Transcriptome sequencing of transgelin-2 inhibiting high glucose induced microglia inflammation / 中华眼底病杂志

Objective:To analyze the change of differential genes and signaling pathways in high glucose induced BV2 cells, and to explore the mechanism of transgelin-2 (TAGLN2) regulating cellular inflammatory response and metabolic process.Methods:An experimental study. The cultured BV2 cells were divided into mannitol treatment (Man) group, glucose treatment (Glu) group, overexpression control Glu treatment (Con) group, overexpression TAGLN2 Glu treatment group, silence control Glu treatment (shCon Glu) group, and silence TAGLN2 Glu treatment (shTAGLN2 Glu) group. Cells in the Man group were cultured in modified Eagle high glucose medium (DMEM) containing 25 mmol/L mannitol and 25 mmol/L glucose, cells in other groups (Glu group, Con Glu group, TAGLN2 Glu group, shCon Glu group and shTAGLN2 Glu group) were cultured in DMEM medium containing 50 mmol/L glucose. After 24 hours of cells culture, transcriptome sequencing of cells in each group were performed using high-throughput sequencing technology, and significantly differentially expressed genes (DEG) were screened. |log 2 (fold change)|≥1 and P≤0.05 were adopted as criteria to screen for DEG. Gene Ontology (GO) function enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis and protein-protein interaction network analysis were performed. Real-time polymerase chain reaction (RT-PCR) was used to detect the relative expression level of DEG mRNA. The data between groups were compared by independent sample t-test. Results:When compared with Man group, a total of 517 differentially expressed genes were screened in Glu group, which including 277 up-regulated genes and 240 down-regulated genes. KEGG pathway enrichment analysis showed that the up-regulated genes were significantly enriched in immune system processes such as nuclear factor (NF)-κB signal pathway, Jak-signal transducers and activators of transcription (STAT) signal pathway, while down-regulated genes were significantly enriched in glycosaminoglycan degradation and glyceride metabolic pathway. Compared with Con Glu group, a total of 480 DEG were screened in TAGLN2 Glu group, among which 147 up-regulated and 333 down-regulated genes were detected. Up-regulated genes were significantly enriched in the metabolic processes of fatty acid, glyceride and pyruvate, while down-regulated genes were significantly enriched in immune system processes such as NF-κB signal pathway, Jak-STAT signal pathway and tumor necrosis factor (TNF) signal pathway. Compared with shCon Glu group, a total of 582 DEG were screened in shTAGLN2 Glu group, among which 423 up-regulated and 159 down-regulated genes were detected. Up-regulated DEG were significantly enriched in immune system processes such as TNF signal pathway and chemokine signal pathway, while down-regulated DEG were significantly enriched in pattern recognition receptor signal pathway. RT-PCR results showed that the relative expression levels of DEG mRNA Card11 ( t=13.530), Icos ( t=3.482), Chst3 ( t=6.949), Kynu ( t=5.399), interleukin (IL)-1β ( t=2.960), TNF-α ( t=5.800), IL-6 ( t=3.130), interferon-γ ( t=7.690) and IL-17 ( t=6.530) in the TAGLN2 Glu treatment group were decreased significantly compared with Con Glu group, and the difference was statistically significant. Conclusion:TAGLN2 can inhibit glucose induced microglia inflammation by NF-κB and Jak-STAT signaling pathways, Card11, Icos, Chst3 and Kynu play an important role in the anti-inflammatory process of TAGLN2.

Compared non-mydriatic two-field 45° fundus imaging methods with non-mydriatic two ultra-widefield fundus imaging in a large-scale diabetic retinopathy screening / 中华眼底病杂志

Objective:To compare the consistency and difference of non-mydriatic two-field 45° ultra-wide field Optos and Clarus500 fundus imaging in a large-scale diabetic retinopathy (DR) screening.Methods:A diagnostic methodology study. From November 2020 to August 2021, 526 eyes of 277 patients with type 2 diabetes who diagnosed in Department of Ophthalmology, Henan Provincial People's Hospital were included in the study. Among them, there were 175 males with 328 eyes and 102 females with 198 eyes; the age was 53±10 years old. The same experienced technician performed the non-mydriatic dual-field 45° fundus imaging and the non-mydriatic ultra-wide-angle imaging system Optos, Clarus500 single-field fundus imaging examination on the patient on the same day, and obtained the dual-field 45° fundus image and Optos, Clarus500 single-field fundus image. The Optos and Clarus500 single-field fundus images in the same area as the dual-field 45° fundus image were captured by Photoshop software, and the Optos and Clarus500 dual-field fundus images were obtained. Subsequently, two experienced ophthalmologists performed interpretation and DR grading of the 5 groups of images, respectively. Images with inconsistent grading results were interpreted by a third ophthalmologist and used as the final grading result. In order to avoid the mydriatic dual-field 45° imaging interpretation results as the standard, the consistency and detection rate difference of the two ultra-wide-angle imaging systems in the rapid DR screening results were evaluated. The weighted Kappa ( κ) test was used to analyze the consistency of DR diagnosis between dual-field 45° fundus imaging and Optos and Clarus500 fundus imaging; χ2 test was used to compare the detection rates of DR between different imaging systems. Results:Compared with the dual-field 45° fundus image, the Clarus500 single-field had a higher DR detection rate ( χ2=24.965, P<0.001), and the Optos dual-field fundus image had a lower DR detection rate ( χ2=49.559, P<0.001). Compared with the DR detection rate of dual-field 45° fundus image, Optos single-field fundus image, Clarus500 double-field fundus image had no significant difference ( χ2=2.572, 0.649; P=0.109, 0.421). Compared with Optos, Clarus500 single-field and dual-field fundus images DR detection rate, the difference was statistically significant ( χ2=43.214, 61.216; P<0.001). Consistency assessment of DR grading results: dual-field 45° fundus images and Clarus500 dual-field fundus images ( κ value=0.932, 95% confidence interval ( CI) 0.907-0.956) were highly consistent; dual-field 45° fundus images and Optos single-field fundus images [ κ value=0.474, 95% CI 0.417-0.532], Optos dual-field fundus image ( κ value=0.495, 95% CI 0.438-0.551), Optos dual-field fundus image ( κ value=0.495, 95% CI 0.438-0.551) and Clarus500 dual-field fundus image ( κ value=0.452, 95% CI 0.395-0.506) were moderately consistent; dual-field 45°fundus images and Clarus500 single-field fundus images ( κ value=0.354, 95% CI 0.303-0.403) and Optos single-field fundus images and Clarus500 single-field fundus images ( κ value=0.347, 95% CI 0.287-0.393) showed general agreement. Conclusions:Compared with Optos dual-field fundus image, dual-field 45°fundus image and Clarus500 dual-field fundus image have high consistency in the grading results of DR rapid screening. Compared with Optos single-field fundus image, the detection rate of the DR of Clarus500 single-field fundus image is higher.

The progress and problems of the fundus multimodal imaging / 中华眼底病杂志

The update of the cognition of fundus diseases is inseparable from the rapid development of fundus multimodal imaging. Especially in recent years, the application of wide and ultra-wide fundus photography, ultra-wide fundus fluorescein angiography, indocyanine green angiography, fundus autofluorescence and optical coherence tomography angiography contribute to observe the peripheral retinopathy more directly. The application of adaptive optics and fluorescence lifetime imaging ophthalmoscopy contribute to have a further understanding of fundus diseases at the cellular and metabolic level. Multimodal imageing reflect the pathological characteristics of the diseases from different angles and levels. At the same time, the digitization and intelligence of fundus images are also developing rapidly. However, there are some problems that the ophthalmologists needs to consider further, such as the correctly understanding the use of multimodal imaging, the application of artificial intelligence, and how to sum up from the images.

Bioinformatics analysis of transcriptome sequencing of early hypoxia damage in photoreceptor 661W cell line / 中华眼底病杂志

Objective:To analyze the early changes of gene expression levels and signaling pathways in 661W cell line under hypoxic conditions and to find potential functional target genes.Methods:The cultured mouse 661W cells were divided into hypoxia treatment group and normoxia control group. Cells in the hypoxia treatment group were cultured in a three-gas incubator with volume fraction of 1% and 5% CO 2 at 37 ℃. Cells in the normoxia control group were cultured in an incubator at 37 ℃ with volume fraction of 5% CO 2. High-throughput sequencing technology was used to sequence the transcriptome of 661W cell treated with hypoxia and normoxia for 4 hours to screen for differentially expressed genes (DEG). Clustering heat map analysis, gene ontology (GO) functional enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis and protein-protein interaction network (PPI) analysis were performed. The reverse transcription-polymerase chain reaction (RT-PCR) was used to verify the accuracy of the sequencing results. Results:A total of 506 differentially expressed genes were screened, including 459 up-regulated genes and 47 down-regulated genes. GO functional enrichment analysis showed that the main biological processes of DEG were the cell's response to hypoxia, glycolysis, negative regulation of cell proliferation and apoptosis. hypoxia inducible factor (HIF)-1α pathway, glycolysis, Forkhead box O (FoxO) pathway, Insulin signaling pathway and Adenosine 5′-monophosphate-activated protein kinase (AMPK) pathway were involved in the above process. PPI analysis results showed that hub genes related to hypoxia were Aldoa, Aldoc, Gpi1, Hk2, Hk1, Pfkl, Pfkp, Vhl, Fbxo10 and Fbxo27. The RT-PCR results showed that the relative expression levels of 15 DEG mRNA in the hypoxic treatment group were higher than that of the normoxic control group, and the difference was statistically significant ( P<0.05). The mRNA expression levels of N-myc downstream-regulated gene-1 ( Ndrg1 ), Mt1, and vascular endothelial growth factor A ( VEGFA) were time-dependent on hypoxia. Conclusions:Under hypoxia, DEG is mainly related to glucose metabolism, cell response to hypoxia, regulation of proliferation and apoptosis. HIF-1α pathway, glycolysis, FoxO pathway and AMPK pathway are involved in the early changes of 661W cells under hypoxia. Aldoa, Aldoc, Gpi1, Hk2, Hk1, Pfkl, Pfkp, Vhl, Fbxo10, Fbxo27 may play key roles in the response of 661W cells to hypoxia. Ndrg1, Mt1 and VEGFA could be potential functional target genes for the study of ischemia and hypoxia-related fundus diseases.

Transcriptomics analysis of hypoxia-induced retinal pigment epithelium cell injury / 中华实验眼科杂志

Objective:To analyze differentially expressed genes (DEGs) and the changes of signal pathways in human retinal pigment epithelium cells (ARPE-19) under hypoxic and normoxic conditions and to explore the biological mechanism of hypoxia-induced ARPE-19 cell damage via transcriptome sequencing (RNA-seq) and bioinformatics technology.Methods:The ARPE-19 cells were divided into the hypoxia treatment group and the normoxia control group treated with 1% and 21% O 2 by volume for 8, 24, 48, 72 hours, respectively.The relative expression levels of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α) mRNA were detected with real-time fluorescent quantitative PCR at different time points.RNA-seq and bioinformatics analysis were performed at 8 hours and 24 hours after hypoxia and normoxia treatment.DEGs were screened out under the conditions of |log 2FC|≥1 and P≤0.05.Then the cluster heat map analysis, Gene Ontology (GO) functional enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis and protein-protein interaction network analysis were also carried out.Real-time fluorescent quantitative PCR was employed at 24 hours after hypoxia to detect the relative mRNA expression of genes that might be related to hypoxia in DEGs.Cell viability kit was used to verify and compare the damage effect of hypoxia on ARPE-19 cells at different time points between the two groups. Results:The relative mRNA expression levels of VEGF at 8, 24, 48 and 72 hours after hypoxia treatment and the relative HIF-1α mRNA expression levels at 8, 24 and 48 hours after hypoxia treatment were significantly higher than those of the normoxia control group (all at P<0.05). There were large differences in the mRNA expression levels at 8-hour and 24-hour treatment between the two groups.A total of 62 significant DEGs were screened between the hypoxia treatment group and the normoxia control group after 8-hour hypoxia treatment, among which 45 genes were significantly up-regulated and 17 genes were significantly down-regulated.A total of 255 significant DEGs were screened out between the hypoxia treatment group and the normoxia control group after 24-hour hypoxia treatment, among which 228 genes were significantly up-regulated and 27 genes were significantly down-regulated.The GO functional analysis of DEGs was mainly enriched in processes such as protein degradation, nucleotide biosynthesis, and material transport.KEGG pathway analysis was mainly enriched in PI3K-Akt, cGMP-PKG, and other signaling pathways closely related to metabolism, cell cycle, cell growth, and apoptosis.The core genes HPCA, MT3 and NOS3 were found by protein-protein interaction network analysis.Real-time fluorescent quantitative PCR test results showed that after 24-hour hypoxia treatment, the mRNA expression levels of hypoxia related genes DEPP1, NPPB, PDZK1, HILPDA, TCEA3, NDRG1 and RORC in ARPE-19 cells were significantly increased and the mRNA expression levels of TFRC and NQO1 were significantly decreased (all at P<0.05). The cell morphology was normal and the growth state was good without dead cells after 8-hour and 24-hour hypoxia treatment in ARPE-19 cells.There were dead cells after 48-hour hypoxia treatment, and the number of dead cells was increased at 72 hours after hypoxia treatment. Conclusions:The PI3K-Akt and cGMP-PKG signaling pathways related to metabolism may be involved in hypoxia-induced injury of ARPE-19 cells.Core genes of HPCA, MT3 and NOS3 can be used as functional target genes and play key roles in hypoxia response of cells.

Interpretation of the 2019 Posterior Vitreous Detachment, Retinal Breaks, and Lattice Degeneration Preferred Practice Pattern / 中华实验眼科杂志

Posterior vitreous detachment (PVD), retinal breaks, and lattice degeneration are common problems in ophthalmic clinical practice, which not only cause disturbance to patients' life-quality, but also increase the risk of retinal detachment and vitreoretinal traction.In September 2019, the American Academy of Ophthalmology published Posterior Vitreous Detachment, Retinal Breaks, and Lattice Degeneration Preferred Practice Pattern (PPP). Based on clinical evidence, this PPP provides authoritative guidance for the definition, epidemiological background, diagnosis and treatment of these diseases.This PPP also gives definite solution for treatment and follow-up of different sub-types.This article provides introduction and interpretation of this PPP.

The clinical efficacy of lamellar hole-associated epiretinal proliferation flap insertion and autologous blood for degenerative type lamellar macular hole / 中华眼底病杂志

Objective To observe the efficacy of lamellar hole-associated epiretinal proliferation (LHEP) flap insertion and autologous blood for degenerative type lamellar macular hole (LMH).Methods Retrospective case review.Twenty-eight eyes of 28 patients with LMH were enrolled in this study.There were 2 males (2 eyes) and 26 females (26 eyes).Best corrected visual acuity (BCVA),medical optometry,slit-lamp biomicroscop,indirect ophthalmoscope,spectral domain optical coherence tomography,b-scan ultrasonography and axial length detection were performed on all patients.Logarithm of the minimum angle of resolution (logMAR) was used to calculate visual acuity.There were 10 eyes (35.7％) with degenerative type LMH (flap insertion group) and LHEP.There were 18 eyes (64.3％) with tractional type LMH (general group).The differences of BCVA,AL,horizontal hole diameter from retina and lens state between two groups were not significant (P＞ 0.05).The differences of horizontal hole diameter of internal limiting membrane (ILM),central foveal thickness (CFT) and integrity of ellipsoidal zone between two groups were significant (P＜ 0.05).LHEP flap insertion and autologous blood without ILM peeling were used in eyes of flap insertion group.Vitrectomy combined ILM peeling were used in eyes of general group.The follow-up was ranged from 3 to 14 months.The changes of CFT,central foveal form and logMAR BCVA were observed.Results At latest follow-up,the BCVA of flap insertion group and general group were 0.34±0.27,0.31±0.29;which significantly better than the preoperative BCVA (Z=-3.519,-4.945;P＜ 0.001).The CFT of flap insertion group and general group were (200.10±58.78),(226.61±70.49) μm.There was no difference between pre-and post-operative CFT in eyes of general group (Z=-1.455,P=0.146).There was significant difference between pre-and post-operative CFT in eyes of flap insertion group (Z=-2.798,P=0.005).In flap insertion group,regular recovery of the foveal contour occurred in 9 eyes (90.0％),improvement in 1 eyes (10.0％).In general group,regular recovery of the foveal contour occurred in 10 eyes (55.6％),improvement in 8 eyes (44.4％).The closure rate of LMH were 100％ both in two groups.Conclusion LHEP flap insertion and autologous blood is an effective treatment of degenerative type LMH.

Disputes revolved about surgeries of macular vitreoretinal interface abnormalities in highly myopic eyes / 中华眼底病杂志

Macular vitreoretinal interface abnormalities in highly myopic eyes are among the most vision-threatening diseases associated with macular retinal schisis and macular holes.To relieve the traction of the posterior vitreous cortex and to recover the anatomy of fovea for good central vision are the keys to successful repair.However,there are many controversial issues in the efficacy of the surgerical procedures including gas injection,scleral buckling and vitrectomy.How to evaluate these different surgeries and to establish standard surgical procedure options for macular vitreoretinal interface abnormalities in highly myopic eyes needs to be explored.

The visual prognostic factors in vitreoretinal surgery for myopic foveoschisis / 中华眼底病杂志

Objective To observe the clinical effects of vitreoretinal surgery for myopic foveoschisis,evaluate the visual prognostic factors.Methods Twenty-seven eyes of 23 patients with myopic foveoschisis underwent vitreoretinal surgery were analyzed retrospectively. All the patients had undergone the examinations of best corrected visual acuity (BCVA),intraocular pressure,slit lamp microscope,direct ophthalmoscope,binocular indirect ophthalmoscope, A or B ultrasonic scan and optical coherence tomography (OCT).Follow-up duration varied from 6 to 36 months,with the mean of (19.4± 10.03)months.The visual acuity,retinal reattachment and the complications were observed.Logistic regression analysis was performed to predict the prognosis of visual acuity.Results Twenty-one eyes (77.78％) were anatomically reattached, six eyes (22.22％) were not anatomically reattached. Postoperative BCVA improved in 24 eyes (88.89％),reduced or remained unchanged in three eyes (11.11％). No ocular complications such as fundus hemorrhage,low or high intraocular pressure,endophthalmitis were found.The preoperative BCVA (OR =9.11,P =0.007),axial length (OR =0.31, P =0.038) and the photoreceptor inner and outer segment (IS/OS) junction line continuity (OR=4.32,P=0.001) are closely related to visual prognosis.Conclusions Vitreoretinal surgery is an effective approach to treat myopic foveoschisis with both anatomical and visual improvement.The preoperative BCVA,axial length and IS/OS line continuity are closely related to visual prognosis.

EFFECTS OF TETRAMETHYLPYRAZINE ON RETINA OF RABBITS WITHEXPERIMENTAL GLAUCOMA / 药物分析学报

Objective To observe the effects of tetramethyipyrazine(TMP) on retina to find out whether it can protect retina from glaucomatous damage. Methods Twenty-four rabbits were randomly divided into four groups. One eye of each rabbit was model eye induced by 2％ methylcellulous, and the other was control eye. Normal saline, TMP, timolol and a combination of timolol and TMP were administrated to group A, B, C and D respectively. At the end of 4th week, eyes were excavated for light and electron microscopic study. Results The numbers of ganglion cells (P ＜0. 01) and bipolar cells (P ＜0. 01) in model eye were different significantly between group A and B. In group A, the model eye ganglion cells were karyopyknosis, chromatin margination and nuclear membrane rupture; some in ner nuclear cells dcveloped marked lytic changes; outer segment appeared disorganized; but group B changed slight ly. Conclusion The results suggest that TMP may protect retina from glaucomatous damage.

The m RNA expression of proinflammatory factors in periretinal membrane of proliferative vitreoretinopathy / 中华眼底病杂志

Objective To observe the expression of proinflammatory factors messenger RNA (mRNA) in periretinal membrane of proliferative vitreoretinopathy. Methods Fourteen specimens of periretinal membrane were collected during vitrectomy, and they were made into paraffin sections.The presence of mRNA coding for IL-1,IL-6,IL-8 and TNF alpha was observed by in situ hybridization(ISH) with biotin labeled oligonuclotide probes respectively.The eyeball after corneal grafting was made as normal control. Results No expression of proinflammatory factors mRNA was found in normal human retina.Positive staining was present in 5 specimens.In these specimens, IL-1? mRNA was found in 3 specimens and TNF? mRNA was found in 3 specimens,there is 1 specimen expressed IL-8 mRNA and 3 specimens expressed IL-6 mRNA.In these positive specimens, one contained cells expressing mRNA for IL-1 ? beta and IL-6, and one exhibited cells expressing mRNA for IL-1??IL-8 and TNF ?,two membranes possessed positive cells for IL-6 and TNF? mRNA, one membrane contained IL-1? mRNA positive cells only. Conclusion These findings suggest that these cytokines may be locally produced by cells infiltrating epiretinal membranes. The expression of IL-1?, IL-6, IL-8 and TNF? mRNA within retinal membranes provides further evidence of a pathogenic role of these cytokines in proliferative vitreoretinopathy.

Interleukine 1 receptor antigonist inhibits proinflammatory factor production by human retinal pigment epithelial cells in vitro / 中华眼底病杂志

Objective To investigate production of interleukine 6 (IL 6) and interleukine 8 (IL 8) by retinal pigment epithelial (RPE) cells and its inhibition by interleukine 1 receptor antigonist(IL 1ra). Methods Cultured human RPE cells was treated with interleukine 1 ? (IL 1?, 10 ng/ml) and/or IL 1ra ( IL 1ra, 1、10、100 ng/ml). IL 6 and IL 8 mRNA and protein expression were detected by ELISA, immunohistochemistry and in situ hybridization (ISH) assay. Results IL 6 and IL 8 in conditioned media of RPE cells in controls was 2 000 pg/ml and 5 000 pg/ml respectively after stimulation of IL 1? for 8 h. IL 1ra (100 ng/ml) significantly inhibited IL 6 (300 pg/ml, t=8.011, P