OBJECTIVE: During diabetes, retinal microglial cells are activated to release inflammatory cytokines that initiate neuronal loss and blood–retinal barrier breakdown seen in diabetic retinopathy (DR). The mechanism by which diabetes activates microglia to release those inflammatory mediators is unclear and was therefore elucidated.RESEARCH DESIGN AND METHODS: Microglia activation was characterized in streptozocin-injected rats and in isolated microglial cells using immunofluorescence, enzyme-linked immunosorbent assay, RT-PCR, and Western blot analyses.RESULTS: In 8-week diabetic retina, phospho-extracellular signal–related kinase (ERK) and P38 mitogen-activated protein kinases were localized in microglia, but not in Mueller cells or astrocytes. At the same time, Amadori-glycated albumin (AGA)-like epitopes were featured in the regions of microglia distribution, implicating a pathogenic effect on microglial activation. To test this, diabetic rats were treated intravitreally with A717, a specific AGA-neutralizing antibody, or murine IgG. Relative to nondiabetic rats, diabetic rats (IgG-treated) manifested 3.9- and 7.9-fold increases in Iba-1 and tumor necrosis factor (TNF)-α mRNAs, respectively. Treatment of diabetic rats with A717 significantly attenuated overexpression of these mRNAs. Intravitreal injection of AGA per se in normal rats resulted in increases of Iba-1 expression and TNF-α release. Guided by these results, a cultured retinal microglia model was developed to study microglial response after AGA treatment and the mechanistic basis behind this response. The results showed that formation of reactive oxygen species and subsequent activation of ERK and P38, but not Jun NH2-terminal kinase, are molecular events underpinning retinal microglial TNF-α release during AGA treatment.CONCLUSIONS: These results provide new insights in understanding the pathogenesis of early DR, showing that the accumulated AGA within the diabetic retina elicits the microglial activation and secretion of TNF-α. Thus, intervention trials with agents that neutralize AGA effects may emerge as a new therapeutic approach to modulate early pathologic pathways long before the occurrence of vision loss among patients with diabetes.

BACKGROUND AND PURPOSE Up-regulation of thioredoxin interacting protein (TXNIP), an endogenous inhibitor of thioredoxin (Trx), compromises cellular antioxidant and anti-apoptotic defences and stimulates pro-inflammatory cytokines expression, implying a role for TXNIP in apoptosis. Here we have examined the causal role of TXNIP expression in mediating retinal neurotoxicity and assessed the neuroprotective actions of verapamil, a calcium channel blocker and an inhibitor of TXNIP expression. EXPERIMENTAL APPROACH Retinal neurotoxicity was induced by intravitreal injection of NMDA in Sprague–Dawley rats, which received verapamil (10 mg·kg⁻¹, p.o.) or vehicle. Neurotoxicity was examined by terminal dUTP nick-end labelling assay and ganglion cell count. Expression of TXNIP, apoptosis signal-regulating kinase 1 (ASK-1), NF-κB, p38 MAPK, JNK, cleaved poly-ADP-ribose polymerase (PARP), caspase-3, nitrotyrosine and 4-hydroxy-nonenal were examined by Western and slot-blot analysis. Release of TNF-α and IL-1β was examined by elisa. KEY RESULTS NMDA injection enhanced TXNIP expression, decreased Trx activity, causing increased oxidative stress, glial activation and release of TNF-α and IL-1β. Enhanced TXNIP expression disrupted Trx/ASK-1 inhibitory complex leading to release of ASK-1 and activation of the pro-apoptotic p38 MAPK/JNK pathway, as indicated by cleaved PARP and caspase-3 expression. Treatment with verapamil blocked these effects. CONCLUSION AND IMPLICATIONS Elevated TXNIP expression contributed to retinal neurotoxicity by three different mechanisms, inducing release of inflammatory mediators such as TNF-α and IL-1β, altering antioxidant status and disrupting the Trx-ASK-1 inhibitory complex leading to activation of the p38 MAPK/JNK apoptotic pathway. Targeting TXNIP expression is a potential therapeutic target for retinal neurodegenerative disease.

Sodium nitrite, a food additive that is used as a color fixative and preservative for meats and fish, has been reported to have adverse health effects due to increased oxidative stress that could be harmful to different organs including the liver. Meanwhile, silymarin protects against hepatotoxicity caused by a variety of agents, on account of its antioxidative and anti-inflammatory effects. We therefore examined the impact of dietary silymarin on sodium nitrite-induced liver damage in rats. Fifty adult male Sprague-Dawley rats received 80 mg/kg sodium nitrite in the presence or absence of silymarin (10 and 25 mg/kg). Hepatic proinflammatory cytokines (TNF-α and IL-1β), hepatic fibrosis marker (MCP-1 and TGF-β1), mitochondrial activity marker (cytochrome C oxidase) and c-reactive protein (CRP) levels were measured. Hepatic apoptosis was assessed through determination of caspase-3 activity and DNA fragmentation. We found that oral sodium nitrite enhanced oxidative stress with subsequent increases in TNF-α (2-fold), IL-1β (4-fold), MCP-1 (4-fold), TGF-β1 (3-fold) and CRP (4-fold). In addition, sodium nitrite brings about reduced cytochrome C oxidase and enhanced caspase-3 activity and DNA fragmentation. Daily treatment with silymarin markedly ameliorated all these effects. Silymarin ameliorated the impairment of hepatic function in rats that had ingested sodium nitrite. Silymarn possesses antioxidant, anti-inflammatory, antifibrotic and anti-apoptotic effects.

Aims/hypothesis Recent studies have demonstrated that cannabinoid-1 (CB₁) receptor blockade ameliorated inflammation, endothelial and/or cardiac dysfunction, and cell death in models of nephropathy, atherosclerosis and cardiomyopathy. However the role of CB₁ receptor signalling in diabetic retinopathy remains unexplored. Using genetic deletion or pharmacological inhibition of the CB₁ receptor with SR141716 (rimonabant) in a rodent model of diabetic retinopathy or in human primary retinal endothelial cells (HREC) exposed to high glucose, we explored the role of CB₁ receptors in the pathogenesis of diabetic retinopathy. Methods Diabetes was induced using streptozotocin in C57BL/6J Cb ₁ (also known as Cnr1)^+/+ and Cb ₁ ^−/− mice aged 8 to 12 weeks. Samples from mice retina or HREC were used to determine: (1) apoptosis; (2) activity of nuclear factor kappa B, intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), poly (ADP-ribose) polymerase and caspase-3; (3) content of 3-nitrotyrosine and reactive oxygen species; and (4) activation of p38/Jun N-terminal kinase/mitogen-activated protein kinase (MAPK). Results Deletion of CB₁ receptor or treatment of diabetic mice with CB₁ receptor antagonist SR141716 prevented retinal cell death. Treatment of diabetic mice or HREC cells exposed to high glucose with SR141716 attenuated the oxidative and nitrative stress, and reduced levels of nuclear factor κB, ICAM-1 and VCAM-1. In addition, SR141716 attenuated the diabetes- or high glucose-induced pro-apoptotic activation of MAPK and retinal vascular cell death. Conclusions/interpretation Activation of CB₁ receptors may play an important role in the pathogenesis of diabetic retinopathy by facilitating MAPK activation, oxidative stress and inflammatory signalling. Conversely, CB₁ receptor inhibition may be beneficial in the treatment of this devastating complication of diabetes.