Sample multiplexing for retinal single-cell RNA-sequencing.

Rare cell populations can be challenging to characterize using microfluidic single-cell RNA sequencing (scRNA-seq) platforms. Typically, the population of interest must be enriched and pooled from multiple biological specimens for efficient collection. However, these practices preclude the resolution of sample origin together with phenotypic data and are problematic in experiments in which biological or technical variation is expected to be high (e.g., disease models, genetic perturbation screens, or human samples). One solution is sample multiplexing whereby each sample is tagged with a unique sequence barcode that is resolved bioinformatically. We have established a scRNA-seq sample multiplexing pipeline for mouse retinal ganglion cells using cholesterol-modified-oligos and utilized the enhanced precision to investigate cell type distribution and transcriptomic variance across retinal samples. As single cell transcriptomics are becoming more widely used to research development and disease, sample multiplexing represents a useful method to enhance the precision of scRNA-seq analysis.

Comprehensive single-cell atlas of the mouse retina.

Single-cell RNA sequencing (scRNA-seq) has advanced our understanding of cellular heterogeneity by characterizing cell types across tissues and species. While several mouse retinal scRNA-seq datasets exist, each dataset is either limited in cell numbers or focused on specific cell classes, thereby hindering comprehensive gene expression analysis across all retina types. To fill the gap, we generated the largest retinal scRNA-seq dataset to date, comprising approximately 190,000 single cells from C57BL/6J mouse retinas, enriched for rare population cells via antibody-based magnetic cell sorting. Integrating this dataset with public datasets, we constructed the Mouse Retina Cell Atlas (MRCA) for wild-type mice, encompassing over 330,000 cells, characterizing 12 major classes and 138 cell types. The MRCA consolidates existing knowledge, identifies new cell types, and is publicly accessible via CELLxGENE, UCSC Cell Browser, and the Broad Single Cell Portal, providing a user-friendly resource for the mouse retina research community.

Comprehensive single-cell atlas of the mouse retina.

Single-cell RNA sequencing (scRNA-seq) has advanced our understanding of cellular heterogeneity at the single-cell resolution by classifying and characterizing cell types in multiple tissues and species. While several mouse retinal scRNA-seq reference datasets have been published, each dataset either has a relatively small number of cells or is focused on specific cell classes, and thus is suboptimal for assessing gene expression patterns across all retina types at the same time. To establish a unified and comprehensive reference for the mouse retina, we first generated the largest retinal scRNA-seq dataset to date, comprising approximately 190,000 single cells from C57BL/6J mouse whole retinas. This dataset was generated through the targeted enrichment of rare population cells via antibody-based magnetic cell sorting. By integrating this new dataset with public datasets, we conducted an integrated analysis to construct the Mouse Retina Cell Atlas (MRCA) for wild-type mice, which encompasses over 330,000 single cells. The MRCA characterizes 12 major classes and 138 cell types. It captured consensus cell type characterization from public datasets and identified additional new cell types. To facilitate the public use of the MRCA, we have deposited it in CELLxGENE, UCSC Cell Browser, and the Broad Single Cell Portal for visualization and gene expression exploration. The comprehensive MRCA serves as an easy-to-use, one-stop data resource for the mouse retina communities.

Intraocular Pressure and Eyedrop Usage Reduction with Intracameral Bimatoprost Implant.

Purpose: Sustained intraocular drug delivery devices are being developed to lower intraocular pressure (IOP) and improve adherence in patients with glaucoma. The purpose of this study was to assess the IOP and eyedrop usage reduction effects of intracameral bimatoprost implants. Methods: We retrospectively reviewed the records of 46 eyes from 38 patients who received an intracameral implant containing 10 µg of bimatoprost as a replacement or addition to their existing eyedrop regimen and investigated IOP, eyedrop usage, and adverse effects. Results: Patients were followed for an average of 274 ± 104 (mean ± standard deviation) days after implant. Mean reduction in IOP (mmHg) at 3 months ±30 days, 6 months ±60 days, and 12 months ±90 days postoperation compared to baseline was 1.26 ± 2.53 (P = 0.002), 0.93 ± 4.71 (P = 0.098), and 1.35 ± 5.24 (P = 0.053), respectively. Reduction in eyedrops at 3 months ±30 days, 6 months ±60 days, and 12 months ±90 days postoperation compared to baseline were 0.62 ± 0.49 (P < 0.001), 0.55 ± 0.73 (P < 0.001), and 0.51 ± 0.71 (P < 0.001), respectively. Fifteen eyes (32.6%) experienced implant failure, defined as either restarting IOP-lowering eyedrops or undergoing surgical intervention, at an average of 260 ± 122 days after implant. Conclusions: While some patients eventually experienced implant failure, intracameral bimatoprost implants may result in fewer adverse reactions and successfully lower IOP and eyedrop burden over a longer period than previously reported.

Evaluation of retinal nerve fibre layer thickness as a possible measure of diabetic retinal neurodegeneration in the EPIC-Norfolk Eye Study.

BACKGROUND/AIMS: Markers to clinically evaluate structural changes from diabetic retinal neurodegeneration (DRN) have not yet been established. To study the potential role of peripapillary retinal nerve fibre layer (pRNFL) thickness as a marker for DRN, we evaluated the relationship between diabetes, as well as glycaemic control irrespective of diabetes status and pRNFL thickness. METHODS: Leveraging data from a population-based cohort, we used general linear mixed models (GLMMs) with a random intercept for patient and eye to assess the association between pRNFL thickness (measured using GDx) and demographic, systemic and ocular parameters after adjusting for typical scan score. GLMMs were also used to determine: (1) the relationship between: (A) glycated haemoglobin (HbA1c) irrespective of diabetes diagnosis and pRNFL thickness, (B) diabetes and pRNFL thickness and (2) which quadrants of pRNFL may be affected in participants with diabetes and in relation to HbA1c. RESULTS: 7076 participants were included. After controlling for covariates, inferior pRNFL thickness was 0.94 µm lower (95% CI -1.28 µm to -0.60 µm), superior pRNFL thickness was 0.83 µm lower (95% CI -1.17 µm to -0.49 µm) and temporal pRNFL thickness was 1.33 µm higher (95% CI 0.99 µm to 1.67 µm) per unit increase in HbA1c. Nasal pRNFL thickness was not significantly associated with HbA1c (p=0.23). Similar trends were noted when diabetes was used as the predictor. CONCLUSION: Superior and inferior pRNFL was significantly thinner among those with higher HbA1c levels and/or diabetes, representing areas of the pRNFL that may be most affected by diabetes.

Selective vulnerability of the intermediate retinal capillary plexus precedes retinal ganglion cell loss in ocular hypertension.

Introduction: Glaucoma, a disease of retinal ganglion cell (RGC) injury and potentially devastating vision loss, is associated with both ocular hypertension (OHT) and reduced ocular blood flow. However, the relationship between OHT and retinal capillary architecture is not well understood. In this project, we studied microvasculature damage in mice exposed to mild levels of induced OHT. Methods: Mild OHT was induced with the microbead model for 2 weeks. At this time point, some retinas were immunostained with CD31 (endothelium), Collagen IV (basement membrane), and RBPMS (RGCs) for z-stack confocal microscopy. We processed these confocal images to distinguish the three retinal capillary plexi (superficial, intermediate, and deep). We manually counted RGC density, analyzed vascular complexity, and identified topographical and spatial vascular features of the retinal capillaries using a combination of novel manual and automated workflows. Other retinas were dissociated and immunopanned to isolate RGCs and amacrine cells (ACs) for hypoxia gene array analysis. Results: RGC counts were normal but there was decreased overall retinal capillary complexity. This reduced complexity could be explained by abnormalities in the intermediate retinal capillary plexus (IRCP) that spared the other plexi. Capillary junction density, vessel length, and vascular area were all significantly reduced, and the number of acellular capillaries was dramatically increased. ACs, which share a neurovascular unit (NVU) with the IRCP, displayed a marked increase in the relative expression of many hypoxia-related genes compared to RGCs from the same preparations. Discussion: We have discovered a rapidly occurring, IRCP-specific, OHT-induced vascular phenotype that precedes RGC loss. AC/IRCP NVU dysfunction may be a mechanistic link for early vascular remodeling in glaucoma.

Total retinal thickness: a neglected factor in the evaluation of inner retinal thickness.

AIM: To determine whether macular retinal nerve fibre layer (mRNFL) and ganglion cell-inner plexiform layer (GC-IPL) thicknesses vary by ethnicity after accounting for total retinal thickness. METHODS: We included healthy participants from the UK Biobank cohort who underwent macula-centred spectral domain-optical coherence tomography scans. mRNFL and GC-IPL thicknesses were determined for groups from different self-reported ethnic backgrounds. Multivariable regression models adjusting for covariables including age, gender, ethnicity and refractive error were built, with and without adjusting for total retinal thickness. RESULTS: 20237 participants were analysed. Prior to accounting for total retinal thickness, mRNFL thickness was on average 0.9 µm (-1.2, -0.6; p<0.001) lower among Asians and 1.5 µm (-2.3, -0.6; p<0.001) lower among black participants compared with white participants. Prior to accounting for total retinal thickness, the average GC-IPL thickness was 1.9 µm (-2.5, -1.4; p<0.001) lower among Asians compared with white participants, and 2.4 µm (-3.9, -1.0; p=0.001) lower among black participants compared with white participants. After accounting for total retinal thickness, the layer thicknesses were not significantly different among ethnic groups. When considered as a proportion of total retinal thickness, mRNFL thickness was ~0.1 and GC-IPL thickness was ~0.2 across age, gender and ethnic groups. CONCLUSIONS: The previously reported ethnic differences in layer thickness among groups are likely driven by differences in total retinal thickness. Our results suggest using layer thickness ratio (retinal layer thicknesses/total retinal thickness) rather than absolute thickness values when comparing retinal layer thicknesses across groups.

Abnormal perception of pattern-induced flicker colors in subjects with glaucoma.

Pattern-induced flicker colors (PIFCs) are subjective colors that can be elicited with rotation of an achromatic stimulus such as the Benham disk. The perceptive mechanisms underlying PIFCs are not well-understood, but are thought to be generated primarily by retinal cell types which may be dysfunctional in glaucoma. Using a custom computer-based system, we tested PIFC perception across several Benham disk parameters, including the rates of acceleration and deceleration, rotational direction, and image contrast in both control and glaucoma subjects. We defined the Benham perception limit (BPL) during acceleration as the rotational speed at which PIFCs were first detected (Benham perception limit for acceleration) and the BPL during deceleration as the rotational speed at which PIFCs were extinguished (Benham perception limit for deceleration). In general, we found that glaucoma subjects perceived PIFCs less frequently than control subjects. For all subjects, we found that slower rates of acceleration and deceleration resulted in a lower Benham perception limit for acceleration and a higher Benham perception limit for deceleration, suggesting that PIFCs were both more easily detected and extinguished. Finally, subjects with glaucoma required increased rotational speeds during acceleration to detect PIFCs under certain conditions. Further study is needed to determine if these findings can be used to enhance clinical detection strategies.

Cerebrospinal fluid and ophthalmic disease.

PURPOSE OF REVIEW: The purpose of this review is to discuss the contemporary body of literature examining the relationship between cerebrospinal fluid (CSF) and ophthalmic disease. This review focuses on diseases that have a pathogenesis related to the translaminar pressure difference, defined as the pressure difference between the orbital subarachnoid space (OSAS) and the intraocular pressure. The diseases discussed include glaucoma, idiopathic intracranial hypertension, and spaceflight associated neuro-ocular syndrome. RECENT FINDINGS: The relationship between cerebrospinal and ophthalmic disease has been investigated for over 100 years. Recent research provides insight into the mechanisms that dictate CSF circulation in the OSAS and how alterations in these mechanism lead to disease. This review discusses these recent findings and their relationship to major ophthalmic diseases. SUMMARY: The recent findings provide insight into diseases that have pathogenic mechanisms that are not fully understood. This information will help physicians gain a clearer understanding of the relationship between CSF and ophthalmic disease and guide future research.

Detecting retinal neurodegeneration in people with diabetes: Findings from the UK Biobank.

IMPORTANCE: Efforts are underway to incorporate retinal neurodegeneration in the diabetic retinopathy severity scale. However, there is no established measure to quantify diabetic retinal neurodegeneration (DRN). OBJECTIVE: We compared total retinal, macular retinal nerve fiber layer (mRNFL) and ganglion cell-inner plexiform layer (GC-IPL) thickness among participants with and without diabetes (DM) in a population-based cohort. DESIGN/SETTING/PARTICIPANTS: Cross-sectional analysis, using the UK Biobank data resource. Separate general linear mixed models (GLMM) were created using DM and glycated hemoglobin as predictor variables for retinal thickness. Sub-analyses included comparing thickness measurements for patients with no/mild diabetic retinopathy (DR) and evaluating factors associated with retinal thickness in participants with and without diabetes. Factors found to be significantly associated with DM or thickness were included in a multiple GLMM. EXPOSURE: Diagnosis of DM was determined via self-report of diagnosis, medication use, DM-related complications or glycated hemoglobin level of ≥ 6.5%. MAIN OUTCOMES AND MEASURES: Total retinal, mRNFL and GC-IPL thickness. RESULTS: 74,422 participants (69,985 with no DM; 4,437 with DM) were included. Median age was 59 years, 46% were men and 92% were white. Participants with DM had lower total retinal thickness (-4.57 µm, 95% CI: -5.00, -4.14; p<0.001), GC-IPL thickness (-1.73 µm, 95% CI: -1.86, -1.59; p<0.001) and mRNFL thickness (-0.68 µm, 95% CI: -0.81, -0.54; p<0.001) compared to those without DM. After adjusting for co-variates, in the GLMM, total retinal thickness was 1.99 um lower (95% CI: -2.47, -1.50; p<0.001) and GC-IPL was 1.02 µm lower (95% CI: -1.18, -0.87; p<0.001) among those with DM compared to without. mRNFL was no longer significantly different (p = 0.369). GC-IPL remained significantly lower, after adjusting for co-variates, among those with DM compared to those without DM when including only participants with no/mild DR (-0.80 µm, 95% CI: -0.98, -0.62; p<0.001). Total retinal thickness decreased 0.40 µm (95% CI: -0.61, -0.20; p<0.001), mRNFL thickness increased 0.20 µm (95% CI: 0.14, 0.27; p<0.001) and GC-IPL decreased 0.26 µm (95% CI: -0.33, -0.20; p<0.001) per unit increase in A1c after adjusting for co-variates. Among participants with diabetes, age, DR grade, ethnicity, body mass index, glaucoma, spherical equivalent, and visual acuity were significantly associated with GC-IPL thickness. CONCLUSION: GC-IPL was thinner among participants with DM, compared to without DM. This difference persisted after adjusting for confounding variables and when considering only those with no/mild DR. This confirms that GC-IPL thinning occurs early in DM and can serve as a useful marker of DRN.

Gout and open-angle glaucoma risk in a veteran population.

PURPOSE: A history of gout, arthritis due to hyperuricemia, has been associated with decreased risk for neurodegenerative diseases such as Parkinson's disease. We performed a population-based case-control study in the US Department of Veterans Affairs (VA) medical centers nationwide to assess if gout or hyperuricemia is similarly associated with the ocular neurodegenerative condition glaucoma. METHODS: We used ICD-9 codes to identify a nationwide cohort of patients examined at VA healthcare eye clinics between 2000 and 2015 with a diagnosis of open-angle glaucoma (OAG) or of glaucoma suspect. We used incidence density matching to choose controls. We used multivariable logistic regression to examine associations between a history of gout and uric acid (UA) levels on relative risk of OAG or glaucoma suspect. RESULTS: There were 1,144,428 OAG or glaucoma suspect cases and 1,144,428 matched controls. Veterans with a history of gout had a small significant decreased risk of OAG compared to controls (ORadjusted(adj) = 0.985, 95% CI: 0.974-0.996). Treated gout was similarly associated with small decreased risk (ORadj = 0.963, 95% CI: 0.950-0.976). A small subset of patients (11.9% of cases and 13.2% of controls) had UA labs available; veterans with the highest median UA levels (> 7.29 mg/dL) did not have statistically significant differences in relative OAG risk (ORadj = 1.014, 95% CI: 0.991-1.036). CONCLUSION: Prospective research in other cohorts is needed to confirm our findings in veterans suggesting a history of gout is associated with a small decreased relative risk of glaucoma.

Optimized culture of retinal ganglion cells and amacrine cells from adult mice.

Cell culture is widely utilized to study the cellular and molecular biology of different neuronal cell populations. Current techniques to study enriched neurons in vitro are primarily limited to embryonic/neonatal animals and induced pluripotent stem cells (iPSCs). Although the use of these cultures is valuable, the accessibility of purified primary adult neuronal cultures would allow for improved assessment of certain neurological diseases and pathways at the cellular level. Using a modified 7-step immunopanning technique to isolate for retinal ganglion cells (RGCs) and amacrine cells (ACs) from adult mouse retinas, we have successfully developed a model of neuronal culture that maintains for at least one week. Isolations of Thy1.2+ cells are enriched for RGCs, with the isolation cell yield being congruent to the theoretical yield of RGCs in a mouse retina. ACs of two different populations (CD15+ and CD57+) can also be isolated. The populations of these three adult neurons in culture are healthy, with neurite outgrowths in some cases greater than 500µm in length. Optimization of culture conditions for RGCs and CD15+ cells revealed that neuronal survival and the likelihood of neurite outgrowth respond inversely to different culture media. Serially diluted concentrations of puromycin decreased cultured adult RGCs in a dose-dependent manner, demonstrating the potential usefulness of these adult neuronal cultures in screening assays. This novel culture system can be used to model in vivo neuronal behaviors. Studies can now be expanded in conjunction with other methodologies to study the neurobiology of function, aging, and diseases.

Intraocular Pressure Elevation Compromises Retinal Ganglion Cell Light Adaptation.

Purpose: Functional adaptation to ambient light is a key characteristic of retinal ganglion cells (RGCs), but little is known about how adaptation is affected by factors that are harmful to RGC health. We explored adaptation-induced changes to RGC physiology when exposed to increased intraocular pressure (IOP), a major risk factor for glaucoma. Methods: Wild-type mice of both sexes were subjected to 2 weeks of IOP elevation using the bead model. Retinas were assessed using a multielectrode array to record RGC responses to checkerboard white noise stimulation under both scotopic and photopic light levels. This information was used to calculate a spike-triggered average (STA) for each RGC with which to compare between lighting levels. Results: Low but not high IOP elevation resulted in several distinct RGC functional changes: (1) diminished adaptation-dependent receptive field (RF) center-surround interactions; (2) increased likelihood of a scotopic STA; and (3) increased spontaneous firing rate. Center RF size change with lighting level varied among RGCs, and both the center and surround STA peak times were consistently increased under scotopic illumination, although none of these properties were impacted by IOP level. Conclusions: These findings provide novel evidence that RGCs exhibit reduced light-dependent adaptation and increased excitability when IOP is elevated to low but not high levels. These results may reveal functional changes that occur early in glaucoma, which can potentially be used to identify patients with glaucoma at earlier stages when intervention is most beneficial.

Single transient intraocular pressure elevations cause prolonged retinal ganglion cell dysfunction and retinal capillary abnormalities in mice.

Transient intraocular pressure (IOP) elevations are likely to occur in certain forms of glaucoma and after intravitreal injections to treat various retinal diseases. However, the impact of these transient IOP elevations on the physiology of individual retinal ganglion cells (RGCs) is unknown. In this report, we explore how transient IOP elevations in mice affect RGC physiology, RGC anatomy, and retinal arteriole and capillary structure. Transient IOP elevation was induced in 12-week old wild type C57BL6J mice by injecting sodium hyaluronate into the anterior chamber. IOP was measured immediately after the injection and again 1 and 7 days later. Average peak IOP after injection was ~50 mmHg and subsequent IOPs returned to normal. RGC physiology was assessed with a multielectrode array (MEA) by calculating a spike triggered average (STA) at the same time points. RGC counts and retinal vascular structure were assessed 14 days after injection with immunohistochemistry to label RGCs and blood vessels. Transient IOP elevation caused a marked reduction of scotopic STA presence and delayed center and surround STA peak times that did not recover. Transient IOP elevation also caused a reduced photopic receptive field size and spontaneous firing rate, both of which showed some recovery with time. Transient IOP elevation also induced vascular remodeling: the number of capillary branches was decreased within the superficial and intermediate vascular plexi. RGC counts, retinal arteriole diameter, and deep capillary plexus branching were unaffected. These previously unappreciated findings suggest that transient IOP elevation may cause unrecognized and potentially long-term pathology to RGCs and associated neurovascular units which should be accounted for in clinical practice.

Modeling a potential SANS countermeasure by experimental manipulation of the translaminar pressure difference in mice.

The spaceflight-associated neuro-ocular syndrome (SANS), which may present after prolonged exposure to microgravity, is thought to occur due to elevated intracranial pressure (ICP). Intracranial pressure interacts with intraocular pressure (IOP) to define the translaminar pressure difference (TLPD; IOP-ICP). We combined inducible models of ICP and IOP elevation in mice to interrogate the relationships among ICP, IOP, and TLPD, and to determine if IOP elevation could mitigate the phenotypes typically caused by elevated ICP and thereby serve as a countermeasure for SANS. Ten C57BL6J mice of both genders underwent experimental elevation of ICP via infusion of artificial cerebrospinal fluid into the subarachnoid space. One eye also underwent experimental elevation of IOP using the bead injection model. Intraocular pressure and ICP were monitored for 2 weeks. Optokinetic-based contrast sensitivity was measured at baseline and after 2 weeks, and post-mortem studies of optic nerve and retina anatomy were performed. Photopic contrast sensitivity was reduced more in IOP elevated than control eyes. Scotopic contrast sensitivity was reduced similarly in IOP elevated and control eyes. However, the pattern of scotopic vision loss was not uniform in IOP elevated eyes; there was minimal loss in eyes that most closely approximated the normal TLPD. Optic nerve axon loss, increased optic nerve disorganization, and retinal ganglion cell loss all occurred similarly between IOP elevated and control eyes. Elevation of IOP in eyes with elevated ICP may counterbalance some effects on vision loss but exacerbate others, suggesting complex relationships among IOP, ICP, and TLPD.

Reticular Epithelial Edema: An Uncommon Side Effect of ROCK/NET Inhibitor Netarsudil.

A 66-year-old female with advanced primary open-angle glaucoma and Descemet's stripping endothelial keratoplasty OD with previously noted inferior stromal edema presented with a 1-month history of progressive decreased visual acuity after starting netarsudil twice daily. Her best-corrected visual acuity was 20/80 OD and no light perception OS. The right cornea was notable for inferior small epithelial bullae in a reticular pattern from 2 to 9 o'clock encroaching on the visual axis involving both sides of the graft-host junction. The reticular epithelial edema resolved upon discontinuation of netarsudil and best-corrected visual acuity improved to 20/50 but was limited by persistent stromal edema. We report a patient with a history of a partially decompensated Descemet's stripping endothelial keratoplasty who develops reticular epithelial corneal edema after starting netarsudil. This unique pattern of edema may present in the setting of preexisting endothelial cell dysfunction when netarsudil is used, a complication not noted in the Food and Drug Administration (FDA) trials.

Author Correction: Characterization of Retinal Ganglion Cell and Optic Nerve Phenotypes Caused by Sustained Intracranial Pressure Elevation in Mice.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Transcriptomic profiles of retinal ganglion cells are defined by the magnitude of intraocular pressure elevation in adult mice.

Elevated intraocular pressure (IOP) is the major risk factor for glaucoma, a sight threatening disease of retinal ganglion cells (RGCs) and their axons. Despite the central importance of IOP, details of the impact of IOP elevation on RGC gene expression remain elusive. We developed a 4-step immunopanning protocol to extract adult mouse RGCs with high fidelity and used it to isolate RGCs from wild type mice exposed to 2 weeks of IOP elevation generated by the microbead model. IOP was elevated to 2 distinct levels which were defined as Mild (IOP increase >1 mmHg and <4 mmHg) and Moderate (IOP increase ≥4 mmHg). RNA sequencing was used to compare the transcriptional environment at each IOP level. Differentially expressed genes were markedly different between the 2 groups, and pathway analysis revealed frequently opposed responses between the IOP levels. These results suggest that the magnitude of IOP elevation has a critical impact on RGC transcriptional changes. Furthermore, it is possible that IOP-based set points exist within RGCs to impact the direction of transcriptional change. It is possible that this improved understanding of changes in RGC gene expression can ultimately lead to novel diagnostics and therapeutics for glaucoma.

Retinal Neurodegeneration as an Early Manifestation of Diabetic Eye Disease and Potential Neuroprotective Therapies.

PURPOSE OF REVIEW: Diabetic retinopathy (DR) is a major cause of visual impairment and blindness throughout the world. Microvascular changes have long been regarded central to disease pathogenesis. In recent years, however, retinal neurodegeneration is increasingly being hypothesized to occur prior to the vascular changes classically associated with DR and contribute to disease pathogenesis. RECENT FINDINGS: There is growing structural and functional evidence from human and animal studies that suggests retinal neurodegeneration to be an early component of DR. Identification of new therapeutic targets is an ongoing area of research with several different molecules undergoing testing in animal models for their neuroprotective properties and for possible use in humans. Retinal neurodegeneration may play a central role in DR pathogenesis. As new therapies are developed, it will be important to develop criteria for clinically defining retinal neurodegeneration. A standardization of the methods for monitoring neurodegeneration along with more sensitive means of detecting preclinical damage is also needed.

Mild Intraocular Pressure Elevation in Mice Reveals Distinct Retinal Ganglion Cell Functional Thresholds and Pressure-Dependent Properties.

Elevation of intraocular pressure (IOP) causes retinal ganglion cell (RGC) dysfunction and death and is a major risk factor for glaucoma. We used a bead injection technique to increase IOP in mice of both genders by an average of ∼3 mmHg for 2 weeks. This level of IOP elevation was lower than that achieved in other studies, which allowed for the study of subtle IOP effects. We used multielectrode array recordings to determine the cellular responses of RGCs exposed to this mild degree of IOP elevation. We found that RGC photopic receptive field (RF) center size and whole-field RGC firing rates were unaffected by IOP elevation. In contrast, we found that the temporal properties of RGC photopic responses in the RF center were accelerated, particularly in ON sustained cells. We also detected a loss of antagonistic surround in several RGC subtypes. Finally, spontaneous firing rate, interspike interval variance, and contrast sensitivity were altered according to the magnitude of IOP exposure and also displayed an IOP-dependent effect. Together, these results suggest that individual RGC physiologic parameters have unique IOP-related functional thresholds that exist concurrently and change following IOP elevation according to specific patterns. Furthermore, even subtle IOP elevation can impart profound changes in RGC function, which in some cases may occur in an IOP-dependent manner. This system of overlapping functional thresholds likely underlies the complex effects of elevated IOP on the retina.SIGNIFICANCE STATEMENT Retinal ganglion cells (RGCs) are the obligate output neurons of the retina and are injured by elevated intraocular pressure (IOP) in diseases such as glaucoma. In this study, a subtle elevation of IOP in mice for 2 weeks revealed distinct IOP-related functional thresholds for specific RGC physiologic parameters and sometimes showed an IOP-dependent effect. These data suggest that overlapping IOP-related thresholds and response profiles exist simultaneously in RGCs and throughout the retina. These overlapping thresholds likely explain the range of RGC responses that occur following IOP elevation and highlight the wide capacity of neurons to respond in a diseased state.