Joanna A. Phipps

Neuronal and Glial Cell Abnormality as Predictors of Progression of Diabetic Retinopathy

Diabetes is known to cause significant alterations in the retinal vasculature. Indeed, diabetic retinopathy is the leading cause of blindness in those of working age. Considerable evidence is emerging that indicates that retinal neurons are also altered during diabetes. Moreover, many types of neuronal deficits have been observed in animal models and patients prior to the onset of vascular compromise. Such clinical tools as the flash ERG, multifocal ERG, colour vision, contrast sensitivity and short-wavelength automated perimetry, all provide novel means whereby neuronal dysfunction can be detected at early stages of diabetes. The underlying mechanisms that lead to neuronal deficits are likely to be broad. Retinal glial cells play an essential role in maintaining the normal function of the retina. There is accumulating evidence that Müller cells are abnormal during diabetes. They are known to become gliotic, display altered potassium siphoning, glutamate and GABA uptake and are also known to express several modulators of angiogenesis. This review will examine the evidence that neurons and glia are altered during diabetes and the relationship these changes have with vascular compromise.

The renin-angiotensin system in retinal health and disease: Its influence on neurons, glia and the vasculature.

Renin-Angiotensin System is classically recognized for its role in the control of systemic blood pressure. However, the retina is recognized to have all the components necessary for angiotensin II formation, suggestive of a role for Angiotensin II in the retina that is independent of the systemic circulation. The most well described effects of Angiotensin II are on the retinal vasculature, with roles in vasoconstriction and angiogenesis. However, it is now emerging that Angiotensin II has roles in modulation of retinal function, possibly in regulating GABAergic amacrine cells. In addition, Angiotensin II is likely to have effects on glia. Angiotensin II has also been implicated in retinal vascular diseases such as Retinopathy of Prematurity and diabetic retinopathty, and more recently actions in choroidal neovascularizaiton and glaucoma have also emerged. The mechanisms by which Angiotensin II promotes angiogensis in retinal vascular diseases is indicative of the complexity of the RAS and the variety of cell types that it effects. Indeed, these diseases are not purely characterized by direct effects of Angiotensin II on the vasculature. In retinopathy of prematurity, for example, blockade of AT1 receptors prevents pathological angiogenesis, but also promotes revascularization of avascular regions of the retina. The primary site of action of Angiotensin II in this disease may be on retinal glia, rather than the vasculature. Indeed, blockade of AT1 receptors prevents glial loss and promotes the re-establishment of normal vessel growth. Blockade of RAS as a treatment for preventing the incidence and progression of diabetic retinopathy has also emerged based on a series of studies in animal models showing that blockade of the RAS prevents the development of a variety of vascular and neuronal deficits in this disease. Importantly these effects may be independent of actions on systemic blood pressure. This has culminated recently with the completion of several large multi-centre clinical trials that showed that blockade of the RAS may be of benefit in some at risk patients with diabetes. With the emergence of novel compounds targeting different aspects of the RAS even more effective ways of blocking the RAS may be possible in the future.

Early Inner Retinal Astrocyte Dysfunction during Diabetes and Development of Hypoxia, Retinal Stress, and Neuronal Functional Loss

PURPOSE Neuronal and glial alterations precede the overt vascular change that characterizes diabetic retinopathy. Because retinal astrocytes modulate neuronal and vascular function, this study investigated the time course of astrocyte, Müller cell, and neuronal change during diabetes to determine whether astrocytes may play an early role in diabetic retinopathy. METHODS Sprague-Dawley rats were rendered diabetic via streptozotocin and neuronal and glial changes were assessed after 2-10 weeks. Astrocyte change was investigated using connexin-26 immunolabeling, whereas connexin-26 and -43 gene expressions were quantified using real-time PCR. Hypoxia was measured by pimonidazole labeling and the expression of hypoxia-inducible factor-1 alpha (HIF-1α) was quantified using Western blot. Müller cell gliosis was assessed by glial fibrillary acidic protein immunolabeling and retinal function assessed using the electroretinogram. RESULTS Astrocyte connexin-26 and -43 gene and protein expression decreased after 4 weeks of diabetes, before significant astrocyte loss. At the same time, the retina became hypoxic, with increased HIF-1α expression and pimonidazole labeling in the ganglion cell layer. This coincided with a decrease in ganglion cell function. After 6 weeks of diabetes, Müller cell gliosis became more evident and there were additional functional deficits in photoreceptoral and amacrine cell responses. CONCLUSIONS These findings suggest that early changes in astrocytes are coincident with inner retinal hypoxia and ganglion cell functional deficits, whereas Müller cell gliosis and more extensive decreases in neuronal function occur later. Astrocytes may play an early and key role in changes in retinal vasculature and inner retinal dysfunction in diabetes.

Dysfunction of retinal neurons and glia during diabetes

Diabetic retinopathy is the leading cause of blindness in those of working age. It is well known that the retinal vasculature is altered during diabetes. More recently, it has emerged that neuronal and glial dysfunction occurs in those with diabetes. Current research is directed at understanding these neuronal and glial changes because they may be an early manifestation of disease processes that ultimately lead to vascular abnormality. This review will highlight the recent advances in our understanding of the neuronal and glial changes that occur during diabetes.

Plasma Kallikrein Mediates Angiotensin II Type 1 Receptor–Stimulated Retinal Vascular Permeability

Hypertension is a leading risk factor for the development and progression of diabetic retinopathy and contributes to a variety of other retinal diseases in the absence of diabetes mellitus. Inhibition of the renin-angiotensin system has been shown to provide beneficial effects against diabetic retinopathy, both in the absence and presence of hypertension, suggesting that angiotensin II (Ang II) and the Ang II type 1 receptor may contribute to retinal vascular dysfunction. We investigated the effects of the Ang II type 1 receptor antagonist candesartan on retinal vascular permeability (RVP) in normotensive rats with streptozotocin-induced diabetes mellitus and in rats with Ang II–induced hypertension. We showed that candesartan treatment decreased diabetes mellitus– and Ang II–stimulated RVP by 58% (P<0.05) and 79% (P<0.05), respectively, compared with untreated controls, suggesting that activation of the Ang II type 1 receptor contributes to blood-retinal barrier dysfunction. We found that plasma kallikrein levels are increased in the retina of rats with Ang II–stimulated hypertension and that intravitreal injection of either plasma kallikrein or bradykinin is sufficient to increase RVP. We showed that a novel small molecule inhibitor of plasma kallikrein, 1-benzyl-1H-pyrazole-4-carboxylic acid 4-carbamimidoyl-benzylamide, delivered systemically via a subcutaneous pump, decreased Ang II–stimulated RVP by 70% (P<0.05) and ameliorates Ang II–induced hypertension, measured from the carotid artery by telemetry, but did not reduce Ang II–induced retinal leukostasis. These findings demonstrate that activation of the Ang II type 1 receptor increases RVP and suggest that systemic plasma kallikrein inhibition may provide a new therapeutic approach for ameliorating blood-retinal barrier dysfunction induced by hypertension.

The kallikrein–kinin system in diabetic retinopathy: Lessons for the kidney

Diabetic retinopathy and diabetic nephropathy are common microvascular complications of diabetes. The kallikrein-kinin system (KKS) has been implicated in the development of both conditions, and, in particular, bradykinin and its receptors have been shown to exert angiogenic and proinflammatory actions. Several of the key processes that underlie the development of diabetic retinopathy, such as increased vascular permeability, edema, neovascularization, and inflammatory changes, have been associated with the KKS, and recent work has shown that components of the KKS, including plasma kallikrein, factor XIIa, and high-molecular-weight kininogen, are present in the vitreous of people with diabetic retinopathy. The role of the KKS in the development of diabetic nephropathy is controversial, with both adverse and protective effects of bradykinin and its receptors reported. The review examines the role of the KKS in pathways central to the development of diabetic retinopathy and compares this with reported actions of this system in diabetic nephropathy. The possibility of therapeutic intervention targeting bradykinin and its receptors as treatment for diabetic microvascular conditions is considered.

Ccl2/Cx3cr1 knockout mice have inner retinal dysfunction but are not an accelerated model of AMD.

PURPOSE The chemokine, Ccl2, and the fractalkine receptor, Cx3cr1, have both been implicated in the pathogenesis of age related macular degeneration (AMD), with mice lacking both genes exhibiting features of AMD by 3 months of age. However, recent reports indicate that this ascribed phenotype is due to the presence of a retinal degeneration mutation (crb1(rd8/rd8), rd8) on the background strain. Our aim was to characterize the retinal effects of lack of Ccl2 and Cx3cr1 (Ccl2(-/-)/Cx3cr1(EGFP/EGFP), CDKO-mice), in mice without the rd8 mutation. METHODS Nine-month-old, CDKO and wildtype C57blk6J mice were investigated for retinal fundus appearance and histology. The function of the rod and cone pathways was assessed using the ERG. RESULTS The CDKO mice did not develop lesions in the retinal fundus, and the ultrastructure of Bruchs membrane and the RPE were similar to that of C57blk6J mice. From the ERG, there was no change in the amplitude of the rod photoreceptor response, or in the rod or cone post-photoreceptor b-wave. However, the rod and cone ERG oscillatory potentials were significantly reduced in the CDKO animals, a phenotype apparent in Cx3cr1(EGFP/EGFP)- but not Ccl2(-/-)-founder lines. This correlated with aberrant amacrine cell morphology in the CDKO mice. In addition, Müller cells were gliotic and microglial morphology subtly altered, indicative of retinal stress. CONCLUSIONS These results suggest that in the absence of the rd8 mutation, the CDKO-mouse has a mild inner retinal phenotype characterized by altered amacrine cell function, but that it is not an accelerated model of AMD.

Angiotensin AT1 receptor antagonism ameliorates murine retinal proteome changes induced by diabetes.

Diabetic retinopathy is the most common microvascular complication caused by diabetes mellitus and is a leading cause of vision loss among working-age adults in developed countries. Understanding the effects of diabetes on the retinal proteome may provide insights into factors and mechanisms responsible for this disease. We have performed a comprehensive proteomic analysis and comparison of retina from C57BL/6 mice with 2 months of streptozotocin-induced diabetes and age-matched nondiabetic control mice. To explore the role of the angiotensin AT1 receptor in the retinal proteome in diabetes, a subgroup of mice were treated with the AT1 antagonist candesartan. We identified 1792 proteins from retinal lysates, of which 65 proteins were differentially changed more than 2-fold in diabetic mice compared with nondiabetic mice. A majority (72%) of these protein changes were normalized by candesartan treatment. Most of the significantly changed proteins were associated with metabolism, oxidative phosphorylation, and apoptotic pathways. An analysis of the proteomics data revealed metabolic and apoptotic abnormalities in the retina from diabetic mice that were ameliorated with candesartan treatment. These results provide insight into the effects of diabetes on the retina and the role of the AT1 receptor in modulating this response.

Fast psychophysical procedures for clinical testing.

Introduction: Psychophysical methods are used in clinical settings to obtain estimates of visual performance. Such methods should be fast and accurate, yet robust to the corrupting effects of false responses.

Studying Age-Related Macular Degeneration Using Animal Models

ABSTRACT Over the recent years, there have been tremendous advances in our understanding of the genetic and environmental factors associated with the development of age-related macular degeneration (AMD). Examination of retinal changes in various animals has aided our understanding of the pathogenesis of the disease. Notably, mouse strains, carrying genetic anomalies similar to those affecting humans, have provided a foundation for understanding how various genetic risk factors affect retinal integrity. However, to date, no single mouse strain that develops all the features of AMD in a progressive age-related manner has been identified. In addition, a mutation present in some background strains has clouded the interpretation of retinal phenotypes in many mouse strains. The aim of this perspective was to describe how animals can be used to understand the significance of each sign of AMD, as well as key genetic risk factors.