Ординатура / Офтальмология / Английские материалы / Eye, Retina, and Visual System of the Mouse_Chalupa, Williams_2008

Moreover, the availability of antibodies and reagents to conduct molecular studies is very limited for dogs. Mice develop degenerate, acellular capillaries in the retina, a critical step in the progression of diabetic retinopathy, as well as some other lesions characteristic of the early stages of retinopathy (Feit-Leichman et al., 2005). The mouse model has a number of advantages over larger models, including lesser space requirements and cost. The mouse also allows genetic manipulation and relatively easy administration of limited pharmacological therapies.

Anatomical differences between the mouse and human retina do exist. The mouse retina has multiple arteries emanating from the optic nerve that feed the different regions of the retina (figure 45.1), whereas the human retina receives its blood supply from one main artery, the ophthalmic artery, which divides only after entering the retina. In addition, retinas from mice (as well as all other nonprimate species) differ from the human retina in that only humans and primates possess a macula (see figure 45.1).

Initial investigations using the mouse as a model of diabetic retinopathy yielded controversial results, but, more recent results have shown the mouse model to reliably develop capillary degeneration, pericyte loss, and capillary basement membrane thickening (Feit-Leichman et al., 2005; Hammes et al., 2002; Joussen et al., 2004; Midena et al., 1989). Diabetes can be induced experimentally using drugs such as streptozotocin and alloxan to disrupt pancreatic beta cells, thus reproducing an insulinopenia like that of type I diabetes. Several strains of mice (including the C57BL/6JIns2Akita [Akita], db/db, KK.Cg-Ay/J [KKAY], and NOD strains) develop diabetes spontaneously and have microvascular changes of the retina consistent with nonproliferative retinopathy (capillary degeneration, pericyte loss, thickening of capillary basement membrane) over a period of 6–9 months (Barber et al., 2005; Cheung et al., 2005; FeitLeichman et al., 2005; Naeser and Agren, 1978; Ning et al.,

2004; Whetzel et al., 2006). The Akita and NOD strains are models of type I diabetes, and db/db and KKAY strains are models of type II diabetes.

Retinal function, as demonstrated by the electroretinogram (ERG), becomes impaired in diabetic mice and other models (Barile et al., 2005; Zheng et al., 2007). Whether or not diabetes causes degeneration of retinal neurons in mice is an area of controversy. Some investigators have reported extensive degeneration of retinal ganglion cells (RGCs) in C57BL/6J mice after diabetes duration as little as 14 weeks (Martin et al., 2004), whereas others have not detected retinal neurodegeneration in that strain even after durations of up to 1 year (Feit-Leichman et al., 2005). Retinal neurodegeneration has been detected in a different diabetic mouse strain, the Akita. Whether or not neuronal cell loss plays an important role in diabetic retinopathy and eventual visual impairment remains an open question.

To date, diabetic mice have not been found to develop preretinal neovascularization secondary to diabetes. (Likewise, neither experimentally diabetic dogs, cats, rats, nor other species have been found to develop the proliferative retinopathy; Kern and Mohr, 2007.) The relatively short duration of diabetes in most studies and the resulting modest extent of capillary degeneration in studies of mice are believed to be the reason that neovascularization has not been observed in these models. Advanced proliferative retinopathy similar to that seen in diabetic patients does develop in nondiabetic mice, however, under specific stresses or gene alterations (Lai et al., 2005; Ruberte et al., 2004; Smith et al., 1999). This is discussed further in a later section.

The mouse models used to study the nonproliferative vascular lesions, neurodegenerative changes, and proliferative stages of diabetic retinopathy are detailed in the next section. Each model is evaluated based on the histopathological changes observed and the pathogenic mechanisms tested using these models.

Figure 45.1 The retinal vasculature of the mouse, following isolation by proteolytic digestion of the formalin-fixed retina. See color plate 45.

Mouse models of vascular lesions of nonproliferative retinopathy

Chemically Induced Diabetes The C57BL/6J mouse is the strain of mouse most used in studies of diabetic retinopathy. Typically, diabetes is induced as a result of a 3- to 5-day course of streptozotocin (50–75 mg/kg of body weight), although other single-dose regimens exist. Several days after chemical induction, randomly sampled blood glucose levels of 250 mg/dL or higher denote diabetes. Long-term monitoring of glycohemoglobin (HbA1c or GHb) ensures comparable degrees of hyperglycemia among experimental groups. Vascular changes consistent with diabetic retinopathy, including acellular capillaries, pericyte loss, and capillary cell apoptosis, become apparent beginning about 6 months after the onset of diabetes (figure 45.2)

(Feit-Leichman et al., 2005). The number of acellular capillaries and pericyte ghosts increases with diabetes duration (figure 45.3). Maintaining diabetes for 12 months or more is difficult, owing to increasing mortality, but administration of insulin in doses sufficient to prevent body weight loss but not reduce hyperglycemia (0.1–0.2 U; 2–3 times per week) seems to reduce mortality. Several characteristic lesions of early diabetic retinopathy in humans (including microaneurysms and retinal hemorrhages) have not been reproducibly detected in diabetic mice over the 6–12 months that the animals commonly are studied.

Spontaneous Diabetes

Akita mouse. The Akita mouse develops type I diabetes spontaneously due to a dominant point mutation in the insulin 2 gene (Barber et al., 2005). Heterozygote male mice show hyperglycemia and hypoinsulinemia by 4 weeks of age. By 12 weeks of age, the diabetic mice develop increased vascular permeability compared to sibling wild-type mice. Acellular capillary formation and pericyte loss become statistically greater than in nondiabetic animals at about 6 months of diabetes, and increase with longer diabetes duration (Barber et al., 2005).

db/db mouse. The db/db mouse is a genetic model of type II diabetes. A mutation in the leptin receptor gene leads to obesity and subsequent obesity-induced type II diabetes mellitus. These mice have been reported to develop an increased endothelial cell:pericyte ratio (Midena et al., 1989). This was interpreted as a reduction in the number of capillary pericytes, but this ratio could also increase as a result of endothelial cell proliferation (Cuthbertson and Mandel, 1986). Diabetes-induced vessel leakage, degeneration of capillaries, thickening of retinal capillary basement membrane, and increased expression of vascular endothelial growth factor (VEGF, the protein encoded by gene Vegfa) and platelet/ endothelial cell adhesion molecule-1 (PECAM-1, the protein encoded by gene Pecam1) have been detected at 15 months of age (Cheung et al., 2005).

db/db mice were crossed with apolipoprotein E (Apoe)- deficient mice to generate insulin-deficient and hyperlipidemic animals (Barile et al., 2005). The superimposition of hyperlipidemia on diabetes resulted in accelerated development of acellular capillaries and pericyte loss at 6 months of diabetes (Barile et al., 2005). This same hyperglycemic, hyperlipidemic model was used to study the role of advanced glycation end products (AGEs) in the development of diabetic retinopathy. Diabetes-induced capillary degeneration and pericyte loss could be inhibited by the administration of soluble receptors for AGEs (sRAGE) daily from 8 weeks to 6 months of diabetes (Barile et al., 2005), thus suggesting that AGEs play a role in the development of retinal histopathology in this model. Consistent with the conclusions of that study, the diabetes-induced thickening of retinal capillary basement membranes in the outer plexiform layer (OPL) of the retina from diabetic db/db mice was not observed after intraperitoneal injection with antibodies against glycated albumin (Clements et al., 1998). Whether or not this therapeutic approach also had effects on other aspects of the microvascular disease was not determined.

Aldose reductase deficiency in db/db mice, achieved by crossing db/db mice and aldose reductase null mutation mice, resulted in less of the diabetes-induced increase in vascular permeability, VEGF expression, and PECAM-1 expression than that seen in control db/db mice (Cheung et al., 2005). The effect of aldose reductase deficiency on the development of retinal histopathology has not been reported to date in diabetic mice, but results in other species have been mixed (Dagher et al., 2004; Engerman and Kern, 1993).

KKAY mouse. The KKAY mouse develops diabetes mellitus when fed a high fat diet, thus, it is considered a genetic model of type II diabetes mellitus (Siracusa, 1994). Control animals are fed a standard diet. After 3 months of diabetes, KKAY mice demonstrated microvascular changes that included variable basement membrane thickening, mitochondrial swelling, capillary peripheral cell edema and

degeneration, and endothelial cell hyperplasia, as detected by transmission electron microscopy (Ning et al., 2004). These animals have not been studied with respect to susceptibility to developing capillary degeneration, microaneurysms, or other characteristic vascular histopathology.

NOD mouse. The nonobese diabetic mouse (NOD) develops diabetes through an autoimmune-mediated destruction of pancreatic beta cells (Leiter et al., 1987). The resulting insulitis leads to spontaneous hyperglycemia, again modeling type I diabetes in humans. Retinas from the NOD mouse have not yet been examined for the development of the characteristic microvascular lesions of diabetic retinopathy.

ob/ob mouse. ob/ob mice are leptin deficient, and phenotypically are obese by 4 weeks of age. Regression of pancreatic islets results in severe diabetes and early death when on the C57BLKS background. In control animals (C57BL/6J background), the mice are only transiently hyperglycemic, blood glucose returning to normal by 14–16 weeks. Retinas from these obese, hyperglycemic mice were morphologically the same as in littermate controls, with a similar endothelial: pericyte ratio and absence of microaneuryms (Naeser and Agren, 1978).

Nondiabetic Mouse Models That Develop

Diabetic-Like Retinopathy

Galactose feeding. Feeding nondiabetic mice a high-galactose diet results in systemic elevation of blood galactose levels, but glucose levels remain normal. This model confirms the causative role of elevated hexose in the pathogenesis of diabetic retinopathy, since the other metabolic alterations characteristic of insulin deficiency, including altered lipid metabolism, are not observed (Engerman and Kern, 1984). In mice fed 30% galactose, the retinopathy can be followed for up to 24 months because the animals generally are quite healthy. Mice (C57BL/6J or BALB/cJ) fed 30% galactose develop a diabetic-like retinopathy with acellular capillaries, pericyte ghosts, basement membrane thickening, and occasional saccular microaneurysms ( Joussen et al., 2004; Kern and Engerman, 1996). The advantages of this model are that experimental galactosemia is easy to establish and the animals require less care than diabetic animals. A possible deficiency of the model is that galactosemic retinopathy might differ in several ways from that in diabetes (Frank et al., 1997; Kern et al., 2000; Mohr et al., 2002). Moreover, the cost of the galactose diet can be expensive for large experimental groups.

Plasminogen activator inhibitor-1(PAI-1). PAI-1 regulates serine proteases known as plasminogen activators (PAs). PAs convert plasminogen to plasmin, which then degrades fibrin, as well as basement membrane components such as laminin and fibronectin. A transgenic mouse model was developed

with the human PAI-1 gene sequence (SERPINE1) under regulation of the metallothionein promoter (Grant et al., 2000). Administration of ZnSO4, which allows for continuous expression of the transgene, resulted in increased PAI-1 immunoreactivity in the retinal capillaries. Electron microscopy demonstrated increased basement membrane thickening in the PAI-1-overexpressing mice compared to control animals. Whole-mount retinal analysis revealed an increased endothelial cell:pericyte ratio (even in the absence of diabetes), suggesting that PAI-1 might contribute to alterations in vascular structure. Degeneration of capillaries in diabetes has not been determined in this model.

Platelet-derived growth factor(PDGF-B). PDGF-B (encoded by gene Pdgfb) is an important factor in pericyte recruitment, and studies support a role for pericytes in the formation and stabilization of vessels. Mice that are heterozygous for Pdgfb (i.e., with one functional allele) have fewer pericytes and a significant increase in the number of acellular capillaries in their retinas compared to wild-type littermates (Hammes et al., 2002). Under diabetic conditions, the retinopathy of the Pdgfb heterozygous mice was worsened, with increased formation of acellular capillaries and lesions regarded as microaneurysms. In addition, studies of nondiabetic mice deficient in endothelium-specific PDGF-B demonstrated pericyte deficiency associated with capillaries of variable diameter, density, and ring structure, as well as microaneurysms (Enge et al., 2002). This suggests an important role for PDGF-B in the development of diabetic retinopathy.

Use of mouse models to study diabetes-induced retinal neurodegeneration

Of the many experimental models of developing retinal microvascular lesions described in the previous section, only a few have been examined for neurodegenerative changes to date. The mouse models evaluated thus far are reviewed in this section. Techniques to quantify neurodegenerative changes in the retina include counting the number of cells in the ganglion cell layer (GCL) per length of retina, quantifying the number of retinal neurons that are immunopositive for activated caspase-3 (encoded by gene Casp3) or TUNEL stain, or measuring the thickness of the retina or neuroretinal layers.

C57BL/6J mouse. The C57BL/6J mouse strain has been evaluated for diabetes-induced neurodegeneration in the retina. Some investigators reported a 20%–25% reduction in cells of the GCL of the retina as early as 14 weeks of diabetes (Martin et al., 2004). In contrast, others did not detect any GC loss at diabetes durations of up to 1 year (FeitLeichman et al., 2005). No explanation for these different conclusions has yet been put forth. Retinal glia cells also have

been reported to show signs that suggest cell death in diabetes (Kusner et al., 2004). Translocation of glyceraldehyde-3- phosphate dehydrogenase (GAPDH) to the nucleus, which is strongly associated with cell death in cerebral injury (Tatton et al., 2000), has also been demonstrated in retinal Müller cells of diabetic mice and humans (Kusner et al., 2004).

Akita mouse. In the Akita mouse at 22 weeks of hyperglycemia, neurodegenerative changes detected included a reduction in (1) the thickness of the inner plexiform layer in the central and peripheral portions of the retina, (2) the thickness of the inner nuclear layer, and (3) the number of nuclei in the RGC layer (Barber et al., 2005). Although morphological alterations in the astrocytes and microglia were observed, no changes in glial fibrillary acidic protein (GFAP) immunoreactivity were noted. After 4 weeks of diabetes, caspase-3 immunoreactivity increased, suggesting increased apoptosis of some cells of the neuroglial retina.

db/db mouse. At 15 months of age, db/db mice have been reported to demonstrate accelerated ganglion cell apoptosis and glial cell activation as detected by GFAP staining (Cheung et al., 2005).

At 6 months of diabetes, db/db mice crossed with Apoedeficient mice had abnormal function of retinal neurons as characterized by abnormal electroretinograms (ERGs) (Barile et al., 2005).

Aldose reductase deficiency in the db/db mouse resulted in a reduction in the number of caspase-3 immunoreactive cells and GFAP-positive cells compared to the wild-type db/db diabetics (Cheung et al., 2005).

KKAY mouse. In this model, neurodegeneration was quantified by the use of TUNEL staining of ultrathin sections of fixed retinas at 1 and 3 months of diabetes (Ning et al., 2004). An increased rate of neuronal cell loss in the ganglion cell in diabetic compared to control animals has been reported. The number of apoptotic cells increased with the duration of diabetes in both the RGC layer and the inner nuclear layer.

Biochemical changes in the mouse retina that contribute to the development of diabetic retinopathy

In both clinical trials and experimental studies of diabetic models, hyperglycemia has been found to be closely associated with the development of the spectrum of lesions regarded as diabetic retinopathy (Engerman and Kern, 1995; Zhang et al., 2001). Diabetes-induced death of retinal capillary and neuroglial cells likely is due to (1) metabolic abnormalities within those cells or (2) extracellular events, such as capillary obstruction by leukocytes, platelets, or erythrocytes, or receptor-mediated processes initiated by binding of AGE or

insulin-like growth factor I (IGF-I) (figure 45.4). Oxidative stress, protein kinase C, the sequelae of nonenzymatic glycation, altered metabolism of sugar through glycolysis and the polyol pathways, and inflammatory processes have been postulated to be intracellular abnormalities contributing to the pathogenesis of the retinopathy ( Joussen et al., 2004; Kowluru, 2002; Moore et al., 2003; Ruberte et al., 2004; Vlassara, 2001). The respective contributions of these sequelae of hyperglycemia to the pathogenesis of diabetic retinopathy remain under investigation.

A comparison of metabolic abnormalities in the retinas of diabetic mice and rats has demonstrated both similarities and differences. At 2 months’ duration of diabetes, diabetesinduced reductions in glutathione and increases in nitric oxide, lipid peroxide, and protein kinase C activity in the mouse retina paralleled results in the rat model (Kowluru, 2002). Another study compared the mouse and rat models using a different panel of biochemical changes at 6 weeks of diabetes (Obrosova et al., 2006). Whereas the rat model showed enhanced VEGF expression, increased poly(ADPribosyl)ation, and decreased antioxidant enzyme activities, the mouse model reflected none of these changes. However, documentation of long-term hyperglycemia was not presented in the latter publication, and differences in the severity of hyperglycemia between the studies might have contributed to the differences between the rat and mouse models. Overall, it seems that the biochemical alterations in the mouse model are less severe than in the rat model, although in both models animals develop a similar severity of capillary degeneration at approximately the same duration of diabetes (6–8 months).

The administration of pharmacological therapies that can inhibit a particular biochemical defect or the induction of diabetes in mice having selective gene deficiencies has pro-

Figure 45.4 Leukocyte adherence, or leukostasis (arrows), in the retinal microvasculature of the diabetic mouse. See color plate 47.

vided novel insights into the pathogenesis of the early stages of diabetic retinopathy. Several of these areas of investigation are summarized here.

Advanced glycation end products. AGEs form and accumulate in the retinal microvascular cells of diabetic animals and have been linked to generation of oxidative stress and activation of NFkB, a transcription factor that regulates expression of a variety of pro-inflammatory proteins, including inducible nitric oxide synthase (iNOS, which is encoded by gene Nos2) and intracellular adhesion molecule-1 (ICAM-1, the protein encoded by gene Icam1) (Goldin et al., 2006; Vlassara, 2001). To assess in vivo the role of AGEs in retinal microvascular disease, nondiabetic C57BL/6J mice received 7 daily intraperitoneal injections of AGE-albumin or albumin alone as control (Moore et al., 2003). The mice receiving AGE-albumin demonstrated a threefold increase in NFkB expression compared to control animals, as well as a significant increase in Icam1 mRNA, leukocyte adherence, and blood-retinal barrier breakdown. Moreover, hyperlipidemic diabetic mice were protected from capillary degeneration, pericyte loss, and neural dysfunction by administration of sRAGE (a soluble form of the receptor for AGE, which is a therapy that reduces binding of AGEs to cellular receptors) (Barile et al., 2005).

Aldose reductase. Aldose reductase (AR) is the first enzyme in the polyol pathway. During hyperglycemia, excess glucose is converted by AR to sorbitol and then potentially to fructose by sorbitol dehydrogenase. AR activity is increased in diabetes, although more modestly so in the mouse retina than in the rat retina (Kowluru, 2002; Obrosova et al., 2006). Transgenic mice overexpressing human AR (AKR1B1) fed a galactose-rich diet for 7 days demonstrated occlusion of the retinochoroidal vessels. AR (encoded by gene Akr1b3)- deficient mice crossed with db/db mice were protected from diabetes-associated microvascular and neurodegenerative changes (Cheung et al., 2005).

Angiopoietin. Pericyte loss is heralded as an early morphological change in diabetic retinopathy, in part because pericytes are believed to regulate endothelial proliferation. Evidence indicates that angiopoietin-1 and -2 regulate vessel sprouting and regression through pericyte recruitment. Both angiopoietin-1 and -2 have been evaluated in mouse models for roles in diabetic retinopathy pathogenesis. At 16 weeks of diabetes, mice injected via tail vein with an adenovirus coding for human angiopoietin-1 (gene ANGPT1) demonstrated less leukocyte adherence, reduced endothelial cell damage or death (as detected by propidium iodide staining), and inhibition of blood-retinal barrier breakdown ( Joussen et al., 2002). Recently, heterozygous deficiency of angiopoi- etin-2 (mouse gene Angpt2) in Ang-2 lacZ knock-in mice

prevented loss of pericytes and acellular capillary formation at 26 weeks of diabetes (Hammes et al., 2004). This suggests that angiopoietin-1 and -2 are involved in various proposed pathogenic mechanisms leading to diabetic retinopathy.

Eicosanoids. Eicosanoids, metabolites of arachidonic acid, are known inflammatory mediators. 5-Lipoxygenase (Alox5) products, including leukotriene B4 and leukotriene C4/ D4/E4, function in leukocyte recruitment and vascular permeability, respectively. Over a period of 9 months of diabetes, Alox5 deficiency inhibited the degeneration of retinal capillaries (Gubitosi-Klug and Kern, 2006). In addition, diabetes-induced leukostasis (Gubitosi-Klug and Kern, 2006) and superoxide generation (Gubitosi-Klug and Kern, 2008) in the retina were significantly inhibited in the Alox5-deficient mice compared to diabetic wild-type controls.

Intracellular adhesion molecule-1 and CD18. ICAM-1 (encoded by gene Icam1) and its ligand CD18 (encoded by gene Itgb2) are involved in the adherence of leukocytes to the vascular endothelium, especially in inflammatory conditions. The role of these molecules and leukostasis in diabetic retinopathy was explored using ICAM-1- and CD18-deficient mice ( Joussen et al., 2004). In both the streptozotocin-induced diabetic and galactosemic models, permeability of the retinal vasculature and the number of leukocytes adhering to the retinal vasculature were less in the diabetic ICAM-1- or CD18-deficient mice than in diabetic wild-type controls. Of note, capillary degeneration and pericyte loss also were inhibited in these genetically deficient animals compared to wild-type diabetic controls ( Joussen et al., 2004).

Inducible nitric oxide synthase. Nitric oxide (NO) is an important signaling molecule in many physiological processes such as inflammation and cell survival. It is synthesized by two types of nitric oxide synthase, one that is expressed constitutively and another that is regulated or induced (iNOS) by cellular stimuli such as cytokines. After 9 months of diabetes, mice deficient in iNOS (encoded by gene Nos2) had developed significantly less degeneration of retinal capillaries than wild-type diabetic control animals (Zheng et al., 2007). Diabetic iNOS-deficient mice had less leukostasis and less production of nitric oxide, PGE2, and superoxide.

Interleukin-1b. IL-1β (encoded by gene IL1b) is a proinflammatory cytokine that acts through IL-1β receptors on cells. The role of IL-1β in the development of diabetic retinopathy has been investigated in two mouse models. Inhibition of caspase-1 activity (the enzyme that activates IL-1β) by administration of minocycline inhibited retinal levels of IL-1β and inhibited the degeneration of retinal capillaries in C57BL/6J mice having chemically induced diabetes or

galactosemia (Vincent and Mohr, 2007). As a further confirmation of the role of IL-1β in degeneration of retinal capillaries, mice lacking the IL-1β receptor likewise were protected from degeneration of retinal capillaries in diabetes (Vincent and Mohr, 2007).

Superoxide dismutase. It has been demonstrated in rats (Du et al., 2003) and mice (unpublished results) that diabetes induces an increase in superoxide production. Retinal mitochondrial superoxide dismutase activity has been found to be subnormal in diabetic rats (Du et al., 2003), likely contributing to this increase in superoxide. A recent report examined the effect of mitochondrial superoxide dismutase (encoded by gene Sod2) overexpression on retinal oxidative and nitrative stress parameters at 7 weeks of diabetes duration (Kowluru et al., 2006). In contrast to wild-type diabetic mice, diabetic mice overexpressing Sod2 did not show subnormal glutathione levels or elevated nitrotyrosine levels. Possible effects on the development of retinal lesions in diabetes have not been reported to date.

Mouse models of proliferative retinopathy

To date, diabetic mice have not been found to develop proliferative (preretinal neovascularization) retinopathy without the superimposition of some other stress. Thus, studies of retinal neovascularization have relied on other pathological processes that result in new vessel growth in the retina. The nondiabetic models of retinal neovascularization are being used as models of the neovascular process in diabetic proliferative retinopathy. Available evidence suggests that VEGFmediated neovascularization in diabetes is similar to that underlying other conditions of retinal neovascularization, such as retinopathy of prematurity. Non-VEGF-mediated mechanisms of neovascularization have been identified recently, and the ability of the nondiabetic and acute models to reproduce the complexity of all the stimuli of neovascularization induced by diabetes remains to be learned.

Oxygen-induced retinopathy. This model differs from that of diabetic retinopathy in that the retina in oxygen-induced retinopathy is immature and poorly differentiated. Newborn mice, typically C57BL/6J, are exposed from P7 to P12 to hyperoxic conditions, during which interval vascular development is arrested. The mice then are returned to room air, the retinas thus becoming relatively “hypoxic” as a result of the inadequate vascularization of the retina. Preretinal neovascularization and upregulation of VEGF develop during the subsequent days in room air (Smith et al., 1994).

Using this mouse model, multiple in vivo studies have been done to investigate candidate antiangiogenic drugs. For example, inhibition of neovascularization has been achieved by intravitreal injection of soluble VEGF receptor/IgG

fusion proteins, Vegfa antisense oligonucleotides, neutralizing VEGF antibodies, soluble erythropoietin receptor, and intravitreal injection of MAE 87, a receptor tyrosine kinase inhibitor (Agostini et al., 2005; Aiello et al., 1995; Robinson et al., 1996; Sone et al., 1999; Watanabe et al., 2005). In addition, insulin receptor and IGF-I receptor gene deficient mice are protected from hypoxia-induced retinal neovascularization (Kondo et al., 2003). Lentivirus-mediated expression of angiostatin, a derivative of plasminogen, also inhibited this hypoxia-induced retinal neovascularization (Igarashi et al., 2003).

An important role of nitric oxide in the neovascular response and retinal development has been demonstrated in mice lacking iNOS. Oxygen-induced retinopathy induced less apoptosis and thinning in the inner nuclear layer and retina, respectively, of newborn iNOS knockout mice than in wild-type controls (Sennlaub et al., 2002). Fewer TUNELpositive cells were found in the avascular retina and ONL and GCLs of the retina of these animals. Decreased 3- nitrotyrosine production also was detected in retinas from the iNOS-deficient mice compared to wild-type controls. The iNOS inhibitor 1400W injected intravitreally gave similar results with less apoptosis in the INL and less intravitreal neovascularization.

Increased cyclooxygenase-2 (COX-2) activity also has been implicated in angiogenesis in tumors, and neovascularization in cornea and retina. In the mouse model of oxygen-induced retinopathy, preretinal (intravitreal) neovascularization was inhibited using selective COX-2 inhibitors (Sennlaub et al., 2003). Surprisingly, COX-2 (encoded by gene Ptgs2)-deficient mice showed an increased area of retinal nonperfusion, suggesting that COX-2 also might have a vasoprotective effect in the ischemic retina (Cryan et al., 2006). This was not confirmed using the COX-2 inhibitor (Sennlaub et al., 2003).

Endothelial cell interactions with extracellular matrix proteins, such as fibronectin, are a critical step in angiogenesis. This integrin-mediated adhesive interaction can be inhibited by small peptides, such as the α-defensins, which are activated during inflammatory processes. Administration of α- defensins systemically or locally blocked neovascularization in the oxygen-induced retinopathy mouse model (Economopoulou et al., 2005).

ob/ob mice develop less neovascularization than controls in the oxygen-induced retinopathy model. Opposite of these results in leptin-deficient mice, overexpression of leptin in this mouse model worsened neovascularization (Suganami et al., 2004). The role of leptin in the development of diabetes-induced retinal neovascularization remains to be clarified.

VEGF overexpression. VEGF has been implicated in diabetic retinopathy and is a known potent angiogenic factor in

normal retinal development (Saint-Geniez and D’Amore, 2004). Intravitreal injection of human VEGF (VEGFA) causes severe neovascularization in several animal models (Ozaki et al., 1997; Tolentino et al., 1996; Wong et al., 2001). Transgenic mice in which VEGFA expression was under the control of the mouse rhodopsin promoter developed intraretinal and subretinal neovascularization ranging from mild to severe (Lai et al., 2005; Vinores et al., 2000). VEGFA expression directed by a lens-specific promoter resulted in some abnormal growth of vessels on the retinal surface but lacked extension of the vessels into the vitreous, which is characteristic of the preretinal neovascularization in diabetes. The more severe phenotype included more extensive subretinal neovascularization with hemorrhages and retinal detachment (Lai et al., 2005).

Insulin-like growth factor-I. IGF-I (encoded by gene IgfI ) stimulates retinal endothelial cell growth, and IGF-I signaling through the IGF-I receptor leads to increased VEGF levels (Smith et al., 1999). Retinas from nondiabetic mice that overexpress an IgfI transgene reportedly demonstrate lesions characteristic of nonproliferative diabetic retinopathy, including pericyte loss, thickened capillary basement membrane, and intraretinal microvascular abnormalities. In addition, these models have been reported to progress to proliferative retinopathy and retinal detachment (Ruberte et al., 2004).

Summary

The use of mice to model diabetic retinopathy has allowed investigators to creatively test the contributions of various mechanisms in the pathogenesis of early, nonproliferative diabetic retinopathy (e.g., the roles of various pro-inflamma- tory mediators) and more advanced proliferative retinopathy (e.g., VEGF). Studies using pharmacological therapies to inhibit retinopathy have generated considerable excitement, but the lack of absolute specificity of most available pharmacological therapies complicates the interpretation of how they might be acting. Genetically modified mice will continue to be an important tool to further dissect the pathogenesis of retinopathy. Additional studies in the mouse hold the potential of offering new insight into the pathogenesis of diabetic retinopathy, and thus a means to identify new therapeutic approaches to inhibit the retinopathy.

REFERENCES

Agostini, H., Boden, K., Unsold, A., Martin, G., Hansen, L., Fiedler, U., Esser, N., and Marme, D. (2005). A single local injection of recombinant VEGF receptor 2 but not of Tie2 inhibits retinal neovascularization in the mouse. Curr. Eye Res. 30(4):249–257.

Aguilar, E., Friedlander, M., and Gariano, R. F. (2003). Endothelial proliferation in diabetic retinal microaneurysms. Arch. Ophthalmol. 121(5):740–741.

Aiello, L. P., Pierce, E. A., Foley, E. D., Takagi, H., Chen, H., Riddle, L., N. Ferrara, N., King, G. L., and Smith, L. E. (1995). Suppression of retinal neovascularization in vivo by inhibition of vascular endothelial growth factor (VEGF) using soluble VEGF-receptor chimeric proteins. Proc. Natl. Acad. Sci. U.S.A. 92(23):10457–10461.

Barber, A. J., Antonetti, D. A., Kern, T. S., Reiter, C. E., Soans, R. S., Krady, J. K., Levison, S. W., Gardner, T. W., and Bronson, S. K. (2005). The Ins2Akita mouse as a model of early retinal complications in diabetes. Invest. Ophthalmol. Vis. Sci. 46(6):2210–2218.

Barber, A. J., Lieth, E., Khin, S. A., Antonetti, D. A., Buchanan, A. G., and Gardner, T. W. (1998). Neural apoptosis in the retina during experimental and human diabetes: Early onset and effect of insulin. J. Clin. Invest. 102(4):783–791.

Barile, G. R., Pachydaki, S. I., Tari, S. R., Lee, S. E., Donmoyer, C. M., Ma, W., Rong, L. L., Buciarelli, L. G., Wendt, T., et al. (2005). The RAGE axis in early diabetic retinopathy. Invest. Ophthalmol. Vis. Sci. 46(8):2916–2924.

Bloodworth, J. M., Jr., and Molitor, D. L. (1965). Ultrastructural aspects of human and canine diabetic retinopathy. Invest. Ophthalmol. 4(6):1037–1048.

Bresnick, G. H., and Palta, M. (1987a). Predicting progression to severe proliferative diabetic retinopathy. Arch. Ophthalmol. 105 (6):810–814.

Bresnick, G. H., and Palta, M. (1987b). Oscillatory potential amplitudes: Relation to severity of diabetic retinopathy. Arch. Ophthalmol. 105(7):929–933.

Cheung, A. K., Fung, M. K., Lo, A. C., Lam, T., So, K. F., Chung, S. S., and Chung, S. K. (2005). Aldose reductase deficiency prevents diabetes-induced blood-retinal barrier breakdown, apoptosis, and glial reactivation in the retina of db/db mice. Diabetes 54(11):3119–3125.

Clements, R. S., Jr., Robison, W. G., Jr., and Cohen, M. P. (1998). Anti-glycated albumin therapy ameliorates early retinal microvascular pathology in db/db mice. J. Diabetes Complications 12(1):28–33.

Cogan, D. G., and Kuwabara, T. (1967). The mural cell in perspective. Arch. Ophthalmol. 78(2):133–139.

Cryan, L. M., Pidgeon, G. P., Fitzgerald, D. J., and O’Brien, C. J. (2006). COX-2 protects against thrombosis of the retinal vasculature in a mouse model of proliferative retinopathy. Mol. Vis. 12:405–414.

Cunha-Vaz, J., Faria de Abreu, J. R., and Campos, A. J. (1975). Early breakdown of the blood-retinal barrier in diabetes. Br. J. Ophthalmol. 59(11):649–656.

Cuthbertson, R. A., and Mandel, T. E. (1986). Anatomy of the mouse retina: Endothelial cell-pericyte ratio and capillary distribution. Invest. Ophthalmol. Vis. Sci. 27(11):1659–1664.

Dagher, Z., Park, Y. S., Asnaghi, V., Hoehn, T., Gerhardinger, C., and Lorenzi, M. (2004). Studies of rat and human retinas predict a role for the polyol pathway in human diabetic retinopathy. Diabetes 53(9):2404–2411.

Davis, M., Kern, T., and Rand, L. (1977). Diabetic retinopathy. In P. Zimmet and R. DeFronzo (Eds.), International textbook of diabetic retinopathy. New York: John Wiley and Sons.

The Diabetes Control and Complications Trial Research Group. (1993). The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulindependent diabetes mellitus. N. Engl. J. Med. 329(14):977–986.

Du, Y., Miller, C. M., and Kern, T. S. (2003). Hyperglycemia increases mitochondrial superoxide in retina and retinal cells.

Free Radic. Biol. Med. 235(11):1491–1499.

Economopoulou, M., Bdeir, K., Cines, D. B., Fogt, F., Bdeir, Y., Lubkowski, J., Lu, W., Preissner, K. T., Hammes, H. P., et al. (2005). Inhibition of pathologic retinal neovascularization by alpha-defensins. Blood 106(12):3831–3838.

Enge, M., Bjarnegard, M., Gerhardt, H., Gustafsson, E., Kalen, M., Asker, N., Hammes, H. P., Shani, M., Fassler, R., et al. (2002). Endothelium-specific platelet-derived growth factor-B ablation mimics diabetic retinopathy. EMBO J. 21(16): 4307–4316.

Engerman, R. L. (1989). Pathogenesis of diabetic retinopathy. Diabetes 38(10):1203–1206.

Engerman, R. L., and Bloodworth, J. M., Jr. (1965). Experimental diabetic retinopathy in dogs. Arch. Ophthalmol. 73:205– 210.

Engerman, R. L., and Kern, T. S. (1984). Experimental galactosemia produces diabetic-like retinopathy. Diabetes 33(1):97–100.

Engerman, R. L., and Kern, T. S. (1993). Aldose reductase inhibition fails to prevent retinopathy in diabetic and galactosemic dogs. Diabetes 42(6):820–825.

Engerman, R. L., and Kern, T. S. (1995). Retinopathy in galactosemic dogs continues to progress after cessation of galactosemia. Arch. Ophthalmol. 113(3):355–358.

Feit-Leichman, R. A., Kinouchi, R., Takeda, R. M., Fan, Z., Mohr, S., Kern, T. S., and Chen, D. F. (2005). Vascular damage in a mouse model of diabetic retinopathy: Relation to neuronal and glial changes. Invest. Ophthalmol. Vis. Sci. 46(11):4281– 4287.

Frank, R. N., et al. (1997). An aldose reductase inhibitor and aminoguanidine prevent vascular endothelial growth factor expression in rats with long-term galactosemia. Arch. Ophthalmol. 115(8):1036–1047.

Goldin, A., Amin, R., Kennedy, A., and Hohman, T. C. (2006). Advanced glycation end products: Sparking the development of diabetic vascular injury. Circulation 114(6):597–605.

Grant, M. B., Spoerri, P. E., Player, D. W. D., Bush, M., Ellis, E. A., Caballero, S., and Robison, W. G. (2000). Plasminogen activator inhibitor (PAI)-1 overexpression in retinal microvessels of PAI-1 transgenic mice. Invest. Ophthalmol. Vis. Sci. 41(8):2296– 2302.

Gubitosi-Klug, R., and Kern, T. (2006). A role for 5-Lipoxygenase in diabetic retinopathy. Presented at the annual meeting of the ADA, Washington, DC.

Gubitosi-Klug, R., and Kern, T. (2008). [5-Lipoxygenase, but not 12/5-lipoxygenase, contributes to degeneration of retinal capillaries in a mouse model of diabetic retinopathy.] Unpublished results.

Hammes, H. P., Lin, J., Renner, O., Shani, M., Lundqvist, A., Betsholtz, C., Brownlee, M., and Deutsch, U. (2002). Pericytes and the pathogenesis of diabetic retinopathy. Diabetes 51(10):3107–3112.

Hammes, H. P., Lin, J., Wagner, P., Feng, Y., Vom Hagen, F., Krzizok, T., Renner, O., Breier, G., Brownlee, M., et al. (2004). Angiopoietin-2 causes pericyte dropout in the normal retina: Evidence for involvement in diabetic retinopathy. Diabetes 53(4):1104–1110.

Igarashi, T., Miyake, K., Kato, K., Watanabe, A., Ishizaki, M., Ohara, K., and Shimada, T. (2003). Lentivirus-mediated expression of angiostatin efficiently inhibits neovascularization in a murine proliferative retinopathy model. Gene Ther. 10(3):219– 226.

Jandeleit-Dahm, K., and Cooper, M. E. (2006). Hypertension and diabetes: Role of the renin-angiotensin system. Endocrinol. Metab. Clin. North Am. 35(3):469–490, vii.

Joussen, A. M., Poulaki, V., Le, M. L., Koizumi, K., Esser, C., Janicki, H., Schraermeyer, U., Kociok, N., Fauser, S., et al. (2004). A central role for inflammation in the pathogenesis of diabetic retinopathy. FASEB J. 18(12):1450–1452.

Joussen, A. M., Poulaki, V., Tsujikawa, A., Qin, W., Qaum, T., Xu, W., Moromizato, Y., Bursell, S. E., Wiegand, S. J., et al. (2002). Suppression of diabetic retinopathy with angiopoietin-1. Am. J. Pathol. 160(5):1683–1693.

Kern, T. S., and Engerman, R. L. (1996). A mouse model of diabetic retinopathy. Arch. Ophthalmol. 114(8):986–990.

Kern, T. S., and Mohr, S. (2007). Nonproliferative diabetic retinopathy. In A. M. Joussen, T. W. Gardner, B. Kirchhof, and S. J. Ryan (Eds.), Ratinal vascular disease. Heidelberg: Springer.

Kern, T. S., Tang, J., Mizutani, M., Kowluru, R. A., Nagaraj, R. H., Romeo, G., Podesta, F., and Lorenzi, M. (2000). Response of capillary cell death to aminoguanidine predicts the development of retinopathy: Comparison of diabetes and galactosemia. Invest. Ophthalmol. Vis. Sci. 41(12):3972–3978.

Kohner, E. M., and Henkind, P. (1970). Correlation of fluorescein angiogram and retinal digest in diabetic retinopathy. Am. J. Ophthalmol. 69(3):403–414.

Kondo, T., Vicent, D., Suzuma, K., Yanagisawa, M., King, G. L., Holzenberger, M., and Kahn, C. R. (2003). Knockout of insulin and IGF-1 receptors on vascular endothelial cells protects against retinal neovascularization. J. Clin. Invest. 111(12): 1835–1842.

Kowluru, R. A. (2002). Retinal metabolic abnormalities in diabetic mouse: Comparison with diabetic rat. Curr. Eye Res. 24(2):123–128.

Kowluru, R. A., Kowluru, V., Xiong, Y., and Ho, Y. S. (2006). Overexpression of mitochondrial superoxide dismutase in mice protects the retina from diabetes-induced oxidative stress. Free Radic. Biol. Med. 41(8):1191–1196.

Kusner, L. L., Sarthy, V. P., and Mohr, S. (2004). Nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase: A role in high glucose-induced apoptosis in retinal Mˇuller cells. Invest. Ophthalmol. Vis. Sci. 45(5):1553–1561.

Lai, C. M., Dunlop, S. A., May, L. A., Gorbatov, M., Brankov, M., Shen, W. Y., Binz, N., Lai, Y. K., Graham, C. E., et al. (2005). Generation of transgenic mice with mild and severe retinal neovascularisation. Br. J. Ophthalmol. 89(7):911–916.

Leiter, E. H., Prochazka, M., and Coleman, D. L. (1987). The non-obese diabetic (NOD) mouse. Am. J. Pathol. 128(2):380– 383.

Lieth, E., Gardner, T. W., Barber, A. J., and Antonetti, D. A. (2000). Retinal neurodegeneration: Early pathology in diabetes.

Clin. Exp. Ophthalmol. 28(1):3–8.

Martin, P. M., Roon, P., Van Ells, T. K., Ganapathy, V., and Smith, S. B. (2004). Death of retinal neurons in streptozotocininduced diabetic mice. Invest. Ophthalmol. Vis. Sci. 45(9):3330– 3336.

Midena, E., Segato, T., Radin, S., di Giorgio, G., Meneghini, F., Piermarocchi, S., and Belloni, A. S. (1989). Studies on the retina of the diabetic db/db mouse. I. Endothelial cell-pericyte ratio. Ophthalmic Res. 21(2):106–111.

Mohr, S., Xi, X., Tang, J., and Kern, T. S. (2002). Caspase activation in retinas of diabetic and galactosemic mice and diabetic patients. Diabetes 51(4):1172–1179.

Moore, T. C., Moore, J. E., Kaji, Y., Frizzell, N., Usui, T., Poulaki, V., Campbell, I. L., Stitt, A. W., Gardiner, T. A.,

et al. (2003). The role of advanced glycation end products in retinal microvascular leukostasis. Invest. Ophthalmol. Vis. Sci. 44 (10):4457–4464.

Naeser, P., and Agren, A. (1978). Morphology and enzyme activities of the retinal capillaries in mice with the obese-hyperglycae- mic syndrome (gene symbol ob). Acta. Ophthalmol. (Copenh.) 56 (4):607–616.

Ning, X., Baoyu, Q., Yuzhen, L., Shuli, S., Reed, E., and Li, Q. Q. (2004). Neuro-optic cell apoptosis and microangiopathy in KKAY mouse retina. Int. J. Mol. Med. 13(1):87–92.

Obrosova, I. G., Drel, V. R., Kumagai, A. K., Szabo, C., Pacher, P., and Steven, M. J. (2006). Early diabetes-induced biochemical changes in the retina: Comparison of rat and mouse models. Diabetologia 49:2525–2233.

Ozaki, H., Hayashi, H., Vinores, S. A., Moromizato, Y., Campochiaro, P. A., and Oshima, K. (1997). Intravitreal sustained release of VEGF causes retinal neovascularization in rabbits and breakdown of the blood-retinal barrier in rabbits and primates. Exp. Eye Res. 64(4):505–517.

Robinson, G. S., Pierce, E. A., Rook, S. L., Foley, E., Webb, R., and Smith, L. E. (1996). Oligodeoxynucleotides inhibit retinal neovascularization in a murine model of proliferative retinopathy. Proc. Natl. Acad. Sci. U.S.A. 93(10):4851–4856.

Ruberte, J., Ayuso, E., Navarro, M., Carretero, A., Nacher, V., Haurigot, V., George, M., Llombart, C., Casellas, A., et al. (2004). Increased ocular levels of IGF-1 in transgenic mice lead to diabetes-like eye disease. J. Clin. Invest. 113(8):1149– 1157.

Saint-Geniez, M., and D’Amore, P. A. (2004). Development and pathology of the hyaloid, choroidal and retinal vasculature. Int. J. Dev. Biol. 48(8–9):1045–1058.

Sennlaub, F., Courtois, Y., and Goureau, O. (2002). Inducible nitric oxide synthase mediates retinal apoptosis in ischemic proliferative retinopathy. J. Neurosci. 22(10):3987–3993.

Sennlaub, F., Valamanesh, F., Vazquez-Tello, A., El-Asrar, A. M., Checchin, D., Brault, S., Gobeil, F., Beauchamp, M. H., Mwaikambo, B., et al. (2003). Cyclooxygenase-2 in human and experimental ischemic proliferative retinopathy. Circulation 108(2):198–204.

Siracusa, L. D. (1994). The agouti gene: Turned on to yellow. Trends Genet. 10(12):423–428.

Smith, L. E., Shen, W., Perruzzi, C., Soker, S., Kinose, F., Xu, X., Robinson, G., Driver, S., Bischoff, J., et al. (1999). Regulation of vascular endothelial growth factor-dependent retinal neovascularization by insulin-like growth factor-1 receptor. Nat. Med. 5(12):1390–1395.

Smith, L. E., Wesolowski, E., McLellan, A., Kostyk, S. K., D’Amato, R., Sullivan, R., and D’Amore, P. A. (1994). Oxygeninduced retinopathy in the mouse. Invest. Ophthalmol. Vis. Sci. 35(1):101–111.

Sone, H., Kawakami, Y., Segawa, T., Okuda, Y., Sekine, Y., Honmura, S., Segawa, T., Suzuki, H., Yamashita, K., et al. (1999). Effects of intraocular or systemic administration of neutralizing antibody against vascular endothelial growth factor on the murine experimental model of retinopathy. Life Sci. 65(24):2573–2580.

Suganami, E., Takagi, H., Ohashi, H., Suzuma, K., Suzuma, I., Oh, H., Watanabe, D., Ojima, T., Suganami, T., et al. (2004). Leptin stimulates ischemia-induced retinal neovascularization: possible role of vascular endothelial growth factor expressed in retinal endothelial cells. Diabetes 53(9):2443–2448.

Tatton, W. G., Chalmers-Redman, R. M., Elstner, M., Leesch, W., Jagodzinski, F. B., Stupak, D. P., Sugrue, M. M., and

Tatton, N. A. (2000). Glyceraldehyde-3-phosphate dehydrogenase in neurodegeneration and apoptosis signaling. J. Neural Transm. Suppl. 2000(60):77–100.

Tolentino, M. J., Miller, J. W., Gragoudas, E. S., Jakobiec, F. A., Flynn, E., Chatzistefanou, K., Ferrara, N., and Adamis, A. P. (1996). Intravitreous injections of vascular endothelial growth factor produce retinal ischemia and microangiopathy in an adult primate. Ophthalmology 103(11):1820–1828.

Vincent, J. A., and Mohr, S. (2007). Inhibition of caspase-1/ interleukin-1β signaling prevents degeneration of retinal capillaries in diabetes and galactosemia. Diabetes 56(1):224–230.

Vinores, S. A., Seo, M. S., Okamoto, N., Ash, J. D., Wawrousek, E. F., Xiao, W. H., Hudish, T., Derevjanik, N. L., and Campochiaro, P. A. (2000). Experimental models of growth factor-mediated angiogenesis and blood-retinal barrier breakdown. Gen. Pharmacol. 35(5):233–239.

Vlassara, H. (2001). The AGE-receptor in the pathogenesis of diabetic complications. Diabetes Metab. Res. Rev. 17(6):436–443.

Watanabe, D., Suzuma, K., Matsui, S., Kurimoto, M., Kiryu, J., Kita, M., Suzuma, I., Ohashi, H., Ojima, T., et al. (2005). Erythropoietin as a retinal angiogenic factor in proliferative diabetic retinopathy. N. Engl. J. Med. 353(8):782–792.

Whetzel, A. M., Bolick, D. T., Srinivasan, S., Macdonald, T. M., Morris, M. A., Ley, K., and Hedrick, C. C. (2006). Sphingosine-1 phosphate prevents monocyte/endothelial interactions in type 1 diabetic NOD mice through activation of the S1P1 receptor. Circ. Res. 99(7):731–739.

Wong, C. G., Rich, K. A., Liaw, L. H., Hsu, H. T., and Berns, M. W. (2001). Intravitreal VEGF and bFGF produce florid retinal neovascularization and hemorrhage in the rabbit. Curr. Eye Res. 22(2):140–147.

Yamaoka, T., Nishimura, C., Yamashita, K., Itakura, M., Yamada, T., Fujimoto, J., and Kokai, Y. (1995). Acute onset of diabetic pathological changes in transgenic mice with human aldose reductase cDNA. Diabetologia 38(3):255–261.

Zhang, L., Krzentowski, G., Albert, A., and Lefebvre, P. J. (2001). Risk of developing retinopathy in Diabetes Control and Complications Trial type 1 diabetic patients with good or poor metabolic control. Diabetes Care 24(7):1275–1279.

Zheng, L., Du, Y., Miller, C., Gubitosi-Klug, R. A., Kern, T. S., Ball, S., and Berkowitz, R. A. (2007). Critical role of inducible nitric oxide synthase in degeneration of retinal capillaries in mice with streptozotocin-induced diabetes. Diabetologia 50(9):1987–1996.