Objective:To investigate the effect of advanced glycation end products (AGEs) on ferroptosis in human retinal pigment epithelium (RPE) cell cultured in vitro. Methods:ARPE-19 cell lines were cultured in DMEM medium containing 10% fetal bovine serum (FBS), and the 3rd to 6th generations of cells were used for further study.Cell activity was detected by using the cell counting kit 8 after ARPE-19 were cultured with AGEs at 0, 50, 100, 200, 400 μg/ml for 48 hours.The cells were cultured with 200 μg/ml AGEs for 48 hours.Cell lipid peroxidation level was measured by Lipid Peroxidation Assay Kit (Bodipy 581/591 C11) combined with flow cytometry.The relative mRNA and protein expression levels of solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4) were determined by quantitative real-time PCR (qRT-PCR) and Western blot.The morphology of mitochondria was observed by transmission electron microscope.Results:The activity of ARPE-19 cells decreased with increasing AGEs concentration, and the overall difference of ARPE-19 activity among 0, 50, 100, 200, 400 μg/ml AGEs groups was statistically significant ( F=6.21, P<0.01).The cell activity of ARPE-19 cells in 200 and 400 μg/ml AGEs groups was lower than that in control group (both P<0.05).Flow cytometry showed that the fluorescence intensity in AGEs group was 622.0±11.3, which was significantly higher than 487.7±12.8 in control group ( t=6.809, P=0.002).qRT-PCR showed that the mRNA and protein relative expression levels of SLC7A11 and GPX4 were lower in AGEs group than those in control group (mRNA: t=3.72, 7.14, both P<0.05; protein: t=6.20, 5.15, both P<0.05).Transmission electron microscopy showed that mitochondria in AGEs group shrank with significantly reduced volume, decreased mitochondrial cristae, and increased mitochondrial membrane density. Conclusions:AGEs can induce ferroptosis in ARPE-19 cultured in vitro.

Objective:To investigate the effect of advanced glycation end products (AGEs) on ferroptosis in human retinal pigment epithelium (RPE) cell cultured in vitro. Methods:ARPE-19 cell lines were cultured in DMEM medium containing 10% fetal bovine serum (FBS), and the 3rd to 6th generations of cells were used for further study.Cell activity was detected by using the cell counting kit 8 after ARPE-19 were cultured with AGEs at 0, 50, 100, 200, 400 μg/ml for 48 hours.The cells were cultured with 200 μg/ml AGEs for 48 hours.Cell lipid peroxidation level was measured by Lipid Peroxidation Assay Kit (Bodipy 581/591 C11) combined with flow cytometry.The relative mRNA and protein expression levels of solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4) were determined by quantitative real-time PCR (qRT-PCR) and Western blot.The morphology of mitochondria was observed by transmission electron microscope.Results:The activity of ARPE-19 cells decreased with increasing AGEs concentration, and the overall difference of ARPE-19 activity among 0, 50, 100, 200, 400 μg/ml AGEs groups was statistically significant ( F=6.21, P<0.01).The cell activity of ARPE-19 cells in 200 and 400 μg/ml AGEs groups was lower than that in control group (both P<0.05).Flow cytometry showed that the fluorescence intensity in AGEs group was 622.0±11.3, which was significantly higher than 487.7±12.8 in control group ( t=6.809, P=0.002).qRT-PCR showed that the mRNA and protein relative expression levels of SLC7A11 and GPX4 were lower in AGEs group than those in control group (mRNA: t=3.72, 7.14, both P<0.05; protein: t=6.20, 5.15, both P<0.05).Transmission electron microscopy showed that mitochondria in AGEs group shrank with significantly reduced volume, decreased mitochondrial cristae, and increased mitochondrial membrane density. Conclusions:AGEs can induce ferroptosis in ARPE-19 cultured in vitro.

Objective:To investigate the inhibitory effect of indocyanine green (ICG) on biological behavior and transdifferentiation of human lens epithelial cells (HLECs) and its mechanism.Methods:HLECs were divided into blank control group, 5% glucose solution (GS) group and 0.5% ICG group, 1.5% ICG group and 2.5% ICG group, which were treated with balanced salt solution, 5% GS and 0.5%, 1.5% and 2.5% ICG solutions for 3 minutes, respectively, and then were incubated in fresh medium for 24 hours.The apoptosis level of HLECs was detected by flow cytometry.The expression levels of apoptosis-related proteins, Bcl-2-associated X protein (Bax), B-cell lymphoma-2 (Bcl-2), caspase-3 and caspase-9 were detected by Western blot.Cell proliferation was detected via the cell counting kit-8 (CCK-8) assay and 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay.The migration ability of HLECs was detected by cell scratch assay.Cell migration and invasion were determined by Transwell assays.The expression levels transdifferentiation-related proteins, α-smooth muscle actin (α-SMA), nerve calcium adhesion protein (N-cadherin), fibronectin (FN) and vimentin were assessed by Western blot.Results:The apoptosis rates of blank control group, 5% GS group, 0.5% ICG group, 1.5% ICG group and 2.5% ICG group were (4.35±0.60)%, (4.63±0.19)%, (8.17±0.69)%, (13.90±0.33)% and (23.08±1.12)%, with a statistically significant difference in the overall comparison ( F=412.74, P<0.05). The apoptosis rate was significantly higher in 0.5% ICG group, 1.5% ICG group and 2.5% ICG group than in blank control group and 5% GS group (all at P<0.05). The relative expressions of caspase-3, caspase-9 and Bax proteins were significantly higher in 0.5% ICG group, 1.5% ICG group and 2.5% ICG group than in blank control group and 5% GS group, and the relative expression of Bcl-2 protein was lower in 1.5% ICG group and 2.5% ICG group than in blank control group and 5% GS group, and the differences were statistically significant (all at P<0.05). The rate of EdU-positive cells was significantly lower in 0.5% ICG group, 1.5% ICG group and 2.5% ICG groups than in blank control group and 5% GS group (all at P<0.05). The survival rate of cells was significantly lower in 0.5% ICG group, 1.5% ICG group and 2.5% ICG group than in blank control group and 5% GS group (all at P<0.05). The migration rates of scratch cells were significantly lower in 0.5% ICG group, 1.5% ICG group and 2.5% ICG group than in blank control group and 5% GS group, and the differences were statistically significant (all P<0.05). The number of migrating cells and the number of invading cells were significantly lower in 0.5% ICG group, 1.5% ICG group and 2.5% ICG group than in blank control group and 5% GS group (all at P<0.05). The relative expressions of α-SMA, N-cadherin and FN were significantly lower in 0.5% ICG group, 1.5% ICG group and 2.5% ICG group than in blank control group and 5% GS group, and the relative expression of vimentin was lower in 1.5% ICG group and 2.5% ICG group than in blank control group and 5% GS group, and the differences were statistically significant (all at P<0.05). Conclusions:ICG can promote HLECs apoptosis and inhibit HLECs proliferation, migration, invasion and transdifferentiation in a concentration-dependent manner.

The internal limiting membrane located at vitreoretinal interface is formed by the contiguous basement membranes of Müller cells.Nowadays, vitrectomy combined with internal limiting membrane peeling has been widely used in many operations involving macular area.Although its clinical efficacy and safety have been demonstrated, it lacks the necessary histological support.At the same time, many studies have shown that the internal limiting membrane plays different roles in the occurrence of different diseases.Current studies have found that the proliferation of inflammatory cells, glial cells and vitreous cells leads to the physiological dysfunction of the vitreoretinal interface, and the internal limiting membrane can also become a scaffold for the proliferation of myofibroblasts, which will lead to the occurrence of macular diseases.This article reviewed the histological research of internal limiting membrane in terms of diabetic retinopathy, idiopathic macular hole and idiopathic macular epiretinal membrane, hoping to better understand the internal limiting membrane under pathological conditions and to confirm the safety and necessity of internal limiting membrane peeling from ultrastructure.

Objective:To investigate the effect of distilled water on the viability of human lens epithelial cells (LECs) cultured in vitro. Methods:A total of 156 anterior capsule specimens were collected from 156 patients (156 eyes) who were diagnosed with age-related cataract during phacoemulsification combined with intraocular lens implantation from May to December 2020 in Drum Tower Hospital, the Affiliated Hospital of Nanjing University Medical School.The 156 specimens were divided into 312 small pieces.Of the 312 pieces, 157 pieces were divided into normal control group (23 pieces), positive control group (10 pieces), balanced salt solution (BSS) immersion group (61 pieces) and distilled water immersion group (63 pieces) using computer-generated random numbers.Normal control group received no treatment.Positive control group was directly fixed with a mass fraction of 4% histiocytes fixative solution.For the 61 pieces in BSS immersion group, 20 pieces were soaked for 1 minute, 21 pieces for 2 minutes, and 20 pieces for 3 minutes.For the 63 pieces in distilled water immersion group, 20 pieces were soaked for 1 minute, 23 pieces for 2 minutes, and 20 pieces for 3 minutes.Another 125 pieces were selected to simulate the cataract aspiration-irrigation according to the treatment in BSS immersion group and distilled water immersion group respectively, plus rinse in a bottle containing BSS at a height of 70 cm for 1 minute.Cell viability was detected by trypan blue-eosin staining.LECs density, dead cell count, cell death rate and percentage of shedding (%) were calculated.Of the remaining 30 pieces, every 15 pieces were divided into normal control group, BSS immersion group, and distilled water immersion for 1, 2 and 3 minutes groups, with 3 pieces in each group.BSS immersion group was immersed for 3 minutes, and the other four groups were treated as mentioned above, and the LECs structure of the four groups was observed by light microscopy and transmission electron microscopy.This study adhered to the Declaration of Helsinki.The study protocol was approved by the Ethics Committee of Drum Tower Hospital, the Affiliated Hospital of Nanjing University Medical School (No.2019-248-01). Written informed consent was obtained from each subject.Results:The boundaries of LECs in BSS treatment groups were clear, and there was no significant difference in morphology compared with normal control group.With time increasing, LECs in distilled water treatment groups gradually swelled, and the boundaries of dead cells were not clear.There were significant differences in LECs density, dead LECs count and LECs mortality ( F=13.459, 98.918, 130.600; all at P<0.001). The LECs density was lower in 2-minute and 3-minute distilled water treatment groups than in normal control group, showing statistically significant differences (both at P<0.05). The dead LECs count and LECs mortality were higher in 1-minute, 2-minute and 3-minute distilled water treatment groups than in normal control group and BSS treatment groups for the same time, and the differences were statistically significant (all at P<0.05). Only a few shed LECs were seen in normal control group, 1-minute, 2-minute and 3-minute BSS treatment groups, and BSS immersion combined rinse group.After different time of soaking, there were more shed LECs in distilled water immersion combined rinse group, and the range of LECs shedding increased with the extension of distilled water immersion.There was a significant difference in the shedding percentage of LECs among different groups ( F=123.670, P<0.001). The shedding percentages of LECs at different time points were higher in distilled water immersion groups and distilled water immersion combined rinse groups than in normal control group, and the difference was statistically significant (all at P<0.05). The shedding percentage of LECs increased significantly in distilled water immersion groups with the extension of immersion.Light microscopy showed that the cells were destroyed in 1-minute, 2-minute and 3-minute distilled water treatment groups, and some LECs shed in the 2-minute and 3-minute treatment groups.Transmission electron microscopy showed cell lysis and destruction, suborganelles swelling, disruption of intercellular junctions in 1-minute, 2-minute and 3-minute distilled water treatment groups, loose attachment between cells and capsule in the 2-minute and 3-minute treatment groups, and cell detachment from capsule in the 3-minute treatment group. Conclusions:Distilled water immersion leads to LECs death in a time-dependent manner, and distilled water immersion combined with rinse can remove LECs on the lens capsule.

Objective:To compare the one-year postoperative visual quality after trifocal intraocular lens (IOL) implantation and monofocal IOL implantation.Methods:A cohort study was conducted.Forty-one eyes from 41 age-related cataract patients who underwent phacoemulsification extraction combined with IOL implantation in Nanjing Drum Tower Hospital from May 2017 to June 2018 were enrolled.The patients were divided into trifocal IOL group (20 eyes) receiving ZEISS AT LISA tri 839MP trifocal IOL implantation and monofocal IOL group (21 eyes) receiving ZEISS 603P monofocal IOL implantation according to their willingness.One year after surgery, uncorrected distant visual acuity (UCDVA), uncorrected intermediate visual acuity (UCIVA), uncorrected near visual acuity (UCNVA), best corrected distance visual acuity (BCDVA), distance corrected intermediate visual acuity (DCIVA) and distance corrected near visual acuity (DCNVA) were detected in both groups.The patient point spread function (PSF), modulation transfer function (MTF) cutoff frequency, Strehl ratio (SR), OQAS Ⅱ values at 100%, 20%, and 9% contrast (OV 100%, OV 20%, OV 9%) and objective scattering index (OSI) were measured by OQAS Ⅱ.Wavefront aberrations including total aberration (TA), total high order aberrations (tHOAs), spherical aberration, coma, trefoil aberration, total low order aberrations (tLOAs), defocus, and astigmatism were evaluated with the iTrace visual function analyzer.All aberrations were represented by root mean square.The visual acuity of operative eyes was measured with a phoropter, and defocus curves were drawn with visual acuity better than 0.5 LogMAR.The incidence of posterior capsular opacification (PCO) in the IOL region was quantitatively analyzed by Sellman method.Visual function was scored by visual function index (VF-14). This study adhered to the Declaration of Helsinki.The study protocol was approved by an Ethics Committee of Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School (No.2018-219-01). Written informed consent was obtained from each subject prior to any medical examination.Results:One year after the operation, UCIVA, UCNVA, DCIVA, and DCNVA of trifocal IOL group were significantly better than those of monofocal IOL group, and the differences were statistically significant (all at P<0.001). OQAS Ⅱ visual quality indicators showed that the MTF cutoff frequency, SR, OV 100%, and OSI values of trifocal IOL group were significantly higher than those of monofocal IOL group, showing statistically significant differences (all at P<0.001). No significant difference in wavefront aberrations was found between the two groups (all at P>0.05). Defocus curve showed that the LogMAR visual acuity of patients at -1.0 D, -1.5 D, -2.0 D, -2.5 D, -3.0 D, and -3.5 D (namely, 1 m, 66 cm, 50 cm, 40 cm, 33 cm, and 29 cm) in monofocal IOL group were significantly better than those in trifocal IOL group (all at P<0.05). There was a higher incidence of PCO in trifocal IOL group than monofocal IOL group, with a statistically significant difference ( χ 2=41.0, P<0.001). The VF-14 score of trifocal IOL group was 87.99±1.09, which was significantly higher than 81.49±1.67 of monofocal IOL group ( t=10.301, P<0.001). Conclusions:One year after trifocal IOL implantation, the full range of vision, subjective and objective visual quality of eyes are better than eyes implanted with monofocal IOL.

Objective:To investigate and evaluate the safety of intravitreal injection of recombinant human endostatin (rh-endostatin) with different concentrations in rabbit eyes.Methods:Thirty healthy adult New Zealand white rabbits were enrolled with the right eyes selected as experimental eyes, and were randomly divided into five groups by random distribution of computer numbers, with 6 eyes in each group.The rabbits in the normal control group were given no treatment, and the rabbits in the normal saline group, 0.125 mg rh-endostatin group, 0.250 mg rh-endostatin group and 0.500 mg rh-endostatin group were treated with 100 μl of normal saline, 0.125 mg/100 μl, 0.250 mg/100 μl and 0.500 mg/100 μl rh-endostatin according to grouping, respectively.The anterior segment and fundus of the experimental eyes were examined using slit lamp biomicroscope and indirect ophthalmoscope, and the intraocular pressure (IOP) of the experimental eyes were measured with iCARE handheld tonometer before injection and 1 day, 3, 7, 14, 30 and 60 days after injection.Optical coherence tomography (OCT) examination was performed before the intravitreal injection and 7, 30, and 60 days after injection, respectively.Flash electroretinogram was performed before intravitreal injection and 14 days and 60 days after injection.The rabbits were sacrificed by euthanasia at 60th day after injection.Three experimental eyes of each group were dissected and made into paraffin section, and histopathological staining was used to detect the retinal structural changes.The retinal tissue was separated from the other three study eyes in each group, and the transmission electron microscope was employed to observe the ultrastructural changes of the retina.All animal experiments were performed in adherence to the Regulations of the State and the Animal Center of Yangzhou University Medical College for the Use of Animals in Research.Results:After intravitreal injection, no obvious anterior or posterior chamber change was observed by slit lamp microscopy in all groups at any time point.Flocculent seepage was observed in one eye of the 0.125 mg and 0.500 mg rh-endostatin group, respectively, which was then absorbed completely on the 7th and 14th day.OCT examination showed no abnormal light reflection or morphological changes in fundus of day after injection in all the groups.There was no significant difference in IOP, a-wave and b-wave amplitude among all the groups at different time points ( Fgroup=0.134, 0.101, 0.476; Ftime=1.709, 2.479, 1.706; all at P>0.05). Neither light nor electron microscopy showed any retinal damage in any group. Conclusions:Intravitreal injection of rh-endostatin is safe at the dosage of 0.125-0.500 mg in rabbits.

Objective:To observe the effect of recombinant human vascular endostatin(ES) on retinal barrier related proteins in early streptozotocin (STZ)-induced diabetic rats.Methods:Diabetes rat model was induced by STZ.Two weeks after the model was successfully constructed, 36 diabetic model rats were randomly divided into the model group, 1.0 μl ES group, 2.5 μl ES group, 5.0 μl ES group, 2.5 μl bevacizumab group, and the combination therapy group, with 6 rats in each group.Different doses of recombinant human ES and 2.5 μl bevacizumab were injected into the vitreous cavity of the right eye according to the grouping.Six normal rats were selected as the blank control group.At 4 weeks after intravitreal injection, the retinal tissue of the right eye in each group was collected, and the expression levels of inter cellular cell adhesion molecule-1(ICAM-1), vascular cell adhesion molecule-1(VCAM-1), occludin, claudin-5, vascular endothelial growth factor(VEGF) and other proteins in retinal tissue were detected by Western blot assay.The use and care of animals was in accordance with the regulations for the administration of experimental animals.Results:The diabetic model rats showed polydipsia, polyuria, polyphagia and other typical diabetic symptoms, the body quality decreased significantly, and the success rate of modeling was 100%.Western blot results showed that the expression levels of VCAM-1, ICAM-1 and VEGF in the blank group, the 2.5 μl ES group, the 5.0 μl ES group, the bevacizumab group, the combination therapy group were significantly lower than those in the model group, while the expression levels of claudin-5 and occludin were significantly higher than those in the model group, the differences were statistically significant (all at P<0.05). The relative expression of occludin protein in the 1.0 μl ES group was significantly higher than that in the model group(0.23±0.02 vs.0.13±0.02), while the relative expression of ICAM-1 and VEGF was significantly lower than that in the model group(0.53±0.01 vs.0.81±0.01; 0.57±0.00 vs.0.86±0.00), the differences were statistically significant (all at P<0.05). As the dose of ES increased, the relative expressions of claudin-5 and occludin protein tended to increase, while the relative expressions of VCAM-1, ICAM-1 and VEGF tended to decrease. Conclusions:Recombinant human vascular endostatin can directly or indirectly reduce the release of inflammatory factors VCAM-1 and ICAM-1 and inhibit the expression of VEGF, thereby reduce the loss of retinal tight junction.

Idiopathic macular hole (IMH) refers to full thickness defects of retinal neuroepithelial layer in macular area without clear reasons,and the combination of pars plana vitrectomy (PPV) with internal limiting membrane peeling (ILMP) is a standard procedure for macular hole.This technique can improve anatomical success and reduce the tangential forces,and thus accelerating the macular hole closure.With increasing use of ILMP and vital dye,the controversial issue of the intentional ILMP has arisen.First,the earliest change in the macula after ILMP is postoperative swelling of the arcuate retinal nerve fiber layer and dissociated optic nerve fiber layer occurs later in the postoperative period;second,retinal thickness modification,such as the thinning of retinal nerve fiber layer (RNFL),ganglial cell layer (GCL) and inner plexiform layer (IPL);third,displacement of foveal area toward optic disc and decrease of the foveal avascular zone area decrease retinal sensitivity and changes of the focal macular electroretinogram.This article reviewed the effects of ILMP during macular hole surgery on retinal anatomical and functional outcomes.

Objective: To reveal the pathogenic mutations in Chinese families with idiopathic congenital nystagmus(ICN) Methods: Six families with ICN were recruited from Subei People's Hospital.DNA was extracted from peripheral blood samples of all participants.All coding and exon-intronic boundary regions of the targeted gene FRMD7 were amplified with PCR and sequenced using Sanger sequencing to detect potential pathogenic mutations.This study followed the Helsinki Declaration and was approved by the Ethics Committee of Subei People's Hospital (NO.2015KY-126). All patients or their guardians signed informed consent. Results: Three mutations (c.902A>G, c.1944T>A and 1945G>T) were screened in two families after co-segregation validation of intrafamilial genotype-phenotype, c.1944T>A and 1945G>T were newly detected mutations which were not detected in 100 normal controls.No significant mutations were found in the FRMD7 coding region and adjacent splicing sites in the probands of the other four families. Conclusions Two novel pathogenic mutations of FRMD7 are discovered, which expands the pathogenic mutational spectrum of FRMD7 gene causing ICN.