The Role of Microglia in Diabetic Retinopathy
There is growing evidence that chronic inflammation plays a role in both the development and progression of diabetic retinopathy. There is also evidence that molecules produced as a result of hyperglycemia can activate microglia. However the exact contribution of microglia, the resident immune cells of the central nervous system, to retinal tissue damage during diabetes remains unclear. Current data suggest that dysregulated microglial responses are linked to their deleterious effects in several neurological diseases associated with chronic inflammation. As inflammatory cytokines and hyperglycemia disseminate through the diabetic retina, microglia can change to an activated state, increase in number, translocate through the retina, and themselves become the producers of inflammatory and apoptotic molecules or alternatively exert anti-inflammatory effects. In addition, microglial genetic variations may account for some of the individual differences commonly seen in patient’s susceptibility to diabetic retinopathy.
It is generally acknowledged that central nervous system (CNS) disorders involve microglial activation and that progression and resolution of many diseases depend in part on the actions of microglia. As resident inflammatory cells of the CNS, microglia potentially modulate inflammatory processes. However, microglial functions do not happen in isolation, but in concert with the activities of neurons, glial, and vascular cells. Diabetic retinopathy (DR) is the leading cause of vision loss in individuals 20–75 years of age. It remains a frightening prospect to patients and physicians as the cause(s) remain unclear. While DR has been described classically as a microvascular disease, recent evidence suggests that changes to retinal microglia are an early feature of retinopathy [1–6]. Clinically, DR is classified as nonproliferative (NPDR) and proliferative diabetic retinopathy (PDR). NPDR exhibits damage to retinal vasculature, leaky blood vessels, and associated mobilization of blood components into the retina. Proliferative DR associates with growth of blood vessels on the surface of the retina. Although diabetic macular edema (DME) can cause vision loss in both NPDR and PDR, the severe angiogenesis in proliferative disease can result in retinal detachment with a potential to cause total blindness. The loss of retinal neurons and the loss of contrast sensitivity in diabetes have only recently been described [1–9]. The study of microglia at different stages of diabetes, and its interaction with peripheral leukocytes and retinal cells, is crucial to provide insights into mechanisms of damage and repair. The use of genetic and induced models of diabetes, in combination with histochemical and imaging approaches, has provided instrumental information in the role of microglia during retinal diseases. Understanding the role of microglia in the diabetic retina is essential in comprehending the inflammatory components during disease progression.
1.1. Microglial Phenotypes
Immune surveillance is the most common function associated with microglia during healthy and diseased states. Several studies have elegantly shown that microglia are continuously surveying their microenvironment by extending and retracting their highly motile processes [14, 15]. This property is critical to elicit prompt responses to injury or infection. Hanisch and Kettenmann hypothesize that microdamage in the brain such as microaneurism or the incipient demise of a single neuron can be monitored and repairs can be initiated without triggering a more massive activated state [16–18]. Phagocytosis of cellular debris is an important task of microglia and is tightly linked to their immune surveillant behavior. The activation of microglia in response to neural damage involves proliferative, morphological, immunoreactive, and migratory changes [14, 19, 20]. Studies have suggested that the severity of the microglia response is dependent on the gravity of the neural damage; additionally, the microglia response can be either neural-protective or toxic [16, 21–28]. Graded changes in the morphological appearance of microglia are often used to distinguish surveillant or highly ramified microglia from activated or amoeboid microglia. The amoeboid state refers to cells with larger cell bodies and thicker processes and is usually correlated with activation of microglia in response to CNS insults such as autoimmune inflammation, neuronal injury, cancer, infection, or hyperglycemia. In the retina, under normal conditions, surveillant microglia are localized to the inner and outer plexiform layers and are absent from the outer nuclear layer (Table 1) [10, 29]. Retinal microglia activation correlates with neuronal damage induced by retinopathies such as autoimmune reactions, ocular infections, ischemia, neural injury,and cytokine exposure but direct evidence of microglial mediated neurotoxicity is missing . Interestingly, during normal CNS development, microglia possess an amoeboid morphology and as microglia become mature they transition to a ramified form . However, there are instances in which microglial activation occurs without evident cellular transformation. For example, low doses of systemic lipopolysaccharide (LPS) induce the production of proinflammatory cytokines without any apparent change in microglial morphology . Therefore, microglial responses are shaped by an arsenal of signal transduction pathways that lead to an effector phenotype associated with neuroprotection or neurotoxicity. Disregulated microglial responses could be the result of a triggering receptor or the loss of a constitutive inhibitory signal [33, 34]. Ultimately, the balance between these contrasting outcomes guides tissue repair or exacerbates injury.
1.2. Hyperglycemia, Inflammation, and Microglia
It has been suspected that chronic inflammation played a role in DR since 1964 when Powell found that high dose aspirin used to treat rheumatoid arthritis was also helpful in treating DR . Several molecular changes associated with chronic inflammation have been detected in the diabetic retina and there is growing evidence that this plays a role in both the development and progression of DR [36–38]. Various mechanisms including ischemia, hypoxia, hyperglycemia, dyslipidemia, advanced glycation end products (AGE), and endoplasmic reticular (ER) stress have been hypothesized to contribute to the inflammatory component of DR [39, 40]. Chronic inflammation has also been linked to diabetic nephropathy and peripheral neuropathy . Since there is significant evidence that chronic inflammation plays a role in diabetic retinopathy and microglia are the resident immune cells in the retina, it is likely that unleashed microglial activation may play a role in the neurotoxicity and tissue damage in the diabetic retina. Microglia may function in acute inflammation protecting the CNS, but have also been linked to deleterious effects on the CNS when they exhibit chronic inflammation in diseases such as HIV, multiple sclerosis, and Alzheimer’s . Yang et al. defines DR as a “chronic, low-grade inflammatory disease of the retina” . Since inflammatory conditions present in diabetes can activate microglia, they further state that “activated microglia thereby stimulate a cycle of inflammation that recruits leukocytes, causes vascular breakdown, and directly induces glial dysfunction and neuronal cell death through the release of cytotoxic substances” .
It is well established that hyperglycemia results in increased cellular oxidative stress. This can be either through (1) direct generation of reactive oxygen species (ROS), (2) alteration of the oxidation-reduction balance, that is, increase of polyol pathway flux reducing NADPH reservoirs and/or conversion of oxidized glutathione to glutathione, (3) increase of AGE formation, protein kinase C (PKC), or (4) superoxide overproduction by the mitochondrial electron transport chain [43–45]. It is also known that hyperglycemia induced ROS can cause nuclear factor kappa B (NF-B) translocation to the nucleus [46, 47]. Many of these pathways work to elevate NF-B, which when activated produces many inflammatory cytokines including tumor necrosis factor alpha (TNF-), interleukins 1 beta, 6, and 8 (IL-1, IL-6, IL-8), vascular cell adhesion molecule 1 (VCAM-1), and intercellular adhesion molecule 1 (ICAM-1) [43, 48–50]. Wautier et al. found increased AGE formation on the erythrocytes of human subjects with diabetes, which, when cultured with human umbilical cord endothelial cells, caused an increase in the generation of ROS and activation of NF-B. This increase in ROS was reversed by antioxidants [46, 51, 52]. Such soluble mediators have the potential to activate perivascular microglia . Furthermore, it is known that AGE concentration increases in the retina during diabetes [50, 53]. Wang et al. found that AGE exposure activated microglia, as measured by NF-B translocation, increased ROS production and promoted TNF- mRNA production and release of TNF- from retinal microglia . The increase in ROS can also locally damage vessels. TNF- was found to enhance the expression levels of adhesion molecules such as ICAM-1 and VCAM . Therefore, TNF- production may trigger leukocyte infiltration to retinal vessels with resulting vascular inflammation . The breakdown of the blood-retinal barrier (BRB) is an early development in the progression of DR . This leads to increases in AGE concentration throughout the retina, resulting in vessel and neuronal damage [50, 55]. Increased exposure of inflammatory agents, such as AGE to the microglia, as a result of early BRB breakdown, results in activation of perivascular microglia. This activation leads to a release of microglial products further breaking down the BRB and an increase in retinal chronic inflammation [50, 56, 57]. However, the contribution of hematogenous cells such as neutrophils, T cells, or B cells is still yet to be determined.
As previously mentioned nonenzymatic glycoxidation of proteins, AGE formation, is elevated in oxidative stress including diabetes [46, 58]. AGE formation involves the irreversible glycation of long-lived tissue proteins which can alter their conformations and their physiological functions . The receptor for AGE (RAGE) is a member of the cell surface molecule immunoglobulin superfamily which responds to multiple ligands including AGE, amyloid, amphoterin, and S100/calgranulins. The receptor tends to accumulate where the concentration of its ligand is the highest and contains a short cytosolic tail, which appears to be crucial for cellular activation, and therefore has important functions in signal transduction. Leakiness in bovine aortic endothelial monolayers induced by diabetic red cells and blood brain barrier vascular permeability in streptozotocin-induced (STZ) diabetic rats was reduced by inhibiting the AGE-RAGE interaction using either anti-RAGE antibody or soluble RAGE (sRAGE) which lacks the transmembrane portion [46, 52]. This permeability was also blocked with the antioxidants vitamin E and probucol . Wautier et al. report that AGE-RAGE binding generates ROS through activation of NADPH oxidase . Furthermore, RAGE expression has been found in microglia [61, 62]. Wong et al. report that AGE-RAGE induces microglial activation and oxygen free radicals serve as second messengers in AGE-RAGE inflammatory signal transduction pathways . AGE-RAGE interactions have been linked to tumor angiogenesis. Thus, it is reasonable that RAGE-mediated cellular interactions of chronic inflammation and angiogenesis render tissues susceptible to diabetic complications .
2. Microglia in Human and Animal Models of Diabetic Retinopathy
2.1. Histopathological Evidence of Microglial Involvement in Diabetic Retinopathy
Microglia activation in different forms of DR has been studied in healthy controls and DR . Microglia labeling with antibodies against human microglial markers CD45, CD68, and HLA-DR, a major histocompatibility complex II antigen, was evaluated in individuals with no diabetes (Table 2), background (Table 3), pre-proliferative (Table 4), proliferative DR (Table 5), and diabetic macular edema (DME) . Microglia were generally increased in number (proliferation) and in size and found mainly associated with retinal vasculature, dilated veins, cotton-wool spots, and around microaneurisms in the inner retina. The eyes with proliferative retinopathy had an increase in activated microglia in the ganglion cell layer (GCL) and the neovascular area of the vitreous. In the DME eyes, proliferated microglia were found throughout the retina and subretinal space .
2.2. Morphological and Molecular Studies of Retinal Microglia in Various Animal Models of DR
Zeng et al. using STZ-induced diabetic rat model propose an association between microglia and neuronal cells . After 1 month of diabetes symptoms, microglia appeared hypertrophic and their numbers had increased significantly. Interestingly the number of microglia peaked at 4 to 6 months in the inner plexiform layer (IPL) coinciding with the decrease in the number of neuronal cells. Additionally, microglia were observed in the outer plexiform layer (OPL) at 4 months, but at 14–16 months, activated microglia were observed also in the outer nuclear layer (ONL) and photoreceptor layer (PRL) of the outer retina. The authors suggest that these microglia were attracted to these areas by damage to neuronal cells and photoreceptors, respectively. Initial signals eliciting microglial activation within the inner retina were likely from cell death in the nerve fiber layer (NFL) and inner nuclear layer (INL) . Also the activation, proliferation, and trafficking of microglia in both the inner and outer retina in the Goto-Kakizaki rat model (GK) has been reported . Interestingly, Omri et al. found the development of transcellular pores lined with occludin, caveolin-1 (CAV-1), and protein kinase C zeta (PKC ) in RPE cells which reached a maximum at 6 months of hyperglycemia in streptozotocin (STZ) induced diabetic mice. By 12 months the number of pores is reduced in diabetic mice, but increased non-diabetic controls. Interestingly, this reduction correlated with an increased population of microglia/macrophages in the outer retina and also with microglial migration through a disorganization of the intracellular RPE tight-junctions. Rhodamine-liposomes were injected in the vitreous helping to identify microglial migration through the retina into the choroid. A PKC inhibitor reduced accumulation of microglia and macrophages implicating PKC as a factor in the diabetic retina and suggests that manipulation of microglia activity might be therapeutic in the treatment of diabetic retinopathy. Furthermore, while retinal microglia express MHC class II markers and ramified microglia have many features similar to ocular dendritic antigen-presenting cells (APCs), there remains doubt as to whether they can function as antigen presenting cells [10, 66, 67].
The cooperative function of microglia and hematogenous macrophages was defined more recently in the removal of dead photoreceptors resulting from light damage in susceptible (albino) mice models. Joly et al. exposed mice injected with green fluorescent protein (GFP) positive bone marrow cells or GFP-labeled microglia to focal blue light . Blood borne macrophages entered the retina via the optic nerve and ciliary body and migrated to the damaged area. Resident microglia were activated throughout the retina but assumed the phagocytic phenotype only at the site of photic injury. The macrophages, which had ingested debris, including rhodopsin from photoreceptor outer segments, were observed to leave the retina via the optic nerve head. The authors suggest that the departing macrophages could reach the spleen and raise an immune response against specific retinal proteins.
The role of microglia in initiating chronic neuroinflammation in the diabetic retina has been recently reviewed by Abcouwer . Once activated, microglia can assume any of the several phenotypic states, some inflammatory, others anti-inflammatory. In diabetic retinas, macrophages of hematogenous origin are also present in damaged portions of the retina. Future research should aim to distinguish the relative importance of these two retinal monocytic populations in the chronic inflammatory response to diabetes. Therapeutic approaches to diabetic inflammation and homeostasis could employ signals to modify microglial and/or macrophage function.
3. Molecular Mechanisms Linking Hyperglycemia and Microglial Activation
3.1. Classic Molecular Mechanisms
Cytokines considered markers of inflammation in DR include TNF-, IL-1, IL-6, IL-8, CRP, and chemokine (C-C motif) ligand 2 (CCL2). These cytokines are produced chiefly by activated immune cells, although resident glial cells such as astrocytes have the potential to also secrete a wide array of chemokines [37, 70]. Cyclooxygenase (COX) enzymes whose end-products are prostaglandins, thromboxanes, and leukotrienes have also been associated with DR [37, 71, 72]. In fact, thromboxanes and prostaglandins have been associated with the development of angiogenesis [37, 71, 73].
It is well known that elevated levels of IL-1, IL-6, gamma-interferon (-IFN), and TNF- cytokines activate microglia [56, 74]. Zorena et al. report that higher than normal serum levels of CRP, IL-6, and TNF- have been found in children with type 1 diabetes and NPDR [37, 75]. Several studies have given conflicting reports over the role of CRP in DR [76–82]. Correlations to plasma levels of TNF-, IL-1, IL-8, and CCL2 and vitreous levels of TNF-, IL-1, IL-6, IL-8, and CCL2 have been found for both NPDR and PDR [37, 38, 83–86]. In children and adolescents with type 1 diabetes, serum TNF-, vascular endothelial growth factor (VEGF), AGE concentration, and urinary albumin excretion were all predictive of retinal microvascular disease . Similarly Ben-Mahmud et al. found a correlation between the levels of plasma TNF- and the degree of DR in patients with either type 1 and type 2 diabetes . Diabetic retinopathy is typically treated as a vascular disease, and thus proangiogenic factors such as vascular endothelial growth factor (VEGF) have been shown to be elevated in DR rodent models and in the vitreous of human patients leading to increased leakage through retinal vascular walls [88, 89].
Molecules released by activated retinal microglia include glutamate, proteases, leukotrienes, IL-1, IL-3, IL-6, TNF-, VEGF, lymphotoxin, macrophage inflammatory protein 1 (MIP-1), matrix metalloproteinases (MMPs), and other ROS (Table 6) [56, 57, 92–97]. Glutamate, proteases, leukotrienes, IL-1, IL-6, TNF-, VEGF, lymphotoxin MMPs, and ROS have all been linked to diabetic retinopathy and a role for MIP-1, IL-1, and IL-3 in angiogenesis has been established in ischemic mouse models [56, 85, 98–102]. IL-1 is a trigger of the neuroinflammatory cascade [90, 103]. Wang et al. found increased production of IL-1, TNF-, and nitric oxide (NO) produced into the culture medium by rat retinal microglia that had been stimulated with LPS . Intracellular calcium levels can regulate release of microglia inflammatory mediators such as IL-1, TNF-, and NO . De-Oliveria-Simoes-Pereria et al. found that when rat retinal cells were cultured in high glucose conditions, purinergic P2 receptors in microglia were upregulated, causing a calcium influx and release of additional microglial and associated proinflammatory mediators seen in the early stages of DR . This release of inflammatory mediators and neurotransmitters may also contribute to early retinal neuron death associated with diabetes . TNF- and IL-1 are known to increase caspase 3 activity, inducing endothelial cell apoptosis [40, 105, 106]. Microglia are considered the early primary source of IL-1 in CNS injury, infection, or inflammation. Cardona et al. found that systemic inflammation induces IL-1 as a mediator of microglial neurotoxicity in mice lacking the inhibitory fractalkine receptor [103, 107]. While Liu et al. found lack of IL-1 production from brain microglial cultures in hyperglycemia, Yang et al. found increased retinal production of IL-18, TNF-, and IL-1 in hyperglycemic rats which was reduced with the anti-inflammatory and suppressor of microglia baicalein [42, 90].
Yoshida et al. found activated NF-B in pericytes, vascular endothelial cells, macrophages, and microglia in a C57BL/6N hypoxia-induced mice model of neovascularization . They also propose that NF-b activation is required for retinal angiogenesis and that inhibition of NF-b could be used to ameliorate neuronal cell death in PDR . Inhibition of NF-b by the antioxidant resveratrol, a naturally occurring phenol that increases the action of superoxide dismutase (SOD), resulted in decreased serum levels of IL-1, IL-6, and TNF-. However, the direct correlation between NF-b inhibition and neuronal damage has not been addressed [43, 48].
Microglia in rat retinas become activated soon after the onset of hyperglycemia and some studies indicate that inhibition of microglial activation correlates with neuronal protection in the diabetic retina [2, 6, 56]. Krady et al. found the microglia at this early stage in the inner retina become transformed to an activated amoeboid state, with few or no processes, corresponding to an increase in retinal expression of IL-1 and TNF- mRNA . They found a 6-fold increase in TNF- mRNA and a 4.5 fold increase in IL-1 mRNA. Diabetic rats treated soon after STZ injection with minocycline did not show this increase in IL-1 and TNF- mRNA. In addition, in microglial cell culture the addition of minocycline prevented an increase in the expression of COX-2 mRNA induction by TNF- and caspase 3 activation was blocked with a resulting decrease in retinal neuron death . It is known that minocycline has anti-inflammatory effects separate from its antimicrobial effects . Minocycline is a semisynthetic tetracycline derivative found to cross the blood-brain/retinal barrier and inhibit microglial proliferation and activation by inhibiting the p38 MAPK pathway . Minocycline inhibits matrix MMPs, nitric oxide synthases (NOS), COX-2, phospholipase A2, and (IL-1-) converting enzyme (ICE) mRNA, each of which is thought to be produced by activated microglia .
Microglia are known to produce COX-2 [56, 110]. As mentioned earlier, the use of COX inhibitors such as aspirin in diabetes has had questionable results with the early work of Powell showing promising results [35, 111]. The Dipyridamole Aspirin Microangiography of Diabetes Study Group (DAMAD) in 1989 found a small reduction in the number of microaneursyms (MAs) in an aspirin treated group (990 mg daily), while the Early Treatment of Diabetic Retinopathy Research Group (ETDRS), in 1991, found no significant difference (650 mg daily) . ETDRS found no significant help from aspirin usage in the development of high-risk DR, the risk of vision loss, or the development of vitreous hemorrhage . Kern and Engerman found a significant inhibition in the development of acellular capillaries and retinal hemorrhages in diabetic dogs using 40 mg/kg/day aspirin (considered the maximum dose for dogs), but insignificant effect on the number of MAs or pericyte ghosts . Acellular capillaries lose their pericytes and then their endothelial cells, leaving empty tubes formed by basement membrane before eventually losing perfusion . Talahalli et al. found increased leukotriene production from retinal “glial” cells only when stimulated by precursor exogenous leukotriene from bone marrow derived cells. Whole retina lysates from diabetic mice exhibited significantly more LTA4 hydrolase, which converts leukotriene A (LTA4) to leukotriene B (LTB4), than nondiabetic mice . Therefore the use of nonsteroidal anti-inflammatory drugs (NSAIDS) is questionable for long term management of DR.
The hypothesis that glycated products trigger cytokine release by microglia and initiate a circle of inflammation is supported by the following observation. Using STZ induced diabetic rats, TNF- colocalized to Iba-1+ microglia but not to Muller cells or astrocytes . In vitro cultures showed that TNF- production was induced by glycated albumin and cytokine production and blocked by the ERK and p38MAPK inhibitors . The link between increased TNF- serum levels and activation of the glycosylating enzyme 2GlcNac-T provides evidence of the complex pathways induced by hyperglycemia . The involvement of glycation and enzyme-mediated glycosylation therefore awaits further investigation.
The role of NO in the diabetic retina is still not clear. NO can be generated from L-arginine by catalysis of reactions involving nitric oxide synthase expressed by neurons (nNOS), endothelial (eNOS), and induced on inflammatory cells (iNOS). NO regulates vascular tone and can also react with superoxide to form peroxynitrate which can damage proteins, lipids, and DNA. Increased expression of iNOS has been found in diabetic retinas of human patients and in rodent models . Blocking iNOS with aminoguanidine inhibited AGE formation and decreased hyperglycemia induced microvascular anomalies in dogs, rats, and mice [35, 36, 59, 118, 119]. Diabetic iNOS-deficient mice exhibited decreased capillary degeneration, pericyte ghost, superoxide production, and reduced leukostasis . Increased eNOS expression has also been associated with retinal vascular complication in diabetic mouse models . Therefore, the effects of iNOS and eNOS in the retina may act synergistically to promote chemotaxis and tissue damage and may explain the pathology associated with extravasation in PDR.
3.2. Other Modifiers of Retinal Inflammation
Pigment epithelial-derived factor (PEDF), a protein exhibiting both antiangiogenic and neuroprotective properties, has been shown to effectively modulate diabetic retinal complications such as neurodegeneration, inflammation, and microvascular insult. Furthermore, when topically applied to mice, the PEDF peptide PEDF78-121 (P78) prevented microglia activation and retinal ganglion cell dropout, ultimately leading to vascular stability. Insulin-2 Akita () have been used as a model of type 1 diabetes due to a mutation in the insulin 2 gene that leads to an abnormal protein structure and aggregation leading to death of pancreatic beta cells . It is well established that the mouse model develops significant retinal pathology as shown by quantifiable vision deficits in an optomotor behavior . Several cytokines, IFN-γ, TNF-, IL-3, IL-6, IL-10, and IL-12, were increased in the vitreous of diabetic mice (see Table 6). P78 reduced the quantity of all of these cytokines, while another PEDF peptide, PEDF60-77 (P60), reduced all but IL-2 and TNF- .
Among other mediators the chemokine fractalkine (CX3CL1 or FKN) has been shown to modulate microglial function. FKN exists as a cell membrane-bound or soluble protein . In the CNS it is constitutively produced by neurons while its receptor CX3CR1 is only expressed by microglia [107, 124]. FKN and its receptor play an integral role in the process of both inflammation and neurotoxic responses . Cardona et al. report that CX3CR1 in vivo “governs critical components of the microglial response to systemic inflammation” . Hatori et al. report in cell culture the application of nanomolar concentrations of soluble CX3CL1 to the treating media increases the proliferation of microglia 3 fold . In addition, a deficiency of microglial CX3CR1 induces neurotoxicity and an increase in inflammatory cytokines such as IL-1, IFN-γ, and IL-17 [107, 125]. Diabetic mice with microglial CX3CR1 deficiency exhibit increased numbers of vitreal and subretinal macrophages and also showed severe morphological retinal microglial network abnormalities . While the precise role of CX3CR1 on the retina remains unclear, four allelic variants of this gene have been found in 20–30% of the population. One of these variants, the M280, has been associated with enhanced susceptibility to age-related macular degeneration (AMD) [127–129]. Does this variant also result in higher susceptibility to DR?
3.3. Genetic Variations in Microglial Responses
Gene expression profiling has offered clues as to the potential regulators of the effector responses of microglia. Some of the most interesting potential targets are chemokines and other myeloid specific transmembrane receptors. The team of immunologist and computational biologist of the Immunological Genome Project (ImmGen) performed gene expression and regulatory networks analyses in tissue resident macrophages . They reported a high degree of diversity in gene expression among dendritic cells and macrophages isolated from peritoneum, spleen, lung, and microglia. A few of the transcripts that were upregulated in microglia and linked to diabetes are discussed. Serpinf1 (serpin peptidase inhibitor, clade F) or PEDF is reported to promote neuronal survival and differentiation and to be a potent inhibitor of angiogenesis . Plasma levels of secreted protein acidic and rich in cysteine (SPARC) associated with insulin resistance, dyslipidemia, and inflammation during gestational diabetes mellitus . Since SPARC is a widely expressed profibrotic protein that modulates tissue physiology by altering cell-extracellular matrix interactions, it has the potential to mediate microglia cell proliferation and migration. IL-7r was studied in a Spanish cohort of patients with type 1 diabetes and the homozygous IL-7r allele rs1445898 showed a trend towards a protective effect based on the genotype and an association to early onset diabetes . The adenosine A3 receptor (Adora3) binds adenosine, a phosphohydrolytic derivate of ATP, and mediates microglial migration [134, 135]. Also, A2A adenosine receptor engagement led to TNF- release by retinal microglia, and treatment with A2AAR antagonist resulted in a marked decrease in diabetes-induced retinal cell death [136, 137].
In conclusion, the ImmGen study provides novel information to further our understanding of microglia regulation during retinopathies. Several genes have been associated with susceptibility to eye diseases but how genetic backgrounds affecting microglia effect functions, neuronal damage, and inflammation awaits further investigation.
4. The Effects of Diabetes Treatments on Microglia
The nature of retinal microglia under normal and reactive conditions, and how phenotypic transformations affect microglia function, is not well understood. Recent studies have focused on the effects that current therapies such as laser photocoagulation and growth factor inhibitor injections have on microglia. In this section we discuss recent findings regarding the effects of light induced retinal treatments and the use of anti-VEGF therapy, steroids, and tetracycline inhibitors.
4.1. Light-Induced Retinal Damage and Laser Photocoagulation
It is known that prolonged exposure to bright light induces retinal damage and photoreceptor degeneration [138–140]. However, the effects that light induced photoreceptor degeneration has on microglia cells are not well understood. A study conducted by Santos et al. demonstrated that microglia were activated in the retina of mice with light induced photoreceptor degeneration. Microglia were shown to change from a ramified to an amoeboid morphology and to migrate from inner retinal layers to the outer nuclear layer. Additionally, the activated retinal microglia expressed CD11b, CD45, F4/80, and SRA consistent with immunophenotypic activation . Additionally, increases in signal peptides such as CCL2, MIP-1, and TNF- have been noted in a study of light-induced photodegeneration. These signals or their analogs induce apoptosis of photoreceptor cells in the ONL [26, 142]. The dynamic activation of microglia in response to light induced retinal degeneration suggests a damaging inflammatory response at the photoreceptor cell layer. However, microglia can also be neuroprotective thus the nature and function of these cells after light induced retinal damage remain unclear.
Laser retinal photocoagulation is routinely used in the clinical setting for treatment of eye diseases such as proliferative diabetic retinopathy. Laser retinal treatment delivers varied degrees of coagulation through the retinal thickness; these vary from mild, moderate, or heavy depending on the retinal layers involved. The objective is to use light energy to cauterize blood vessels, coagulate tissue to reduce oxygen consumption, and prevent the formation of new vasculature and its effects, such as retinal detachment. However, the effect of retinal photocoagulation on microglia activation has not been determined. A study using ex vivo live retinal imaging in conjunction with laser treatments describes microglial behavior in response to focal retinal injury. In this study, 50–100 μm photocoagulation injuries were induced using an argon laser platform in mice retina explants. Using time-lapse confocal microscopy, microglia were observed to change from a ramified, symmetrical, and arbor-shaped cell to a polarized morphology. The microglia morphology was observed to become less branched and extended fewer but longer processes toward the site of injury; these microglia were also reported to acquire migratory drive toward the injury. Additionally, other activated microglia were reported to gain an amoeboid morphology and migrate toward the laser induced retinal injury, and some microglia were observed to maintain their normal ramified arbor morphology . The observations in this study suggest that retinal microglia may be in multiple states of activity and may transform from being at rest to a dynamic active state after focal laser treatment.
In a report by Song et al., the activation of microglia and the intracellular pathways involved in activation were studied in response to low-level laser therapy (He-Ne 632.8 nm, 64 mw/cm2). Using a tissue culture system, the investigators showed that low-level laser therapy could activate Src and may lead to microglial phagocytic activity. Additionally, it is suggested that activation of Src kinases may downregulate the expression of proinflammatory cytokines and NO. In this experiment, low-level laser therapy was delivered to cultured SH-SY5Y neuroblastoma cells. Thus it is possible, as the author mentions, that the microglia in the culture system may have responded to malignancy markers, in addition to the laser treatment . Thus, to determine the effects of low-level laser therapy in a more physiological system, future studies could benefit from the use of primary neuronal or microglial cultures and in vivo photocoagulation models of variable degrees.
4.2. Anti-VEGF Injections
It is well documented that blocking VEGF has inhibitory effects on neovascularization. However, the effects that blocking VEGF receptors have on retinal microglia are not well understood; recently, studies have begun to elucidate information regarding the control of microglia infiltration by targeting VEGF receptors. Huang et al. demonstrated that blocking of the chemoattractant receptors VEGFR1 and VEGFR2 reduced microglia/macrophage infiltration in laser-induced choroidal neovascularization (CNV) . This report shows that VEGFR1 and its endogenous ligands were expressed early in the CNV development process, while VEGFR2 expression appeared in late stages. Additionally, blocking of VEGFR1 inhibited infiltration of activated microglia at early and late stages of CNV, while VEGFR2 was also reported to inhibit microglia infiltration but only at the late stage of CNV development . This study suggests a potential use for VEGF receptor blockers and their inhibition of the microglia inflammatory response as a therapeutic approach.
4.3. Steroid Therapy
Corticosteroids have potent anti-inflammatory effects and have a long history of use in the eye. Given the apparent role of inflammation in the pathogenesis of DR and DME, intravitreal steroids have been utilized for the treatment of DME. The mode of action is unclear, but it has been suggested to be through multiple mechanisms including their ability to inhibit expression of VEGF [146, 147]. The effects of steroids on microglia under DR and DME conditions remain unknown; however, other ocular disease models are starting to elucidate the effects of intravitreal corticosteroid delivery on microglia.
For example, in a study focused on N-methyl D-aspartate (NMDA) induced glaucoma, Singhal et al. observed a reduction in the number of microglia induced by the loss of the retina ganglion cells when triamcinolone acetonide (TA) was used along with the NMDA. Then Muller cells from the Muller cell line MIO-M1 were transplanted into the eyes of the glaucoma model. A reduction of transplant induced activated microglia was observed in eyes that had been treated with TA . In a separate study, Shen et al. delivered TA by intraocular injection to a transgenic model which mimics the type of damage that might occur in AMD or DR. In this model the results of Muller cell ablation are retina degeneration with photoreceptor loss, vascular leaks, and retinal neovascularization. This study reported that TA prevented photoreceptor degeneration and inhibited activation of microglial and Muller cells. Additionally, TA attenuated Muller cell loss and inhibited overexpression of p75 neurotrophin receptor (NTR), TNF-, and proneurotrophin (NT) and the activation of p53 and p38/stress-activated protein kinase (SAPK) signaling pathways. It is unclear whether TA inhibited these reactions from microglia, Muller cells, or both . Treatment with TA also prevented the development of retinal vascular lesions and inhibited fluorescein leakage from established vascular lesions. Furthermore, in a study using retina degeneration model, S334ter-line-3, the corticosteroid fluocinolone acetonide (FA) was implanted and delivered to the vitreous. Electrophysiology results from this study show that the ERG a and ERG b waves are preserved in experimental eyes over nontreated controls. The thickness of the ONL was also reported to be between 22% and 25% higher in the eyes that were treated with the corticosteroid. Finally, changes in microglia cells were examined on retinal whole mounts. Activated microglia were found in the ILM and the photoreceptor cell layer. The activated microglia count was reported to be 4 times lower in the FA treated eyes than the control samples . Thus the effects of corticosteroids on microglia seem to be of those of an anti-inflammatory agent preventing microglia activation, attenuation of proinflammatory mediator expression, and modulation of immune signals. Cortisol is known to reduce the activated microglial expression of iNOS mRNA as well as reducing the amount of iNOS, NO, COX-2, and TNF-  Additionally, corticosteroids have a protective effect, which may be the result of their ability to attenuate overexpression of VEGF and transcription factors that regulate proapototic genes.
4.4. Inhibition of Microglia as a Therapeutic Therapy
As mentioned previously, the tetracycline derivatives minocycline and doxycycline have been linked to inhibition of inflammation and prevention of microglial activation in the retina [56, 151, 152]. Recent clinical trials have investigated the inhibition of microglia as a method to inhibit DR. Cukras et al. performed a prospective, nonrandomized, and uncontrolled single center pilot study to evaluate the use of oral minocycline on 5 human subjects with DME that had previously had focal laser photocoagulation, but in whom DME had persisted . At six months they found a modest increase in best-corrected visual acuity (BCVA), a 5–10% reduction in central subfield retinal thickness (CST) on optical coherence tomography (OCT), and a decrease in the area of late leakage on fluorescein angiography (FA) . The authors note that this improvement, though small, compared favorably with the control arm of the Ranibizumab in Diabetic Macular Edema (RESOLVE) study at 6 months [153, 154]. Recently, Scott et al. performed a randomized, double-masked, 24-month clinical trial using doxycycline monohydrate orally on 30 patients who had at least one eye with severe NPDR or PDR eyes rated as ETDRS less than high risk [113, 155]. While they found no significant difference between those taking the doxycycline and the control group on white/white visual field testing, contrast sensitivity, visual acuity, or anatomic factors, foveal frequency doubling perimetry (FDT) was improved at 6, 12, and 24 months after treatment . Foveal FDT has been determined to be an accurate measure of inner retinal function and has high diagnostic test sensitivity for subjects with NPDR and PDR . However, another randomized, double-masked, 24-month clinical trial using doxycycline monohydrate in 33 patients with mild to moderate NPDR reported no significant difference between control and treatment groups using the previously mentioned test including foveal FDT [113, 157]. Studies with an increased number of patients may clarify in the future the broad application of these treatments. Therefore, there is an urgent need to find additional therapeutic alternatives for the management of DR.
5. Conclusions and Perspectives
Many have proposed that chronic inflammation, including cytokine and chemokine release, cell death, increased vascular permeability, neovascularization, and repair attempts play a role in DR, not only in early changes, but also during the late proliferative stage. It is now established that the loss of retinal ganglion cells occurs early in the DR process. Microglia have been linked to neuronal loss in several neurodegenerative diseases by a mechanism of chronic inflammation. Therefore, it is probable that disregulated microglial activation also functions in the loss of these retinal cells. We propose that some of the first signals of hyperglycemia are picked up by the perivascular microglia from capillary blood or the inner retinal parenchymal microglia, which are exposed from the vitreous. This may be either from hyperglycemia itself or from a systemic deluge of inflammatory cytokines, AGE, or ROS which have been found to increase in the capillary blood and vitreous (Figure 1). The effects of VEGF may elevate exposure to inflammatory mediators in the retinal capillaries. This leakage leads to microglial activation resulting in production of glutamate, IL-1, TNF-, MMPs, and NOS. IL-1 and TNF- lead to production of caspase 3, which along with glutamate, results in neurotoxicity of retinal ganglion cells. Caspase 3 also can be toxic to retinal capillary endothelial cells and pericytes. TNF- leads to production of ICAM-1 and VCAM, leading to increased leakage of macrophages through capillary walls, sustaining chronic inflammation. COX-2 is also a product of IL-1 and TNF- stimulation. MMP remodeling of capillary basement membranes can further play a role in vessel destruction. Activation of NF-B as well as its release of inflammatory cytokines is linked to both early and late stages of diabetic retinopathy. Stages of the inflammatory pathway mediated through ROS, AGE-RAGE, NF-B blockers, or genetic manipulation of fractalkine could be targeted to reduce chronic activation and retinal translocation of microglia in the diabetic retina.
It has also become obvious that people react differently to hyperglycemia. Some develop extremely morbid cases of DR and some seem to go unscathed. It is well established that control of systemic factors such as blood glucose level and blood pressure plays significant roles, but genetic factors likely alter the variability of DR morbidity when systemic factors seem to be similar. As pointed out in this paper, certain genetic variations may regulate the microglial responses to diabetes. Certainly, other yet unknown genetic factors are likely playing a role in this degenerative process.
Microglial responses to treatment of DR including anti-VEGF therapies, panretinal photocoagulation, steroids, and tetracycline derivatives are not yet well investigated but clearly may play a role in how patients respond to these treatments. Studies have focused on microglia behavior under light-induced retinal damage and laser treatments and the role that anti-VEGF treatments have on retinal microglia. Although microglia are activated and migrate toward the site of insult, the effects of the morphological and phenotypic changes of activated microglia on the damaged and surrounding tissue remain unknown. Additionally, to appreciate the effects of these treatments on retinal microglia under disease conditions, the presence of activated microglia due to the initial insult and prior to treatments should be considered in future studies. Available studies performed to determine the effect of anti-VEGF treatments have shown a decrease in activated microglia due to treatment. However, these studies have been performed in the absence of diabetic insult. Retinal photocoagulation has been demonstrated to induce activation of microglia, but variable phenotypes have been observed. The roles of the different phenotypes of activated microglia observed after retinal photocoagulation have yet to be determined. Additionally, it is good to remember that the functions of microglia do not happen in isolation but likely influence and are influenced by neighboring cells such as astrocytes, neurons, and the blood-retina barrier. Investigators must determine how DR treatments affect the actions of microglia and in turn how the actions of microglia can be modified to enhance the benefits of treatments.
Conflict of Interests
The authors declare that there is no conflict of interests regarding the publication of this paper.
The authors would like to thank Pamela Forbes, Teresa Estrada, and Suzanne Ferimer for their research assistance, Brandi Betts for organizational help, and Dr. Mark Tso for reviewing and offering suggestions on the paper. This work was supported in parts by Ashbel Smith Professorship (DMA), UTSA MBRS-RISE PhD research training program (2R25GM060655-15 to ASM), The San Antonio Area Foundation (Grant 201135345 to AEC), and National Institutes of Health (Grants G12MD007591 to AT and SC1GM095426 to AEC).
G. H. Bresnick, “Diabetic retinopathy viewed as a neurosensory disorder,” Archives of ophthalmology, vol. 104, no. 7, pp. 989–990, 1986.View at: Publisher Site | Google Scholar
E. Rungger-Brändle, A. A. Dosso, and P. M. Leuenberger, “Glial reactivity, an early feature of diabetic retinopathy,” Investigative Ophthalmology and Visual Science, vol. 41, no. 7, pp. 1971–1980, 2000.View at: Google Scholar
H. P. Hammes, H. J. Federoff, and M. Brownlee, “Nerve growth factor prevents both neuroretinal programmed cell death and capillary pathology in experimental diabetes,” Molecular Medicine, vol. 1, no. 5, pp. 527–534, 1995.View at: Google Scholar
A. J. Barber, E. Lieth, S. A. Khin, D. A. Antonetti, A. G. Buchanan, and T. W. Gardner, “Neural apoptosis in the retina during experimental and human diabetes: early onset and effect of insulin,” Journal of Clinical Investigation, vol. 102, no. 4, pp. 783–791, 1998.View at: Publisher Site | Google Scholar
E. Lieth, T. W. Gardner, A. J. Barber, and D. A. Antonetti, “Retinal neurodegeneration: early pathology in diabetes,” Clinical & Experimental Ophthalmology, vol. 28, no. 1, pp. 3–8, 2000.View at: Publisher Site | Google Scholar
X. Zeng, Y. Ng, and E. Ling, “Neuronal and microglial response in the retina of streptozotocin-induced diabetic rats,” Visual Neuroscience, vol. 17, no. 3, pp. 463–471, 2000.View at: Publisher Site | Google Scholar
M. A. S. di Leo, S. Caputo, B. Falsini et al., “Nonselective loss of contrast sensitivity in visual system testing in early type I diabetes,” Diabetes Care, vol. 15, no. 5, pp. 620–625, 1992.View at: Publisher Site | Google Scholar
A. A. Dosso, E. R. Bonvin, Y. Morel, A. Golay, J. P. Assal, and P. M. Leuenberger, “Risk factors associated with contrast sensitivity loss in diabetic patients,” Graefe's Archive for Clinical and Experimental Ophthalmology, vol. 234, no. 5, pp. 300–305, 1996.View at: Publisher Site | Google Scholar
A. Harris, O. Arend, R. P. Danis, D. Evans, S. Wolf, and B. J. Martin, “Hyperoxia improves contrast sensitivity in early diabetic retinopathy,” British Journal of Ophthalmology, vol. 80, no. 3, pp. 209–213, 1996.View at: Publisher Site | Google Scholar
L. Chen, P. Yang, and A. Kijlstra, “Distribution, markers, and functions of retinal microglia,” Ocular Immunology and Inflammation, vol. 10, no. 1, pp. 27–39, 2002.View at: Publisher Site | Google Scholar
A. M. Santos, R. Calvente, M. Tassi et al., “Embryonic and postnatal development of microglial cells in the mouse retina,” The Journal of Comparative Neurology, vol. 506, no. 2, pp. 224–239, 2008.View at: Publisher Site | Google Scholar
M. A. Cuadros and J. Navascués, “The origin and differentiation of microglial cells during development,” Progress in Neurobiology, vol. 56, no. 2, pp. 173–189, 1998.View at: Publisher Site | Google Scholar
P. E. Tan, P. K. Yu, C. Balaratnasingam et al., “Quantitative confocal imaging of the retinal microvasculature in the human retina,” Investigative Ophthalmology and Visual Science, vol. 53, no. 9, pp. 5728–5736, 2012.View at: Publisher Site | Google Scholar
D. Davalos, J. Grutzendler, G. Yang et al., “ATP mediates rapid microglial response to local brain injury in vivo,” Nature Neuroscience, vol. 8, no. 6, pp. 752–758, 2005.View at: Publisher Site | Google Scholar
A. Nimmerjahn, F. Kirchhoff, and F. Helmchen, “Neuroscience: Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo,” Science, vol. 308, no. 5726, pp. 1314–1318, 2005.View at: Publisher Site | Google Scholar
U. K. Hanisch and H. Kettenmann, “Microglia: active sensor and versatile effector cells in the normal and pathologic brain,” Nature Neuroscience, vol. 10, no. 11, pp. 1387–1394, 2007.View at: Publisher Site | Google Scholar
C. Giaume, F. Kirchhoff, C. Matute, A. Reichenbach, and A. Verkhratsky, “Glia: the fulcrum of brain diseases,” Cell Death and Differentiation, vol. 14, no. 7, pp. 1324–1335, 2007.View at: Publisher Site | Google Scholar
V. Hughes, “Microglia: The constant gardeners,” Nature, vol. 485, no. 7400, pp. 570–572, 2012.View at: Publisher Site | Google Scholar
G. W. Kreutzberg, “Microglia: a sensor for pathological events in the CNS,” Trends in Neurosciences, vol. 19, no. 8, pp. 312–318, 1996.View at: Publisher Site | Google Scholar
B. R. Tambuyzer, P. Ponsaerts, and E. J. Nouwen, “Microglia: gatekeepers of central nervous system immunology,” Journal of Leukocyte Biology, vol. 85, no. 3, pp. 352–370, 2009.View at: Publisher Site | Google Scholar
G. Raivich, M. Bohatschek, C. U. A. Kloss, A. Werner, L. L. Jones, and G. W. Kreutzberg, “Neuroglial activation repertoire in the injured brain: graded response, molecular mechanisms and cues to physiological function,” Brain Research Reviews, vol. 30, no. 1, pp. 77–105, 1999.View at: Publisher Site | Google Scholar
A. Y. Lai and K. G. Todd, “Differential regulation of trophic and proinflammatory microglial effectors is dependent on severity of neuronal injury,” Glia, vol. 56, no. 3, pp. 259–270, 2008.View at: Publisher Site | Google Scholar
E. Polazzi and A. Contestabile, “Reciprocal interactions between microglia and neurons: from survival to neuropathology,” Reviews in the Neurosciences, vol. 13, no. 3, pp. 221–242, 2002.View at: Google Scholar
R. S. Roque, C. J. Imperial, and R. B. Caldwell, “Microglial cells invade the outer retina as photoreceptors degenerate in Royal College of Surgeons rats,” Investigative Ophthalmology and Visual Science, vol. 37, no. 1, pp. 196–203, 1996.View at: Google Scholar
E. H. Hughes, F. C. Schlichtenbrede, C. C. Murphy et al., “Generation of activated sialoadhesin-positive microglia during retinal degeneration,” Investigative Ophthalmology & Visual Science, vol. 44, no. 5, pp. 2229–2234, 2003.View at: Publisher Site | Google Scholar
H. Zeng, X. Zhu, C. Zhang, L. Yang, L. Wu, and M. O. M. Tso, “Identification of sequential events and factors associated with microglial activation, migration, and cytotoxicity in retinal degeneration in rd mice,” Investigative Ophthalmology and Visual Science, vol. 46, no. 8, pp. 2992–2999, 2005.View at: Publisher Site | Google Scholar
T. F. Ng and J. W. Streilein, “Light-induced migration of retinal microglia into the subretinal space,” Investigative Ophthalmology and Visual Science, vol. 42, no. 13, pp. 3301–3310, 2001.View at: Google Scholar
T. Harada, C. Harada, S. Kohsaka et al., “Microglia-Müller glia cell interactions control neurotrophic factor production during light-induced retinal degeneration,” The Journal of Neuroscience, vol. 22, no. 21, pp. 9228–9236, 2002.View at: Google Scholar
J. M. Provis, C. M. Diaz, and P. L. Penfold, “Microglia in human retina: a heterogeneous population with distinct ontogenies,” Perspectives on Developmental Neurobiology, vol. 3, no. 3, pp. 213–222, 1996.View at: Google Scholar
E. Schuetz and S. Thanos, “Microglia-targeted pharmacotherapy in retinal neurodegenerative diseases,” Current Drug Targets, vol. 5, no. 7, pp. 619–627, 2004.View at: Publisher Site | Google Scholar
J. Boya, J. Calvo, and A. Prado, “The origin of microglial cells,” Journal of Anatomy, vol. 129, no. 1, pp. 177–186, 1979.View at: Google Scholar
A. Sierra, A. C. Gottfried-Blackmore, B. S. Mcewen, and K. Bulloch, “Microglia derived from aging mice exhibit an altered inflammatory profile,” GLIA, vol. 55, no. 4, pp. 412–424, 2007.View at: Publisher Site | Google Scholar
U. K. Hanisch, “Microglia as a source and target of cytokine activities in the brain,” in Microglia in the Regenerating and Degenerating Central Nervous System, W. J. Streit, Ed., Springer, New York, NY, USA, 2010.View at: Google Scholar
D. van Rossum and U. K. Hanisch, “Microglia,” Metabolic Brain Disease, vol. 19, no. 3-4, pp. 393–411, 2004.View at: Publisher Site | Google Scholar
E. U. Powell and R. Field, “Diabetic retinopathy and rheumatoid arthritis,” The Lancet, vol. 284, no. 7349, pp. 17–18, 1964.View at: Publisher Site | Google Scholar
T. S. Kern, “Contributions of inflammatory processes to the development of the early stages of diabetic retinopathy,” Experimental Diabetes Research, vol. 2007, Article ID 95103, 2007.View at: Publisher Site | Google Scholar
K. Kaul, A. Hodgkinson, J. M. Tarr, E. M. Kohner, and R. Chibber, “Is inflammation a common retinal-renal-nerve pathogenic link in diabetes?” Current diabetes reviews, vol. 6, no. 5, pp. 294–303, 2010.View at: Publisher Site | Google Scholar
S. Kojima, T. Yamada, and M. Tamai, “Quantitative analysis of interleukin-6 in vitreous from patients with proliferative vitreoretinal diseases,” Japanese Journal of Ophthalmology, vol. 45, no. 1, pp. 40–45, 2001.View at: Publisher Site | Google Scholar
J. Tang and T. S. Kern, “Inflammation in diabetic retinopathy,” Progress in Retinal and Eye Research, vol. 30, no. 5, pp. 343–358, 2011.View at: Publisher Site | Google Scholar
W. Zhang, H. Liu, M. Al-Shabrawey, R. Caldwell, and R. Caldwell, “Inflammation and diabetic retinal microvascular complications,” Journal of Cardiovascular Disease Research, vol. 2, no. 2, pp. 96–103, 2011.View at: Publisher Site | Google Scholar
F. González-Scarano and G. Baltuch, “Microglia as mediators of inflammatory and degenerative diseases,” Annual Review of Neuroscience, vol. 22, pp. 219–240, 1999.View at: Publisher Site | Google Scholar
L. Yang, H. Sun, L. Wu et al., “Baicalein reduces inflammatory process in a rodent model of diabetic retinopathy,” Investigative Ophthalmology and Visual Science, vol. 50, no. 5, pp. 2319–2327, 2009.View at: Publisher Site | Google Scholar
F. G. Soufi, M. Vardyani, R. Sheervalilou, M. Mohammadi, and M. H. Somi, “Long-term treatment with resveratrol attenuates oxidative stress pro-inflammatory mediators and apoptosis in streptozotocin-nicotinamide-induced diabetic rats,” General Physiology and Biophysics, vol. 31, no. 4, pp. 431–438, 2012.View at: Publisher Site | Google Scholar
J. L. Rains and S. K. Jain, “Oxidative stress, insulin signaling, and diabetes,” Free Radical Biology and Medicine, vol. 50, no. 5, pp. 567–575, 2011.View at: Publisher Site | Google Scholar
X. L. Du, D. Edelstein, L. Rossetti et al., “Hyperglycemia-induced mitochondrial superoxide overproduction activates the hexosamine pathway and induces plasminogen activator inhibitor-1 expression by increasing Sp1 glycosylation,” Proceedings of the National Academy of Sciences of the United States of America, vol. 97, no. 22, pp. 12222–12226, 2000.View at: Publisher Site | Google Scholar
D. M. Stern, S. D. Yan, S. F. Yan, and A. M. Schmidt, “Receptor for advanced glycation endproducts (RAGE) and the complications of diabetes,” Ageing Research Reviews, vol. 1, no. 1, pp. 1–15, 2002.View at: Publisher Site | Google Scholar
T. Nishikawa, D. Edelstein, X. L. Du et al., “Normalizing mitochondrial superoxide production blocks three pathways of hyperglycaemic damage,” Nature, vol. 404, no. 6779, pp. 787–790, 2000.View at: Publisher Site | Google Scholar
P. Palsamy and S. Subramanian, “Ameliorative potential of resveratrol on proinflammatory cytokines, hyperglycemia mediated oxidative stress, and pancreatic β-cell dysfunction in streptozotocin-nicotinamide-induced diabetic rats,” Journal of Cellular Physiology, vol. 224, no. 2, pp. 423–432, 2010.View at: Publisher Site | Google Scholar
A. Yoshida, S. Yoshida, T. Ishibashi, M. Kuwano, and H. Inomata, “Suppression of retinal neovascularization by the NF-κB inhibitor pyrrolidine dithiocarbamate in mice,” Investigative Ophthalmology and Visual Science, vol. 40, no. 7, pp. 1624–1629, 1999.View at: Google Scholar
A. L. Wang, A. C. H. Yu, Q. H. He, X. Zhu, and M. O. M. Tso, “AGEs mediated expression and secretion of TNF alpha in rat retinal microglia,” Experimental Eye Research, vol. 84, no. 5, pp. 905–913, 2007.View at: Publisher Site | Google Scholar
J. L. Wautier, M. P. Wautier, A. M. Schmidt et al., “Advanced glycation end products (AGEs) on the surface of diabetic erythrocytes bind to the vessel wall via a specific receptor inducing oxidant stress in the vasculature: a link between surface-associated AGEs and diabetic complications,” Proceedings of the National Academy of Sciences of the United States of America, vol. 91, no. 16, pp. 7742–7746, 1994.View at: Publisher Site | Google Scholar
J. L. Wautier, C. Zoukourian, O. Chappey et al., “Receptor-mediated endothelial cell dysfunction in diabetic vasculopathy: soluble receptor for advanced glycation end products blocks hyperpermeability in diabetic rats,” Journal of Clinical Investigation, vol. 97, no. 1, pp. 238–243, 1996.View at: Publisher Site | Google Scholar
J. L. Wautier and P. J. Guillausseau, “Advanced glycation end products, their receptors and diabetic angiopathy,” Diabetes and Metabolism, vol. 27, no. 5 I, pp. 535–542, 2001.View at: Google Scholar
G. A. Limb, A. H. Chignell, W. Green, F. LeRoy, and D. C. Dumonde, “Distribution of TNFα and its reactive vascular adhesion molecules in fibrovascular membranes of proliferative diabetic retinopathy,” British Journal of Ophthalmology, vol. 80, no. 2, pp. 168–173, 1996.View at: Publisher Site | Google Scholar
Q. Xu, T. Qaum, and A. P. Adamis, “Sensitive blood-retinal barrier breakdown quantitation using Evans blue,” Investigative Ophthalmology and Visual Science, vol. 42, no. 3, pp. 789–794, 2001.View at: Google Scholar
J. K. Krady, A. Basu, C. M. Allen et al., “Minocycline reduces proinflammatory cytokine expression, microglial activation, and caspase-3 activation in a rodent model of diabetic retinopathy,” Diabetes, vol. 54, no. 5, pp. 1559–1565, 2005.View at: Publisher Site | Google Scholar
A. L. Wang, A. C. Yu, L. T. Lau et al., “Minocycline inhibits LPS-induced retinal microglia activation,” Neurochemistry International, vol. 47, no. 1-2, pp. 152–158, 2005.View at: Publisher Site | Google Scholar
J. W. Baynes and S. R. Thorpe, “Role of oxidative stress in diabetic complications: a new perspective on an old paradigm,” Diabetes, vol. 48, no. 1, pp. 1–9, 1999.View at: Publisher Site | Google Scholar
B. J. Davis, J. M. Forbes, M. C. Thomas et al., “Superior renoprotective effects of combination therapy with ACE and AGE inhibition in the diabetic spontaneously hypertensive rat,” Diabetologia, vol. 47, no. 1, pp. 89–97, 2004.View at: Publisher Site | Google Scholar
M. Wautier, O. Chappey, S. Corda, D. M. Stern, A. M. Schmidt, and J. Wautier, “Activation of NADPH oxidase by AGE links oxidant stress to altered gene expression via RAGE,” The American Journal of Physiology—Endocrinology and Metabolism, vol. 280, no. 5, pp. E685–E694, 2001.View at: Google Scholar
L. F. Lue, D. G. Walker, L. Brachova et al., “Involvement of microglial receptor for advanced glycation endproducts (RAGE)in Alzheimer's disease: identification of a cellular activation mechanism,” Experimental Neurology, vol. 171, no. 1, pp. 29–45, 2001.View at: Publisher Site | Google Scholar
S. Dukic-Stefanovic, J. Gasic-Milenkovic, W. Deuther-Conrad, and G. Münch, “Signal transduction pathways in mouse microglia N-11 cells activated by advanced glycation endproducts (AGEs),” Journal of Neurochemistry, vol. 87, no. 1, pp. 44–55, 2003.View at: Publisher Site | Google Scholar
A. Wong, S. Dukic-Stefanovic, J. Gasic-Milenkovic et al., “Anti-inflammatory antioxidants attenuate the expression of inducible nitric oxide synthase mediated by advanced glycation endproducts in murine microglia,” European Journal of Neuroscience, vol. 14, no. 12, pp. 1961–1967, 2001.View at: Publisher Site | Google Scholar
H. Y. Zeng, W. R. Green, and M. O. M. Tso, “Microglial activation in human diabetic retinopathy,” Archives of Ophthalmology, vol. 126, no. 2, pp. 227–232, 2008.View at: Publisher Site | Google Scholar
S. Omri, F. Behar-Cohen, Y. De Kozak et al., “Microglia/macrophages migrate through retinal epithelium barrier by a transcellular route in diabetic retinopathy: role of PKC in the Goto Kakizaki rat model,” The American Journal of Pathology, vol. 179, no. 2, pp. 942–953, 2011.View at: Publisher Site | Google Scholar
J. Gehrmann, R. B. Banati, and G. W. Kreutzberg, “Microglia in the immune surveillance of the brain: human microglia constitutively express HLA-DR molecules,” Journal of Neuroimmunology, vol. 48, no. 2, pp. 189–198, 1993.View at: Publisher Site | Google Scholar
P. L. Penfold, J. M. Provis, and S. C. K. Liew, “Human retinal microglia express phenotypic characteristics in common with dendritic antigen-presenting cells,” Journal of Neuroimmunology, vol. 45, no. 1-2, pp. 183–192, 1993.View at: Publisher Site | Google Scholar
S. Joly, M. Francke, E. Ulbricht et al., “Resident microglia and bone marrow immigrants remove dead photoreceptors in retinal lesions,” The American Journal of Pathology, vol. 174, no. 6, pp. 2310–2323, 2009.View at: Publisher Site | Google Scholar
S. F. Abcouwer, “Angiogenic factors and cytokines in diabetic retinopathy,” Journal of Clinical and Cellular Immunology, supplement 1, no. 11, pp. 1–12, 2013.View at: Publisher Site | Google Scholar
W. Panenka, H. Jijon, L. M. Herx et al., “P2X7-like receptor activation in astrocytes increases chemokine monocyte chemoattractant protein-1 expression via mitogen-activated protein kinase,” Journal of Neuroscience, vol. 21, no. 18, pp. 7135–7142, 2001.View at: Google Scholar
A. M. A. El-Asrar, “Role of inflammation in the pathogenesis of diabetic retinopathy,” Middle East African Journal of Ophthalmology, vol. 19, no. 1, pp. 70–74, 2012.View at: Publisher Site | Google Scholar
A. M. Abu El-Asrar, L. Missotten, and K. Geboes, “Expression of cyclo-oxygenase-2 and downstream enzymes in diabetic fibrovascular epiretinal membranes,” British Journal of Ophthalmology, vol. 92, no. 11, pp. 1534–1539, 2008.View at: Publisher Site | Google Scholar
T. Kuwano, S. Nakao, H. Yamamoto et al., “Cyclooxygenase 2 is a key enzyme for inflammatory cytokine-induced angiogenesis,” The FASEB Journal, vol. 18, no. 2, pp. 300–310, 2004.View at: Publisher Site | Google Scholar
G. I. Liou, “Diabetic retinopathy: role of inflammation and potential therapies for anti-inflammation,” World Journal of Diabetes, vol. 1, no. 1, pp. 12–18, 2010.View at: Publisher Site | Google Scholar
K. Zorena, J. Myśliwska, M. Myśliwiec et al., “Serum TNF-α level predicts nonproliferative diabetic retinopathy in children,” Mediators of Inflammation, vol. 2007, Article ID 92196, 5 pages, 2007.View at: Publisher Site | Google Scholar
M. V. van Hecke, J. M. Dekker, G. Nijpels et al., “Inflammation and endothelial dysfunction are associated with retinopathy: the Hoorn Study,” Diabetologia, vol. 48, no. 7, pp. 1300–1306, 2005.View at: Publisher Site | Google Scholar
A. M. W. Spijkerman, M.-A. Gall, L. Tarnow et al., “Endothelial dysfunction and low-grade inflammation and the progression of retinopathy in Type 2 diabetes,” Diabetic Medicine, vol. 24, no. 9, pp. 969–976, 2007.View at: Publisher Site | Google Scholar
D. S. N. T. Le, R. Miles, P. J. Savage et al., “The association of plasma fibrinogen concentration with diabetic microvascular complications in young adults with early-onset of type 2 diabetes,” Diabetes Research and Clinical Practice, vol. 82, no. 3, pp. 317–323, 2008.View at: Publisher Site | Google Scholar
T. T. Nguyen, E. Alibrahim, F. M. A. Islam et al., “Inflammatory, hemostatic, and other novel biomarkers for diabetic retinopathy: The multi-ethnic study of atherosclerosis,” Diabetes Care, vol. 32, no. 9, pp. 1704–1709, 2009.View at: Publisher Site | Google Scholar
B. E. K. Klein, M. D. Knudtson, M. Y. Tsai, and R. Klein, “The relation of markers of inflammation and endothelial dysfunction to the prevalence and progression of diabetic retinopathy: wisconsin epidemiologic study of diabetic retinopathy,” Archives of Ophthalmology, vol. 127, no. 9, pp. 1175–1182, 2009.View at: Publisher Site | Google Scholar
M. T. Schram, N. Chaturvedi, C. G. Schalkwijk, J. H. Fuller, and C. D. A. Stehouwer, “Markers of inflammation are cross-sectionally associated with microvascular complications and cardiovascular disease in type 1 diabetes—the EURODIAB prospective complications study,” Diabetologia, vol. 48, no. 2, pp. 370–378, 2005.View at: Publisher Site | Google Scholar
L. S. Lim, E. Shyong Tai, P. Mitchell et al., “C-reactive protein, body mass index, and diabetic retinopathy,” Investigative Ophthalmology and Visual Science, vol. 51, no. 9, pp. 4458–4463, 2010.View at: Publisher Site | Google Scholar
T. Yuuki, T. Kanda, Y. Kimura et al., “Inflammatory cytokines in vitreous fluid and serum of patients with diabetic vitreoretinopathy,” Journal of Diabetes and its Complications, vol. 15, no. 5, pp. 257–259, 2001.View at: Publisher Site | Google Scholar
G. A. Limb, L. Webster, H. Soomro, S. Janikoun, and J. Shilling, “Platelet expression of tumour necrosis factor-alpha (TNF-α), TNF receptors and intercellular adhesion molecule-1 (ICAM-1) in patients with proliferative diabetic retinopathy,” Clinical and Experimental Immunology, vol. 118, no. 2, pp. 213–218, 1999.View at: Publisher Site | Google Scholar
N. Demircan, B. G. Safran, M. Soylu, A. A. Ozcan, and S. Sizmaz, “Determination of vitreous interleukin-1 (IL-1) and tumour necrosis factor (TNF) levels in proliferative diabetic retinopathy,” Eye, vol. 20, no. 12, pp. 1366–1369, 2006.View at: Publisher Site | Google Scholar
E. Cicik, H. Tekin, S. Akar et al., “Interleukin-8, nitric oxide and glutathione status in proliferative vitreoretinopathy and proliferative diabetic retinopathy,” Ophthalmic Research, vol. 35, no. 5, pp. 251–255, 2003.View at: Publisher Site | Google Scholar
K. Zorena, M. Kula, E. Malinowska, D. Raczyńska, M. Myśliwiec, and K. Raczyńska, “Threshold serum concentrations of tumour necrosis factor alpha (TNFα) as a potential marker of the presence of microangiopathy in children and adolescents with type 1 diabetes mellitus (T1DM),” Human Immunology, vol. 74, no. 1, pp. 75–81, 2013.View at: Publisher Site | Google Scholar
R. B. Caldwell, M. Bartoli, M. A. Behzadian et al., “Vascular endothelial growth factor and diabetic retinopathy: Pathophysiological mechanisms and treatment perspectives,” Diabetes/Metabolism Research and Reviews, vol. 19, no. 6, pp. 442–455, 2003.View at: Publisher Site | Google Scholar
X. Wang, G. Wang, and Y. Wang, “Intravitreous Vascular Endothelial Growth Factor and Hypoxia-Inducible Factor 1a in Patients With Proliferative Diabetic Retinopathy,” The American Journal of Ophthalmology, vol. 148, no. 6, pp. 883–889, 2009.View at: Publisher Site | Google Scholar
Y. Liu, M. Costa, and C. Gerhardinger, “IL-1β is upregulated in the diabetic retina and retinal vessels: cell-specific effect of high glucose and IL-1β autostimulation,” PLoS ONE, vol. 7, no. 5, Article ID e36949, 2012.View at: Publisher Site | Google Scholar
Y. Liu, L. F. Leo, C. McGregor, A. Grivitishvili, C. J. Barnstable, and J. Tombran-Tink, “Pigment epithelium-derived factor (PEDF) peptide eye drops reduce inflammation, cell death and vascular leakage in diabetic retinopathy in Ins2(Akita) mice,” Molecular Medicine, vol. 18, pp. 1387–1401, 2012.View at: Google Scholar
J. Gehrmann, Y. Matsumoto, and G. W. Kreutzberg, “Microglia: intrinsic immuneffector cell of the brain,” Brain Research Reviews, vol. 20, no. 3, pp. 269–287, 1995.View at: Publisher Site | Google Scholar
C. A. Colton and D. L. Gilbert, “Production of superoxide anions by a CNS macrophage, the microglia,” FEBS Letters, vol. 223, no. 2, pp. 284–288, 1987.View at: Publisher Site | Google Scholar
R. B. Banati, G. Rothe, G. Valet, and G. W. Kreutzberg, “Detection of lysosomal cysteine proteinases in microglia: flow cytometric measurement and histochemical localization of cathepsin B and L,” Glia, vol. 7, no. 2, pp. 183–191, 1993.View at: Publisher Site | Google Scholar
M. Sayyah, I. T. Najafabadi, S. Beheshti, and S. Majzoob, “Lipopolysaccharide retards development of amygdala kindling but does not affect fully-kindled seizures in rats,” Epilepsy Research, vol. 57, no. 2-3, pp. 175–180, 2003.View at: Publisher Site | Google Scholar
J. P. Godbout, B. M. Berg, K. W. Kelley, and R. W. Johnson, “α-tocopherol reduces lipopolysaccharide-induced peroxide radical formation and interleukin-6 secretion in primary murine microglia and in brain,” Journal of Neuroimmunology, vol. 149, no. 1-2, pp. 101–109, 2004.View at: Publisher Site | Google Scholar
S. G. Kremlev, R. L. Roberts, and C. Palmer, “Differential expression of chemokines and chemokine receptors during microglial activation and inhibition,” Journal of Neuroimmunology, vol. 149, no. 1-2, pp. 1–9, 2004.View at: Publisher Site | Google Scholar
D. J. Kauffmann, J. C. van Meurs, D. A. Mertens, E. Peperkamp, C. Master, and M. E. Gerritsen, “Cytokines in vitreous humor: interleukin-6 is elevated in proliferative vitreoretinopathy,” Investigative Ophthalmology and Visual Science, vol. 35, no. 3, pp. 900–906, 1994.View at: Google Scholar
B. M. Ben-Mahmud, W. H. Chan, R. M. Abdulahad et al., “Clinical validation of a link between TNF-α and the glycosylation enzyme core 2 GlcNAc-T and the relationship of this link to diabetic retinopathy,” Diabetologia, vol. 49, no. 9, pp. 2185–2191, 2006.View at: Publisher Site | Google Scholar
S. Doehmen, V. Poulaki, K. Koizumi, K. Kirchhof, and A. M. Joussen, “The TNF-Inhibitor Enbrel prevents early diabetic retinopathy changes in vivo,” Investigative Ophthalmology & Visual Science, vol. 43, supplement 1, p. U301, 2002, abstract no. 1332.View at: Google Scholar
L. P. Aiello, R. L. Avery, P. G. Arrigg et al., “Vascular endothelial growth factor in ocular fluid of patients with diabetic retinopathy and other retinal disorders,” New England Journal of Medicine, vol. 331, no. 22, pp. 1480–1487, 1994.View at: Publisher Site | Google Scholar
S. Yoshida, A. Yoshida, T. Ishibashi, S. G. Elner, and V. M. Elner, “Role of MCP-1 and MIP-1α in retinal neovascularization during postischemic inflammation in a mouse model of retinal neovascularization,” Journal of Leukocyte Biology, vol. 73, no. 1, pp. 137–144, 2003.View at: Publisher Site | Google Scholar
N. J. Rothwell and G. N. Luheshi, “Interleukin 1 in the brain: biology, pathology and therapeutic target,” Trends in Neurosciences, vol. 23, no. 12, pp. 618–625, 2000.View at: Publisher Site | Google Scholar
T. D. O. S. Pereira, G. N. F. da Costa, A. R. S. Santiago, A. F. Ambrósio, and P. F. M. dos Santos, “High glucose enhances intracellular Ca2+ responses triggered by purinergic stimulation in retinal neurons and microglia,” Brain Research, vol. 1316, pp. 129–138, 2010.View at: Publisher Site | Google Scholar
R. A. Kowluru and S. Odenbach, “Role of interleukin-1β in the pathogenesis of diabetic retinopathy,” The British Journal of Ophthalmology, vol. 88, no. 10, pp. 1343–1347, 2004.View at: Publisher Site | Google Scholar
C. A. Aveleira, C. M. Lin, S. F. Abcouwer, A. F. Ambrósio, and D. A. Antonetti, “TNF-α signals through PKC/NF-κB to alter the tight junction complex and increase retinal endothelial cell permeability,” Diabetes, vol. 59, no. 11, pp. 2872–2882, 2010.View at: Publisher Site | Google Scholar
A. E. Cardona, E. P. Pioro, M. E. Sasse et al., “Control of microglial neurotoxicity by the fractalkine receptor,” Nature Neuroscience, vol. 9, no. 7, pp. 917–924, 2006.View at: Publisher Site | Google Scholar
Y. He, S. Appel, and W. Le, “Minocycline inhibits microglial activation and protects nigral cells after 6-hydroxydopamine injection into mouse striatum,” Brain Research, vol. 909, no. 1-2, pp. 187–193, 2001.View at: Publisher Site | Google Scholar
T. Tikka, B. L. Fiebich, G. Goldsteins, R. Keinänen, and J. Koistinaho, “Minocycline, a tetracycline derivative, is neuroprotective against excitotoxicity by inhibiting activation and proliferation of microglia,” Journal of Neuroscience, vol. 21, no. 8, pp. 2580–2588, 2001.View at: Google Scholar
D. Y. Lu, C. H. Tang, Y. H. Chen, and I. H. Wei, “Berberine suppresses neuroinflammatory responses through AMP-activated protein kinase activation in BV-2 microglia,” Journal of Cellular Biochemistry, vol. 110, no. 3, pp. 697–705, 2010.View at: Publisher Site | Google Scholar
T. S. Kern and R. L. Engerman, “Pharmacological inhibition of diabetic retinopathy: aminoguanidine and aspirin,” Diabetes, vol. 50, no. 7, pp. 1636–1642, 2001.View at: Publisher Site | Google Scholar
DAMAD Study Group, “Effect of aspirin alone and aspirin plus dipyridamole in early diabetic retinopathy. A multicenter randomized controlled clinical trial,” Diabetes, vol. 38, no. 4, pp. 491–498, 1989.View at: Publisher Site | Google Scholar
Early Treatment of Diabetic Retinopathy Research Group, “Effects of aspirin treatment on diabetic retinopathy. ETDRS report number 8. Early Treatment Diabetic Retinopathy Study Research Group,” Ophthalmology, vol. 98, supplement 5, pp. 757–765, 1991.View at: Google Scholar
J. G. Lawrenson, “Histology and pathogenesis of diabeti retinopathy,” in Diabetic Eye Disease, A. R. Rudnicka and J. Birch, Eds., Butterworth-Heinemann, Woburn, Mass, USA, 2000.View at: Google Scholar
R. Talahalli, S. Zarini, N. Sheibani, R. C. Murphy, and R. A. Gubitosi-Klug, “Increased synthesis of leukotrienes in the mouse model of diabetic retinopathy,” Investigative Ophthalmology and Visual Science, vol. 51, no. 3, pp. 1699–1708, 2010.View at: Publisher Site | Google Scholar
A. S. Ibrahim, M. M. El-Shishtawy, A. Peña Jr., and G. I. Liou, “Genistein attenuates retinal inflammation associated with diabetes by targeting of microglial activation,” Molecular Vision, vol. 16, pp. 2033–2042, 2010.View at: Google Scholar
L. Zheng, Y. Du, C. Miller et al., “Critical role of inducible nitric oxide synthase in degeneration of retinal capillaries in mice with streptozotocin-induced diabetes,” Diabetologia, vol. 50, no. 9, pp. 1987–1996, 2007.View at: Publisher Site | Google Scholar
R. Nagai, D. B. Murray, T. O. Metz, and J. W. Baynes, “Chelation: A fundamental mechanism of action of AGE inhibitors, AGE breakers, and other inhibitors of diabetes complications,” Diabetes, vol. 61, no. 3, pp. 549–559, 2012.View at: Publisher Site | Google Scholar
D. Edelstein and M. Brownlee, “Mechanistic studies of advanced glycosylation end product inhibition by aminoguanidine,” Diabetes, vol. 41, no. 1, pp. 26–29, 1992.View at: Google Scholar
Q. Li, A. Verma, P. Han et al., “Diabetic eNOS-knockout mice develop accelerated retinopathy,” Investigative Ophthalmology and Visual Science, vol. 51, no. 10, pp. 5240–5246, 2010.View at: Publisher Site | Google Scholar
A. J. Barber, D. A. Antonetti, T. S. Kern et al., “The Ins2Akita mouse as a model of early retinal complications in diabetes,” Investigative Ophthalmology and Visual Science, vol. 46, no. 6, pp. 2210–2218, 2005.View at: Publisher Site | Google Scholar
N. P. Akimov and R. C. Rentería, “Spatial frequency threshold and contrast sensitivity of an optomotor behavior are impaired in the Ins2 Akita mouse model of diabetes,” Behavioural Brain Research, vol. 226, no. 2, pp. 601–605, 2012.View at: Publisher Site | Google Scholar
D. N. Cook, S. C. Chen, L. M. Sullivan et al., “Generation and analysis of mice lacking the chemokine fractalkine,” Molecular & Cellular Biology, vol. 21, no. 9, pp. 3159–3165, 2001.View at: Publisher Site | Google Scholar
K. Hatori, A. Nagai, R. Heisel, J. K. Ryu, and S. U. Kim, “Fractalkine and fractalkine receptors in human neurons and glial cells,” Journal of Neuroscience Research, vol. 69, no. 3, pp. 418–426, 2002.View at: Publisher Site | Google Scholar
J. A. Garcia, P. A. Pino, M. Mizutani et al., “Regulation of adaptive immunity by the fractalkine receptor during autoimmune inflammation,” The Journal of Immunology, vol. 191, no. 3, pp. 1063–1072, 2013.View at: Publisher Site | Google Scholar
J. M. Kezic, X. Chen, E. P. Rakoczy, and P. G. McMenamin, “The effects of age and Cx3cr1 deficiency on retinal microglia in the Ins2Akita diabetic mouse,” Investigative Ophthalmology and Visual Science, vol. 54, no. 1, pp. 854–863, 2013.View at: Publisher Site | Google Scholar
D. H. McDermott, A. M. Fong, Q. Yang et al., “Chemokine receptor mutant CX3CR1-M280 has impaired adhesive function and correlates with protection from cardiovascular disease in humans,” Journal of Clinical Investigation, vol. 111, no. 8, pp. 1241–1250, 2003.View at: Publisher Site | Google Scholar
L. Ziegler-Heitbrock and T. P. Hofer, “Toward a refined definition of monocyte subsets,” Frontiers in Immunology, vol. 4, article 23, 2013.View at: Google Scholar
C. Auffray, D. Fogg, M. Garfa et al., “Monitoring of blood vessels and tissues by a population of monocytes with patrolling behavior,” Science, vol. 317, no. 5838, pp. 666–670, 2007.View at: Publisher Site | Google Scholar
E. L. Gautiar, T. Shay, J. Miller et al., “Gene-expression profiles and transcriptional regulatory pathways that underlie the identity and diversity of mouse tissue macrophages,” Nature Immunology, vol. 13, no. 11, pp. 1118–1128, 2012.View at: Publisher Site | Google Scholar
A. Böhm, A.-M. Ordelheide, J. Machann et al., “Common genetic variation in the SERPINF1 locus determines overall adiposity, obesity-related insulin resistance, and circulating leptin levels,” PLoS ONE, vol. 7, no. 3, Article ID e34035, 2012.View at: Publisher Site | Google Scholar
L. Xu, F. Ping, J. Yin et al., “Elevated plasma SPARC levels are associated with insulin resistance, dyslipidemia, and inflammation in gestational diabetes mellitus,” PLoS ONE, vol. 8, Article ID e81615, 2013.View at: Google Scholar
J. L. Santiago, B. Z. Alizadeh, A. Martínez et al., “Study of the association between the CAPSL-IL7R locus and type 1 diabetes,” Diabetologia, vol. 51, no. 9, pp. 1653–1658, 2008.View at: Publisher Site | Google Scholar
S. Nishat, H. Shabir, A. S. Azmi, and H. R. Ansari, “A3 adenosine receptor: a plausible therapeutic target for cardio-protection in diabetes,” Recent Patents on Cardiovascular Drug Discovery, vol. 7, no. 1, pp. 59–70, 2012.View at: Publisher Site | Google Scholar
K. Ohsawa, T. Sanagi, Y. Nakamura, E. Suzuki, K. Inoue, and S. Kohsaka, “Adenosine A3 receptor is involved in ADP-induced microglial process extension and migration,” Journal of Neurochemistry, vol. 121, no. 2, pp. 217–227, 2012.View at: Publisher Site | Google Scholar
A. S. Ibrahim, M. M. El-shishtawy, W. Zhang, R. B. Caldwell, and G. I. Liou, “A2A adenosine receptor (A2AAR) as a therapeutic target in diabetic retinopathy,” The American Journal of Pathology, vol. 178, no. 5, pp. 2136–2145, 2011.View at: Publisher Site | Google Scholar
G. I. Liou, S. Ahmad, M. Naime, N. Fatteh, and A. S. Ibrahim, “Role of adenosine in diabetic retinopathy,” Journal of Ocular Biology, Diseases, and Informatics, vol. 4, no. 1-2, pp. 19–24, 2011.View at: Publisher Site | Google Scholar
M. A. Manister, “Retinal injuries from light: mechanisms, hazards and prevention,” in Retina, S. J. Ryan, A. P. Schachat, C. P. Wilkinson, D. R. Hinton, S. R. Sadda, and P. Wiedemann, Eds., pp. 1857–1870, Elsevier, London, UK, 2005.View at: Google Scholar
M. Rózanowska and T. Sarna, “Light-induced damage to the retina: role of rhodopsin chromophore revisited,” Photochemistry and Photobiology, vol. 81, no. 6, pp. 1305–1330, 2005.View at: Publisher Site | Google Scholar
A. Wenzel, C. Grimm, M. Samardzija, and C. E. Remé, “Molecular mechanisms of light-induced photoreceptor apoptosis and neuroprotection for retinal degeneration,” Progress in Retinal and Eye Research, vol. 24, no. 2, pp. 275–306, 2005.View at: Publisher Site | Google Scholar
A. M. Santos, D. Martín-Oliva, R. M. Ferrer-Martín et al., “Microglial response to light-induced photoreceptor degeneration in the mouse retina,” Journal of Comparative Neurology, vol. 518, no. 4, pp. 477–492, 2010.View at: Publisher Site | Google Scholar
C. Zhang, J. Shen, T. T. Lam et al., “Activation of microglia and chemokines in light-induced retinal degeneration,” Molecular Vision, vol. 11, pp. 887–895, 2005.View at: Google Scholar
J. E. Lee, K. J. Liang, R. N. Fariss, and W. T. Wong, “Ex vivo dynamic imaging of retinal microglia using time-lapse confocal microscopy,” Investigative Ophthalmology and Visual Science, vol. 49, no. 9, pp. 4169–4176, 2008.View at: Publisher Site | Google Scholar
S. Song, F. Zhou, and W. R. Chen, “Low-level laser therapy regulates microglial function through Src-mediated signaling pathways: implications for neurodegenerative diseases,” Journal of Neuroinflammation, vol. 9, article 219, 2012.View at: Publisher Site | Google Scholar
H. Huang, R. Parlier, J. Shen, G. A. Lutty, and S. A. Vinores, “VEGF receptor blockade markedly reduces retinal microglia/macrophage infiltration into laser-induced CNV,” PLoS ONE, vol. 8, no. 8, Article ID e71808, 2013.View at: Publisher Site | Google Scholar
S. Matsuda, F. Gomi, Y. Oshima, M. Tohyama, and Y. Tano, “Vascular endothelial growth factor reduced and connective tissue growth factor induced by triamcinolone in ARFE19 cells under oxidative stress,” Investigative Ophthalmology and Visual Science, vol. 46, no. 3, pp. 1062–1068, 2005.View at: Publisher Site | Google Scholar
H. Zhou, L. Yang, H. Li et al., “Downregulation of VEGF mRNA expression by triamcinolone acetonide acetate-loaded chitosan derivative nanoparticles in human retinal pigment epithelial cells,” International Journal of Nanomedicine, vol. 7, pp. 4649–4660, 2012.View at: Publisher Site | Google Scholar
S. Singhal, J. M. Lawrence, T. E. Salt, P. T. Khaw, and G. A. Limb, “Triamcinolone attenuates macrophage/microglia accumulation associated with NMDA-induced RGC death and facilitates survival of Müller stem cell grafts,” Experimental Eye Research, vol. 90, no. 2, pp. 308–315, 2010.View at: Publisher Site | Google Scholar
W. Shen, S. R. Lee, J. Araujo, S. H. Chung, L. Zhu, and M. C. Gillies, “Effect of glucocorticoids on neuronal and vascular pathology in a transgenic model of selective Muller cell ablation,” Glia, vol. 62, no. 7, pp. 1110–1124.View at: Publisher Site | Google Scholar
I. V. Glybina, A. Kennedy, P. Ashton, G. W. Abrams, and R. Iezzi, “Intravitreous delivery of the corticosteroid fluocinolone acetonide attenuates retinal degeneration in S334ter-4 Rats,” Investigative Ophthalmology and Visual Science, vol. 51, no. 8, pp. 4243–4252, 2010.View at: Publisher Site | Google Scholar
K. L. Kirkwood, L. M. Golub, and P. G. Bradford, “Non-antimicrobial and antimicrobial tetracyclines inhibit IL-6 expression in murine osteoblasts,” Annals of the New York Academy of Sciences, vol. 878, pp. 667–670, 1999.View at: Publisher Site | Google Scholar
T. J. Federici, “The non-antibiotic properties of tetracyclines: clinical potential in ophthalmic disease,” Pharmacological Research, vol. 64, no. 6, pp. 614–623, 2011.View at: Publisher Site | Google Scholar
C. A. Cukras, P. Petrou, E. Y. Chew, C. B. Meyerle, and W. T. Wong, “Oral minocycline for the treatment of diabetic macular edema (DME): results of a phase I/II clinical study,” Investigative Ophthalmology and Visual Science, vol. 53, no. 7, pp. 3865–3874, 2012.View at: Publisher Site | Google Scholar
P. Massin, F. Bandello, J. G. Garweg et al., “Safety and efficacy of ranibizumab in diabetic macular edema (RESOLVE study): a 12-month, randomized, controlled, double-masked, multicenter phase II study,” Diabetes Care, vol. 33, no. 11, pp. 2399–2405, 2010.View at: Publisher Site | Google Scholar
I. U. Scott, G. R. Jackson, D. A. Quillen et al., “Effect of doxycycline vs placebo on retinal function and diabetic retinopathy progression in patients with severe nonproliferative or non-high-risk proliferative diabetic retinopathy: a randomized clinical trial,” JAMA Ophthalmology, vol. 132, no. 5, pp. 535–543, 2014.View at: Google Scholar
G. R. Jackson, I. U. Scott, D. A. Quillen, L. E. Walter, and T. W. Gardner, “Inner retinal visual dysfunction is a sensitive marker of non-proliferative diabetic retinopathy,” British Journal of Ophthalmology, vol. 96, no. 5, pp. 699–703, 2012.View at: Publisher Site | Google Scholar
I. U. Scott, G. R. Jackson, D. A. Quillen, R. Klein, J. Liao, and T. W. Gardner, “Effect of doxycycline vs placebo on retinal function and diabetic retinopathy progression in mild to moderate nonproliferative diabetic retinopathy: a randomized proof-of-concept clinical trial,” JAMA Ophthalmology, 2014.View at: Publisher Site | Google Scholar