Retinal Ganglion Cell Function Research Articles

To explore the protective effect and mechanism of KLF7 overexpression on retinal ganglion cells after optic nerve crush in mice.

To investigate the protective effect and mechanism of Krüppel-like factor 7 (KLF7) on retinal ganglion cells (RGCs) after optic nerve crush (ONC). Ninety 10-week-old C57BL/6J mice were randomly assigned to five groups. The blank control group (group A), IVT (intravitreal injection) of KLF7 (group B), IVT of phosphate-buffered saline after ONC (group C), IVT of KLF7 after ONC (group D), and IVT of green fluorescent protein after ONC (group E). Retinal electroretinography and immunofluorescence staining were performed to observe the function and survival rate of RGCs on days 3 and 7 after ONC. The Western Blot determined the expressions of JNK 1, ERK, P38, NF-κB, IL-1, IL-6, and TNF-α seven days after ONC. After ONC, KLF7 gradually increased from day 3 to day 14, with the peak noted a peak on day 7. On day 7 after ONC, the RGC survival rate in Group D was significantly higher than in groups C (P = 0.028) and E (P = 0.007). The negative PhNR wave amplitude significantly decreased in groups C (P = 0.03) and E (P = 0.04) compared to group D. Moreover, group D also showed significantly higher ERK levels among the groups (all P < 0.01); NF-κB, IL-1, IL-6, and TNF-α expression decreased significantly in group D compared with groups C and E (all P < 0.01). Overexpression of KLF7 improved the survival rate and function of RGCs in mice ONC models by activating the ERK pathway and inhibiting relevant inflammatory factors. KLF7 may be a promising protective factor against optic nerve damage.

Progressive Optic Neuropathy in Hydrocephalic Ccdc13 Mutant Mice Caused by Impaired Axoplasmic Transport at the Optic Nerve Head.

Optic nerve head (ONH) atrophy is frequently associated with hydrocephalic conditions. Cerebrospinal fluid (CSF)-containing meninges form a subarachnoid space that terminates at the ONH, which physically impacts it. This study aims to characterize optic neuropathy in congenital hydrocephalic mice with genetic disruption of the Ccdc13 gene. The ccdc13 germline knockout mice were generated. The hydrocephalus phenotype and subarachnoid space surrounding the optic nerve were evaluated using routine histology and Evans blue stain. Optic neuropathy was examined with immunohistochemistry and transmission electron microscopy (TEM). Axon transport was indicated by cholera toxin subunit B (CTB) fluorescence conjugate. Retinal function was evaluated by electroretinography (ERG), and Ccdc13 expression was revealed by a knock-in Gfp reporter. Ccdc13 mutant mice manifested hydrocephalus at birth. ONH displacement, or negative cupping, and enlarged subarachnoid space at the optic terminus occurred as early as 1 month after birth. Intraocular pressure (IOP) was normal. Optic neuropathy was first observed at the ONH, followed by a distal-to-proximal progression of optic nerve pathology indicated by alteration of axonal ultrastructure and deposition of unphosphorylated neurofilament heavy chain. Anterograde axonal transport was also hampered. Retinal ganglion cell (RGC) function was compromised as early as postnatal day 21 (P21), along with reduced neurofilament heavy chain expression. Optic neuropathy caused by disruption of Ccd13 was non-cell autonomous, stemming from hydrocephalus with presumed high intracranial pressure (ICP), which physically impacts the ONH by increasing the translaminar pressure gradient. We provided knowledge of optic neuropathy from a congenital mouse model for hydrocephalus. The hydrocephalus in mice could damage the ONH by increasing the translaminar pressure gradient and negative cupping, leading to impairment in axoplasmic transport and RGC pathology. Our findings highlight the importance of the interplay between IOP and ICP in the development of glaucoma.

NFATc4 Knockout Promotes Neuroprotection and Retinal Ganglion Cell Regeneration After Optic Nerve Injury.

Retinal ganglion cells (RGCs), neurons transmitting visual information via the optic nerve, fail to regenerate their axons after injury. The progressive loss of RGC function underlies the pathophysiology of glaucoma and other optic neuropathies, often leading to irreversible blindness. Therefore, there is an urgent need to identify the regulators of RGC survival and the regenerative program. In this study, we investigated the role of the family of transcription factors known as nuclear factor of activated T cells (NFAT), which are expressed in the retina; however, their role in RGC survival after injury is unknown. Using the optic nerve crush (ONC) model, widely employed to study optic neuropathies and central nervous system axon injury, we found that NFATc4 is specifically but transiently up-regulated in response to mechanical injury. In the injured retina, NFATc4 immunolocalized primarily to the ganglionic cell layer. Utilizing NFATc4-/- and NFATc3-/- mice, we demonstrated that NFATc4, but not NFATc3, knockout increased RGC survival, improved retina function, and delayed axonal degeneration. Microarray screening data, along with decreased immunostaining of cleaved caspase-3, revealed that NFATc4 knockout was protective against ONC-induced degeneration by suppressing pro-apoptotic signaling. Finally, we used lentiviral-mediated NFATc4 delivery to the retina of NFATc4-/- mice and reversed the pro-survival effect of NFATc4 knockout, conclusively linking the enhanced survival of injured RGCs to NFATc4-dependent mechanisms. In summary, this study is the first to demonstrate that NFATc4 knockout may confer transient RGC neuroprotection and decelerate axonal degeneration after injury, providing a potent therapeutic strategy for optic neuropathies.

The relationship between ON-OFF function and OCT structural and angiographic parameters in early diabetic retinal disease.

This study measured associations between ON and OFF functional indicators and structural optical coherence tomography (OCT) and OCT angiography (OCTA) markers in diabetic retinal disease. Fifty-four participants with type 1 or type 2 diabetes (mean age = 34.1 years; range 18-60) and 48 age-matched controls (mean age = 35.4 years, range 18-59) underwent visual psychophysical testing, OCT and OCTA retinal imaging. Psychophysical tasks measuring (A) contrast increment and decrement sensitivity and (B) response times to increment and decrement targets were assessed as surrogate measures of ON and OFF retinal ganglion cell function. The group with diabetes had worse foveal contrast increment and decrement thresholds (p = 0.04) and were slower to search for increment and decrement targets relative to controls (p = 0.009). Individuals with diabetes had a less circular foveal avascular zone (FAZ) (p < 0.001) but did not differ from controls in foveal vessel density and FAZ area. Functional and structural outcome measures related to the peripheral retina were also comparable between those with and without diabetes. Functional responses to increments and decrements were not significantly correlated with FAZ circularity or vessel density in individuals with diabetes. Diabetic retinal disease results in impaired performance on measures of inferred ON and OFF pathway function in addition to vascular deficits measurable with OCTA. Future longitudinal studies may determine the temporal relationship between these deficits, and whether they predict future diabetic retinopathy.

Use of Electrophysiology in the Diagnosis of Optic Nerve Disorders

Visual electrodiagnostic tests are non invasive, easily applicable that enable objective evaluation of the functions of the visual pathways. The most preferred among these test are; electroretinography (ERG), pattern electroretinography (PERG), electrooculography (EOG) and visual evoked potential (VEP). ERG is used to evaluate retinal functions especially rod and cone responses. PERG is used to evaluate functions of macular and retinal ganglion cells. EOG is used to evaluate retinal pigment epithelium functions. VEP is preferred method for evaluating the visual pathways up to the optic nerve and occipital cortex as a whole. Photopic negative response (PhNR) is originates from the electrical activity of amacrine and glial cells and also mostly retinal ganglion cells. In this study, VEP, PERG and PhNR tests which are the most preferred in the diagnosis of optic nerve disorders, will be mainly mentioned.

Effect of brimonidine on visual indices in patients with acute optic neuritis: A single blind randomized clinical trial

PurposeThis study investigates brimonidine's potential effect on visual functions, particularly contrast sensitivity (CS), an indicator of retinal ganglion cell function. MethodsIn this single-blind, randomized clinical trial, 60 patients (aged 23–56) with first-episode acute optic neuritis within seven days of symptom onset were randomly assigned to brimonidine or control groups. The intervention group received brimonidine three times daily for three months, while the control group received synthetic tears with the same dosage and frequency. Primary outcomes were changes in CS, visual acuity (VA), and color vision at one and three months post-treatment. Repeated measures ANOVA was used to assess statistically significant and partial eta squared (η2) values, mean differences, and clinically significance important were reported. ResultsAll participants completed the study without complications. VA improved significantly in both groups by follow-up end (p < 0.001), with significant improvement from first to third month only in the brimonidine group (p < 0.001). The mean VA difference between groups was not statistically and clinically significant. CS showed statistically significant improvement within both groups (p < 0.001) and between groups (p < 0.001), with a large effect size (partial η2 = 0.28). The mean CS difference between groups (14.5) was clinically considerable. No significant changes in color vision were observed between groups (p = 0.96). ConclusionBrimonidine significantly improved contrast sensitivity compared to placebo and was well-tolerated. Its neuroprotective effects suggest it may be beneficial in treating optic neuritis and preserving retinal ganglion cell function. Trial registrationProspectively registered at Iranian Clinical Trial Registration; Registration date 3 December 2022; Registration number: IRCT20221127056631N1

GABAergic Retinal Ganglion Cells Projecting to the Superior Colliculus Mediate the Looming-Evoked Flight Response.

The looming stimulus-evoked flight response to approaching predators is a defensive behavior in most animals. However, how looming stimuli are detected in the retina and transmitted to the brain remains unclear. Here, we report that a group of GABAergic retinal ganglion cells (RGCs) projecting to the superior colliculus (SC) transmit looming signals from the retina to the brain, mediating the looming-evoked flight behavior by releasing GABA. GAD2-Cre and vGAT-Cre transgenic mice were used in combination with Cre-activated anterograde or retrograde tracer viruses to map the inputs to specific GABAergic RGC circuits. Optogenetic technology was used to assess the function of SC-projecting GABAergic RGCs (scpgRGCs) in the SC. FDIO-DTA (Flp-dependent Double-Floxed Inverted Open reading frame-Diphtheria toxin) combined with the FLP (Florfenicol, Lincomycin & Prednisolone) approach was used to ablate or silence scpgRGCs. In the mouse retina, GABAergic RGCs project to different brain areas, including the SC. ScpgRGCs are monosynaptically connected to parvalbumin-positive SC neurons known to be required for the looming-evoked flight response. Optogenetic activation of scpgRGCs triggers GABA-mediated inhibition in SC neurons. Ablation or silencing of scpgRGCs compromises looming-evoked flight responses without affecting image-forming functions. Our study reveals that scpgRGCs control the looming-evoked flight response by regulating SC neurons via GABA, providing novel insight into the regulation of innate defensive behaviors.

Altered Functional Responses of the Retina in B6 Albino Tyrc/c Mice.

Mammals with albinism present low visual discrimination ability and different proportions of certain retinal cell subtypes. As the spatial resolution of the retina depends on the visual field sampling by retinal ganglion cells (RGCs) based on the convergence of upstream cell inputs, it could be affected in albinism and thus modify the RGC function. We used the Tyrc/c line, a mouse model of oculocutaneous albinism type 1 (OCA1), carrying a tyrosinase mutation, and previously characterized by a total absence of pigment and severe visual deficits. To assess their retinal function, we recorded the light responses of hundreds of RGCs ex vivo using multi-electrode array (MEA). We estimated the receptive field (RF)-center diameter of Tyr+/c and Tyrc/c RGCs using a checkerboard stimulation before simultaneously stimulating the center and surround of RGC RFs with full-field flashes. Following checkerboard stimulation, the RF-center diameters of RGCs were indistinguishable between Tyrc/c and Tyr+/c retinas. Nevertheless, RGCs from Tyrc/c retinas presented more OFF responses to full-field flashes than RGCs from Tyr+/c retinas. Unlike Tyr+/c retinas, very few OFF-center RGCs switched polarity to ON or ON-OFF responses after full-field flashes in Tyrc/c retinas, suggesting a different surround suppression in these retinas. The retinal output signal is affected in Tyrc/c retinas, despite intact RF-center diameters of their RGCs. Adaptive mechanisms during development are probably responsible for this change in RGC responses, related to the absence of ocular pigments.

Effects of Acute High-Altitude Exposure on Morphology and Function of Retinal Ganglion Cell in Mice.

High altitude retinopathy (HAR) is a retinal functional disorder caused by inadequate adaptation after exposure to high altitude. However, the cellular and molecular mechanisms underlying retinal dysfunction remain elusive. Retinal ganglion cell (RGC) injury is the most important pathological basis for most retinal and optic nerve diseases. Studies focusing on RGC injury after high-altitude exposure (HAE) are scanty. Therefore, the present study sought to explore both functional and morphological alterations of RGCs after HAE. A mouse model of acute hypobaric hypoxia was established by mimicking the conditions of a high altitude of 5000 m. After HAE for 2, 4, 6, 10, 24, and 72hours, the functional and morphological alterations of RGCs were assessed using retinal hematoxylin and eosin (H&E) sections, retinal whole mounts, transmission electron microscopy (TEM), and the photopic negative response (PhNR) of the electroretinogram. Compared with the control group, the thickness of the ganglion cell layer and retinal nerve fiber layer increased significantly, RGC loss remained significant, and the amplitudes of a-wave, b-wave, and PhNR were significantly reduced after HAE. In addition, RGCs and their axons exhibited an abnormal ultrastructure after HAE, including nuclear membrane abnormalities, uneven distribution of chromatin in the nucleus, decreased cytoplasmic electron density, widening and vacuolization of the gap between axons, loosening and disorder of myelin sheath structure, widening of the gap between myelin sheath and axon membrane, decreased axoplasmic density, unclear microtubule and nerve fiber structure, and abnormal mitochondrial structure (mostly swollen, with widened membrane gaps and reduced cristae and vacuolization). The study findings confirm that the morphology and function of RGCs are damaged after HAE. These findings lay the foundation for further study of the specific molecular mechanisms of HAR and promote the effective prevention.

Electroretinographic changes in the inner and outer retinal layers before and after intravenous chemotherapy for retinoblastoma.

To study the inner and outer retinal functions using a full-field electroretinogram (ERG) before and after intravenous chemotherapy (IVC) in children with retinoblastoma (RB). Of the 11 eyes, seven had RB and four were normal. All children were examined under anesthesia using a handheld ERG machine with a standard protocol - light-adapted single-flash ERG (fERG), photopic single-flash 3.0- and 30-Hz flickers, and photopic negative response (PhNR) amplitudes at 72 ms (P72). The amplitudes and peak times were compared before and after IVC. Post-chemotherapy tumor regressed in all seven eyes. Of the seven eyes, the fERG peak time (a-wave) was delayed in two eyes (29%), whereas the b-wave was delayed in six eyes (86%). The fERG amplitude height for a- and b-waves decreased in five eyes (71%) and six eyes (86%), respectively. In addition, photopic flicker 30-Hz b-wave peak time delayed in five eyes (71%), whereas the b-wave amplitude height decreased in six eyes (86%). Simultaneously, the P72 amplitude height decreased in six eyes (86%), whereas the P-ratio increased in all seven eyes (100%). In comparison, the ERG responses improved in three of the four contralateral normal eyes. Overall, the cone function improved in two eyes (29%), whereas cone bipolar cell and retinal ganglion cell (RGC) function improved in one eye (14%) each. Comparison of light-adapted ERG changes before and after IVC showed reduced amplitudes and delayed peak times for both a and b waveforms, as well as reduced PhNR amplitude attributable to bipolar and RGC injury.

Longitudinal changes in retinal ganglion cell function in optic pathway glioma evaluated by photopic negative response

Photopic negative response (PhNR), an index of retinal ganglion cell (RGC) function, is impaired in patients with optic pathway gliomas (OPGs). The aim of this longitudinal study was to evaluate whether PhNR deteriorates over time in OPG patients. Fourteen pediatric patients affected by OPG (4 males and 10 females, mean age 12.4 ± 5.7 years, 8 with neurofibromatosis type 1 [NF1]) with ≥12 months of follow-up and ≥2 evaluations, were included in this retrospective study. All patients had received chemotherapy, with or without OPG surgical resection, at least 5 years prior to the study. At baseline, all patients underwent a complete ophthalmological examination. Follow-up included clinical examination and PhNR measurement as well as brain MRI (according to pediatric oncologist indications) every 6 or 12 months. Mean follow-up duration was 16.7 ± 7.5 months (range 12–36 months). Photopic electroretinograms were elicited by 2.0 cd-s/m2 Ganzfeld white flashes presented on a steady 20 cd/m2 white background. The PhNR amplitude was measured as the difference between baseline and the maximal negative amplitude (minimum) of the negative wave, following the photopic b-wave. Compared to baseline, mean PhNR amplitude was significantly decreased at the end of follow-up (p = 0.008). NF1-related OPGs exhibited a decline in PhNR amplitude (p = 0.005) and an increase in PhNR peak-time during the follow-up (p = 0.013), whereas sporadic OPGs showed no significant changes. Tumor size remained stable in all patients on MRI. PhNR amplitude decreased over the observation period, suggesting progressive RGC dysfunction in NF1-related pediatric OPGs, despite stable size on MRI imaging. PhNR could serve as a non-invasive objective tool for assessing longitudinal changes in RGC function in the clinical management of childhood OPG.

Lycium ruthenicum water extract preserves retinal ganglion cells in chronic ocular hypertension mouse models.

Lycium ruthenicum Murray (LR), known as "black goji berry" or "black wolfberry", is widely utilized in chinese herbal medicine. LR fruit showed its antioxidant and/or anti-inflammation activity in treating cardiac injury, experimental colitis, nonalcoholic fatty liver disease, fatigue, and aging. Glaucoma is the leading cause of irreversible blindness. Besides elevated intraocular pressure (IOP), oxidative stress and neuroinflammation were recognized to contribute to the pathogenesis of glaucoma. This study investigated the treatment effects of LR water extract (LRE) on retinal ganglion cells (RGCs) threatened by sustained IOP elevation in a laser-induced chronic ocular hypertension (COH) mouse model and the DBA/2J mouse strain. The antioxidation and anti-inflammation effects of LRE were further tested in the H2O2-challenged immortalized microglial (IMG) cell line in vitro. LRE oral feeding (2g/kg) preserved the function of RGCs and promoted their survival in both models mimicking glaucoma. LRE decreased 8-hydroxyguanosine (oxidative stress marker) expression in the retina. LRE reduced the number of Iba-1+ microglia in the retina of COH mice, but not in the DBA/2J mice. At the mRNA level, LRE reversed the COH induced HO-1 and SOD-2 overexpressions in the retina of COH mice. Further in vitro study demonstrated that LRE pretreatment to IMG cells could significantly reduce H2O2 induced oxidative stress through upregulation of GPX-4, Prdx-5, HO-1, and SOD-2. Our work demonstrated that daily oral intake of LRE can be used as a preventative/treatment agent to protect RGCs under high IOP stress probably through reducing oxidative stress and inhibiting microglial activation in the retina.

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.

Topical administration of GLP-1 eyedrops improves retinal ganglion cell function by facilitating presynaptic GABA release in early experimental diabetes.

Diabetic retinopathy is a prominent cause of blindness in adults, with early retinal ganglion cell (RGC) loss contributing to visual dysfunction or blindness. In the brain, defects in y-aminobutyric acid (GABA) synaptic transmission are associated with pathophysiological and neurodegenerative disorders, whereas glucagon-like peptide-1 (GLP-1) has demonstrated neuroprotective effects. However, it is not yet clear whether diabetes causes alterations in inhibitory input to RGCs and whether and how GLP-1 protects against neurodegeneration in the diabetic retina through regulating inhibitory synaptic transmission to RGCs. In the present study, we used the patch-clamp technique to record GABA subtype A receptor-mediated miniature inhibitory postsynaptic currents (mIPSCs) in RGCs from streptozotocin-induced diabetes model rats. We found that early diabetes (4 weeks of hyperglycemia) decreased the frequency of GABAergic mIPSCs in RGCs without altering their amplitude, suggesting a reduction in the spontaneous release of GABA to RGCs. Topical administration of GLP-1 eyedrops over a period of 2 weeks effectively countered the hyperglycemia-induced downregulation of GABAergic mIPSC frequency, subsequently enhancing the survival of RGCs. Concurrently, the protective effects of GLP-1 on RGCs in diabetic rats were eliminated by topical administration of exendin-9-39, a specific GLP-1 receptor antagonist, or SR95531, a specific antagonist of the GABA subtype A receptor. Furthermore, extracellular perfusion of GLP-1 was found to elevate the frequencies of GABAergic mIPSCs in both ON- and OFF-type RGCs. This elevation was shown to be mediated by activation of the phosphatidylinositol-phospholipase C/inositol 1,4,5-trisphosphate receptor/Ca2+/protein kinase C signaling pathway downstream of GLP-1 receptor activation. Moreover, multielectrode array recordings revealed that GLP-1 functionally augmented the photoresponses of ON-type RGCs. Optomotor response tests demonstrated that diabetic rats exhibited reductions in visual acuity and contrast sensitivity that were significantly ameliorated by topical administration of GLP-1. These results suggest that GLP-1 facilitates the release of GABA onto RGCs through the activation of GLP-1 receptor, leading to the de-excitation of RGC circuits and the inhibition of excitotoxic processes associated with diabetic retinopathy. Collectively, our findings indicate that the GABA system has potential as a therapeutic target for mitigating early-stage diabetic retinopathy. Furthermore, the topical administration of GLP-1 eyedrops represents a non-invasive and effective treatment approach for managing early-stage diabetic retinopathy.

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.

Inner limiting Membrane Peel Extends In vivo Calcium Imaging of Retinal Ganglion Cell Activity Beyond the Fovea in Non-Human Primate.

High resolution retinal imaging paired with intravitreal injection of a viral vector coding for the calcium indicator GCaMP has enabled visualization of activity dependent calcium changes in retinal ganglion cells (RGCs) at single cell resolution in the living eye. The inner limiting membrane (ILM) is a barrier for viral vectors, restricting transduction to a ring of RGCs serving the fovea in both humans and non-human primates (NHP). We evaluate peeling the ILM prior to intravitreal injection as a strategy to expand calcium imaging beyond the fovea in the NHP eye in vivo. Five Macaca fascicularis eyes (age 3-10y; n=3 individuals; 2M, 1F) underwent vitrectomy and 5 to 6-disc diameter ILM peel centered on the fovea prior to intravitreal delivery of 7m8:SNCG:GCaMP8s. Calcium responses from RGCs were recorded using a fluorescence adaptive optics scanning laser ophthalmoscope. In all eyes GCaMP was expressed throughout the peeled area, representing a mean 8-fold enlargement in area of expression relative to a control eye. Calcium recordings were obtained up to 11 degrees from the foveal center. RGC responses were comparable to the fellow control eye and showed no significant decrease over the 6 months post ILM peel, suggesting that RGC function was not compromised by the surgical procedure. In addition, we demonstrate that activity can be recorded directly from the retinal nerve fiber layer. This approach will be valuable for a range of applications in visual neuroscience including pre-clinical evaluation of retinal function, detecting vision loss, and assessing the impact of therapeutic interventions.

Neuroprotective effects of apigenin on retinal ganglion cells in ischemia/reperfusion: modulating mitochondrial dynamics in in vivo and in vitro models

BackgroundRetinal ischemia/reperfusion (RIR) is implicated in various forms of optic neuropathies, yet effective treatments are lacking. RIR leads to the death of retinal ganglion cells (RGCs) and subsequent vision loss, posing detrimental effects on both physical and mental health. Apigenin (API), derived from a wide range of sources, has been reported to exert protective effects against ischemia/reperfusion injuries in various organs, such as the brain, kidney, myocardium, and liver. In this study, we investigated the protective effect of API and its underlying mechanisms on RGC degeneration induced by retinal ischemia/reperfusion (RIR).MethodsAn in vivo model was induced by anterior chamber perfusion following intravitreal injection of API one day prior to the procedure. Meanwhile, an in vitro model was established through 1% oxygen and glucose deprivation. The neuroprotective effects of API were evaluated using H&E staining, spectral-domain optical coherence tomography (SD-OCT), Fluoro-Gold retrograde labeling, and Photopic negative response (PhNR). Furthermore, transmission electron microscopy (TEM) was employed to observe mitochondrial crista morphology and integrity. To elucidate the underlying mechanisms of API, the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, flow cytometry assay, western blot, cell counting kit-8 (CCK-8) assay, lactate dehydrogenase (LDH) assay, JC-1 kit assay, dichlorofluorescein-diacetate (DCFH-DA) assay, as well as TMRE and Mito-tracker staining were conducted.ResultsAPI treatment protected retinal inner plexiform layer (IPL) and ganglion cell complex (GCC), and improved the function of retinal ganglion cells (RGCs). Additionally, API reduced RGC apoptosis and decreased lactate dehydrogenase (LDH) release by upregulating Bcl-2 and Bcl-xL expression, while downregulating Bax and cleaved caspase-3 expression. Furthermore, API increased mitochondrial membrane potential (MMP) and decreased extracellular reactive oxygen species (ROS) production. These effects were achieved by enhancing mitochondrial function, restoring mitochondrial cristae morphology and integrity, and regulating the expression of OPA1, MFN2, and DRP1, thereby regulating mitochondrial dynamics involving fusion and fission.ConclusionAPI protects RGCs against RIR injury by modulating mitochondrial dynamics, promoting mitochondrial fusion and fission.

ISCEV standard for clinical pattern electroretinography (2024 update)

The pattern electroretinogram (PERG) is a localized retinal response evoked by a contrast-reversing pattern, usually a black and white checkerboard, which provides information about macular and retinal ganglion cell function. This document, from the International Society for Clinical Electrophysiology of Vision (ISCEV; www.iscev.org) presents an updated and revised Standard for clinical PERG testing. This replaces the 2013 and all earlier versions. Minimum protocols for basic PERG stimuli, recording methods and reporting are specified, to promote consistency of methods for diagnosis and monitoring purposes, while responding to evolving clinical practices and technology. The main changes in the updated ISCEV Standard for clinical PERG include expanded guidance about large stimulus fields, stimulus parameters for simultaneous PERG and pattern visual evoked potential recording, baseline drift correction, and use of consistent ambient room lighting. These changes aim to provide a clinically relevant document about current practice which will facilitate good quality recordings and inter-laboratory comparisons.

Effect of eccentric fixation on the steady-state pattern electroretinogram

PurposeThe steady-state pattern electroretinogram (ssPERG) is used to assess retinal ganglion cell function in a variety of research contexts and diagnostic applications. In certain groups of patients or study participants, stable central fixation of the stimulus is not guaranteed. The present study aimed at assessing the effects of misfixation on the ssPERG response to checkerboard reversal stimuli.MethodsUsing two check sizes (0.8° and 15°), we compared ssPERG responses for several amounts of fixation deviation, ranging from 0° to 19° horizontally and from 0° to 14° diagonally. The stimulus area extended to 15° eccentricity, stimulus reversal rate was 15/s.ResultsUp to around 7° eccentricity, there was no sizable effect of fixation deviation under most conditions. Effects were somewhat larger for nasal than for temporal deviation, in particular for small checks. Diagonal deviation was associated with a response to luminance onset/offset at 7.5 Hz (subharmonic of the reversal rate), most prominently when the interior of a large check was fixated.ConclusionGenerally, moderate inaccuracies of fixation do not have a sizable effect on ssPERG amplitude. However, with large checks, the luminance response has to be considered.

Dysregulated 24 h melatonin secretion associated with intrinsically photosensitive retinal ganglion cell function in diabetic retinopathy: a cross-sectional study.

The aim of this study was to explore whether diabetic retinopathy is associated with alterations of the circadian system, and to examine the role of reduced intrinsically photosensitive retinal ganglion cell (ipRGC) function. Participants with type 2 diabetes, with diabetic retinopathy (n=14) and without diabetic retinopathy (n=9) underwent 24 h blood sampling for melatonin and cortisol under controlled laboratory conditions. ipRGC function was inferred from the post-illumination pupil response (PIPR). Habitual sleep duration, efficiency and variability were assessed by actigraphy. Participants with diabetic retinopathy compared to participants without diabetic retinopathy had smaller PIPR (p=0.007), lower 24 h serum melatonin output (p=0.042) and greater day-to-day sleep variability (p=0.012). By contrast, 24 h cortisol profiles, sleep duration and efficiency were similar in both groups. Six individuals with diabetic retinopathy had no detectable dim-light melatonin onset. PIPR correlated with 24 h mean melatonin levels (r=0.555, p=0.007). ipRCG dysfunction in diabetic retinopathy is associated with disruptions of the 24 h melatonin rhythm, suggesting circadian dysregulation in diabetic retinopathy.