Generating ESC-Derived RGCs for Cell Replacement Therapy.

In several ocular diseases, degeneration of retinal neurons can lead to permanent blindness. Transplantation of stem cell (SC)-derived RGCs has been proposed as a potential therapy for RGC loss. Although there are reports of successful cases of SC-derived RGC transplantation, achieving long-distance regeneration and functional connectivity remains a challenge. To address these hurdles, retinal organoids are being used to study the regulatory mechanism of stem cell transplantation. Here we present a modified protocol for differentiating human embryonic stem cells (ESCs) into retinal organoids and transplanting organoid-derived RGCs into the murine eyes.

The assessment of structural and functional test results for early detection of hydroxychloroquine macular toxicity.

PURPOSE: To assess structural (optical coherence tomography, fundus autofluorescence) and functional (contrast sensitivity and visual field) test results which were used for detecting early retinal changes in patients using oral hydroxychloroquine. METHODS: Patients using oral hydroxychloroquine for at least one year were divided into two groups according to the duration of drug use. Groups 1 and 2 consisted of patients with drug use for more than 5 years and 1-5 years, respectively. The drug-using groups were compared with the control group. The mean retinal nerve fiber layer (RNFL), central macular thickness (CMT), ganglion cell-inner plexiform layer (GC-IPL), static 10-2 visual field, fundus autofluorescence (FAF) imaging, and contrast sensitivity tests were performed and statistically compared between groups. RESULTS: Median and temporal quadrant RNFL thicknesses were found to be statistically significantly lower in the drug groups. In the drug groups, the GC-IPL sectoral and mean thicknesses were found to be statistically lower in all quadrants. Central macular thickness was also found to be similar in all three groups. There was no significant difference between the groups in visual field parameters. Macular FAF images were significantly higher in the drug users, but there was no significant difference between the three groups in foveal FAF images. Contrast sensitivity measurements were significantly lower in the drug groups than in the control group at all spatial frequencies except 6 and 18 cycles/degree. CONCLUSIONS: The combined use of structural and functional tests in patients using hydroxychloroquine provides useful information in detecting early retinal changes.

Inner retinal layer thickness alterations in adult and pediatric patients with neurofibromatosis 1.

This study compared the thickness of each intraretinal layer in patients with neurofibromatosis 1 (NF1) and controls to analyze the association between intraretinal layer thickness and visual function. The macular spectral-domain optical coherence tomography volumetric dataset obtained from 68 eyes (25 adult eyes, 43 pediatric eyes) with NF1 without optic glioma and 143 control eyes (100 adult eyes, 43 pediatric eyes) was used for image auto-segmentation. The intraretinal layers segmented from the volumetric data included the macular retinal nerve fiber layer (RNFL), ganglion cell-inner plexiform layer (GCIPL), inner nuclear layer, outer plexiform layer, outer nuclear layer, and photoreceptor layer. Cases and controls were compared after adjusting for age, sex, refractive error, and binocular use. The association between retinal layer thickness and visual acuity was also analyzed. The GCIPL was significantly thinner in both adult and pediatric patients with NF1 compared with healthy controls. Average RNFL and GCIPL thicknesses were associated with visual acuity in adult patients with NF1. In pediatric patients, average GCIPL thickness was associated with visual acuity. These results suggest that changes in the inner retinal layer could be a biomarker of the structural and functional status of patients with NF1.

Changes in Intrinsically Photosensitive Retinal Ganglion Cells, Dopaminergic Amacrine Cells, and Their Connectivity in the Retinas of Lid Suture Myopia.

Purpose: This study investigates alterations in intrinsically photosensitive retinal ganglion cells (ipRGCs) and dopaminergic amacrine cells (DACs) in lid suture myopia (LSM) rats. Methods: LSM was induced in rats by suturing the right eyes for 4 weeks. Double immunofluorescence staining of ipRGCs and DACs in whole-mount retinas was performed to analyze changes in the density and morphology of control, LSM, and fellow eyes. Real-time quantitative PCR and Western blotting were used to detect related genes and protein expression levels. Results: Significant myopia was induced in the lid-sutured eye, but the fellow eye was not different to control. Decreased ipRGC density with paradoxically increased overall melanopsin expression and enlarged dendritic beads was observed in both the LSM and fellow eyes of the LSM rat retinas. In contrast, DAC changes occurred only in the LSM eyes, with reduced DAC density and tyrosine hydroxylase (TH) expression, sparser dendritic processes, and fewer varicosities. Interestingly, contacts between ipRGCs and DACs in the inner plexiform layer (IPL) and the expression of pituitary adenylate cyclase-activating polypeptide (PACAP) and vesicular monoamine transporter protein 2 (VMAT2) mRNA were decreased in the LSM eyes. Conclusions: The ipRGCs and DACs in LSM rat retinas undergo multiple alterations in density, morphology, and related molecule expressions. However, the ipRGC changes alone appear not to be required for the development of myopia, given that myopia is only induced in the lid-sutured eye, and they are unlikely alone to drive the DAC changes. Reduced contacts between ipRGCs and DACs in the LSM eyes may be the structural foundation for the impaired signaling between them. PACAP and VMAT2, strongly associated with ipRGCs and DACs, may play important roles in LSM through complex mechanisms.

Using Multi-Layer Perceptron Driven Diagnosis to Compare Biomarkers for Primary Open Angle Glaucoma.

Purpose: To use neural network machine learning (ML) models to identify the most relevant ocular biomarkers for the diagnosis of primary open-angle glaucoma (POAG). Methods: Neural network models, also known as multi-layer perceptrons (MLPs), were trained on a prospectively collected observational dataset comprised of 93 glaucoma patients confirmed by a glaucoma specialist and 113 control subjects. The base model used only intraocular pressure, blood pressure, heart rate, and visual field (VF) parameters to diagnose glaucoma. The following models were given the base parameters in addition to one of the following biomarkers: structural features (optic nerve parameters, retinal nerve fiber layer [RNFL], ganglion cell complex [GCC] and macular thickness), choroidal thickness, and RNFL and GCC thickness only, by optical coherence tomography (OCT); and vascular features by OCT angiography (OCTA). Results: MLPs of three different structures were evaluated with tenfold cross validation. The testing area under the receiver operating characteristic curve (AUC) of the models were compared with independent samples t-tests. The vascular and structural models both had significantly higher accuracies than the base model, with the hemodynamic AUC (0.819) insignificantly outperforming the structural set AUC (0.816). The GCC + RNFL model and the model containing all structural and vascular features were also significantly more accurate than the base model. Conclusions: Neural network models indicate that OCTA optic nerve head vascular biomarkers are equally useful for ML diagnosis of POAG when compared to OCT structural biomarker features alone.

Peripapillary Retinal Nerve Fiber Layer and Macular Ganglion Cell-Inner Plexiform Layer Parameters and Their Associated Factors in Cynomolgus Macaques.

Purpose: The purpose of this study was to define the normal range of peripapillary retinal nerve fiber layer (pRNFL), macular ganglion cell layer (mGCL), and macular inner plexiform layer (mIPL) thickness in cynomolgus macaques, and explore their inter-relationship and correlation with age, refractive errors, and axial length (AL). Methods: In this cross-sectional study, we measured biometric and refractive parameters, and pRNFL/mGCL/mIPL thickness in 357 healthy cynomolgus macaques. Monkeys were divided into groups by age and spherical equivalent (SE). Correlation and regression analyses were used to explore the relationship between pRNFL and mGCL/mIPL thickness, and their correlation with the above parameters. Results: The mean age, SE, and AL were 14.46 ± 6.70 years, -0.96 ± 3.23 diopters (D), and 18.39 ± 1.02 mm, respectively. The mean global pRNFL thickness was 95.06 ± 9.42 µm (range = 54-116 µm), with highest values in the inferior quadrant, followed by the superior, temporal, and nasal quadrants (P < 0.001). Temporal pRNFL thickness correlated positively with age (r = 0.218, P < 0.001) and AL (r = 0.364, P < 0.001), and negatively with SE (r = -0.270, P < 0.001). In other quadrants, pRNFL thickness correlated negatively with age and AL, but positively with SE. In the multivariable linear regression model, adjusted for sex and AL, age (ß = -0.350, P < 0.001), and SE (ß = 0.206, P < 0.001) showed significant associations with global pRNFL thickness. After adjusting for age, sex, SE, and AL, pRNFL thickness positively correlated with mGCL (ß = 0.433, P < 0.001) and mIPL thickness (ß = 0.465, P < 0.001). Conclusions: The pRNFL/mGCL/mIPL thickness distribution and relationship with age, AL, and SE in cynomolgus macaques were highly comparable to those in humans, suggesting that cynomolgus monkeys are valuable animal models in ophthalmic research.

Association of Retinal Nerve Fiber Layer Thinning With Elevated High Density Lipoprotein Cholesterol in UK Biobank.

Purpose: The purpose of this study was to investigate the association between retinal nerve fiber layer (RNFL) thickness and high-density lipoprotein cholesterol (HDL-C) in a healthy population. Methods: This cross-sectional study included 31,738 UK Biobank participants with high quality optical coherence tomography (OCT) images, excluding those with neurological or ocular diseases. The locally estimated scatterplot smoothing (LOESS) curve and multivariable piecewise linear regression models were applied to assess the association between HDL-C and RNFL thickness, and HDL-C subclasses were further analyzed using nuclear magnetic resonance (NMR) spectroscopy. Results: Multivariate piecewise linear regression revealed that high HDL-C levels (>1.7 mmol/L in women or > 1.5 mmol/L in men) were associated with thinner RNFL thickness (women: ß = -0.13, 95% confidence interval [CI] = -0.23 to -0.02, P = 0.017; male: ß = -0.23, 95% CI = -0.37 to -0.10, P = 0.001). Conversely, a significant positive association between HDL-C and RNFL thickness was observed when HDL-C was between 1.4 and 1.7 mmol/L for female participants (ß = 0.13, 95% CI = 0.02 to 0.24, P = 0.025). NMR analysis showed that these associations are potentially driven by distinct HDL-C subclasses. Conclusions: This study revealed an association between HDL-C levels and retinal markers of neurodegenerative diseases, suggesting that elevated HDL-C may serve as a new risk factor for neurodegenerative conditions. These findings may contribute to the implementation of preventive interventions and improved patient outcomes.

Thorny and Tufted Retinal Ganglion Cells Express the Transcription Factor Forkhead Proteins Foxp1 and Foxp2 in Marmoset (Callithrix jacchus).

The transcription factor forkhead/winged-helix domain proteins Foxp1 and Foxp2 have previously been studied in mouse retina, where they are expressed in retinal ganglion cells named F-mini and F-midi. Here we show that both transcription factors are expressed by small subpopulations (on average less than 10%) of retinal ganglion cells in the retina of the marmoset monkey (Callithrix jacchus). The morphology of Foxp1- and Foxp2-expressing cells was revealed by intracellular DiI injections of immunofluorescent cells. Foxp1- and Foxp2-expressing cells comprised multiple types of wide-field ganglion cells, including broad thorny cells, narrow thorny cells, and tufted cells. The large majority of Foxp2-expressing cells were identified as tufted cells. Tufted cells stratify broadly in the middle of the inner plexiform layer. They resemble broad thorny cells but their proximal dendrites are bare of branches and the distal dendrites branch frequently forming dense dendritic tufts. Double labeling with calretinin, a previously established marker for broad thorny and narrow thorny cells, showed that only a small proportion of ganglion cells co-expressed calretinin and Foxp1 or Foxp2 supporting the idea that the two markers are differentially expressed in retinal ganglion cells of marmoset retina.

Cellular and circuit remodeling of the primate foveal midget pathway after acute photoreceptor loss.

The retinal fovea in human and nonhuman primates is essential for high acuity and color vision. Within the fovea lies specialized circuitry in which signals from a single cone photoreceptor are largely conveyed to one ON and one OFF type midget bipolar cell (MBC), which in turn connect to a single ON or OFF midget ganglion cell (MGC), respectively. Restoring foveal vision requires not only photoreceptor replacement but also appropriate reconnection with surviving ON and OFF MBCs and MGCs. However, our current understanding of the effects of cone loss on the remaining foveal midget pathway is limited. We thus used serial block-face electron microscopy to determine the degree of plasticity and potential remodeling of this pathway in adult Macaca fascicularis several months after acute photoreceptor loss upon photocoagulation. We reconstructed MBC structure and connectivity within and adjacent to the region of cone loss. We found that MBC dendrites within the scotoma retracted and failed to reach surviving cones to form new connections. However, both surviving cones and ON and OFF MBC dendrites at the scotoma border exhibited remodeling, suggesting that these neurons can demonstrate plasticity and rewiring at maturity. At six months postlesion, disconnected OFF MBCs clearly lost output ribbon synapses with their postsynaptic partners, whereas the majority of ON MBCs maintained their axonal ribbon numbers, suggesting differential timing or extent in ON and OFF midget circuit remodeling after cone loss. Our findings raise rewiring considerations for cell replacement approaches in the restoration of foveal vision.

Evaluation of choroidal thickness and optic disc parameters in patients with chronic phase gout.

PURPOSE: This study aims to assess the disparities in choroidal thickness and optic disc parameters between individuals diagnosed with chronic gout and an age- and gender-matched control cohort. METHODS: This cross-sectional study involved 30 gout patients receiving treatment at the Rheumatology clinic, alongside 30 healthy control individuals matched for age and gender. A comprehensive ophthalmological assessment, encompassing visual acuity measurement, intraocular pressure evaluation, slit-lamp biomicroscopy, and dilated fundus examination, was conducted for all participants. Peripapillary retinal nerve fiber layer (RNFL), ganglion cell complex (GCC), and subfoveal choroidal thickness (SFCT) were quantified utilizing Spectral Domain Optical Coherence Tomography. RESULTS: The mean age within the study group was 54.53 ± 9.43 years, while the control group's mean age was 53.20 ± 10.36 years. In both the gout and control cohorts, there were 28 men and 2 women. No significant differences were observed in age and gender between the groups. Gout patients manifested thinner RNFL and GCC across all quadrants; however, statistically significant thinning was only evident in the nasal and inferior quadrants for RNFL. Despite a thinner SFCT observed in gout patients compared to controls, this discrepancy did not attain statistical significance. CONCLUSION: Chronic phase gout patients may display alterations in optic disc and macular parameters, alongside potential variations in choroidal thickness. Nevertheless, more controlled studies encompassing a larger participant pool are imperative to substantiate our findings.

Detecting Visual Field Worsening From Optic Nerve Head and Macular Optical Coherence Tomography Thickness Measurements.

Purpose: Compare the use of optic disc and macular optical coherence tomography measurements to predict glaucomatous visual field (VF) worsening. Methods: Machine learning and statistical models were trained on 924 eyes (924 patients) with circumpapillary retinal nerve fiber layer (cp-RNFL) or ganglion cell inner plexiform layer (GC-IPL) thickness measurements. The probability of 24-2 VF worsening was predicted using both trend-based and event-based progression definitions of VF worsening. Additionally, the cp-RNFL and GC-IPL predictions were combined to produce a combined prediction. A held-out test set of 617 eyes was used to calculate the area under the curve (AUC) to compare cp-RNFL, GC-IPL, and combined predictions. Results: The AUCs for cp-RNFL, GC-IPL, and combined predictions with the statistical and machine learning models were 0.72, 0.69, 0.73, and 0.78, 0.75, 0.81, respectively, when using trend-based analysis as ground truth. The differences in performance between the cp-RNFL, GC-IPL, and combined predictions were not statistically significant. AUCs were highest in glaucoma suspects using cp-RNFL predictions and highest in moderate/advanced glaucoma using GC-IPL predictions. The AUCs for the statistical and machine learning models were 0.63, 0.68, 0.69, and 0.72, 0.69, 0.73, respectively, when using event-based analysis. AUCs decreased with increasing disease severity for all predictions. Conclusions: cp-RNFL and GC-IPL similarly predicted VF worsening overall, but cp-RNFL performed best in early glaucoma stages and GC-IPL in later stages. Combining both did not enhance detection significantly. Translational Relevance: cp-RNFL best predicted trend-based 24-2 VF progression in early-stage disease, while GC-IPL best predicted progression in late-stage disease. Combining both features led to minimal improvement in predicting progression.

Optimization of an Ischemic Retinopathy Mouse Model and the Consequences of Hypoxia in a Time-Dependent Manner.

The retina is one of the highest metabolically active tissues with a high oxygen consumption, so insufficient blood supply leads to visual impairment. The incidence of related conditions is increasing; however, no effective treatment without side effects is available. Furthermore, the pathomechanism of these diseases is not fully understood. Our aim was to develop an optimal ischemic retinopathy mouse model to investigate the retinal damage in a time-dependent manner. Retinal ischemia was induced by bilateral common carotid artery occlusion (BCCAO) for 10, 13, 15 or 20 min, or by right permanent unilateral common carotid artery occlusion (UCCAO). Optical coherence tomography was used to follow the changes in retinal thickness 3, 7, 14, 21 and 28 days after surgery. The number of ganglion cells was evaluated in the central and peripheral regions on whole-mount retina preparations. Expression of glial fibrillary acidic protein (GFAP) was analyzed with immunohistochemistry and Western blot. Retinal degeneration and ganglion cell loss was observed in multiple groups. Our results suggest that the 20 min BCCAO is a good model to investigate the consequences of ischemia and reperfusion in the retina in a time-dependent manner, while the UCCAO causes more severe damage in a short time, so it can be used for testing new drugs.

The people behind the papers - Kevin Emmerich and Jeff Mumm.

Retinal regeneration has been mostly studied after widespread tissue injury, but it is not well understood how the retina regenerates at the cellular level following loss of specific cell types. In a new study, Jeff Mumm and colleagues selectively ablate retinal ganglion cells in zebrafish and find that the retina elicits different genetic responses in a context-dependent manner to replace lost cells. To find out more about the story behind the paper, we caught up with first author Kevin Emmerich and corresponding author Jeff Mumm, Associate Professor in Ophthalmology at Johns Hopkins University.

Hebbian instruction of axonal connectivity by endogenous correlated spontaneous activity.

Spontaneous activity refines neural connectivity prior to the onset of sensory experience, but it remains unclear how such activity instructs axonal connectivity with subcellular precision. We simultaneously measured spontaneous retinal waves and the activity of individual retinocollicular axons and tracked morphological changes in axonal arbors across hours in vivo in neonatal mice. We demonstrate that the correlation of an axon branch's activity with neighboring axons or postsynaptic neurons predicts whether the branch will be added, stabilized, or eliminated. Desynchronizing individual axons from their local networks, changing the pattern of correlated activity, or blocking N-methyl-d-aspartate receptors all significantly altered single-axon morphology. These observations provide the first direct evidence in vivo that endogenous patterns of correlated neuronal activity instruct fine-scale refinement of axonal processes.

Evaluation of thickness of individual macular retinal layers in diabetic eyes from optical coherence tomography.

The effect of diabetes mellitus (DM) on individual retinal layers remains incompletely understood. We evaluated the intra-retinal layer thickness alterations in 71 DM eyes with no diabetic retinopathy (DR), 90 with mild DR, and 63 with moderate DR without macular edema, using spectral-domain optical coherence tomography (SD-OCT) and the Iowa Reference Algorithm for automated retinal layer segmentation. The average thickness of 10 intra-retinal layers was then corrected for ocular magnification using axial length measurements, and pairwise comparisons were made using multivariable linear regression models adjusted for gender and race. In DM no DR eyes, significant thinning was evident in the ganglion cell layer (GCL; p < 0.001), inner nuclear layer (INL; p = 0.001), and retinal pigment epithelium (RPE; p = 0.014) compared to normal eyes. Additionally, mild DR eyes exhibited a thinner inner plexiform layer (IPL; p = 0.008) than DM no DR eyes. Conversely, moderate DR eyes displayed thickening in the INL, outer nuclear layer, IPL, and retinal nerve fiber layer (all p ≤ 0.002), with notably worse vision. These findings highlight distinctive patterns: early diabetic eyes experience thinning in specific retinal layers, while moderate DR eyes exhibit thickening of certain layers and slightly compromised visual acuity, despite the absence of macular edema. Understanding these structural changes is crucial for comprehending diabetic eye complications.

Lycium barbarum glycopeptide promotes neuroprotection in ET-1 mediated retinal ganglion cell degeneration.

BACKGROUND: Vascular dysregulation is one of the major risk factors of glaucoma, and endothelin-1 (ET-1) may have a role in the pathogenesis of vascular-related glaucoma. Fruit extract from Lycium Barbarum (LB) exhibits anti-ageing and multitarget mechanisms in protecting retinal ganglion cells (RGC) in various animal models. To investigate the therapeutic efficacy of LB glycoproteins (LbGP) in ET-1 induced RGC degeneration, LbGP was applied under pre- and posttreatment conditions to an ET-1 mouse model. Retina structural and functional outcomes were characterised using clinical-based techniques. METHODS: Adult C57BL/6 mice were randomly allocated into four experimental groups, namely vehicle control (n = 9), LbGP-Pretreatment (n = 8), LbGP-Posttreatment (day 1) (n = 8) and LbGP-Posttreatment (day 5) (n = 7). Oral administration of LbGP 1 mg/Kg or PBS for vehicle control was given once daily. Pre- and posttreatment (day 1 or 5) were commenced at 1 week before and 1 or 5 days after intravitreal injections, respectively, and were continued until postinjection day 28. Effects of treatment on retinal structure and functions were evaluated using optical coherence tomography (OCT), doppler OCT and electroretinogram measurements at baseline, post-injection days 10 and 28. RGC survival was evaluated by using RBPMS immunostaining on retinal wholemounts. RESULTS: ET-1 injection in vehicle control induced transient reductions in arterial flow and retinal functions, leading to significant RNFL thinning and RGC loss at day 28. Although ET-1 induced a transient loss in blood flow or retinal functions in all LbGP groups, LbGP treatments facilitated better restoration of retinal flow and retinal functions as compared with the vehicle control. Also, all three LbGP treatment groups (i.e. pre- and posttreatments from days 1 or 5) significantly preserved thRNFL thickness and RGC densities. No significant difference in protective effects was observed among the three LbGP treatment groups. CONCLUSION: LbGP demonstrated neuroprotective effects in a mouse model of ET-1 induced RGC degeneration, with treatment applied either as a pretreatment, immediate or delayed posttreatment. LbGP treatment promoted a better restoration of retinal blood flow, and protected the RNFL, RGC density and retinal functions. This study showed the translational potential of LB as complementary treatment for glaucoma management.

Protocadherin 19 regulates axon guidance in the developing Xenopus retinotectal pathway.

Protocadherin 19 (Pcdh19) is a homophilic cell adhesion molecule and is involved in a variety of neuronal functions. Here, we tested whether Pcdh19 has a regulatory role in axon guidance using the developing Xenopus retinotectal system. We performed targeted microinjections of a translation blocking antisense morpholino oligonucleotide to knock down the expression of Pcdh19 selectively in the central nervous system. Knocking down Pcdh19 expression resulted in navigational errors of retinal ganglion cell (RGC) axons specifically at the optic chiasm. Instead of projecting to the contralateral optic tectum, RGC axons in the Pcdh19-depleted embryo misprojected ipsilaterally. Although incorrectly delivered into the ipsilateral brain hemisphere, these axons correctly reached the optic tectum. These data suggest that Pcdh19 has a critical role in preventing mixing of RGC axons originating from the opposite eyes at the optic chiasm, highlighting the importance of cell adhesion in bundling of RGC axons.

Oral nicotinamide provides robust, dose-dependent structural and metabolic neuroprotection of retinal ganglion cells in experimental glaucoma.

A compromised capacity to maintain NAD pools is recognized as a key underlying pathophysiological feature of neurodegenerative diseases. NAD acts as a substrate in major cell functions including mitochondrial homeostasis, cell signalling, axonal transport, axon/Wallerian degeneration, and neuronal energy supply. Dendritic degeneration is an early marker of neuronal stress and precedes cell loss. However, little is known about dendritic structural preservation in pathologic environments and remodelling in mature neurons. Retinal ganglion cell dendritic atrophy is an early pathological feature in animal models of the disease and has been demonstrated in port-mortem human glaucoma samples. Here we report that a nicotinamide (a precursor to NAD through the NAD salvage pathway) enriched diet provides robust retinal ganglion cell dendritic protection and preserves dendritic structure in a rat model of experimental glaucoma. Metabolomic analysis of optic nerve samples from the same animals demonstrates that nicotinamide provides robust metabolic neuroprotection in glaucoma. Advances in our understanding of retinal ganglion cell metabolic profiles shed light on the energetic shift that triggers early neuronal changes in neurodegenerative diseases. As nicotinamide can improve visual function short term in existing glaucoma patients, we hypothesize that a portion of this visual recovery may be due to dendritic preservation in stressed, but not yet fully degenerated, retinal ganglion cells.

Inhibition of angiogenesis by the secretome from iPSC-derived retinal ganglion cells with Leber's hereditary optic neuropathy-like phenotypes.

The blood supply in the retina ensures photoreceptor function and maintains regular vision. Leber's hereditary optic neuropathy (LHON), caused by the mitochondrial DNA mutations that deteriorate complex I activity, is characterized by progressive vision loss. Although some reports indicated retinal vasculature abnormalities as one of the comorbidities in LHON, the paracrine influence of LHON-affected retinal ganglion cells (RGCs) on vascular endothelial cell physiology remains unclear. To address this, we established an in vitro model of mitochondrial complex I deficiency using induced pluripotent stem cell-derived RGCs (iPSC-RGCs) treated with a mitochondrial complex I inhibitor rotenone (Rot) to recapitulate LHON pathologies. The secretomes from Rot-treated iPSC-RGCs (Rot-iPSC-RGCs) were collected, and their treatment effect on human umbilical vein endothelial cells (HUVECs) was studied. Rot induced LHON-like characteristics in iPSC-RGCs, including decreased mitochondrial complex I activity and membrane potential, and increased mitochondrial reactive oxygen species (ROS) and apoptosis, leading to mitochondrial dysfunction. When HUVECs were exposed to conditioned media (CM) from Rot-iPSC-RGCs, the angiogenesis of HUVECs was suppressed compared to those treated with CM from control iPSC-RGCs (Ctrl-iPSC-RGCs). Angiogenesis-related proteins were altered in the secretomes from Rot-iPSC-RGC-derived CM, particularly angiopoietin, MMP-9, uPA, collagen XVIII, and VEGF were reduced. Notably, GeneMANIA analysis indicated that VEGFA emerged as the pivotal angiogenesis-related protein among the identified proteins secreted by health iPSC-RGCs but reduced in the secretomes from Rot-iPSC-RGCs. Quantitative real-time PCR and western blots confirmed the reduction of VEGFA at both transcription and translation levels, respectively. Our study reveals that Rot-iPSC-RGCs establish a microenvironment to diminish the angiogenic potential of vascular cells nearby, shedding light on the paracrine regulation of LHON-affected RGCs on retinal vasculature.

Macrophages Modulate Optic Nerve Crush Injury Scar Formation and Retinal Ganglion Cell Function.

Purpose: Optic nerve (ON) injuries can result in vision loss via structural damage and cellular injury responses. Understanding the immune response, particularly the role of macrophages, in the cellular response to ON injury is crucial for developing therapeutic approaches which affect ON injury repair. The present study investigates the role of macrophages in ON injury response, fibrotic scar formation, and retinal ganglion cell (RGC) function. Methods: The study utilizes macrophage Fas-induced apoptosis (MaFIA) mice to selectively deplete hematogenous macrophages and explores the impact macrophages have on ON injury responses. Histological and immunofluorescence analyses were used to evaluate macrophage expression levels and fibrotic scar formation. Pattern electroretinogram (PERG) recordings were used to assess RGC function as result of ON injury. Results: Successful macrophage depletion was induced in MaFIA mice, which led to reduced fibrotic scar formation in the ON post-injury. Despite an increase in activated macrophages in the retina, RGC function was preserved, as demonstrated by normal PERG waveforms for up to 2 months post-injury. The study suggests a neuroprotective role for macrophage depletion in ON damage repair and highlights the complex immune response to ON injury. Conclusions: To our knowledge, this study is the first to use MaFIA mice to demonstrate that targeted depletion of hematogenous macrophages leads to a significant reduction in scar size and the preservation of RGC functionality after ON injury. These findings highlight the key role of hematogenous macrophages in the response to ON injury and opens new avenues for therapeutic interventions in ON injuries. Future research should focus on investigating the distinct roles of macrophage subtypes in ON injury and potential macrophage-associated molecular targets to improve ON regeneration and repair.