Long noncoding RNA ZFAS1: A novel anti-apoptotic target in Fuchs endothelial corneal dystrophy.

Fuchs endothelial corneal dystrophy (FECD) is the leading cause of endothelial keratoplasty without efficacious drug treatment. Recent studies have emphasized the involvement of epigenetic regulation in FECD development. Long non-coding RNAs (lncRNAs) are recognized as crucial epigenetic regulators in diverse cellular processes and ocular diseases. In this study, we revealed the expression patterns of lncRNAs using high-throughput sequencing technology in FECD mouse model, and identified 979 significantly dysregulated lncRNAs. By comparing the data from FECD human cell model, we obtained a series of homologous lncRNAs with similar expression patterns, and revealed that these homologous lncRNAs were enriched in FECD related biological functions, with apoptosis (mmu04210) showing the highest enrichment score. In addition, we investigated the role of lncRNA zinc finger antisense 1 (ZFAS1) in apoptotic process. This study would broaden our understanding of epigenetic regulation in FECD development, and provide potential anti-apoptotic targets for FECD therapy.

Intravitreal injection of mitochondrial DNA induces cell damage and retinal dysfunction in rats.

BACKGROUND: Retinal neurodegeneration is induced by a variety of environmental insults and stresses, but the exact mechanisms are unclear. In the present study, we explored the involvement of cytosolic mitochondrial DNA (mtDNA), resulting in the cGAS-STING dependent inflammatory response and apoptosis in retinal damage in vivo. METHODS: Retinal injury was induced with white light or intravitreal injection of lipopolysaccharide (LPS). After light- or LPS-induced injury, the amount of cytosolic mtDNA in the retina was detected by PCR. The mtDNA was isolated and used to transfect retinas in vivo. WB and real-time PCR were used to evaluate the activation of cGAS-STING pathway and the levels of apoptosis-associated protein at different times after mtDNA injection. Retinal cell apoptosis rate was detected by TUNEL staining. Full-field electroretinography (ERG) was used to assess the retinal function. RESULTS: Light injury and the intravitreal injection of LPS both caused the leakage of mtDNA into the cytoplasm in retinal tissue. After the transfection of mtDNA in vivo, the levels of cGAS, STING, and IFN-ß mRNAs and the protein levels of STING, phosph-TBK1, phospho-IRF3, and IFN-ß were upregulated. mtDNA injection also induced the activation of caspase 3 and caspase 9. BAX and BAK were increased at both the mRNA and protein levels. The release of cytochrome c from the mitochondria to the cytosol was increased after mtDNA injection. The wave amplitudes on ERG decreased and retinal cell apoptosis was detected after mtDNA injection. CONCLUSIONS: Cytosolic mtDNA triggers an inflammatory response. It also promotes apoptosis and the dysfunction of the retina.

Characteristics and Visual Prognosis of Pediatric Open Globe Injury in Shanghai, China.

INTRODUCTION: The aim of the study was to describe the characteristics of open globe injury (OGI) and the relationship between the complications and visual outcomes in children with this type of injury. METHODS: This was a retrospective chart review of 1,664 children, under the age of 16 years, who were hospitalized for OGI between January 1, 2007, and December 31, 2015. Each patient's age, sex, cause and agent of injury, complications, visual acuity, and classification of ocular trauma were collected for review and analysis. RESULTS: The mean age was 5.6 ± 3.4 years. Right eyes were particularly vulnerable to injury (right eye:left eye ratio = 1.2:1). Traumatic cataract was the most common complication. The average initial and final best corrected visual acuity were logarithm of the minimum angle of resolution (logMAR) 2.04 ± 0.78 and logMAR 1.74 ± 0.88, respectively. Logistic regression analysis showed that hyphema (odds ratio [OR] = 1.850), iris prolapse (OR = 1.702), vitreous hemorrhage (OR = 9.703), retinal detachment (OR = 11.938), endophthalmia (OR = 5.377), intraocular foreign body (OR = 3.346), and initial visual acuity <0.05 (OR = 9.017) were risk factors for visual acuity <0.05 at hospital discharge. CONCLUSION: OGI was most frequent in preschool children and boys. Right eyes were more vulnerable than left eyes. Poor visual outcomes were associated with hyphema, iris prolapse, vitreous hemorrhage, retinal detachment, endophthalmia, intraocular foreign body, and an initial visual acuity <0.05.

Observation of varicella zoster virus-induced acute retinal necrosis: viral load detection and visual outcome.

OBJECTIVES: To observe the changes of viral load in aqueous humour samples and visual outcomes in varicella zoster virus (VZV)-induced acute retinal necrosis (ARN). METHODS: Observational retrospective study. Medical records and viral load measured by real-time quantitative polymerase chain reaction (qPCR) of 20 eyes with VZV-induced ARN were reviewed. RESULTS: The mean viral load at presentation was 5.7 × 107 ± 9.7 × 107 copies/mL. An initial plateau phase for viral load lasting up to 2 weeks occurred in most eyes (18 eyes, 90%). In the following logarithmic reduction phase, the mean slope of the decline in viral load was -0.103 ± 0.029 log/day, and the expected time for half reduction of the initial viral load was 3.2 ± 1.0 days. At the end of the first 8-week's antiviral treatment, the viral load was below detection threshold in all 20 eyes (100.0%). The mean logarithm of the minimum angle of resolution (logMAR) best-corrected visual acuity (BCVA) improved from 1.1 ± 0.7 (Snellen equivalent 20/250) to 0.7 ± 0.6 (Snellen equivalent 20/100) after a follow-up of 8.6 ± 2.0 months. Thirteen of the 20 eyes (65.0%) suffered retinal detachment and underwent vitrectomy. The initial viral load was the independent predictive factor of logMAR BCVA at the last follow-up (ß = 0.745, P < 0.001). CONCLUSIONS: The observation of viral load changes by qPCR was useful for better monitoring of therapeutic efficacy and deciding needed antiviral duration in VZV-induced ARN patients.

Intravitreal injection of mitochondrial DNA induces cell damage and retinal dysfunction in rats

BACKGROUND: Retinal neurodegeneration is induced by a variety of environmental insults and stresses, but the exact mechanisms are unclear. In the present study, we explored the involvement of cytosolic mitochondrial DNA (mtDNA), resulting in the cGAS-STING dependent inflammatory response and apoptosis in retinal damage in vivo. METHODS: Retinal injury was induced with white light or intravitreal injection of lipopolysaccharide (LPS). After light-or LPS-induced injury, the amount of cytosolic mtDNA in the retina was detected by PCR. The mtDNA was isolated and used to transfect retinas in vivo. WB and real-time PCR were used to evaluate the activation of cGAS-STING path-way and the levels of apoptosis-associated protein at different times after mtDNA injection. Retinal cell apoptosis rate was detected by TUNEL staining. Full-field electroretinography (ERG) was used to assess the retinal function. RESULTS: Light injury and the intravitreal injection of LPS both caused the leakage of mtDNA into the cytoplasm in retinal tissue. After the transfection of mtDNA in vivo, the levels of cGAS, STING, and IFN-ß mRNAs and the protein levels of STING, phosph-TBK1, phospho-IRF3, and IFN-ß were upregulated. mtDNA injection also induced the activation of caspase 3 and caspase 9. BAX and BAK were increased at both the mRNA and protein levels. The release of cytochrome c from the mitochondria to the cytosol was increased after mtDNA injection. The wave amplitudes on ERG decreased and retinal cell apoptosis was detected after mtDNA injection. CONCLUSIONS: Cytosolic mtDNA triggers an inflammatory response. It also promotes apoptosis and the dysfunction of the retina.

Changes in Retinal Perfusion in Leber's Hereditary Optic Neuropathy: An Optical Coherence Tomography-Angiography Study.

INTRODUCTION: We aimed to study the change in the retinal perfusion in Leber's hereditary optic neuropathy (LHON). METHODS: Fourteen patients (28 eyes) diagnosed with LHON and 14 healthy controls (28 eyes) were enrolled. The retinal vessel densities in the parafoveal and peripapillary areas were measured with optical coherence tomography-angiography. RESULTS: In the subacute LHON patients, the parafoveal superficial capillary plexus (SCP) and inner retinal thickness (IRT) were significantly reduced in all sectors compared with the controls (all p < 0.05), and the radial peripapillary capillary (RPC) network was significantly reduced in the temporal and inferior temporal sectors compared with the controls (p < 0.05). In the chronic LHON patients, the SCP and IRT were significantly lower in all sectors than in the controls (all p < 0.05), the RPC vessel density and thickness were significantly lower in all sectors than in the controls and lower in the temporal, superior temporal, inferior temporal, and nasal sectors than in the subacute-stage patients (all p < 0.05). CONCLUSION: The retinal structure and the perfusion of the macular and peripapillary areas are reduced in subacute LHON, and the retinal structure and the perfusion of the peripapillary area are further reduced in chronic LHON.

SHP-2-Induced Activation of c-Myc Is Involved in PDGF-B-Regulated Cell Proliferation and Angiogenesis in RMECs.

Background: Aberrant neovascularization resulting from inappropriate angiogenic signaling is closely related to many diseases, such as cancer, cardiovascular disease, and proliferative retinopathy. Although some factors involved in regulating pathogenic angiogenesis have been identified, the molecular mechanisms of proliferative retinopathy remain largely unknown. In the present study, we determined the role of platelet-derived growth factor-B (PDGF-B), one of the HIF-1-responsive gene products, in cell proliferation and angiogenesis in retinal microvascular endothelial cells (RMECs) and explored its regulatory mechanism. Methods: Cell counting kit-8 (CCK-8), bromodeoxyuridine (BrdU) incorporation, tube formation, cell migration, and Western blot assays were used in our study. Results: Our results showed that PDGF-B promoted cell proliferation and angiogenesis by increasing the activity of Src homology 2 domain-containing tyrosine phosphatase 2 (SHP-2) in RMECs, which was attenuated by the inhibition of PDGF receptor (PDGFR) or SHP-2 knockdown. Moreover, activation of c-Myc was involved in the processes of PDGF-B/SHP-2-driven cell proliferation in RMECs. The promoting effects of PDGF-B/SHP-2 on c-Myc expression were mediated by the Erk pathway. Conclusion: These results indicate that PDGF-B facilitates cell proliferation and angiogenesis, at least in part, via the SHP-2/Erk/c-Myc pathway in RMECs, implying new potential treatment candidates for retinal microangiopathy.

Mitochondrial DNA drives noncanonical inflammation activation via cGAS-STING signaling pathway in retinal microvascular endothelial cells.

BACKGROUND: Pathological stimuli cause mitochondrial damage and leakage of mitochondrial DNA (mtDNA) into the cytosol, as demonstrated in many cell types. The cytosolic mtDNA then drives the activation of noninfectious inflammation. Retinal microvascular endothelial cells (RMECs) play an important role in the inner endothelial blood-retinal barrier (BRB). RMEC dysfunction frequently occurs in posterior-segment eye diseases, causing loss of vision. In this study, we investigated the involvement of cytosolic mtDNA in noninfectious immune inflammation in RMECs under pathological stimuli. METHODS: RMECs were stimulated with 100 ng/ml lipopolysaccharide (LPS), 200 µM hydrogen peroxide (H2O2), or 25 mM D-glucose. After 24 h, immunofluorescent staining was used to detect the opening of the mitochondrial permeability transition pore (MPTP). Cytosolic mtDNA was detected with immunofluorescent staining and PCR after stimulation. mtDNA was then isolated and used to transfect RMECs in vitro, and the protein levels of cGAS were evaluated with western blotting. Real-time PCR was used to examine cGAS mRNA expression levels at different time points after mtDNA stimulation. The activation of STING was detected with immunofluorescent staining 6 h after mtDNA stimulation. Western blotting was used to determine the expression of STING and IFNß, the phosphorylation status of TBK1, IRF3, and nuclear factor-κB (NF-κB) P65, and the nuclear translocation of IRF3 and NF-κB P65 at 0, 3, 6, 12, and 24 h. The mRNA expression of proinflammatory cytokines CCL4, CXCL10, and IFNB1, and transcription factor IRF1 were determined with real-time PCR, together with the concentrations of intercellular adhesion molecule 1 (ICAM-1) mRNA. RESULTS: Pathological stimuli caused mtDNA to leak into the cytosol by opening the MPTP in RMECs after 24 h. Cytosolic mtDNA regulated the expression of cGAS and the distribution of STING in RMECs. It promoted ICAM-1, STING and IFNß expression, TBK1, IRF3, and NF-κB phosphorylation and the nuclear translocation in RMECs at 12 and 24 h after its transfection. The mRNAs of proinflammatory cytokines CCL4, CXCL10, and IFNB1, and transcription factor IRF1 were significantly elevated at 12 and 24 h after mtDNA stimulation. CONCLUSIONS: Pathological stimulation induces mtDNA escape into the cytosol of RMECs. This cytoplasmic mtDNA is recognized by the DNA sensor cGAS, increasing the expression of inflammatory cytokines through the STING-TBK1 signaling pathway. Video Abstract. (MP4 37490 kb).

MicroRNA-29b-3p inhibits cell proliferation and angiogenesis by targeting VEGFA and PDGFB in retinal microvascular endothelial cells.

Purpose: Excessive angiogenesis, also known as neovascularization, has considerable pathophysiologic roles in several retinal diseases, including retinopathy of prematurity, diabetic retinopathy, and exudative age-related macular degeneration. Accumulated evidence has revealed that miRNAs play important roles in endothelial cell dysfunction and angiogenesis. However, the role of microRNA-29b-3p (miR-29b-3p) in retinal angiogenesis is still unclear. Therefore, we investigated whether and how miR-29b-3p affects the function of retinal microvascular endothelial cells (RMECs). Methods: The overexpression and inhibition of miR-29b-3p were achieved by transfecting rat RMECs with an miR-29b-3p mimic and inhibitor, respectively. The proliferation, migration, and angiogenesis of RMECs were evaluated using a Cell Counting Kit-8 assay, Ki67 staining, western blotting (of proliferating cell nuclear antigen, cyclin A2, cyclin D1, and cyclin E1), wound healing test, and tube formation assay. The expression levels of vascular endothelial growth factor A (VEGFA) and platelet-derived growth factor B (PDGFB) were examined with quantitative real-time PCR and western blotting, respectively. Results: Overexpression of miR-29b-3p statistically significantly inhibited the function of RMECs in cell proliferation and angiogenesis, while inhibition of miR-29b-3p increased the proliferative and angiogenic activities of RMECs. Moreover, VEGFA and PDGFB, as the targets of miR-29b-3p, were statistically significantly downregulated by the miR-29b mimic, whereas the miR-29b-3p inhibitor had the opposite effects. Conclusions: miR-29b-3p negatively regulates RMEC proliferation and angiogenesis, at least partly by targeting VEGFA and PDGFB. These data may provide a potential therapeutic strategy for treating ocular neovascular diseases.

Synthetic Glucocorticoid-Induced Leucine Zipper Peptide Inhibits Lipopolysaccharide-Induced Ocular Inflammation in Rats.

PURPOSE: To demonstrate the anti-inflammatory action of a synthetic glucocorticoid-induced leucine zipper (GILZ98-134) peptide (GILZ-p) in a model of endotoxin-induced uveitis (EIU) in rats. METHODS: The EIU model was induced in Sprague Dawley rats with an intravitreal injection of lipopolysaccharide (LPS). Synthetic GILZ-p was injected intravitreally 6 h after the LPS injection. To evaluate the anti-inflammatory effects of GILZ-p, the inflammatory response in the anterior chamber and iris of the rat eyes was evaluated with a slitlamp microscope on days 0, 1, 2, 3, and 4 after GILZ-p injection. The retinal expression of inflammatory cytokines was measured on days 0, 1, 2, 3, and 4 after GILZ-p injection. Müller cell gliosis was also detected at planned time points after GILZ-p injection. RESULTS: Anterior segment inflammation peaked at 24 h after LPS injection in the EIU model. Compared with the controls, intravitreal GILZ-p significantly suppressed LPS-induced anterior segment inflammation in the EIU rats. The levels of retinal inflammatory factors IL-1ß, TNF-α, MCP-1, and ICAM-1 were simultaneously reduced by the intravitreal GILZ-p injection. The expression of vimentin in the EIU retina was significantly reduced by GILZ-p, and the downregulated aquaporin 4 in the EIU retina was significantly restored by GILZ-p. CONCLUSION: The synthetic GILZ-p inhibited the inflammatory reaction in the EIU model and may have utility in the treatment of inflammatory ocular disease.

Synthesized glucocorticoid-induced leucine zipper peptide inhibits photoreceptor apoptosis and protects retinal function in light-induced retinal degeneration model.

BACKGROUND: This study aimed to investigate the neuroprotective function of a synthesized glucocorticoid-induced leucine zipper peptide (GILZ-p) in a light-induced retinal degeneration model. METHODS: The GILZ98-134 peptide was synthesized and injected intravitreally into Sprague Dawley rats. Retinal injury was then induced in the rats by exposing their eyes to constant white light (5000 lux) for 24 h. The activation of retinal caspases-9/3 and the release of cytochrome c from the mitochondria to the cytosol were measured at 1, 3, 5 and 7 d after light injury. Photoreceptor apoptosis was evaluated with terminal-deoxynucleotidyl-transferase-mediated deoxyuridine triphosphate-biotin nick end labelling (TUNEL) staining at 3 d after injury. Haematoxylin and eosin staining and electroretinography were used to observe the changes in the retinal morphology and function, respectively, at 7 and 14 d after light injury. RESULTS: The intravitreally injected synthesized GILZ-p successfully penetrated to the retina and significantly inhibited the activation of retinal caspase-3 and caspase-9 at 1, 3, 5 and 7 d after light injury, and reduced the number of TUNEL-positive photoreceptors at 3 d after light injury. GILZ-p pre-treatment also alleviated cytochrome c release and rescued mitochondria-mediated apoptosis after injury. Simultaneously, GILZ-p pre-treatment also mitigated the light-induced thinning of the outer nuclear layer and the loss of retinal function at 7 and 14 d after light injury, respectively. CONCLUSIONS: The synthesized GILZ-p prevented light-induced photoreceptor apoptosis and protected retinal function from degeneration, and is therefore a potential therapeutic option for degenerative retinal diseases.

Endogenous Fungal Endophthalmitis Associated with Genitourinary Procedures.

Purpose: To summarize the characteristics of endogenous fungal endophthalmitis (EFE) after genitourinary procedures. Methods: Medical records of patients diagnosed with EFE after genitourinary procedures from a single center during a 6-year period were reviewed. Results: Nineteen eyes of 15 patients were included. The interval time between procedure to symptom was 3.6 ± 3.6 weeks. As the initial treatment, 9/19 eyes underwent primary vitrectomy and 10/19 eyes underwent intravitreal antifungal injection . Candida albicans was the pathogen in 15 of 19 eyes. Systemic treatment with itraconazole was used in all patients. LogMAR best corrected visual acuity improved from 2.2 ± 0.9 to 0.9 ± 1.2 after treatment (p = 0.002) in 15 eyes that were followed-up for an average of 4.9 ± 2.1 years. Conclusion: Genitourinary procedure is a predisposing factor for EFE. Candida albicans is the predominant pathogen. Normative systemic and local antifungal treatments improved the final visual outcome.

RETINAL TELANGIECTASIA IN PATIENTS WITH PATHOLOGIC MYOPIA: A CASE SERIES.

PURPOSE: To describe the characteristics of retinal telangiectasia in eyes with pathologic myopia. METHODS: The study included 10 patients (18 eyes) who were diagnosed with pathologic myopia combined with retinal telangiectasia. The patients visited our retinal clinic every 3 months. Nine eyes underwent vitrectomy for vision-threatening complications after diagnosis. All eyes underwent comprehensive ophthalmologic examinations including multimodal retinal imaging at presentation and at each follow-up. RESULTS: Retinal telangiectasia in pathologic myopia was characterized by saccular aneurysmal dilatation of the capillary bed without hard exudates in color fundus photographs and hyporeflective saccular structure in infrared reflectance fundus photographs, and it was filled in the early retinal arteriovenous phase with minimal dye leakage in the late phase of fundus fluorescein angiography. Spectral domain optical coherence tomography and optical coherence tomographic angiography showed that retinal telangiectasia was primarily located in the superficial retina, together with myopic traction maculopathy. In the 9 eyes that underwent vitrectomy, the retinal telangiectasia regressed within 3 months of surgery. Retinal telangiectasia remained stable in the other nine eyes, but these eyes were at risk of spontaneous bleeding. CONCLUSION: Retinal telangiectasia is a relatively quiescent and uncommon disorder in patients with pathologic myopia that might be closely related to myopic traction maculopathy.

Light-Induced Lipocalin 2 Facilitates Cellular Apoptosis by Positively Regulating Reactive Oxygen Species/Bim Signaling in Retinal Degeneration.

Purpose: Lipocalin 2 (LCN2) is reported to be one of the key regulators of cell survival and death; however, its effect on retinal degeneration is unclear. Therefore, we aimed to investigate the role of LCN2 and its underlying mechanisms in light-induced retinal degeneration. Methods: A recombinant lentivirus expressing a short hairpin RNA targeting LCN2 mRNA and a recombinant lentivirus overexpressing LCN2 were used to downregulate and upregulate retinal LCN2, respectively. Seven days after intravitreal injection of the lentiviruses, rats were exposed to blue light (2500 lux) for 24 hours. Retinal function and morphology were evaluated with ERG and hematoxylin-eosin staining, respectively. TUNEL staining was used to detect apoptotic cells. The levels of reactive oxygen species (ROS) were evaluated with dihydroethidium labeling. Western blotting and real-time PCR were used to examine protein and mRNA expression levels, respectively. Results: Retinal LCN2 expression was significantly upregulated after light exposure. Light exposure reduced the amplitudes of a- and b-waves on the ERG and the thickness of the outer nuclear layer and promoted photoreceptor apoptosis. These phenomena were clearly attenuated by LCN2 knockdown, whereas LCN2 overexpression had the opposite effects. The overexpression of LCN2 facilitated photoreceptor apoptosis by increasing ROS generation and Bim expression. On the opposite, LCN2 knockdown mitigated the generation of light-exposure-induced ROS and the activation of the Bim-mediated mitochondrial apoptotic pathway. Conclusions: Light-induced LCN2 is a proapoptotic factor in the retina, and LCN2 knockdown protects photoreceptors from apoptosis by inhibiting ROS production and Bim expression. LCN2 is a potential therapeutic target for light-induced retinal degeneration.

Intrusion of Dacron suture knot 15 years after scleral buckling.

BACKGROUND: We present a case of intrusion of a suture knot 15 years after scleral buckling surgery. CASE PRESENTATION: A 62-year-old woman with high myopia had undergone scleral buckling surgery in her left eye 15 years previously for rhegmatogenous retinal detachment. She recently displayed highly elevated intraocular pressure, with hyphema and vitreous hemorrhage. After the blood was cleared, a ring-shaped protrusion was noted around the equator of the eyeball, with a blue suture knot standing out on its surface and extending into the vitreous cavity at 5 o'clock. The suture knot was removed successfully. Mass spectrometry revealed that the material of the suture was polyethylene terephthalate, or Dacron. One week later, at the place where the suture knot had been located, the choroidal and retinal tissue disappeared and the silicone buckle remained an uncovered intrusion, whereas the rest of the retina was still attached. CONCLUSIONS: The suture knot was possibly the one used to close the drainage port for subretinal fluid, which was covered by the encircling band. During the buckling procedure, covering a nonabsorbable suture, which is usually placed where the sclera is compromised by trauma or the surgical incision, with an encircling band may lead to the intrusion of the suture. Therefore, a soft absorbable suture may be preferable, if possible.

Effect of Phosphorylated-Extracellular Regulated Kinase 1/2 Inhibitor on Retina from Light-induced Photoreceptor Degeneration.

BACKGROUND: The demonstrated role of mitogen-activated protein kinase (MAPK) in both cell apoptosis and the inflammation pathway makes it an attractive target for photoreceptor protection. The aim of this study was to investigate the protective effects of MAPK antagonists against photoreceptor degeneration and retinal inflammation in a rat model of light-induced retinal degeneration. METHODS: Sprague Dawley rats were treated with intravitreal injections of MAPK antagonists, inhibitors of p-P38, phosphorylated-extracellular regulated kinase (p-ERK) 1/2, and p-c-Jun N-terminal kinase (JNK) just before they were assigned to dark adaptation. After dark adaptation for 24 h, rats were exposed to blue light (2500 lux) in a light box for 24 h, and then returned to the normal 12-h light/12-h dark cycle. Samples were collected at different time points. MAPK expression during light exposure was examined with immunofluorescence. Photoreceptor death was detected with histopathology and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. The expression of retinal p-ERK1/2, caspase 3, activated caspase 3, tumor necrosis factor (TNF)-α, and interleukin (IL)-1ß was examined by Western blotting. Differences between groups were evaluated using unpaired one-way analysis of variance and least significant difference post hoc tests. RESULTS: MAPKs (P38, ERK1/2, and p-JNK) were phosphorylated and activated in the light injury groups, compared with normal group, and their expressions were mainly elevated in the outer nuclear layer (ONL). Among the selected MAPK antagonists, only the p-ERK1/2 inhibitor attenuated the loss of photoreceptors and the thinning of ONL in light injury groups. Besides, p-ERK1/2 inhibitor refrained light-induced photoreceptor apoptosis, which was presented by TUNEL positive cells. Light injury significantly increased the expression of p-ERK1/2 (1.12 ± 0.06 vs. 0.57 ± 0.08, t = 9.99, P < 0.05; 1.23 ± 0.03 vs. 0.57 ± 0.08, t = 11.90, P < 0.05; and 1.12 ± 0.12 vs. 0.57 ± 0.08, t = 9.86, P < 0.05; F = 49.55, P < 0.001), and induced caspase 3 activating (0.63 ± 0.06 vs. 0.14 ± 0.05, t = 13.67, P < 0.05; 0.74 ± 0.05 vs. 0.14 ± 0.05, t = 16.87, P < 0.05; and 0.80 ± 0.05 vs. 0.14 ± 0.05, t = 18.57, P < 0.05; F = 100.15, P < 0.001), compared with normal group. The p-ERK1/2 inhibitor significantly reduced p-ERK1/2 overexpression (0.61 ± 0.06 vs. 1.12 ± 0.06, t = -9.26, P < 0.05; 0.77 ± 0.06 vs. 1.23 ± 0.03, t = -8.29, P < 0.05; and 0.68 ± 0.03 vs. 1.12 ± 0.12, t = -7.83, P < 0.05; F = 49.55, P < 0.001) and downregulated caspase 3 activating (0.23 ± 0.04 vs. 0.63 ± 0.06, t = -11.24, P < 0.05; 0.43 ± 0.03 vs. 0.74 ± 0.05, t = -8.86, P < 0.05; and 0.58 ± 0.03 vs. 0.80 ± 0.05, t = -6.17, P < 0.05; F = 100.15, P < 0.001), compared with light injury group. No significant change in the total level of caspase 3 was seen in different groups (F = 0.56, P = 0.75). As for inflammation, light injury significantly increased the expression of TNF-α (0.42 ± 0.04 vs. 0.25 ± 0.05, t = 5.99, P < 0.05; 0.65 ± 0.03 vs. 0.25 ± 0.05, t = 14.87, P < 0.05; and 0.86 ± 0.04 vs. 0.25 ± 0.05, t = 22.58, P < 0.05; F = 160.27, P < 0.001) and IL-1ß (0.24 ± 0.01 vs. 0.19 ± 0.02, t = 2.33, P < 0.05; 0.35 ± 0.02 vs. 0.19 ± 0.02, t = 7.97, P < 0.05; and 0.48 ± 0.04 vs. 0.19 ± 0.02, t = 14.69, P < 0.05; F = 77.29, P < 0.001), compared with normal group. P-ERK1/2 inhibitor significantly decreased the overexpression of TNF-α (0.22 ± 0.02 vs. 0.42 ± 0.04, t = -7.40, P < 0.05; 0.27 ± 0.02 vs. 0.65 ± 0.03, t = -14.27, P < 0.05; and 0.33 ± 0.03 vs. 0.86 ± 0.04, t = -19.58, P < 0.05; F = 160.27, P < 0.001) and IL-1ß (0.13 ± 0.03 vs. 0.24 ± 0.01, t = -5.77, P < 0.05; 0.17 ± 0.01 vs. 0.22 ± 0.02, t = -9.18, P < 0.05; and 0.76 ± 0.05 vs. 0.48 ± 0.04, t = -13.12, P < 0.05; F = 77.29, P < 0.001), compared with light injury group. CONCLUSION: The p-ERK1/2 inhibitor might protect the retina from light-induced photoreceptor degeneration and retinal inflammation.

Microglia enhanced the angiogenesis, migration and proliferation of co-cultured RMECs.

BACKGROUND: Attention is increasingly being given to microglia-related inflammation in neovascular diseases, such as diabetic retinopathy and age-related macular disease. Evidence shows that activated microglia contribute to disruption of the blood-retinal barrier, however, the mechanism is unclear. In this study, we aimed to clarify whether and how microglia affect the function of retinal microvascular endothelial cells (RMECs). METHODS: We activated microglia by Lipopolysaccharides (LPS) stimulation. After co-culturing static or activated microglia with RMECs using the Transwell system, we evaluated the function of RMECs. Vascular endothelial growth factor-A (VEGF-A) and platelet-derived growth factor-BB (PDGF-BB) levels in the supernatant from the lower chamber were evaluated by ELISA. Angiogenesis, migration, and proliferation of RMECs were assessed by tube formation, wound healing, and WST-1 assays. The expression levels of tight junction proteins (ZO-1 and occludin) and endothelial markers (CD31 and CD34) were examined by Western blot analysis. RESULTS: We successfully established an LPS-activated microglia model and co-culture system of static or activated microglia with RMECs. In the co-culture system, we showed that microglia, especially activated microglia stimulated VEGF-A and PDGF-BB expression, enhanced angiogenesis, migration, proliferation, and permeability, and altered the phenotype of co-cultured RMECs. CONCLUSIONS: Microglia, especially activated microglia, play important roles in angiogenesis and maintenance of vascular function hemostasis in the retinal microvasculature. The mechanism needs further investigation and clarification.

Area of the cone interdigitation zone in healthy Chinese adults and its correlation with macular volume.

BACKGROUND: Numerous studies have suggested that the integrity of the cone interdigitation zone (IZ) could be considered to be a marker of photoreceptor damage and its recovery. However, little is known about the IZ in healthy eyes. Our present study was to measure the cone IZ area by optical coherence tomography (OCT), and determine its distribution in healthy adults. METHODS: This was a cross-sectional non-interventional study. We involved a group of 158 emmetropic or low myopic (from -3D to + 0.5D) eyes in 97 healthy adult volunteers. All subjects underwent thorough ophthalmologic examinations and the posterior pole was scanned by OCT. The cone IZ area in healthy adults and its correlation with macular volume and other factors was analyzed. RESULTS: The cone IZ was visible and clear in all 158 eyes, and the IZ area was successfully measured by 6 radical scans centered on the fovea. The mean IZ area was 30.22 ± 12.70 mm2, and ranged from 5.91 to 57.47 mm2. The IZ area exhibited a normal distribution (P = 0.635) with 95% confidence interval of 28.06-32.29 mm2. The IZ area was significantly correlated with the retinal and outer nuclear layer (ONL) volumes within the macula. CONCLUSIONS: The cone IZ area could be measured using a commercially available OCT system. The IZ area showed high variability among healthy adults, and this might be related to the variability in the photoreceptor distribution in healthy adults.

A Synthesized Glucocorticoid- Induced Leucine Zipper Peptide Inhibits Retinal Müller Cell Gliosis.

Purpose: The anti-inflammatory activities of protein glucocorticoid-induced leucine zipper (GILZ) have been demonstrated in vivo and in vitro. Here, we examined the potential effect of a synthetic peptide derived from the leucine zipper motif and proline-rich region of GILZ on suppressing inflammatory responses in primary cultured rat Müller cells. Methods: Peptides were selected from amino acids 98-134 of the GILZ protein (GILZ-p). Solid-phase peptide synthesis was used to generate the cell-penetrating peptide TAT, which was bound to the amino terminus of GILZ-p. Primary cultured retinal Müller cells were stimulated with lipopolysaccharide (LPS) alone or in combination with different concentrations of GILZ-p, and the interaction of GILZ-p with nuclear factor (NF)-κB p65 in Müller cells was investigated by western blotting, immunoprecipitation, and immunofluorescence. The expression of the Müller cell gliosis marker glial fibrillary acidic protein (GFAP), functional protein aquaporin (AQP)-4, and the inflammatory cytokines interleukin (IL)-1ß, tumor necrosis factor (TNF) α, intercellular adhesion molecule (ICAM)-1, and monocyte chemoattractant protein (MCP)-1 was measured by Western Blotting. The concentration of those cytokines in culture medium was measured by using Enzyme-Linked Immunosorbent Assay. Results: The synthesized GILZ-p, which was water-soluble, entered cells and bound with NF-κB p65, inhibiting p65 nuclear translocation. GILZ-p inhibited the LPS-induced expression of GFAP, IL-1ß, TNFα, ICAM-1, and MCP-1 in Müller cells and prevented the LPS-induced downregulation of AQP4. Conclusions: These results indicate that GILZ-p interacted with NF-κB p65 and suppressed p65 nuclear translocation, thereby inhibiting inflammatory cytokine release and Müller cell gliosis.

Lipocalin 2 Suppresses Ocular Inflammation by Inhibiting the Activation of NF-κß 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.