Translational Vision Science & Technology

AimThe ERG b-wave is primarily attributed to ON bipolar cell activity, while the contribution of the OFF pathway and the differential role of voltage-gated sodium (NaV) channels in these pathways remain unclear. This study investigated whether pharmacological modulation of NaV channels differentially alters ON and OFF cone bipolar cell responses and ERG b-wave amplitudes. MethodsDark- and light-adapted ERGs were recorded from rats across stimulus intensities spanning rod, mixed rod-cone, and cone pathways (1-1000 lux). ON and OFF cone bipolar cell pathways were pharmacologically isolated using intravitreal cis-PDA. NaV channel activity was modulated via intravitreal administration of lidocaine and lamotrigine (blockers) and veratridine (agonist). Changes in b-wave amplitudes were analysed to assess pathway-specific effects. ResultsBoth lidocaine and lamotrigine significantly globally reduced ERG b-wave across all stimulus intensities, confirming a role for NaV channels in bipolar cell signalling. Pathway isolation revealed differential effects: lidocaine predominantly suppressed ON pathway, whereas lamotrigine preferentially reduced OFF pathway responses. In contrast, veratridine enhanced both ON and OFF pathway activity. These findings indicate that NaV channel activity in ON and OFF cone bipolar cells can be independently and differentially modulated. ConclusionThe ERG b-wave reflects integrated contributions from both ON and OFF cone bipolar cells. Differential NaV channel modulation alters these pathways distinctly, highlighting sodium channel-mediated mechanisms as potential targets for physiologically relevant retinal stimulation strategies in degenerative retinal conditions.

PurposeTo describe the effect of myopic eye growth on the structure and distribution of astrocytes, vasculature and ganglion cell thickness, critical for inner retinal tissue homeostasis and survival. MethodsAstrocyte and capillary distribution, retinal nerve fiber (RNFL) and ganglion cell layer (GCL) thicknesses were assessed using immunochemistry and spectral domain optical coherence tomography on eleven retinas of juvenile common marmosets (Callithrix Jacchus), six of which were induced with lens-induced myopia (refraction, Rx: -7.01{+/-}1.8D). Five untreated age-matched juvenile marmoset retinas were used as controls (Rx: -0.74{+/-}0.4D). ResultsAs control marmoset eyes grew normally, there was an age-related increase in astrocyte numbers associated with RNFL thickening. Marmosets with induced myopia did not show this trend and, on the contrary, had reduced astrocyte numbers, increased positive GFAP immunopositive staining, thinner RNFL, lower peripheral capillary branching, and increased numbers of string vessels. ConclusionThe myopic changes in retinal astrocytes, vasculature, and ganglion cell layer thickness suggest a reorganization of the astrocyte and vascular templates during myopia development and progression. Whether these adaptations are beneficial or harmful to the retina remains to be investigated. Summary StatementThis article provides new information on how progressive myopia affects key elements of the retinal neurovascular unit.

This study characterizes a fluorescent Slc17a6-tdTomato neuronal reporter mouse line offering strong labeling in axons throughout the optic nerve, dendrites and soma in 99% of retinal ganglion cells (RGCs). The model facilitates neuronal assessment ex vivo with wholemounts quantified to show neurodegeneration following optic nerve crush or elevated IOP as related to glaucoma, in vitro with robust Ca2+ responses to P2X7 receptor stimulation in neuronal cultures, and in vivo using a confocal scanning laser ophthalmoscope (cSLO). While the tdTomato signal showed strong overlap with RGC markers, BRN3A and RBPMS, there was no cross-labeling of displaced amacrine cells in the ganglion cell layer. Controls indicated no impact of Slc17a6-tdTomato expression on light-dependent neuronal function, as determined with a microelectrode array (MEA), or on structure, as measured with optical coherence tomography (OCT). In summary, this novel neuronal reporter mouse model offers an effective means to increase the efficiency for real-time, specific visualization of retinal ganglion cells. It holds substantial promise for enhancing our understanding of RGC pathology in glaucoma and other diseases of the optic nerve, and could facilitate the screening of targeted therapeutic interventions for neurodegeneration. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/599589v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@13db8dforg.highwire.dtl.DTLVardef@1345f10org.highwire.dtl.DTLVardef@14d34e1org.highwire.dtl.DTLVardef@1cacb87_HPS_FORMAT_FIGEXP M_FIG Graphical abstract. Fluorescent identification of axons, dendrites and soma of neuronal retinal ganglion cells with a genetic marker as a tool for facilitating the study of neurodegeneration. Puttipong Sripinun, Wennan Lu, Sergei Nikonov, Suhani Patel, Sarah Hennessy, Claire H. Mitchell*. This study delves into a new mouse model, featuring a fluorescent Slc17a6-tdTomato neuronal reporter. This model effectively labels axons in the optic nerve, as well as dendrites and soma in 99% of retinal ganglion cells (RGCs). This allows for both in vitro and in vivo assessment of neurodegeneration, offering a practical tool for real-time, precise visualization of RGCs, with potential applications in various fields of neuroscience and neurology. Created by Biorender.com. C_FIG

PurposeTo investigate the repeatability of a combined Dual-Scheimpflug placido disc corneal topographer (Zeimer Galilei G4) with respect to keratometric indices used to monitor progression of keratoconus (KCN). MethodsPatients with KCN were prospectively enrolled. For each eye lacking history of corneal surgery, 5 measurements were taken in succession. Eyes in which 3 or more measurements could be obtained (defined by the devices 4 image quality metrics) were included in the analysis. The repeatability limits (RL) and interclass correlation coefficients (ICC) were calculated for various parameters. Results32 eyes from 25 patients met all image quality metrics, and 54 eyes from 38 patients met at least 3/4 criteria (all except the placido image quality metric). RLs for key parameters when 4/4 or [≥]3/4 image quality metrics were met included: 0.37 and 0.77 diopters (D) for steep simulated keratometry, 0.79 and 1.65 D for maximum keratometry, 13.80 and 13.88 degrees for astigmatism axis, 0.64 and 0.56 m for vertical coma magnitude, and 3.76 and 3.84 m for thinnest pachymetry, respectively. The ICCs for all parameters were excellent [above 0.87 except for spherical aberration (0.77)]. ConclusionsThe dual-Scheimpflug placido disc corneal topographer is highly repeatable in quantifying parameters used in monitoring KCN. Excellent placido images are difficult to capture in eyes with KCN, but when available, increase the reliability of the measurements. The RLs may be especially helpful in detecting progression in mild KCN when interventions such as corneal cross-linking or intrastromal corneal ring segments are most beneficial.

PurposeTo train and evaluate segmentation-free 3D convolutional neural network (3DCNN) models for estimating visual field (VF) from optical coherence tomography (OCT) images and to independently assess the longitudinal variability and progression detection capabilities of Humphrey Field Analyzer (HFA) measurements and OCT-based estimated VF (OCT-VF) in a diverse clinical population. DesignRetrospective multicenter study. Participants13,366 patients (24,313 eyes) underwent HFA tests (24-2, trimmed 30-2, or 10-2 test patterns) and macular OCT imaging at five ophthalmic institutions. The dataset included 129,007 paired OCT-VF data points representing various ocular conditions. MethodsWe trained segmentation-free 3DCNN models using comprehensive OCT datasets without disease-specific exclusions, employing 10-fold cross-validation to estimate VF thresholds and mean deviation (MD). Unlike previous studies, we independently assessed both OCT-VF and HFA measurements by creating separate longitudinal datasets with standardized measurement counts and observation periods for comparative analysis, enabling direct evaluation of clinical reliability. We analyzed absolute residual variability from regression lines using jackknife resampling, applied Bonferroni correction for multiple comparisons, and used Spearmans correlation for progression analysis. Main Outcome MeasuresOCT-VF and HFA VF agreement, residual variability, progression detection rates, and progression rate correlations. ResultsOCT-VF and HFA VF showed correlations (Pearsons r: 24-2 thresholds 0.863, MD 0.924; 10-2 thresholds 0.881, MD 0.939; all p < 0.001). OCT-VF demonstrated significantly lower residual variability than HFA for all parameters (OCT-VF vs. HFA: 0.58 vs. 1.12 dB for 24-2 MD; 0.70 vs. 1.12 dB for 10-2 MD; all p < 0.001). This advantage persisted across all test points (mean variability reduction: 60.4% for 24-2; 55.1% for 10-2), age groups, and most severity levels. OCT-VF identified more progression events (24-2 MD: 113% more, 10-2 MD: 48.6% more). MD slopes showed correlations between OCT-VF and HFA (Pearsons r: 24-2 MD 0.831, 10-2 MD 0.863; all p < 0.001). ConclusionsThe segmentation-free 3DCNN models objectively estimated VF from OCT images with significantly lower longitudinal variability than performance-dependent HFA measurements across diverse ocular conditions. The lower variability of OCT-VF enhances statistical power for progression detection, suggesting its clinical potential as a complementary tool derived from routine OCT data, decreasing measurement noise, and enabling more timely therapeutic interventions.

Glaucoma is a progressive optic neuropathy characterized by retinal ganglion cell loss, often leading to visual impairment before measurable deficits in visual acuity occur. Contrast sensitivity (CS) provides critical insight into visual function, particularly under low-contrast conditions. This study investigates the pattern of contrast sensitivity loss in glaucoma using a reaction time (RT) paradigm, which reflects the interval between stimulus presentation and response initiation. Forty subjects (20 glaucoma patients, 20 healthy controls) aged 16 to 40 were tested using the Contrast Sensitivity Test Suite software with three stimuli (number, blinking square, jumping square). Reaction time increased as contrast decreased, with glaucoma patients showing significantly longer RTs across all stimuli compared to controls (p < 0.01). Regression analyses indicated that dysfunction in slow sustained (magnocellular) channels primarily accounts for low-contrast sensitivity deficits in glaucoma. These findings support the use of contrast sensitivity and reaction time measurements as sensitive functional markers for early glaucomatous changes, even in individuals with normal visual acuity.

PurposeMultiple mouse lines using constitutive or inducible Cre recombinase expression have taken advantage of the early optic field expression of the Rax/Rx gene and its subsequent, progressive restriction to retinal progenitors, retinal pigmented epithelium and the optic stalk. Among these, the constitutive transgenic lines, Rx3-Cre and Rax-Cre BAC Tg, are currently used by vision researchers. Here we directly compare their prenatal ocular and extra-ocular Cre activities. MethodsRx3-Cre or Rax-Cre BAC transgenic male mice were mated to the Cre-dependent lineage tracer Ai9/tdTomato. The resulting live and fixed tissue expression for each line was evaluated at multiple stages of development, from the optic vesicle stage onward. Both whole embryo and immunolabeled cryosectioned material were digitally imaged. ResultsRx3-Cre recapitulates endogenous RAX protein expression at the optic vesicle and optic cup stages, but there is also ectopic Cre activity in periocular mesenchyme (POM), within PITX2- and PECAM-1-expressing subpopulations. We also show that ectopic Cre activity can include germline expression. Besides a small ectopic domain in developing limbs, the Rax-Cre BAC mouse driver fully recapitulates endogenous RAX expression in the embryonic and postnatal eye. ConclusionsWe provide a systematic analysis of two Rax-Cre drivers during embryonic and postnatal eye development that are very useful to recapitulate severe congenital eye diseases. Data presented here strongly support the inclusion of lineage reporting and evaluation of littermates lacking a Cre transgene, whenever conditional targeting strategies are used in studies of optic vesicle-derived tissues.

Handheld optical coherence tomography (OCT) systems have shown promises to provide detailed evaluation of the pediatric retina. It is less stressful for preterm infants compared to binocular indirect ophthalmoscopy and shows promises for screening for retinopathy of prematurity. There are currently only one commercially available non-contact handheld OCT systems, and no commercially available widefield handheld OCT systems. Here we report and evaluate the first use of the Theia Imaging Investigational handheld OCT system with widefield lens (T1-W, Theia Imaging, Durham, NC) in pediatric subjects. We compare the OCT en face view to handheld widefield or tabletop ultra-widefield fundus photographs and evaluate the ability to visualize the vitreoretinal interface, intraretinal, subretinal and choroidal features on cross-sectional OCT scans.

PurposeTo test the feasibility of simultaneous steady-state pattern electroretinogram (PERG) and intraocular pressure (IOP) measurements with an IOP sensor and to test a model for IOP manipulation during lateral decubitus positioning (LDP) and its impact on the PERG. DesignA prospective, observational study. Methods15 healthy controls and 15 treated glaucoma patients participated in the study. 8 patients had an intraocular IOP sensor (eyemate-IO(R), Implandata Ophthalmic Products GmbH) in the right eye (GLAIMP) and 7 had no sensor and with glaucoma in the left eye. (1) We tested the feasibility of simultaneous IOP and PERG recordings by comparing PERGs with and without simultaneous IOP-read out in GLAIMP. (2) All participants were positioned in the following order: sitting1 (S1), right LDP (LDR), sitting2 (S2), left LDP (LDL) and sitting3 (S3). For each position, PERG amplitudes and IOP were determined with rebound tonometry (Icare(R) TA01i) in all participants without the IOP sensor. ResultsElectromagnetic intrusions of IOP sensor readout onto steady-state PERG-recordings had, due to different frequency ranges, no relevant effect on PERG amplitudes. IOP and PERG measures were affected by LDP, e.g., IOP was increased during LDR vs S1 in the lower eyes of GLAIMP and controls (P < 0.001 and P < 0.05, respectively) and PERG amplitude was decreased (P < 0.05 and P < 0.01, respectively). ConclusionsDuring LDP, IOP and PERG measurements changed more in the lower eye. IOP changes induced by LDP may be a model for studying the interaction of IOP and ganglion cell function.

PurposeStandard automated perimetry (SAP) is the gold standard for functional assessment in glaucoma. SAP can be too demanding for some groups of patients. Continuous visual stimulus tracking (SONDA: Standardized Oculomotor and Neurological Disorders Assessment) simplifies the perimetric task to following a moving stimulus on a screen. In this study we evaluated the screening performance of SONDA-based eye movement perimetry (SONDA-EMP) in glaucoma. To explore generalizability, we evaluated an experimental setup (SONDA-Eyelink) and a clinic-ready version (SONDA-Neon). MethodsSONDA-Eyelink and SONDA-Neon measurements were performed in 100 cases with glaucoma (36, 36, and 28 with early, moderate, and severe glaucoma, respectively) and 100 age-similar controls. Participants monocularly tracked a moving stimulus (Goldmann size III) at 40% contrast (both setups) and 160% (SONDA-Eyelink). Eye movements were continuously recorded. Outcome was the agreement between gaze and stimulus position. We used previously collected glaucoma case-control data to build a continuous glaucoma screening score. This score was used for an ROC-analysis applied to the current, independently collected dataset. We predefined good screening performance as: at 95% specificity, a sensitivity of at least 50%, 90%, and 100% for early, moderate, and severe glaucoma, respectively. ResultsAt 95% specificity, the sensitivity of SONDA-Eyelink was 58, 94, and 100% at 40% contrast and 56, 97, and 100% at 160% contrast for early, moderate, and severe glaucoma, respectively. Sensitivity was 53, 94, and 100% for SONDA-Neon. ConclusionsSONDA-EMP is a novel, fast, and intuitive method to screen for visual function loss in glaucoma.

Purpose: To compare hypopyon detection using anterior segment optical coherence tomography (ASOCT) versus slit lamp examination (SLE) in microbial keratitis, and to evaluate intra-and inter-grader agreement for ASOCT hypopyon measurement. Methods: Two masked graders independently evaluated ASOCT images for hypopyon presence or absence in eyes with microbial keratitis, with disagreements resolved by consensus. A subset of hypopyon eyes underwent triplicate height measurement using two methods (endothelial length, vertical height). Cohen's kappa, intraclass correlation coefficients (ICC), sensitivity, and specificity were calculated comparing diagnostic performance of ASOCT versus SLE. Results: Inter-grader agreement for hypopyon detection on ASOCT was excellent (k=0.94; 95% CI 0.84-1.00) and intra-grader agreement was excellent (k=0.89-1.00). ASOCT detected hypopyon in 67.1% of eyes versus 57.0% by SLE (sensitivity 83.0%, specificity 96.2% using ASOCT as reference). Intra-grader reproducibility was excellent for both endothelial length and vertical height measurements (ICC 0.977-0.996). Inter-grader agreement was good for endothelial length (ICC 0.831) and vertical height (ICC 0.827), though a statistically significant inter-grader bias was identified for vertical height only (Wilcoxon p=0.008). Conclusions: ASOCT detected hypopyon with greater sensitivity than SLE and demonstrated excellent intra-grader and good inter-grader measurement reproducibility. Endothelial length showed slightly superior inter-grader concordance to vertical height measurement.

ObjectiveTo assess the feasibility of remotely training glaucoma patients to take a ten-session clustered virtual reality (VR) visual field test (VVP-10) at home, analyze results for test-retest variability, and assess correspondence with conventional perimetry. DesignCross-sectional study. Subjects21 subjects with glaucoma were enrolled and included in the feasibility assessment of remote training. 36 eyes were used for test-retest analysis and determination of concordance with Humphrey Visual Field (HVF) testing. MethodsSubjects were provided with a mobile VR headset containing the VVP-10 test software and trained remotely via video conferencing. Subjects were instructed to complete ten sessions over a 14-day period. Main Outcome MeasuresFeasibility was determined by the number of subjects who were able to independently complete VVP-10 over the 14-day period after one remote training session. Intraclass correlation coefficient (ICC) of average fraction seen across ten sessions and standard error (SE) for the mean were primary outcome measures for assessing test-retest variability. Correlation with HVF mean sensitivity (MS) across eyes, was a secondary outcome measure. Results20 subjects (95%) successfully completed the VVP-10 test series after one training session. ICC of VVP-10 was 0.95 (95% CI [0.92, 0.97]). Mean SE in units of fraction seen was 0.012. The Spearman correlations of VVP-10 average fraction seen versus HVF MS were 0.88 (95% CI [0.66, 0.99]) for moderate to advanced glaucoma eyes, and decreased to 0.68 (95% CI [0.29, 0.94]) when all eyes were included. ConclusionsRemote training of patients at home is feasible and subsequent remote clustered visual field testing using VVP-10 by patients on their own without any further interactions with caregivers or study staff was possible. At-home VVP-10 results demonstrated low test-retest variability. Future studies must be conducted to determine if VVP-10, taken at home as convenient for the patient, may be a viable supplement to provide equivalent or complementary results to that of standard in-clinic assessment of visual function in glaucoma.

PurposeAtoh7 is a transiently expressed developmental transcription factor that gives rise to the seven major retinal cell types. Despite this broad lineage, Atoh7 is only required for retinal ganglion cell (RGC) genesis and survival, even though a significant portion of RGCs are Atoh7 negative based on lineage tracing in mice, suggesting a cell nonautonomous role for Atoh7 in the genesis and survival of all RGCs. Atoh7 function is conserved in zebrafish, yet the full retinal lineage, including the RGC population, has remained unidentified. Therefore, we sought to determine the atoh7 retinal lineage in wild type and atoh7 mutant zebrafish retinas. MethodsWe generated atoh7:iCre transgenic zebrafish and in combination with the established ubi:Switch lineage trace permanently labeled cells that represent the atoh7 lineage. A combination of in vivo live imaging and immunohistochemical techniques were used to validate atoh7:iCre transgene expression and the atoh7 lineage in embryonic, larval, and adult retinas as well as the adult brain. ResultsThe atoh7:iCre;ubi:Switch transgene combination successfully recapitulated the onset of endogenous atoh7 expression and transgene fluorophores persisted into adulthood labeling the atoh7 lineage. Most notably, we determined 79% of total RGCs in the wild type retina come from atoh7+ progenitor cells, a greater number than reported in the mouse retina. In atoh7 mutant retinas, we confirmed a complete loss of RGCs and observed a statistically significant increase in the proportion of atoh7+/Pax6+ amacrine cells, as well as an increase in the total number of Prox1+ bipolar cells. Interestingly, we discovered atoh7+ cells located outside the eye in other areas of the central nervous system. ConclusionsThese data demonstrate the presence of atoh7 positive and negative retinal cell types in the zebrafish retina, including RGCs, highlighting the potential to study survival mechanisms of atoh7 negative RGCs and fate switch paradigms using zebrafish retinal development models.

The development of non-invasive live ocular imaging and electrophysiological test systems for rodent eyes provides new tools for not only averaged analysis of the entire retina but also the ability to see, test, and compare different subregions of the same retina. These new capabilities provide the possibility for more detailed examinations of local structural and functional relationships within a single eye and the ability to also follow changes longitudinally over time. We have developed protocols based around the Micron-III/IV retinal imaging camera system for combining fluorescent imaging of the neural retinal micro-vasculature by FA (fluorescein angiography), imaging of all neural retinal layers by SD-OCT (Spectral-Domain Ocular Coherence Tomography), and focal "spot" light-targeted electroretinography (Focal-ERG) to relate the local neurovascular unit structure to the inner (photoreceptor) and outer-retinal electrical response to light stimulation. For demonstration purposes we have used the popular mouse oxygen induced retinopathy (OIR) model, which causes radial central patches of retinal neuron loss mostly in zones away from and between the primary retinal arteries and veins. In this model, the loss of central microvasculature is induced developmentally in mouse litters exposed to 75% oxygen from age P7 to P11. Return to room air on P12, causes several days of retinal ischemia during which neurons, mostly of the inner retina, perish. Bipolar and ganglion cell death ends as neovascular growth revascularizes the central retina. This model provides for non-uniform retinal damage as well as gradual progression and resolution over time. The OIR model was used to generate regions of inner retinal neuron loss in B6.Cg-TgThy1-YFP mice. Using image-guided focal-ERG, the dark-adapted mixed rod-cone light response was compared using stimulation of small circular (0.27 mm diameter) target areas located in the central retinas of the same eyes (OIR and control). The same areas of the same retinas were followed over three ages after revascularization (P21, P28 and P42). ConclusionsCombined FA and SD-OCT imaging can provide local geographic specific information on retinal structural changes and be used to select different retinal areas within the same eye for testing of local light response. This analysis strategy can be employed for studies with rodent disease models that do not uniformly impact the entire retinal area. Combining these techniques would also be useful for testing gene and cell replacement therapies in retinal degeneration models where typically a small zone of the retina is treated. Both treated and untreated retinal zones within the eye can be followed non-invasively over many weeks. SUMMARYMouse models utilized for retinal disease research including retinal vascular models can display nonuniform changes over the entire retina. Damage or loss of retinal layers and retinal neurons due to hypoxia can impact some retinal areas while leaving adjacent regions unaltered. Combining vascular imaging by fluoresceine angiography, vascular imaging and retinal layer imaging by SD-OCT, and focal-ERG provides us with new tools to examine retinal structure-function relationships within a single retina.

BackgroundThe visual field (VF) test results of many eyes with glaucoma progress despite treatment. This suggests that some eyes are either untreated or that the management of intraocular pressure (IOP) does not influence the outcome. In this work, we explore whether future VF parameters can be predicted from a baseline optical coherence retinal nerve fibre layer (OCT-RNFL) scan using a deep learning model. MethodsThe model was developed using 1792 eyes from 1610 patients, and externally validated on 151 eyes from a second centre using the same Zeiss Cirrus machine and 281 eyes from a third centre using scans obtained from a different (Heidelberg Spectralis) machine. The Vision Transformers (ViT)-based regression model was trained on baseline OCT-RNFL scans to predict three key VF indices (follow-up interval: 4.74 {+/-} 2.59 years). Model performance was evaluated using Mean Absolute Error (MAE) and Root Mean Square Error (RMSE), with 95% confidence intervals (CI). ResultsThe model achieved an overall MAE of 2.07 (95% CI: 1.91-2.22) and RMSE of 2.87 (95% CI: 2.60-3.14) on the internal validation set. On external validation, the model showed comparable performance with an MAE of 2.07 (95% CI: 1.8-2.35) for the external validation (Zeiss OCT) cohort and 2.11 (95% CI: 1.93-2.31) for the external validation (Heidelberg OCT) cohort. Saliency maps revealed that the inner and outer RNFL layers were key structures in driving the models predictions. ConclusionsOur ViT-based regression model effectively predicts key VF indices objectively from a single OCT-RNFL scan, with strong performance across two OCT devices, offering a novel tool for predicting glaucoma progression.

PurposeDespite popularity of optical coherence tomography (OCT) in glaucoma studies, its unclear how well OCT-derived metrics compare to traditional measures of retinal ganglion cell (RGC) abundance. Here, Diversity Outbred (J:DO) mice are used to directly compare ganglion cell complex (GCC)-thickness measured by OCT to metrics of retinal anatomy measured ex vivo with retinal wholemounts or optic nerve cross sections. MethodsJ:DO mice (n = 48) underwent OCT and fundoscopic exams, with GCC-thickness measured using automated segmentation. Following euthanasia, RGC axons were quantified from para-phenylenediamine-stained optic nerve cross sections and RGC somas from BRN3A-immunolabeled retinal wholemounts with total cellularity assessed by TO-PRO or hematoxylin nuclear staining. ResultsJ:DO tissues lacked overt disease. GCC-thickness (62.4 {+/-} 3.7 {micro}m), RGC abundance (3,097 {+/-} 515 BRN3A+ nuclei/mm2; 45,533 {+/-} 9,077 axons), and total inner retinal cell abundance (6,952 {+/-} 810 nuclei/mm2) varied broadly. GCC-thickness correlated significantly to RGC somal density (r = 0.46) and axon number (r = 0.49), whereas total inner retinal cellularity did not. Retinal area (20.3 {+/-} 2.4 mm2) and optic nerve (0.09 {+/-} 0.02 mm2) cross-sectional area varied widely. Sex did not significantly influence any of these metrics. In bilateral comparisons, GCC-thickness (r = 0.89), inner retinal cellularity (r = 0.47), and RGC axon abundance (r = 0.72) all correlated significantly. ConclusionsAmongst outbred mice with widely variable phenotypes, OCT-derived measurements of GCC thickness correlate significantly to RGC abundance and axon number. The extensive phenotypic variability exhibited by J:DO mice make them a powerful resource for studies of retinal anatomy using quantitative genetics.

ObjectiveTo examine how binocularly asymmetric glaucomatous visual field damage affects processing of binocular disparity across the visual field. DesignCase-control study. Participants and ControlsA sample of 18 patients with primary open-angle glaucoma, 16 age-matched controls, and 13 young controls. MethodsParticipants underwent standard clinical assessments of binocular visual acuity, binocular contrast sensitivity, stereoacuity, and perimetry. We employed a previously validated psychophysical procedure to measure how sensitivity to binocular disparity varied across spatial frequencies and visual field sectors, i.e. with full-field stimuli spanning the central 21{degrees} of the visual field, and with stimuli restricted to annular regions spanning 0{degrees}-3{degrees}, 3{degrees}-9{degrees} or 9{degrees}-21{degrees}. Main Outcome MeasuresWe verified the presence of binocularly asymmetric glaucomatous visual field damage by comparing--between the two eyes-- the mean deviation values obtained from the Humphrey Field Analyzer (HFA) 24-2 test. To assess the spatial-frequency tuning of disparity sensitivity across the visual field of patients and controls, we fit disparity sensitivity data to log-parabola models and compared fitted model parameters. Lastly, we employed disparity sensitivity measurements from restricted visual field conditions to model different possible scenarios regarding how disparity information is combined across visual field sectors. We adjudicated between the potential mechanisms by comparing model predictions to the observed patterns of disparity sensitivity with full-field stimuli. ResultsThe interocular difference in HFA 24-2 mean deviation was greater in glaucoma patients compared to both young and age-matched controls (ps=.01). Across participant groups foveal regions preferentially processed disparities at finer spatial scales, whereas periphery regions were tuned for coarser scales (p<.001). Disparity sensitivity also decreased from the fovea to the periphery (p<.001) and across participant groups (ps<.01). Finally, similar to controls, glaucoma patients exhibited near-optimal disparity integration, specifically at low spatial frequencies (p<.001). ConclusionsContrary to the conventional view that glaucoma spares central vision, we find that glaucomatous damage causes a widespread loss of disparity sensitivity across both foveal and peripheral regions. Despite these losses, cortical integration mechanisms appear to be well preserved, suggesting that glaucoma patients make the best possible use of their remaining binocular function.

The retinal pigment epithelium (RPE) is critical for maintaining outer retinal barrier homeostasis. In age-related macular degeneration (AMD), the RPE can undergo a dedifferentiation process that includes tight junction (TJ) loss and displacement of zonula occludens-1 (ZO-1), which may impair structural and functional integrity of the RPE barrier and contribute to disease pathogenesis. Our objective was to develop an automated and sensitive quantification method for TJ aberrations in an RPE immunofluorescence imaging assay, following treatment with TNF or TGF{beta}2. However, quantifying ZO-1 morphological changes in the RPE using standard image analysis methods did not provide a satisfactory assay window. To address this challenge, we developed an imaging assay to quantify ZO-1 changes using a machine learning approach, enabling enhanced phenotypic characterization of the ZO-1 changes in RPE cells and improved assay sensitivity. We were also able to capture and quantify the reversal of these changes using etanercept, an TNF inhibitor, with this imaging assay. Our findings indicated that this machine learning ZO-1 quantification assay could serve as a potential phenotypic readout for RPE dedifferentiation and enabling large-scale mechanistic studies.

PurposeTo determine the repeatability of a clinical pupillometer in healthy participants with a 5 and 30-minute test-retest break for both Swinging-Flashlight and Low-High Luminance approach. Methods20 healthy participants (mean age: 27 years {+/-}5; 9 females) placed their heads into the devices headrest and fixated a central spot. A Swinging-Flashlight approach evoked a pupillary light reflex, stimulating alternatingly 8 times each eye by a brief diffuse white light flash followed by a continuous measurement of constriction and dilation of the direct and consensual pupils. For the Low-High Luminance setting, continuous measurements of pupil diameters for a 5 second low luminance display followed by a 5 second high luminance white light. Pupillary light reflex parameters for each test and each eye were calculated by the device. Repeatability was investigated after a 5-minute break time and during a control experiment for 21 participants after a 30-minute break for each approach in counterbalanced order using Bland-Altman analysis. ResultsOverall, many parameters for both Swinging-Flashlight and Low-High Luminance approach showed retest biases for all pupil light reflex parameters after a 5-minute test-retest break. These biases were almost completely reduced after a 30-minute break between test and retest for both approaches. The test-retest variabilities as expressed using the Coefficient of Repeatability was reduced after 30-minutes for the majority of the Low-High Luminance results but not for results of the Swinging-Flashlight method. ConclusionsClinical pupillometry includes the Swinging Flashlight Test (SFL) and Low-High Luminance assays. In SFL, many pupillary dynamic metrics showed test-retest bias at a 5-minute interval, whereas relative afferent pupillary defect (RAPD) measurements remained unbiased at both 5 and 30-minute intervals. Similarly, Low-High Luminance testing showed minimal bias after 30 minutes but exhibited bias at the 5-minute interval. Thus, when evaluating multiple pupillary parameters in scientific or clinical settings, RAPD is less susceptible to bias, while other metrics require longer intervals between testing and retesting.

PurposeTo develop and test a deep learning (DL) algorithm for detecting referable glaucoma in the Los Angeles County (LAC) Department of Health Services (DHS) teleretinal screening program. MethodsFundus photographs and patient-level labels of referable glaucoma (defined as cup-to-disc ratio [CDR] [&ge;] 0.6) provided by 21 trained optometrist graders were obtained from the LAC DHS teleretinal screening program. A DL algorithm based on the VGG-19 architecture was trained using patient-level labels generalized to images from both eyes. Area under the receiver operating curve (AUC), sensitivity, and specificity were calculated to assess algorithm performance using an independent test set that was also graded by 13 clinicians with one to 15 years of experience. Algorithm performance was tested using reference labels provided by either LAC DHS optometrists or an expert panel of 3 glaucoma specialists. Results12,098 images from 5,616 patients (2,086 referable glaucoma, 3,530 non-glaucoma) were used to train the DL algorithm. In this dataset, mean age was 56.8 {+/-} 10.5 years with 54.8% females and 68.2% Latinos, 8.9% Blacks, 2.7% Caucasians, and 6.0% Asians. 1,000 images from 500 patients (250 referable glaucoma, 250 non-glaucoma) with similar demographics (p [&ge;] 0.57) were used to test the DL algorithm. Algorithm performance matched or exceeded that of all independent clinician graders in detecting patient-level referable glaucoma based on LAC DHS optometrist (AUC = 0.92) or expert panel (AUC = 0.93) reference labels. Clinician grader sensitivity (range: 0.33-0.99) and specificity (range: 0.68-0.98) ranged widely and did not correlate with years of experience (p [&ge;] 0.49). Algorithm performance (AUC = 0.93) also matched or exceeded the sensitivity (range: 0.78-1.00) and specificity (range: 0.32-0.87) of 6 LAC DHS optometrists in the subsets of the test dataset they graded based on expert panel reference labels. ConclusionsA DL algorithm for detecting referable glaucoma developed using patient-level data provided by trained LAC DHS optometrists approximates or exceeds performance by ophthalmologists and optometrists, who exhibit variable sensitivity and specificity unrelated to experience level. Implementation of this algorithm in screening workflows could help reallocate eye care resources and provide more reproducible and timely glaucoma care.