Chieh Allen Lee

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.

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.

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.

MyD88-Dependent Pathways in Leukocytes Affect the Retina in Diabetes

Background Previous studies by us and other have provided evidence that leukocytes play a critical role in the development of diabetic retinopathy, suggesting a possible role of the innate immune system in development of the retinopathy. Since MyD88 is a convergence point for signaling pathways of the innate immune system (including Toll-Like Receptors (TLRs) and interleukin-1ß (IL-1ß)), the purpose of this study was to assess the role of MyD88 and its dependent pathways on abnormalities that develop in retina and white blood cells related to diabetic retinopathy. Methods C57BL/6J mice were made diabetic with streptozotocin. Chimeric mice were generated in which MyD88-dependent pathways were deleted from bone marrow-derived only. Mice were sacrificed at 2 mos of diabetes for assessment of, leukostasis, albumin accumulation in neural retina, leukocyte-mediated killing of retinal endothelial cells, and cytokine/chemokine generation by retinas of diabetic mice in response to TLR agonists, Results IL-6 and CXCL1 were generated in retinas from diabetic (but not nondiabetic mice) following incubation with Pam3CysK/TLR2, but incubation with other TLR ligands or IL-1ß did not induce cytokine production in retinas from nondiabetic or diabetic mice. Diabetes-induced abnormalities (leukostasis, ICAM-1 expression on the luminal surface of the vascular endothelium, retinal superoxide generation) were significantly inhibited by removing either MyD88 or the signaling pathways regulated by it (TLRs 2 and 4, and IL-1ß) from bone marrow-derived cells only. Leukocyte-mediated killing of endothelial cells tended to be decreased in the marrow-derived cells lacking TLR2/4, but the killing was significantly exacerbated if the marrow cells lacked MyD88 or the receptor for IL-1ß (IL-1ßr). Conclusions MyD88-dependent pathways play an important role in the development of diabetes-induced inflammation in the retina, and inhibition of MyD88 might be a novel target to inhibit early abnormalities of diabetic retinopathy and other complications of diabetes.

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 Ca2+ 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.—Du, Y., Cramer, M., Lee, C. A., Tang, J., Muthusamy, A., Antonetti, D. A., Jin, H., Palczewski, K., Kern, T. S. Adrenergic and serotonin receptors affect retinal superoxide generation in diabetic mice: relationship to capillary degeneration and permeability. FASEB J. 29, 2194‐2204 (2015). www.fasebj.org

Retinylamine Benefits Early Diabetic Retinopathy in Mice.

Background: The development of diabetic retinopathy (DR) is incompletely understood. Administered retinylamine is stored in the retinal pigmented epithelium (RPE) where it affects the ocular visual cycle. Results: Retinylamine inhibited vascular and neural lesions of early DR. Conclusion: Both the RPE and visual cycle are novel targets for the inhibition of DR. Significance: Vision-related processes can contribute to DR. 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.

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.

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.