Glaucomatous Optic Nerve Head Research Articles

Structural Features of Optic Nerve Head in Glaucoma with Single-hemispheric and Bi-hemispheric Visual Field Defects

Purpose: To compare the structural features of the optic nerve head (ONH) in moderate glaucoma patients with single-hemispheric and bi-hemispheric visual field (VF) defects.Methods: Patients with open-angle glaucoma (OAG) and a VF mean deviation between -6 and -12 dB were retrospectively enrolled. Based on VF defect patterns, eyes were classified into two groups: those with single-hemispheric VF defects and those with bi-hemispheric VF defects. Structural features analyzed included Bruch’s membrane opening area, lamina cribrosa (LC) thickness, temporal β-zone parapapillary atrophy with and without Bruch's membrane (βPPA<sub>+BM</sub>, βPPA<sub>-BM</sub>, respectively), and peripapillary retinal nerve fiber layer thickness using spectral-domain optical coherence tomography.Results: Out of 137 eyes from 124 patients, 80 eyes had single-hemispheric VF defects and 57 eyes had bi-hemispheric VF defects. The bi-hemispheric group exhibited longer axial length (<i>p</i> = 0.038), thinner central corneal thickness (<i>p</i> = 0.003), thinner LC (<i>p</i> < 0.001), and larger temporal βPPA<sub>+BM</sub> (<i>p</i> = 0.002) compared to the single-hemispheric group.Conclusions: Among Korean OAG patients with moderate VF defects, those with bi-hemispheric VF defects showed more structurally vulnerable optic discs characterized by longer axial lengths, thinner central corneal thickness, thinner LC, and larger β PPA+BM compared to those with single-hemispheric VF defects. These findings underline the importance of evaluating ONH structures in monitoring glaucoma progression particularly in patients with extensive VF damage.

The Role of αvβ3 Integrin in Lamina Cribrosa Cell Mechanotransduction in Glaucoma.

Purpose: Glaucoma, one of the leading causes of irreversible blindness, is a common progressive optic neuropathy characterised by visual field defects and structural changes to the optic nerve head (ONH). There is extracellular matrix (ECM) accumulation and fibrosis of the lamina cribrosa (LC) in the ONH, and consequently increased tissue stiffness of the LC connective tissue. Integrins are cell surface proteins that provide the key molecular link connecting cells to the ECM and serve as bidirectional sensors transmitting signals between cells and their environment to promote cell adhesion, proliferation, and remodelling of the ECM. Here, we investigated the expression of αVβ3 integrin in glaucoma LC cell, and its effect on stiffness-induced ECM gene transcription and cellular proliferation rate in normal (NLC) and glaucoma (GLC) LC cells, by down-regulating αVβ3 integrin expression using cilengitide (a known potent αVβ3 and αVβ5 inhibitor) and β3 integrin siRNA knockdown. Methods: GLC cells were compared to age-matched controls NLC to determine differential expression levels of αVβ3 integrin, ECM genes (Col1A1, α-SMA, fibronectin, vitronectin), and proliferation rates. The effects of αVβ3 integrin blockade (with cilengitide) and silencing (with a pool of four predesigned αVβ3 integrin siRNAs) on ECM gene expression and proliferation rates were evaluated using both reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blotting in the human NLC cells cultured on soft (4 kPa) and stiff (100 kPa) substrate and in GLC cells grown on standard plastic plates. Results: αVβ3 integrin gene and protein expression were enhanced (p < 0.05) in GLC cells as compared to NLC. Both cilengitide and siRNA significantly reduced αVβ3 expression in GLC. When NLC were grown in the stiff substrate, cilengitide and siRNA also significantly reduced the increased expression in αVβ3, ECM components, and proliferation rate. Conclusions: Here, we provide evidence of cilengitide- and siRNA-mediated silencing of αVβ3 integrin expression, and inhibition of ECM synthesis in LC cells. Therefore, αVβ3 integrin may be a promising target for the development of novel anti-fibrotic therapies for treating the LC cupping of the ONH in glaucoma.

Complement Factor B Inhibition or Deletion Is Not Sufficient to Prevent Neurodegeneration in a Murine Model of Glaucoma.

Purpose: Activation of the classical complement pathway is thought to contribute to the development and progression of glaucoma. The role of alternative complement or amplification pathways in glaucoma is not well understood. We evaluated complement factor B (FB) expression in postmortem human ocular tissues with or without glaucoma and the effect of FB inhibition and deletion in a mouse ocular hypertensive model of glaucoma induced by photopolymerized hyaluronic acid glycidyl methacrylate (HAGM). Methods: Human CFB mRNA in human eyes was assessed by RNAscope and TaqMan. HAGM model was performed on C57BL6/J mice. The effect of FB in HAGM model was evaluated with an oral FB inhibitor and Cfb-/- mice. Complement mRNA and proteins in mouse eyes were assessed by TaqMan and western blot, respectively. Results: CFB mRNA in human glaucomatous macular neural retina and optic nerve head was upregulated. Cfb mRNA is also upregulated in the HAGM model. Oral FB inhibitor, ED-79-GX17, dosed daily at 200 mg/kg for 3 days after intraocular pressure (IOP) induction in wild-type mice showed complement inhibition in ocular tissues and significantly inhibited systemic complement levels. Daily dosing of ED-79-GX17 for 30 days or Cfb deletion was also unable to prevent retinal ganglion cell or axon loss 30 days after IOP induction in mice. Conclusion: The alternative complement component FB may not substantially contribute to RGC loss in the HAGM mouse glaucoma model despite upregulation of Cfb expression and activation of the alternative pathway. The relevance of these findings to human glaucoma remains to be determined.

Postmortem Analysis of Optic Nerve Head Vascularization in an Individual With Glaucoma.

Reduced ocular perfusion likely contributes to glaucomatous damage at the optic nerve head (ONH). In recent decades, investigators have focused heavily on ocular perfusion pressure and other factors affecting blood flow to the eye. Comparatively, far less attention has been focused on the blood vessels themselves. Here, we asked whether glaucomatous individuals exhibit anatomical deficiencies (i.e., fewer blood vessels) in their ONH blood supply. To answer this question, we performed a systematic literature review to (1) determine how many studies have reported measuring blood vessels in the ONH and (2) whether these studies reported differences in blood vessel quantity. Additionally, we report a method for quantifying blood vessels in ex vivo human ONH preparations, including an ONH from an individual with glaucoma. Our results show that only two studies in the past 50 years have published data concerning blood vessel density in glaucomatous ONHs. Interestingly, both studies reported decreased blood vessel density in glaucoma. Consistent with this finding, we also report reduced blood vessel numbers in the superolateral quadrant of a glaucomatous individual's ONH. Vascularity in the three remaining quadrants was similar to control. Together, our findings raise the interesting possibility that individuals with a relatively sparse ONH blood supply are more likely to develop glaucoma. Future studies with larger sample sizes and more thorough quantification are necessary to determine the link more accurately between glaucoma and the blood supply to the ONH.

Impact of a short online course on the accuracy of non-ophthalmic diabetic retinopathy graders in recognising glaucomatous optic nerves in Vietnam

PurposeTo test an online training course for non-ophthalmic diabetic retinopathy (DR) graders for recognition of glaucomatous optic nerves in Vietnam.MethodsThis was an uncontrolled, experimental, before-and-after study in which 43 non-ophthalmic...

Astrocytes in the Optic Nerve Are Heterogeneous in Their Reactivity to Glaucomatous Injury.

The optic nerve head is thought to be the site of initial injury to retinal ganglion cell injury in glaucoma. In the initial segment of the optic nerve directly behind the globe, the ganglion cell axons are unmyelinated and come into direct contact to astrocytes, suggesting that astrocytes may play a role in the pathology of glaucoma. As in other parts of the CNS, optic nerve head astrocytes respond to injury by characteristic changes in cell morphology and gene expression profile. Using RNA-sequencing of glaucomatous optic nerve heads, single-cell PCR, and an in-vivo assay, we demonstrate that an up-regulation of astrocytic phagocytosis is an early event after the onset of increased intraocular pressure. We also show that astrocytes in the glial lamina of the optic nerve are apparently functionally heterogeneous. At any time, even in naïve nerves, some of the cells show signs of reactivity-process hypertrophy, high phagocytic activity, and expression of genetic markers of reactivity whereas neighboring cells apparently are inactive. A period of increased intraocular pressure moves more astrocytes towards the reactive phenotype; however, some cells remain unreactive even in glaucomatous nerves.

Mechanical strain in the mouse astrocytic lamina increases after exposure to recombinant trypsin.

The responses of astrocytes in the optic nerve head (ONH) to mechanical and biochemical stimuli are important to understanding the degeneration of retinal ganglion cell axons in glaucoma. The ONH in glaucoma is vulnerable to stress produced by the intraocular pressure (IOP). Notably, after three days of elevated IOP in a mouse model, the junctions between the astrocytic processes and the peripapillary sclera were altered and the structural compliance of the ONH increased. In order to simulate this aspect of glaucomatous remodeling, explanted mouse eyes were treated with TrypLE, a recombinant trypsin enzyme. Treatment with TrypLE caused the periphery of the astrocytic lamina to contract radially by 0.044±0.038. Transmission electron microscopy showed that TrypLE caused a separation of the end-feet of the astrocyte processes from the basement membrane at the junction with the sclera. Inflation testing after treatment with TrypLE caused an increased strain response in the astrocytic lamina compared to the strain response before treatment. The greatest increase was in the radial Green-Lagrange strain, Err = 0.028±0.009, which increased by 340%. The alterations in the microstructure and in the strain response of the astrocytic lamina reported in mouse experimental glaucoma were partially reproduced by experimental treatment of mouse eyes with TrypLE. The results herein suggest that separation of junctions between the astrocyte processes and the sclera may be instrumental in increasing the structural compliance of the ONH after a period of elevated IOP. STATEMENT OF SIGNIFICANCE: Astrocytes of the optic nerve of the eye spread out from edge to edge across the optic nerve in a region referred to as the astrocytic lamina. In an experimental model of glaucoma caused by elevated eye-pressure, there is disruption of the connections between astrocytes and the edge of the astrocytic lamina. We caused a similar event in the lamina by incubating explanted mouse eyes with an enzyme. Disruption of the astrocyte connections to the edge of their tissue caused the tissue to stretch more when we increased the eye-pressure, compared to the control tissue. This work is the first on the tissue of the optic nerve to demonstrate the importance of cell connections in preventing the over-stretching of the astrocytic lamina.

Role of Epithelial-to-Mesenchymal Transition of Retinal Pigment Epithelial Cells in Glaucoma Cupping

Optic nerve head (ONH) cupping is a clinical feature of glaucoma associated with extracellular matrix (ECM) remodelling and lamina cribrosa (LC) fibrosis. Peripapillary atrophy (PPA) occurs commonly in glaucoma, and is characterised by the loss of retinal pigment epithelium (RPE) adjacent to the ONH. Under pro-fibrotic conditions, epithelial cells throughout the body can differentiate into fibroblast-like cells through epithelial-to-mesenchymal transition (EMT) and contribute to ECM fibrosis. This is investigated here in the context of glaucoma and PPA. Human-donor ONH sections were assessed for the presence of the RPE cell-specific marker RPE65 using immunofluorescence. We examined the EMT response of ARPE-19 cells to the following glaucoma-related stimuli: cyclic mechanical stretch, mechanical stiffness, transforming growth factor beta (TGFβ), and tumour necrosis factor alpha (TNFα). The gene expression was measured using the PCR of the epithelial tight junction marker zona occludens 1 (ZO-1) and the mesenchymal markers alpha smooth muscle actin (αSMA) and vimentin. A scratch assay was used to assess the ARPE-19 migration. Significant RPE-65 staining was demonstrated in the glaucomatous ONH. The cyclic stretching and substrate stiffness of the ARPE-19 cells caused a significant decrease in ZO-1 (p = 0.04), and an increase in αSMA (p = 0.04). The scratch assays demonstrated increased migration of ARPE19 in the presence of TNFα (p = 0.02). Furthermore, ARPE-19 cells undergo an EMT-like transition (gain of αSMA, loss of ZO-1 and increased migration) in response to glaucomatous stimuli. This suggests that during PPA, RPE cells have the potential to migrate into the ONH and differentiate into fibroblast-like cells, contributing to glaucomatous ONH cupping.

Alteration of neurofilament heavy chain and its phosphoforms reveals early subcellular damage beyond the optic nerve head in glaucoma.

Retinal ganglion cells (RGCs) axon loss at the site of optic nerve head (ONH) is long believed as the common pathology in glaucoma since different types of glaucoma possessing different characteristic of intraocular pressure, and this damage was only detected at the later stage. To address these disputes and detect early initiating events underlying RGCs, we firstly detected somatic or axonal change and compared their difference in acute and chronic phase of primary angle-closed glaucoma (PACG) patient using optical coherence tomography (OCT), then an axonal-enriched cytoskeletal protein neurofilament heavy chain and its phosphoforms (NF-H, pNF-H) were utilized to reveal spatio-temporal undetectable damage insulted by acute and chronic ocular hypertension (AOH, COH) in two well characterized glaucoma mice models. In clinic, we detected nonhomogeneous changes such as ONH and soma of RGCs presenting edema in acute phase but atrophy in chronic one by OCT. In AOH animal models, an increase expression of NF-H especially its phosphorylation modification was observed as early as 4 h before RGCs loss, which presented as somatic accumulation in the peripheral retina and at the sites of ONH. In contrast, in microbeads induced COH model, NF-H and pNF-H reduced significantly, these changes firstly occurred as NF-H or pNF-H disconnection at ONH and optic nerve after 2 weeks when the intraocular pressure reaching the peak; Meanwhile, we detected aqueous humor pNF-H elevation after AOH and slight reduction in the COH. Together, our data supports that early alteration of NF-H and its phosphoforms would reveal undetectable subcellular damage consisting of peripheral somatic neurofilament compaction, impaired axonal transport and distal axonal disorganization of cytoskeleton beyond the ONH, and identifies two distinct axonal degeneration which were Wallerian combination with retrograde degeneration in acute PACG and retrograde degeneration in the chronic one.

Normotensive Glaucoma in the Fellow Eye of Patient with Unilateral Pseudoexfoliation

To investigate the characteristics of normotensive glaucoma (NTG) in the fellow eye of patients with unilateral pseudoexfoliation syndrome (PXS). This study is a retrospective chart review. We included 313 patients with NTG. Using the 1:1 matched propensity score, only 94 well-matched patients were selected. A total of 47 NTG patients who had PXS in their contralateral eye (PXS group) and 47 NTG patients who did not (control group) were compared. The propensity score was matched based on age, mean intraocular pressure (IOP), baseline retinal nerve fiber layer (RNFL) thickness and baseline mean deviation (MD) of visual field (VF) score. The diagnosis of NTG was based on the presence of glaucomatous optic nerve head injury with VF defect, intraocular pressure less than 22 mmHg, open angles and no pseudoexfoliation material. The PXS group had a higher ratio of males (34.0%) than the control group (17.0%). No significant differences were observed between the two groups in terms of CCT, axial length, untreated baseline IOP, baseline PSD of VF, systemic blood pressure and follow-up duration. The rate of RNFL thinning was significantly faster in the PXS group (-1.88 ± 2.83 μm/year) compared with the control group (-0.27 ± 5.29 μm/year) (p = 0.02). The progression rate of VF MD was slightly faster in PXS than in the control group, but there was no statistically significant difference (PXS group, -0.33 ± 0.90 dB/year; control group, -0.11 ± 0.84 dB/year; p = 0.236). NTG eyes with PXS showed faster RNFL thinning than did control NTG eyes.

Optical coherence angiography of optic disc in eyes with primary open‐angle glaucoma and normal tension glaucoma with equal levels of structural damage

AbstractPurpose: To compare visual field (VF), nerve fibre and ganglion cell loss and changes in peripapillary vasculature in age matched patients with normal‐tension (NTG) and high tension glaucoma (HTG) at an equal level of glaucomatous structural damage of the optic nerve head (ONH).Methods: For the present study a retrospective analysis of patients with NTG and HTG was performed. One eye of each patient was randomly assigned for further analysis. To provide a reliable case by case match of HTG and NTG eyes, an equal optic disk size as measured by available spectral‐domain OCT system (RTVue XR OCT Avanti System; Optovue Inc., Fremont, CA) and an equal estimated excavation of the optic disk were chosen. Optic nerve head parameters, retinal nerve fibre layer (RNFL) ganglion cell (GCC) thickness and vessel densities (VD) in the peripapillary area were measured automatically by OCT with a respective software. Forty eyes with NTG, were matched according to the same glaucomatous ONH damage based on cup to disc ratio rim area and disc size with 40 eyes with HTG.Results: Mean age of patients was 68.5 ± 12 (27–84) years. The visual field index, RNFL, GCC thickness and peripapillary VDs in the HTG eyes were similar to those in the NTG eyes (mean VFI 79.6 vs. 78.8 p = 0.84, mean RNFL thickness 71.5 vs. 72.05 μm p = 0.87, mean GCC thickness 76.2 vs. 74.5 μm p = 0.62, mean peripapillary VD 43.6 vs. 43.8% p = 0.87). There was no difference in sectoral RNFL thickness and peripapillary VDs between HTG and NTG. In both groups, univariate regression analysis using the Pearson correlation coefficient showed VFI significantly correlated with RNFL thickness (NTG r = 0.423 p = 0.006 HTG r = 0.514 p = 0.002), and peripapillary VD (NTG r = 0.793 p &lt; 0.001 HTG p = 0.534 p = 0.001) in both groups but with GCC thickness (NTG r = 0.585 p &lt; 0.001) only in eyes with NTG.Conclusions: At an equal level of glaucomatous structural damage of the ONH indicated by cup to disk ratio and RNFL thickness, NTG patients seem to have same losses of peripapillary vasculature. Visual field index correlated with RNFL thickness and peripapillary vessel density in both groups.

TLR4 signaling modulates extracellular matrix production in the lamina cribrosa.

The optic nerve head (ONH) is a place of vulnerability during glaucoma progression due to increased intraocular pressure damaging the retinal ganglion cell axons. The molecular signaling pathways involved in generating glaucomatous ONH damage has not been fully elucidated. There is a great deal of evidence that pro-fibrotic TGFβ2 signaling is involved in modulating the ECM environment within the lamina cribrosa (LC) region of the ONH. Here we investigated the role of signaling crosstalk between the TGFβ2 pathway and the toll-like receptor 4 (TLR4) pathway within the LC. ECM deposition was examined between healthy and glaucomatous human ONH sections, finding increases in fibronectin and fibronectin extra domain A (FN-EDA) an isoform of fibronectin known to be a damage associated molecular pattern (DAMP) that can activate TLR4 signaling. In human LC cell cultures derived from healthy donor eyes, inhibition of TLR4 signaling blocked TGFβ2 induced FN and FN-EDA expression. Activation of TLR4 by cellular FN (cFN) containing the EDA isoform increased both total FN production and Collagen-1 production and this effect was dependent on TLR4 signaling. These studies identify TGFβ2-TLR4 signaling crosstalk in LC cells of the ONH as a novel pathway regulating ECM and DAMP production.

Evidence of an AnnexinA4 mediated plasma membrane repair response to biomechanical strain associated with glaucoma pathogenesis.

Glaucoma is a common neurodegenerative blinding disease that is closely associated with chronic biomechanical strain at the optic nerve head (ONH). Yet, the cellular injury and mechanosensing mechanisms underlying the resulting damage have remained critically unclear. We previously identified Annexin A4 (ANXA4) from a proteomic analyses of human ONH astrocytes undergoing pathological biomechanical strain that mimics glaucomatous conditions. Annexins are a family of calcium-dependent phospholipid binding proteins with key functions in plasma membrane repair (PMR); an active mechanism to limit and mend cellular injury that involves membrane and cytoskeletal reorganizations. However, a role for direct membrane damage and PMR has not been well studied in the context of biomechanical strain, such as that associated with glaucoma. Here we report that this moderate strain surprisingly damages cell membranes to increase permeability in a calcium-dependent manner, and induces rapid aggregation of ANXA4 at injury sites. ANXA4 loss-of-function increases permeability, while exogenous ANXA4 reduces it. Furthermore, ANXA4 aggregation is associated with F-actin dynamics in vitro, and remarkably this interaction and aggregation signature is also observed in the glaucomatous ONH in patient samples. Together these studies link moderate biomechanical strain with direct membrane damage and actin dynamics, and identify an active PMR role for ANXA4 in new model of cell injury associated with glaucoma pathogenesis.

Inter-eye Comparison of the Lamina Cribrosa Depth in Patients with Bilateral Normal-tension Glaucoma with Asymmetrical Damage

Purpose: To evaluate the association between lamina cribrosa depth (LCD) and the severity of glaucomatous damage in patients with normal-tension glaucoma (NTG).Methods: The optic nerve heads (ONHs) of patients with bilateral NTG showing asymmetrical damage were scanned using spectral-domain optical coherence tomography. LCDs were measured on ONH horizontal B-scan images at three locations equidistant across the vertical optic disc diameter and compared between the more damaged and contralateral eyes. Conditional logistic regression analysis was performed to identify ocular risk factors associated with more severe damage between the eyes.Results: One hundred and four eyes of 52 patients with bilateral NTG were included. The mean age was 66.0 ± 15.0 years; there were 31 males and 21 females. The more damaged eyes exhibited a higher baseline intraocular pressure (IOP) (p &lt; 0.001), a thinner global retinal nerve fiber layer thickness (p &lt; 0.001), and worse visual field mean deviation (p &lt; 0.001) and a pattern standard deviation (p &lt; 0.001), than the contralateral eyes. In contrast, we found no significant inter-eye difference in either the spherical equivalent or the axial length. The average LCD was significantly larger in the more damaged eyes (529.4 ± 116.7 vs. 482.9 ± 107.5 μm, p &lt; 0.001). On conditional logistic regression analysis, the higher the baseline IOP (p = 0.006) and the larger the LCD (p = 0.003), the higher the risk of having more severe damage compared to the contralateral eye under similar systemic conditions.Conclusions: The LCD was significantly larger in the more damaged eyes of patients with bilateral NTG, suggesting that LC deformation might be associated with glaucomatous ONH damage in NTG eyes.

Engineering a 3D hydrogel system to study optic nerve head astrocyte morphology and behavior

In glaucoma, astrocytes within the optic nerve head (ONH) rearrange their actin cytoskeleton, while becoming reactive and upregulating intermediate filament glial fibrillary acidic protein (GFAP). Increased transforming growth factor beta 2 (TGF β2) levels have been implicated in glaucomatous ONH dysfunction. A key limitation of using conventional 2D culture to study ONH astrocyte behavior is the inability to faithfully replicate the in vivo ONH microenvironment. Here, we engineer a 3D ONH astrocyte hydrogel to better mimic in vivo mouse ONH astrocyte (MONHA) morphology, and test induction of MONHA reactivity using TGF β2. Primary MONHAs were isolated from C57BL/6J mice and cell purity confirmed. To engineer 3D cell-laden hydrogels, MONHAs were mixed with photoactive extracellular matrix components (collagen type I, hyaluronic acid) and crosslinked for 5 minutes using a photoinitiator (0.025% riboflavin) and UV light (405-500 nm, 10.3 mW/cm2). MONHA-encapsulated hydrogels were cultured for 3 weeks, and then treated with TGF β2 (2.5, 5.0 or 10 ng/ml) for 7 days to assess for reactivity. Following encapsulation, MONHAs retained high cell viability in hydrogels and continued to proliferate over 4 weeks as determined by live/dead staining and MTS assays. Sholl analysis demonstrated that MONHAs within hydrogels developed increasing process complexity with increasing process length over time. Cell processes connected with neighboring cells, coinciding with Connexin43 expression within astrocytic processes. Treatment with TGF β2 induced reactivity in MONHA-encapsulated hydrogels as determined by altered F-actin cytoskeletal morphology, increased GFAP expression, and elevated fibronectin and collagen IV deposition. Our data sets the stage for future use of this 3D biomimetic ONH astrocyte-encapsulated hydrogel to investigate astrocyte behavior in response to injury.

Topographic Relationships among Deep Optic Nerve Head Parameters in Patients with Primary Open-Angle Glaucoma

Purpose: To investigate the topographic relationships among the deep optic nerve head (ONH) parameters representing myopic axial elongation or changes in the lamina cribrosa (LC) in patients with primary open-angle glaucoma (POAG). Methods: Among patients with POAG who visited the clinic between January 2015 and March 2017, the following deep ONH parameters were measured using spectral-domain optical coherence tomography (SD-OCT): externally oblique border tissue (EOBT) length, ONH tilt angle, optic canal (OC) obliqueness, and anterior LC insertion depth (ALID). In addition, the angular locations of the maximal value of each parameter were measured. We analyzed the correlations between the parameters, correlations with axial length (AL), and the spatial correspondence with glaucomatous ONH damage. Results: A total of 100 eyes with POAG were included in the analysis. The EOBT length, ONH tilt angle, and OC obliqueness were correlated with each other and with AL, whereas ALID showed less correlation with the other parameters and AL. The angular location where the three AL-related parameters had maximum values was also correlated with the predominant region of the glaucomatous ONH damage, while the angular location of the deepest ALID showed less correlation. Conclusions: Among the deep ONH parameters, the AL-related parameters EOBT length, ONH tilt angle, and OC obliqueness showed strong spatial correspondence with glaucomatous ONH damage, whereas the LC-related parameter ALID was less correlated with both AL and the region with glaucomatous ONH damage. Further studies are needed to determine how these differences affect glaucomatous ONH change.

Mitochondrial Dysfunction in Primary Open-Angle Glaucoma Characterized by Flavoprotein Fluorescence at the Optic Nerve Head

To investigate the presence of flavoprotein fluorescence (FPF) at the optic nerve head (ONH) rim as a marker of mitochondrial dysfunction in primary open-angle glaucoma (POAG) and control eyes. Retrospective cross-sectional study of patients recruited from the New York Eye and Ear Infirmary of Mount Sinai. A total of 86 eyes (50 eyes of 30 patients with POAG and 36 eyes of 20 control participants) were enrolled. The presence of POAG was defined by circumpapillary retinal nerve fiber layer thickness (cpRNFLT) of less than the bottom fifth percentile of the normative database, glaucomatous ONH changes, and visual field defects on 24-2 tests. Primary open-angle glaucoma and control eyes were imaged using the OcuMet Beacon. A 23°× 23° infrared scan was obtained, and an FPF scan was performed within a capture field spanning 13° in diameter. The ONH margins on the infrared image were identified by software algorithms. Then, FPF was measured within an elliptical annulus around the ONH rim, with the inner and outer boundaries corresponding to 0.5 to 1.1 times the ONH rim size. Flavoprotein fluorescence at the OHN rim in POAG and control eyes. Differences in FPF between POAG and control eyes were characterized through mixed-effects logistic regression, adjusted for age and interocular pressure. Flavoprotein fluorescence was significantly higher in POAG versus control eyes, with a mean ± SD of 46.4 ± 27.9 versus 28.0 ± 11.7 (P < 0.001), respectively. Among POAG eyes, FPF showed correlation to visual field mean deviation (P < 0.001), visual field pattern standard deviation (P= 0.003), and cpRNFLT (P= 0.001) on linear mixed-effects models. Higher FPF in POAG versus control eyes suggests the presence of mitochondrial dysfunction at the ONH rim in eyes with glaucomatous damage. The degree of FPF corresponds to disease severity, as measured by visual field and nerve fiber layer thickness metrics. Thus, FPF may represent a metabolic indicator of disease status that reveals the extent of injury in glaucoma.

Mechanosensitive channel inhibition attenuates TGFβ2-induced actin cytoskeletal remodeling and reactivity in mouse optic nerve head astrocytes

Astrocytes within the optic nerve head undergo actin cytoskeletal rearrangement early in glaucoma, which coincides with astrocyte reactivity and extracellular matrix (ECM) deposition. Elevated transforming growth factor beta 2 (TGFβ2) levels within astrocytes have been described in glaucoma, and TGFβ signaling induces actin cytoskeletal remodeling and ECM deposition in many tissues. A key mechanism by which astrocytes sense and respond to external stimuli is via mechanosensitive ion channels. Here, we tested the hypothesis that inhibition of mechanosensitive channels will attenuate TGFβ2-mediated optic nerve head astrocyte actin cytoskeletal remodeling, reactivity, and ECM deposition. Primary optic nerve head astrocytes were isolated from C57BL/6J mice and cell purity was confirmed by immunostaining. Astrocytes were treated with vehicle control, TGFβ2 (5 ng/ml), GsMTx4 (a mechanosensitive channel inhibitor; 500 nM), or TGFβ2 (5 ng/ml) + GsMTx4 (500 nM) for 48 h. FITC-phalloidin staining was used to assess the formation of f-actin stress fibers and to quantify the presence of crosslinked actin networks (CLANs). Cell reactivity was determined by immunostaining and immunoblotting for GFAP. Levels of fibronectin and collagen IV deposition were also quantified. Primary optic nerve head astrocytes were positive for the astrocyte marker GFAP and negative for markers for microglia (F4/80) and oligodendrocytes (OSP1). Significantly increased %CLAN-positive cells were observed after 48-h treatment with TGFβ2 vs. control in a dose-dependent manner. Co-treatment with GsMTx4 significantly decreased %CLAN-positive cells vs. TGFβ2 treatment and the presence of f-actin stress fibers. TGFβ2 treatment significantly increased GFAP, fibronectin, and collagen IV levels, and GsMTx4 co-treatment ameliorated GFAP immunoreactivity. Our data suggest inhibition of mechanosensitive channel activity as a potential therapeutic strategy to modulate actin cytoskeletal remodeling within the optic nerve head in glaucoma.

Describing the Structural Phenotype of the Glaucomatous Optic Nerve Head Using Artificial Intelligence

To develop a novel deep-learning approach that can describe the structural phenotype of the glaucomatous optic nerve head (ONH) and can be used as a robust glaucoma diagnosis tool. Retrospective, deep-learning approach diagnosis study. We trained a deep-learning network to segment 3 neural-tissue and 4 connective-tissue layers of the ONH. The segmented optical coherence tomography images were then processed by a customized autoencoder network with an additional parallel branch for binary classification. The encoder part of the autoencoder reduced the segmented optical coherence tomography images into a low-dimensional latent space (LS), whereas the decoder and the classification branches reconstructed the images and classified them as glaucoma or nonglaucoma, respectively. We performed principal component analysis on the latent parameters and identified the principal components (PCs). Subsequently, the magnitude of each PC was altered in steps and reported how it impacted the morphology of the ONH. The image reconstruction quality and diagnostic accuracy increased with the size of the LS. With 54 parameters in the LS, the diagnostic accuracy was 92.0 ± 2.3% with a sensitivity of 90.0 ± 2.4% (at 95% specificity), and the corresponding Dice coefficient for the reconstructed images was 0.86 ± 0.04. By changing the magnitudes of PC in steps, we were able to reveal how the morphology of the ONH changes as one transitions from a "nonglaucoma" to a "glaucoma" condition. Our network was able to identify novel biomarkers of the ONH for glaucoma diagnosis. Specifically, the structural features identified by our algorithm were found to be related to clinical observations of glaucoma.

Large-scale machine-learning-based phenotyping significantly improves genomic discovery for optic nerve head morphology

SummaryGenome-wide association studies (GWASs) require accurate cohort phenotyping, but expert labeling can be costly, time intensive, and variable. Here, we develop a machine learning (ML) model to predict glaucomatous optic nerve head features from color fundus photographs. We used the model to predict vertical cup-to-disc ratio (VCDR), a diagnostic parameter and cardinal endophenotype for glaucoma, in 65,680 Europeans in the UK Biobank (UKB). A GWAS of ML-based VCDR identified 299 independent genome-wide significant (GWS; p ≤ 5 × 10−8) hits in 156 loci. The ML-based GWAS replicated 62 of 65 GWS loci from a recent VCDR GWAS in the UKB for which two ophthalmologists manually labeled images for 67,040 Europeans. The ML-based GWAS also identified 93 novel loci, significantly expanding our understanding of the genetic etiologies of glaucoma and VCDR. Pathway analyses support the biological significance of the novel hits to VCDR: select loci near genes involved in neuronal and synaptic biology or harboring variants are known to cause severe Mendelian ophthalmic disease. Finally, the ML-based GWAS results significantly improve polygenic prediction of VCDR and primary open-angle glaucoma in the independent EPIC-Norfolk cohort.