Relationships between Intraocular Pressure, Effective Filtration Area and Morphological Changes in the Trabecular Meshwork of Steroid-Induced Ocular Hypertensive Mouse Eyes

We investigated whether an inverse relationship exists between intraocular pressure (IOP) and effective filtration area (EFA) in the trabecular meshwork (TM) in a steroid-induced ocular hypertensive (SIOH) mouse model and the morphological changes associated with the reduction of EFA. C57BL/6 mice (n = 15 per group) received either 0.1% dexamethasone (DEX) or saline eye drops twice daily for five weeks. IOP was measured weekly. Fluorescent tracers were injected into the anterior chamber to label EFA at the endpoint. Injected eyes were fixed and processed for confocal microscopy. EFA in the TM was analyzed. Light and electron microscopy were performed in high- and low-tracer regions of six eyes per group. The mean IOP was ~4 mm Hg higher in DEX-treated than saline-treated control eyes (p < 0.001) at the endpoint. EFA was reduced in DEX-treated eyes compared to controls (p < 0.01) and negatively correlated with IOP (R2 = 0.38, p = 0.002). Reduced thickness of juxtacanalicular tissue (JCT) and increased abnormal extracellular matrix in the JCT were found to be associated with reduced EFA. Our data confirm the inverse relationship between EFA and IOP, suggesting that morphological changes in the JCT contribute to the reduction of EFA, thus elevating IOP in SIOH mouse eyes.

Deep learning based models to study the effect of glaucoma genes on angle dysgenesis in-vivo

Objective: To predict the presence of Angle Dysgenesis on Anterior Segment Optical Coherence Tomography (ADoA) using deep learning and to correlate ADoA with mutations in known glaucoma genes. Design: A cross-sectional observational study. Participants: Eight hundred, high definition anterior segment optical coherence tomography (ASOCT) B-scans were included, out of which 340 images (One scan per eye) were used to build the machine learning (ML) model and the rest were used for validation of ADoA. Out of 340 images, 170 scans included PCG (n=27), JOAG (n=86) and POAG (n=57) eyes and the rest were controls. The genetic validation dataset consisted of another 393 images of patients with known mutations compared with 320 images of healthy controls Methods: ADoA was defined as the absence of Schlemm's canal(SC), the presence of extensive hyper-reflectivity over the region of trabecular meshwork or a hyper-reflective membrane (HM) over the region of the trabecular meshwork. Deep learning was used to classify a given ASOCT image as either having angle dysgenesis or not. ADoA was then specifically looked for, on ASOCT images of patients with mutations in the known genes for glaucoma (MYOC, CYP1B1, FOXC1 and LTBP2). Main Outcome measures: Using Deep learning to identify ADoA in patients with known gene mutations. Results: Our three optimized deep learning models showed an accuracy > 95%, specificity >97% and sensitivity >96% in detecting angle dysgenesis on ASOCT in the internal test dataset. The area under receiver operating characteristic (AUROC) curve, based on the external validation cohort were 0.91 (95% CI, 0.88 to 0.95), 0.80 (95% CI, 0.75 to 0.86) and 0.86 (95% CI, 0.80 to 0.91) for the three models. Amongst the patients with known gene mutations, ADoA was observed among all the patients with MYOC mutations, as it was also observed among those with CYP1B1, FOXC1 and with LTBP2 mutations compared to only 5% of those healthy controls (with no glaucoma mutations). Conclusions: Three deep learning models were developed for a consensus-based outcome to objectively identify ADoA among glaucoma patients. All patients with MYOC mutations had ADoA as predicted by the models.

Trabecular Meshwork Motion Profile from Pulsatile Pressure Transients: A New Platform to Simulate Transitory Responses in Humans and Nonhuman Primates

Trabecular meshwork (TM) motion abnormality is the leading cause of glaucoma. With technique limitations, how TM moves is still an enigma. This study describes a new laboratory platform to investigate TM motion responses to ocular transients in ex vivo eyes. The anterior segments of human cadaver and primate eyes were mounted in a perfusion system fitting. Perfusion needles were placed to establish mean baseline pressure. A perfusion pump was connected to the posterior chamber and generated an immediate transient pressure elevation. A phase-sensitive optical coherent tomography system imaged and quantified the TM motion. The peak-to-peak TM displacements (ppTMD) were determined, a tissue relaxation curve derived, and a time constant obtained. This study showed that the ppTMD increased with a rise in the pulse amplitude. The ppTMD was highest for the lowest mean pressure of 16 mmHg and decreased with mean pressure increase. The pulse frequency did not significantly change ppTMD. With a fixed pulse amplitude, an increase in mean pressure significantly reduced the time constant of recoil from maximum distension. Our research platform permitted quantitation of TM motion responses to designed pulse transients. Our findings may improve the interpretation of new TM motion measurements in clinic, aiding in understanding mechanisms and management.

Pathogenesis of Increased Intraocular Pressure in Primary Open‑Angle Glaucoma: Literature Review

Introduction. Despite the high socio-economic significance of primary open-angle glaucoma (POAG), the etiotropic and pathogenetic treatment of this disorder has not yet been implemented, since the mechanisms of the development of this disease are not fully known and understood. One of the topical issues is the pathogenesis of ophthalmic hypertension in POAG.Purpose. To summarize the currently known mechanisms of ophthalmic hypertension in POAG to search for potential molecular targets for pathogenetic pharmacotherapy.Materials and methods. Analysis of publications on PubMed, Medline and eLibrary.Results. Ophthalmic hypertension in POAG emerges due to increased resistance to aqueous humor (AH) outflow. It is caused by increased stiffness of Schlemm’s canal endothelium and trabecular meshwork as a result of changes in the structure and biomechanical properties of its cells and extracellular matrix. These changes are determined by the interaction of signaling molecules in the form of a pathological circle, the main links of which are TGF-β2 and its receptor, Smad 2/3/4, YAP/TAZ, sFRP-1 and CTGF.Conclusion. The cause of ophthalmic hypertension in POAG is fibrosis of the trabecular meshwork. This pathological process is based on the interaction of proteins, the main of which are TGF-β2 and its receptor, Smad 2/3/4, YAP/TAZ, sFRP-1 and CTGF. These molecules can become promising targets for the pathogenetic pharmacological therapy of POAG.

Primary Human Trabecular Meshwork Model for Pseudoexfoliation

The lack of an animal model or an in vitro model limits experimental options for studying temporal molecular events in pseudoexfoliation syndrome (PXF), an age related fibrillopathy causing trabecular meshwork damage and glaucoma. Our goal was to create a workable in vitro model of PXF using primary human TM (HTM) cell lines simulating human disease. Primary HTM cells harvested from healthy donors (n = 3), were exposed to various concentrations (5 ng/mL, 10 ng/mL, 15 ng/mL) of transforming growth factor-beta1 (TGF-β1) for different time points. Morphological change of epithelial–mesenchymal transition (EMT) was analyzed by direct microscopic visualization and immunoblotting for EMT markers. Expression of pro-fibrotic markers were analyzed by quantitative RT-PCR and immunoblotting. Cell viability and death in treated cells was analyzed using FACS and MTT assay. Protein complex and amyloid aggregate formation was analyzed by Immunofluorescence of oligomer11 and amyloid beta fibrils. Effect of these changes with pharmacological inhibitors of canonical and non-canonical TGF pathway was done to analyze the pathway involved. The expression of pro-fibrotic markers was markedly upregulated at 10 ng/mL of TGF-β1 exposure at 48–72 h of exposure with associated EMT changes at the same time point. Protein aggregates were seen maximally at these time points that were found to be localized around the nucleus and in the extracellular matrix (ECM). EMT and pro-fibrotic expression was differentially regulated by different canonical and non-canonical pathways suggesting complex regulatory mechanisms. This in vitro model using HTM cells simulated the main characteristics of human disease in PXF like pro-fibrotic gene expression, EMT, and aggregate formation.

Reduction in trabecular meshwork stem cell content in donor eyes with primary open angle glaucoma

We previously identified and characterized human trabecular meshwork stem cells (TMSCs) based on high expression of ABCG2/p75 positivity and high nucleus to cytoplasmic ratio. These TMSCs expressing high ABCG2 and p75 were located to the insert region of the human TM. Additionally, we demonstrated an age-related reduction in the TMSC content which was significantly associated with TM cell loss. In continuation, this study was aimed to determine the TMSC content in glaucomatous donor eyes wherein a drastic reduction in TM cellularity has already been reported. Anterior segments from known glaucomatous (n = 6) and age-matched normal (n = 8) donors were dissected into four quadrants. A minimum of three sections from each quadrant were used for histopathological analysis as well as immunostaining. Analysis of hematoxylin and eosin-stained sections from glaucomatous tissues revealed a decrease in total TM cellularity, thickening of trabecular beams, fusion of trabeculae, absence of patent Schlemm’s canal compared to age-matched controls. In addition, the TM thickness at various positions of the meshwork and the coronal as well as the meridional diameters of the Schlemm’s canal were observed to be significantly reduced in glaucomatous eyes. Further, sections from both the groups were immunostained for universal stem cell marker ABCG2 and neural crest derived stem cell marker p75. The images were acquired using Leica SP8 confocal microscope. Quantification of total TM cellularity based on nuclear counterstain (mean ± SD) using ImageJ identified 69.33 ± 12.77 cells/section in control eyes. In glaucomatous donors, the TM cellularity was found to be reduced significantly to 41.83 ± 9.0 (p = 0.0007). In addition, a reduction in the percentage of TMSCs (cells with high ABCG2 expression and p75 positivity) was evident in glaucomatous donors (0.14 ± 0.17%) compared to age-matched controls (4.73 ± 5.46%) (p = 0.064). Thus, the present study confirmed the significant decline in TM cellularity and a reducing trend in the TMSC content, though this reduction was non-significant in glaucomatous donor eyes. Further studies are essential to elucidate the role of TMSCs in the pathogenesis of primary open angle glaucoma.