Corneal re-innervation following refractive surgery treatments

Laser refractive surgery is one of the most performed surgical procedures in the world. Although regarded safe and efficient, it has side effects. All of the laser based refractive surgical procedures invoke corneal nerve injury to some degree. The impact of this denervation can range from mild discomfort to neurotrophic corneas. Currently, three techniques are widely used for laser vision correction: small incision lenticule extraction, laser-assisted keratomileusis in situ and photorefractive keratotomy. Each of these techniques affects corneal innervation differently and has a different pattern of nerve regeneration. The purpose of this review is to summarize the different underlying mechanisms for corneal nerve injury and compare the different patterns of corneal reinnervation

Prospects and Challenges of Translational Corneal Bioprinting

Corneal transplantation remains the ultimate treatment option for advanced stromal and endothelial disorders. Corneal tissue engineering has gained increasing interest in recent years, as it can bypass many complications of conventional corneal transplantation. The human cornea is an ideal organ for tissue engineering, as it is avascular and immune-privileged. Mimicking the complex mechanical properties, the surface curvature, and stromal cytoarchitecure of the in vivo corneal tissue remains a great challenge for tissue engineering approaches. For this reason, automated biofabrication strategies, such as bioprinting, may offer additional spatial control during the manufacturing process to generate full-thickness cell-laden 3D corneal constructs. In this review, we discuss recent advances in bioprinting and biomaterials used for in vitro and ex vivo corneal tissue engineering, corneal cell-biomaterial interactions after bioprinting, and future directions of corneal bioprinting aiming at engineering a full-thickness human cornea in the lab

Immunopanning purification and long-term culture of human retinal ganglion cells

Purpose: To establish a robust method to isolate primary retinal ganglion cells (RGCs) from human fetal retina for long-term culture while maintaining neuronal morphology and marker protein expression. Methods: A total of six human retinas were obtained from aborted fetuses at 10 to 12 weeks of gestation with informed consent from mothers. RGCs were isolated and purified by a modified two-step immunopanning procedure. The cells were maintained in a serum-free defined medium supplemented with brain-derived neurotrophic factor, ciliary neutrophic factor, and forskolin. The viable RGCs and the extent of neurite outgrowth were examined by calcein-acetoxymethylester assay. Expression of RGC markers was studied by immunocytochemistry. Results: Primary RGCs from human fetal retinas were isolated and maintained in vitro for one month with substantial neurite elongation. In cell culture, almost 70% of the isolated cells attached, spread, and displayed numerous dendrites. They were immunoreactive to RGC-specific markers (Thy-1, TUJ-1, and Brn3a) and negative for glial fibrillary acidic protein and amacrine cells marker HPC-1. Conclusions: Human RGCs were successfully isolated and maintained in long-term culture. This can serve as an ideal model for biologic, toxicological, and genomic assays of human RGCs in vitr

Multiple gene polymorphisms analysis revealed a different profile of genetic polymorphisms of primary open-angle glaucoma in northern Chinese

Purpose: To evaluate the individual and interactive effects of polymorphisms in the myocilin (MYOC), optineurin (OPTN), WD repeat domain 36 (WDR36), and apolipoprotein E (APOE) genes on primary open-angle glaucoma (POAG) in northern Chinese. Methods: Northern Chinese study subjects, 176 POAG patients and 200 controls, were recruited for screening of the coding exons and splicing regions of MYOC. Five single nucleotide polymorphisms (SNPs) in OPTN (M98K, R545Q, IVS5+38T>G, IVS8-53T>C, and IVS15+10G>A), one SNP in WDR36 (IVS5+30C>T) as well as the APOE promoter and epsilon 2/epsilon 3/epsilon 4 polymorphisms were also examined. Association analysis was performed by using chi(2) analysis. High-order gene-gene interaction was also analyzed using the multifactor dimensionality reduction (MDR) method. Results: In MYOC, 22 variants were identified. Four of them were novel but found in controls only. The missense mutation, Val53Ala, is likely a glaucoma causing mutation, accounting for 0.6% of cases. No individual polymorphism in OPTN, WDR36, or APOE was associated with POAG. MDR analysis identified a best 6-factor model for POAG: MYOC IVS2+35A>G, OPTN Met98Lys, OPTN IVS5+38T>G, OPTN IVS8-53T>C, WDR36 IVS5+30C>T, and APOE -491A>T. Conclusions: The association pattern between the genes, MYOC, OPTN, WDR36, and APOE, and POAG in northern Chinese is different from that of southern Chinese. Disease-causing mutations in MYOC accounted for a small proportion of northern Chinese POAG patients. Common polymorphisms in these genes were not associated with POAG individually but might interactively contribute to the disorder, supporting a polygenic etiology.Biochemistry & Molecular BiologyOphthalmologySCI(E)20ARTICLE9-1189-981

AC and AG Dinucleotide Repeats in the PAX6 P1 Promoter are Associated with High Myopia

Purpose: The PAX6 gene, located at the reported myopia locus MYP7 on chromosome 11p13, was postulated to be associated with myopia development. This study investigated the association of PAX6 with high myopia in 379 high myopia patients and 349 controls. Methods: High myopia patients had refractive errors of –6.00 diopters or greater and axial length longer than 26 mm. Control subjects had refractive errors less than –1.00 diopter and axial length shorter than 24 mm. The P1 promoter, all coding sequences, and adjacent splice-site regions of the PAX6 gene were screened in all study subjects by polymerase chain reaction and direct sequencing. PAX6 P1 promoter-luciferase constructs with variable AC and AG repeat lengths were prepared and transfected into human ARPE-19 cells prior to assaying for their transcriptional activities. Results: No sequence alterations in the coding or splicing regions showed an association with high myopia. Two dinucleotide repeats, (AC)m and (AG)n, in the P1 promoter region were found to be highly polymorphic and significantly associated with high myopia. Higher repeat numbers were observed in high myopia patients for both (AC)m (empirical p = 0.013) and (AG)n (empirical p = 0.012) dinucleotide polymorphisms, with a 1.327-fold increased risk associated with the (AG)n repeat (empirical p = 0.016; 95% confidence interval: 1.059–1.663). Luciferase-reporter analysis showed elevated transcription activity with increasing individual (AC)m and (AG)n and combined (AC)m(AG)n repeat lengths. Conclusions: Our results revealed an association between high myopia and AC and AG dinucleotide repeat lengths in the PAX6 P1 promoter, indicating the involvement of PAX6 in the pathogenesis of high myopia

Effect of Indocyanine Green and Illumination on Gene Expression in Human Retinal Pigment Epithelial Cells

PURPOSE. To investigate the biological effects of indocyanine green (ICG) and acute illumination on human retinal pigment epithelial (RPE) cells. METHODS. Three concentrations (0, 0.25, and 2.5 mg/mL) of ICG were applied to ARPE19 cells for 1 minute. After isotonic rinsing, the cells were irradiated with a light beam with a wavelength spectrum from 400 to 800 nm and an output of 1850 lumens for 15 minutes. The cells were collected at timed intervals for the investigation of cell death and expression of stress-response genes by reverse transcription-polymerase chain reaction, immunofluorescence, and Western blot analysis. RESULTS. After ICG incubation, photoreactive changes were observed in the RPE cells. A reduction in cellular viability and considerable shrinkage of the cells were observed. The expressions of the apoptosis-related genes p53 and bax and the cell cycle arrest protein p21 were upregulated in cells treated with both ICG and light. Of the early-response genes, the expression of c-fos was specifically enhanced by light, with additive effects from the presence of ICG. Such stimulatory effects on these gene expressions were greater at 2.5 mg/mL than at 0.25 mg/mL ICG. CONCLUSIONS. ICG in the presence of acute illumination can elicit cell-cycle arrest and even apoptosis in RPE cells. The establishment of a safety level in the application of ICG in the region of 0.25 mg/mL is recommended. (Invest Ophthalmol Vis Sci