effects of pupillary constriction on frequency doubling perimetry in a group of normal subjects. Methods: Eighteen healthy volunteers participated in the study. Only one eye per patient (right eye) underwent frequency doubling perimetry (Full Threshold C-20 strategy). For the second session, one drop of 2% pilocarpine was administered to the volunteers' right eye and the examination was repeated after 60 minutes. Results: Sixty minutes after administration of 2% pilocarpine, there was a significant reduction of the pupillary diameter from 4.22 ± 0.17 mm to 1.55 ± 0.51 mm (p<0.05). There was a significant reduction of the mean retinal sensibility after pupillary constriction. The threshold sensitivity of the central 5º worsened by 5.67 ± 2.49 dB; the area between 2.5º and 10º worsened by 4.49 ± 2.73 dB; and the area between 10º and 20º worsened by 5.10 ± 3.55 dB (p<0.01). A reduction of 4.06 ± 2.67 dB was observed in the mean deviation, as well as an increase of 0.64 ± 0.94 dB in the pattern standard deviation (p<0.01). No differences were observed regarding the number of fixation losses, false-positive and false-negatives responses, and duration of the examination. Conclusion: Changes in pupillary diameter may produce significant declines in threshold sensitivities of the 20º visual field tested by frequency doubling perimetry. These results suggest that is important to maintain a constant pupillary diameter in seriate examinations.

Influência da redução medicamentosa da pressão intra-ocular na medida da espessura da camada de fibras nervosas da retina de olhos hipertensos e glaucomatosos pela polarimetria de varredura a laser

PURPOSE: To evaluate changes in retinal nerve fiber layer thickness as measured by scanning laser polarimetry (SLP) after the use of medication to reduce intraocular pressure (IOP) in glaucomatous or ocular hypertensive patients. METHODS: The authors prospectively enrolled 37 eyes of 37 patients in whom IOP was reduced by more than 25% after the use of medication. The images were obtained before and 15 to 30 days after the introduction of medication. The SLP parameters measured before and after the use of medication were compared using paired Student's t Test. RESULTS: The mean IOP was significantly reduced from 26.57±4.23 mmHg to 16.54 ±2.92 mmHg after the use of medication (p<0.05). None of the 10 SLP analyzed parameters was significantly affected by the reduction of IOP with medication (p>0.05). CONCLUSION: The retinal nerve fiber layer thickness, as measured by SLP, is not affected by the reduction of IOP with medication in patients with glaucoma or ocular hypertension.

Sensitivity and specificity of machine learning classifiers for glaucoma diagnosis using Spectral Domain OCT and standard automated perimetry

PURPOSE: To evaluate the sensitivity and specificity of machine learning classifiers (MLCs) for glaucoma diagnosis using Spectral Domain OCT (SD-OCT) and standard automated perimetry (SAP). METHODS: Observational cross-sectional study. Sixty two glaucoma patients and 48 healthy individuals were included. All patients underwent a complete ophthalmologic examination, achromatic standard automated perimetry (SAP) and retinal nerve fiber layer (RNFL) imaging with SD-OCT (Cirrus HD-OCT; Carl Zeiss Meditec Inc., Dublin, California). Receiver operating characteristic (ROC) curves were obtained for all SD-OCT parameters and global indices of SAP. Subsequently, the following MLCs were tested using parameters from the SD-OCT and SAP: Bagging (BAG), Naive-Bayes (NB), Multilayer Perceptron (MLP), Radial Basis Function (RBF), Random Forest (RAN), Ensemble Selection (ENS), Classification Tree (CTREE), Ada Boost M1(ADA),Support Vector Machine Linear (SVML) and Support Vector Machine Gaussian (SVMG). Areas under the receiver operating characteristic curves (aROC) obtained for isolated SAP and OCT parameters were compared with MLCs using OCT+SAP data. RESULTS: Combining OCT and SAP data, MLCs' aROCs varied from 0.777(CTREE) to 0.946 (RAN).The best OCT+SAP aROC obtained with RAN (0.946) was significantly larger the best single OCT parameter (p<0.05), but was not significantly different from the aROC obtained with the best single SAP parameter (p=0.19). CONCLUSION: Machine learning classifiers trained on OCT and SAP data can successfully discriminate between healthy and glaucomatous eyes. The combination of OCT and SAP measurements improved the diagnostic accuracy compared with OCT data alone.

Psychophysical measurements of luminance and chromatic spatial and temporal contrast sensitivity in Duchenne muscular dystrophy

In children with Duchenne muscular dystrophy, color vision losses have been related to dystrophin deletions downstream of exon 30, which affect a dystrophin isoform, Dp260, present in the retina. To further evaluate visual function in DMD children, we measured spatial, temporal, and chromatic red-green and blue-yellow contrast sensitivity in two groups of DMD children with gene deletion downstream and upstream of exon 30. Psychophysical spatial contrast sensitivity was measured for low, middle, and high spatial frequencies with achromatic gratings and for low and middle frequencies with red-green and blue-yellow chromatic gratings. Temporal contrast sensitivity was also measured with achromatic stimuli. A reduction in sensitivity at all spatial luminance contrasts was found for the DMD patients with deletion downstream of exon 30. Similar results were found for temporal luminance contrast sensitivity. Red-green chromatic contrast sensitivity was reduced in DMD children with deletion downstream of exon 30, whereas blue-yellow chromatic contrast sensitivity showed no significant differences. We conclude that visual function is impaired in DMD children. Furthermore, we report a genotype-phenotype relationship because the visual impairment occurred in children with deletion downstream but not upstream of exon 30, affecting the retinal isoform of dystrophin Dp260.

Comparison of full-field electroretinogram in diabetic and non diabetic dogs with cataracts

Being the commonest ocular disorder, dense cataracts disable fundoscopic examination and the diagnosis of retinal disorders, which dogs may be predisposed. The aim of this study was to compare the electroretinographic responses recorded according to the International Society for Clinical Electrophysiology of Vision human protocol to evaluate retinal function of diabetic and non diabetic dogs, both presenting mature or hypermature cataracts. Full-field electroretinogram was recorded from 66 dogs, with ages varying from 6 to 15 years old allocated into two groups: (1) CG, non diabetic cataractous dogs, and (2) DG, diabetic cataractous dogs. Mean peak-to-peak amplitude (microvolts) and b-wave implicit time (milliseconds) were determined for each of the five standard full-field ERG responses (rod response, maximal response, oscillatory potentials, single-flash cone response and 30 Hz flicker). Comparing CG to DG, ERGs recorded from diabetic dogs presented lower amplitude and prolonged b-wave implicit time in all ERG responses. Prolonged b-wave implicit time was statistically significant (p< 0.05) at 30 Hz flicker (24.0 ms versus 22.4 ms). These data suggests full-field ERG is capable to record sensible alterations, such as flicker's implicit time, being useful to investigate retinal dysfunction in diabetic dogs.

Padronização do eletrorretinograma de campo total em cães

Eletrorretinograma (ERG) é o meio diagnóstico objetivo e não-invasivo para avaliar a função retiniana e detectar precocemente, em várias espécies, lesões nas suas camadas mais externas. As indicações mais comuns para ERG em cães são: avaliação pré-cirúrgica de pacientes com catarata, caracterização de distúrbios que causam cegueira, além de servir como importante modelo para o estudo da distrofia retiniana que acomete o homem. Vários são os fatores que podem alterar o ERG tais como: eletrorretinógrafo, fonte de estimulação luminosa, tipo do eletrodo, tempo de adaptação ao escuro, tamanho pupilar, opacidade de meios e protocolo de sedação ou anestesia; além da espécie, raça e idade. Objetivou-se com este estudo padronizar o ERG para cães submetidos à sedação, seguindo o protocolo da International Society for Clinical Electrophysiology of Vision (ISCEV), utilizando Ganzfeld e eletrodos Burian Allen. Foram realizados 233 eletrorretinogramas em cães, 147 fêmeas e 86 machos, com idades entre um e 14 anos. Dos 233 cães examinados, 100 apresentavam catarata em diferentes estágios de maturação, 72 eram diabéticos e apresentavam catarata madura ou hipermadura, 26 apresentaram eletrorretinograma compatível com degeneração retiniana progressiva, três apresentaram eletrorretinograma compatível com síndrome da degeneração retiniana adquirida subitamente e 32 não apresentaram lesão retiniana capaz de atenuar as respostas do ERG, sendo considerados normais quanto à função retiniana. A sedação foi capaz de produzir boa imobilização do paciente sem rotacionar o bulbo ocular, permitindo adequada estimulação retiniana bilateralmente, com auxílio do Ganzfeld. O sistema eletrodiagnóstico Veris registrou com sucesso e simultaneamente de ambos os olhos, as cinco respostas preconizadas pela ISCEV. Como o ERG de campo total tornou-se exame fundamental na rotina oftalmológica, sua padronização é indispensável quando se objetiva comparar resultados de laboratórios distintos. A confiabilidade e reprodutibilidade deste protocolo foi demonstrada com a obtenção de registros de ótima qualidade utilizando protocolo padrão da ISCEV, eletrorretinógrafo Veris, Ganzfeld e eletrodos Burian Allen nos cães submetidos à sedação.

Active Dendrites Enhance Neuronal Dynamic Range

Since the first experimental evidences of active conductances in dendrites, most neurons have been shown to exhibit dendritic excitability through the expression of a variety of voltage-gated ion channels. However, despite experimental and theoretical efforts undertaken in the past decades, the role of this excitability for some kind of dendritic computation has remained elusive. Here we show that, owing to very general properties of excitable media, the average output of a model of an active dendritic tree is a highly non-linear function of its afferent rate, attaining extremely large dynamic ranges (above 50 dB). Moreover, the model yields double-sigmoid response functions as experimentally observed in retinal ganglion cells. We claim that enhancement of dynamic range is the primary functional role of active dendritic conductances. We predict that neurons with larger dendritic trees should have larger dynamic range and that blocking of active conductances should lead to a decrease in dynamic range.

Expression and cellular localization of microRNA-29b and RAX, an activator of the RNA-dependent protein kinase (PKR), in the retina of streptozotocin-induced diabetic rats

Purpose: The apoptosis of retinal neurons plays a critical role in the pathogenesis of diabetic retinopathy (DR), but the molecular mechanisms underlying this phenomenon remain unclear. The purpose of this study was to investigate the cellular localization and the expression of microRNA-29b (miR-29b) and its potential target PKR associated protein X (RAX), an activator of the pro-apoptotic RNA-dependent protein kinase (PKR) signaling pathway, in the retina of normal and diabetic rats. Methods: Retinas were obtained from normal and diabetic rats within 35 days after streptozotocin (STZ) injection. In silico analysis indicated that RAX is a potential target of miR-29b. The cellular localization of miR-29b and RAX was assessed by in situ hybridization and immunofluorescence, respectively. The expression levels of miR-29b and RAX mRNA were evaluated by quantitative reverse transcription PCR (qRT-PCR), and the expression of RAX protein was evaluated by western blot. A luciferase reporter assay and inhibition of endogenous RAX were performed to confirm whether RAX is a direct target of miR-29b as predicted by the in silico analysis. Results: We found that miR-29b and RAX are localized in the retinal ganglion cells (RGCs) and the cells of the inner nuclear layer (INL) of the retinas from normal and diabetic rats. Thus, the expression of miR-29b and RAX, as assessed in the retina by quantitative RT-PCR, reflects their expression in the RGCs and the cells of the INL. We also revealed that RAX protein is upregulated (more than twofold) at 3, 6, 16, and 22 days and downregulated (70%) at 35 days, whereas miR-29b is upregulated (more than threefold) at 28 and 35 days after STZ injection. We did not confirm the computational prediction that RAX is a direct target of miR-29b. Conclusions: Our results suggest that RAX expression may be indirectly regulated by miR-29b, and the upregulation of this miRNA at the early stage of STZ-induced diabetes may have a protective effect against the apoptosis of RGCs and cells of the INL by the pro-apoptotic RNA-dependent protein kinase (PKR) signaling pathway.