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Purpose: To investigate the role of Ca²(+) in lipofuscin formation in human retinal pigment epithelial (RPE) cells that phagocytize bovine photoreceptor outer segments (POSs).Methods: Cultured human RPE cells fed with 2 × 10⁷per l bovine POS were treated with flunarizine, an antagonist of Ca²(+) channel, or/and centrophenoxine, a lipofuscin scavenger. The Ca²(+) changes and lipofuscin formation were measured with fluoresence dye Fluo-3/AM ester, laser scanning confocal microscopy (LSCM) and flow cytometry (FCM). The activity of RPE cells was measured by methyl thiazolyl tetrazolium (MTT) assay and argyrophilic nucleolar organizer regions (AgNORs) assay.Results: The Ca²(+) fluorescence intensity (CFI) of RPE cells fed with POS was significantly increased compared with the controls (165.36 ± 29.92 U). It reached a peak with 777.33 ± 63.86 U (P<0.01) at 12 h, and then decreased but still maintained a high level of 316.90 ± 36.07 U (P<0.01) for 4 days. Flunarizine and centrophenoxine significantly decreased the Ca²(+) overload to 227.18 ± 14.00 U at 12 h and 211.06 ± 20.45 U at 4 days. FCM confirmed these changes. The drugs also showed an inhibitory effect on the lipofuscin formation. The proliferation rate of the cells fed with POS increased significantly. Both drugs had inhibitory effects on the activity of the cultured cells. This tendency was confirmed by AgNORs assay.Conclusions: The Ca²(+) inflow initiated lipofuscin accumulation in RPE cells fed with POS. Flunarizine and centrophenoxine can decrease Ca²(+) overload and lipofuscin formation in RPE cells, accompanied by maintaining cellular vitality. [ABSTRACT FROM AUTHOR]

Purpose: We aimed to evaluate the impacts of the different types of posterior staphyloma (PS) in high myopia on parapapillary microvasculature and parapapillary atrophy (PPA, i.e., γ-zone and δ-zone) with optical coherence tomography angiography., Methods: This cross-sectional study included 152 high myopic eyes (152 participants) with γ-zone. After matching, high myopic eyes were stratified into PS (n = 33) and non-PS (n = 33) groups. PS types, parapapillary microvasculature characteristics, the prevalence of non-juxtapapillary microvasculature dropout (NJ-MvD), the longest radial width from the optic disc edge to retinal pigment epithelium atrophy edge (PPA-w), γ-zone, and δ-zone with changes in visual field and best-corrected visual acuity (BCVA) were evaluated and analyzed., Results: According to Curtin's classification, we identified five types of PS included: primary types I, II, III, and compound types VII, and IX. Eyes with type II showed a higher optic disc tilted ratio and larger PPA-w compared to other primary PS. Subjects with type IX were the oldest. Eyes with types VII and IX had wider γ-zone and δ-zone, worse visual field outcomes, poorer BCVA, and a higher incidence of NJ-MvD compared to primary PS. Choroidal and intrascleral vessels were associated with NJ-MvD and were located near or connected to the circle of Zinn-Haller., Conclusions: Eyes with high myopia and PS show wider PPA, affecting the γ-zone and δ-zone, and are at a higher risk of developing NJ-MvD. Specifically, eyes with PS types VII and IX are more susceptible to visual field damage and decreased BCVA. These results highlight the importance of impact of PS on PPM and PPA in high myopia.

Purpose: To observe the effects of oxidative stress on vascular endothelial growth factor (VEGF) and connections of lens epithelial cells., Methods: Human lens epithelium of patients with age-related cataract (ARC), both SRA01/04 cells and whole mice lens stimulated by H 2 O 2 were employed. VEGF in human aqueous humor of ARC-patients and the supernatant of SRA01/04 cells was determined by ELISA. The expressions of VEFG in human lens epithelium were detected by immunofluorescence staining. Multiple linear regression analysis and spearman rank-order correlation were used to determine the associations between VEGF and parameters of ARC individuals. In H 2 O 2 -induced SRA01/04 cells, Catalase (CAT), PP1 (inhibitor of c-Src kinase) and Avastin (VEGF antibody) were used to inhibit the effects of H 2 O 2 , activation of c-Src kinase and VEGF, which were detected by Western blot. The alterations of ZO-1 and N-cadherin were tested by immunofluorescence staining and Western blot. In H 2 O 2 -induced whole lens, the changes of opacification area in different treatment of inhibitors were observed., Results: The secretion of VEGF in aqueous humor and expression of VEGF in the lens epithelium of ARC patients increased significantly with age. In H 2 O 2 -induced SRA01/04 cells, the VEGF in the supernatant was increased with the culture duration and the dose of H 2 O 2 . The expressions of p-Src 418 and VEGF were also up-regulated, whereas the expressions of ZO-1 and N-cadherin were down-regulated. CAT effectively prevented these changes induced by H 2 O 2 , while PP1 inhibited not only p-Src 418 but also up-regulation of VEGF, Avastin partially inhibited VEGF up-regulation. Both PP1 and Avastin prevented down-regulation of ZO-1 and N-cadherin, respectively, but Avastin combined with PP1 had no significant synergistic effects. In H 2 O 2 -induced cataract, CAT prevented development of opacification area effectively, and PP1 and Avastin did partially., Conclusions: Oxidative stress disrupts connections of lens epithelial cells by activating c-Src/VEGF, inhibiting which may prevent cataract.

Aim: To assess the long-term efficacy and safety of yttrium-aluminum garnet (YAG) laser vitreolysis for vision degrading myodesopsia (VDM) caused by posterior vitreous detachment (PVD)., Methods: This retrospective study reviewed VDM patients of PVD type undergoing YAG laser vitreolysis. The baseline demographic information, the patterns of floaters, the number of floaters, and the subjective improvement of floater sympotoms (ranging from 0 to 100%) from medical records were collected. Significant improvement was defined as a relief of floater symptoms of ≥50% at the final visit. The long-term efficacy and safety of YAG laser vitreolysis were analyzed. The risk factors linked to significant improvement of floater symptoms were defined using univariate and multivariate logistic regression analyses., Results: The final analysis included 221 patients with VDM. The mean age of patients was 61.08±7.74y, and the mean length of follow-up was 21.38±5.61mo. Totally 57.01% of patients experienced a significant improvement in their floater symptoms after YAG laser therapy, and none of them developed delayed retinal abnormalities such as retinal tears or detachments. Age (OR=1.049, 95%CI=1.007-1.092, P =0.021) was identified as a significant risk factor for significant improvement in VDM., Conclusion: YAG laser vitreolysis is an effective and secure treatment for PVD-type VDM, and patients of advanced age are more likely to get favorable outcomes., (International Journal of Ophthalmology Press.)

Aim: To access the agreement of intraocular pressure (IOP) values obtained from biomechanically corrected tonometer [Corvis ST (CST)], non-contact tonometer (NCT), and Goldmann applanation tonometer (GAT) in children with NCT measured-IOP (NCT-IOP) values of 22 mm Hg or more, and related factors., Methods: A total of 51 eyes with NCT-IOP≥22 mm Hg in children aged 7 to 14y were examined and IOP was measured by CST, NCT, and GAT. Based on GAT measured IOP (GAT-IOP), ocular hypertension (OHT) group (≥22 mm Hg, 24 eyes) and the non-OHT group (<22 mm Hg, 27 eyes) were defined. We compared the agreement of the three measurements, i.e. , CST measured IOP (CST-IOP), GAT-IOP, and NCT-IOP, and further analyzed the correlation between the differences in tonometry readings, central corneal thickness (CCT), axial length (AL), optic disc rim volume, and age., Results: Compared with the OHT group, thicker CCT, larger rim volume, and higher differences between NCT-IOP and GAT-IOP, were found in the non-OHT group. The differences between CST-IOP and GAT-IOP were lower than the differences between NCT-IOP and GAT-IOP in both groups. The mean differences in CST-IOP and GAT-IOP were 1.26 mm Hg (95% limit of agreement ranged from 0.1 to 2.41 mm Hg, OHT group) and 1.20 mm Hg (95% limit of agreement ranged from -0.5 to 3.00 mm Hg, non-OHT group), and the mean differences in NCT and GAT were 3.90 mm Hg (95% limit of agreement ranged from -0.19 to 9.70 mm Hg, OHT group) and 6.00 mm Hg (95% limit of agreement ranged from 1.50 to 10.50 mm Hg, non-OHT group). The differences between CST-IOP and GAT-IOP were not related to CCT, age, and AL in both groups; while the differences between NCT-IOP and GAT-IOP were related to CCT in the OHT group ( r =0.93, P <0.001) and to CCT and AL in the non-OHT group ( r =0.66, P <0.001, r =-0.81, P <0.001)., Conclusion: The accuracy of NCT in the diagnosis of pediatric OHT is low. The agreement of CST-IOP and GAT-IOP was significantly higher in children with and without OHT than in those with NCT-IOP and GAT-IOP. Therefore, CST can be used as a good alternative for IOP measurement in children. The impacts of CCT and AL on NCT measurement need to be fully considered when managing childhood IOP., (International Journal of Ophthalmology Press.)

Aim: To assess the long-term outcomes of treating macular edema (ME) associated with central retinal vein occlusion (CRVO) with a regimen of "5+ pro re nata (PRN)"., Methods: This retrospective study included 27 eyes of 27 patients with ME associated with non-ischemic CRVO (non-iCRVO group, n =15) and ischemic CRVO (iCRVO group, n =12). The eyes were treated with five consecutive intravitreal injections of conbercept or ranibizumab, followed by reinjections as needed or PRN. Retinal laser photocoagulation or intravitreal dexamethasone implants (DEX) were implemented in both groups when necessary. The best-corrected visual acuity (BCVA, logMAR) and central retinal thickness (CRT) were recorded at baseline, at 1, 2, 3, 4, 5, 6, and 12mo, and at the final visit. The efficacy rates of BCVA and CRT before and after treatment were calculated. The number of injections at each visit and the incidence of adverse events were also recorded., Results: The patients, aged 59.4±15.1y, were followed up for 24.7±8.8mo (range: 15-42mo). After treatment, BCVA improved significantly from 1.04±0.56 logMAR at baseline to 0.59±0.36 logMAR ( P =0.038) at the final visit in all patients. Both the non-iCRVO and the iCRVO groups achieved improved BCVA compared to the baseline at all visit points, but there was no statistical significance ( P =0.197 and 0.33, respectively). The mean CRT was statistically reduced compared to baseline at all visit points in all the eyes and in both groups (all P <0.001). The apparent effective rate was 22.22% for BCVA and 37.04% for CRT after the first injection, 48.15% for BCVA and 62.96% for CRT after 5 consecutive injections, and 74.08% for BCVA and 100% for CRT at the end of follow up. The average number of injections in all patients was 9.0±2.4 at 12mo and 14.9±8.1 finally with no statistical significance between both groups ( P >0.05). Laser treatment was applied to all eyes in the iCRVO group, while only 5 patients in the non-iCRVO group. Six patients in the non-iCRVO group and 3 patients in the iCRVO group had a drug switch. DEX was applied to 4 eyes in the non-iCRVO group and 5 eyes in the iCRVO group., Conclusion: The 5+PRN anti-vascular endothelial growth factor (VEGF) regimen is found to be safe and effective for both iCRVO and non-iCRVO, especially in the iCRVO group. The best regimen for such patients needs to be further investigated. Adjuvant laser therapy and DEX are necessary in some cases., (International Journal of Ophthalmology Press.)

Aim: To assess the efficacy versus the adverse effects of various concentrations of atropine in the prevention of myopia in Asian children., Methods: Databases (PubMed, EMBASE, the Cochrane Library and Web of science) were comprehensively searched from inception to April 2022. Types of studies included were randomized clinical trials (RCTs). The published languages were limited to English. Two researchers assessed the quality of included studies independently using Cochrane risk of bias tool based on the Cochrane Handbook for Systematic Reviews of Interventions. Funnel plots and Egger's test were used for detection of publication bias. Meta-analyses were conducted using STATA (version 15.0; StataCorp)., Results: A total of 15 RCTs involving 2268 patients were included in the study. In the atropine group, spherical equivalent progressed at a significantly lower rate [weighted mean difference (WMD)=0.39, 95% confidence interval (CI): 0.23, 0.54] than in the control group. A WMD of 0.15 mm was associated with less axial elongation (95%CI -0.19, -0.10). Different doses showed statistically significant differences ( P <0.05) and an improved effect could result from a higher concentration. Changes in photopic pupil size and mesopic pupil size in atropine group is 0.70 mm (95%CI: 0.33, 1.06) and 0.38 mm (95%CI: 0.22, 0.54) more than the control group. In the present Meta-analysis, no changes in accommodative amplitude (AA) were associated with atropine administration. Atropine administration increased the risk of adverse effects by 1.37 times., Conclusion: Concentrations of less than 1% atropine are able to effectively retard diopter and axis growth of myopia in Asian children in a dose-dependent manner. Meanwhile, it caused pupil enlargement, but induced no change in the AA within this range. Further study is required to determine the dosage needed to achieve maximum efficacy and minimal side effects., (International Journal of Ophthalmology Press.)

Aim: To evaluate the difference and the correlation between the concentrations of cytokines in the aqueous humor of eyes with macular edema secondary to diabetic retinopathy (DR) or retinal vein occlusion (RVO)., Methods: This is a retrospective case control study. The aqueous humor samples were collected during intravitreal injection of anti-vascular endothelial growth factor (VEGF) for patients diagnosed with macular edema secondary to DR (DME) or RVO (RVO-ME) at Xijing Hospital from August 2021 to July 2022. Meanwhile, aqueous humor samples during vitrectomy from patients with idiopathic macular hole (IMH) were also collected and served as controls. The aqueous humor concentrations of VEGF, platelet-derived factor (PDGF), interleukin (IL)-6, IL-8, IL-18, tumor necrosis factor-α (TNF-α) and monocyte chemoattractant protein 1 (MCP-1) were measured with Human Premixed Multi-Analyte Kit (Luminex). The difference of the aqueous cytokines and the correlation between the two diseases were analyzed., Results: A total of 40 eyes of 38 patients were enrolled in the study, including 13 eyes of 11 DME patients (DME group), 16 eyes of 16 RVO-ME patients (RVO-ME group) and 11 eyes of 11 IMH patients (control group). The VEGF, PDGF, IL-6, IL-8, and MCP-1 levels of the aqueous humor were higher in both DME and RVO-ME groups compared with the control group (all P <0.05), the levels of TNF-α was higher in the DME group than in the control group ( P <0.05). The VEGF, IL-6, MCP-1, and TNF-α levels in the aqueous humor were significantly higher in the DR group than those in the RVO group (all P <0.05). Correlation analyses revealed that there were complex positive correlations between IL-6, IL-8, IL-18, MCP-1, and TNF-α levels in the aqueous humor of eyes with two diseases., Conclusion: Although ischemic and inflammatory factors are similarly involved in the pathogenesis of DME and RVO-ME, the roles of these factors are more significant or more likely to be activated in DR patients, suggesting different treatment strategies should be considered for the two diseases., (International Journal of Ophthalmology Press.)

The vitreous body, the largest intraocular component, plays a key role in eye development, refraction, cell barrier function, oxygen metabolism and the pathogenesis of assorted diseases. Age, refraction and systemic diseases can cause vitreous metabolic abnormalities. With the continuous development of vitrectomy techniques and equipment, vitreous injections and vitrectomies have increased over the recent decades. However, the normal oxygen tension gradient in the vitreous helps to protect the lens and anterior chamber angle from oxidative stress damage, whereas the increased vitreous oxygen tension around lens and the trabecular meshwork after vitrectomy. It may lead to postoperative nuclear cataract and increase the risk for glaucoma. As a conventional procedure, scleral buckling holds several advantages over vitrectomy in selected cases. This review raises concerns regarding the function of the vitreous and encourages conducting vitreous interventions prudently if it is possible., (International Journal of Ophthalmology Press.)

At present, the incidence of infectious endophthalmitis after cataract surgery has been significantly reduced, but it is still a serious complication. Removal or not of the intraocular lens (IOL) during vitrectomy in cases with a moderate or severe inflammation is controversial. In order to call upon more discussion, we publish the article entitled "Timely vitrectomy without intraocular lens removal for acute endophthalmitis after cataract surgery" written by Guo et al in this issue. With recent advanced vitrectomy techniques, and critical measures for management of risk factors related to occurrence of infection, IOL remaining during timely vitrectomy for acute endophthalmitis can possibly be safe and effective in selected cases., (International Journal of Ophthalmology Press.)

Aim: To evaluate the long-term anatomical and visual outcomes of drusenoid pigment epithelial detachment (D-PED) in intermediate age-related macular degeneration (AMD) eyes treated with 577 nm yellow subthreshold micropulse laser (SML)., Methods: In this retrospective study, 21 eyes of 16 patients with D-PED in intermediate AMD were consecutively included and assessed. All the eyes were treated with 577 nm SML in several sessions according to D-PED growth status. The logarithm of the minimum angle of resolution (logMAR) best-corrected visual acuity (BCVA) were assessed at the initial visit and after treatment. Spectral-domain optical coherence tomography (SD-OCT) was performed to evaluate the D-PED lifecycle by volumetric calculations. Regression analysis was used to determine the breakpoint, growth, and collapse rate of the D-PED lesions. The progression to advanced AMD was also documented., Results: All the eyes were treated with SML for 2.9±1.0 sessions. The mean follow-up period was 25.3±12.6mo. The BCVA was stable from the baseline to final visit. All the eyes were categorized into two groups according to the anatomical changes of the D-PED lesion: the collapse group ( n =6, 28.6%) and non-collapse group ( n =15, 71.4%). The change in logMAR BCVA did not differ significantly between the collapse group 0.00 (-0.31, 0.85) and non-collapse group 0.00 (0.00, 0.00; P =1). Regression analysis showed that the growth rate was significantly higher in the collapse group (0.090±0.095 mm 3 /mo) than in the non-collapse group (0.025±0.035 mm 3 /mo; P <0.001). One eye (4.8%) developed macular neovascularization at 11mo after SML treatment in the non-collapse group. Three eyes (14.3%) developed geographic atrophy (GA) in the collapse group., Conclusion: Compared to the natural course of D-PED reported by previous studies, our results preliminarily show that SML can alleviate visual loss and possibility of progression to advanced AMD in eyes with D-PED in intermediate AMD. A controlled clinical trial needs to further verify the benefit of the intervention., (International Journal of Ophthalmology Press.)

The vitreous body, the largest intraocular component, plays a key role in eye development, refraction, cell barrier function, oxygen metabolism and the pathogenesis of assorted diseases. Age, refraction and systemic diseases can cause vitreous metabolic abnormalities. With the continuous development of vitrectomy techniques and equipment, vitreous injections and vitrectomies have increased over the recent decades. However, the normal oxygen tension gradient in the vitreous helps to protect the lens and anterior chamber angle from oxidative stress damage, whereas the increased vitreous oxygen tension around lens and the trabecular meshwork after vitrectomy may lead to postoperative nuclear cataract and a high incidence of open angle glaucoma. As a conventional procedure, scleral buckling holds several advantages over vitrectomy in selected cases. This review raises concerns regarding the function of the vitreous, and encourages conducting vitreous interventions prudently., (International Journal of Ophthalmology Press.)

Exercise can reduce the incidence of stress-related mental diseases, such as depression and anxiety. Control group was neither exposed to CVMS nor TRE (noCVMS/noTRE). Females were tested and levels of serum17-beta-oestradiol (E 2 ), estrogen receptors α immunoreactive neurons (ERα-IRs), estrogen receptors β immunoreactive neurons (ERβ-IRs) and oxytocin immunoreactive neurons (OT-IRs) were measured. The results showed there's increased anxiety-like behaviors for mice from CVMS/noTRE, CVMS/higher speed TRE (CVMS/HTRE) and noCVMS/HTRE groups when they were put in open field and elevated maze tests. They had lower serum E 2 levels than mice from CVMS/low-moderate speed TRE (CVMS/LMTRE), noCVMS/LMTRE and noCVMS/noTRE groups. The three groups of CVMS/noTRE, CVMS/HTRE and noCVMS/HTRE mice had more ERα-IRs and less ERβ-IRs in the medial preoptic area (mPOA), bed nucleus of the stria terminalis (BNST) and medial amygdala (MeA), hypothalamic paraventricular nucleus (PVN) and supraoptic nucleus (SON). The number of OT-IRs in PVN and SON of CVMS/noTRE, CVMS/HTRE and noCVMS/HTRE mice was also lower than that of mice from CVMS/LMTRE, noCVMS/LMTRE and noCVMS/noTRE groups. Interestingly, CVMS/LMTRE and noCVMS/LMTRE mice were similar to noCVMS/noTRE mice in that they did not show anxiety, while CVMS/HTRE and noCVMS/HTRE mice did not, which were similar to the mice in CVMS/noTRE. We propose that LMTRE instead of HTRE changes the serum concentration of E 2 . ERβ/ERα ratio and OT level in the brain may be responsible for the decrease in anxiety-like behavior in female mice exposed to anxiety-inducing stress conditions., Competing Interests: The authors declare no competing financial interests., (© 2021 Published by Elsevier Ltd on behalf of International Brain Research Organization.)

Metformin (MET), a first-line oral agent used to treat diabetes, exerts its function mainly by activating adenosine monophosphate-activated protein. The accumulation of oxidized phospholipids in the outer layer of the retina plays a key role in retinal pigment epithelium (RPE) cells death and the formation of choroidal neovascularization (CNV), which mean the development of age-related macular degeneration (AMD). Recent studies have shown that MET can regulate lipid metabolism, inhibit inflammation, and prohibit retinal cell death and CNV formation due to various pathological factors. Here, newly discovered functions of MET that may be used for the prevention and treatment of AMD were reviewed., (International Journal of Ophthalmology Press.)

The incidences of open angle glaucoma (OAG) and high myopia are increasing concomitantly. Considering the aging population and concurrent rapid increase in the number of individuals with myopia, the risk of visual defects caused by highly myopic OAG is likely to increase dramatically over the next few decades. However, precise screening and diagnosis of OAG is challenging because of the tilt and rotation of the optic disc, as well as extensive β-zone parapapillary atrophy in highly myopic eyes. Recent advances in optical coherence tomography (OCT) and OCT angiography (OCTA) technologies imply that both modalities are promising tools for the detection of highly myopic OAG. Notably, the diagnosis of OAG remains to be determined with the longitudinal changes of functional damages ( e.g. visual field defect, visual electrophysiological changes). We herein describe some aspects of microvascular and microstructural pathology in patients with highly myopic OAG and proposes a framework for the development of novel diagnostic and therapeutic strategies., (International Journal of Ophthalmology Press.)

Aim: To investigate whether intravitreal injection of oxidized low-density lipoprotein (OxLDL) can promote laser-induced choroidal neovascularization (CNV) formation in mice and the mechanism involved, thereby to develop a better animal model., Methods: C57BL6/J mice were randomized into three groups. Immediately after CNV induction with 532 nm laser photocoagulation, 1.0 µL of OxLDL [100 µg/mL in phosphate-buffered saline (PBS)] was intravitreally injected, whereas PBS and the same volume low-density lipoprotein (LDL; 100 µg/mL in PBS) were injected into the vitreous as controls. Angiogenic and inflammatory cytokines were measured by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting (WB) after 5d, and CNV severity was analyzed by choroid flat mount and immunofluorescence staining after 1wk. In vitro , retinal pigment epithelial (RPE) cell line (ARPE19) were treated with OxLDL (LDL as control) for 8h. Angiogenic and inflammatory cytokine levels were measured. A specific inhibitor of lectin-like oxidized low-density lipoprotein receptor 1 (LOX1) was used to evaluate the role of LOX1 in this process., Results: At 7d after intravitreal injection of 1 µL (100 µg/mL) OxLDL, T15-labeled OxLDL was mainly deposited around the CNV area, and the F4/80-labeled macrophages, the CD31-labeled vascular endothelial cells number and CNV area were increased. Meanwhile, WB and qRT-PCR results showed that vascular endothelial growth factor (VEGF), CC chemokine receptor 2 (CCR2), interleukin-6 (IL-6), IL-1β, and matrix metalloproteinase 9 (MMP9) expressions were increased, which was supported by in vitro experiments in RPE cells. LOX1 inhibitors significantly reduced expressions of inflammatory factors IL-1β and VEGF., Conclusion: A modified laser-induced CNV animal model is established with intravitreal injection of 1 µL (100 µg/mL) of OxLDL at 7d, which at least partially through LOX1. This animal model can be used as a simple model for studying the role of OxLDL in age-related macular degeneration., (International Journal of Ophthalmology Press.)

Aim: To evaluate the value of ultra-wide field (UWF) imaging in the management of traumatic retinopathy under the condition of corneal scar or fixed small pupil after complicated ocular trauma., Methods: Twenty-eight patients (28 eyes) with complicated ocular trauma were enrolled in the study from June 2016 to May 2017, including 19 males and 9 females with age ranged from 11 to 64 (43.42±12.62)y. All patients were treated with secondary vitrectomy after emergency operation for wound repair of open ocular trauma. Direct ophthalmoscopy and 45-degree fundus photography were taken at each time point of follow up for comparison of findings with UWF images. Routine eye examination including visual acuity, intraocular pressure, slit lamp examination were performed and analyzed as well., Results: Among the 28 traumatized eyes, the positive rate for identification of traumatic retinopathed was 32.1% (9 cases), 14.9% (5 cases), and 85.7% (24 cases) with direct ophthalmoscopy, 45-degree fundus photography, and UWF imaging, respectively. The detective rate of UWF imaging under the condition of corneal scar or fixed small pupil was statistically greater than that of 45-degree fundus photography and direct ophthalmoscopy (Bonferroni correction, P <0.001). UWF image was obtained in 19 eyes with opaque corneal scar, otherwise their fundus could not be seen by conventional methods. The additional findings of traumatic retinopathies by UWF imaging included periretinal membranes or pre-retinal proliferating strip, retinal holes, hemorrhage in the vitreous or sub-retinal space., Conclusion: UWF imaging is superior to traditional fundus photography in the evaluation of traumatic retinopathies under the condition of corneal scar or fixed small pupil after complicated ocular trauma.

Aim: To investigate the findings of infrared fundus autofluorescence (IR-AF) and spectral-domain optical coherence tomography (SD-OCT) in eyes with optic disc melanocytoma (ODM)., Methods: IR-AF findings and those of other ophthalmologic imaging examinations, including short-wave autofluorescence (SW-AF), fluorescein angiography (FA), fundus color photography, and SD-OCT of 8 eyes of 8 consecutive cases with ODM were assessed., Results: The ODMs in all cases (100%) presented similar IR-AF, SW-AF, and FA findings. On IR-AF images, ODMs showed outstanding hyper-AF with well-defined outline. On SW-AF images, the area of ODMs presented as hypo-AF. FA images revealed the leaking retinal telangiectasia on the surface of the ODMs. On SD-OCT images in 8 cases (100%), the ODMs were sloped with highly reflective surface, which were disorganized retina and optic nerve layers. In 7 cases (87.5%), peripapillary choroids were involved. The melanocytomas of 8 cases (100%) presented as optically empty spaces. Vitreous seeds were found in one case (12.5%)., Conclusion: IR-AF imaging may provide a new modality to evaluate the pathologic features of ODMs, and together with SW-AF imaging, offers a new tool to study biological characteristics associated with ODMs. SD-OCT is a valuable tool in delimitating the tumor extension and providing morphological information about the adjacent retinal tissue.

The natural vitreous body has a fine structure and complex functions. The imitation of the natural vitreous body by vitreous substitutes is a challenging work for both researchers and ophthalmologists. Gases, silicone oil, heavy silicone oil and hydrogels, particularly the former two vitreous substitutes are clinically widely used with certain complications. Those, however, are not real artificial vitreous due to lack of structure and function like the natural vitreous body. This article reviews the situations, challenges, and future directions in the development of vitreous substitutes, particularly the experimental and clinical use of a new artificial foldable capsular vitreous body.

Objective: To investigate the regulatory effects of ninjurin-1 on adhesion of myeloid cells in the retina at the early stage of diabetic rats., Methods: Experimental study. The rat diabetic model was induced by intraperitoneal injection of streptozotocin. After 2 months of diabetes induction, 27 diabetic rats were randomly chosen and assigned to 3 groups, including diabetes and phosphate buffered saline (PBS) injection group (group B), diabetes and anti-Ninj-1 injection group (group C) and diabetes and anti-IgG injection group (group D), with 9 rats in each group. Nine age matched health rats were chosen as control group (group A). Retinal leukostasis was quantified with acridine orange leukocyte fluorography. Retinal myeloid cell infiltration activity was measured by enzyme linked immunosorbent assay of myeloperoxidase (MPO). The differences of the mean values among the four groups were analyzed by one-factor analysis of variance. The multiple comparisons of the mean values among the four groups were analyzed by LSD-t analysis., Results: According to the results of the acridine orange leukocyte fluorography, the numbers of leukocyte adhesion in the four groups were 49.66 ± 13.51, 153.66 ± 20.43, 85.33 ± 15.03 and 156.33 ± 11.53, respectively. The differences among them were significant (F = 143.34, P = 0.000). The numbers of leukocyte adhesion in the group C were significantly lower than that in group B (P = 0.000, 95%CI: -82.68 - -53.98). The levels of retinal MPO in the four groups were (15.66 ± 2.08), (27.66 ± 2.51), (18.02 ± 2.01) and (26.66 ± 3.21) µg/L, respectively. The differences among them were significant (F = 17.61, P = 0.010). The level of retinal MPO in the group C was significantly lower than that in group B (P = 0.010, 95%CI: -14.37 - -4.95)., Conclusions: Ninj-1 may play a role in the mediation of the adhesion of myeloid cell to the rat retina of early-stage of diabetes in vivo.

Animal models of diabetes mellitus (DM) are vital for research on pathogenesis and pharmaceutical therapy of diabetic retinopathy (DR). At present, diabetic animal models include chemicals or dietary-induced models, transgenic or gene knockout mice, and spontaneous mice, etc. Among these models, rat model induced by streptozotocin is simple and imminent with high repeatability, which made it the most popular one. However, no available diabetic model could reproduce all vascular and neural pathological changes observed in human non-proliferative and proliferative DR. It is also known that not all DM models could lead to DR. In addition, features and severity of retinopathy in the model depend on animal kinds, animal lifespan, time course of the disease and induction methods. Therefore, researchers should consider characteristics and limitations of different models while choosing suitable DM model based on research objectives and resources. It is particularly emphasized that the results from animal models do not always fit human conditions.

Although the resource devoted for scientific research increased substantially in recent years, the quality of our basic and clinical study still leaves much to be improved. Besides the shortcomings in administration of research funding, our researchers, as the entity of scientific work, should fully recognize that innovation and application are vital for basic and clinical research in ophthalmology. It is emphasized that the principles and methods in an innovative study, including comprehensive grasp for up-to-date information, proposal of a key project, collaboration of multi-disciplines, through design of a study, and acceleration of application in practice, should be followed in order to make new advances in our cause in ophthalmology.

Purpose: Anti-vascular endothelial growth factor (VEGF) agents have recently been used intravitreally during the perioperative period for proliferative diabetic retinopathy (PDR). However, the mechanism of theraputic effects of the agents remains unclear. This study aimed to investigate the effects of intravitreal bevacizumab (IVB) on retinal vascular endothelial cells and expressions of VEGF and hypoxia inducible factor-1α (HIF-1α) in PDR., Methods: Twenty-four patients with PDR were enrolled and randomized to two groups. Twelve eyes of 12 patients of each group received either an intravitreal injection of 1.25 mg bevacizumab or a sham injection 6 days before vitrectomy. Neovascular membranes (NVMs) were collected during pars plana vitrectomy. The numbers of vascular endothelial cells in the NVMs were counted after staining with hematoxylin and eosin and von Willebrand. The expressions of VEGF and HIF-1α in the NVMs were detected through immunohistochemistry. Ten epiretinal membrane specimens from patients with proliferative vitreoretinopathy (PVR) without IVB treatment were set as an additional control., Results: The number of vascular endothelial cells in NVMs of the IVB pretreated group was significantly lower than that of the sham group (21.5±3.94 versus 41.33±7.44, p=0.003). The IVB pretreated group also showed significantly lower levels of VEGF and HIF-1α in NVMs than those of the sham group (P(HIF-1α)=0.02, P(VEGF)<0.001). A stepwise regression analysis showed that IVB was a significant negative predictor for the numbers of vascular endothelial cells (β=-0.89, p<0.001) and the expressions of VEGF (β=-0.85, p<0.001) and HIF-1α (β=-0.64, p=0.001) in PDR patients. Epiretinal membranes of the PVR group showed negative staining of VEGF and HIF-1α., Conclusions: Pretreatment with IVB in patients with PDR significantly decreased vascular endothelial cells and expressions of VEGF and HIF-1α, which further supports preoperative use of IVB in such patients.

Purpose: This study aimed at identifying the expression of functional formyl peptide receptor (FPR)-1 in human retinal pigment epithelium (hRPE) cells and to evaluate the role of FPR in regulation of wound closure of the hRPE monolayer under electric fields (EFs)., Methods: The expression of FPR in hRPE cells was analyzed with an immunofluoresence labeling assay and RT-PCR. Cultured wounded hRPE monolayers were exposed to EFs with free serum, 20%, serum, and a classical FPR agonist, N-formyl-Met-Leu-Phe (fMLF), respectively, for 3 hours. Cell monolayer migrations were traced using an image analyzer. Expressions of cell junction molecules were measured by RT-PCR, and the ultrastructure of cell junctions was observed with transmission electron microscopy (TEM)., Results: The expression of functional FPR was observed and localized along actin filaments in cellular lamellipodia and filopodia. EFs and fMLF significantly increased the migration rates of the wounded RPE monolayer. The migrating distances of monolayers were 24.262 ± 6.82 μm, 40.243 ± 5.069 μm, and 56.926 ± 7.821 μm in cells with free serum, 20% serum, and fMLF under EFs at 3 hours, respectively (P < 0.01). The mRNA expressions of connexin 43(Cx43) and zonula occludens (ZO)-1 were detected in hRPE cells. TEM revealed that cell junctions formed between hRPE cells in the monolayer., Conclusions: These results showed for the first time that functional FPR expresses in hRPE cells and that activation of FPR enhances migration of the wounded hRPE monolayer. The mRNA expressions and ultrastructures of cell junctions further demonstrated the RPE sheet as a monolayer migrating under EFs.

Aims: Direct current electric fields (EFs) can induce directed cell migration in a wide variety of cells, and this has been proven to be of importance in wound healing. Here we observed the effects of EFs on cultured human retinal pigment epithelial (RPE) cells and explored the possible involvement of integrin β1 subunit signaling in the process., Methods: Cultured human RPE cells were exposed to an EF at 5 V/cm for 3 h. The rate and directionality of cell migration were quantified. The distribution of integrin β1 subunit was measured by immunohistochemistry and the expression of integrin β1 subunit and phosphorylated focal adhesion kinase (FAK) was determined by PCR and Western blotting. Experiments were performed in the presence or absence of anti-integrin β1 subunit antibody., Results: During exposure to an EF at 5 V/cm for 3 h, the separated human RPE cells and wounded RPE monolayer demonstrated a cathodal-directed migration. The distribution of integrin β1 subunit in the cells was also polarized to the cathode, and the expression in mRNA and its protein level were obviously increased. Furthermore, exposure to EFs of 5 V/cm triggered the phosphorylation of FAK in human RPE cells. In contrast, blocking of integrin β1 subunit suppressed the directed migration of RPE cells and reduced the activation of FAK in EFs., Conclusions: These findings indicate that EF exposure results in directed migration of the separated RPE cells and RPE monolayer. These effects may partially act through the activation of integrin β1 subunit signaling., (Copyright © 2010 S. Karger AG, Basel.)

Aim: To study the effect of Rac1 on the induction of HIF-1α in choroidal neovascularization (CNV) in mice., Methods: One hundred C57BL/6J mice were laser photocoagulated to induce CNV, fifty mice of that were selected randomly for intravitreal injection of Rac1 inhibitor NSC23766 solution (1µL). After laser photocoagulation, fundus fluorescein angiography (FFA) was performed to verify the growth of CNV, Immunohistochemistry and Western blot were used to detect HIF-1α and Rac1 in posterior segment of eye globes., Results: FFA verified that incidence of CNV was significantly reduced in the eyes with NSC23766 injection comparing with that of eyes without NSC23766 injection (P<0.01). Immunohistochemistry detected that HIF-1α and Rac1 mainly expressing in the new fibrovascular tissue. Western blot showed that HIF-1α and Rac1 was highly increased in tissue explants of retinal pigment epithelium (RPE) and choroid without NSC23766 injection. But for tissue explants of RPE and choroid with NSC23766 injection, both the expression of HIF-1α and Rac1 were inhibited., Conclusion: Rac1 is crucial to activate HIF-1 regulating the growth of CNV, and its inhibition may have potential therapeutic value.

Background: Human N-Myc downstream regulated gene2 (NDRG2), a novel gene has been cloned and shown to be related to a number of cellular processes, including proliferation, differentiation, stress, and apoptosis. NDRG2 has also been linked to age-related Alzheimer's disease. Since the role of this gene in senescence is limited, we have investigated the potential role of NDRG2 in human lens epithelial cells (HLECs), a paradigm implicated in age-related cataract., Methodology/principal Findings: Cultured HLECs (SRA01/04) were subjected to prolonged exposure to low dose of H(2)O(2) to simulate senescence. After being exposed to 50 µM H(2)O(2) for 2 weeks, HLECs senescent-morphological changes appeared, cell viability decreased dramatically, cell proliferation reduced from 37.4% to 16.1%, and senescence-associated β-galactosidase activity increased from 0 to 90.3%. Ndrg2 protein expression was also significantly increased in these senescent cells. To induce overexpression of NDRG2, SRA01/04 cells were infected with the adenoviral vector of NDRG2. In these cells, overexpression of NDRG2 resulted in a fibroblast-like appearance and the cell viability decreased about 20%. In addition, the NDRG2-overexpression cells demonstrated 20% lower viability when exposed to 50-200 µM H(2)O(2) for acute oxidative stress. Furthermore, the expression of NDRG2 from age-related cataracts was up-regulated 2-fold at both mRNA and protein levels compared with the clear lenses., Conclusions/significance: NDRG2 is up regulated not only in the ageing process of HLECs in vitro but also in the cells from human age-related cortical cataract in vivo. Up-regulation of NDRG2 induces cell morphological changes, reduces cell viability, and especially lowers cellular resistance to oxidative stress. NDRG2-mediated affects in HLECs may associate with age-related cataract formation.

With an increasing number of new cases of tuberculosis every year, the incidence of tuberculous uveitis presents a rising trend. The disease is associated with a wide spectrum of clinical manifestations. The common clinical presentations appear to be anterior uveitis, choroidal tubercles, multifocal choroiditis, rarely serpiginous-like choroiditis, subretinal abscesses or suspected ocular tumors. Macular edema can be the only ocular manifestation of tuberculosis. With absence of proper diagnostic standard, it usually leads to missed diagnosis, misdiagnosis, and delayed diagnosis of tuberculous uveitis which can result in severe consequences such as vision loss, blind, and even eye enucleation. New technology such as PCR and Interferon-Gamma release assays can be helpful for the diagnosis of tuberculous uveitis. The early diagnosis and standard treatment of tuberculous uveitis are in urgent need.

Purpose: To explore the feasibility of bone marrow mesenchymal stem cells (MSCs) transdifferentiating into corneal epithelial cells in a limbal stem cell deficiency (LSCD) model in rats., Methods: Rat MSCs were isolated and purified using a gradient isolation procedure. The cells were induced by rat corneal stromal cells (CSCs) in a transwell co-culture system. The induced MSCs were identified by immunofluorescence staining, flow cytometry, and scanning electron microscopy (SEM). A corneal LSCD model was produced in the right eyes of 48 rats by alkali injury. The eyes of 12 rats without any transplant served as controls (Group 1). Amniotic membranes (AM; Group 2), uninduced MSCs (Group 3), or MSCs induced by CSCs (Group 4), were transplanted onto the cornea of the model (n=12 each). The therapeutic effects of the four groups were evaluated by slit lamp observation, hematoxylin and eosin staining, immunohistochemistry staining, and confocal laser corneal microscopy., Results: Cultivated MSCs were positive for CD29, CD44, and CD90, but negative for CD34, CD45, CD133, and CK12, with typical MSCs characteristics revealed by SEM. After co-culture with CSCs, the induced MSCs expressed positive staining for CK12 with corneal epithelial cell characteristics confirmed by SEM; the induced MSCs were unchanged on the amnion. Compared with the other three groups, the corneal opacity, fluorescence staining, and neovascularization grades were significantly decreased in the induced MSCs group, both on postoperative week four and ten., Conclusion: MSCs induced by CSCs can transdifferentiate into corneal epithelial cells in vitro. The induced MSCs on an amniotic membrane have remarkable effects on the treatment of corneal alkali burn and the reconstruction of the corneal surface of rats.

Background: It has been hypothesized that emulsification of silicone oil might stimulate preretinal membrane formation and proliferative vitreoretinopathy. This study aimed to test the effects of vesicles of silicone oil (VSO), which were prepared by a novel membrane emulsification technique, on the migration and proliferation of human retinal pigment epithelial (hRPE) cells in vitro., Methods: VSO were produced by extrusion of the oil through a Supor membrane (Pall Life Sciences, Port Washington, NY, USA) with 0.45 microm pore size under a pressure of 0.1 MPa. hRPE cells were incubated with stained 25% VSO for 24 h to assess whether hRPE cells could phagocytize VSO. A wound-healing model was used by denuding cells from a glass slide to assess the migration of hRPE cells. The cells were then incubated with 25%, 50% and 100% VSO with or without serum for up to 72 h. The number of cells that had entered the denuded area was counted under a microscope. To assess the proliferative activity, the cells were incubated with 25%, 50% and 100% VSO, with or without serum, for 24 h. A total of 100 nuclei were examined for each slide, and the numbers of stained argyrophilic nucleolar organizer regions (AgNORs) in the nuclei were measured., Results: The mean vesicle diameter of VSO prepared was 4.25 +/- 0.77 microm. The stained 25% VSO were phagocytized by hRPE cells. After the cells cultured with serum-free Dulbecco's Modified Eagle Medium (DMEM) were incubated with VSO, the numbers of migratory cells at higher concentrations (50% and 100%) of VSO were significantly decreased (48.9 +/- 5.37 and 10.6 +/- 3.03 respectively) compared to controls (69.9 +/- 9.88; P < 0.01). After the cells cultured with serum-free DMEM were incubated with VSO, the number of AgNORs in nucleus at 100% VSO was significantly increased compared to controls (3.1 +/- 0.72 vs 1.6 +/- 0.6; P < 0.01)., Conclusion: The result indicated that VSO inhibited the migration but stimulated the proliferation of hRPE cells.

Background: Rhegmatogenous retinal detachment and proliferative vitreoretinopathy (PVR) are eye diseases that are characterized by mechanical stress involving stretching of the retinal pigment epithelial (RPE) cells by the vitreous or the hyperplastic membranes. Here, we assessed whether mechanical force could change the expression of matrix metalloproteinases (MMPs) in RPE cells via the mitogen-activated protein kinase (MAPK) pathway., Methods: Collagen-coated magnetite beads and magnetic fields were used to apply tensile forces to cultured RPE cells at focal adhesions. Activation of the MAPK, including extracellular signal-regulated protein kinase (ERK), c-jun N-terminal kinase (JNK), and p38 were determined over a time course from 5 to 30 min by Western-blot analysis. Activation of p38 was also tested using immunofluorescence staining. The mRNA levels of MMP-2, MMP-9, tissue inhibitor of MMP (TIMP)-2 and fibronectin (FN) were analyzed by RT-PCR. Active MMP-2 and MMP-9 were demonstrated by zymography. MMP-2 secretion was evaluated by enzyme immunoassay., Results: Stimulation of RPE cells with mechanical stress did not change the total protein expression of the MAPK proteins ERK, JNK, and p38. However, of the three kinases, only active p38 showed an increased protein expression which was also shown by a 2.8-fold increase in immunofluorescence staining at 5 min following mechanical stress stimulation. This increase in active p38 expression was blocked by treating the cells with the p38 inhibitor SB203580. FN mRNA increased 2.4-fold at 15 min and MMP-2 mRNA increased 2.1-fold at 4 h. MMP-2 secretion increased 1.5-fold at 4 h and 1.9-fold at 12 h. The expression of MMP-2 and FN, and the activation and secretion of MMP-2, were inhibited in the presence of SB203580. The mRNA expression of MMP-9 and TIMP-2 did not change throughout., Conclusions: This study shows that mechanical stress upregulates MMP-2 and FN expression through activation of the p38 pathway. The increase in MMP-2 levels evoked by mechanical force may contribute to the remodeling of the extracellular matrix around RPE cells, weakening the interlinkage and membrane attachment between RPE cells, and facilitate cellular migration.

Purpose: The migration of retinal pigment epithelium (RPE) cells is an initial step in the development of proliferative vitreoretinopathy (PVR). We investigated the expression of connective tissue growth factor (CTGF) in an in vitro model of wound healing and effects of recombinant human CTGF (rhCTGF) on modulating migration and Ca(2+) signaling in RPE cells., Methods: Cultured human RPE monolayers were used to establish a wound-healing model. Western blot and in situ hybridization were used to detect the CTGF expression in RPE cells. Migration of RPE cells was measured under the stimulation of rhCTGF alone or in combination with dexamethasone (DEX) or 8-Br-cAMP. To determine the concentration of cytoplasmic-free Ca(2+) ([Ca(2+)]i) responding to CTGF, the fluo-3/AM-loaded RPE cells were observed with a laser scanning confocal microscope., Results: The CTGF expression first increased after being wounded in RPE cells, then reached a peak and maintained at a high level. The positive expression was mainly at the edge of scrape and in motile RPE cells. rhCTGF-stimulated RPE cells migrated in a dose-dependent manner, and both DEX and 8-Br-cAMP could significantly inhibit the CTGF-induced migrations. CTGF induced a (Ca(2+))i elevation in RPE cells in a concentration-dependent manner. Moreover, stimulation of RPE cells with CTGF and DEX or 8-Br-cAMP counteracted the elevation of (Ca(2+))i induced by CTGF., Conclusions: The CTGF expression could be induced by an in vitro model of scrape wounding. rhCTGF stimulated the migration and Ca(2+) signal pathway in RPE cells in a dose-dependent manner, and DEX and 8-Br-cAMP suppressed this effect. Our results indicate that CTGF is involved in the wound-healing process and plays an important role in the pathogenesis of intraocular proliferative diseases.

Purpose: To observe the effects of electric fields (EFs) on the migration of human retinal pigment epithelial (RPE) cells and to explore possible related mechanisms., Methods: Cultured human RPE cells were exposed to EFs, and images of the cells were obtained at regular intervals to evaluate the cell migration and viability. Distribution of F-actin and beta1 integrin was measured by immunohistochemistry. Expression of beta1 integrin was determined by PCR and Western blotting., Results: Exposure to EFs resulted in a cathodal-directed migration of RPE cells and a cathodal accumulation of F-actin and beta1 integrin in the cells. EF stimulation increased expression of beta1 integrin both in mRNA and protein levels. These effects could be inhibited by cytochalasin B., Conclusion: EFs induce directed migration of RPE cells, and this effect is, to some extent, regulated by the interaction between cytoskeleton and integrins.

Age-related macular degeneration (AMD) remains high incidence and accounts for a main cause of blindness in ageing people, but its mechanism is still poorly understood. Ageing and associated dysfunction of retinal pigment epithelial (RPE) cells were believed to be the pathological onset of AMD. 20S proteasome has been tightly correlated with cell ageing due to its fundamental role in maintaining cellular homeostasis, but its implication in the ageing process of human RPE cells was seldom concerned. This study aimed to demonstrate the interconnections between 20S proteasome and ageing RPE cells by characterizing age-dependent alterations of the 20S proteasome in primarily cultured human RPE cells. For this purpose, a replicative ageing RPE cell model was established and validated through testing the cell viability, beta-galactosidase activity and cellular autofluorescence. Decline in chymotrypsin-like, peptidylglutamyl-peptide hydrolase and trypsin-like activities of the 20S proteasome was detected in aged RPE cells through degradation of fluorogenic substrates. Immunofluorescence assay revealed that the 20S proteasome was concentrated in RPE nucleus, and redistributed partly to the peri-nuclear regions in old RPE passages. These age-dependent changes of the 20S complex were accompanied with a significantly increased fluorescent intensity of intracellular oxidized proteins. Further analysis of the proteasome-to-oxidized protein ratio indicated a preferred protection of the RPE nuclear proteins by the 20S proteasome, which also subsided remarkably as a function of the cell ageing. In conclusion, we demonstrated functional impairment and redistribution of the 20S proteasome with age in human RPE cells and supposed these alterations impactful on the process of RPE cell ageing and furthermore on the pathogenesis of AMD. Future researches on the mechanism of these alterations and the pathways to manipulate their effects are still strongly recommended.

Background: Retinal pigment epithelial (RPE) cells and choroidal microvascular endothelial cells (CECs) are the main cells involved in choroidal neovascularization (CNV), and hypoxia plays an important role in CNV formation via hypoxia inducible factor 1 (HIF-1). Our aim was to evaluate the role of HIF-1 in human RPE cells with regard to proliferation, migration and tube formation of CECs under hypoxia., Methods: RPE cells were cultured under chemical hypoxia induced by 200 muM CoCl(2), and RNA interference (RNAi) technique was used to knock down HIF-1alpha gene in RPE cells. mRNA and protein expression of HIF-1alpha and VEGF in RPE cells were investigated by real-time RT-PCR and Western blot. Three kinds of coculture models were used to observe the effects of RPE cells transfected by short hairpin RNA (shRNA)-expressing plasmid DNA (pDNA) (pshHIF-1alpha) on the proliferation, migration and tube formation of CECs respectively., Results: Transfection of shRNA-expressing pDNA targeting HIF-1alpha to RPE cells resulted in the knock down of HIF-1alpha gene and reduction of the corresponding mRNA and protein of HIF-1alpha and VEGF under hypoxia. Consequently, the proliferation, migration and tube formation of CECs were significantly inhibited by the knocked-down RPE cells compared with the control in the coculture system. The proliferation rates of CECs decreased by 40.2%, 36.6% and 36.8% on days 3, 4 and 5 respectively. Migration reduced by 49.6% at 5 h, and tube formation decreased by 40.4% at 48 h., Conclusion: RNAi of HIF-1alpha in RPE cells can inhibit angiogenesis in vitro and provide a possible strategy for treatment of choroidal neovascularization diseases by targeting HIF-1alpha.

Objective: To evaluate the efficacy of lamellar keratoplasty in the rabbit using a graft of lyophilized acellular porcine corneal stroma (APCS)., Animal Studied: Twelve adult 2-2.5 kg Zealand white rabbits were studied., Procedure: The cell components of the porcine cornea were removed by the means of enzymatic digestion, freezing, and thawing and then APCS was lyophilized. The 6.5 mm diameter APCS was implanted on a 6.0-mm diameter keratectomy wound each of 12 rabbits. The postoperative clinical and histological evaluations were performed in the early, intermediate, and late periods., Results: All corneal wounds healed. Ten of the 12 grafts of APCS were integrated completely with the receptive cornea except two grafts scraped partially off by the eyelid. The blepharospasm, ocular discharge, and edema of the cornea were marked 1 week after transplantation. New vessels invaded the graft after week 2 and regressed after week 8. The cornea became transparent gradually. The histological evaluation showed that the epithelium on the graft stratified normally post surgery. The keratocytes of the recipient grew into the graft and were proliferative at week 4. The inflammatory cells and new vessels were observed before week 8. The fibrosis in the graft was revealed at week 4 and lessened at week 8. The histological structure of the cornea after surgery was similar to the normal cornea at week 32., Conclusions: APCS can recover the integrity of the rabbit's cornea and become transparent in vivo. APCS is an effective graft for lamellar keratoplasty in the rabbit.

Purpose: To investigate whether rhegmatogenous retinal detachment (RRD) alters intraocular soluble syndecan-1 levels., Methods: In all, 39 samples of subretinal fluid (SRF) and 10 samples of vitreous fluid from RRD patients were collected. Using ELISA, soluble syndecan-1 levels were detected, and potential correlations between syndecan-1 levels with clinical parameters were analyzed., Results: Soluble syndecan-1 in the vitreous fluid (2.577+/-0.578 ng/ml) and in the SRF (1.499+/-0.184 ng/ml) from eyes with RRD enhanced significantly compared to that of the controls (0.224+/-0.095 ng/ml) (p<0.0001 and p=0.006). An increase in the syndecan-1 concentrations in SRF samples correlated with a longer duration of retinal detachment (r=0.716, p<0.0001) and a younger age (r= -0.341, p=0.017)., Conclusions: RRD was found to be associated with a significant increase of soluble syndecan-1 in the vitreous fluid and SRF. In SRF, an enhanced soluble syndecan-1 concentration correlated positively with the duration of retinal detachment and inversely with the age of patients.

Purpose: To determine the effects of pericytes and angiopoietin-1 on the expression of occludin and zonula occludens-1 (ZO-1) in retinal endothelial cells (ECs) under both normoxic and hypoxic conditions., Methods: Rat primary retinal microvascular ECs were cultured under normoxia or hypoxia in either absence or presence of pericytes conditioned medium (PCM). PCM was pretreated with or without angiopoietin-1 neutralizing antibody. Immuofluorescent staining, Western blot and RT-PCR were used to detect the alterations of occludin and ZO-1 expression., Results: Under normoxia, PCM strengthened occludin and ZO-1 immunofluorescent staining at cytomembrane as well as increased their expression at both protein and mRNA level. When pretreated with angiopoietin-1 neutralizing antibody, occludin upregulation induced by PCM was significantly blocked at protein level (62%) and mRNA level (34%). Under hypoxia, the continuity of occludin and ZO-1 staining at cell boundaries was disrupted consistent with a decrease of their protein level by 31 and 27%, respectively. Also occludin and ZO-1 mRNA level decreased by 46 and 57%, respectively. PCM was observed to partially increase expression of occludin at protein and mRNA level. Angiopoietin-1 antibody slightly inhibited (16%) PCM induced occludin mRNA increase under hypoxia., Conclusion: Pericytes improved the integrity of endothelial barrier through inducing occludin and ZO-1 expression at protein and mRNA level under normoxia. Under hypoxia, pericytes could partially reverse occludin decrease. These protecting effects of pericytes on endothelial barrier were at least in part mediated by angiopoietin-1.

Objectives: This study investigated whether integrin-linked kinase (ILK) is involved in the pathogenesis of diabetic retinopathy, by analyzing the expression and activity of ILK in the retina from a streptozotocin (STZ)-induced rat model of diabetes., Methods: ILK expression in the retina from both control and STZ-induced diabetic rats was measured by reverse transcription polymerase chain reaction, immunohistochemistry and Western blot analysis. The expressions of Akt and FOXO1A, the downstream molecules of ILK, were also examined., Results: The present study showed that the STZ-induced diabetes was associated with the increase in the vascular permeability in the retina. This elevated vascular permeability increased with the progression of diabetic retinopathy. Meanwhile, the results also showed that the expression of ILK increased in protein and mRNA levels in the retina of STZ-induced diabetic rats. Immunohistochemistry showed that immunostaining of ILK was localized in the outer plexiform layer (OPL), the inner nuclear layer (INL), the inner plexiform layer (IPL), the ganglion cell layer (GCL) and the retinal microvasculature of rats. However, the expression of Akt was reduced in the retinas at 8 and 12 weeks and increased in the retinas at 4 weeks after induction of diabetes. Meanwhile, the expression of the FOXO1A protein increased in the retinas at 8 and 12 weeks and decreased in the retinas at 4 weeks after induction of diabetes. The FOXO1A immunostaining was also observed in the retinal microvasculature and in the OPL, INL, IPL and GCL of rat retinas., Conclusion: These results indicate that diabetes affects the expression of ILK in the retina. ILK may be involved in the diabetes-induced damage and/or alterations of neural and microvascular structures.

Purpose: To investigate the role played by E26 transformation-specific-1 (Ets-1), a transcription factor, and extracellular signal-regulated kinase 1/2 (ERK1/2) in the expression of vascular endothelial growth factor (VEGF), and the interaction of Ets-1 and ERK1/2 in the retina of diabetic rats., Methods: Diabetes was induced in rats by an intraperitoneal injection of streptozotocin (STZ). To follow the time course in the expression of Ets-1, phosphorylated ERK1/2 (pERK1/2), and VEGF, rats were killed at 1, 2, 4, and 8 weeks after the injection of STZ, and total proteins were extracted from the isolated retinas. An adenovirus vector encoding dominant-negative Ets-1 and an inhibitor of PD98059 was injected intravitreally to investigate the effects of Ets-1 blockade and ERK1/2 inhibition on the expression of VEGF. Four weeks after the first intravitreal injection, total proteins and total RNA were extracted from the retinas for Western blot and Northern blot analyses., Results: The expression of Ets-1, pERK1/2, and VEGF in the retina increased in a time-dependent manner after STZ injection. The phosphorylation of ERK1/2 and protein level of VEGF were significantly reduced following intravitreal Ets-1. Inhibition of ERK1/2 phosphorylation resulted in a significant reduction in the expression of Ets-1 and the level of VEGF protein., Conclusions: These results indicate that in the retina of STZ-induced diabetic rats: (1) the alterations of Ets-1, pERK1/2, and VEGF are approximately synchronized; (2) the phosphorylation of ERK1/2 is regulated by the expression of Ets-1; (3) the production of Ets-1 protein is dependent on the ERK1/2 pathway, and (4) the protein level of VEGF is regulated by both Ets-1 expression and ERK1/2 phosphorylation. We propose that VEGF, Ets-1, and ERK1 act synergistically in the development of diabetic retinopathy., (Copyright (c) 2007 S. Karger AG, Basel.)

Background: Migration and proliferation of retinal pigment epithelial (RPE) cells play an important role in proliferative vitreoretinopathy. Epidermal growth factor receptor (EGFR) is a cell surface receptor with intrinsic tyrosine kinase activity. The engagement of the receptor by its ligand can induce intracellular mitogenic signal transduction pathways and stimulate proliferation, migration and differentiation of cells. This experiment aimed to investigate the activation and role of EGFR signal transduction pathway in proliferation of human RPE cells., Methods: Cultured human RPE cells of the 3rd to 6th passages were studied by colorimetric assay for cellular growth and survival (MTT assay) to test the effects of EGF (0.1, 1, 10, 50, and 100 ng/ml) and fetal bovine serum (FBS) on proliferation of human RPE cells. An in vitro wound healing model was also set up, and the number of cells that had entered the denuded area was counted. The human RPE cells were cultured for 3 days with 0.1% FBS, 10% FBS, 10 ng/ml EGF + 0.1% FBS and a combination of EGF and 10% FBS, respectively. Immunohistochemical staining and in situ hybridization were used to observe the expressions of EGFR protein and mRNA, respectively. Activation of mitogen-activated protein kinase (MAPK) was detected by immunohistochemical method with specific antiphosphorylated extracellular signal-regulated kinase (ERK)1/2 antibody., Results: EGF stimulated proliferation and migration of cultured human RPE cells in a concentration-dependent manner. The maximum of the proliferation rate of RPE cells was 81.8% with EGF at a concentration of 10-100 ng/ml of EGF in serum-free Dulbecco's modified essential medium (DMEM) and 122.7% at a concentration of 1-10 ng/ml of EGF in 5% FBS DMEM (p < 0.001); there was a significant difference between serum-free DMEM groups and 5% FBS DMEM groups. The maximum of the migration rate of the cells was 438.9% at a concentration of 10-100 ng/ml of EGF in 10% FBS DMEM, 147% with 10% FBS, and only 36% with EGF in 0.1% FBS at the concentration of 10 ng/ml (p < 0.001). EGF promoted the expression of EGFR protein and mRNA in RPE cells. FBS cooperated with EGF in the stimulation of EGFR expression, and it had a stronger effect in the process than EGF alone. After 3 days of incubation with EGF, phosphorylated ERK1/2 was detectable in the nucleus of RPE cells, whereas cells presented immunostaining positive for phosphorylated ERK1/2 in the cytoplasm before stimulation, indicating that EGF could induce MAPK nuclear translocation., Conclusion: EGF could induce EGF-EGFR-MAPK signal transduction pathway in human RPE cells in a concentration-dependent manner in vitro, which may play a key role in the activation of human RPE cell proliferation and migration., (Copyright (c) 2007 S. Karger AG, Basel.)

Objective: To observe the changes of mitogen activated protein kinase (MAPK) signaling pathways of human retinal pigment epithelial cells( RPEs) bound with beads under stretch caused by the force from magnetic field in vitro., Methods: Ferric oxide microparticles, coAted with collagen, were added to dishes containing substrate-attached RPEs. After incubation, the cell layer bound with beads was laid in a magnetic field, the cells were stretched by the magnetic force. The activation status of the extracellular signal-regulated protein kinase (ERK), c-jun N-terminal kinase (JNK), and p38 in RPEs was determined over a time course from 3 to 30 minutes with western-blot analysis. To examine the role of p38 kinase in the response to stretching, cells were grown for 30 minutes in the presence or absence of inhibitor of p38 (SB203580). The changes of the expression of active p38 kinase were observed with fluorescence staining., Results: Total ERK, JNK, and p38 were detected in RPEs. Active ERK and p38 were detected but active JNK was not detected. Activation of ERK was unchanged during the time course. In contrast, p38 activation was barely detected in the normal cells, but this stress-activated protein kinase exhibited a robust activation after 5 minutes in the magnetic field. SB203580 blocked the p38 activation during stretch stimulation. The stretch stimulation also increased the fluorescence staining of active p38., Conclusion: A magnetic field can affect RPEs bound with beads. The effect may be partially through p38 signaling pathway.

Objective: To observe the experimental results and histopathological changes of acellular xenogenic dermal matrix (X-ADM) and allogeneic sclera used as wrapping materials of hydroxy apatite (HA) ocular implants in New Zealand white rabbits., Methods: Twenty-four rabbits received unilateral eye enucleating and the sockets were implanted with HA spherical implants wrapped with either acellular xenogenic dermal matrix or allogeneic sclera at random. The rabbits were examined for inflammation and implant exposure and sacrificed at 1, 2, 4, 6, 8 and 12 weeks after implantation. The sockets with the grafts were exenterated and the specimens were assessed histopathologically and ultrastructurally with light or transmission electron microscopy for the changes in inflammation reaction and vascularization., Results: Compared to allogeneic sclera at the same stage of implantation, acellular xenogenic dermal matrix demonstrated more active and earlier growth of fibroblasts and new vessels with abundant collagen deposition. There were few inflammatory cells and no rejection was found., Conclusion: This experiment showed that the acellular xenogenic dermal matrix, with fast neovascularization and low immunity, can be an ideal material of ocular implant and a good substitute for allogeneic sclera.

Objective: To observe changes of the cytoskeleton of cultured human retinal pigment epithelium (hRPE) cells in a mechanical stress model in vitro., Methods: Ferric oxide beads, coated with collagen, were added to dishes containing substrate-attached hRPE cells. After incubation, the cells were washed to remove unbound beads. The cells were pretreated by actin polymerization inhibitor cytochalasin D (0.05 mmol/L, 25 min). Before and 4, 8, 12 and 24 h after vertical magnetic force application the cells were stained for actin and vimentin using immunity fluorescence double labelled technique and analyzed with confocal microscopy., Results: Four hours after force application, the polarity of hRPE cells was changed, and actin was polymerized into filaments in array of them along with the direction of the force. Cytoskeleton filaments were located mainly around the nucleus and the ferric oxide beads. Pretreatment with cytochalasin D, the same changes as described above occurred 8 h after magnetic force application., Conclusions: Mechanical force could induce changes of distribution of cytoskeleton in hRPE cells, which are some biological characteristics of muscle cells, and this may facilitate the cellular migration and proliferation.

Objective: To investigate the expressions of monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8) of cultured human retinal pigment epithelial (hRPE) cells under mechanical stress in vitro, so as to mimic and understand the role of these 2 inflammatory cytokines in the early stage of development of primary retinal detachment., Methods: Ferric oxide beads coated with collagen were added to the culture plate containing wall-attaching hRPE cells and then the cells were incubated and washed to remove the unbound beads. A magnet was put to the culture plate to provide vertical magnetic force. Cytochalasin D (CD) was added to the culture fluid to inhibit the phagocytizing, secreting, and moving functions of the hRPE. Before the experiment and 15 min, 0.5 h, 1 h, 4 h, and 8 h the beginning of experiment, the MCP-1 mRNA and IL-8 mRNA in the hRPE cells were measured with reverse transcriptase polymerse chain reaction (RT-PCR), and the MCP-1 and IL-8 protein expression in the supernatants was tested with enzyme-linked immunosorbent assay (ELISA) kits., Results: MCP-1 and IL-8 mRNA were expressed at very low levels in the RPE cells and supernatant not exposed to magnetic force. After exposure to magnetic force, both MCP-1 and IL-8 mRNA demonstrated "double peaks" expression. Their first peak levels appeared within 0.5 h, being 3.30 ng/L +/- 0.12 ng/L and 1.88 ng/L +/- 0.08 ng/L respectively. The first peaks of MCP-1 and IL-8 protein expression were within 1 h, being 552.05 ng/L +/- 7.64 ng/Land 236.67 ng/L +/- 14.30 ng/L respectively, and the second peaks appeared about 4 hours later. After cytochalasin D pretreated, the expression and secretion of both MCP-1 and IL-8 of the hRPE cells were significantly decreased to the levels of 2.36 ng/L +/- 0.27 ng/L and 1.64 ng/L +/- 0.08 ng/L, and 353.80 ng/L +/- 16.68 ng/L and 101.86 ng/L +/- 15.92 ng/L respectively., Conclusion: Mechanical stress induces the RPE cells to express MCP-1 and IL-8, and this effect was inhibited in part by pretreatment of CD, indicating that the cytoskeleton may be involved in the effect and that these two inflammatory cytokines take a part in the early stage of development of primary retinal detachment.