Regulation of Adrenergic, Serotonin, and Dopamine Receptors to Inhibit Diabetic Retinopathy: Monotherapies versus Combination Therapies.

We compared monotherapies and combinations of therapies that regulate G-protein-coupled receptors (GPCRs) with respect to their abilities to inhibit early stages of diabetic retinopathy (DR) in streptozotocin-diabetic mice. Metoprolol (MTP; 0.04-1.0 mg/kg b.wt./day), bromocriptine (BRM; 0.01-0.1 mg/kg b.wt./day), doxazosin (DOX; 0.01-1.0 mg/kg b.wt./day), or tamsulosin (TAM; 0.05-0.25 mg/kg b.wt./day) were injected individually daily for 2 months in dose-response studies to assess their effects on the diabetes-induced increases in retinal superoxide and leukocyte-mediated cytotoxicity against vascular endothelial cells, both of which abnormalities have been implicated in the development of DR. Each of the individual drugs inhibited the diabetes-induced increase in retinal superoxide at the higher concentrations tested, but the inhibition was lost at lower doses. To determine whether combination therapies had superior effects over individual drugs, we intentionally selected for each drug a low dose that had little or no effect on the diabetes-induced retinal superoxide for use separately or in combinations in 8-month studies of retinal function, vascular permeability, and capillary degeneration in diabetes. At the low doses used, combinations of the drugs generally were more effective than individual drugs, but the low-dose MTP alone totally inhibited diabetes-induced reduction in a vision task, BRM or DOX alone totally inhibited the vascular permeability defect, and DOX alone totally inhibited diabetes-induced degeneration of retinal capillaries. Although low-dose MTP, BRM, DOX, or TAM individually had beneficial effects on some endpoints, combination of the therapies better inhibited the spectrum of DR lesions evaluated. SIGNIFICANCE STATEMENT: The pathogenesis of early stages of diabetic retinopathy remains incompletely understood, but multiple different cell types are believed to be involved in the pathogenic process. We have compared the effects of monotherapies to those of combinations of drugs that regulate GPCR signaling pathways with respect to their relative abilities to inhibit the development of early diabetic retinopathy.

Diabetic Retinopathy: Retina-Specific Methods for Maintenance of Diabetic Rodents and Evaluation of Vascular Histopathology and Molecular Abnormalities.

Diabetic retinopathy is a major cause of visual impairment, which continues to increase in prevalence as more and more people develop diabetes. Despite the importance of vision, the retina is one of the smallest tissues in the body, and specialized techniques have been developed to study retinopathy. This article summarizes several methods used to (i) induce diabetes in mice, (ii) maintain the diabetic animals throughout the months required for development of typical vascular histopathology, (iii) evaluate vascular histopathology of diabetic retinopathy, and (iv) quantitate abnormalities implicated in the development of the retinopathy.

Retinylamine Benefits Early Diabetic Retinopathy in Mice.

Recent evidence suggests an important role for outer retinal cells in the pathogenesis of diabetic retinopathy (DR). Here we investigated the effect of the visual cycle inhibitor retinylamine (Ret-NH2) on the development of early DR lesions. Wild-type (WT) C57BL/6J mice (male, 2 months old when diabetes was induced) were made diabetic with streptozotocin, and some were given Ret-NH2 once per week. Lecithin-retinol acyltransferase (LRAT)-deficient mice and P23H mutant mice were similarly studied. Mice were euthanized after 2 (WT and Lrat(-/-)) and 8 months (WT) of study to assess vascular histopathology, accumulation of albumin, visual function, and biochemical and physiological abnormalities in the retina. Non-retinal effects of Ret-NH2 were examined in leukocytes treated in vivo. Superoxide generation and expression of inflammatory proteins were significantly increased in retinas of mice diabetic for 2 or 8 months, and the number of degenerate retinal capillaries and accumulation of albumin in neural retina were significantly increased in mice diabetic for 8 months compared with nondiabetic controls. Administration of Ret-NH2 once per week inhibited capillary degeneration and accumulation of albumin in the neural retina, significantly reducing diabetes-induced retinal superoxide and expression of inflammatory proteins. Superoxide generation also was suppressed in Lrat(-/-) diabetic mice. Leukocytes isolated from diabetic mice treated with Ret-NH2 caused significantly less cytotoxicity to retinal endothelial cells ex vivo than did leukocytes from control diabetics. Administration of Ret-NH2 once per week significantly inhibited the pathogenesis of lesions characteristic of early DR in diabetic mice. The visual cycle constitutes a novel target for inhibition of DR.

Adrenergic and serotonin receptors affect retinal superoxide generation in diabetic mice: relationship to capillary degeneration and permeability.

Reactive oxygen species play an important role in the pathogenesis of diabetic retinopathy. We studied the role of adrenergic and serotonin receptors in the generation of superoxide by retina and 661W retinal cells in high glucose and of the α1-adrenergic receptor (AR) on vascular lesions of the retinopathy in experimentally diabetic C57Bl/6J mice (and controls) after 2 and 8 months. Compared with 5 mM glucose, incubating cells or retinal explants in 30 mM glucose induced superoxide generation. This response was reduced or ablated by pharmacologic inhibition of the α1-AR (a Gq-coupled receptor) or Gs-coupled serotonin (5-HT2, 5-HT4, 5-HT6, and 5-HT7) receptors or by activation of the Gi-coupled α2-AR. In elevated glucose, the α1-AR produced superoxide via phospholipase C, inositol triphosphate-induced Ca(2+) release, and NADPH oxidase, and pharmacologic inhibition of these reactions prevented the superoxide increase. Generation of retinal superoxide, expression of proinflammatory proteins, and degeneration of retinal capillaries in diabetes all were significantly inhibited with daily doxazosin or apocynin (inhibitors of α1-AR and NADPH oxidase, respectively), but increased vascular permeability was not significantly affected. Adrenergic receptors, and perhaps other GPCRs, represent novel targets for inhibiting the development of important features of diabetic retinopathy.

Metanx and early stages of diabetic retinopathy.

PURPOSE: l-Methylfolate, pyridoxal 5'-phosphate, and methylcobalamin, individually have been reported to have beneficial effects on diabetes-induced defects. The possibility that combining these therapeutic approaches might have additional benefit led us to investigate the effect of Metanx against development of early stages of diabetic retinopathy in a mouse model. METHODS: C57BL/6J mice were made diabetic with streptozotocin, and some were given Metanx (a combination food product) mixed in the food at a dose of 5 mg/kg of body weight. Mice were killed at 2 months and 10 months of study for assessment of retinal function, retinal vascular histopathology, accumulation of albumin in neural retina, and biochemical and physiological abnormalities in retina. RESULTS: Two months of diabetes significantly increased leukostasis within retinal vessels and superoxide generation by the retina. Diabetes also significantly increased expression of intercellular adhesion molecule-1 (ICAM-1) and phosphorylation of IκB. Daily consumption of Metanx significantly inhibited all of these abnormalities. Ten months of diabetes significantly increased the degeneration of retinal capillaries and impaired visual function (spatial frequency threshold (SFT) and a parameter of contrast sensitivity) compared to nondiabetic controls. Daily consumption of Metanx for 10 months inhibited impairment of SFT but had no significant beneficial effect on capillary degeneration, pericyte loss, or the estimate of contrast sensitivity. CONCLUSIONS: Metanx inhibited a diabetes-induced defect in retinal spatial frequency threshold and inhibited measures of oxidative stress and inflammation. It had no significant effect on contrast sensitivity or retinal capillary degeneration. Nutritional management with Metanx may help inhibit diabetes-induced defects in visual function.

Diabetes-induced impairment in visual function in mice: contributions of p38 MAPK, rage, leukocytes, and aldose reductase.

PURPOSE: Visual function is impaired in diabetes, but molecular causes of this dysfunction are not clear. We assessed effects of diabetes on visual psychophysics in mice, and tested the effect of therapeutic approaches reported previously to inhibit vascular lesions of the retinopathy. METHODS: We used the optokinetic test to assess contrast sensitivity and spatial frequency threshold in diabetic C57Bl/6J mice and age-matched nondiabetic controls between 2 and 10 months of diabetes. Contributions of p38 MAP kinase (MAPK), receptor for advanced glycation end products (RAGE), leukocytes, and aldose reductase (AR) to the defect in contrast sensitivity were investigated. Cataract, a potential contributor to reductions in vision, was scored. RESULTS: Diabetes of 2 months' duration impaired contrast sensitivity and spatial frequency threshold in mice. The defect in contrast sensitivity persisted for at least 10 months, and cataract did not account for this impairment. Diabetic mice deficient in AR were protected significantly from development of the diabetes-induced defects in contrast sensitivity and spatial frequency threshold. In contrast, pharmacologic inhibition of p38 MAPK or RAGE, or deletion of inducible nitrous oxide synthase (iNOS) from bone marrow-derived cells did not protect the visual function in diabetes. CONCLUSIONS: Diabetes reduces spatial frequency threshold and contrast sensitivity in mice, and the mechanism leading to development of these defects involves AR. The mechanism by which AR contributes to the diabetes-induced defect in visual function can be probed by identifying which molecular abnormalities are corrected by AR deletion, but not other therapies that do not correct the defect in visual function.

Low-intensity far-red light inhibits early lesions that contribute to diabetic retinopathy: in vivo and in vitro.

PURPOSE: Treatment with light in the far-red to near-infrared region of the spectrum (photobiomodulation [PBM]) has beneficial effects in tissue injury. We investigated the therapeutic efficacy of 670-nm PBM in rodent and cultured cell models of diabetic retinopathy. METHODS: Studies were conducted in streptozotocin-induced diabetic rats and in cultured retinal cells. Diabetes-induced retinal abnormalities were assessed functionally, biochemically, and histologically in vivo and in vitro. RESULTS: We observed beneficial effects of PBM on the neural and vascular elements of retina. Daily 670-nm PBM treatment (6 J/cm(2)) resulted in significant inhibition in the diabetes-induced death of retinal ganglion cells, as well as a 50% improvement of the ERG amplitude (photopic b wave responses) (both P < 0.01). To explore the mechanism for these beneficial effects, we examined physiologic and molecular changes related to cell survival, oxidative stress, and inflammation. PBM did not alter cytochrome oxidase activity in the retina or in cultured retinal cells. PBM inhibited diabetes-induced superoxide production and preserved MnSOD expression in vivo. Diabetes significantly increased both leukostasis and expression of ICAM-1, and PBM essentially prevented both of these abnormalities. In cultured retinal cells, 30-mM glucose exposure increased superoxide production, inflammatory biomarker expression, and cell death. PBM inhibited all of these abnormalities. CONCLUSIONS: PBM ameliorated lesions of diabetic retinopathy in vivo and reduced oxidative stress and cell death in vitro. PBM has been documented to have minimal risk. PBM is noninvasive, inexpensive, and easy to administer. We conclude that PBM is a simple adjunct therapy to attenuate the development of diabetic retinopathy.

Exclusion of aldose reductase as a mediator of ERG deficits in a mouse model of diabetic eye disease.

Streptozotocin (STZ)-induced diabetes is associated with reductions in the electrical response of the outer retina and retinal pigment epithelium (RPE) to light. Aldose reductase (AR) is the first enzyme required in the polyol-mediated metabolism of glucose, and AR inhibitors have been shown to improve diabetes-induced electroretinogram (ERG) defects. Here, we used control and AR -/- mice to determine if genetic inactivation of this enzyme likewise inhibits retinal electrophysiological defects observed in a mouse model of type 1 diabetes. STZ was used to induce hyperglycemia and type 1 diabetes. Diabetic and age-matched nondiabetic controls of each genotype were maintained for 22 weeks, after which ERGs were used to measure the light-evoked components of the RPE (dc-ERG) and the neural retina (a-wave, b-wave). In comparison to their nondiabetic controls, wildtype (WT) and AR -/- diabetic mice displayed significant decreases in the c-wave, fast oscillation, and off response components of the dc-ERG but not in the light peak response. Nondiabetic AR -/- mice displayed larger ERG component amplitudes than did nondiabetic WT mice; however, the amplitude of dc-ERG components in diabetic AR -/- animals were similar to WT diabetics. ERG a-wave amplitudes were not reduced in either diabetic group, but b-wave amplitudes were lower in WT and AR -/-diabetic mice. These findings demonstrate that the light-induced responses of the RPE and outer retina are disrupted in diabetic mice, but these defects are not due to photoreceptor dysfunction, nor are they ameliorated by deletion of AR. This latter finding suggests that benefits observed in other studies utilizing pharmacological inhibitors of AR might have been secondary to off-target effects of the drugs.

Beneficial effects of a novel RAGE inhibitor on early diabetic retinopathy and tactile allodynia.

PURPOSE: The receptor for advanced glycation end products (RAGE) has been implicated in the pathogenesis of numerous complications of diabetes. We assessed the effect of a novel RAGE fusion protein inhibitor on retinal histopathology and nerve function, and on retinal inflammation and oxidative stress. METHODS: C57BL/6J mice were made diabetic with streptozotocin, and some were given a RAGE fusion protein (10, 100, or 300 µg per mouse 3 times per week). Mice were sacrificed at 2 months and 10 months into the study to assess retinal vascular histopathology, accumulation of albumin in the neural retina, cell loss in the ganglion cell layer, and biochemical and physiologic abnormalities in the retina. Tactile allodynia (light touch) was measured on a paw of each animal at 2 months. RESULTS: Leukostasis, expression of the intercellular adhesion molecule-1 (ICAM-1), accumulation of albumin in the neural retina, and nitration of retinal proteins were significantly increased in the retinas of mice diabetic for 2 months. The number of degenerate retinal capillaries was significantly increased in mice diabetic for 10 months, compared to the nondiabetic controls. Diabetes also enhanced sensitivity of peripheral nerves to tactile allodynia. All three doses of the RAGE fusion protein inhibited capillary degeneration, accumulation of albumin in the neural retina, nitration of retinal proteins, and tactile allodynia, demonstrating that biologically meaningful levels of the drug reached the retina. RAGE inhibition did tend to inhibit diabetes-induced retinal leukostasis and ICAM-1 expression (previously postulated to be important in the pathogenesis of retinopathy), but these effects were not statistically significant for the use of the lower doses of the drug that normalized the vascular histopathology. CONCLUSIONS: Inhibition of RAGE blocked the development of important lesions of diabetic retinopathy, but these beneficial effects seemed not to be mediated via leukostasis. RAGE inhibition also blocked the development of sensory allodynia in diabetes. RAGE is an important therapeutic target to inhibit the development of vascular and neural complications of diabetes.

Effects of p38 MAPK inhibition on early stages of diabetic retinopathy and sensory nerve function.

Purpose. p38 mitogen-activated protein kinase (MAPK) is known to play a regulatory role in inflammatory processes in disease. Inflammation has been linked also to the development of diabetic retinopathy in rodents. This study was conducted to evaluate the effect of a p38 MAPK inhibitor on the development of early stages of diabetic retinopathy in rats. Methods. Streptozotocin-diabetic rats were assigned to two groups-treated with the p38 MAPK inhibitor PHA666859 (Pfizer, New York, NY) and untreated-and compared with age-matched nondiabetic control animals. Results. At 2 months of diabetes, insulin-deficient diabetic control rats exhibited significant increases in retinal superoxide, nitric oxide (NO), cyclooxygenase (COX)-2, and leukostasis within retinal microvessels. All these abnormalities were significantly inhibited by the p38 MAPK inhibitor (25 mg/kgBW/d). At 10 months of diabetes, significant increases in the number of degenerate (acellular) capillaries and pericyte ghosts were measured in control diabetic rats versus those in nondiabetic control animals, and pharmacologic inhibition of p38 MAPK significantly inhibited all these abnormalities (all P < 0.05). This therapy also had beneficial effects outside the eye in diabetes, as evidenced by the inhibition of a diabetes-induced hypersensitivity of peripheral nerves to light touch (tactile allodynia). Conclusions. p38 MAPK plays an important role in diabetes-induced inflammation in the retina, and inhibition of p38 MAPK offers a novel therapeutic approach to inhibiting the development of early stages of diabetic retinopathy and other complications of diabetes.

Markers of glycemic control in the mouse: comparisons of 6-h- and overnight-fasted blood glucoses to Hb A1c.

The present studies examined the relationship between fasting blood glucose and Hb A(1c) in C57BL/6J, DBA/2J, and KK/HlJ mice with and without diabetes mellitus. Daily averaged blood glucose levels based on continuous glucose monitoring and effects of 6-h vs. overnight fasting on blood glucose were determined. Daily averaged blood glucose levels were highly correlated with Hb A(1c), as determined with a hand-held automated device using an immunodetection method. R(2) values were 0.90, 0.95, and 0.99 in KK/HIJ, C57BL/6J, and DBA/2J, respectively. Six-hour fasting blood glucose correlated more closely with the level of daily averaged blood glucose and with Hb A(1c) than did blood glucose following an overnight fast. To validate the immunoassay-determined Hb A(1c), we also measured total glycosylated hemoglobin using boronate HPLC. Hb A(1c) values correlated well with total glycosylated hemoglobin in all three strains but were relatively lower than total glycosylated hemoglobin in diabetic DBA/2J mice. These results show that 6-h fasting glucose provides a superior index of glycemic control and correlates more closely with Hb A(1c) than overnight-fasted blood glucose in these strains of mice.