Rod-Shaped Mesoporous Zinc-Containing Bioactive Glass Nanoparticles: Structural, Physico-Chemical, Antioxidant, and Immuno-Regulation Properties.

Bioactive glass nanoparticles (BGNs) are applied widely in tissue regeneration. Varied micro/nanostructures and components of BGNs have been designed for different applications. In the present study, nanorod-shaped mesoporous zinc-containing bioactive glass nanoparticles (ZnRBGNs) were designed and developed to form the bioactive content of composite materials for hard/soft tissue repair and regeneration. The nanostructure and components of the ZnRBGNs were characterized, as were their cytocompatibility and radical-scavenging activity in the presence/absence of cells and their ability to modulate macrophage polarization. The ZnRBGNs possessed a uniform rod shape (length ≈ 500 nm; width ≈ 150 nm) with a mesoporous structure (diameter ≈ 2.4 nm). The leaching liquid of the nanorods at a concentration below 0.5 mg/mL resulted in no cytotoxicity. More significant improvements in the antioxidant and M1-polarization-inhibiting effects and the promotion of M2 polarization were found when culturing the cells with the ZnRBGNs compared to when culturing them with the RBGNs. The doping of the Zn element in RBGNs may lead to improved antioxidant and anti-inflammatory effects, which may be beneficial in tissue regeneration/repair.

[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.

Role of NF-kappaB and MAPKs in light-induced photoreceptor apoptosis.

PURPOSE: To elucidate the role of nuclear factor kappa B (NF-kappaB) and mitogen-activated protein kinases (MAPKs) in light-induced apoptosis of photoreceptors in culture and to explore the potential inhibitory effect of minocycline and sulforaphane on apoptosis. METHODS: Apoptosis of 661W cells was induced by exposure to light and was detected by terminal dUTP transferase nick end labeling (TUNEL). The mRNA expression and protein production of 10 chemokines and noxious factors were examined by reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). The protein expression of the p65 subunit of NF-kappaB, and the MAPKs p-p38, p-p44/42, and p-JNK were examined by Western blot and immunofluorescence analyses. RESULTS: After exposure to light for 4 hours, 60% to 70% of the 661W cells underwent apoptosis. The expression of five selected chemokines and noxious factors was upregulated. The protein expression of the p65 subunit of NF-kappaB was downregulated, and the expression of the MAPKs p-p38, p-p44/42, and p-JNK was upregulated. Pretreatment with SB203580 for 1 hour inhibited light-induced upregulation of p-p38 and inhibited photoreceptor apoptosis. Pretreatment with minocycline or sulforaphane for 1 hour inhibited light-induced downregulation of the NF-kappaB p65 subunit and inhibited photoreceptor apoptosis. CONCLUSIONS: Apoptotic photoreceptors secrete chemokines and noxious factors to induce an immunologic response. The NF-kappaB and MAPK pathways both are involved in light-induced 661W photoreceptor apoptosis. Minocycline and sulforaphane inhibit light-induced photoreceptor apoptosis, partly through an NF-kappaB-dependent mechanism, but not through the MAPK pathway.

Minocycline delayed photoreceptor death in rds mice through iNOS-dependent mechanism.

PURPOSE: To elucidate the role of activated microglia and nitric oxide (NO) in photoreceptor apoptosis in rds mice, and to investigate the effect of minocycline treatment on rds mice. METHODS: Photoreceptor apoptosis in rds mice was detected by terminal dUTP transferase nick end labeling (TUNEL). Retinal microglial cells were identified by CD11b antibody. The mRNA expression of inducible nitric oxide synthase (iNOS) and chemokines were examined by reverse transcription polymerase chain reaction (RT-PCR) assay. The protein expression of iNOS was examined by immunohistochemistry and Western blotting analysis. The rds mice were treated intra-peritoneally from the second postnatal day (P2) with minocycline. RESULTS: Accompanying photoreceptor degeneration in rds mice, microglia were activated and immigrated from inner retinal layer (IRL) to outer nuclear layer (ONL), and the expression of iNOS was up-regulated. Minocycline treatment reduced the iNOS expression and decreased the initial photoreceptor apoptosis, but did not provide long term ameliorative effect on the photoreceptor cell loss of rds mice. CONCLUSIONS: NO played a major role in the initial photoreceptor apoptosis in rds mice. The migration of activated microglia to the ONL contributed to the subsequent photoreceptor cell death; minocycline treatment ameliorated the photoreceptor apoptosis in rds mice, and this protective effect was partly through iNOS-suppressive mechanism.

Minocycline and sulforaphane inhibited lipopolysaccharide-mediated retinal microglial activation.

PURPOSE: To elucidate the inhibitory effect of minocycline and sulforaphane on lipopolysaccharide (LPS)-induced retinal microglial activation and the mechanisms through which they exerted their inhibitory effects. METHODS: Primary retinal microglial cultures were exposed to LPS with or without minocycline and sulforaphane. The mRNA expression of monocyte chemotactic protein (MCP)-1, MCP-3, macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, eotaxin, regulated upon activation normal T-cell expressed and secreted (RANTES) protein, and interleukin (IL)-10 were examined by reverse transcription polymerase chain reaction (RT-PCR) assay. The mRNA expression of inducible nitric oxide synthase (iNOS) and subsequent nitric oxide (NO) production were examined by RT-PCR assay and Griess reagent assay. Protein expression of the p65 subunit of nuclear factor-kappaB (NF-kappaB) and p-p38, p-p44/42 and p-JNK mitogen-activated protein kinases (MAPKs) were examined by Western blot and immunofluorescent analysis. RESULTS: Cultured retinal microglial cells were activated following exposure to LPS. The mRNA expression and protein production of eotaxin, RANTES, and IL-10 and the mRNA expression of iNOS and subsequent NO production were upregulated. The protein expression of p-p38, p-JNK, and the p65 subunit of NF-kappaB were also upregulated. However, the protein expression of p-p44/42 was not significantly changed. Pretreatment with minocycline or sulforaphane for 1 h before LPS administration inhibited LPS-induced microglial morphological change and inhibited LPS-induced upregulation of p-p38, but had no effect on the expression of p-p44/42, p-JNK, and the p65 subunit of NF-kappaB. CONCLUSIONS: Minocycline and sulforaphane inhibited LPS-induced retinal microglial activation, Western blot and immunofluorescent studies showed decreased p-p38 MAPK expression. We suggested that the inhibitory effect of minocycline and sulforaphane was partly through a p38 MAPK-dependent mechanism.

A possible mechanism of microglia-photoreceptor crosstalk.

PURPOSE: The goal of this study was to explore the relationship between photoreceptor apoptosis and retinal microglial activation. METHODS: A murine photoreceptor cell line (661W cells) was exposed to LPS-treated microglial cell conditioned medium (MGCM), and cell viability was assessed by terminal dUTP transferase nick end labeling (TUNEL) and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. In addition, microglia were exposed to culture media from light-damaged 661W photoreceptor cells (PRCM), and microglial activation was assessed morphologically by phase contrast microscopy. Reverse transcription polymerase chain reaction was used to examine mRNA levels of several chemokines and noxious factors in the MGCM-treated photoreceptor cells and the PRCM-treated microgial. Western blotting was used to analyze NF-kappaB p65 subunit, phosphorylated MAPKs p38, p44/42 (Erk1/2), and c-Jun N-terminal kinase (JNK). RESULTS: Our results showed 37% of 661W cells underwent apoptosis following exposure to MGCM for 24 h. MGCM-induced death was associated with down-regulation of chemokine expression (i.e., eotaxin and RANTES), upregulation of inflammatory mediators (i.e., MIP-1alpha, MIP-1beta, IL-10, iNOS, and TNF-alpha), and increased phosphorylation of p38, p44/p42, and JNK. Retinal microglia acquired an activated phenotype after exposure to PRCM for 24 h. Microglial activation was accompanied by increased NF-kappaB p65 expression, increased phosphorylation of p38 and JNK, and upregulation of chemokines (i.e., eotaxin and RANTES) and inflammatory mediators (i.e., iNOS and IL-10). CONCLUSIONS: Light-damaged photoreceptors release immunological signaling molecules that attract microglia, resulting in microglial activation and subsequent further degeneration of remaining photoreceptors. These results also suggest that p38, p44/42, and JNK may regulate glial-induced photoreceptor death and that p38, JNK, and NF-kappaB may regulate photoreceptor-induced microglial activation.

Sinomenine inhibits activation of rat retinal microglia induced by advanced glycation end products.

Diabetic retinopathy 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 retina injury following an inflammatory stimulus. Cytokines, released by activated microglia, regulate the influx of inflammatory cells to the damaged area. Thus, therapeutic strategy to reduce cytokine expression in microglia would be neuroprotective. Sinomenine, an alkaloid isolated from the stem and root of Sinomenium acutum, has long been recognized as an anti-inflammatory drug for rheumatoid arthritis and also inhibits macrophage activation. In this study, we activated retinal microglia in culture with advanced glycation end products (AGEs) treatment and attempted to determine whether sinomenine could reduce the production of cytokines from the activated microglia at both gene and protein levels. Changes in inflammatory cytokines, TNF alpha, IL-1 beta and IL-6, were measured by semi-quantitative RT-PCR and enzyme-linked immunosorbent assay (ELISA) both in the presence and absence of AGEs. The effect of sinomenine on levels of reactive oxygen species (ROS) and the nuclear translocation of NF-kB p65 were studied with a laser confocal scanning microscope. AGEs treatment induced a significant release of TNF alpha, IL-1 beta, and IL-6 from retinal microglia. Sinomenine could inhibit release of these cytokines. Sinomenine attenuated ROS production in a dose-dependent fashion and reduced the nuclear translocation of NF-kB p65 in AGEs-activated retinal microglia in culture.

Cyclosporine-loaded microspheres for treatment of uveitis: in vitro characterization and in vivo pharmacokinetic study.

PURPOSE: A sustained intraocular level of immunosuppressive drug is desirable for the treatment of uveitis and other intraocular immune disorders. The objective of the present investigation was to assess the suitability of cyclosporine-loaded poly(lactic-co-glycolic acid) microspheres (CyS-PLGA-MS) to achieve this goal. METHODS: A solvent-evaporation method was used in the preparation of CyS-PLGA-MS. These microspheres were characterized for drug loading, entrapment efficiency, and in vitro release by high-performance liquid chromatography, particle size by phase-contrast light microscopy and surface morphology by scanning electron microscopy. The 3H-CyS-PLGA-MS suspension was injected into the vitreous body of healthy rabbits, and the concentration of cyclosporine in various ocular tissues and blood at predetermined intervals was measured by a scintillation counting technique and the pharmacokinetic parameters were calculated. Intravitreous administration of 3H-CyS solution was conducted as the control. RESULTS: The CyS-PLGA-MS was produced, with drug-loading ranging from 11% to 16% and a high entrapment efficiency from 86% to 98%. Microspheres were discrete, spherical particles with a diameter of approximately 50 microm. The CyS was constantly and slowly released from microspheres in the in vitro release experiment. Compared with CyS solution, microspheres prolonged the release of CyS and maintained therapeutic CyS concentrations for at least 65 days in disease-related tissues such as the choroid-retina and iris-ciliary body. The percentage of CyS released in vitro correlated well with the CyS distribution rate in vivo. CONCLUSIONS: CyS-PLGA-MS, displaying sustained intraocular release of CyS and showing advantages over CyS solution, may meet clinical needs more efficiently.

Therapeutic and toxicological evaluations of cyclosporine a microspheres as a treatment vehicle for uveitis in rabbits.

AIM: This study was undertaken to investigate the therapeutic efficacy and the toxicity of the intravitreal biodegradable poly(dl-lactide-co-glycolide)co-polymer microspheres containing cyclosporin A (CsA-PLGA-MS) on experimental uveitis in rabbits. METHODS: CsA-PLGA-MS that had been prepared by a solvent evaporation approach were characterized for morphology, particle size, entrapment efficiency, and in vitro release profile of CsA-PLGA-MS. Therapeutic efficacy of the CsA-PLGA-MS was evaluated by scoring of the inflammation, aqueous leukocyte counting, aqueous protein determination, and histological examination in the experimental rabbits with artificial uveitis induced by the injection of lipopolysaccharide. The toxicity was investigated by slit-lamp examination, indirect ophthalmoscopy, and electroretinography (ERG) in the noninflamed rabbit eye. RESULTS: The CsA-PLGA-MS were spherical in shape, with an average particle size of nearly 50 microm and an entrapment efficiency of more than 80%. The compositions of the formulation that was most effective in the in vivo studies included CsA, PLGA, and 3% Pluronic F68. In vitro released cyclosporine A from the optimized microspheres was approximately 25% during the 60-day incubation at 37 degrees C. It was demonstrated that the intravitreal injection of the optimized CsA-PLGA-MS decreased significantly the severity of the inflammatory signs, cellular infiltrate, aqueous leukocyte counts, and protein levels in the eyes of experimental rabbits with uveitis, compared to other formulations. Also, the preparation did not cause obvious toxicity in the noninflamed eyes of rabbits, except that the ERG b-wave amplitude for the test eyes was reversibly depressed, compared to those of the control eyes at 2 weeks, which almost recovered at the end of 6 weeks. CONCLUSIONS: The CsA-PLGA-MS preparation might be useful in the treatment of patients with severe chronic posterior uveitis who cannot tolerate systemic or periocular therapy.

[Progress of researches on lysozyme and its expression in Oncomelania hupensis].

Lysozyme generally exists in animals, plants and microorganisms, and it is used as a natural anti-infection material and one of the important non-specific immune factors in organisms. This paper reviews the progress of researches on its classification, gene structure and function, and expression regulation in Oncomelania hupensis, and on the factors affecting its activities in recent years, in order to further discuss its distribution in O. hupensis.

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.

[Establishment of a model of retinal microglial cells activation in vitro].

OBJECTIVE: To develop a method of retinal microglial cell culture to study the function of the microglial cell in diabetic retinopathy. METHODS: Microglia were activated with LPS. Immunocytochemistry, con-focal microscopy, flow cytometry, MTT and ELISA were applied to observe the morphological characters, quantity, and functional changes of the microglia. RESULTS: The purity of the microglia was up to 96% as determined by immunostaining and flow cytometry. Some morphological changes of microglia were observed after treatment with LPS, but their quantity kept stable. Cytokine TNF-alpha released from microglia increased significantly. CONCLUSION: The isolated microglial cells are pure by using this culture system, which would provide a valuable tool for studying mechanisms of microglial alterations in diabetic retinopathy.

Functional reconstruction of rabbit corneal epithelium by human limbal cells cultured on amniotic membrane.

PURPOSE: To investigate the phenotype of fetal and adult human limbal cells cultured on human amniotic membrane and the ability of cultured adult human limbal cells to repair limbal stem cell deficiency in a rabbit model. METHODS: Human adult and fetal limbal cells were isolated and cultured either on plastic plates or on human amniotic membrane. Connexin43, p63, and keratins 3 and 12 (K3 and K12) were detected by immunofluorescence and RT-PCR. Limbal stem cell deficiency was established in rabbits using chemical ablation and mechanical debridement. Cultured adult human limbal cells were transplanted onto rabbit corneas one month after injury, then fixed and imbedded in paraffin forty days later. Immunofluorescent staining of human-nuclear antigen, p63, K3, and connexin43 identified human-specific cells, progenitor cells, and differentiated corneal epithelial cells, respectively. RESULTS: Adult and fetal cultured limbal cells appeared similar in morphology. RT-PCR results showed that cells cultured from the human adult and fetal limbal area expressed both p63 and K12, whereas cells from central adult epithelium expressed K12 only. Immunofluorescent staining showed that more cells were p63 positive when cultured on human amniotic membrane than on plastic. Double staining for p63 and connexin43 showed some p63-positive cells co-expressing connexin43. After transplantation of adult human limbal cells cultured on human amniotic membrane, injured rabbit corneas were completely reconstructed exhibiting epithelial integrity, improved corneal clarity, and little or no neovascularization. The majority of repopulated epithelial cells expressed anti-human nuclear antibody. Cells expressing p63 occurred throughout the new epithelium. CONCLUSIONS: During healing, expression of p63 is not limited to epithelial stem cells but may also mark transient amplifying progenitor cells. Culture on human amniotic membrane suppresses differentiation of limbal epithelial cells and promotes the proliferation of p63 expressing cells. Amniotic membrane-cultured human limbal cells fully reconstructed rabbit corneas having limbal stem cell deficiency, with human cells providing most of the cells of the new epithelium. Expression p63 is distributed throughout the reconstructed tissue.

[Effects of Nomega-nitro-L-arginine on photoreceptor apoptosis in inherited retinal degeneration of RCS rats].

OBJECTIVE: To investigate inducible nitric oxide synthase(iNOS) activity of retina and the effects of N(omega)-nitro-L-arginine(N-Arg) on photoreceptor apoptosis in inherited retinal degeneration of Royal College of Surgeons (RCS) rats. METHODS: iNOS activity was assayed in the whole retinal homogenates of RCS rats and Wistar rats by monitoring the conversion rate of (3)H-arginine to (3)H-citrulline. Intravitreal injection of the NOS inhibitor, N(omega)-nitro-L-arginine(N-Arg), in one lateral eye on postnatal days 17 (P17), P22, P27 and P32 was performed, while the other lateral eye was treated with PBS by intravitreal injection as controls. Then the retinas of the RCS rats were studied by TdT-mediated biotin-dUTP nick-end labeling (TUNEL) for apoptosis on P38. RESULTS: The enzymatic activity of iNOS was elevated in RCS rat retinas on P25. In RCS rats on P38, the percent area of apoptotic photoreceptor nuclei and the thickness of rod and cone layer in the treated group were significantly reduced compared with the controls, while the thickness of outer nuclear layer (ONL) was increased. CONCLUSION: The inhibitor of NOS might supply a potential medicine for inherited retinal degeneration.

[Excimer retreatment for undercorrection or regression after laser in situ keratomileusis].

OBJECTIVE: To evaluate the results of excimer retreatment for undercorrection or regression after laser in situ keratomileusis (LASIK). METHODS: Eight-eight eyes received retreatment for undercorrection or regression after first LASIK (FLASIK) in 2 149 eyes in our photorefractive keratotectomy (PRK)/LASIK center from March, 1996 to July, 1999. They were divided into 2 groups according to their degrees of pre-FLASIK myopia spherical equivalents. Group I was 10.00 D (47 eyes). We analyzed the factors of FLASIK which led to the re-LASIK (RLASIK), and observed the mean spherical equivalents (MSE), the ratio within +/- 1.00 D of emmetropia, the uncorrected visual acuity (UCVA), the best corrected visual acuity (BCVA) and the complication after RLASIK for at least 1 year follow-up study. RESULTS: The retreatment ratio was 4.1% among the 2 149 eyes. In group II, the ratio was 2.3 times that of the group I. In the comparison with other eyes, the eyes received RLAISK had no difference in sex, laterality and age (P > 0.05). Over 50.0% had pre-FLASIK diopters >/= 10.00 D, and 73.9% had astigmatism correction in FLASIK. After RLASIK, no undercorrection or regression occurred in group I, but there was undercorrection in group II. The ratio within +/- 1.00 D of emmetropia after 1 year was 68.3% in group I and 51.1% in group II. The UCVA above 0.5 was over 90.0% in all eyes. Eight eyes (9.0%) lost two or more lines of BCVA. The serious complication after RLASIK was keratoconus of 3 eyes in group II. CONCLUSIONS: The risk factors which lead to retreatment after FLASIK are high myopia with astigmatism before FLKSIK and the reaction to the operation. In this study, RLKSIK is safe, effective, predictable and accurate for -10.00 D in FLASIK.

AGEs mediated expression and secretion of TNF alpha in rat retinal microglia.

Diabetic retinopathy induces an inflammatory response in the retina characterized by an increase in inflammatory cytokines and the activation of microglia. The degree of microglia activation may influence the extent of retina injury following retinal metabolic stress. We have previously shown that DR rats have elevated levels of advanced glycation end products (AGEs) in their blood. We have also suggested that AGEs might be involved in microglial activation and production of tumor necrosis factor alpha (TNF alpha). In this study, we attempted to confirm that AGEs induce the release of TNF alpha from rat retinal microglia using an in vitro microglia culture system, and concurrently to explore the mediating mechanisms. AGEs increased the protein secretion and mRNA expression of TNF alpha in cultured rat retinal microglia. These effects of AGEs were primarily mediated by reactive oxygen species (ROS). Furthermore, the inhibitors for mitogen-activated protein kinases (MAPK; p38, JNK and ERK 1/2) and nuclear factor-kB (NF-kB) could significantly decrease AGEs-induced TNF alpha release. AGEs-activated microglia showed an increase of NF-kB p65 nuclear translocation. These observations indicated that pathophysiological levels of AGEs may alter rat retinal microglia function by up-regulating TNF alpha expression and release via enhanced formation of intracellular ROS. AGEs-induced ROS subsequently activates MAPK (p38, JNK and ERK1/2) and NF-kB.