Spectral domain-optical coherence tomography (SD-OCT) has become an essential tool for assessing ocular tissues in live subjects and conducting research on ocular development, health, and disease. The processing of SD-OCT images, particularly those from non-mammalian species, is a labor-intensive manual process due to a lack of automated analytical programs. This paper describes the development and implementation of a novel computer algorithm for the quantitative analysis of SD-OCT images of live teleost eyes. Automated segmentation processing of SD-OCT images of retinal layers was developed using a novel algorithm based on thresholding. The algorithm measures retinal thickness characteristics in a large volume of imaging data of teleost ocular structures in a short time, providing increased accuracy and repeatability of SD-OCT image analysis over manual measurements. The algorithm also generates hundreds of retinal thickness measurements per image for a large number of images for a given dataset. Meanwhile, heat mapping software that plots SD-OCT image measurements as a color gradient was also created. This software directly converts the measurements of each processed image to represent changes in thickness across the whole retinal scan. It also enables 2D and 3D visualization of retinal thickness across the scan, facilitating specimen comparison and localization of areas of interest. The study findings showed that the novel algorithm is more accurate, reliable, and repeatable than manual SD-OCT analysis. The adaptability of the algorithm makes it potentially suitable for analyzing SD-OCT scans of other non-mammalian species.
Purpose: This study sought to investigate the association of molecular markers with chronic ocular sequelae in Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN).
Methods: One hundred SJS/TEN patients (200 eyes) with confirmed diagnosis were enrolled between July 2011 and July 2015 from a tertiary eye-care hospital, and their clinical histories were noted. Each eye was scored for severity of manifestation on a scale of 0-5. Peripheral blood samples were collected for DNA followed by screening for interleukin (IL-4, IL-13, IL-4R) polymorphisms, HLA-A locus allele typing, and sera to detect levels of the apoptotic markers granulysin and sFas L.
Results: Of the 100 enrolled patients (53 males/47 females; age range: 6-58 years), the incriminating drugs were non-steroidal anti-inflammatory (52%), antibiotics (10%), sulphonamides (8%), anti-epileptics (6%), and unknown (24%). Significant differences in the frequencies of IL-4R polymorphism, HLA-A*3301, HLA-A*02, and HLA-A*2402 alleles, and elevated levels of granulysin and sFas L were observed in patients compared to controls. The ocular complications of conjunctival keratinization (p=0.004) showed an association with IL-13 promoter region (IL-13a) genotypes.
Conclusions: The study highlights the possible association of interleukin-13 with severity-graded chronic sequelae and the role of HLA-A alleles- HLA-A*3301, HLA-A*02, and HLA-A*2402 in SJS/TEN causation and manifestation. Screening of these alleles may help caregivers to identify markers associated with severe and lifelong ocular complications, and help in appropriate treatment and management of the condition.
Objective: To investigate the effect of sodium polyanethol sulfonate (SPS) on herpes simplex virus type 1 (HSV-1) infection in vitro.
Methods: Human corneal epithelial (HCE-T) cells and Vero cells were infected with HSV-1 [HSV-1 f strain, HSV-1f; HSV-1-H129 with green fluorescent protein (GFP) knock-in, HSV-1g]. SPS was added to the culture medium at various concentrations in time-of-addition assay. Experiments including photography of fluorescence in HSV-1g or plaque formation by HSV-1f, western blot assays, real-time RT-PCR assays, cytopathic effect inhibition assays, cytotoxicity assays, and viral absorption and penetration assays were performed to explore the antiviral effect and mechanism of the compounds.
Results: We identified that SPS reduced the replication of HSV-1 in HCE-T and Vero cells in a dose-dependent manner. HSV-1g fluorescence was reduced by 66.3% and 65.4% in HCE-T and Vero cells, respectively, after treatment with 0.4 µg/ml SPS. Furthermore, the viral fluorescence intensities were inhibited by SPS in a dose-dependent manner when the viruses or cells were preincubated with SPS. Relative levels of the ICP4 protein and VP16 mRNA were decreased by SPS in a dose-dependent manner. Moreover, the IC50 values of SPS for HSV-1g and HSV-1f in HCE-T cells were 0.69±0.09 μg/ml and 1.63±0.44 μg/ml, respectively. Even 10,000 µg/ml SPS had no obvious cytotoxicity toward HCE-T and Vero cells. Importantly, viral absorption and penetration assays showed that the relative fluorescence intensity of HSV-1g was significantly reduced by SPS in a dose-dependent manner in the absorption test, but no change was observed in the penetration test.
Conclusions: SPS inhibits HSV-1 replication in HCE-T and Vero cells, indicating that SPS has the potential for treating HSV-1 infection, particularly HSV-1 keratitis.
Purpose: We designed a study to find theoretical evidence for the induction, movement, fusion, proliferation, and safety of human adipose mesenchymal stem cells (hADSCs) in intraocular application.
Methods: HADSCs were induced to confirm that they can express the characteristics of endothelial cells (ECs) in vitro. HADSCs were intraocularly injected into oxygen-induced retinopathy (OIR) mice to check the movement, fusion, proliferation, and prognosis in vivo. Electron microscopy was used to check retinal changes to confirm the safety of hADSCs in intraocular application.
Results: After induction, hADSCs expressed von Willebrand Factor (vWF), the cell marker of ECs. The hADSCs were distributed above the retina after an intravitreal injection in the OIR mice. The injected cells did not fuse with the retina and gathered in the central and peripheral areas, which is the lesion area of the OIR model. Five days after the hADSC intravitreal injection, the area of neovascularization was reduced by 94.83% compared with that of the OIR group. Hematologic staining and electron microscopy did not show noticeable proliferation and degeneration of the retina.
Conclusions: This study provides evidence for the intraocular application of hADSCs.
Purpose: The development of biomaterials provides potent promise for the regeneration of neuroretinal cells in degenerative eye diseases and retinal tissue engineering. Biomimetic three-dimensional (3D) microenvironments and specific growth factors motivate the differentiation of human retinal pigment epithelial (hRPE) cells toward a retinal neural lineage. In this study, we evaluated alginate/gelatin (A/G) as a substrate for the culture of hRPE cells.
Methods: hRPE cells were isolated from neonatal human cadaver globes and cultivated on A/G substrate under different culture conditions, including 30% human amniotic fluid (HAF), 10% fetal bovine serum (FBS), and serum-free Dulbecco's modified Eagle's medium/nutrient mixture F-12 (DMEM/F12). The proliferation of cells in different culture conditions was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and a cell proliferation assay. Immunocytochemistry and real-time PCR were performed to evaluate the effect of the substrate on hRPE cell differentiation.
Results: A significant increase in the cell proliferation rate was observed in hRPE cells cultivated on an A/G substrate. Continuous observations demonstrated that hRPE cells formed densely packed, suspended spheroids in DMEM/F12 culture conditions, with dominant transdifferentiation into amacrine cells. Small adherent clusters of hRPE cells in HAF- and FBS-treated cultures represented dedifferentiation toward retinal progenitor cells. These cultures generated amacrine, rod photoreceptors, and bipolar cells.
Conclusions: These findings indicated that A/G substrate induced neural retinal cell propagation in cultures and would therefore be promising for RPE-based tissue engineering studies.
Purpose: Strabismus (STBMS) is a multifactorial ocular disorder in children that leads to misalignment of the eyes. Insulin-like growth factor 1 (IGF1) has been shown to be involved in the development of extraocular muscles and myopia; however, data are limited on the genetic associations of IGF1 with STBMS in Pakistan.
Methods: Two hundred seventy-four STBMS cases and 272 unaffected controls were recruited, and their DNA was extracted. Two IGF1 single nucleotide polymorphisms, rs6214 and rs5742632, were genotyped using PCR-restriction fragment length polymorphism. Univariate logistic regression analysis was performed to determine the association of these single nucleotide polymorphisms with STBMS, and the results were adjusted for age and sex. In addition, 26 extraocular muscle tissues were collected from patients with STBMS undergoing squint correction surgery, along with 3 deceased control samples. IGF1 mRNA expression was measured by quantitative PCR; the Mann-Whitney U test was applied, and fold change was calculated. Logistic regression analysis was applied to determine the association of RNA expression and fold change with genotype.
Results: Multivariate logistic regression analysis revealed that rs5742632 (odds ratio [95% confidence interval] = 1.05[1.01-1.06], p = 0.03) is associated with STBM. Moreover, rs6214 (1.03[1.01-1.05], p = 0.03) and rs5742632 (1.09[1.04-1.11], p = 0.04) were associated with exotropia. Statistically, no significant difference in IGF1 mRNA expression in the extraocular muscles between the STBMS cases and the controls was observed.
Conclusions: IGF1 polymorphisms rs5742632 (A>G) and rs6214 (C>T) are plausible risk factors for the development of exotropia. However, the physiologic mechanism requires further evaluation.
Purpose: To identify and characterize properties of αA- and αB-crystallins' low molecular weight peptides (molecular weight [Mr] < 5 kDa) that were present in a 62-year-old human nuclear cataract, but not in normal 62-year-old human lenses.
Methods: Low molecular weight peptides (< 5 kDa) were isolated with a trichloroacetic acid (TCA) solubilization method from water-soluble (WS) and water-insoluble (WI) proteins of nuclear cataractous lenses of a 62-year-old donor and normal human lenses from an age-matched donor. Five commercially synthesized peptides (found only in cataractous lenses and not in normal lenses) were used to determine their chaperone and antichaperone activity and aggregation properties.
Results: Mass spectrometric analysis showed 28 peptides of αA-crystallin and 38 peptides of αB-crystallin were present in the cataractous lenses but not in the normal lenses. Two αA peptides (named αAP1 and αAP2; both derived from the αA N-terminal domain (NTD) region) and three αB peptides (named αBP3, αBP4, and αBP5, derived from the αB NTD-, core domain (CD), and C-terminal extension (CTE) regions, respectively) were commercially synthesized. αAP1 inhibited the chaperone activity of αA- and αB-crystallins, but the other four peptides (αAP2, αBP3, αBP4, and αBP5) exhibited mixed effects on chaperone activity. Upon incubation with human WS proteins and peptides in vitro, the αBP4 peptide showed higher aggregation properties relative to the αAP1 peptide. During in vivo experiments, the cell-penetrating polyarginine-labeled αAP1 and αBP4 peptides showed 57% and 85% aggregates, respectively, around the nuclei of cultured human lens epithelial cells compared to only 35% by a scrambled peptide.
Conclusions: The antichaperone activity of the αAP1 peptide and the aggregation property of the αBP4 peptide with lens proteins could play a potential role during the development of lens opacity.
Purpose: The purpose of this study was to analyze genetic and nongenetic associations with reticular pseudodrusen (RPD) in patients with and without age-related macular degeneration (AMD).
Methods: This case-control study included 2,719 consecutive subjects from the prospective multicenter European Genetic Database (EUGENDA). Color fundus photographs and optical coherence tomography (OCT) scans were evaluated for the presence of AMD and RPD. Association of RPD with 39 known AMD polymorphisms and various nongenetic risk factors was evaluated. Stepwise backward variable selection via generalized linear models (GLMs) was performed based on models including the following: a) age, sex, and genetic factors and b) all predictors. Receiver operating characteristic (ROC) curves and the areas under the curve (AUCs) were determined.
Results: RPD were present in 262 cases (no AMD, n = 9 [0.7%; early/intermediate AMD, n = 75 [12.4%]; late AMD, n = 178 [23.8%]). ROC analysis of the genetic model including age, APOE rs2075650, ARMS2 rs10490924, CFH rs800292, CFH rs12144939, CFI rs10033900, COL8A1 rs13081855, COL10A1 rs3812111, GLI3 rs2049622, and SKIV2L rs4296082 revealed an AUC of 0.871. Considering all possible predictors, backward selection revealed a slightly different set of genetic factors, as well as the following nongenetic risk factors: smoking, rheumatoid arthritis, steroids, antiglaucomatous drugs, and past sunlight exposure; the results showed an AUC of 0.886.
Conclusions: RPD share a variety of genetic and nongenetic risk factors with AMD. Future AMD grading systems should integrate RPD as an important risk phenotype.
Purpose: Primary angle-closure glaucoma (PACG) is a leading cause of blindness. Despite tremendous human effort and financial input, no definitive causative gene has been identified either through genome-wide association or Mendelian family studies. In the current study, novel candidate genes for PACG were investigated by studying the variants of nanophthalmos-associated genes.
Methods: A case-control study was conducted that included 45 PACG patients and 12 normal controls with short axial length (AL, less than 23.5 mm but more than 20.5 mm). Whole-exome sequencing (WES) was performed to screen the variants in previously identified nanophthalmos-associated genes, as well as other risk genes.
Results: The age range of the 45 PACG patients was 24 to 80 years, with an average AL of 21.87±0.65 mm (range: 20.54-23.45 mm) in the right eye and 21.89±0.64 mm (range 20.60-23.23 mm) in the left eye. Four novel myelin regulatory factor (MYRF) gene missense variants (p.G117S, p.H1057R, p.H230R, and p.R316C) were identified in four out of the 45 enrolled PACG patients, respectively. No MYRF or other nanophthalmos-associated gene variants were detected in the 12 normal controls.
Conclusions: An appropriate approach was adopted to investigate the genetics of PACG through nanophthalmos-associated genes. A genetic variant, MYRF, was identified in four out of 45 PACG patients, which might be a novel candidate gene for PACG.
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