Microscopic hyperspectral imaging studies of normal and diabetic retina of rats

A microscopic hyperspectral imager was developed based on the microscopic technology and the spectral imaging technology. Some microscopic hyperspectral images of retina sections of the normal, the diabetic, and the treated rats were collected by the new imager. Single-band images and pseudo-color images of each group were obtained and the typical transmittance spectrums were ex-tracted. The results showed that the transmittance of outer nuclear layer cells of the diabetic group was generally higher than that of the normal. A small absorption peak appeared near the 180th band in the spectrum of the diabetic group and this peak weakened or disappeared in the spectrum of the treated group. Our findings indicate that the microscopic hyperspectral images include wealthy information of retina sections which is helpful for the ophthalmologist to reveal the pathogenesis of diabetic reti-nopathy and explore the therapeutic effect of drugs.     

Vascular changes precede tomographic changes in diabetic eyes without retinopathy and improve artificial intelligence diagnostics

The purpose of this study was to evaluate early vascular and tomographic changes in the retina of diabetic patients using artificial intelligence (AI). The study included 74 age‐matched normal eyes, 171 diabetic eyes without retinopathy (DWR) eyes and 69 mild non‐proliferative diabetic retinopathy (NPDR) eyes. All patients underwent optical coherence tomography angiography (OCTA) imaging. Tomographic features (thickness and volume) were derived from the OCTA B‐scans. These features were used in AI models. Both OCT and OCTA features showed significant differences between the groups (P < .05). However, the OCTA features indicated early retinal changes in DWR eyes better than OCT (P < .05). In the AI model using both OCT and OCTA features simultaneously, the best area under the curve of 0.91 ± 0.02 was obtained (P < .05). Thus, the combined use of AI, OCT and OCTA significantly improved the early diagnosis of diabetic changes in the retina.     

Glucose-mediated de novo lipogenesis in photoreceptors drives early diabetic retinopathy

Diabetic retinopathy (DR) is an increasingly frequent cause of blindness across populations; however, the events that initiate pathophysiology of DR remain elusive. Strong preclinical and clinical evidence suggests that abnormalities in retinal lipid metabolism caused by diabetes may account for the origin of this disease. A major arm of lipid metabolism, de novo biosynthesis, is driven by elevation in available glucose, a common thread binding all forms of vision loss in diabetes. Therefore, we hypothesized that aberrant retinal lipid biogenesis is an important promoter of early DR. In murine models, we observed elevations of diabetes-associated retinal de novo lipogenesis ∼70% over control levels. This shift was primarily because of activation of fatty acid synthase (FAS), a rate-limiting enzyme in the biogenic pathway. Activation of FAS was driven by canonical glucose-mediated disinhibition of acetyl-CoA carboxylase, a major upstream regulatory enzyme. Mutant mice expressing gain-of-function FAS demonstrated increased vulnerability to DR, whereas those with FAS deletion in rod photoreceptors maintained preserved visual responses upon induction of diabetes. Excess retinal de novo lipogenesis—either because of diabetes or because of FAS gain of function—was associated with modestly increased levels of palmitate-containing phosphatidylcholine species in synaptic membranes, a finding with as yet uncertain significance. These findings implicate glucose-dependent increases in photoreceptor de novo lipogenesis in the early pathogenesis of DR, although the mechanism of deleterious action of this pathway remains unclear.     

An in vitro cell model to study microglia activation in diabetic retinopathy

Microglial activation has been studied extensively in diabetic retinopathy. We have previously detected activation and migration of microglia in 8-week-old diabetic rat retinas. It is widely acknowledged that microglia-mediated inflammation contributes to the progression of diabetic retinopathy. However, existing cell models do not explore the role of activated microglia in vitro. In this study, microglia were subject to various conditions mimicking diabetic retinopathy, including high glucose, glyoxal, and hypoxia. Under high glucose or glyoxal treatment, microglia demonstrated only partially functional changes, while under hypoxia, microglia became fully activated showing enlarged cell bodies, enhanced migration and phagocytosis as well as increased production of pro-inflammatory factors such as cyclooxygenase-2 (COX-2), interleukin-1β (IL-1β), and inducible nitric oxide synthase (iNOS). The data indicate that hypoxia-treated microglia is an optimal in vitro model for exploration of microglia activation in diabetic retinopathy.     

Mitochondrial dysfunction in diabetes and the regulatory roles of antidiabetic agents on the mitochondrial function

The prevalence of type 2 diabetes mellitus (T2DM) is increasing rapidly with its associated morbidity and mortality. Many pathophysiological pathways such as oxidative stress, inflammatory responses, adipokines, obesity-induced insulin resistance, improper insulin signaling, and beta cell apoptosis are associated with the development of T2DM. There is increasing evidence of the role of mitochondrial dysfunction in the onset of T2DM, particularly in relation to the development of diabetic complications. Here, the role of mitochondrial dysfunction in T2DM is reviewed together with its modulation by antidiabetic therapeutic agents, an effect that may be independent of their hypoglycemic effect.     

CaMKII regulates pericyte loss in the retina of early diabetic mouse

Inducible nitric oxide synthase (iNOS) is an essential mediator in diabetic vascular lesions and known to be regulated by activation of Ca²⁺/calmodulin-dependent protein kinase II (CaMKII). The aim of this study was to investigate whether CaMKII affects iNOS-mediated pericyte death in the retina of diabetic mice with early stage disease. Total- and phospho-CaMKII, iNOS, and active caspase-3 protein levels were assessed by Western blotting, and CaMKII activity was measured by kinase assay. iNOS-related pericyte death was assessed by double immunofluorescent staining for iNOS and α-smooth muscle actin, followed by the TUNEL assay. Autocamtide-2-related inhibitory peptide (AIP), a specific inhibitor of CaMKII, was injected into the right vitreous 2 days before sacrifice of mice, to examine the effect of CaMKII inactivation in diabetic retinas. The levels of total- and phospho-CaMKII, iNOS, and active caspase-3 protein, and CaMKII activity were significantly increased in the diabetic retinas compared with those of control retinas. Furthermore, TUNEL-positive signals colocalized with iNOS-immunoreactive pericytes in the same retinas. However, inactivation of CaMKII by AIP treatment inhibited all these changes, which was accompanied by less pericyte loss. Our results demonstrate that CaMKII contributes to iNOS-related death of pericytes in the diabetic retina and that inactivation of this enzyme may be a potential treatment for retinal vascular lesion.     

Association between miRNAs expression and signaling pathways of oxidative stress in diabetic retinopathy

Diabetic retinopathy (DR) is a major cause of vision reduction in diabetic patients. Hyperglycemia is a known instigator for the development of DR, even though the role of oxidative stress pathways in the pathogenesis of DR is established. The studies indicate that microRNAs (miRNAs) are significant to the etiology of DR; changes in miRNAs expression levels may be associated with onset and progression of DR. In addition, miRNAs have emerged as a useful disease marker due to their availability and stability in detecting the severity of DR. The relationship between miRNAs expression levels and oxidative stress pathways has been investigated in several studies. The aim of this study is the examination of function and expression levels of target miRNAs in oxidative stress pathway and pathogenesis of diabetic retinopathy.     

Plasma angiogenesis and oxidative stress markers in patients with diabetic retinopathy

Abstract

Background: Neovascularization in the retina and hyperglycaemia-induced oxidative stress are implicated in the pathogenesis of diabetic retinopathy (DR). In this study, we hypothesized that the plasma angiogenic and oxidative stress markers associated with these derangements could aid in the screening of diabetic patients who are at an increased risk of developing retinopathy.

Methods: This study included normal (n?=?148), type2 diabetes without retinopathy (DNR; n?=?148), proliferative DR (PDR; n?=?74) and non-PDR (NPDR; n?=?148) subjects. Plasma concentrations of vascular endothelial growth factor-A (VEGF-A), hypoxia-inducible factor-1α (HIF-1α), matrix metalloproteinase-9 (MMP-9), pigment epithelium-derived factor (PEDF), nitric oxide (NO), soluble receptors for advanced glycation end products (sRAGE), malondialdehyde (MDA) and protein thiols were estimated.

Results: A statistically significant increase was observed in the plasma concentrations of pro-angiogenic factors and markers of oxidative stress in both retinopathy groups. By contrast, the concentrations of anti-angiogenic factors and antioxidants were decreased significantly in these groups. Receiver operating characteristic analysis indicated that the plasma thresholds of HIF-1α and PEDF can be suitable markers in case of NPDR. However, in PDR, HIF-1α, NO, MMP-9 and PEDF showed high sensitivity and specificity.

Conclusions: The factors associated with hypoxia, matrix degradation and angiogenic inhibition play a crucial role in predicting DR.     

Role of histone modification and DNA methylation in signaling pathways involved in diabetic retinopathy

Retinopathy, characterized by an alteration of the retinal microvasculature, is a common complication of diabetes mellitus. These changes can cause increased permeability and alter endothelial cell proliferation, edema, and abnormal neovascularization and eventually result in blindness. The pathogenesis of diabetic retinopathy (DR) is complicated, involving many factors/mediators such as genetic susceptibility, microRNAs, and cytokines. One of the factors involved in DR pathogenesis is epigenetic changes that can have a key role in the regulation of gene expression; these include microRNAs, histone modifications, and methylation of DNA. The main epigenetic modifications are DNA methylation and posttranslational modifications of the histones. Generally, the studies on epigenetics can provide new opportunities to investigate the molecular basis of diseases with complicated pathogenesis, including DR, and provide essential insights into the potential design of strategies for its treatment. The aim of this study is an investigation of DR pathogenesis and epigenetic modifications that involve in DR development.     

Epigenetic control of early neurodegenerative events in diabetic retinopathy by the histone deacetylase SIRT6

Diabetic retinopathy (DR ) is one of the common complications associated with diabetes mellitus and the leading cause of blindness worldwide. Recent research has demonstrated that DR is not only a microvascular disease but may be a result of neurodegenerative processes. Moreover, glucose‐induced neuron and glial cell damage may occur shortly after the onset of diabetes which makes the disease hard to diagnose at early stages. SIRT 6, a NAD ‐dependent sirtuin deacylase, modulates aging, energy metabolism, and neurodegeneration. In previous studies we showed that SIRT 6 deficiency causes major retinal transmission defects, changes in the expression of glycolytic genes, and elevated levels of apoptosis. Given the importance of glucose availability for retinal function and the critical role of SIRT 6 in modulating glycolysis, we aimed to analyze SIRT 6 participation in the molecular machinery that regulates the development of experimental DR . Using non‐obese diabetic mice, we determined by western blot that 2 weeks after the onset of the disease, high glucose concentrations induced retinal increase in a neovascularization promoting factor (vascular endothelial growth factor, VEGF ), and the loss of a neuroprotective factor (brain‐derived neurotrophic factor, BDNF) associated with reduced levels of SIRT 6 and increased acetylation levels of its substrates (H3K9 and H3K56) suggesting a deregulation of key neural factors. Noteworthy, retinas from CNS conditionally deleted SIRT 6 mice showed a resemblance to diabetic retinas exhibiting lower protein levels of BDNF factor and increased protein levels of VEGF . Moreover, cultured Müller glial cells subjected to high glucose concentrations exhibited decreased levels of SIRT 6 and increased levels of H3K56 acetylation. In addition, the increment of VEGF levels induced by high glucose was reverted by the over‐expression of SIRT 6 in this cell type. Accordingly, siRNA experiments showed that, when SIRT 6 was silenced, VEGF levels increased. Our findings suggest that epigenetically regulated neurodegenerative events may occur at an early diabetic stage prior to the characteristic proliferative and vascular changes observed at a later diabetic stage.

     

Role of histone acetylation in the development of diabetic retinopathy and the metabolic memory phenomenon

Hyperglycemia is considered as one of the major determinants in the development of diabetic retinopathy, but the progression of retinopathy resists arrest after hyperglycemia is terminated, suggesting a metabolic memory phenomenon. Diabetes alters the expression of retinal genes, and this continues even after good glycemic control is re‐instituted. Since the expression of genes is affected by chromatin structure that is modulated by post‐translational modifications of histones, our objective is to investigate the role of histone acetylation in the development of diabetic retinopathy, and in the metabolic memory phenomenon. Streptozotocin‐induced rats were maintained either in poor glycemic control (PC, glycated hemoglobin, GHb >11%) or good glycemic control (GC, GHb <6%) for 12 months, or allowed to be in PC for 6 months followed by in GC for 6 months (PC‐GC). On a cellular level, retinal endothelial cells, the target of histopathology of diabetic retinopathy, were incubated in 5 or 20 mM glucose for 4 days. Activities of histone deacetylase (HDAC) and histone acetyltransferase (HAT), and histone acetylation were quantified. Hyperglycemia activated HDAC and increased HDAC1, 2, and 8 gene expressions in the retina and its capillary cells. The activity HAT was compromised and the acetylation of histone H3 was decreased. Termination of hyperglycemia failed to provide any benefits to diabetes‐induced changes in retinal HDAC and HAT, and histone H3 remained subnormal. This suggests “in principle” the role of global acetylation of retinal histone H3 in the development of diabetic retinopathy and in the metabolic memory phenomenon associated with its continued progression. J. Cell. Biochem. 110: 1306–1313, 2010. © 2010 Wiley‐Liss, Inc.     

Increase in mitochondrial DNA mutations impairs retinal function and renders the retina vulnerable to injury

Mouse models that accumulate high levels of mitochondrial DNA (mtDNA) mutations owing to impairments in mitochondrial polymerase γ (PolG) proofreading function have been shown to develop phenotypes consistent with accelerated aging. As increase in mtDNA mutations and aging are risk factors for neurodegenerative diseases, we sought to determine whether increase in mtDNA mutations renders neurons more vulnerable to injury. We therefore examined the in vivo functional activity of retinal neurons and their ability to cope with stress in transgenic mice harboring a neural‐targeted mutant PolG gene with an impaired proofreading capability (Kasahara, et al. (2006) Mol Psychiatry 11 (6):577–93, 523). We confirmed that the retina of these transgenic mice have increased mtDNA deletions and point mutations and decreased expression of mitochondrial oxidative phosphorylation enzymes. Associated with these changes, the PolG transgenic mice demonstrated accelerated age‐related loss in retinal function as measured by dark‐adapted electroretinogram, particularly in the inner and middle retina. Furthermore, the retinal ganglion cell–dominant inner retinal function in PolG transgenic mice showed greater vulnerability to injury induced by raised intraocular pressure, an insult known to produce mechanical, metabolic, and oxidative stress in the retina. These findings indicate that an accumulation of mtDNA mutations is associated with impairment in neural function and reduced capacity of neurons to resist external stress in vivo, suggesting a potential mechanism whereby aging central nervous system can become more vulnerable to neurodegeneration.     

Glial cell regulation of neuronal activity and blood flow in the retina by release of gliotransmitters

Astrocytes in the brain release transmitters that actively modulate neuronal excitability and synaptic efficacy. Astrocytes also release vasoactive agents that contribute to neurovascular coupling. As reviewed in this article, Müller cells, the principal retinal glial cells, modulate neuronal activity and blood flow in the retina. Stimulated Müller cells release ATP which, following its conversion to adenosine by ectoenzymes, hyperpolarizes retinal ganglion cells by activation of A1 adenosine receptors. This results in the opening of G protein-coupled inwardly rectifying potassium (GIRK) channels and small conductance Ca²⁺-activated K⁺ (SK) channels. Tonic release of ATP also contributes to the generation of tone in the retinal vasculature by activation of P2X receptors on vascular smooth muscle cells. Vascular tone is lost when glial cells are poisoned with the gliotoxin fluorocitrate. The glial release of vasoactive metabolites of arachidonic acid, including prostaglandin E₂ (PGE₂) and epoxyeicosatrienoic acids (EETs), contributes to neurovascular coupling in the retina. Neurovascular coupling is reduced when neuronal stimulation of glial cells is interrupted and when the synthesis of arachidonic acid metabolites is blocked. Neurovascular coupling is compromised in diabetic retinopathy owing to the loss of glial-mediated vasodilation. This loss can be reversed by inhibiting inducible nitric oxide synthase. It is likely that future research will reveal additional important functions of the release of transmitters from glial cells.     

Proteome map of normal rat retina and comparison with the proteome of diabetic rat retina: new insight in the pathogenesis of diabetic retinopathy

We have employed proteomics to establish a proteome map of the normal rat retina. This baseline map was then used for comparison with the early diabetic rat retinal proteome. Diabetic rat retinae were obtained from Dark Agouti rats after 10 wk of streptozotocin-induced hyperglycaemia. Extracted proteins from normal and diabetic rat retinae were separated and compared using 2-DE. A total of 145 protein spots were identified in the normal rat retina using MALDI-MS and database matching. LC-coupled ESI-MS increased the repertoire of identified proteins by 23 from 145 to 168. Comparison with early diabetic rat retinae revealed 24 proteins unique to the diabetic gels, and 37 proteins absent from diabetic gels. Uniquely expressed proteins identified included the HSPs 70.1A and 8, and platelet activating factor. There were eight spots with increased expression and 27 with decreased expression on diabetic gels. Beta catenin, phosducin and aldehyde reductase were increased in expression in diabetes whilst succinyl coA ligase and dihydropyrimidase-related protein were decreased. Identification of such changes in protein expression has given new insights and a more comprehensive understanding of the pathogenesis of diabetic retinopathy, widening the scope of potential avenues for new therapies for this common cause of blindness.     

Lack of relationship between Alu repetitive elements in angiotensin converting enzyme and the severity of diabetic retinopathy

BackgroundAngiotensin-converting enzyme (ACE) stimulates angiogenesis that leads to the development of diabetic retinopathy (DR). Alu repetitive elements in ACE gene increase the expression of this enzyme. We investigated the frequency of Alu repetitive elements, insertion/deletion (I/D) polymorphism, in angiotensin-converting enzyme among diabetic retinopathy patients and whether this polymorphism is associated with the severity of retinopathy in Jordanians with type 2 diabetes.MethodsA total of 277 subjects participated in this case/ control study (100 diabetic patients without DR, 82 diabetic patients with DR, and 95 healthy control). Blood samples were withdrawn, followed by DNA extraction. Alu repetitive elements were examined by polymerase chain reaction followed by gel electrophoresis.ResultsThe genotype and allele frequencies among diabetic patients, were close to healthy controls (genotypes, II 44.4 vs. 44.7%, ID 44.4 vs. 42.6%, DD 12.2 vs. 12.8%, P = 0.402 and 0.677 respectively, alleles, I 65.6 vs. 66%, D 34.4 vs. 34%, P=0.863). Complicated diabetics with retinopathy showed similar genotype and allele frequency to those without complications. The severity of diabetic retinopathy in affected individuals was not correlated with I/D polymorphism (P=0.862).ConclusionsWe conclude that the presence of Alu repetitive elements did not increase the development or progression risk to retinopathy in Jordanian type 2 diabetic patients. No association between I or D alleles with the severity of DR was detected.     

Diabetes-induced nitrative stress in the retina, and correction by aminoguanidine

Aminoguanidine inhibits the development of retinopathy in diabetic animals, but the mechanism remains unclear. Inasmuch as aminoguanidine is a relatively selective inhibitor of the inducible isoform of nitric oxide synthase (iNOS), we have investigated the effects of hyperglycemia on the retinal nitric oxide (NO) pathway in the presence and absence of aminoguanidine. In vivo studies utilized retinas from experimentally diabetic rats treated or without aminoguanidine for 2 months, and in vitro studies used bovine retinal endothelial cells and a transformed retinal glial cell line (rMC-1) incubated in 5 mm and 25 mm glucose with and without aminoguanidine (100 microg/mL). NO was detected as nitrite and nitrate, and nitrotyrosine and iNOS were detected using immunochemical methods. Retinal homogenates from diabetic animals had greater than normal levels of NO and iNOS (p < 0.05), and nitrotyrosine was greater than normal, especially in one band immunoprecipitated from retinal homogenates. Oral aminoguanidine significantly inhibited all of these increases. Nitrotyrosine was detected immunohistochemically only in the retinal vasculature of non-diabetic and diabetic animals. Retinal endothelial and rMC-1 cells cultured in high glucose increased NO and NT, and aminoguanidine inhibited both increases in rMC-1 cells, but only NT in endothelial cells. Hyperglycemia increases NO production in retinal cells, and aminoguanidine can inhibit this abnormality. Inhibition of diabetic retinopathy by aminoguanidine might be mediated in part by inhibition of sequelae of NO production.