| Background1.Blood-retinal barrier(BRB)breakdown and vascular leakage is the leading cause of blindness of diabetic retinopathy(DR).Hyperglycemia-induced oxidative stress and in ammation are primary pathogenic factors of this severe DR complication.An effective interventional modality against the pathogenic factors during early DR is needed to curb BRB breakdown and vascular leakage.2.Retinal neovascularization(RNV)occurs in multiple eye diseases,and can lead to bleeding and even retinal detachment.Therefore,inhibition of pathological RNV is becoming crucial to the treatment of ocular diseases.Research has shown that ?-melanocyte-stimulating hormone(?-MSH)inhibits retinal angiogenesis during physiological development,however,the effects of ?-MSH on pathological RNV remain unknown.Purpose1.This study sought to examine the protective effects of ?-Melanocyte-stimulating hormone(?-MSH)on early diabetic retina against vascular hyperpermeability,electrophysiological dysfunction,and morphological deterioration in a rat model of diabetes and probe the mechanisms underlying the ?-MSH's anti-hyperpermeability in both rodent retinas and simian retinal vascular endothelial cells(RF6A).2.This study sought to examine the protective effects of ?-Melanocyte-stimulating hormone(?-MSH)on early diabetic retina,probe the protective mechanisms underlying the ?-MSH's anti-hyperpermeability in both rodent retinas and simian retinal vascular endothelial cells(RF6A).3.To study the effects of intravitreal injection of ?-MSH at different concentrations on pathological RNV in a mouse model of oxygen-induced retinopathy(OIR).Methods1 Sprague Dawley rats were injected through tail vein with streptozotocin to induce diabetes.The rats were intravitreally injected with ?-MSH or saline at Week 1 and 3 after hyperglycemia.In another 2 weeks,Evans blue assay,transmission electron microscopy,electroretinogram(ERG),and hematoxylin and eosin(H&E)staining were performed to examine the protective effects of ?-MSH in diabetic retinas.2.The expression of pro-inflammatory factors and tight junction at m RNA and protein levels in retinas was analyzed.Finally,the ?-MSH's anti-hyperpermeability was confirmed in a high glucose(HG)-treated RF6 A cell monolayer transwell culture by transendothelial electrical resistance(TEER)measurement and auorescein isothiocyanate-Dextran assay.Universal or specific melanocortin receptor(MCR)blockers were also employed to elucidate the MCR subtype mediating ?-MSH's protection.3.Healthy C57BL/6J pups of hygiene grade were randomly divided into OIR+?-MSH-1,OIR+?-MSH-5,OIR+?-MSH-10,OIR,and normal control groups at postnatal day 7(P7),with 8 pups in each group.The ?-MSH intervention groups and OIR group were exposed to high oxygen(75±2 %)for 5 d,then maintained under normal air condition for another 5 d;whereas the normal control group was raised under normoxia for 10 d.Retro-orbital injection of high molecular weight(MW)fluorescein isothiocyanate-dextran(FITC-dextran,average MW 2,000 k D)was performed on P17 mice.The retina whole mounts were prepared to reveal retinal vasculature and quantify relative area of vessel obliteration.The mouse eyeballs were subjected to paraffin sections and hematoxylin-eosin staining,and the averaged number of pre-retinal nuclei per section was quantified.Results1.Evans blue assay showed that BRB breakdown and vascular leakage was detected,and rescued by ?-MSH both qualitatively and quantitatively in early diabetic retinas;electron microscopy revealed substantially improved retinal and choroidal vessel ultrastructures in ?-MSH-treated diabetic retinas;scotopic ERG suggested partial rescue of functional defects by ?-MSH in diabetic retinas;and H&E staining revealed signi cantly increased thickness of all layers in ?-MSH-treated diabetic retinas.2.Mechanistically,?-MSH corrected aberrant transcript and protein expression of pro-in ammatory factor and tight junction genes in the diseased retinas;moreover,it prevented abnormal changes in TEER and permeability in HG-stimulated RF6 A cells,and this anti-hyperpermeability was abolished by a universal MCR blocker or an antagonist speci c to MC4 R.3.FITC-dextran-labeled retinal whole mounts showed that the relative vessel obliteration area in normal control,OIR,OIR+?-MSH-1,OIR+?-MSH-5,and OIR+?-MSH-10 groups were 0.00%±0.00%,23.01±3.39%,18.14%±7.20%,15.64%±7.07%,and 7.62%±6.52%,respectively.There was statistical significance in the relative avascular area among the groups(F=19.635,P<0.05).The relative avascular area in the OIR group was significantly greater than that in the normal control and OIR+?-MSH-10 groups(t=22.946,P<0.001;t=4.293,P<0.01),the differences between the OIR group and those two groups were of statistical significance.The results of histopathological examinations showed that the averaged number of pre-retinal nuclei per section in normal control,OIR,OIR+?-MSH-1,OIR+?-MSH-5,and OIR+?-MSH-10 groups were 0.00±0.00 ? 11.45±4.26 ?6.35±2.34?4.96±1.79,and 1.03±1.25,respectively.There was statistical significance in the averaged number of pre-retinal nuclei per section among the groups(F=147.87,P<0.05).The number of averaged pre-retinal nuclei per section in the OIR group was significantly higher than the normal control,OIR+?-MSH-1,OIR+?-MSH-5,and OIR+?-MSH-10 groups,the differences between the OIR and these groups had statistical significance(all P<0.001).Conclusions1.This study showed previously undescribed protective effects of ?-MSH on inhibiting BRB breakdown and vascular leakage,improving electrophysiological functions and morphology in early diabetic retinas,which may be due to its down-regulating pro-inflammatory factors and augmenting tight junctions.?-MSH acts predominantly on MC4 R to antagonize hyperpermeability in retinal microvessel endothelial cells.2.This study showed previously undescribed protective effects of ?-MSH on inhibiting BRB breakdown and vascular leakage,improving electrophysiological functions and morphology in early diabetic retinas,which may be due to its down-regulating pro-inflammatory factors and augmenting tight junctions.?-MSH acts predominantly on MC4 R to antagonize hyperpermeability in retinal microvessel endothelial cells.3.?-MSH reduces the relative area of vessel obliteration and the averaged number of pre-retinal nuclei in the retinas of OIR mouse model,and the inhibitory effects of this peptide on the pathological RNV are dose-dependent. |
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