Faim knockout leads to gliosis and late-onset neurodegeneration of photoreceptors in the mouse retina.

Fas Apoptotic Inhibitory Molecule protein (FAIM) is a death receptor antagonist and an apoptosis regulator. It encodes two isoforms, namely FAIM-S (short) and FAIM-L (long), both with significant neuronal functions. FAIM-S, which is ubiquitously expressed, is involved in neurite outgrowth. In contrast, FAIM-L is expressed only in neurons and it protects them from cell death. Interestingly, FAIM-L is downregulated in patients and mouse models of Alzheimer's disease before the onset of neurodegeneration, and Faim transcript levels are decreased in mouse models of retinal degeneration. However, few studies have addressed the role of FAIM in the central nervous system, yet alone the retina. The retina is a highly specialized tissue, and its degeneration has proved to precede pathological mechanisms of neurodegenerative diseases. Here we describe that Faim depletion in mice damages the retina persistently and leads to late-onset photoreceptor death in older mice. Immunohistochemical analyses showed that Faim knockout (Faim-/- ) mice present ubiquitinated aggregates throughout the retina from early ages. Moreover, retinal cells released stress signals that can signal to Müller cells, as shown by immunofluorescence and qRT-PCR. Müller cells monitor retinal homeostasis and trigger a gliotic response in Faim-/- mice that becomes pathogenic when sustained. In this regard, we observed pronounced vascular leakage at later ages, which may be caused by persistent inflammation. These results suggest that FAIM is an important player in the maintenance of retinal homeostasis, and they support the premise that FAIM is a plausible early marker for late photoreceptor and neuronal degeneration.

Beneficial Effects of Glucagon-Like Peptide-1 (GLP-1) in Diabetes-Induced Retinal Abnormalities: Involvement of Oxidative Stress.

BACKGROUND: Hyperglycemia-induced oxidative stress plays a key role in diabetic complications, including diabetic retinopathy. The main goal of this study was to assess whether the topical administration (eye drops) of glucagon-like peptide-1 (GLP-1) has any effect on oxidative stress in the retina. METHODS: db/db mice were treated with eye drops of GLP-1 or vehicle for three weeks, with db/+ mice being used as control. Studies included the assessment by western blot of the antioxidant defense markers CuZnSOD, MnSOD, glutathione peroxidase and reductase; immunofluorescence measurements of DNA/RNA damage, nitro tyrosine and Ki67 and Babam2 proteins. RESULTS: GLP-1 eye drops protected from oxidative stress by increasing the protein levels of glutathione reductase, glutathione peroxidase and CuZnSOD and MnSOD in diabetic retinas. This was associated with a significant reduction of DNA/RNA damage and the activation of proteins involved in DNA repair in the retina (Babam2) and Ki67 (a biomarker of cell proliferation). CONCLUSIONS: GLP-1 modulates the antioxidant defense system in the diabetic retina and has a neuroprotective action favoring DNA repair and neuron cells proliferation.

SOCS1-Derived Peptide Administered by Eye Drops Prevents Retinal Neuroinflammation and Vascular Leakage in Experimental Diabetes.

Current treatments for diabetic retinopathy (DR) target late stages when vision has already been significantly affected. Accumulating evidence suggests that neuroinflammation plays a major role in the pathogenesis of DR, resulting in the disruption of the blood-retinal barrier. Suppressors of cytokine signaling (SOCS) are cytokine-inducible proteins that function as a negative feedback loop regulating cytokine responses. On this basis, the aim of the present study was to evaluate the effect of a SOCS1-derived peptide administered by eye drops (2 weeks) on retinal neuroinflammation and early microvascular abnormalities in a db/db mouse model. In brief, we found that SOCS1-derived peptide significantly reduced glial activation and neural apoptosis induced by diabetes, as well as retinal levels of proinflammatory cytokines. Moreover, a significant improvement of electroretinogram parameters was observed, thus revealing a clear impact of the histological findings on global retinal function. Finally, SOCS1-derived peptide prevented the disruption of the blood-retinal barrier. Overall, our results suggest that topical administration of SOCS1-derived peptide is effective in preventing retinal neuroinflammation and early microvascular impairment. These findings could open up a new strategy for the treatment of early stages of DR.

New Insights into the Mechanisms of Action of Topical Administration of GLP-1 in an Experimental Model of Diabetic Retinopathy.

The main goals of this work were to assess whether the topical administration of glucagon-like peptide-1 (GLP-1) could revert the impairment of the neurovascular unit induced by long-term diabetes (24 weeks) in diabetic mice and to look into the underlying mechanisms. For that reason, db/db mice were treated with eye drops of GLP-1 or vehicle for 3 weeks. Moreover, db/+ mice were used as control. Studies performed in vivo included electroretinogramand the assessment of vascular leakage by using Evans Blue. NF-κB, GFAP and Ki67 proteins were analyzed by immunofluorescence (IF). Additionally, caspase 9, AMPK, IKBα, NF-κB, AKT, GSK3, ß-catenin, Bcl-xl, and VEGF were analyzed by WB. Finally, VEGF, IL-1ß, IL-6, TNF-α, IL-18, and NLRP3 were studied by reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence. We found that topical administration of GLP-1 reverted reactive gliosis and albumin extravasation, and protected against apoptosis and retinal dysfunction. Regarding the involved mechanisms, GLP-1 exerted an anti-inflammatory action by decreasing NF-κB, inflammosome, and pro-inflammatory factors. In addition, it also decreased VEGF expression. Furthermore, GLP-1 promoted cell survival by increasing the anti-apoptotic protein Bcl-xl and the signaling pathway Akt/GSK3b/ß-catenin. Finally, Ki67 results revealed that GLP-1 treatment could induce neurogenesis. In conclusion, the topical administration of GLP-1 reverts the impairment of the neurovascular unit by modulating essential pathways involved in the development of diabetic retinopathy (DR). These beneficial effects on the neurovascular unit could pave the way for clinical trials addressed to confirm the effectiveness of GLP-1 in early stages of DR.

Topical Administration of Bosentan Prevents Retinal Neurodegeneration in Experimental Diabetes.

Experimental evidence suggests that endothelin 1 (ET-1) is involved in the development of retinal microvascular abnormalities induced by diabetes. The effects of ET-1 are mediated by endothelin A- and B-receptors (ETA and ETB). Endothelin B-receptors activation mediates retinal neurodegeneration but there are no data regarding the effectiveness of ETB receptor blockage in arresting retinal neurodegeneration induced by diabetes. The main aim of the present study was to assess the usefulness of topical administration of bosentan (a dual endothelin receptor antagonist) in preventing retinal neurodegeneration in diabetic (db/db) mice. For this purpose, db/db mice aged 10 weeks were treated with one drop of bosentan (5 mg/mL, n = 6) or vehicle (n = 6) administered twice daily for 14 days. Six non-diabetic (db/+) mice matched by age were included as the control group. Glial activation was evaluated by immunofluorescence using specific antibodies against glial fibrillary acidic protein (GFAP). Apoptosis was assessed by TUNEL method. A pharmacokinetic study was performed in rabbits. We found that topical administration of bosentan resulted in a significant decrease of reactive gliosis and apoptosis. The results of the pharmacokinetic study suggested that bosentan reached the retina through the trans-scleral route. We conclude that topical administration of bosentan was effective in preventing neurodegeneration in the diabetic retina and, therefore, could be a good candidate to be tested in clinical trials.

Effects of Liposomal Formulation of Citicoline in Experimental Diabetes-Induced Retinal Neurodegeneration.

Diabetic retinopathy (DR) has been classically considered a microcirculatory disease of the retina. However, there is growing evidence to suggest that retinal neurodegeneration is also an early event in the pathogenesis of DR. Citicoline has been successfully used as a neuroprotective agent in the treatment of glaucoma but their effects on DR remain to be elucidated. On this basis, the main aim of the present study was to evaluate the effect of topical administration of citicoline in liposomal formulation on retinal neurodegeneration in db/db mouse and to investigate the underlying mechanisms of action. The treatment (citicoline or vehicle) was topically administered twice daily for 15 days. Retinal analyses were performed in vivo by electroretinography and ex vivo by using Western blot and immunofluorescence measurements. We found that the liposomal formulation of citicoline prevented glial activation and neural apoptosis in the diabetic retina. The main mechanism implicated in these beneficial effects were the inhibition of the downregulation of synaptophysin and its anti-inflammatory properties by means of preventing the upregulation of NF-κB and TNF-α (Tumor Necrosis Factor α) induced by diabetes. Overall, these results suggest that topical administration of citicoline in liposomal formulation could be considered as a new strategy for treating the early stages of DR.

Calcium dobesilate prevents the oxidative stress and inflammation induced by diabetes in the retina of db/db mice.

AIM: Calcium dobesilate (CaD) is beneficial in early stages of diabetic retinopathy (DR), but its mechanisms of action remains to be elucidated. The aim was to investigate the effect of CaD on proinflammatory cytokines and oxidative stress. METHODS: db/db mice were randomly assigned to daily oral treatment with CaD (200mg/kg/day) or vehicle for 15days. Biomarkers of oxidative stress (dihydroethidium, malondialdehyde), NF-κB, and proinflammatory cytokines (IL-1ß, IL-6, IL-8, TNF-α, MCP-1) were examined in the retina by immunohistochemical analysis. Cultures of human retinal endothelial cells (HRECs) were used for complementary experiments. RESULTS: CaD significantly reduced the biomarkers of oxidative stress in the retina of db/db mice. In addition, CaD prevented the increase of NF-κB, IL-6, IL-8, TNF-α and MCP-1 induced by diabetes. CaD inhibited the activation of NF-kß induced by IL-1ß by preventing IKKB-α phosphorylation in HRECs and reduced the upregulation of IL-6 and IL-18 induced by TNF-α in a dose-dependent manner. CONCLUSION: Our results suggest that antioxidant and antiinflammatory effects are crucial in accounting for the effectiveness of CaD for treating DR.

Topical administration of DPP-IV inhibitors prevents retinal neurodegeneration in experimental diabetes.

AIMS/HYPOTHESIS: The main aims of the present study were: (1) to assess the expression and content of dipeptidyl peptidase IV (DPP-IV) in human and db/db mouse retinas, and in human vitreous fluid; and (2) to determine whether the topical administration of the DPP-IV inhibitors (DPP-IVi) would prevent retinal neurodegeneration and vascular leakage in db/db mice by reducing endogenous glucagon-like peptide 1 (GLP-1) degradation. METHODS: To assess the expression and content of DPP-IV, human samples of vitreous fluid and retinas were obtained from participants with type 2 diabetes (n = 8) and age-matched non-diabetic individuals (n = 8), as well as from db/db (n = 72) and db/+ (n = 28) mice. The interventional study, which included 72 db/db mice, consisted of the topical administration (eye drops) of saxagliptin, sitagliptin or vehicle for 14 days. DPP-IV mRNA levels were assessed by RT-PCR, and protein content was measured by ELISA or western blotting. GLP-1 was assessed by immunofluorescence, and its downstream effector exchange protein activated by cAMP-1 (EPAC-1) was used as a measure of GLP-1 receptor activation. Retinal analyses were performed in vivo by electroretinography and ex vivo by RT-PCR (Epac-1, Iba-1 [also known as Aif1]), western blotting (EPAC-1, glial fibrillar acidic protein [GFAP], glutamate-aspartate transporter [GLAST]) and immunofluorescence measurements (GLP-1, GFAP, ionised calcium binding adaptor molecule 1 [IBA-1], TUNEL, GLAST, albumin and collagen IV). Glutamate was quantified by HPLC. In addition, vascular leakage was examined by the Evans Blue method. RESULTS: DPP-IV was present in human vitreous fluid but in a range 100-fold less than in plasma. Both mRNA levels and protein content were much lower in the retina than in the liver or bowel, but were significantly higher in retinal pigment epithelium (RPE) from diabetic donors in comparison to non-diabetic donors (p < 0.05). Topical treatment with DPP-IVi prevented glial activation, apoptosis and vascular leakage induced by diabetes in db/db mice (p < 0.05). Moreover, it also significantly prevented diabetes-induced functional abnormalities in the electroretinogram. A significant increase of both GLP-1 and EPAC-1 was found after treatment with DPP-IVi (p < 0.05). Furthermore, GLAST downregulation induced by diabetes was prevented, resulting in a significant reduction of extracellular glutamate concentrations. All these effects were observed without any changes in blood glucose levels. CONCLUSIONS/INTERPRETATION: The topical administration of DPP-IVi is effective in preventing neurodegeneration and vascular leakage in the diabetic retina. These effects can be attributed to an enhancement of GLP-1, but other mechanisms unrelated to the prevention of GLP-1 degradation cannot be ruled out.

Calcium Dobesilate Prevents Neurodegeneration and Vascular Leakage in Experimental Diabetes.

PURPOSE: The mechanisms involved in the reported beneficial effects of Calcium dobesilate monohydrate (CaD) for the treatment of diabetic retinopathy (DR) remain to be elucidated. The main aim of the present study is to examine whether CaD prevents early events in the pathogenesis of DR such as neurodegeneration and vascular leakage. In addition, putative mediators of both neurodegeneration (glutamate/GLAST, ET-1/ETB receptor) and early microvascular impairment (ET-1/ETA receptor, oxidative stress, VEGF, and the PKC-delta-p38 MAPK pathway) have been examined. METHODS: Diabetic (db/db) mice were randomly assigned to daily oral treatment with CaD (200 mg/Kg/day) (n = 12) or vehicle (n = 12) for 14 days. In addition, 12 non-diabetic (db/+) mice matched by age were used as the control group. Functional abnormalities were assessed by electroretinography. Neurodegeneration and microvascular abnormalities were evaluated by immunohistochemistry and Western blot. Glutamate was determined by HPLC. RESULTS: CaD significantly decreased glial activation and apoptosis and produced a significant improvement in the electroretinogram parameters. Mechanistically, CaD prevented the diabetes-induced up-regulation of ET-1 and its cognate receptors (ETA-R and ETB-R), which are involved in microvascular impairment and neurodegeneration, respectively. In addition, treatment with CaD downregulated GLAST, the main glutamate transporter, and accordingly prevented the increase in glutamate. Finally, CaD prevented oxidative stress, and the upregulation of VEGF and PKC delta-p38 MAPK pathway induced by diabetes, thus resulting in a significant reduction in vascular leakage. CONCLUSIONS: Our findings demonstrate for the first time that CaD exerts neuroprotection in an experimental model of DR. In addition, we provide first evidence that CaD prevents the overexpression of ET-1 and its receptors in the diabetic retina. These beneficial effects on the neurovascular unit could pave the way for clinical trials addressed to confirm the effectiveness of CaD in very early stages of DR.