RESUMO
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.
Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Retinopatia Diabética/tratamento farmacológico , Inflamação/tratamento farmacológico , Proteína 1 Supressora da Sinalização de Citocina/farmacologia , Animais , Retinopatia Diabética/genética , Retinopatia Diabética/patologia , Modelos Animais de Doenças , Eletrorretinografia , Humanos , Inflamação/genética , Inflamação/patologia , Camundongos , Soluções Oftálmicas/farmacologia , Peptídeos/química , Peptídeos/farmacologia , Retina/efeitos dos fármacos , Retina/patologia , Proteína 1 Supressora da Sinalização de Citocina/químicaRESUMO
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.
Assuntos
Citidina Difosfato Colina/uso terapêutico , Diabetes Mellitus Experimental/complicações , Retinopatia Diabética/tratamento farmacológico , Retinopatia Diabética/etiologia , Fármacos Neuroprotetores/uso terapêutico , Retina/efeitos dos fármacos , Administração Tópica , Animais , Citidina Difosfato Colina/administração & dosagem , Retinopatia Diabética/patologia , Lipossomos , Masculino , Camundongos , Fármacos Neuroprotetores/administração & dosagem , Nootrópicos/administração & dosagem , Nootrópicos/uso terapêutico , Retina/patologiaRESUMO
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.
Assuntos
Bosentana/uso terapêutico , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/tratamento farmacológico , Retinopatia Diabética/prevenção & controle , Administração Tópica , Animais , Apoptose/efeitos dos fármacos , Retinopatia Diabética/metabolismo , Endotelina-1/metabolismo , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Técnicas In Vitro , Masculino , CoelhosRESUMO
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.
Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Inibidores da Dipeptidil Peptidase IV/uso terapêutico , Retina/efeitos dos fármacos , Retina/patologia , Adamantano/análogos & derivados , Adamantano/uso terapêutico , Animais , Diabetes Mellitus Experimental/fisiopatologia , Dipeptídeos/uso terapêutico , Eletrorretinografia , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Fosfato de Sitagliptina/uso terapêuticoRESUMO
(1) Background: Previous studies have identified disparities in stroke care and outcomes by sex. Therefore, the main objective of this study was to evaluate the average cost of stroke care and the existence of differences in care provision by biological sex. (2) Methods: This observational study adhered to the recommendations of the STROBE statement. The calculation of costs was performed based on the production cost of the service or the rate paid for a set of services, depending on the availability of the corresponding information. (3) Results: A total of 336 patients were included, of which 47.9% were women, with a mean age of 73.3 ± 11.6 years. Women were typically older, had a higher prevalence of hypertension (p = 0.005), lower pre-stroke proportion of mRS 0-2 (p = 0.014), greater stroke severity (p < 0.001), and longer hospital stays (p = 0.017), and more were referred to residential services (p = 0.001) at 90 days. Women also required higher healthcare costs related to cardiovascular risk factors, transient ischemic strokes, institutionalization, and support needs; in contrast, they necessitated lower healthcare costs when undergoing endovascular therapy and receiving rehabilitation services. The unadjusted averaged cost of stroke care was EUR 22,605.66 (CI95% 20,442.8-24,768.4), being higher in women [p = 0.027]. The primary cost concept was hospital treatment (38.8%), followed by the costs associated with dependence and support needs (36.3%). At one year post-stroke, the percentage of women not evaluated for a degree of dependency was lower (p = 0.008). (4) Conclusions: The total unadjusted costs averaged EUR 22,605.66 (CI95% EUR 20,442.8-24,768.4), being higher in women compared to men. The primary cost concept was hospital treatment (38.8%), followed by the costs associated with dependence and support needs (36.3%).
RESUMO
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.
RESUMO
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.
Assuntos
Dobesilato de Cálcio/farmacologia , Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2/complicações , Retinopatia Diabética/prevenção & controle , Estresse Oxidativo/genética , Retina/metabolismo , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Sobrevivência Celular , Células Cultivadas , Diabetes Mellitus Tipo 2/metabolismo , Retinopatia Diabética/diagnóstico , Retinopatia Diabética/etiologia , Eletrorretinografia , Ensaio de Imunoadsorção Enzimática , Regulação da Expressão Gênica , Hemostáticos/farmacologia , Humanos , Marcação In Situ das Extremidades Cortadas , Masculino , Camundongos , Microscopia de Fluorescência , Estresse Oxidativo/efeitos dos fármacos , RNA/genética , Reação em Cadeia da Polimerase em Tempo Real , Retina/efeitos dos fármacos , Retina/patologia , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia , Fator A de Crescimento do Endotélio Vascular/biossíntese , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
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.
Assuntos
Anti-Inflamatórios não Esteroides/uso terapêutico , Dobesilato de Cálcio/uso terapêutico , Retinopatia Diabética/prevenção & controle , Fármacos Neuroprotetores/uso terapêutico , Estresse Oxidativo/efeitos dos fármacos , Retina/efeitos dos fármacos , Retinite/prevenção & controle , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Antioxidantes/farmacologia , Antioxidantes/uso terapêutico , Biomarcadores/metabolismo , Dobesilato de Cálcio/farmacologia , Células Cultivadas , Cruzamentos Genéticos , Citocinas/antagonistas & inibidores , Citocinas/metabolismo , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/tratamento farmacológico , Retinopatia Diabética/imunologia , Retinopatia Diabética/metabolismo , Retinopatia Diabética/patologia , Humanos , Quinase I-kappa B/metabolismo , Masculino , Camundongos Mutantes , Camundongos Transgênicos , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Distribuição Aleatória , Retina/imunologia , Retina/metabolismo , Retina/patologia , Retinite/complicações , Retinite/imunologia , Retinite/metabolismoRESUMO
Retinal neurodegeneration is an early event in the pathogenesis of diabetic retinopathy (DR). Since glucagon-like peptide 1 (GLP-1) exerts neuroprotective effects in the central nervous system and the retina is ontogenically a brain-derived tissue, the aims of the current study were as follows: 1) to examine the expression and content of GLP-1 receptor (GLP-1R) in human and db/db mice retinas; 2) to determine the retinal neuroprotective effects of systemic and topical administration (eye drops) of GLP-1R agonists in db/db mice; and 3) to examine the underlying neuroprotective mechanisms. We have found abundant expression of GLP-1R in the human retina and retinas from db/db mice. Moreover, we have demonstrated that systemic administration of a GLP-1R agonist (liraglutide) prevents retinal neurodegeneration (glial activation, neural apoptosis, and electroretinographical abnormalities). This effect can be attributed to a significant reduction of extracellular glutamate and an increase of prosurvival signaling pathways. We have found a similar neuroprotective effect using topical administration of native GLP-1 and several GLP-1R agonists (liraglutide, lixisenatide, and exenatide). Notably, this neuroprotective action was observed without any reduction in blood glucose levels. These results suggest that GLP-1R activation itself prevents retinal neurodegeneration. Our results should open up a new approach in the treatment of the early stages of DR.