Retinal Ganglion Cell Dysfunction Research Articles

Visual Deficits and Diagnostic and Therapeutic Strategies for Neurofibromatosis Type 1: Bridging Science and Patient-Centered Care.

Neurofibromatosis type 1 (NF1) is an inherited autosomal dominant disorder primarily affecting children and adolescents characterized by multisystemic clinical manifestations. Mutations in neurofibromin, the protein encoded by the Nf1 tumor suppressor gene, result in dysregulation of the RAS/MAPK pathway leading to uncontrolled cell growth and migration. Neurofibromin is highly expressed in several cell lineages including melanocytes, glial cells, neurons, and Schwann cells. Individuals with NF1 possess a genetic predisposition to central nervous system neoplasms, particularly gliomas affecting the visual pathway, known as optic pathway gliomas (OPGs). While OPGs are typically asymptomatic and benign, they can induce visual impairment in some patients. This review provides insight into the spectrum and visual outcomes of NF1, current diagnostic techniques and therapeutic interventions, and explores the influence of NF1-OPGS on visual abnormalities. We focus on recent advancements in preclinical animal models to elucidate the underlying mechanisms of NF1 pathology and therapies targeting NF1-OPGs. Overall, our review highlights the involvement of retinal ganglion cell dysfunction and degeneration in NF1 disease, and the need for further research to transform scientific laboratory discoveries to improved patient outcomes.

Slope between positive and negative ERG components in patients with open-angle glaucoma.

To evaluate ERG morphology, in particular the slope between P50 and N95 components of the PERG, as well as between the b-wave and the photopic negative response (PhNR) of the light-adapted (LA) ERG in patients with retinal ganglion cell (RGC) dysfunction due to open-angle glaucoma. The PERG and LA-ERG traces of 16 glaucoma patients and 21 age-similar controls were retrospectively analysed. The ERG signal between the peak of the positive component (P50 and b-wave) towards the negative component (N95 and PhNR) was described by a linear regression y = a + bx, where the parameter b indicated the steepness of the P50-N95 and b-PhNR slope. The P50-N95 slope was less steep in glaucoma patients (-0.079 ± 0.034 vs. -0.166 ± 0.050 in controls, p < 0.001), while the b-PhNR slope was not affected (-4.2 ± 2.1 vs. -4.4 ± 1.2, p = NS). The P50-N95 slope showed strong correlation with PhNR and N95 amplitude (r = -0.68 and -0.92, respectively; p < 0.001), while the b-PhNR slope correlated only with b-wave amplitude (r = -0.66, p < 0.001). The P50-N95 slope is a sensitive indicator of RGC dysfunction in patients with open-angle glaucoma. A similar component of LA-ERG, the b-PhNR slope, is less affected by glaucomatous RGC dysfunction and probably originates from similar retinal mechanisms as the b-wave.

Correlations between Steady-State Pattern Electroretinogram and Humphrey Visual Field Analyzer Global Indices and Their Associations with Retinal Ganglion Cell Layer-Inner Plexiform Layer Thickness in Glaucoma Suspects.

The purpose of this study was to investigate the utility of steady state pattern electroretinogram (ss-PERG) in detecting retinal ganglion cell (RGC) dysfunction in glaucoma suspects (GS) who had normal 24-2 Humphrey Visual Fields (HFA). This was a prospective cohort study of GS patients who were identified based on optic disc appearance with normal HFAs. Patients received a complete eye examination, standard automated perimetry (SAP), optical coherence tomography (OCT), and ss-PERG measurements. The ss-PERG parameters, Magnitude (Mag), Magnitude D (MagD), and MagD/Mag ratio, were examined, along with their relationships between HFA and OCT measurements. Twenty-five patients were included in this study, with a total of 49 eyes. Fifteen eyes had abnormal ss-PERG parameters and when compared to GS eyes with normal ss-PERG parameters, there were significant differences in HFA 24-2, retinal nerve fiber layer (RNFL) thickness, and ganglion cell layer and inner plexiform layer (GCL + IPL) thickness. All ss-PERG parameters were significantly correlated with 24-2 VF mean deviation (MD) and visual field index (VFI), as well as 10-2 VF MD after controlling for age, sex, intraocular pressure, central corneal thickness, and spherical equivalent. When controlled for age, spherical equivalent, and IOP, MagD/Mag ratio significantly contributed to the variance in average GCL + IPL thicknesses, whereas 24-2 VF MD and 10-2 VF MD did not. MagD/Mag ratio also significantly accounted for variance in all macular GCL + IPL sectors, while 10-2 VF MD did not. ss-PERG has significant correlations with HFA global indices and was predictive of GCL + IPL thickness in GS patients. Clinical Significance. ss-PERG may serve as a useful functional tool for detecting and measuring RGC dysfunction in GS. It appears to be more sensitive than HFA in the detection of early changes in GCL + IPL thicknesses and may be helpful to use in conjunction with current diagnostic studies to improve the ability of monitoring GS progression.

Prophylactic nicotinamide treatment protects from rotenone-induced neurodegeneration by increasing mitochondrial content and volume

Leber’s hereditary optic neuropathy (LHON) is driven by mtDNA mutations affecting Complex I presenting as progressive retinal ganglion cell dysfunction usually in the absence of extra-ophthalmic symptoms. There are no long-term neuroprotective agents for LHON. Oral nicotinamide provides a robust neuroprotective effect against mitochondrial and metabolic dysfunction in other retinal injuries. We explored the potential for nicotinamide to protect mitochondria in LHON by modelling the disease in mice through intravitreal injection of the Complex I inhibitor rotenone. Using MitoV mice expressing a mitochondrial-tagged YFP in retinal ganglion cells we assessed mitochondrial morphology through super-resolution imaging and digital reconstruction. Rotenone induced Complex I inhibition resulted in retinal ganglion cell wide mitochondrial loss and fragmentation. This was prevented by oral nicotinamide treatment. Mitochondrial ultrastructure was quantified by transition electron microscopy, demonstrating a loss of cristae density following rotenone injection, which was also prevented by nicotinamide treatment. These results demonstrate that nicotinamide protects mitochondria during Complex I dysfunction. Nicotinamide has the potential to be a useful treatment strategy for LHON to limit retinal ganglion cell degeneration.

Exploring Epigenetic Modifications as Potential Biomarkers and Therapeutic Targets in Glaucoma.

Glaucoma, a complex and multifactorial neurodegenerative disorder, is a leading cause of irreversible blindness worldwide. Despite significant advancements in our understanding of its pathogenesis and management, early diagnosis and effective treatment of glaucoma remain major clinical challenges. Epigenetic modifications, encompassing deoxyribonucleic acid (DNA) methylation, histone modifications, and non-coding RNAs, have emerged as critical regulators of gene expression and cellular processes. The aim of this comprehensive review focuses on the emerging field of epigenetics and its role in understanding the complex genetic and molecular mechanisms underlying glaucoma. The review will provide an overview of the pathophysiology of glaucoma, emphasizing the intricacies of intraocular pressure regulation, retinal ganglion cell dysfunction, and optic nerve damage. It explores how epigenetic modifications, such as DNA methylation and histone modifications, can influence gene expression, and how these mechanisms are implicated in glaucomatous neurodegeneration and contribute to glaucoma pathogenesis. The manuscript discusses evidence from both animal models and human studies, providing insights into the epigenetic alterations associated with glaucoma onset and progression. Additionally, it discusses the potential of using epigenetic modifications as diagnostic biomarkers and therapeutic targets for more personalized and targeted glaucoma treatment.

Ochratoxin A induces mitochondrial dysfunction, oxidative stress, and apoptosis of retinal ganglion cells (RGCs), leading to retinal damage in mice

PurposeOchratoxin A (OTA) contamination of food and feed is a serious problem worldwide. OTA is considered a carcinogen and immunotoxic, nephrotoxic, and neurotoxic mycotoxin. The present study aims to determine the toxic effects of OTA on retinal ganglion cells (RGCs) and assess the resulting impairment of retinal function in mice.MethodsRGC-5 cells were exposed to OTA (100 and 200 μg/L) for 3 days, and the mice were fed OTA-contain (100 and 200 μg/kg) diets for 4 weeks. Antioxidant indices were detected by spectrophotometer. The apoptosis of RGC-5 cells was determined by flow cytometry. Mitochondrial morphology and mitochondrial membrane potential were detected by immunofluorescence. RGC survival was determined by immunofluorescence staining with Brn3a. Flash electroretinography (ERG) was conducted to assess visual function.ResultsThe oxidative-antioxidant balance suggested that OTA-induced severe oxidative stress, including increased reactive oxygen species (ROS) and malondialdehyde (MDA) levels in the OTA-exposed RGC-5 cells, and the reduced activity of superoxide dismutase (SOD) and glutathione-S-transferase (GST) in the OTA exposed group. Furthermore, OTA exposure led to remarkable apoptosis in RGC-5 cells. The mitochondrial detection showed that OTA caused significant mitochondrial membrane potential reduction and mitochondrial fragmentation, which may be the cause of apoptosis of RGC-5 cells. Additionally, in vivo experiments demonstrated that OTA resulted in significant death of RGCs and subsequent retinal dysfunction in mice.ConclusionOchratoxin A induces mitochondrial dysfunction, oxidative stress, and RGCs death in mice.

Visual Improvement Following Glaucoma Surgery

Introduction : Glaucoma is a progressive optic neuropathy and a leading cause of irreversible blindness. The aim of glaucoma treatment is to slow progression and minimize the risk of further damage, not to expect functional improvement. We report the case of a patient who experienced an improvement in vision following glaucoma surgery and review the literature regarding this phenomenon.&#x0D; Case Illustration : A 66-year old female presented with vision loss in the left eye that could only perceive finger counting in one meter. Her intraocular pressure (IOP) measured 51,8 mmHg and she was found to have primary open angle glaucoma. Medical treatment was commenced and she underwent a successful trabeculectomy. Unexpectedly she experienced improvement in vision after treatment. At her recent visit visual acuity was 6/30 in the affected eye.&#x0D; Discussion : Although the mechanism of improved vision cannot be proven it is likely that successful lowering of IOP resulted in some reversal of retinal ganglion cell dysfunction. Some evidence explained that retinal ganglion cells damaged by glaucoma might undergo a period of reversible dysfunction preceding cell death. Furthermore, reversible changes in optic nerve head morphology have been reported following reduction of intraocular pressure. These observations suggest that certain structural and functional improvements may in fact be possible in some patients. The changes of anterior chamber depth, corneal radius of curvature, corneal thickness or corneal edema could be considered as other factors which influence this improvement.&#x0D; Conclusion : Functional improvements may occur following trabeculectomy. Glaucoma surgery should be offered early, and considered even in those with reduced visual acuity.

Loss of Sarm1 reduces retinal ganglion cell loss in chronic glaucoma

Glaucoma is one of the leading causes of irreversible blindness worldwide and vision loss in the disease results from the deterioration of retinal ganglion cells (RGC) and their axons. Metabolic dysfunction of RGC plays a significant role in the onset and progression of the disease in both human patients and rodent models, highlighting the need to better define the mechanisms regulating cellular energy metabolism in glaucoma. This study sought to determine if Sarm1, a gene involved in axonal degeneration and NAD+ metabolism, contributes to glaucomatous RGC loss in a mouse model with chronic elevated intraocular pressure (IOP). Our data demonstrate that after 16 weeks of elevated IOP, Sarm1 knockout (KO) mice retain significantly more RGC than control animals. Sarm1 KO mice also performed significantly better when compared to control mice during optomotor testing, indicating that visual function is preserved in this group. Our findings also indicate that Sarm1 KO mice display mild ocular developmental abnormalities, including reduced optic nerve axon diameter and lower visual acuity than controls. Finally, we present data to indicate that SARM1 expression in the optic nerve is most prominently associated with oligodendrocytes. Taken together, these data suggest that attenuating Sarm1 activity through gene therapy, pharmacologic inhibition, or NAD+ supplementation, may be a novel therapeutic approach for patients with glaucoma.

Retinal Ganglion Cell Functional Recovery after Intraocular Pressure Lowering Treatment Using Prostaglandin Analogs in Glaucoma Suspects: A Prospective Pilot Study.

To evaluate the ability of pattern electroretinogram (PERG) to detect improvement of retinal ganglion cell (RGC) function in glaucoma suspects (GS) after medically reducing intraocular pressure (IOP) using prostaglandin analog drops. Six subjects (eight eyes) received topical IOP lowering treatment based on their clinical examination and were observed at Manhattan Eye, Ear & Throat Hospital over an average of 3.1 ± 2.2 months. During this time, participants underwent a full ophthalmologic exam and were evaluated with a Humphrey visual field analyzer (HFA) 24-2 [24-2 mean deviation (MD), 24-2 pattern standard deviation (PSD), and 24-2 visual field indices (VFI)], Diopsys NOVA PERG optimized for glaucoma [magnitude (Mag), magnitudeD (MagD), and magnitudeD/magnitude ratio (MagD/Mag ratio)] and optical coherence tomography (OCT)-derived average retinal nerve fiber layer thickness (avRNFLT) and average ganglion cell layer + inner plexiform layer (avGCL + IPL) thicknesses at baseline visit (pretreatment) and 3 months later (posttreatment). Goldman applanation tonometry was used to measure IOP at each visit. Paired sample t-tests were conducted to determine the statistical significance of the change in IOP, HFA indices, PERG parameters, and OCT thickness measurements between the two visits. Lowering IOP by 22.29% resulted in a significant increase (32.98 and 15.49%) in MagD [t (7) = -3.174, 95% confidence interval (CI) = -0.53, -0.08, p = 0.016] and MagD/Mag ratio [t (7) = -3.233, 95% CI = -0.20, -0.03, p = 0.014], respectively. There was a positive percentage change for all variables of interest, however, 24-2 MD, Mag, avRNFLT, and GCL+ IPLT did not reach statistical significance. After reducing IOP by 22.29% for a duration of 3.1 months, the PERG parameters, MagD and MagD/Mag ratio, significantly improved by 32.98 and 15.49%, respectively. Pattern electroretinogram (PERG) may be a crucial tool for clinicians to locate a window of opportunity in which degenerating yet viable RGCs could be rescued from irreversible damage. We suggest consideration of PERG as a tool in early retinal ganglion cell (RGC) dysfunction detection as well as for monitoring IOP lowering treatment. Tirsi A, Gliagias V, Sheha H, et al. Retinal Ganglion Cell Functional Recovery after Intraocular Pressure Lowering Treatment Using Prostaglandin Analogs in Glaucoma Suspects: A Prospective Pilot Study. J Curr Glaucoma Pract 2023;17(4):178-190.

Estrogen-induced glial IL-1β mediates extrinsic retinal ganglion cell vulnerability in murine Nf1 optic glioma.

Optic pathway gliomas (OPGs) arising in children with neurofibromatosis type 1 (NF1) can cause retinal ganglion cell (RGC) dysfunction and vision loss, which occurs more frequently in girls. While our previous studies demonstrated that estrogen was partly responsible for this sexually dimorphic visual impairment, herein we elucidate the underlying mechanism. In contrast to their male counterparts, female Nf1OPG mice have increased expression of glial interleukin-1β (IL-1β), which is neurotoxic to RGCs in vitro. Importantly, both IL-1β neutralization and leuprolide-mediated estrogen suppression decrease IL-1β expression and ameliorate RGC dysfunction, providing preclinical proof-of-concept evidence supporting novel neuroprotective strategies for NF1-OPG-induced vision loss.

What OPA1 models teach us about potential therapy

Autosomal dominant optic atrophy (DOA) is the most common inherited optic neuropathy, affecting approximately 1 in 25 000 people. DOA has an insidious onset in early childhood. It presents typically with bilateral central painless vision loss, dyschromatopsia, and optic disc pallor due to gradual retinal ganglion cell (RGC) loss and optic nerve degeneration. 60%–70% of genetically confirmed DOA cases are associated with variants in OPA1. OPA1 is an ubiquitously expressed GTPase that regulates mitochondrial homeostasis through coordination of inner membrane fusion, maintenance of cristae structure, and maintenance of bioenergetic output. Over 250 known pathogenic variants in OPA1 have been reported, with missense variants within the GTPase domain associated with an increased susceptibility to developing DOA plus (DOA+), a multisystemic syndromic form of the disease with extraocular features, including sensorineural hearing loss and ataxia. Current therapeutic options to treat DOA are limited. Idebenone has been shown to protect against visual loss in Leber's hereditary optic neuropathy (LHON). In DOA off ‐label retrospective series show some promise in some patients.Animal models for OPA1 DOA, have been reported, including Drosophila, C elegans, Danio rerio and mouse models. They have cast light on some potential therapeutic modalities. Despite limitations, murine models of DOA are currently indispensable for understanding pathophysiology of disease, drug testing and development. They replicate many symptoms and signs of human disease. However, there are caveats. Chemically induced Complex I deficits are very toxic. Genetic models of mitochondrial mutation affect protein integrity and function and can cause severe pathologies. This is made more problematic if there is a need for aging the mouse to bring out the full phenotype. We shall review the mouse models and describe some features that aid or our understanding of pathophysiological mechanisms. Our research has characterized the earliest morphological changes as a progressive loss of synapses, dendrite retraction and RGC remodelling and loss of connectivity. In the DOA mouse Opa1+/− mouse (B6;C3‐Opa1Q285STOP), heterozygous Opa1+/− mice develop visual defects, on psychophysics (optokinetic response) and electrophysiology (VEP, PhNR), consistent with RGC dysfunction. There is depressed mitochondrial Complex I and IV activity. Visible structural changes and disrupted axon morphology in the optic nerve are seen. We have carried out a randomized, placebo‐controlled trial of the benzoquinone idebenone in our ADOA mouse, which revealed a limited therapeutic effect on RGC dendropathy and visual function.It is apparent that there are large differences between the rodent and primate visual system. The fovea of the retina, a critical component for central vision is one of the only primate‐specific components of the mammalian nervous system and is thus, not found in rodent. For these reasons, there is a strong case to use human cells. We shall review data on induced human pluripotent stem cell (hiPSC) with OPA1 mutation, with or without differentiation to RGCs, as models, and on CRISPRCas9 correction of patient‐derived OPA1 iPSC lines.

Preventing retinal homocysteine dysregulation protects against retinal ganglion cell death

Glaucoma, the most common cause of irreversible blindness, is characterized by progressive dysfunction and loss of retinal ganglion cells. There are no neuroprotective therapies. Metabolic dysfunction is an emerging pathophysiological mechanism in glaucoma with widespread changes to energy homeostasis, yet little is known about non‐energy metabolism. We recently identified elevated retinal homocysteine as a strong early metabolomic signature in a rat model of glaucoma. We tested whether local elevation of homocysteine to the retina compounded glaucoma in the rat model by artificially rising homocysteine through intravitreal injection. Short‐term elevation of homocysteine (from 1 to 15 μM, as has been detected in the vitreous of diabetic retinopathy patients) either alone or in addition to glaucoma does not worsen retinal ganglion cell death, suggesting that local elevation of homocysteine does not contribute to retinal ganglion cell degeneration, but may be an indicator of wider metabolic dysfunction. Homocysteine is central to one‐carbon metabolism, an anabolic pathway with an essential role in generation of methionine and methyl donors (necessary for the epigenetic regulation of gene expression), and glutathione (antioxidant). In a mouse model of glaucoma, we identified early‐ and sustained‐dysregulation of genes involved in one‐carbon metabolism in whole retina. In retinal ganglion cells, a number of these genes change prior to detectable neurodegeneration. These pathways require vitamin B6 and B12 as essential cofactors, and folic acid (B9) and choline are essential precursors, for the metabolism of homocysteine. Genes involved in the transport and utilization of these cofactors and precursors were also significantly dysregulated in the retina, further supporting dysfunctional homocysteine metabolism. Dietary deficiency in these leads to homocysteine accumulation and are well associated with optic neuropathies. We hypothesize that local retinal deficiency in these may contribute to dysfunction in one‐carbon metabolism and impact wider retinal ganglion cell homeostasis. To test this hypothesis, we first supplemented the drinking water of mice with B6 (4.5 mg/kg), folic acid (B9) (1.5 mg/kg), cobalamin (B12) (20 μg/kg), and choline (750 mg/kg) as a prophylactic treatment 7 days prior to intravitreal homocysteine injection. We used a supraphysiological dose of homocysteine (500 μM) that causes spontaneous death of ~10% of retinal ganglion cell death after 7 days in untreated animals. Pre‐treated animals were completely protected from retinal ganglion cell death. Next, we pre‐treated rats in the same way for 7 days prior to induction of our glaucoma model. In untreated rats there was a ~ 40% loss of retinal ganglion cells after 14 days, and this was significantly mitigated in pre‐treated rats (only ~25% loss), demonstrating that these cofactors can provide moderate neuroprotection in a complex, multifactorial neurodegenerative context.

Increasing the number and intensity of shock tube generated blast waves leads to earlier retinal ganglion cell dysfunction and regional cell death

The purpose of this study was to examine the effect of a blast exposure generated from a shock tube on retinal ganglion cell (RGC) function and structure. Mice were exposed to one of three blast conditions using a shock tube; a single blast wave of 20 PSI, a single blast wave of 30 PSI, or three blast waves of 30 PSI given on three consecutive days with a one-day inter-blast interval. The structure and function of the retina were analyzed using the pattern electroretinogram (PERG), the optomotor reflex (OMR), and optical coherence tomography (OCT). The in vivo parameters were examined at baseline, and then again 1-week, 4-weeks, and 16-weeks following blast exposure. The number of surviving RGCs was quantified at the end of the study. Analysis of mice receiving a 20 PSI injury showed decreased PERG and OMR responses 16-weeks post blast, without evidence of changed retinal thickness or RGC death. Mice subjected to a 30 PSI injury showed decreased PERG responses 4 weeks and 16 weeks after injury, without changes in the retinal thickness or RGC density. Mice subjected to 30 PSI X 3 blast exposures had PERG deficits 1-week and 4-weeks post exposure. There was also significant change in retinal thickness 1-week and 16-weeks post blast exposure. Mice receiving 30 PSI X 3 blast injuries had regional loss of RGCs in the central retina, but not in the mid-peripheral or peripheral retina. Overall, this study has shown that increasing the number of blast exposures and the intensity leads to earlier functional loss of RGCs. We have also shown regional RGC loss only when using the highest blast intensity and number of blast injuries.

Topographical Correlation between Structural and Functional Impairment of the Macular Inner Retinal Layers in Multiple Sclerosis Eyes with a History of Optic Neuropathy.

We investigated the potential correlation between morphological and functional parameters describing the rarefaction and dysfunction of retinal ganglion cells (RGCs), located in the macula, in multiple sclerosis eyes with a history of optic neuritis (MS-ON). A total of 19 MS-ON eyes from 19 MS patients (mean age: 44.16 ± 4.66 years; 11 females and 8 males), with a mean disease duration of 10.06 ± 6.12 years and full recovery of visual acuity, and 30 age-similar (mean age: 45.09 ± 5.08 years) healthy eyes were submitted for ophthalmological evaluation using swept-source optical coherence tomography (SS-OCT) and multifocal photopic negative response (mfPhNR) to study the structural and functional features of localized RGCs. Both GCL+ thickness (via SS-OCT) and response amplitude density (RAD) (via mfPhNR) measurements were obtained from annular regions and ETDRS sectors. Morphological and electrophysiological data from the control and MS groups were compared by using an ANOVA test. GCL+ values were correlated with the corresponding RADs derived from almost superimposable areas using Pearson's tests (p < 0.01). In MS-ON eyes, the mean values of macular GCL+-T and mfPhNR RAD detected in all rings and ETDRS sectors were significantly reduced (p < 0.01) when compared with control ones. In addition, when plotting the GCL+-T and mfPhNR RAD individual data from MS-ON eyes, we found statistically significant linear correlations (p < 0.01) when considering responses from both rings and sectors. In conclusion, in MS-ON eyes, a topographical correlation between structural and functional impairment of macular RGCs occurs.

The Inflammasome-Dependent Dysfunction and Death of Retinal Ganglion Cells after Repetitive Intraocular Pressure Spikes.

The dysfunction and selective loss of retinal ganglion cells (RGCs) is a known cause of vision loss in glaucoma and other neuropathies, where ocular hypertension (OHT) is the major risk factor. We investigated the impact of transient non-ischemic OHT spikes (spOHT) on RGC function and viability in vivo to identify cellular pathways linking low-grade repetitive mechanical stress to RGC pathology. We found that repetitive spOHT had an unexpectedly high impact on intraocular homeostasis and RGC viability, while exposure to steady OHT (stOHT) of a similar intensity and duration failed to induce pathology. The repetitive spOHT induced the rapid activation of the inflammasome, marked by the upregulation of NLRP1, NLRP3, AIM2, caspases -1, -3/7, -8, and Gasdermin D (GSDMD), and the release of interleukin-1β (IL-1β) and other cytokines into the vitreous. Similar effects were also detected after 5 weeks of exposure to chronic OHT in an induced glaucoma model. The onset of these immune responses in both spOHT and glaucoma models preceded a 50% deficit in pattern electroretinogram (PERG) amplitude and a significant loss of RGCs 7 days post-injury. The inactivation of inflammasome complexes in Nlrp1-/-, Casp1-/-, and GsdmD-/- knockout animals significantly suppressed the spOHT-induced inflammatory response and protected RGCs. Our results demonstrate that mechanical stress produced by acute repetitive spOHT or chronic OHT is mechanistically linked to inflammasome activation, which leads to RGC dysfunction and death.

A new model of axon degeneration in the mouse optic nerve using repeat intraocular pressure challenge

We characterize a new experimental model for inducing retinal ganglion cell (RGC) dysfunction and degeneration in mice. C57BL/6J mice were subjected to two acute periods of intraocular pressure (IOP) elevation (50 mmHg for 30 min) by cannulation of the anterior chamber. We used full-field electroretinography and visual evoked potentials (VEPs) to measure subsequent changes in retina and optic nerve function, and histochemical techniques to assess RGC survival and optic nerve structure. In 12 month old mice, a single IOP challenge caused loss and subsequent recovery of RGC function over the following 28 days with minimal cell death and no observed axonal damage. A second identical IOP challenge resulted in persistent RGC dysfunction and significant (36%) loss of RGC somas. This was accompanied by a 16.7% delay in the latency and a 27.6% decrease in the amplitude of the VEP. Severe axonal damage was seen histologically with enlargement of axons, myelin disruption, reduced axon density, and the presence of glial scarring. In contrast, younger 3 month old mice when exposed to a single or repeat IOP challenge showed quicker RGC functional recovery after a single challenge and full functional recovery after a repeat challenge with no detectable optic nerve dysfunction. These data demonstrate a highly reproducible and minimally invasive method for inducing RGC degeneration and axonal damage in mice. Resilience of the optic nerve to damage is highly dependent on animal age. The time-defined nature of functional versus structural loss seen in this model stands to facilitate investigation of neuroglial responses in the retina after IOP injury and the associated evaluation of neuroprotective treatment strategies. Further, the model may be used to investigate the impact of aging and the cellular switch between neurorecovery and neurodegeneration.

The diagnostic accuracy of photopic negative responses evoked by broadband and chromatic stimuli in a clinically heterogeneous population

PurposeTo compare the diagnostic accuracy of the photopic negative response (PhNR) elicited by red-blue (RB) and white-white (WW) stimuli, for detection of retinal ganglion cell (RGC) dysfunction in a heterogeneous clinical cohort.MethodsAdults referred for electrophysiological investigations were recruited consecutively for this single-centre, prospective, paired diagnostic accuracy study. PhNRs were recorded to red flashes (1.5 cd·s·m−2) on a blue background (10 cd·m−2) and to white flashes on a white background (the latter being the ISCEV standard LA 3 stimulus). PhNR results were compared with a reference test battery assessing RGC/optic nerve structure and function including optical coherence tomography (OCT) retinal nerve fibre layer thickness and mean RGC volume measurements, fundus photography, pattern electroretinography and visual evoked potentials. Primary outcome measures were differences in sensitivity and specificity of the two PhNR methods.ResultsTwo hundred and forty-three participants were initially enrolled, with 200 (median age 54; range 18–95; female 65%) meeting inclusion criteria. Sensitivity was 53% (95% confidence intervals [CI] 39% to 68%) and 62% (95% CI 48% to 76%), for WW and RB PhNRs, respectively. Specificity was 80% (95% CI 74% to 86%) and 78% (95% CI 72% to 85%), respectively. There was a statistically significant difference between sensitivities (p = 0.046) but not specificities (p = 0.08) of the two methods. Receiver operator characteristic (ROC) area under the curve (AUC) values were 0.73 for WW and 0.74 for RB PhNRs.ConclusionPhNRs to red flashes on a blue background may be more sensitive than white-on-white stimuli, but there is no significant difference between specificities. This study highlights the value and potential convenience of using white-on-white stimuli, already used widely for routine ERG assessment.

Glutamate transporter contribution to retinal ganglion cell vulnerability in a rat model of multiple sclerosis

Glial glutamate transporters actively participate in neurotransmission and have a fundamental role in determining the ambient glutamate concentration in the extracellular space. Their expression is dynamically regulated in many diseases, including experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. In EAE, a downregulation has been reported which may render neurons more susceptible to glutamate excitotoxicity. In this study, we have investigated the expression of GLAST (EAAT1) and GLT-1 (EAAT2) in the retina of Brown Norway rats following induction of myelin oligodendrocyte glycoprotein (MOG)-EAE, which results in retinal ganglion cell (RGC) degeneration and dysfunction. In addition, we tested whether AAV-mediated overexpression of GLAST in the retina can protect RGCs from degeneration. To address the impact of glutamate transporter modulation on RGCs, we performed whole-cell recordings and measured tonic NMDA receptor-mediated currents in the absence and presence of a glutamate-uptake blocker. We report that αOFF-RGCs show larger tonic glutamate-induced currents than αON-RGCs, in line with their greater vulnerability under neuroinflammatory conditions. We further show that increased AAV-mediated expression of GLAST in the retina does indeed protect RGCs from degeneration during the inflammatory disease. Collectively, our study highlights the neuroprotective role of glutamate transporters in the EAE retina and provides a characterization of tonic glutamate-currents of αRGCs. The larger effects of increased extracellular glutamate concentration on the αOFF-subtype may underlie its enhanced vulnerability to degeneration.

Keratin8 Deficiency Aggravates Retinal Ganglion Cell Damage Under Acute Ocular Hypertension.

Keratin 8/18 (KRT8/18), paired members of the intermediate filament family, have shown vital functions in regulating physiological activities more than supporting the mechanic strength for cells and organelles. However, the KRT8/18 presence in retinal ganglion cells (RGCs) and functions on neuroprotection in a mouse model of acute ocular hypertension (AOH) are unknown and worthy of exploration. We identified the existence of KRT8/18 in normal human and mouse retinas and primary RGCs. KRT8/18 levels were detected after AOH modeling. The adeno-associated virus (AAV) system was intravitreally used for selective KRT8 knockdown in RGCs. The histological changes, the loss and dysfunction of RGCs, and the gliosis in retinas were detected. The markers of cell apoptosis and MAPK pathways were investigated. KRT8/18 existed in all retinal layers and was highly expressed in RGCs, and they increased after AOH induction. The KRT8 knockdown in RGCs caused no histopathological changes and RGC loss in retinas without AOH modeling. However, after the KRT8 deficiency, AOH significantly promoted the loss of whole retina and inner retina thickness, the reduction, apoptosis, and dysfunction of RGCs, and the glial activation. Besides, downregulated Bcl-2 and upregulated cleaved-Caspase 3 were found in the AOH retinas with KRT8 knockdown, which may be caused by the increased phosphorylation level of MAPK pathways (JNK, p38, and ERK). The KRT8 deficiency promoted RGC apoptosis and neurodegeneration by abnormal activation of MAPK pathways in AOH retinas. Targeting KRT8 may serve as a novel treatment for saving RCGs from glaucomatous injuries.

Inducible Rbpms-CreERT2 Mouse Line for Studying Gene Function in Retinal Ganglion Cell Physiology and Disease.

Retinal ganglion cells (RGCs) are the sole output neurons conveying visual stimuli from the retina to the brain, and dysfunction or loss of RGCs is the primary determinant of visual loss in traumatic and degenerative ocular conditions. Currently, there is a lack of RGC-specific Cre mouse lines that serve as invaluable tools for manipulating genes in RGCs and studying the genetic basis of RGC diseases. The RNA-binding protein with multiple splicing (RBPMS) is identified as the specific marker of all RGCs. Here, we report the generation and characterization of a knock-in mouse line in which a P2A-CreERT2 coding sequence is fused in-frame to the C-terminus of endogenous RBPMS, allowing for the co-expression of RBPMS and CreERT2. The inducible Rbpms-CreERT2 mice exhibited a high recombination efficiency in activating the expression of the tdTomato reporter gene in nearly all adult RGCs as well as in differentiated RGCs starting at E13.5. Additionally, both heterozygous and homozygous Rbpms-CreERT2 knock-in mice showed no detectable defect in the retinal structure, visual function, and transcriptome. Together, these results demonstrated that the Rbpms-CreERT2 knock-in mouse can serve as a powerful and highly desired genetic tool for lineage tracing, genetic manipulation, retinal physiology study, and ocular disease modeling in RGCs.