Efficacy of subthreshold micropulse laser photocoagulation therapy versus anti-vascular endothelial growth factor therapy for refractory macular edema secondary to non-ischemic branch retinal vein occlusion.

OBJECTIVE: To assess the efficacy of subthreshold micropulse laser photocoagulation (SMLP) therapy versus anti-vascular endothelial growth factor (anti-VEGF) therapy in patients with refractory macular edema (ME) secondary to non-ischemic branch retinal vein occlusion (BRVO). METHODS: This single-center, prospective, nonrandomized, case-control trial involved patients with refractory ME that responded poorly to three or more initial anti-VEGF injections. The patients were examined and divided into two groups according to their chosen treatment: the intravitreal ranibizumab (IVR) group and the SMLP group. Both groups were followed up monthly for 12 months. Therapeutic efficacy and safety were assessed throughout the follow-up period. RESULTS: The IVR group comprised 49 eyes, and the SMLP group comprised 45 eyes. The improvements in the optical coherence tomography findings and visual acuity were comparable between the two groups at the final follow-up. The total number of injections was significantly lower in the SMLP than IVR group. No serious adverse events occurred during the study period. CONCLUSIONS: SMLP therapy is better for patients with central macular thickness (CMT) of ≤400 µm. For patients with CMT of >400 µm, we advise continuation of anti-VEGF agents to reduce ME followed by application of SMLP therapy when CMT has decreased to ≤400 µm.

Integrative RNA-seq and ATAC-seq analyses of phosphodiesterase 6 mutation-induced retinitis pigmentosa.

PURPOSE: Inhibition of poly-ADP-ribose polymerase 1 (PARP1) could relieve phosphodiesterase 6 mutation-induced retinitis pigmentosa (RP). However, the mechanism related to PARP1 overexpression in the RP has not been clarified. We attempted to explore the potential mechanism related to PARP1 regulating RP. METHODS: ATAC-seq and RNA-seq were performed for retina tissues of C3H and rd1 mice. The differentially expressed genes (DEGs) were identified, followed by the construction of PARP1-DEG co-expression and protein-protein interaction (PPI) networks. Gene ontology-biological process and pathway enrichment of DEGs were performed by clusterProfiler software. The overlapped genes that might play regulatory roles in PARP1 expression were mined by integrated analysis of RNA-seq and ATAC-seq data. RESULTS: A total of 1061 DEGs were identified between C3H and rd1 group. Co-expression network was constructed with 313 PARP1-gene co-expression pairs. The down-regulated DEGs were closely related to visual perception and light stimulus-related biological process, while the up-regulated DEGs were significantly enriched in phototransduction and PPAR signaling pathway. PPI network was constructed with 202 nodes and 375 edges, which was clustered into 3 modules. Module 1 genes were closely related to detection of light stimulus, visual perception related biological process and phototransduction pathway (involved with Gnat1/Guca1b/Gnat2/Sag/Pde6g). By integrated analysis of the RNA-seq and ATAC-seq, the overlapped up-regulated genes were Asxl3 and Nyap2, while the down-regulated genes were Tmem136 and Susd3. CONCLUSION: Gnat1 may play a key role in RP development by interacting with PARP1. Susd3 may play a regulatory role in PARP1 expression and affect RP formation.

PI3K/AKT/mTOR signaling participates in insulin-mediated regulation of pathological myopia-related factors in retinal pigment epithelial cells.

BACKGROUND: Insulin positively correlates with the length of the eye axis and is increased in the vitreous and serum of patients with pathological myopia (PM). How insulin influences the physiological process of retinal pigment epithelial (RPE) cells in PM remains unclear. This study aimed to explore the effect of insulin on the ultrastructure and function of RPE cells and the role of PI3K/AKT/mTOR signaling involved in the development of PM. METHODS: The ARPE-19 cells were treated with different concentrations of insulin to analyze the cell morphology, cell viability, the protein level of insulin receptor ß, and the mRNA and protein levels of and PM-related factors (TIMP-2, MMP-2, bFGF, and IGF-1). The ultrastructure of APRE-19 cells was also observed after insulin treatment. Besides, the PI3K/AKT/mTOR signaling was studied with or without the PI3K inhibitor LY294002 in ARPE-19 cells. RESULTS: Insulin enhanced the cell viability of ARPE-19 cells and caused the endoplasmic reticulum to expand and vesiculate, suggesting increased secretion of growth factors and degeneration in ARPE-19 cells. Furthermore, the insulin receptor ß was stimulated with insulin treatment, subsequently, the phosphorylation of AKT and mTOR was positively activated, which was adversely suppressed in the presence of LY294002. The secretion of TIMP-2 and bFGF was significantly decreased, and the secretion of MMP-2 and IGF-1 was highly elevated with insulin treatment depending on the concentration in ARPE-19 cells. Furthermore, the effect of insulin on PM-related proteins was restored with the addition of LY294002. CONCLUSIONS: Our results indicated that insulin regulated the secretion of PM-related factors via the PI3K/AKT/mTOR signaling pathway in retinal pigment epithelial cells, and thus probably promoted the development of PM through transducing regulation signals from retina to choroid and sclera.

MiR-126 overexpression inhibits high glucose-induced migration and tube formation of rhesus macaque choroid-retinal endothelial cells by obstructing VEGFA and PIK3R2.

AIM: The aims of this study are to investigate the relative regulation between miR-126 and VEGF/PI3K/AKT signaling pathway in retinal vascular endothelial cells. METHODS: Rhesus macaque choroid-retinal endothelial cell line (RF/6A) cells were cultured in high glucose to imitate the conditions occurring in DR. First, we detected the expression of miR-126, VEGFA and PIK3R2 in RF/6A cells on the condition of high glucose by q-PCR and western blot. Then, after addition of miR-126 mimics and miR-126 inhibitor, we investigated the function of miR-126 in RF/6A cells by scratch wound, Transwell migration and tube formation assays, and the effect of miR-126 on the expression of VEGFA, PIK3R2 and AKT. Moreover, bioinformatics analysis and luciferase array were used to confirm the direct or specific regulation of miR-126 to VEGFA or PIK3R2. RESULTS: Here, first, we found that high glucose could induce the decrease of miR-126 and the increase of VEGFA and PIK3R2 in RF/6A. Then, by scratch wound, Transwell migration and tube formation assays, we found that miR-126 overexpression could inhibit the migration and sprouting of RF/6A cells induced by high glucose, while knockdown of miR-126 led to the opposite results. Moreover, overexpression of miR-126 inhibited the increased expression of VEGFA, PIK3R2, SDF-1α, VCAM-1, and SPRED1, and the activation of AKT1 induced by high glucose and miR-126 inhibitor caused the opposite results which were determined by q-PCR and western blot. In addition, by luciferase assay, we found that miR-126 could directly negatively regulate VEGFA and PIK3R2. CONCLUSION: Our results suggest that miR-126 overexpression inhibits the migration and sprouting of RF/6A cells induced by high glucose which might possibly be by blocking VEGFA and PIK3R2 in the VEGF/PI3K/AKT signaling pathway.

Nestin is induced by hypoxia and is attenuated by hyperoxia in Müller glial cells in the adult rat retina.

This study investigated the reactive changes in Müller glial cells and astrocytes of the rat retinae, which had been subjected either to hypoxia or to hypoxia followed by hyperoxia treatments. Fifteen rats were used. Ten rats were exposed to 9% O(2) for 2 h. Of these, five rats were killed at 24 h later; the remaining five rats were immediately exposed to 80% O(2) for 2 h and then killed 24 h later. Double immunofluorescence was carried out between nestin and glutamine synthetase (GS) and between glial fibrilary acidic proteins (GFAP) and GS in normal and pathological retinae. Enhanced nestin expression was observed in reactive astrocytes following hypoxia treatment as revealed in whole mount sections. A novel finding was the induction of nestin expression in Müller glial cells. Remarkably, the nestin immunostaining was downregulated to levels comparable to those of the normal rats with immediate hyperoxia treatment. Induced nestin expression by hypoxia colabelled with GFAP in astrocytes, however, remained unaffected after hyperoxia treatment. The induced expression of nestin in Müller glial cells and astrocytes in hypoxia and differential downregulation after hyperoxia treatment suggest a structural plasticity of the cytoskeletal framework of these cells. The differential response after hyperoxia treatment may be related to the functional states of the cells.

Nestin, a new marker, expressed in Müller cells following retinal injury.

PURPOSE: To investigate whether nestin would be a useful marker for retinal injury and also to ascertain a better understanding of the roles of Müller cells in the injured retina by the use of damaged rat retina. METHODS: A total of 33 adult female Wistar rats were used in this study. Three were used as controls and the remaining as retinal injury modes (6 for hypoxia; 15 for experimental glaucoma and 9 for optic nerve transection). Double immunofluorescence labeling was carried out between nestin and glutamine synthetase (GS), and between glial fibrillary acidic protein (GFAP) and GS antisera in normal and pathological retinae. RESULTS: The results showed that there were no nestin nor GFAP staining in mature Müller cells of the normal retina. A major finding was that nestin expression was induced in Müller cells subjected to hypoxia, glaucoma and optic nerve transection. CONCLUSIONS: These results suggest that nestin as well as GFAP (even more sensitive than GFAP) are useful and reliable biomarkers for retinal damage. The more intense expression of nestin, GFAP and GS in the end-feet of Müller cells suggest that they may help to maintain the retinal structural integrity and to enhance functional recovery in various retinal diseases.