Baicalein reduces inflammatory process in a rodent model of diabetic retinopathy.

PURPOSE: This study was designed to elucidate the role of inflammatory process in diabetic retinopathy and to investigate the effect of baicalein treatment on diabetic rat. METHODS: Retinal microglial cells were identified with CD11b antibody, and retinal Müller cells were identified with glial fibrillary acidic protein (GFAP). The gene expression of interleukin (IL)-18, tumor necrosis factor (TNF)-alpha, and IL-1beta was examined by quantitative real-time PCR. The expression of GFAP and vascular endothelial growth factor (VEGF) was examined by quantitative real-time PCR, immunohistochemistry, and Western blot analysis. Vascular permeability was measured in vivo by bovine serum albumin conjugated with FITC. Baicalein was given by oral administration (150 mg/kg/d) with an animal feeding needle beginning 5 days after streptozotocin (STZ) injection. RESULTS: By 24 weeks after onset of diabetes, microglial cells were activated and proliferated, and Müller cells upregulated their GFAP and VEGF expression. Pro-inflammatory factors, including IL-18, TNF-alpha, and IL-1beta, were significantly upregulated. Obvious vascular leakage and abnormality were demonstrated, and ganglion cell loss was significant. Baicalein treatment ameliorated diabetes-induced microglial activation and pro-inflammatory expression, reduced the GFAP and VEGF expression from Müller cells, and significantly reduced vascular abnormality and ganglion cell loss within the retina. CONCLUSIONS: Inflammatory process, characterized by microglial activation and Müller cells dysfunction, was implicated in STZ-induced diabetic retinopathy. Baicalein treatment ameliorated inflammatory process, and therefore inhibited vascular abnormality and neuron loss in diabetic retinas.

[Activation of endoplasmic reticulum stress proteins in light-induced retinal degeneration].

OBJECTIVE: To investigate the expression of endoplasmic reticulum stress proteins in photoreceptor apoptosis in light-induced retinal degeneration. METHODS: Exposure to excessive levels of light induced photoreceptor apoptosis and had been previously used as a model for the study of retinal degeneration. Photoreceptor apoptosis was detected by terminal dUTP transferase nick end labeling (TUNEL). The protein expression levels of ER stress sensors including glucosejregulated protein-78 (GRP78/BiP), caspase-12, phospho-eukaryotic initiation factor 2alpha (eIF2alpha) and phospho- double-stranded RNA-activated protein kinase-like endoplasmic reticulum kinase (PERK) were examined by immunojfluorescence and Western blot analysis. RESULTS: Following light exposure, the protein expression levels of GRP78/BiP, caspase-12, phospho-eIF2alpha and phospho-PERK were up-regulated in a time dependent manner. The up-regulation of these proteins coincided with or preceded the photoreceptor apoptosis. At the peak of their expression, they were mainly located in the photoreceptor inner segments and/or outer nuclear layers (ONL). CONCLUSION: Activation of endoplasmic reticulum stress proteins appears to play an important role in light-induced retinal degeneration. Therefore endoplasmic reticulum stress modulators could become a strong candidate for a therapeutic agent in treatment of these diseases.

Endoplasmic reticulum stress is activated in light-induced retinal degeneration.

Exposure to excessive levels of light induces photoreceptor apoptosis and has previously been used as a model for the study of retinal degeneration. During the light exposure, intracellular calcium levels increase, and reactive oxygen species (ROS) are generated, which have been shown to cause endoplasmic reticulum (ER) stress. In the present study, we investigated the role of ER stress in light-induced photoreceptor apoptosis. Our study demonstrated that, after light exposure, the ER stress sensors including glucose-regulated protein-78 (GRP78/BiP), caspase-12, phospho-eukaryotic initiation factor 2 alpha (eIF2 alpha), and phospho-pancreatic ER kinase (PERK) were significantly up-regulated in a time-dependent manner. The up-regulation of these proteins coincided with or preceded the photoreceptor apoptosis indicated by TUNEL. These data showed that ER stress played an important role in light-induced photoreceptor apoptosis. Therefore, ER stress modulators could be strong candidates as therapeutic agents in the treatment of retinal degenerative diseases.

[Endoplasmic reticulum stress proteins are activated in rd retinal degeneration].

OBJECTIVE: To investigate the expression of endoplasmic reticulum (ER) stress proteins in photoreceptor apoptosis in rd mouse (Pde6bRd1/Rd1). METHODS: Photoreceptor apoptosis in rd mouse was detected by terminal dUTP transferase nick end labeling (TUNEL). The protein expression of ER stress sensors including glucose-regulated protein-78 (GRP78/BiP), caspase-12, phospho-eukaryotic initiation factor 2alpha (eIF2alpha) and phospho-pancreatic ER kinase (PERK) was examined by immunofluorescence and Western Blot analysis. RESULTS: Accompanying photoreceptor apoptosis in rd mouse, protein expression of GRP78/BiP, caspase-12, phospho-eIF2alpha and phospho-PERK was up-regulated in a time dependent manner. The up-regulation of these proteins coincided with or preceded the photoreceptor apoptosis. At the peak of their expression, they were mainly located in the photoreceptor inner segment and/or outer nuclear layer (ONL). CONCLUSION: Activation of ER stress proteins appears to play an important role in rd retinal degeneration. Therefore endoplasmic reticulum stress modulators could be a strong candidate as a therapeutic agent in the treatment of these diseases.

Activation of endoplasmic reticulum stress in degenerating photoreceptors of the rd1 mouse.

PURPOSE: Endoplasmic reticulum (ER) stress has been implicated in a wide variety of neurodegenerative disorders of the central nervous system (CNS). This study was designed to elucidate the role of ER stress in photoreceptor apoptosis in the rd1 mouse. METHODS: Photoreceptor apoptosis in the rd1 mouse was detected by terminal dUTP transferase nick-end labeling (TUNEL). Protein expressions of ER stress sensors, including glucose-regulated protein-78 (GRP78/BiP), caspase-12, phospho-eukaryotic initiation factor 2alpha (eIF2alpha), and phospho-pancreatic ER kinase (PERK), were examined by immunofluorescence and Western blot assays. RESULTS: Accompanying photoreceptor apoptosis in the rd1 mouse, the protein expressions of GRP78/BiP, caspase-12, phospho-eIF2alpha, and phospho-PERK were upregulated in a time-dependent manner. The upregulation of these proteins coincided with or preceded photoreceptor apoptosis. At the peak of their expression, these proteins were primarily located in the photoreceptor inner segments, the outer nuclear layer, or both. CONCLUSIONS: ER stress plays an important role in photoreceptor apoptosis in the rd1 mouse. Therefore, ER stress modulators may be strong candidates as therapeutic agents in the treatment of retinal degenerative diseases.

Identification of sequential events and factors associated with microglial activation, migration, and cytotoxicity in retinal degeneration in rd mice.

PURPOSE: To elucidate the role of activated microglia in the photoreceptor apoptosis of rd mice by identifying sequential events and factors associated with microglial activation, migration, and cytotoxicity during retinal degeneration. METHODS: Photoreceptor apoptosis in rd mice at postnatal days (P)8, 10, 12, 14, 16, and 18 was detected by terminal dUTP transferase nick end labeling (TUNEL). Retinal microglia were identified by CD11b antibody. Expression of chemokine mRNA, including monocyte chemoattractant protein (MCP)-1, MCP-3, macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, regulated on activation normal T-cell expressed and secreted (RANTES), interferon-gamma-inducible 10-kDa protein (IP-10), and fractalkine in the retina were examined by reverse transcription-polymerase chain reaction (RT-PCR) assay. Production of tumor necrosis factor (TNF)-alpha in the dystrophic retina was studied by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry analysis. Microglial expression of TNF-alpha was determined by double immunolabeling. RESULTS: Whereas photoreceptor apoptosis in the rd mice started at P10 and reached a peak at P16, activation and migration of microglial cells were observed at P10 and peaked at P14. The expression of MCP-1, MCP-3, MIP-1alpha, MIP-1beta, and RANTES transcripts were noted at P8 and reached a peak at P12. Production of TNF-alpha was noted in the outer nuclear layer (ONL) of the rd mice at P8 and reached a peak at P12. At the peak of microglial activity, TNF-alpha was predominantly expressed in the activated microglial cells in the ONL. CONCLUSIONS: Activation of microglia, as well as expression of their signaling molecules (chemokines) and microglia-derived toxic factor (TNF-alpha), coincides with or precedes the occurrence of photoreceptor apoptosis, suggesting activated microglia play a major role in retinal degeneration in rd mice. The chemokines MCP-1, MCP-3, MIP-1alpha, MIP-1beta, and RANTES are involved in activation and recruitment of the microglia to the degenerating photoreceptor cell layer. TNF-alpha, produced by the activated microglia, may accentuate the photoreceptor cell death.

Minocycline inhibits LPS-induced retinal microglia activation.

Retinal neurodegenerative disease involves an inflammatory response in the retina characterized by an increase in inflammatory cytokines and activation of microglia. The degree of microglia activation may influence the extent of retinal injury following an inflammatory stimulus. Cytokines released by activated microglia regulate the influx of inflammatory cells to the damaged area. Thus, a therapeutic strategy to reduce cytokine expression in microglia would be neuroprotective. Minocycline, a semisynthetic tetracycline derivative, is known to protect rodent brain from ischemia and to inhibit microglial activation. In this study, we activated retinal microglia in culture with lipopolysaccharide (LPS) and attempted to determine whether minocycline could reduce the production of cytokines from activated microglia at both gene and protein levels. Changes in inflammatory cytokines, TNF-alpha and IL-1beta, were measured by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) in the presence or absence of LPS. We also measured the levels of nitric oxide (NO) by the nitrate reductase method under similar conditions. LPS treatment induced a significant upregulation of the mRNA and release of TNF-alpha, IL-1beta, and NO from retinal microglia. Minocycline inhibited these releases. Thus, minocycline might exert its antiinflammatory effect on microglia by inhibiting the expression and release of TNF-alpha, IL-1beta, and NO.