Ruiping Gu

Macular Perfusion in Healthy Chinese: An Optical Coherence Tomography Angiogram Study

PURPOSE To investigate macular perfusion in healthy Chinese individuals and examine its dependence on age and sex. METHODS Healthy adult Chinese individuals were recruited. Macular perfusion was measured by spectral-domain optical coherence tomography (OCT) using the split-spectrum amplitude-decorrelation angiography (SSADA) algorithm. The parafoveal flow index and vessel area density as well as the area of the foveal capillary-free zone (CFZ) were quantified. RESULTS A total of 76 eyes in 45 subjects were included (20 males and 25 females, mean age 36 ± 11 years). The mean parafoveal flow index was 0.099 ± 0.013; the mean vessel area density was 0.891 ± 0.073; and the mean CFZ area was 0.474 ± 0.172 mm2. All three parameters were significantly correlated with age (flow index: P = 0.00; vessel area density: P = 0.00; CFZ area: P = 0.02). The flow index and vessel area density decreased annually by 0.6% and 0.4%, respectively, and CFZ area increased by 1.48% annually. The CFZ area was larger in females than in males, while all three parameters seemed to change more rapidly with age in males than in females. CONCLUSIONS In healthy Chinese eyes, macular perfusion decreased with increasing age, and decreased more rapidly in males than in females. The application of OCT angiograms may provide a useful approach for monitoring macular perfusion, although caution must be exercised with regard to age- and sex-related variations.

Relationship Between Retinal Perfusion and Retinal Thickness in Healthy Subjects: An Optical Coherence Tomography Angiography Study.

Purpose To investigate the relationship between retinal perfusion and retinal thickness in the peripapillary and macular areas of healthy subjects. Methods Using spectral-domain optic coherence tomography and split-spectrum amplitude decorrelation angiography (SSADA) algorithm, retinal perfusion and retinal thicknesses in the macular and peripapillary areas were measured in healthy volunteers, and correlations among these variables were analyzed. Results Overall, 64 subjects (121 eyes) including 28 males and 36 females with a mean ± SD age of 38 ± 13 years participated. Linear mixed-models showed that vessel area density was significantly correlated with the inner retinal thickness (from the inner limiting membrane to the outer border of the inner nucleus layer; P < 0.05), but not with the thickness of the full retina (P > 0.05) in the parafoveal area. The area of the foveal capillary-free zone was negatively correlated with the inner and full foveal thicknesses (all P < 0.001). In the peripapillary area, the vessel area density was positively correlated with the thickness of the retinal nerve fiber layer (P < 0.001). Conclusions In healthy subjects, retinal perfusion in small vessels was closely correlated with the thickness of the inner retinal layers in both the macular and peripapillary areas.

Activated microglia induce the production of reactive oxygen species and promote apoptosis of co-cultured retinal microvascular pericytes

PurposePericyte apoptosis is a predominant feature of early diabetic retinopathy. In diabetic retinopathy, activated microglia migrate and release proinflammatory cytokines that contribute to disruption of the blood–retinal barrier, neuronal loss, and enhanced ROS production. Reactive oxygen species (ROS) are implicated in pericyte death; however, the mechanism by which activated microglia affect retinal microvascular pericytes is unclear. We hypothesized that activated microglia may promote pericyte apoptosis by enhancing ROS production.MethodsLipopolysaccharide (LPS)-activated microglia and pericytes were co-cultured in a cell culture system. Pericyte ROS production and the mitochondrial membrane potential (ΔΨm) were determined by flow cytometry. The pericyte protein expression levels of NADPH oxidase subunits, uncoupling protein 2, nuclear NF-κB-p65, and caspase-3 were determined by western blotting. One-way ANOVAs were used for statistical analysis.ResultsLPS successfully activated the microglia, as demonstrated by their morphological and phenotype changes and the significant increase in tumor necrosis factor secretion (P < 0.01). Co-culture with activated microglia significantly up-regulated NADPH oxidase subunits (NOX4, NOX2, and NCF1; P < 0.01) and down-regulated uncoupling protein 2 expression (P < 0.01) in pericytes. Pericyte ROS production increased by 20% in the activated microglia co-cultured group, and was inhibited by pretreatment with diphenyleneiodonium, coenzyme Q10, and N-acetylcysteine. The proapoptotic pericyte changes induced by co-culture with activated microglia included a 9.50% decrease in pericyte ΔΨm and significant increases in NF-κB-p65 nuclear translocation (P < 0.01) and activated caspase-3 (P < 0.01). These proapoptotic effects of activated microglia were inhibited by diphenyleneiodonium.ConclusionsOur results are consistent with our hypothesis that activated microglia may promote pericyte apoptosis by enhancing ROS production. Further studies are needed to examine retinal microglia activation and the corresponding changes in pericytes in a rat model of diabetes mellitus.

Glucocorticoid-Induced Leucine Zipper Suppresses ICAM-1 and MCP-1 Expression by Dephosphorylation of NF-κB p65 in Retinal Endothelial Cells.

Purpose Glucocorticoid-induced leucine zipper (GILZ) is involved in anti-inflammatory activities in several animal models and in various cell types. In this study, we explored the role of GILZ in rat retinal vascular endothelial cells. Methods Glucocorticoid-induced leucine zipper overexpression or silencing was established using GILZ overexpressing recombinant lentivirus (OE-GILZ-rLV) or short-hairpin RNA targeting GILZ recombinant lentivirus (shRNA-GILZ-rLV), respectively, in rat primary retinal microvascular endothelial cells (RMECs) and intact retina. Seventy-two hours after transfection, RMECs were stimulated with 1000 ng/mL lipopolysaccharide (LPS), 20 μM isoliensinine (an alkaloid derived from the embryos of Nelumbo nucifera, could enhance the dephosphorylation of p65 at Ser536), or PBS for another 24 hours. Western blotting and immunofluorescence were performed to measure protein expression. The concentrations of intercellular adhesion molecule (ICAM)-1 and monocyte chemoattractant protein (MCP)-1 in the RMEC culture media were measured by ELISA. Results Lipopolysaccharide downregulated GILZ expression in RMECs in a time- and dose-dependent manner, and the decrease in GILZ expression was accompanied by increased ICAM-1 and MCP-1 expression. Glucocorticoid-induced leucine zipper overexpression decreased LPS-induced ICAM-1 and MCP-1 expression, whereas GILZ silencing significantly attenuated the production of both cytokines. Glucocorticoid-induced leucine zipper overexpression also inhibited LPS-induced nuclear factor-κB p65 nuclear translocation in RMECs that was mediated by enhanced p65 dephosphorylation. The dephosphorylation of NF-κB p65 further downregulated ICAM-1 and MCP-1 expression in RMECs. Conclusions Glucocorticoid-induced leucine zipper overexpression inhibited NF-κB p65 nuclear translocation by enhancing p65 dephosphorylation. Exogenous GILZ regulated ICAM-1 and MCP-1 expression, which was probably mediated by enhanced p65 dephosphorylation.

Retina Is Protected by Neuroserpin from Ischemic/Reperfusion-Induced Injury Independent of Tissue-Type Plasminogen Activator.

The purpose of the present study was to investigate the potential neuroprotective effect of neuroserpin (NSP) on acute retinal ischemic/reperfusion-induced (IR) injury. An IR injury model was established by elevating intraocular pressure (IOP) for 60 minutes in wild type and tPA-deficient (tPA-/-) mice. Prior to IR injury, 1 μL of 20 μmol/L NSP or an equal volume of bovine serum albumin (BSA) was intravitreally administered. Retinal function was evaluated by electroretinograph (ERG) and the number of apoptotic neurons was determined via TUNEL labeling. Caspase-3, -8, -9,poly (ADP-ribose) polymerase (PARP)and their cleaved forms were subsequently analyzed. It was found that IR injury significantly damaged retinal function, inducing apoptosis in the retina, while NSP attenuated the loss of retinal function and significantly reduced the number of apoptotic neurons in both wild type and tPA-/- mice. The levels of cleaved caspase-3, cleaved PARP (the substrate of caspase-3) and caspase-9 (the modulator of the caspase-3), which had increased following IR injury, were significantly inhibited by NSP in both wild type and tPA-/- mice. NSP increased ischemic tolerance in the retina at least partially by inhibiting the intrinsic cell death signaling pathway of caspase-3. It was therefore concluded that the protective effect of neuroserpin maybe independent from its canonical interaction with a tissue-type plasminogen activator.

Correlation between Retinal Changes and Visual Function in Late-Stage Vogt-Koyanagi-Harada Disease: An Optical Coherence Tomography Study

Purpose. To characterize the optical coherence tomography (OCT) findings in late-stage Vogt-Koyanagi-Harada (VKH) disease and its correlation with visual function. Methods. The records of patients with late-stage VKH disease (defined as ≥12 months from disease onset) were retrospectively reviewed. The analysis focused on the OCT findings and microperimetry, in addition to the possible correlation between morphology and functional findings. Results. Twenty-nine patients (58 eyes) were included. Mean age at onset was 34.24 ± 10.67 years. The OCT revealed that the outer retina and retinal pigment epithelium (RPE) were mainly affected. These effects included RPE thickening and breakage or disappearance of the cone outer segment tip (COST) line and/or inner segment/outer segment (IS/OS) junction. The COST line and IS/OS results were related to macular function and the interval between symptom onset and initiation of high-dose corticosteroid treatment (all P < 0.01). Eyes with intact COST lines demonstrated intact IS/OS and normal RPE layers as well as better visual function and normal retinal sensitivity. Conclusions. The OCT findings are strongly correlated with macular function, as well as other clinical findings in late-stage VKH. With respect to the COST line and retinal sensitivity especially, the OCT and microperimetry findings may be useful for evaluating later-stage VKH.

Glucocorticoid-Induced Leucine Zipper Protects the Retina From Light-Induced Retinal Degeneration by Inducing Bcl-xL in Rats

Purpose The aim of the present study was to investigate the neuroprotective effects of glucocorticoid-induced leucine zipper (GILZ) in a light-induced retinal degeneration model and to explore the underlying mechanisms. Methods Intravitreal injection of recombinant GILZ-overexpressing lentivirus (OE-GILZ-rLV) and short hairpin RNA targeting GILZ recombinant lentivirus (shRNA-GILZ-rLV) was performed to up- and downregulate retinal GILZ, respectively. Three days after stable transduction, rats were exposed to continuous bright light (5000 lux) for 2 days. Retinal function was assessed by full-field electroretinography (ERG), and the retinal structure was examined for photoreceptor survival and death in rats kept under a 12-hour light:2-hour dark cycle following light exposure. The expression levels of retinal Bcl-xL, caspase-9, and caspase-3 were examined by Western blotting or real-time PCR at 1, 3, 5, and 7 days after light exposure. Results Exposure to bright light downregulated retinal GILZ in parallel with the downregulation of Bcl-xL and the upregulation of active caspase-3. Overexpression of retinal GILZ attenuated the decrease of Bcl-xL and the activation of caspase-9 and caspase-3 at 1, 3, 5, and 7 days after bright light exposure, respectively. GILZ silencing aggravated the downregulation of Bcl-xL induced by bright light exposure. Bright light exposure reduced the amplitude of ERG, increased the number of apoptotic photoreceptor cells, and decreased retinal thickness; and GILZ overexpression could attenuate all these effects. Conclusions Overexpression of GILZ by OE-GILZ-rLV transduction protected the retina from light-induced cellular damage by activating antiapoptotic pathways.

Glucocorticoid-induced leucine zipper overexpression inhibits lipopolysaccharide-induced retinal inflammation in rats

Abstract Glucocorticoid‐induced leucine zipper (GILZ) mediates several effects of glucocorticoids and has important anti‐inflammatory properties. Here, we explored the role of GILZ in inhibiting retinal inflammation. Endotoxin‐induced uveitis (EIU) was established in rats by intravitreal injection of lipopolysaccharide (LPS). GILZ levels decreased in the EIU retina after LPS injection. Retinal GILZ was downregulated by recombinant lentivirus‐delivered short‐hairpin RNA targeting GILZ (shRNA‐GILZ‐rLV) and upregulated by recombinant lentivirus‐mediated GILZ overexpression (Oe‐GILZ‐rLV). GILZ silencing attenuated the anti‐inflammatory effects of intravitreal injection of triamcinolone acetonide (TA) in the EIU retina, as demonstrated by increased retinal interleukin (IL)‐1&bgr;, monocyte chemoattractant protein (MCP)‐1and intercellular cell adhesion molecule‐1 expression at 18 h after TA injection. A Bio‐Plex cytokine assay and western blotting demonstrated that GILZ overexpression inhibited the effects of LPS, downregulating retinal IL‐1&bgr;, MCP‐1, MIP‐1&agr;, and IL‐17 and inhibiting LPS‐induced activation of the retinal toll‐like receptor 4–myeloid differentiation factor 88 signaling pathway. At 48 and 72 h after LPS injection, the clinical score of inflammation was significantly lower in Oe‐GILZ‐rLV–transfected eyes than in blank‐rLV–transfected eyes. Histological examination showed a 67.85% reduction of infiltrating inflammatory cells in the anterior chamber and a 58.97% reduction in vitreous cavity of Oe‐GILZ‐rLV transfected eyes at 48 h after LPS injection. Taken together, our results suggest that GILZ is a novel therapeutic target for the treatment of retinal inflammatory diseases. HighlightsRetinal GILZ was downregulated or upregulated by specific recombinant lentiviruses.Retinal GILZ overexpression attenuated expression of proinflammatory biomarkers.GILZ overexpression reduced retinal IL‐1&bgr;, MCP‐1, MIP‐1&agr;, and IL‐17 expression.GILZ inhibited LPS‐induced activation of the retinal TLR4–MyD88 signaling pathway.

Elevated concentration of cytokines in aqueous in post-vitrectomy eyes.

We aim to study the level of inflammatory cytokines in aqueous of post‐vitrectomy eyes.

Lipocalin 2 Suppresses Ocular Inflammation by Inhibiting the Activation of NF-κβ Pathway in Endotoxin-Induced Uveitis

Background/Aims: Lipocalin 2 (LCN2), an important mediator of a variety of cellular processes, is involved in regulating the inflammatory response, but its roles in different inflammatory diseases are controversial. Because the role of LCN2 in ocular inflammation has been unclear until now, we explored the function of LCN2 in lipopolysaccharide (LPS)-induced ocular inflammation in vivo and in vitro. Methods: Endotoxin-induced uveitis (EIU) was induced in male Sprague Dawley rats by the intravitreal injection of LPS. The expression and location of LCN2 in the retina were detected with western blotting and immunohistochemistry, respectively. We determined the clinical scores for anterior inflammation, quantified the infiltrated inflammatory cells, and measured the pro-inflammatory factors to determine the anti-inflammatory effects of LCN2 in EIU eyes. Cultured primary rat Müller cells were stimulated with LPS and the expression and secretion of LCN2 were measured with real-time PCR, western blotting, and an ELISA. After Müller cells were cotreated with LPS and LCN2 or PBS, the expression and secretion of TNF-α, IL-6, and MCP-1 were examined with realtime PCR, western blotting, and ELISAs. Western blotting and immunofluorescence were used to detect the phosphorylation and cellular distribution of nuclear factor kappaB (NF-κB) subunit p65. Results: In EIU, the expression of LCN2 was significantly upregulated in the retina, especially in the outer nuclear layer (mainly composed of Müller cells). LPS stimulation of cultured Müller cells also markedly elevated LCN2 expression. Intravitreal injection of LCN2 significantly reduced the clinical scores, inflammatory infiltration, and protein leakage in EIU, which correlated with the reduced levels of proinflammatory factors in the aqueous humor and retina. LCN2 treatment also reduced the expression and secretion of TNF-α, IL-6, and MCP-1 in LPS-stimulated Müller cells. LCN2 inhibited the inflammatory response by inhibiting the phosphorylation and translocation of NF-κB p65. Conclusions: LCN2 protects against ocular inflammation, at least in part, by negatively regulating the activation of the NF-κB signaling pathway. LCN2 may be a promising anti-inflammatory therapy for ocular diseases, such as uveitis.