Identification of immune cell subsets involved in retinal ganglion cell damage following blast exposure.

Primary open-angle glaucoma as a causal factor for circadian disruption: Living by the clock, in alignment with external time cues is an important condition for human health and well-being. Periodic changes in the ambient light serve as a key factor to synchronize the endogenously generated circadian rhythms. The retina perceives the photic signals and transmits them to the central body clock, the suprachiasmatic nuclei (SCN), via the retinohypothalamic tract. Primary open angle glaucoma (POAG) is an optic neuropathy, in which disease progression can be monitored by assessing damage to the retinal ganglion cells (RGCs) (Pérez-Rico et al., 2010; Feigl et al., 2011; Kankipati et al., 2011). Damage of retinal ganglion cells, particularly of intrinsically photosensitive RGCs (ipRGCs), is also one of the causes of circadian disruption. Pathophysiological mechanisms of POAG are complex, including elevated intraocular pressure (IOP), which adds mechanical stress, causing damage, dysfunction, and death of the RGCs (Figure 1). Glaucoma progression affects both image-forming and non-image-forming visual functions of RGCs. A central role of ipRGCs is to convey non-image-forming photic information to the clock. Their damage reduces light signaling to the SCN. Already in early stages of glaucoma, ipRGCs are dysfunctional (Pérez-Rico et al., 2010; Feigl et al., 2011; Kankipati et al., 2011).Figure 1: Melatonin potential to counteract complex circadian alterations with aging, neurodegeneration, specifically in glaucoma.ipRGCs: Intrinsically photosensitive retinal ganglion cells; SNPs: single nucleotide polymorphisms.As RGCs are progressively altered, and non-image-forming function is affected, circadian rhythms are disrupted, sleep is impaired, and mood is altered (Graticelli et al., 2015; Gubin et al., 2019, 2021). Circadian rhythm alterations are found in POAG as compared to age-matched healthy peers (Gubin et al., 2019). Circadian disruption worsens in advanced POAG (Gubin et al., 2019, Neroev et al., 2020), correlating to the increasing loss of ipRGCs with disease progression (Obara et al., 2016). Circadian rhythms also change with increasing age. Age-dependent circadian alterations are not necessarily related to retinal damage, as photic transduction to the central clock is not always compromised. When they are related to retinal damage, they can be due to either neurodegenerative ipRGCs damage, or to ipRGC damage caused by increased mean or deregulated circadian IOP. The intriguing principal difference between the presence or absence of retinal damage in aging is that the reduced light transmitted to the SCN by damaged ipRGCs phase-delays circadian rhythms, but ipRGC-uncompromised aging is commonly associated with phase-advanced circadian rhythms (Gubin et al., 2019). Since individual differences in sensitivity to light, and/or in endogenous melatonin production may interfere with this theoretical modeling, the search for specific genetic factors that may determine such individual differences constitutes a promising approach. In conditions where photic entrainment is compromised, not only is the alignment with external time cues altered, so can be the variability of overt physiologic functions. We showed that large inter-individual variability obscured the circadian IOP rhythm in POAG (Neroev et al., 2020). Circadian IOP rhythms had specific alterations manifested in advanced, but not in mild POAG, which were associated with the progressive damage and dysfunction of RGCs. In patients with RGCs' global loss volume above 15%, as assessed by high-definition optical coherence tomography, the 24-hour IOP rhythm peaked during the night, whereas in patients with stable POAG and a two-eye mean RGCs' global loss volume less than 10%, the IOP peaked predominantly during the daytime. Misalignment between circadian rhythms in body temperature and IOP increased as a function of global loss volume loss. Higher nocturnal IOP in POAG may adversely affect the disease state, fostering damage to RGCs (Neroev et al., 2020). Depending on individual genetic factors, these changes may manifest themselves to a different degree. Individual clock properties depend on numerous genetic factors, comprising clock genes and melatonin receptor genes, melatonin nuclear receptor 1b (MTNR1b) in particular, which may account for large individual differences in light sensitivity. Our pilot study of gene polymorphisms in POAG showed that the D-allele of the Angiotensin-converting enzyme holding a deletion of the 16th intron Alu repeat was significantly associated with alterations of the circadian IOP rhythm. It may also account for the resistance to IOP-lowering therapy (Neroev et al., 2020). Endogenous melatonin production in primary open-angle glaucoma: Glaucoma patients experienced reduced post-illumination pupil response (Kankipati et al., 2011) and reduced nocturnal melatonin suppression by light (Pérez-Rico et al., 2010). Clinical evidence for changes in the timing and mean values of endogenous melatonin production in POAG was also evident (reviewed in Gubin et al., 2021): in POAG, salivary melatonin can be lower than in age-matched controls without POAG; even greater alterations were observed In advanced stages of the disease. The main alteration concerned the time of maximal secretion of melatonin. Such altered melatonin production in POAG and other neurodegenerative pathologies can stem from different factors, including diminished light signaling due to a reduced sensitivity to light. The presence of certain gene polymorphisms can increase the susceptibility of carriers to these factors. We investigated 24-hour profiles of salivary melatonin under controlled lighting conditions and analyzed several clock genes and polymorphisms of the melatonin receptor gene MTNR1b (Gubin et al., 2021). Patients diagnosed with stable POAG had unaltered circadian rhythms of salivary melatonin and body temperature, which peaked at the anticipated time. Circadian rhythms of both variables were delayed, however, in patients diagnosed with advanced POAG (Gubin et al., 2019, 2021). Their 24-hour mean value and circadian amplitude of melatonin were also reduced (Gubin et al., 2021). Analysis of selected polymorphisms in clock and melatonin receptor genes revealed that these changes were observed specifically in carriers of the MTNR1B rs10830963 G-allele with advanced POAG. Overt changes of circadian phenotypes in POAG patients occur when several factors are present in combination: for example, when RGC loss exceeds a certain threshold in carriers of those genotypes, known to be associated with a prolonged duration of melatonin production. The MTNR1B rs10830963 G-allele is mainly known for its association with an elevated fasting glucose and the risk of type 2 diabetes, but it is also listed as a factor predicting POAG independently of diabetes (Shen et al., 2016), a fact supporting the assumption that melatonin may have pleiotropic physiological functions in the development of POAG. Melatonin to counteract non-image-forming visual function deterioration in primary open-angle glaucoma: To enhance circadian entrainment, morning light therapy and evening melatonin administration can both be effective. While studies aimed at estimating the merit of morning light therapy or outdoor light exposure in POAG are lacking, some studies provide evidence for a beneficial effect of exogenously administered melatonin in glaucoma and neurodegenerative pathologies (González Fleitas et al., 2021; Gubin et al., 2021). Melatonin transmits environmental light signals, thus facilitating the synchronization of peripheral clocks. It can thus mitigate several conditions such as glaucoma and its progression: disruption of circadian rhythms, compromised sleep, and mood (Tosini et al., 2012; Gubin et al., 2021) (Figure 1). Melatonin improves internal synchronization, ameliorating circadian alignment between local (IOP) and systemic (temperature) circadian rhythms (Gubin et al., 2021), which were progressively desynchronized with greater RGCs loss in POAG (Neroev et al., 2020). Melatonin is produced endogenously with a pronounced 24-hour rhythm governed by the SCN. Peak production occurs at night. Its specific timing may differ among individuals. Exact endogenous factors that predetermine these differences are not known but may include single nucleotide polymorphisms within candidate genes or melatonin receptors that influence sensitivity to light. Melatonin receptors (MTNR1B) are widespread in numerous brain regions. Their structure may determine the specific response to (both endogenous and exogenous) melatonin. We investigated the effect of oral melatonin administration (daily at 10:30 p.m. for 90 days) on the circadian rhythms of IOP, body temperature, and the pattern electroretinogram in patients diagnosed with stable or advanced POAG, also assessing effects on sleep and mood (Gubin et al., 2021). Melatonin administration increased the stability of the circadian body temperature rhythm, improving its alignment with the circadian IOP rhythm. Melatonin decreased IOP to a different extent at different times of the day and decreased the standard deviation of IOP with statistical significance. Larger changes were found in patients with initially higher 24-hour mean values of IOP. Melatonin improved RGCs function in patients with advanced POAG by increasing the amplitude of pattern electroretinogram that correlated positively with the degree of RGCs loss. Melatonin had more pronounced positive effects on sleep and mood in patients with advanced POAG, who had greater damage of their RGCs. Taken together melatonin has the potential to restore disrupted circadian rhythms in POAG. Its systemic effect is distinct from its local effect on the retinal circadian rhythms. Similar to light exposure, physiological effects of melatonin depend on the time of its administration. Personalizing melatonin administration in terms of its timing and dosing, accounting for the genetic profile, is expected to further refine its multiple benefits. Melatonin may provide beneficial effects in POAG stemming from both its ability to reduce IOP and its potential to prevent RGC damage derived from mechanisms of neurodegeneration (Hardeland, 2021) (Figure 1). These effects may not only mitigate circadian disruption but also improve other aspects of health and well-being. Circadian alignment may strengthen human physiological functions and help slow neurodegeneration. The choice of an optimal melatonin dosing, however, is not yet clear (Hardeland, 2021). Consideration of the best timing should be based on internal circadian parameters and on genes that may account for personal differences in melatonin efficacy. Concluding remarks: In assessing disruptions in the non-image-forming visual system in POAG patients, one needs to discriminate between different situations. There may be complex, non-specific changes in circadian rhythms with age. Changes related to neurogenerative disease, including Alzheimer's disease, Parkinson's disease, and POAG, might promote alterations in neural structures: SCN, pineal, retina. Changes specific to POAG include additional RGC damage caused by the elevated IOP, together with abnormal circadian patterns of physiological variables such as IOP, body temperature, pattern electroretinogram, and melatonin (Figure 1). The circadian IOP pattern with relatively higher values during the resting span may foster harmful effects of IOP on RGCs, since tissue sensitivity may vary depending on circadian time (Neroev et al., 2020). Numerous candidate gene polymorphisms may play a role, alone or in combination with others, affecting the susceptibility to POAG itself (as a primary pathology of vision), or POAG-associated alterations of circadian rhythms, sleep, and mood, linked to non-visual pathways. To answer this question, clinical data combined with circadian profiles of melatonin and other physiological variables, chronotype questionnaires, sleep and mood information, to be checked against single nucleotide polymorphisms databases, should be collected on large cohorts. Constructive collaboration among ophthalmologists, chronobiologists, and geneticists is therefore advocated. The present work was supported by the Russian Foundation for Basic Research (grant No. 19-015-00329) (to DG), and by Government of Tyumen District, Decree of 20.11.2020 No. 928-rp (to DG). The authors have no proprietary or commercial interest in any materials discussed in this article.

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

Commonalities of optic nerve injury and glaucoma-induced neurodegeneration: Insights from transcriptome-wide studies

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.

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.

PurposeVisual impairment from normal-tension glaucoma (NTG) poses an increasing burden, yet the underlying mechanism remains unclear. Investigating protective mechanisms for NTG is critical. We aimed to investigate the role of ferroptosis in retinal ganglion cell (RGC) damage in glutamate–aspartate transporter (GLAST) knockout (GLAST−/−) mice, a model for NTG, and also to determine whether inhibiting ferroptosis can provide neuroprotection.MethodsGLAST−/− mice and a glutamate-induced excitotoxicity model in primary RGCs were used to investigate retinal and RGC damage. RNA sequencing identified ferroptosis-related pathways in GLAST−/− retinas. Oxidative stress, lipid peroxidation, and ferroptosis activation were assessed using western blotting and immunofluorescence. Immunohistochemistry (IHC) assessed lipid peroxidation and ferroptosis activation in human retinal tissue. Ferrostatin-1 (Fer-1) was administered to evaluate its neuroprotective effects on RGC survival, retinal thickness, and visual function.ResultsRNA sequencing revealed significant enrichment of ferroptosis-related pathways in GLAST−/− retinas. Both GLAST deletion and glutamate-induced excitotoxicity increased oxidative stress, lipid peroxidation, and ferroptosis activation in RGCs. IHC in human retinas confirmed elevated 4-hydroxynonenal (4-HNE) and acyl-coenzyme A synthetase long-chain family member 4 (ACSL4) expression. Furthermore, Fer-1 treatment significantly reduced lipid peroxidation, thereby attenuating the ferroptosis pathways. This intervention ameliorated RGC loss associated with GLAST deletion, protected retinal structure and thickness, and improved amplitudes of the photopic negative response, a-wave, b-wave, and oscillatory potentials.ConclusionsFerroptosis significantly contributes to RGC and retinal damage in the GLAST-deletion NTG model. Inhibiting ferroptosis with Fer-1 presents a promising therapeutic strategy for protecting visual function in NTG.

The purpose of this study was to test whether functional loss in the glaucomatous macula is characterized by an enlargement of Ricco's area (RA) through the application of a computational model linking retinal ganglion cell (RGC) damage to perimetric sensitivity. One eye from each of 29 visually healthy subjects <40years old, 30 patients with glaucoma, and 20 age-similar controls was tested with a 10-2 grid with stimuli of 5 different area sizes. Structural estimates of point-wise RGC density were obtained from optical coherence tomography (OCT) scans. Structural and functional data from the young healthy cohort were used to estimate the parameters of a computational spatial summation model to generate a template. The template was fitted with a Bayesian hierarchical model to estimate the latent RGC density in patients with glaucoma and age-matched controls. We tested two alternative hypotheses: fitting the data by translating the template horizontally (H1: change in RA) or vertically (H2: loss of sensitivity without a change in RA). Root mean squared error (RMSE) of the model fits to perimetric sensitivity were compared. Ninety-five percent confidence intervals were bootstrapped. The dynamic range of the functional and structural RGC density estimates was denoted by their 1st and 99th percentiles. The RMSE was 2.09 (95% CI = 1.92-2.26) under H1 and 2.49 (95% CI = 2.24-2.72) under H2 (P < 0.001). The average dynamic range for the structural RGC density estimates was only 11% that of the functional estimates. Macular sensitivity loss in glaucoma is better described by a model in which RA changes with RGC loss. Structural measurements have limited dynamic range.

The damage or apoptosis of retinal ganglion cells (RGCs) is one of the leading causes of various blinding eye diseases, such as glaucoma, diabetic retinopathy, optic neuritis, and ischemic optic neuropathy. Oxidative stress is involved in RGCs death. Baicalin, a flavonoid compound extracted from Scutellaria baicalensis, has various beneficial effects, including anti-inflammatory, anti-apoptotic, and antioxidant properties. However, the effects of baicalin on RGCs and the underlying mechanisms require further investigation. In this study, a glutamate-induced oxidative stress damage model of R28 cells and a rat retinal injury model were established to investigate the effects of baicalin on oxidative stress damage to RGCs and try to elucidate the underlying mechanism. In vitro experiments demonstrated that the survival rate of R28 cells after glutamate treatment dropped to 33.4%, while 10μM baicalin significantly inhibited glutamate-induced damage in RGCs (P < 0.001) and enhanced cell viability through decreasing ROS levels, increasing antioxidant enzyme activity, and suppressing the expression of inflammatory factors iNOS, TNF-α, IL-6, and IL-1β (P < 0.001). In vivo, baicalin effectively mitigated structural damage to retinal tissue and RGCs morphology induced by glutamate, increasing the thickness of the retinal ganglion cell layer, improving RGCs density, and reducing overall retinal thinning in rats (P < 0.001) in a time- and dose-dependent effects. Mechanistic studies revealed that glutamate evaluated the phosphorylation levels of JAK/STAT, while baicalin effectively inhibited the activation of the JAK/STAT signaling pathway. This study confirmed that baicalin protects against glutamate-induced oxidative stress damage in RGCs. It effectively alleviates oxidative stress and inflammatory responses, reduces cell apoptosis, and improves the pathological changes in the retina of rat models of RGCs damage, thereby decreasing RGCs death. Further exploration of its mechanism revealed that baicalin effectively inhibits the JAK/STAT signaling pathway, protecting RGCs from oxidative stress damage. This provides an experimental basis for the application of baicalin in the treatment of RGCs damage.

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.

Dimercaprol attenuates oxidative stress-induced damage of retinal ganglion cells in an in vitro and in vivo model of traumatic optic neuropathy.

Retinal ganglion cells (RGCs) loss or degeneration in the retina is a hallmark of many sight-threatening diseases, including glaucoma and retinopathy. In our previous studies, calcium-activated cysteine protease calpain induced RGC damage under hypoxia/reoxygenation in the monkey retina explants, and the calpain inhibitor SNJ-1945 partially inhibited RGC damage. Calcium-independent proteases such as cathepsins exist in the retina, although the involvement of cathepsins in hypoxia-induced RGC damage is unclear. The purpose of the present study is to determine if cathepsins are involved in RGC damage during hypoxia/reoxygenation and to elucidate the role of calcium. The cathepsin inhibitors (Odanacatib and SID26681509) were tested for their specificity against three cathepsins in vitro. Calpain inhibitors (SNJ-1945, PD-151746, ABT-957, and C2I) were tested for their isozyme specificity in vitro. Monkey retinal explants were cultured under hypoxic conditions with 0.3% oxygen in the chamber, followed by reoxygenation. The calpain- or cathepsin-specific inhibitors were added to the hypoxic culture medium. BAPTA and BAPT-AM were also used to determine the calcium requirement in RGC damage. After the cultured period, retinal explants were prepared for flat mounts and retinal lysates. The flat mounted retinas were stained with propidium Iodide (PI) to assess RGC damage and with an antibody specific for calpain-specific α-spectrin break down product 150 kDa (SBDP150). Immunoblotting assays were performed for α-spectrin and calpains. The cathepsin inhibitors were confirmed to be specific to cathepsin without calpain inhibitory effects in vitro. The number of PI-positive RGCs increased in the hypoxic monkey retina; however, cathepsin inhibitors did not mitigate RGC damage. In contrast, RGC damage was completely inhibited by BAPTA and partially by BAPTA-AM. In hypoxic retinas, calpain-specific SBDP150 increased in the nerve fiber layer (NFL). Immunoblotting revealed an increase in SBDP150 and the activation of calpain 1. These changes were inhibited by BAPTA or BAPTA-AM, with BAPTA demonstrating a stronger effect compared to BAPTA-AM. Calpain inhibitors demonstrated lower specificity in vitro than previously reported. In hypoxic retina, each calpain inhibitor alleviated RGC damage and reduced SBDP150-positive staining in NFL. Furthermore, calpain inhibitors attenuated the activation of calpain 1 and the breakdown of α-spectrin, as assessed by immunoblotting. Calcium-independent cathepsins do not contribute to RGC damage in monkey retinal explants cultured under hypoxia/reperfusion. In contrast, calcium influx from extracellular sources plays a critical role in inducing RGC damage. Elevated intracellular calcium levels could activate calpains, leading to RGC damage; however, other calcium-dependent pathways might also be involved in this process.

Background: Ethambutol-induced optic neuropathy (EON) is one of the most compelling adverse effect of tuberculosis treatment. Recovery often occur several months after treatment discontinuation. Unfortunately, some studies noted that nearly half of patients still have permanent visual loss. Early detection before clinical symptoms appear is necessary to prevent this devastating adverse effect. Therefore, this review aims to evaluate the diagnostic value of retinal nerve fiber layer (RNFL) thickness and ganglion cell inner plexiform layer (GCIPL) thickness changes with OCT, pattern and multifocal electroretinogram (ERG) changes during ethambutol treatment as early detection of EON. Methods: A comprehensive search was conducted from electronic databases (PubMed, EBSCO, Google Scholar, and Springerlink) using relevant search terms. Articles from offline resources were also included. Included studies were selected based on predefined inclusion and exclusion criteria. Result: Three studies reported significant thinning of RNFL after ethambutol initiation. Increased RNFL thickness in patients with EON and subclinical EON found in 3 studies. Significant macular GCPIL thinning was noted in 1 study. One study reported shortening of P50 implicit time and reduced N95 wave amplitude in pattern ERG. Conclusion: Macular GCIPL thinning suggested to be the first pathological changes detected on patients with ethambutol treatment. It can be concomitant with thickening of peripapillary RNFL and followed by peripapillary RNFL thinning. Pattern ERG may reveal abnormality due to retinal ganglion cell (RGC) dysfunction before RGC loss.

To better understand pathophysiologic mechanisms underlying pattern electroretinogram (PERG) losses in glaucoma by simulating either retinal ganglion cell (RGC) dysfunction or RGC loss in normal subjects. The steady-state PERG has been recorded in 10 normal subjects (mean age: 31+/-8 y) according to the PERGLA paradigm by means of skin electrodes in response to horizontal gratings (1.7 cycles/degree, 99% contrast, 40 cd/m mean luminance, circular field size 25 degree diameter) alternating 16.28 times/seconds. Simulated RGC dysfunction has been obtained by reducing either contrast and mean luminance or blurring the visual stimulus. Simulated RGC loss has been obtained by reducing stimulus area. Outcome measures were PERG amplitude and phase obtained by discrete Fourier transform of PERG waveforms. Progressive PERG amplitude reductions spanning the entire dynamic range of PERG response could be obtained by progressively reducing stimulus contrast and luminance, blurring the stimulus, and reducing stimulus area. The same variations in stimulus conditions caused phase changes of disparate sign and magnitude. Phase advanced (latency shortened) by reducing stimulus contrast or blurring the stimulus; phase lagged (latency increased) by reducing stimulus luminance; phase remained constant by reducing stimulus area. PERG amplitude and phase are essentially uncoupled, implying that these measures reflect distinct aspects of RGC activity. On the basis of our results and known PERG physiology, we propose a model in which both RGC dendrites and RGC axons contribute to the PERG signal. PERG delays may represent an indication of synaptic dysfunction that is potentially reversible.

Autosomal dominant optic atrophy (ADOA) is an inherited optic neuropathy primarily caused by mutations in OPA1. We identified and defined a spontaneous nonhuman primate (NHP) model of ADOA using rhesus macaques heterozygous for a missense mutation (OPA1A8S). With ocular examinations, ophthalmic imaging, electroretinography, histopathology, immunohistochemistry, and transmission electron microscopy (TEM), we documented retinal nerve fiber layer (RNFL) thinning, retinal ganglion cell (RGC) loss and dysfunction, OPA1 mislocalization, and reduced axonal mitochondrial density in affected macaques. Our investigation revealed substantial phenotypic variability among affected macaques, shedding light on the pathogenesis of ADOA. The retinas were evaluated using techniques such as spectral-domain optical coherence tomography and fundus photography facilitating observation of structural changes in the retina and optic nerve. Thinning of the RNFL and optic nerve head degeneration, hallmark features of ADOA, were observed in affected macaques. Decreased RGC function in the OPA1 heterozygotes was demonstrated with pattern electroretinography. Histopathological analysis and immunohistochemical staining of postmortem retinal tissue suggested RGC loss in the papillomacular bundle, with reduced OPA1 and mitochondria in the RGC axons, indicating dysfunctional mitochondrial dynamics and reduced function consistent with ADOA. Ultrastructural changes were evident with TEM including dysmorphic mitochondria, axonal loss, myelin disruption, and hypertrophic astrocytic processes. The observed similar pattern of RGC loss and dysfunction coupled with phenotypic heterogeneity in our NHP model reflects the clinical variability observed in human ADOA patients indicating that therapeutic interventions in this foveate model will likely translate to the human condition.

BackgroundGlaucoma is an optic neuropathy that is characterized by the loss of retinal ganglion cells (RGCs) initiated by damage to axons in the optic nerve. The degeneration and death of RGCs has been thought to occur in two waves. The first is axogenic, caused by direct insult to the axon. The second is somatic, and is thought to be caused by the production of inflammatory cytokines from the activated retinal innate immune cells. One of the cytokines consistently linked to glaucoma and RGC damage has been TNFα. Despite strong evidence implicating this protein in neurodegeneration, a direct injection of TNFα does not mimic the rapid loss of RGCs observed after acute optic nerve trauma or exposure to excitotoxins. This suggests that our understanding of TNFα signaling is incomplete.MethodsRGC death was induced by optic nerve crush in mice. The role of TNFα in this process was examined by quantitative PCR of Tnfα gene expression, and quantification of cell loss in Tnfα−/− mice or in wild-type animals receiving an intraocular injection of exongenous TNFα either before or after crush. Signaling pathways downstream of TNFα were examined by immunolabeling for JUN protein accumulation or activation of EGFP expression in NFκB reporter mice.ResultsOptic nerve crush caused a modest increase in Tnfα gene expression, with kinetics similar to the activation of both macroglia and microglia. A pre-injection of TNFα attenuated ganglion cell loss after crush, while ganglion cell loss was more severe in Tnfα−/− mice. Conversely, over the long term, a single exposure to TNFα induced extrinsic apoptosis in RGCs. Müller cells responded to exogenous TNFα by accumulating JUN and activating NFκB.ConclusionEarly after optic nerve crush, TNFα appears to have a protective role for RGCs, which may be mediated through Müller cells.