Diagnosing and monitoring diabetic macular edema: structural and functional tests.

Diabetic macular edema remains a major cause of visual impairment in adults despite the use of intensive glycemic control, photocoagulation therapy and new intravitreal drugs in the treatment of this disease. Although early diagnosis and treatment lead to better results, we still have patients who become legally blind. Therefore, better structural and functional characterization of this disease is necessary in order to customize treatment

Neuroretinal activation in diabetes mellitus

Background: Diabetic retinopathy (DR), one of the leading causes of blindness in developed countries, is the major microvascular complication of diabetes mellitus. Recent studies have demonstrated that the alteration of glial cells and the consequent loss of retinal neuronal cells occur before the vascular lesions are clinically detectable. Purpose: To find early biomarkers of glial activation in the aqueous humor (AH) of diabetic patients both in presence and in absence of clinically detectable signs ofDR. Materials and methods:During cataract surgery, 34 patients’ AH samples were collected as follows: 12 healthy subjects, 11 diabetic patients without DR and 11 diabetic patients with nonproliferative diabetic retinopathy (5 without macular edema-ME and 6 with ME). Before surgery, full ophthalmic examination and Spectral-Domain Optical Coherence Tomography (SD-OCT) (Spectralis HRA+OCT, Heildeberg Engineering) were performed in all eyes. The samples were analyzed for the quantification of total proteins by Bradford method, of GFAP, AQP1 and AQP4 by ELISA and of 40 inflammatory cytokines by protein array. Segmentation of retinal layers was also performed. Results:Mean concentration of GFAP, AQP1 e AQP4 was significantly increased in diabetics versus controls (324.44±262.54pg/µg vs 182.34±114.44pg/µg for GFAP; 105.72±15.69pg/µg vs 50.92±20.36pg/µg for AQP1; and 852.03+103.24pg/µg vs 33.58±21.20pg/µg for AQP4, p<0.05). GFAP showed an approximate 0.8 fold increase, AQP1 1.1 fold increase, whereas AQP4 about 24 folds increase in diabetic patients versus controls. When we separately evaluated DR-no ME eyes vs DR-ME eyes, there was a significant decrease in GFAP, AQP1 e AQPR in DR-ME eyes versus DR-no ME eyes, (Tukey Kramer post hoc p<0.05). GFAP and AQP1 showed even a slight fold decrease versus controls. AQP4/AQP1 concentration showed weak and non significant correlation (Tau=0.21, p=0.3) between these biomarkers, despite the trend in increase.Following cytokines were increased in diabetic patients (with or without DR) compared to healthy subjects: GFAP, AQP1, AQP4, IFNy, IL-1a, IL-1b, IL-3, IL-4, IL-10, IL-11, IL-17, TNF-α, TNF-ß, MCP1, MCP2, Eotaxin,Eotaxin 2, RANTES, sTNFRII, GM-CSF, IP-10, MIP1a, MIP1b. RNFL mean thickness was significantly higher in diabetic patients with DR and ME compared to diabetics without ME (both with and without DR) and healthy subjects (respectively 37.3 μm vs 24.3μm vs 26μm 5μm vs 26.8μm), and the same significance was observed in the inner (33.4μm vs 22.0μm vs 25.0μm 24.3μm) and the external (54.7μm vs 36.7μm 34.6μm 38.1μm) rings and in the superior (40.3μm vs. 26.1μm vs 29.2μm vs 29.5μm), inferior (44.3μm vs 27.2μm vs 29.1μm vs 30.1μm) and temporal (26.3μm vs 16.8μm vs 18.9μm vs 18.0μm) sectors.RNFL mean thickness was significantly reduced in diabetics with DR and without ME compared to healthy subjects. Conclusions: 23 different biomarkers of glial activation have been recognized in the AH of diabetic patients even with subclinical DR. These proteins could be used in the future as risk markers of occurrence of DR and could provide useful therapeutic targets for its prevention and therapy

Diabetic Macular Edema With and Without Subfoveal Neuroretinal Detachment: Two Different Morphologic and Functional Entities

To assess specific morphologic and functional characteristics in eyes with diabetic macular edema (DME) with subfoveal neuroretinal detachment (SND+) vs DME without SND (SND-)

Aqueous Humor Biomarkers of M\ufcller Cell Activation in Diabetic Eyes.

PURPOSE: To identify early biomarkers of retinal M\ufcller cell activation in diabetic eyes with or without clinically detectable signs of diabetic retinopathy (DR). METHODS: This study was a cross-sectional comparative case series. The aqueous humor (AH) of 34 eyes was collected in 12 healthy controls, 11 diabetic patients without DR, and 11 diabetic patients with nonproliferative DR. Full ophthalmic examination and spectral-domain optical coherence tomography were performed in all eyes. Glial fibrillary acidic protein (GFAP), aquaporin 1 (AQP1), and aquaporin 4 (AQP4) were quantified in AH samples as biomarkers of M\ufcller cell activity by ELISA. Statistical analysis was performed with ANOVA followed by Tukey-Kramer post hoc test. RESULTS: There was no significant difference in the age among the three groups. Mean concentration of GFAP, AQP1, and AQP4 significantly increased in diabetic eyes versus controls (P < 0.05, for each comparison). Glial fibrillary acidic protein and AQP1 showed an approximate 2-fold increase, whereas AQP4 showed an approximate 25-fold increase in diabetics with DR versus controls. In diabetics without DR, AQP4 showed an approximate 6-fold increase versus controls. CONCLUSIONS: Glial fibrillary acidic protein, AQP1, and AQP4-biomarkers of M\ufcller cell activity-are significantly increased in human eyes with diabetes, confirming that M\ufcller cells are precociously affected by diabetes mellitus

Aflibercept and Ranibizumab modulate retinal pigment epithelial cells function by acting on their cross talk with vascular endothelial cells

Background/Aims: We performed co-culture experiments between human RPE cells (ARPE-19) and human umbilical vascular endothelial cells (HUVEC) in order to evaluate how anti-VEGF drugs could affect NO release, mitochondrial function, the oxidative status, proliferation and migration of RPE cells through modulation of their cross talk with vascular endothelial cells. Materials: The co-culture HUVEC/RPE, was exposed to Ranibizumab/Aflibercept in the absence/presence of the NO synthase (NOS) inhibitor, the phosphatidylinositol 3\u2032-kinase (PI3K), the extracellular-signal-regulated kinases 1/2 (ERK1/2) and the p38 mitogen-activated protein kinase (p38 MAPK) blockers. Specific kits were used for cell viability, mitochondrial membrane potential, NO, ROS and GSH production. Western blot was performed for apoptosis markers, NOS isoforms, and others kinases detection. Cell migration was analyzed by scratch assay, whereas cell proliferation and cell cycle through xCELLigence and flow cytometry. Results: In RPE cells co-cultured with HUVEC in physiological conditions, Aflibercept/Ranibizumab increased NO release in a dose and time-dependent way. Opposite results were obtained in peroxidative conditions. Both anti-VEGF agents were able to prevent the fall of cell viability and mitochondrial membrane potential, an effect which was reduced by various inhibitors, and increased cell migration. Aflibercept/Ranibizumab counteracted the changes of apoptosis markers, NOS expression/activation, PI3K and ERK1/2 activation caused by peroxidation. These results were confirmed by cell cycle analysis. Conclusion: This study has shown new mechanisms at the basis of protective effects elicited by Aflibercept/Ranibizumab in RPE cells. HUVEC stimulated with Aflibercept/Ranibizumab, could release some paracrine factors that can modulate the RPE cells response in both physiologic and peroxidative conditions

Anti-Vascular Endothelial Growth Factors Protect Retinal Pigment Epithelium Cells Against Oxidation by Modulating Nitric Oxide Release and Autophagy

Background/Aims: the anti-vascular endothelial growth factors (VEGF), Aflibercept and Ranibizumab, are used for the treatment of macular degeneration. Here we examined the involvement of nitric oxide (NO), mitochondria function and of apoptosis/autophagy in their antioxidant effects in human retinal pigment epithelium cells (RPE). Methods: RPE were exposed to Ranibizumab/Aflibercept in the absence or presence of NO synthase (NOS) inhibitor and of autophagy activator/blocker, rapamicyn/3-methyladenine. Specific kits were used for cell viability, NO and reactive oxygen species detection and mitochondrial membrane potential measurement, whereas Western Blot was performed for apoptosis/ autophagy markers and other kinases detection. Results: In RPE cultured in physiological conditions, Aflibercept/Ranibizumab increased NO release in a dose and time-dependent way. Opposite results were obtained in RPE pretreated with hydrogen peroxide. Moreover, both the anti-VEGF agents were able to prevent the fall of cell viability and of mitochondrial membrane potential. Those effects were reduced by the NOS inhibitor and 3-methyladenine and were potentiated by rapamycin. Finally, Aflibercept and Ranibizumab counteracted the changes of apoptosis/autophagy markers, NOS, Phosphatidylinositol-3-Kinase/Protein Kinase B and Extracellular signal–regulated kinases 1/2 caused by peroxidation. Conclusion: Aflibercept and Ranibizumab protect RPE against peroxidation through the modulation of NO release, apoptosis and autophagy

Prevalence of Age-Related Macular Degeneration in Europe: The Past and the Future

Purpose Age-related macular degeneration (AMD) is a frequent, complex disorder in elderly of European ancestry. Risk profiles and treatment options have changed considerably over the years, which may have affected disease prevalence and outcome. We determined the prevalence of early and late AMD in Europe from 1990 to 2013 using the European Eye Epidemiology (E3) consortium, and made projections for the future. Design Meta-analysis of prevalence data. Participants A total of 42 080 individuals 40 years of age and older participating in 14 population-based cohorts from 10 countries in Europe. Methods AMD was diagnosed based on fundus photographs using the Rotterdam Classification. Prevalence of early and late AMD was calculated using random-effects meta-analysis stratified for age, birth cohort, gender, geographic region, and time period of the study. Best-corrected visual acuity (BCVA) was compared between late AMD subtypes; geographic atrophy (GA) and choroidal neovascularization (CNV). Main Outcome Measures Prevalence of early and late AMD, BCVA, and number of AMD cases. Results Prevalence of early AMD increased from 3.5% (95% confidence interval [CI] 2.1%–5.0%) in those aged 55–59 years to 17.6% (95%

Neuroretinal activation in diabetes mellitus

Background: Diabetic retinopathy (DR), one of the leading causes of blindness in developed countries, is the major microvascular complication of diabetes mellitus. Recent studies have demonstrated that the alteration of glial cells and the consequent loss of retinal neuronal cells occur before the vascular lesions are clinically detectable. Purpose: To find early biomarkers of glial activation in the aqueous humor (AH) of diabetic patients both in presence and in absence of clinically detectable signs ofDR. Materials and methods:During cataract surgery, 34 patients’ AH samples were collected as follows: 12 healthy subjects, 11 diabetic patients without DR and 11 diabetic patients with nonproliferative diabetic retinopathy (5 without macular edema-ME and 6 with ME). Before surgery, full ophthalmic examination and Spectral-Domain Optical Coherence Tomography (SD-OCT) (Spectralis HRA+OCT, Heildeberg Engineering) were performed in all eyes. The samples were analyzed for the quantification of total proteins by Bradford method, of GFAP, AQP1 and AQP4 by ELISA and of 40 inflammatory cytokines by protein array. Segmentation of retinal layers was also performed. Results:Mean concentration of GFAP, AQP1 e AQP4 was significantly increased in diabetics versus controls (324.44±262.54pg/µg vs 182.34±114.44pg/µg for GFAP; 105.72±15.69pg/µg vs 50.92±20.36pg/µg for AQP1; and 852.03+103.24pg/µg vs 33.58±21.20pg/µg for AQP4, p<0.05). GFAP showed an approximate 0.8 fold increase, AQP1 1.1 fold increase, whereas AQP4 about 24 folds increase in diabetic patients versus controls. When we separately evaluated DR-no ME eyes vs DR-ME eyes, there was a significant decrease in GFAP, AQP1 e AQPR in DR-ME eyes versus DR-no ME eyes, (Tukey Kramer post hoc p<0.05). GFAP and AQP1 showed even a slight fold decrease versus controls. AQP4/AQP1 concentration showed weak and non significant correlation (Tau=0.21, p=0.3) between these biomarkers, despite the trend in increase.Following cytokines were increased in diabetic patients (with or without DR) compared to healthy subjects: GFAP, AQP1, AQP4, IFNy, IL-1a, IL-1b, IL-3, IL-4, IL-10, IL-11, IL-17, TNF-α, TNF-ß, MCP1, MCP2, Eotaxin,Eotaxin 2, RANTES, sTNFRII, GM-CSF, IP-10, MIP1a, MIP1b. RNFL mean thickness was significantly higher in diabetic patients with DR and ME compared to diabetics without ME (both with and without DR) and healthy subjects (respectively 37.3 μm vs 24.3μm vs 26μm 5μm vs 26.8μm), and the same significance was observed in the inner (33.4μm vs 22.0μm vs 25.0μm 24.3μm) and the external (54.7μm vs 36.7μm 34.6μm 38.1μm) rings and in the superior (40.3μm vs. 26.1μm vs 29.2μm vs 29.5μm), inferior (44.3μm vs 27.2μm vs 29.1μm vs 30.1μm) and temporal (26.3μm vs 16.8μm vs 18.9μm vs 18.0μm) sectors.RNFL mean thickness was significantly reduced in diabetics with DR and without ME compared to healthy subjects. Conclusions: 23 different biomarkers of glial activation have been recognized in the AH of diabetic patients even with subclinical DR. These proteins could be used in the future as risk markers of occurrence of DR and could provide useful therapeutic targets for its prevention and therapy.Presupposti dello studio: La retinopatia diabetica (RD), una delle principali cause di cecità nei paesi sviluppati, costituisce la più comune complicanza microvascolare del diabete mellito. Recenti studi hanno dimostrato che l’alterazione delle cellule gliali e la conseguente perdita di quelle neuronali si verificano prima che le lesioni vascolari siano clinicamente rilevabili. Scopo dello studio: Lo scopo dello studio è quello di ricercare biomarkers precoci di attivazione gliale nell’umore acqueo di soggetti diabetici non solo in presenza di segni clinicamente rilevabili di RD, ma anche in loro assenza. Materiali e metodi: In corso di intervento di cataratta, sono stati raccolti i campioni di umore acqueo di 34 pazienti così suddivisi: 12 soggetti sani, 11 pazienti diabetici senza retinopatia diabetica e 11 con retinopatia diabetica non proliferante (di cui 5 senza edema maculare e 6 con edema maculare-ME). Prima dell’intervento, tutti i pazienti sono stati sottoposti a visita oftalmologica completa e tomografia a coerenza ottica di tipo spectral domain (SD-OCT) (Spectralis HRA+OCT, Heildeberg Engineering). Nei 34 campioni è stata effettuata la quantificazione delle proteine totali con metodo Bradford, di GFAP, AQP1 ed AQP4 con test ELISA e di 40 citochine infiammatorie con protein array. E’ stata, inoltre, effettuata la segmentazione degli strati retinici sulle scansioni SD-OCT. Risultati: I valori medi delle concentrazioni di GFAP, AQP1 e AQP4nell’umore acqueo sono risultati significativamente più elevati nei soggetti diabetici rispetto ai controlli sani (p<0.05). L’incremento di GFAP e’ stato di circa 0.8 volte, di AQP1 di 1.1 volte e di AQP4 di circa 24 volte nei soggetti diabetici rispetto ai controlli. Le concentrazioni di GFAP, AQP1 e AQP4 sono risultate significativamente ridotte nei soggetti diabetici con ME rispetto ai diabetici senza ME, (Tukey Kramer post hoc, p<0.05). La concentrazione nell’umore acqueo, è risultata significativamente maggiore nei pazienti diabetici (con e senza RD) rispetto ai soggetti sani per le seguenti citochine: GFAP, AQP1, AQP4, IFNy, IL-1a, IL-1b, IL-3, IL-4, IL-10, IL-11, IL-17, TNF- α, TNF-ß, MCP1, MCP2, Eotaxin, Eotaxin 2, RANTES, sTNFRII, GM-CSF, IP-10, MIP1a, MIP1b.Lo spessore maculare medio di RNFL è risultato significativamente maggiore nei pazienti diabetici con RD e ME rispetto ai diabetici senza ME (con e senza RD) ed ai soggetti sani; lo stesso rapporto è stato osservato negli anelli interno ed esterno e nei settori superiore, inferiore e temporale. Lo spessore maculare medio di RNFL è risultato significativamente ridotto nei diabetici con RD e senza ME rispetto ai soggetti sani. Conclusioni: Sono stati riconosciuti nell’umore acqueo di soggetti diabetici 23 diversi biomarkers proteici di attivazione gliale presenti sin dallo stadio subclinico della RD. Questi potranno essere utilizzati in futuro come marcatori di rischio per l’insorgenza di tale complicanza microvascolare e costituire degli utili bersagli terapeutici per la sua prevenzione e cura

Quantitative Color Fundus Autofluorescence in Patients with Diabetes Mellitus

A new short wavelength confocal blue-light 450 nm-fundus autofluorescence (color-FAF) allows for visualization of minor fluorophores (e.g., advanced glycation end products, AGEs), besides lipofuscin. The aim of the present pilot study was to quantitatively evaluate color-FAF in patients with diabetes mellitus (DM) and to correlate these data with different stages of retinal disease severity. Optical coherence tomography and color-FAF images of 193 patients/eyes and 18 controls were analyzed using a custom software for quantification of the long (red) and short (green) wavelength components of the emission spectrum (REFC/GEFC). Measurements were performed in nine quadrants of the 6-mm ETDRS macular grid. Foveal GEFC and REFC intensities were higher in patients with DM compared to controls (p = 0.015 and p = 0.006 respectively) and in eyes with center involving diabetic macular edema (DME) compared to eyes without DME (p &lt; 0.001). A positive correlation was found between GEFC and REFC intensities and central retinal thickness, r = 0.37 (p &lt; 0.001) and r = 0.42 (p &lt; 0.001), respectively. No differences were found in color-FAF among different DR severity groups. Quantitative color-FAF could become helpful for the metabolic evaluation of retina in patients with DM and in DME; however, further histologic and immunohistochemical studies on distribution of different retinal fluorophores in DM are needed to better understand its role