Fractalkine-induced microglial vasoregulation occurs within the retina and is altered early in diabetic retinopathy.

Local blood flow control within the central nervous system (CNS) is critical to proper function and is dependent on coordination between neurons, glia, and blood vessels. Macroglia, such as astrocytes and Müller cells, contribute to this neurovascular unit within the brain and retina, respectively. This study explored the role of microglia, the innate immune cell of the CNS, in retinal vasoregulation, and highlights changes during early diabetes. Structurally, microglia were found to contact retinal capillaries and neuronal synapses. In the brain and retinal explants, the addition of fractalkine, the sole ligand for monocyte receptor Cx3cr1, resulted in capillary constriction at regions of microglial contact. This vascular regulation was dependent on microglial Cx3cr1 involvement, since genetic and pharmacological inhibition of Cx3cr1 abolished fractalkine-induced constriction. Analysis of the microglial transcriptome identified several vasoactive genes, including angiotensinogen, a constituent of the renin-angiotensin system (RAS). Subsequent functional analysis showed that RAS blockade via candesartan abolished microglial-induced capillary constriction. Microglial regulation was explored in a rat streptozotocin (STZ) model of diabetic retinopathy. Retinal blood flow was reduced after 4 wk due to reduced capillary diameter and this was coincident with increased microglial association. Functional assessment showed loss of microglial-capillary response in STZ-treated animals and transcriptome analysis showed evidence of RAS pathway dysregulation in microglia. While candesartan treatment reversed capillary constriction in STZ-treated animals, blood flow remained decreased likely due to dilation of larger vessels. This work shows microglia actively participate in the neurovascular unit, with aberrant microglial-vascular function possibly contributing to the early vascular compromise during diabetic retinopathy.

The renin-angiotensin system and the retinal neurovascular unit: A role in vascular regulation and disease.

The retina is known to have a local renin-angiotensin system (RAS) and dysfunction in the RAS is often associated with diseases of the retinal vasculature that cause irreversible vision loss. Regulation of the retinal vasculature to meet the metabolic needs of the tissues occurs through a mechanism called neurovascular coupling, which is critical for maintaining homeostatic function and support for neurons. Neurovascular coupling is the process by which support cells, including glia, regulate blood vessel calibre and blood flow in response to neural activity. In retinal vascular diseases, this coupling mechanism is often disrupted. However, the role that angiotensin II (Ang II), the main effector peptide of the RAS, has in regulating both the retinal vasculature and neurovascular coupling is not fully understood. As components of the RAS are located on the principal neurons, glia and blood vessels of the retina, it is possible that Ang II has a role in regulating communication and function between these three cell types, and therefore the capacity to regulate neurovascular coupling. This review focuses on components of the RAS located on the retinal neurovascular unit, and the potential of this system to contribute to blood flow modulation in the healthy and compromised retina.

Failure of Autophagy-Lysosomal Pathways in Rod Photoreceptors Causes the Early Retinal Degeneration Phenotype Observed in Cln6nclf Mice.

Purpose: Vision loss caused by photoreceptor death represents one of the first symptoms in neuronal ceroid lipofuscinosis, a condition characterized by accumulation of intracellular waste. Cln6nclf mice have a naturally occurring mutation in ceroid-lipofuscinosis neuronal (CLN) protein 6 and are a model of this disorder. In order to identify the effect intracellular waste (lipofuscin) accumulation plays in driving retinal degeneration, the time course of degeneration was carefully characterized functionally using the electroretinogram and structurally using histology. Methods: Cln6nclf and C57BL/6J, wild-type, mice were studied at postnatal day 18 (P18), P30, P60, P120, and P240, and retinal degeneration was correlated with changes in the retinal pigment epithelial (RPE) and neuronal autophagy-lysosomal pathways using super-resolution microscopy. Results: In Cln6nclf mice there was significant loss of rod photoreceptor function at P18, prior to photoreceptor nuclei loss at P60. In contrast, cone pathway function was not affected until P240. The loss of rod photoreceptor function correlated with significant disruption of the autophagy-lysosomal degradation pathways within photoreceptors, but not in the RPE or other retinal neurons. Additionally, there was cytosolic accumulation of P62 and undigested mitochondrial-derived, ATP synthase subunit C in the photoreceptor layers of Cln6nclf mice at P30. Conclusions: These results suggest that rod photoreceptors have an increased sensitivity to disturbances in the autophagy-lysosomal pathway and the subsequent failure of mitochondrial turnover, relative to other retinal cells. It is likely that primary failure of the rod photoreceptors rather than the RPE or other retinal neurons underlies the early visual dysfunction that occurs in the Cln6nclf mouse model.

The Role of the Microglial Cx3cr1 Pathway in the Postnatal Maturation of Retinal Photoreceptors.

Microglia are the resident immune cells of the CNS, and their response to infection, injury and disease is well documented. More recently, microglia have been shown to play a role in normal CNS development, with the fractalkine-Cx3cr1 signaling pathway of particular importance. This work describes the interaction between the light-sensitive photoreceptors and microglia during eye opening, a time of postnatal photoreceptor maturation. Genetic removal of Cx3cr1 (Cx3cr1GFP/GFP ) led to an early retinal dysfunction soon after eye opening [postnatal day 17 (P17)] and cone photoreceptor loss (P30 onward) in mice of either sex. This dysfunction occurred at a time when fractalkine expression was predominantly outer retinal, when there was an increased microglial presence near the photoreceptor layer and increased microglial-cone photoreceptor contacts. Photoreceptor maturation and outer segment elongation was coincident with increased opsin photopigment expression in wild-type retina, while this was aberrant in the Cx3cr1GFP/GFP retina and outer segment length was reduced. A beadchip array highlighted Cx3cr1 regulation of genes involved in the photoreceptor cilium, a key structure that is important for outer segment elongation. This was confirmed with quantitative PCR with specific cilium-related genes, Rpgr and Rpgrip1, downregulated at eye opening (P14). While the overall cilium structure was unaffected, expression of Rpgr, Rpgrip1, and centrin were restricted to more proximal regions of the transitional zone. This study highlighted a novel role for microglia in postnatal neuronal development within the retina, with loss of fractalkine-Cx3cr1 signaling leading to an altered distribution of cilium proteins, failure of outer segment elongation and ultimately cone photoreceptor loss.SIGNIFICANCE STATEMENT Microglia are involved in CNS development and disease. This work highlights the role of microglia in postnatal development of the light-detecting photoreceptor neurons within the mouse retina. Loss of the microglial Cx3cr1 signaling pathway resulted in specific alterations in the cilium, a key structure in photoreceptor outer segment elongation. The distribution of key components of the cilium transitional zone, Rpgr, Rpgrip1, and centrin, were altered in retinae lacking Cx3cr1 with reduced outer segment length and cone photoreceptor death observed at later postnatal ages. This work identifies a novel role for microglia in the postnatal maturation of retinal photoreceptors.

Prophylactic laser in age-related macular degeneration: the past, the present and the future.

The presence of drusen in the posterior eye is a hallmark feature of the early stages of age-related macular degeneration and their size is an indicator of risk of progression to vision-threatening forms of the disease. Since the initial observations that laser treatment can resolve drusen, there has been great interest in whether laser treatment can be used to reduce the progression of age-related macular degeneration. In this article, we review the development of lasers for the treatment of those with age-related macular degeneration. We provide an overview of the clinical trial results that demonstrated drusen resolution but that had mixed effects on progression of disease. In addition, we provide a summary of the recent developments in pulsed lasers that are designed to reduce the energy applied to the posterior eye to provide the therapeutic effects of conventional continuous wave lasers while reducing the secondary tissue effects.

The Role of Angiotensin II/AT1 Receptor Signaling in Regulating Retinal Microglial Activation.

Purpose: This study explored whether the proangiogenic factor Angiotensin II (AngII) had a direct effect on the activation state of microglia via the Angiotensin type 1 receptor (AT1-R). Methods: Microglial dynamic activity was investigated in live retinal flatmounts from adult Cx3Cr1+/GFP mice under control, AngII (5 µM) or AngII (5 µM) + candesartan (0.227 µM) conditions. The effects of intravitreal administration of AngII (10 mM) were also investigated at 24 hours, with retinae processed for immunocytochemistry, flow cytometry, or inflammatory quantitative PCR arrays. Results: We found FACS isolated retinal microglia expressed AT1-R. In retinal flatmounts, microglia showed characteristic movement of processes under control conditions. Perfusion of AngII induced an immediate change in process length (-42%, P < 0.05) and activation state of microglia that was ameliorated by AT1-R blockade, suggesting a direct effect of AngII on microglia via the AT1-R. Intravitreal injection of AngII induced microglial activation after 24 hours, which was characterized by increased soma size (23%, P < 0.001) and decreased process length (20%, P < 0.05). Further analysis indicated a significant decrease in the number of microglial contacts with retinal neurons (saline 15.6 ± 2.31 versus AngII 7.8 ± 1.06, P < 0.05). Retinal cytokine and chemokine expression was modulated, indicative of an inflammatory retinal phenotype. Conclusions: We show that retinal microglia express AT1-R and their activation state is significantly altered by the angiogenic factor, AngII. Specifically, AngII may directly activate AT1-Rs on microglia and contribute to retinal inflammation. This may have implications for diseases like diabetic retinopathy where increases in AngII and inflammation have been shown to play an important role.

Loss of Function of P2X7 Receptor Scavenger Activity in Aging Mice: A Novel Model for Investigating the Early Pathogenesis of Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) is a leading cause of irreversible, severe vision loss in Western countries. Recently, we identified a novel pathway involving P2X7 receptor scavenger function expressed on ocular immune cells as a risk factor for advanced AMD. In this study, we investigate the effect of loss of P2X7 receptor function on retinal structure and function during aging. P2X7-null and wild-type C57bl6J mice were investigated at 4, 12, and 18 months of age for macrophage phagocytosis activity, ocular histological changes, and retinal function. Phagocytosis activity of blood-borne macrophages decreased with age at 18 months in the wild-type mouse. Lack of P2X7 receptor function reduced phagocytosis at all ages compared to wild-type mice. At 12 months of age, P2X7-null mice had thickening of Bruchs membrane and retinal pigment epithelium dysfunction. By 18 months of age, P2X7-null mice displayed phenotypic characteristics consistent with early AMD, including Bruchs membrane thickening, retinal pigment epithelium cell loss, retinal functional deficits, and signs of subretinal inflammation. Our present study shows that loss of function of the P2X7 receptor in mice induces retinal changes representing characteristics of early AMD, providing a valuable model for investigating the role of scavenger receptor function and the immune system in the development of this age-related disease.

Localization and Possible Function of P2X Receptors in Normal and Diseased Retinae.

Purines, when present in the extracellular space, can mediate fast neurotransmission in the retina and central nervous system. Over the last decade there has been emerging evidence for the expression of P2X and P2Y receptors in a range of retinal neuronal subtypes. These results have highlighted important roles for purines in modulating specific retinal circuits, including the rod pathway and amacrine cell circuits. Traditionally, synaptic release of adenosine triphosphate (ATP) involves the novel anion vesicular nucleotide transporter, VNUT, which has recently been identified in a single wide-field amacrine cell population. In addition, nontraditional, conductive mechanisms of release have also been described in the retina. In the synapse, the enzymes involved in rapid degradation of purines are present in both plexiform layers of the retina. A role for P2X receptors in retinal diseases has also emerged recently. High concentrations of ATP lead to photoreceptor loss, through mechanisms involving P2X7 receptors. In addition, activation of P2X7 receptors is associated with activation of the inflammasome, a protein complex important for the release of proinflammatory cytokines. P2X receptors, especially P2X7, are emerging as targets to combat retinal disease.

Assessment of retinal function and morphology in aging Ccl2 knockout mice.

PURPOSE: The chemokine Ccl2, or monocyte chemoattractant protein-1 (MCP-1), has previously been identified as playing a potential role in many ocular diseases; however, its role in mice is less clear. We sought to correlate changes in retinal pigment epithelium (RPE) and retinal morphology with changes in function in aging Ccl2(-/-) mice. METHODS: Ccl2(-/-) mice on a C57BL6J background were genotyped for Crb1(rd8/rd8) and were free of this mutation. Ccl2(-/-) mice and wild-type (WT) C57BL6J mice were investigated for changes in the retinal fundus and histology as a function of age. The function of the rod and cone pathways, and the rate of dark adaptation, was assessed using the electroretinogram (ERG) up to 15 months of age. RESULTS: Fifteen-month-old Ccl2(-/-) mice had fundus lesions, more subretinal microglia/macrophages, and an increase in RPE cell size, indicative of RPE cell loss, when compared with WT mice. Within the retina, gross morphology was normal but there was an increase in Müller cell gliosis and microglial activation. These morphological changes in the Ccl2(-/-) RPE/retina did not correlate with a change in either rod or cone ERG pathway function, or with the rate of dark adaptation. CONCLUSIONS: These data show that Ccl2 is important for preserving RPE and glial morphology with age, yet retinal function and gross morphology are maintained. Altered signaling in this chemokine pathway may, however, increase RPE and retinal vulnerability to disease.

Studying age-related macular degeneration using animal models.

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.

Adenosine triphosphate-induced photoreceptor death and retinal remodeling in rats.

Many common causes of blindness involve the death of retinal photoreceptors, followed by progressive inner retinal cell remodeling. For an inducible model of retinal degeneration to be useful, it must recapitulate these changes. Intravitreal administration of adenosine triphosphate (ATP) has recently been found to induce acute photoreceptor death. The aim of this study was to characterize the chronic effects of ATP on retinal integrity. Five-week-old, dark agouti rats were administered 50 mM ATP into the vitreous of one eye and saline into the other. Vision was assessed using the electroretinogram and optokinetic response and retinal morphology investigated via histology. ATP caused significant loss of visual function within 1 day and loss of 50% of the photoreceptors within 1 week. At 3 months, 80% of photoreceptor nuclei were lost, and total photoreceptor loss occurred by 6 months. The degeneration and remodeling were similar to those found in heritable retinal dystrophies and age-related macular degeneration and included inner retinal neuronal loss, migration, and formation of new synapses; Müller cell gliosis, migration, and scarring; blood vessel loss; and retinal pigment epithelium migration. In addition, extreme degeneration and remodeling events, such as neuronal and glial migration outside the neural retina and proliferative changes in glial cells, were observed. These extreme changes were also observed in the 2-year-old P23H rhodopsin transgenic rat model of retinitis pigmentosa. This ATP-induced model of retinal degeneration may provide a valuable tool for developing pharmaceutical therapies or for testing electronic implants aimed at restoring vision.

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 Bruch's 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.

Alternative pathways in the development of diabetic retinopathy: the renin-angiotensin and kallikrein-kinin systems.

Diabetic retinopathy is a common complication of both type 1 and type 2 diabetes and is the leading cause of blindness in people of working age. Current treatment strategies are mostly limited to laser photocoagulation, which restricts proliferative retinopathic changes but also causes irreversible damage to the retina. This review examines two important pathways involved in regulating vascular function and their role in the development of diabetic retinopathy. One, the renin-angiotensin system, is well known and has established angiogenic effects on the retina that increase in diabetic retinopathy. The other, the kallikrein-kinin system, has recently been found to be important in the development of diabetic retinal complications. This review describes the components of the two signalling networks, examines the current animal model studies investigating the role of these pathways in diabetic retinopathy and reviews the clinical studies that have been undertaken examining systemic inhibition of different points in these pathways. These systems are promising targets for therapies aimed at inhibiting the development of diabetic retinopathy and in the future, combination therapies that take advantage of both pathways might result in new treatment options for this debilitating complication of diabetes.

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.

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.

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.

Seizure-related gene 6 (Sez-6) in amacrine cells of the rodent retina and the consequence of gene deletion.

BACKGROUND: Seizure-related gene 6 (Sez-6) is expressed in neurons of the mouse brain, retina and spinal cord. In the cortex, Sez-6 plays a role in specifying dendritic branching patterns and excitatory synapse numbers during development. METHODOLOGY/PRINCIPAL FINDINGS: The distribution pattern of Sez-6 in the retina was studied using a polyclonal antibody that detects the multiple isoforms of Sez-6. Prominent immunostaining was detected in GABAergic, but not in AII glycinergic, amacrine cell subpopulations of the rat and mouse retina. Amacrine cell somata displayed a distinct staining pattern with the Sez-6 antibody: a discrete, often roughly triangular-shaped bright spot positioned between the nucleus and the apical dendrite superimposed over weaker general cytoplasmic staining. Displaced amacrines in the ganglion cell layer were also positive for Sez-6 and weaker staining was occasionally observed in neurons with the morphology of alpha ganglion cells. Two distinct Sez-6 positive strata were present in the inner plexiform layer in addition to generalized punctate staining. Certain inner nuclear layer cells, including bipolar cells, stained more weakly and diffusely than amacrine cells, although some bipolar cells exhibited a perinuclear "bright spot" similar to amacrine cells. In order to assess the role of Sez-6 in the retina, we analyzed the morphology of the Sez-6 knockout mouse retina with immunohistochemical markers and compared ganglion cell dendritic arbor patterning in Sez-6 null retinae with controls. The functional importance of Sez-6 was assessed by dark-adapted paired-flash electroretinography (ERG). CONCLUSIONS: In summary, we have reported the detailed expression pattern of a novel retinal marker with broad cell specificity, useful for retinal characterization in rodent experimental models. Retinal morphology, ganglion cell dendritic branching and ERG waveforms appeared normal in the Sez-6 knockout mouse suggesting that, in spite of widespread expression of Sez-6, retinal function in the absence of Sez-6 is not affected.

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.

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.

Retinal dysfunction in diabetic ren-2 rats is ameliorated by treatment with valsartan but not atenolol.

PURPOSE: To determine whether diabetes leads to retinal neuronal dysfunction in hypertensive transgenic (mRen-2)27 rats (Ren-2), and whether the effect can be prevented by treatment of hypertension with either the angiotensin-1 receptor blocker (AT1-RB) valsartan or the beta1-adrenergic receptor antagonist atenolol. METHODS: Six-week-old Ren-2 rats were made diabetic (streptozotocin 55 mg/kg; n = 34) or remained nondiabetic (0.1 M citrate buffer; n = 43) and studied for 20 weeks. A subset of animals received valsartan (4 mg/kg per day) or atenolol (30 mg/kg per day) by gavage. Sprague-Dawley (SD) rats served as normotensive controls for blood pressure (BP). We evaluated retinal function in all groups with a paired-flash electroretinogram over high light intensities (0.5-2.0 log cd-s . m(-2)), to isolate rod and cone responses. RESULTS: A reduction in amplitude of all electroretinogram components (PIII, PII, OPs, cone PII) was found in nondiabetic Ren-2 compared with nondiabetic SD rats. A further reduction was observed in diabetic Ren-2 rats. Treatment of both nondiabetic and diabetic Ren-2 rats with valsartan or atenolol reduced BP to within normal limits. This reduction produced some improvement in function in treated nondiabetic Ren-2 rats. However, in treated diabetic Ren-2 rats, retinal dysfunction was ameliorated by valsartan but not by atenolol, with a significant improvement (P < 0.05) observed in all components of the electroretinogram, with the exception of the OPs. CONCLUSIONS: These findings suggest that hypertension induces retinal dysfunction that is exacerbated with diabetes and ameliorated by treatment with an AT1-RB, and not just by normalizing BP. These data provide further evidence for the importance of the renin-angiotensin system in development of diabetic complications.