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-s, 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.