Significant Loss Of Retinal Ganglion Cells Research Articles

Impaired axonal transport at the optic nerve head contributes to neurodegeneration in a novel Cre-inducible mouse model of myocilin glaucoma.

Elevation of intraocular pressure (IOP) due to trabecular meshwork (TM) dysfunction, leading to neurodegeneration, is the pathological hallmark of primary open-angle glaucoma (POAG). Impaired axonal transport is an early and critical feature of glaucomatous neurodegeneration. However, a robust mouse model that replicates these human POAG features accurately has been lacking. We report the development and characterization of a novel Cre-inducible mouse model expressing a DsRed-tagged Y437H mutant of human myocilin (Tg.CreMYOCY437H). A single intravitreal injection of HAd5-Cre induced selective MYOC expression in the TM, causing TM dysfunction, reducing outflow facility, and progressively elevating IOP in Tg.CreMYOCY437H mice. Sustained IOP elevation resulted in significant retinal ganglion cell (RGC) loss and progressive axonal degeneration in Cre-induced Tg.CreMYOCY437H mice. Notably, impaired anterograde axonal transport was observed at the optic nerve head before RGC degeneration, independent of age, indicating that impaired axonal transport contributes to RGC degeneration in Tg.CreMYOCY437H mice. In contrast, axonal transport remained intact in ocular hypertensive mice injected with microbeads, despite significant RGC loss. Our findings indicate that Cre-inducible Tg.CreMYOCY437H mice replicate all glaucoma phenotypes, providing an ideal model for studying early events of TM dysfunction and neuronal loss in POAG.

Methylene Blue Reduces Electroretinogram Distortion and Ganglion Cell Death in a Rat Model of Glaucoma.

Glaucoma is the second leading cause of blindness worldwide and is, in most cases, a consequence of elevated intraocular pressure (IOP), ultimately resulting in the death of retinal ganglion cells (RGCs). Current treatments are mostly focused on normalizing IOP, but we propose the additional use of neuroprotective agents, including methylene blue (MB), to block the loss of RGCs. Wistar rats were subjected to episcleral vein cauterization (EVC) in the left eye while the right eye was sham-operated. One week later, they were divided into two groups, which were injected with either 2.0 mg/kg MB or phosphate-buffered saline (PBS), twice a day, for 7 days. Fifteen days after surgery, rats were tested with scotopic electroretinography (ERG) or pattern electroretinography (PERG). After sacrifice, the number of RGCs and the thickness of the inner retina (IR) were evaluated both in the peripheral and central areas of the retina. Scotopic ERG showed a marked reduction (p < 0.0001) on the a- and b-wave amplitude and oscillatory potential (OP) complexity of the eyes subjected to EVC. These parameters were significantly (p < 0.01) restored by the application of MB. PERG indicated that EVC was responsible for a very significant decrease in N2 amplitude (p < 0.0001) and prolongation of N2 implicit time (p < 0.0001). Treatment with MB significantly restored N2 amplitude (p < 0.0001). In parallel with the ERG results, morphological analysis showed a significant loss of RGCs (p < 0.0001) and IR thickness (p < 0.0001) in both the peripheral and central retinas subjected to EVC, which was significantly prevented (p < 0.0001) by MB treatment. We have shown that MB treatment can be effective in preventing physiological and morphological hallmarks of optic neuropathy in a model of ocular hypertension, which faithfully recapitulates human open-angle glaucoma. Due to its high safety profile, this drug could therefore represent a new pharmacologic strategy to prevent vision loss in glaucoma patients.

Mechanisms contributing to inhibition of retinal ganglion cell death by cell permeable peptain-1 under glaucomatous stress

This study assesses the neuroprotective potential of CPP-P1, a conjugate of an anti-apoptotic peptain-1 (P1) and a cell-penetrating peptide (CPP) in in vitro, in vivo, and ex vivo glaucoma models. Primary retinal ganglion cells (RGCs) were subjected to either neurotrophic factor (NF) deprivation for 48 h or endothelin-3 (ET-3) treatment for 24 h and received either CPP-P1 or vehicle. RGC survival was analyzed using a Live/Dead assay. Axotomized human retinal explants were treated with CPP-P1 or vehicle for seven days, stained with RGC marker RBPMS, and RGC survival was analyzed. Brown Norway (BN) rats with elevated intraocular pressure (IOP) received weekly intravitreal injections of CPP-P1 or vehicle for six weeks. RGC function was evaluated using a pattern electroretinogram (PERG). RGC and axonal damage were also assessed. RGCs from ocular hypertensive rats treated with CPP-P1 or vehicle for seven days were isolated for transcriptomic analysis. RGCs subjected to 48 h of NF deprivation were used for qPCR target confirmation. NF deprivation led to a significant loss of RGCs, which was markedly reduced by CPP-P1 treatment. CPP-P1 also decreased ET-3-mediated RGC death. In ex vivo human retinal explants, CPP-P1 decreased RGC loss. IOP elevation resulted in significant RGC loss in mid-peripheral and peripheral retinas compared to that in naive rats, which was significantly reduced by CPP-P1 treatment. PERG amplitude decline in IOP-elevated rats was mitigated by CPP-P1 treatment. Following IOP elevation in BN rats, the transcriptomic analysis showed over 6,000 differentially expressed genes in the CPP-P1 group compared to the vehicle-treated group. Upregulated pathways included CREB signaling and synaptogenesis. A significant increase in Creb1 mRNA and elevated phosphorylated Creb were observed in CPP-P1-treated RGCs. Our study showed that CPP-P1 is neuroprotective through CREB signaling enhancement in several settings that mimic glaucomatous conditions. The findings from this study are significant as they address the pressing need for the development of efficacious therapeutic strategies to maintain RGC viability and functionality associated with glaucoma.

Cell loss in retinal ischemia is associated with increased necroptosis

Retinal ischemia plays acentral pathophysiological role in numerous eye diseases, such as glaucoma. In addition to apoptosis, autophagy, necroptosis and ferroptosis are among the cell death mechanisms of ischemia; however, their role is not clearly understood and controversially discussed. The aim of this study is to gain an improved understanding of the role of alternative cell death mechanisms such as autophagy and necroptosis after retinal ischemia. Based on this, future autophagy-based or necroptosis-based therapeutic approaches could be developed. Retinal ischemia reperfusion was induced in one eye of 6 to 8‑week-old rats by temporarily increasing the intraocular pressure to 140 mm Hg (60 min), followed by reperfusion. The untreated contralateral eye served as acontrol. Retinas after ischemia and control retinas were examined 7days after ischemia immunohistochemically with markers for retinal ganglion cells (RGC), astrocytes (GFAP) as well as an autophagy (LAMP1) and anecroptosis marker (RIPK3) (n = 6/group). Ischemia reperfusion resulted in both significant RGC loss (p ≤ 0.001) and asignificant increase of astrocyte area (p = 0.026) after 7days. Interestingly, the number of autophagic LAMP1 positive cells was unchanged 7days after ischemia (p = 0.272), whereas the number of necroptotic RIPK3 positive cells was significantly increased (p ≤ 0.001). Necroptotic processes appear to be activated 7days after ischemia reperfusion, contributing to retinal cell death and activation of astrocytes. Late autophagic processes are not activated 7days after ischemia. Necroptosis-associated parameters could therefore be targeted as an early therapeutic approach after ischemia in the future.

Caspase-8-mediated inflammation but not apoptosis drives death of retinal ganglion cells and loss of visual function in glaucomaa.

Glaucoma is a complex multifactorial disease where apoptosis and inflammation represent two key pathogenic mechanisms. However, the relative contribution of apoptosis versus inflammation in axon degeneration and death of retinal ganglion cells (RGCs) is not well understood. In glaucoma, caspase-8 is linked to RGC apoptosis, as well as glial activation and neuroinflammation. To uncouple these two pathways and determine the extent to which caspase-8-mediated inflammation and/or apoptosis contributes to the death of RGCs, we used the caspase-8 D387A mutant mouse (Casp8 DA/DA ) in which a point mutation in the auto-cleavage site blocks caspase-8-mediated apoptosis but does not block caspase-8-mediated inflammation. Intracameral injection of magnetic microbeads was used to elevate the intraocular pressure (IOP) in wild-type, Fas deficient Faslpr, and Casp8 DA/DA mice. IOP was monitored by rebound tonometry. Two weeks post microbead injection, retinas were collected for microglia activation analysis. Five weeks post microbead injection, visual acuity and RGC function were assessed by optometer reflex (OMR) and pattern electroretinogram (pERG), respectively. Retina and optic nerves were processed for RGC and axon quantification. Two- and five-weeks post microbead injection, expression of the necrosis marker, RIPK3, was assessed by qPCR. Wild-type, Faslpr, and Casp8 DA/DA mice showed similar IOP elevation as compared to saline controls. A significant reduction in both visual acuity and pERG that correlated with a significant loss of RGCs and axons was observed in wild-type but not in Faslpr mice. The Casp8 DA/DA mice displayed a significant reduction in visual acuity and pERG amplitude and loss of RGCs and axons similar to that in wild-type mice. Immunostaining revealed equal numbers of activated microglia, double positive for P2ry12 and IB4, in the retinas from microbead-injected wild-type and Casp8 DA/DA mutant mice. qPCR analysis revealed no induction of RIPK3 in wild-type or Casp8 DA/DA mice at two- or five-weeks post microbead injection. Our results demonstrate that caspase-8-mediated extrinsic apoptosis is not involved in the death of RGCs in the microbead-induced mouse model of glaucoma implicating caspase-8-mediated inflammation, but not apoptosis, as the driving force in glaucoma progression. Taken together, these results identify the caspase-8-mediated inflammatory pathway as a potential target for neuroprotection in glaucoma.

IL1A enhances TNF-induced retinal ganglion cell death.

Glaucoma is a neurodegenerative disease that leads to the death of retinal ganglion cells (RGCs). A growing body of literature suggests a role for neuroinflammation in RGC death after glaucoma-relevant insults. For instance, it was shown that deficiency of three proinflammatory cytokines, complement component 1, subcomponent q ( C1q ), interleukin 1 alpha ( Il1a ), and tumor necrosis factor ( Tnf ), resulted in near complete protection of RGCs after two glaucoma-relevant insults, optic nerve injury and ocular hypertension. While TNF and C1Q have been extensively investigated in glaucoma-relevant model systems, the role of IL1A in RGC is not as well defined. Thus, we investigated the direct neurotoxicity of IL1A on RGCs in vivo. Intravitreal injection of IL1A did not result in RGC death at either 14 days or 12 weeks after insult. Consistent with previous studies, TNF injection did not result in significant RGC loss at 14 days but did after 12 weeks. Interestingly, IL1A+TNF resulted in a relatively rapid RGC death, driving significant RGC loss two weeks after injection. JUN activation and SARM1 have been implicated in RGC death in glaucoma and after cytokine insult. Using mice deficient in JUN or SARM1, we show RGC loss after IL1A+TNF insult is JUN-independent and SARM1-dependent. Furthermore, RNA-seq analysis showed that RGC death by SARM1 deficiency does not stop the neuroinflammatory response to IL1A+TNF. These findings indicate that IL1A can potentiate TNF-induced RGC death after combined insult is likely driven by a SARM1-dependent RGC intrinsic signaling pathway.

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.

Long-Term Effects on Retinal Structure and Function in a Mouse Endothelin-1 Model of Retinal Ganglion Cell Degeneration.

To study the long-term effects of endothelin-1 (ET-1)-induced retinal pathologies in mouse, using clinically relevant tools. Adult C57BL/6 mice (7-9 weeks old) were intravitreally injected with PBS (n = 10) or 0.25 (n = 8), 0.5 (n = 8), or 1 nmol ET-1 (n = 9) and examined using electroretinogram, optical coherence tomography (OCT), and Doppler OCT at baseline and postinjection days 10, 28, and 56. Retinal ganglion cell (RGC) survival in retinal whole mount was quantified at days 28 and 56. ET-1 induced immediate retinal arterial constriction. The significantly reduced total blood flow and positive scotopic threshold response in the 0.5- and 1-nmol ET-1 groups at day 10 were recovered at day 28. A-wave magnitude was also significantly reduced at days 10 and 28. While a comparable and significant reduction in retinal nerve fiber layer thickness was detected in all ET-1 groups at day 56, the 1-nmol group was the earliest to develop such change at day 28. All ET-1 groups showed a transient inner retinal layer thinning at days 10 and 28 and a plateaued outer layer thickness at days 10 to 56. The 1-nmol group showed a significant RGC loss over all retinal locations examined at day 28 as compared with PBS control. As for the lower-dosage groups, significant RGC density loss at central and midperipheral retina was detected at day 56 when compared with day 28. ET-1 injection in mice resulted in a transient vascular constriction and reduction in retinal functions, as well as a gradual loss of retinal nerve fiber layer and RGC in a dose-dependent manner.

Cerebrospinal Fluid Pressure Reduction Induces Glia-Mediated Retinal Inflammation and Leads to Retinal Ganglion Cell Injury in Rats.

Low intracranial pressure (LICP)-induced translaminar cribrosa pressure difference (TLCPD) elevation has been proven as a risk factor in glaucomatous neurodegeneration, whereas the underlying retinal immune features of LICP-induced retinal ganglion cells (RGC) injury remain elusive. Here, we identified the retinal immune characteristics of LICP rats, and minocycline (Mino) treatment was utilized to analyze its inhibitory role in glia-mediated retinal inflammation of LICP rats. The results showed that retrograde axonal transport was decreased in LICP rats without significant RGC loss, indicating the RGC injury was at an early stage before the morphological loss. The activation of retinal microglia and astrocytes with morphologic and M1 or A1-marker alternations was detected in TLCPD elevation rats, the activation level is more dramatic in HIOP rats than in the LICP rats (P<0.05). Besides, we detected reduced retinal tight junction protein expressions, accompanied by specific imbalance patterns of T lymphocytes in the retina of both LICP and HIOP rats (P<0.05). Further Mino treatment showed an effective inhibitory role in glia-driven inflammatory responses in LICP rats, including improving retrograde axonal transport, inhibiting retinal glial activation and proinflammatory subtype polarization, and alleviating the blood-retina barrier compromise. This study identified the glia-mediated retinal inflammation features triggered by LICP stimulus, and Mino application exhibited an effective role in the inhibition of retinal glia-mediated inflammation in LICP-induced TLCPD elevation rats.

Colour vision changes in patients with pressure differences across the lamina cribrosa

AbstractThe retinal nerve fibre layer (RNFL) thickness parameters describe the integrity of retinal ganglion cells (RGC) and their axons and can be affected in pathological processes involving the optic nerve. Glaucoma and papilledema are two conditions where RGCs and hence RNFL, are damaged due to the pressure differences across the lamina cribrosa. The latter separates the intraocular and intracranial pressure compartments. In glaucoma, the intraocular pressure (IOP) is elevated and in papilledema, the intracranial pressure is elevated. When intracranial pressure is managed, papilledema patients can recover without significant loss of RGC. Conversely irreversible RGC loss can occur in severe cases.Subtle, functional changes that are often not detected clinically can remain following recovery. The detection of such changes require sensitive functional tests and a knowledge of normal age limits for the stimulus attribute investigated.Results of preliminary studies designed to assess how colour vision and rod and cone mediated flicker sensitivity recover in papilledema will be reported for a number of patients. Following recovery, the results often reveal binocular summation effects, even when the differences between the two eyes remain significant. Patients classed clinically as fully recovered continue to show small differences between the two eyes, even when the measured binocular thresholds are within the expected age limits.In summary, the preliminary findings from this investigation show how various attributes of vision are affected during the early stages of the disease and after treatment and recovery from late stage of severe papilledema with loss of RGCs.

Heat Shock Protein Upregulation Supplemental to Complex mRNA Alterations in Autoimmune Glaucoma.

Glaucomatous optic neuropathy is a common cause for blindness. An elevated intraocular pressure is the main risk factor, but also a contribution of the immune system seems likely. In the experimental autoimmune glaucoma model used here, systemic immunization with an optic nerve homogenate antigen (ONA) leads to retinal ganglion cell (RGC) and optic nerve degeneration. We processed retinae for quantitative real-time PCR and immunohistology 28 days after immunization. Furthermore, we performed mRNA profiling in this model for the first time. We detected a significant RGC loss in the ONA retinae. This was accompanied by an upregulation of mRNA expression of genes belonging to the heat shock protein family. Furthermore, mRNA expression levels of the genes of the immune system, such as C1qa, C1qb, Il18, and Nfkb1, were upregulated in ONA animals. After laser microdissection, inner retinal layers were used for mRNA microarrays. Nine of these probes were significantly upregulated in ONA animals (p < 0.05), including Hba-a1 and Cxcl10, while fifteen probes were significantly downregulated in ONA animals (p < 0.05), such as Gdf15 and Wwox. Taken together, these findings provide further insights into the pivotal role of the immune response in glaucomatous optic neuropathy and could help to identify novel diagnostic or therapeutic strategies.

Crystallins Play a Crucial Role in Glaucoma and Promote Neuronal Cell Survival in an In Vitro Model Through Modulating Müller Cell Secretion

PurposeThe aim of this study was to explore the roles of crystallins in the context of aging in glaucoma and potential mechanisms of neuroprotection in an experimental animal model of glaucoma.MethodsIntraocular pressure (IOP) was significantly elevated for 8 weeks in animals at different ages (10 days, 12 weeks, and 44 weeks) by episcleral vein cauterization. Retinal ganglion cells (RGCs) were quantified by anti-Brn3a immunohistochemical staining (IHC). Proteomics using ESI-LTQ Orbitrap XL-MS was used to analyze the presence and abundance of crystallin isoforms the retinal samples, respectively. Neuroprotective property and localization of three selected crystallins CRYAB, CRYBB2, and CRYGB as most significantly changed in retina and retinal layers were determined by IHC. Their expressions and endocytic uptakes into Müller cells were analyzed by IHC and Western blotting. Müller cell secretion of neurotrophic factors into the supernatant following CRYAB, CRYBB2, and CRYGB supplementation in vitro was measured via microarray.ResultsIOP elevation resulted in significant RGC loss in all age groups (P < 0.001). The loss increased with aging. Proteomics analysis revealed in parallel a significant decrease of crystallin abundance – especially CRYAB, CRYBB2, and CRYGB. Significant neuroprotective effects of CRYAB, CRYBB2, and CRYGB after addition to retinal cultures were demonstrated (P < 0.001). Endocytic uptake of CRYAB, CRYBB2, and CRYGB was seen in Müller cells with subsequent increased secretion of various neurotrophic factors into the supernatant, including nerve growth factor, clusterin, and matrix metallopeptidase 9.ConclusionsAn age-dependent decrease in CRYAB, CRYBB2, and CRYGB abundance is found going along with increased RGC loss. Addition of CRYAB, CRYBB2, and CRYGB to culture protected RGCs in vitro. CRYAB, CRYBB2, and CRYGB were uptaken into Müller cells. Secretion of neurotrophic factors was increased as a potential mode of action.

Investigating the role of Ca2+/calmodulin-dependent protein kinase II in the survival of retinal ganglion cells.

Investigating the role of Ca2+/calmodulin-dependent protein kinase II in the survival of retinal ganglion cells.

Complement component 3 from astrocytes mediates retinal ganglion cell loss during neuroinflammation.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) characterized by varying degrees of secondary neurodegeneration. Retinal ganglion cells (RGC) are lost in MS in association with optic neuritis but the mechanisms of neuronal injury remain unclear. Complement component C3 has been implicated in retinal and cerebral synaptic pathology that may precede neurodegeneration. Herein, we examined post-mortem MS retinas, and then used a mouse model, experimental autoimmune encephalomyelitis (EAE), to examine the role of C3 in the pathogenesis of RGC loss associated with optic neuritis. First, we show extensive C3 expression in astrocytes (C3+/GFAP+ cells) and significant RGC loss (RBPMS+ cells) in post-mortem retinas from people with MS compared to retinas from non-MS individuals. A patient with progressive MS with a remote history of optic neuritis showed marked reactive astrogliosis with C3 expression in the inner retina extending into deeper layers in the affected eye more than the unaffected eye. To study whether C3 mediates retinal degeneration, we utilized global C3-/- EAE mice and found that they had less RGC loss and partially preserved neurites in the retina compared with C3+/+ EAE mice. C3-/- EAE mice had fewer axonal swellings in the optic nerve, reflecting reduced axonal injury, but had no changes in demyelination or T cell infiltration into the CNS. Using a C3-tdTomato reporter mouse line, we show definitive evidence of C3 expression in astrocytes in the retina and optic nerves of EAE mice. Conditional deletion of C3 in astrocytes showed RGC protection replicating the effects seen in the global knockouts. These data implicate astrocyte C3 expression as a critical mediator of retinal neuronal pathology in EAE and MS, and are consistent with recent studies showing C3 gene variants are associated with faster rates of retinal neurodegeneration in human disease.

Oxidative Stress and Hypoxia Modify Mitochondrial Homeostasis During Glaucoma.

Aims: Cellular response to hypoxia can include transition from respiration to glycolysis via upregulation of glycolytic enzymes and transporters, as well as mitophagy induction to eliminate surplus mitochondria. Our purpose was to evaluate the impact of hypoxia-inducible factor-1α (HIF-1α) stabilization on mitochondrial homeostasis and oxidative stress in a chronic model of glaucoma. Results: Retina and optic nerve (ON) were evaluated from young and aged DBA/2J (D2) glaucoma model mice and the control strain, the DBA/2-Gpnmb+. Hypoxic retinal ganglion cells (RGCs) were observed in young and aged D2 retina, with a significant increase in HIF-1α protein in the aged D2 retina. Reactive oxygen species observed in young D2 retina and ON were followed by significant decreases in antioxidant capacity in aged D2 retina and ON. HIF-1α targets such as neuron-specific glucose transporter-3 and lactate dehydrogenase were decreased or unchanged, respectively, in aged D2 retina despite an increased hypoxia response in RGCs. Mitochondrial mass was decreased in aged D2 retina concomitant with decreased mitochondrially encoded electron transport chain transcripts despite a stable nuclear-encoded TFAM (mitochondrial transcription factor), suggesting a breakdown in the nuclear-mitochondrial communication. Decreased mitophagy-associated proteins p62 and Rheb were observed in aged D2 retina, although p62 was significantly increased in the aged D2 ON. Innovation and Conclusion: The increased reactive oxygen species concomitant with HIF-1α upregulation despite reduced glucose transporters, mis-match of nuclear- and mitochondrial-encoded transcripts, and signs of reduced mitophagy suggest that retinas from D2 mice with chronic intraocular pressure elevation transition to pseudohypoxia without consistent metabolic reprogramming before significant RGC loss. Antioxid. Redox Signal. 35, 1341-1357.

Differential susceptibility of retinal ganglion cell subtypes in acute and chronic models of injury and disease

Retinal ganglion cells (RGCs) are a heterogeneous population of neurons, comprised of numerous subtypes that work synchronously to transmit visual information to the brain. In blinding disorders such as glaucoma, RGCs are the main cell type to degenerate and lead to loss of vision. Previous studies have identified and characterized a variety of RGC subtypes in animal models, although only a handful of studies demonstrate the differential loss of these RGC subtypes in response to disease or injury. Thus, efforts of the current study utilized both chronic (bead occlusion) and acute (optic nerve crush, ONC) rat models to characterize disease response and differential loss of RGC subtypes. Bead occlusion and ONC retinas demonstrated significant RGC loss, glial reactivity and apoptosis compared to control retinas. Importantly, bead occlusion and ONC retinas resulted in differential subtype-specific loss of RGCs, with a high susceptibility for alpha- and direction selective-RGCs and preferential survival of ipRGCs. Results of this study serve as an important foundation for future experiments focused on the mechanisms resulting in the loss of RGCs in optic neuropathies, as well as the development of targeted therapeutics for RGC subtype-specific neuroprotection.

Hydrogen Sulfide and β-Synuclein Are Involved and Interlinked in the Aging Glaucomatous Retina.

Purpose Glaucoma, one of the leading causes of irreversible blindness worldwide, is a group of disorders characterized by progressive retinal ganglion cell (RGC) loss. Synucleins, a family of small proteins, have been of interest in studies of neurodegeneration and CNS. However, their roles and functions in glaucoma are still not completely understood and remain to be explored. Our previous studies showed that α-synuclein and H2S play a pivotal role in glaucoma. This study aims to (1) elucidate the potential roles and functions of synucleins in glaucoma throughout aging, (2) investigate the interaction between the synucleins and H2S, and better understand the mechanism of H2S in neuroprotection. Methods The chronic IOP elevation model was carried out in 12 animals at different ages (3 months and 14 months), and RGCs were quantified by Brn3a staining. Mass spectrometric-assisted proteomics analysis was employed to measure synuclein levels and H2S producing proteins in retina. Secondly, the acute IOP elevation model was carried out in 12 juvenile animals, with or without intravitreal injection of GYY4137 (a H2S donor). RGCs were quantified along with the abundancy of synucleins. Results RGCs and β-synuclein (SNCB) are significantly changed in old animals. Under chronic IOP elevation, there is a significant RGC loss in old animals, whereas no significant change in young animals; SNCB is significantly downregulated and 3MST is significantly upregulated in young animals due to IOP, while no significant changes in old ones are notable. Under acute IOP elevation (approx. 55 mmHg), a significant RGC loss is observed; exogenous H2S significantly reduced RGC loss and downregulated SNCB levels. Conclusion The present study indicates a strong link between ageing and SNCB regulation. In young animals SNCB is downregulated going along with less RGC loss. Furthermore, increasing endogenous H2S is effective to downregulate SNCB and is neuroprotective against acute IOP elevation.

INOS-inhibitor driven neuroprotection in a porcine retina organ culture model.

Nitrite oxide plays an important role in the pathogenesis of various retinal diseases, especially when hypoxic processes are involved. This degeneration can be simulated by incubating porcine retinal explants with CoCl2. Here, the therapeutic potential of iNOS‐inhibitor 1400W was evaluated. Degeneration through CoCl2 and treatment with the 1400W were applied simultaneously to porcine retinae explants. Three groups were compared: control, CoCl2, and CoCl2 + iNOS‐inhibitor (1400W). At days 4 and 8, retinal ganglion cells (RGCs), bipolar, and amacrine cells were analysed. Furthermore, the influence on the glia cells and different stress markers were evaluated. Treatment with CoCl2 resulted in a significant loss of RGCs already after 4 days, which was counteracted by the iNOS‐inhibitor. Expression of HIF‐1α and its downstream targets confirmed the effective treatment with 1400W. After 8 days, the CoCl2 group displayed a significant loss in amacrine cells and also a drastic reduction in bipolar cells was observed, which was prevented by 1400W. The decrease in microglia could not be prevented by the inhibitor. CoCl2 induces strong degeneration in porcine retinae by mimicking hypoxia, damaging certain retinal cell types. Treatment with the iNOS‐inhibitor counteracted these effects to some extent, by preventing loss of retinal ganglion and bipolar cells. Hence, this inhibitor seems to be a very promising treatment for retinal diseases.

Decreased Amplitude of the Photopic Negative Response (PhNR) in the Ins2Akita Mouse Model of Type 1 Diabetes Mellitus

Diabetic retinopathy (DR) is a common, severe complication of diabetes mellitus. Current clinical detection of DR is based primarily on visible retinal vascular damage, and earlier detection of impending DR might allow earlier treatment to protect function. Recent studies in both animal models and humans with diabetes have shown that degradation of retinal ganglion cell (RGC) function occurs at an early stage, before visible vascular complications and before significant RGC loss. The photopic negative response (PhNR), obtained from full‐field electroretinogram (ERG) with photopic stimuli is thought to arise directly from RGC activity. Here, we hypothesized that PhNR amplitudes are reduced in a type 1 diabetic mouse model, the Ins2Akita mouse, which develops hyperglycemia by 5 weeks of age and visual function and ocular blood flow deficits early in the disease progression, before overtly visible vascular damage. Diabetic (male heterozygotes) and control (male WT littermates) mice were grouped according to age: young, adult, and old (5–6, 9–11, and 12–14 months of age, respectively). ERG recording was performed with 1.62 log cd/m2 white flashes over a constant 40 cd.s/m2 green background (Diagnosys Espion and ColorDome ganzfeld system) using gold ring contact lens electrodes (LKC Techniologies) under isoflurane anesthesia. Following 1 hour adaptation to the green background illumination, PhNR, a‐wave, and b‐wave amplitudes and implicit times were measured in response to 25 10‐msec flashes.The mean PhNR amplitude of the adult diabetic mice was 36% of their age‐matched controls (p &lt; 0.01). For the old diabetic group, the mean PhNR amplitude was 49% of their age‐matched controls (p &lt; 0.05). However, no significant difference was found in the young group. A‐wave amplitudes were decreased in the diabetic adult group, to a value only 41% of their age‐matched controls (p &lt; 0.05). B‐wave amplitudes were decreased in the diabetic old group, to 35% of their age‐matched controls (p &lt; 0.05). However, no significant difference was found among groups for PhNR, a‐wave, or b‐wave implicit times. Our results suggest that PhNR amplitude is altered relatively early in the Ins2Akita mouse model of diabetes. This suggests PhNR measurements could be used to detect diabetic retinopathy at earlier stages than the current clinical standard.Support or Funding InformationNIH R01 EY023290 and UIWRSO Faculty Development GrantThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Quantification of Changes in Visual Function During Disease Development in a Mouse Model of Pigmentary Glaucoma.

We investigated the relationship between visual parameters that are commonly affected during glaucomatous disease progression with functional measures of retina physiology using electroretinography and behavioral measures of visual function in a mouse model of glaucoma. Electroretinogram components measuring retinal ganglion cell (RGC) responses were determined using the non-invasive Ganzfeld flash electroretinography (fERG) to assess RGC loss in a mouse model of glaucoma. Intraocular pressure (IOP), behaviorally assessed measures of visual function, namely visual acuity and contrast sensitivity as well as fERG responses were recorded in 4- and 11-month-old male DBA/2 mice. Scotopic threshold response (STR) and photopic negative response components as well as oscillatory potentials (OPs) were isolated from fERG responses and correlated with IOP, optomotor reflex measurements, and RGC counts. The 11-month-old DBA/2 mice had significantly elevated IOP, reduced visual performance, as assessed behaviorally, significant RGC loss, deficits in standardized fERG responses, reduced STRs, and differences in OP amplitudes and latencies, when compared with 4-month-old mice of the same strain. STRs and OPs correlated with some visual and physiological parameters. In addition, elevated IOP and RGC loss correlated positively with measures of visual function, specifically with surrogate measures of RGC function derived from fERG. Our data suggest that RGC function as well as interactions of RGCs with other retinal cell types is impaired during glaucoma. In addition, a later OP wavelet denoted as OP4 in this study was identified as a very reproducible indicator of loss of visual function in the glaucoma mouse model.