Focused Ultrasound as a Novel Non-Invasive Method for the Delivery of Gold Nanoparticles to Retinal Ganglion Cells.

Purpose: The blood-retinal barrier (BRB) restricts the delivery of intravenous therapeutics to the retina, necessitating innovative approaches for treating retinal disorders. This study sought to explore the potential of focused ultrasound (FUS) to non-invasively deliver intravenously administered gold nanoparticles (AuNPs) across the BRB. FUS-BRB modulation can offer a novel method for targeted retinal therapy. Methods: AuNPs of different sizes and shapes were characterized, and FUS parameters were optimized to permeate the BRB without causing retinal damage in a rodent model. The delivery of 70-kDa dextran and AuNPs to the retinal ganglion cell (RGC) layer was visualized using confocal and two-photon microscopy, respectively. Histological and statistical analyses were conducted to assess the effectiveness and safety of the procedure. Results: FUS-BRB modulation resulted in the delivery of dextran and AuNPs to the RGC and inner nuclear layer. Smaller AuNPs reached the retinal layers to a greater extent than larger ones. The delivery of dextran and AuNPs across the BRB with FUS was achieved without significant retinal damage. Conclusions: This investigation provides the first evidence, to our knowledge, of FUS-mediated AuNP delivery across the BRB, establishing a foundation for a targeted and non-invasive approach to retinal treatment. The results contribute to developing promising non-invasive therapeutic strategies in ophthalmology to treat retinal diseases. Translational Relevance: Modifying the BRB with ultrasound offers a targeted and non-invasive delivery strategy of intravenous therapeutics to the retina.

PallorMetrics: Software for Automatically Quantifying Optic Disc Pallor in Fundus Photographs, and Associations With Peripapillary RNFL Thickness.

Purpose: We sough to develop an automatic method of quantifying optic disc pallor in fundus photographs and determine associations with peripapillary retinal nerve fiber layer (pRNFL) thickness. Methods: We used deep learning to segment the optic disc, fovea, and vessels in fundus photographs, and measured pallor. We assessed the relationship between pallor and pRNFL thickness derived from optical coherence tomography scans in 118 participants. Separately, we used images diagnosed by clinical inspection as pale (n = 45) and assessed how measurements compared with healthy controls (n = 46). We also developed automatic rejection thresholds and tested the software for robustness to camera type, image format, and resolution. Results: We developed software that automatically quantified disc pallor across several zones in fundus photographs. Pallor was associated with pRNFL thickness globally (ß = -9.81; standard error [SE] = 3.16; P < 0.05), in the temporal inferior zone (ß = -29.78; SE = 8.32; P < 0.01), with the nasal/temporal ratio (ß = 0.88; SE = 0.34; P < 0.05), and in the whole disc (ß = -8.22; SE = 2.92; P < 0.05). Furthermore, pallor was significantly higher in the patient group. Last, we demonstrate the analysis to be robust to camera type, image format, and resolution. Conclusions: We developed software that automatically locates and quantifies disc pallor in fundus photographs and found associations between pallor measurements and pRNFL thickness. Translational Relevance: We think our method will be useful for the identification, monitoring, and progression of diseases characterized by disc pallor and optic atrophy, including glaucoma, compression, and potentially in neurodegenerative disorders.

Characterization of the development of the high-acuity area of the chick retina.

The fovea is a small region within the central retina that is responsible for our high acuity daylight vision. Chickens also have a high acuity area (HAA), and are one of the few species that enables studies of the mechanisms of HAA development, due to accessible embryonic tissue and methods to readily perturb gene expression. To enable such studies, we characterized the development of the chick HAA using single molecule fluorescent in situ hybridization (smFISH), along with more classical methods. We found that Fgf8 provides a molecular marker for the HAA throughout development and into adult stages, allowing studies of the cellular composition of this area over time. The radial dimension of the ganglion cell layer (GCL) was seen to be the greatest at the HAA throughout development, beginning during the period of neurogenesis, suggesting that genesis, rather than cell death, creates a higher level of retinal ganglion cells (RGCs) in this area. In contrast, the HAA acquired its characteristic high density of cone photoreceptors post-hatching, which is well after the period of neurogenesis. We also confirmed that rod photoreceptors are not present in the HAA. Analyses of cell death in the developing photoreceptor layer, where rods would reside, did not show apoptotic cells, suggesting that lack of genesis, rather than death, created the "rod-free zone" (RFZ). Quantification of each cone photoreceptor subtype showed an ordered mosaic of most cone subtypes. The changes in cellular densities and cell subtypes between the developing and mature HAA provide some answers to the overarching strategy used by the retina to create this area and provide a framework for future studies of the mechanisms underlying its formation.

Optimizing retinal ganglion cell nuclear staining for automated cell counting.

The retinal ganglion cells (RGCs) serve as the critical pathway for transmitting visual information from the retina to the brain, yet they can be dramatically impacted by diseases such as glaucoma. When investigating disease processes affecting RGCs in mouse models, accurately quantifying affected cells becomes essential. However, the use of pan RGC markers like RBPMS or THY1 presents challenges in accurate total cell counting. While Brn3a serves as a reliable RGC nuclear marker for automated counting, it fails to encompass all RGC subtypes in mice. To address this limitation and enable precise automated counting, our research endeavors to develop a method for labeling nuclei in all RGC subtypes. Investigating RGC subtypes labeled with the nuclear marker POU6F2 revealed that numerous RGCs unlabeled by Brn3a were, in fact, labeled with POU6F2. We hypothesize that using antibodies against both Brn3a and POU6F2 would label virtually all RGC nuclei in the mouse retina. Our experiments confirmed that staining retinas with both markers resulted in the labeling of all RGCs. Additionally, when using the cell body marker RBPMS known to label all mouse RGCs, all RBPMS-labeled cells also exhibited Brn3a or POU6F2 labeling. This combination of Brn3a and POU6F2 antibodies provides a pan-RGC nuclear stain, facilitating accurate automated counting by labeling cell nuclei in the retina.

Retinal ganglion cell topography and spatial resolving power in the pajama cardinalfish Sphaeramia nematoptera (Bleeker, 1856).

We studied the topography of retinal ganglion cells (GCs) and estimated spatial resolving power (SRP) in the pajama cardinalfish Sphaeramia nematoptera (Bleeker, 1856), a relatively small brightly colored fish inhabiting coral reefs and lagoons in the Western Pacific. S. nematoptera is an active night predator feeding on near-bottom animal plankton and benthos. DAPI staining was used to label nuclei of GCs and non-GCs in the inner plexiform and ganglion cell layers. Non-GCs were distinguished from GCs in Nissl-stained retinal wholemounts based on cell size, shape, and staining intensity. The proportion of displaced amacrine cells (DACs) varied from 15.46 ± 1.12 (visual streak [VS]) to 17.99 ± 1.06% (dorsal periphery) (mean ± S.E.M., N = 5); the respective proportions of glial cells were 6.61 ± 0.84 and 5.89 ± 0.76%. Thus, 76%-78% of cells in the ganglion cell layer and inner plexiform layer were GCs. The minimum spatial coverage of GCs (3600-4600 cells/mm2) was detected in the dorsal and ventral periphery. It gradually increased toward the central retina to form a moderate VS. The maximum GC density (11,400-12,400 cells/mm2) was registered in the central portion of the VS. No pronounced concentric retinal specializations were found. The total number of GCs ranged within 595.2-635.9 × 103. The anatomical spatial resolving power was minimum in the ventral periphery (4.91-5.53 cpd) and maximum in the central portion of the VS (8.47-9.07 cpd). The respective minimum separable angles were 0.18-0.20° and 0.11-0.12°. The relatively high spatial resolving power and presence of the VS in the pajama cardinalfish are in line with its highly visual behavior.

A molecular switch for neuroprotective astrocyte reactivity.

The intrinsic mechanisms that regulate neurotoxic versus neuroprotective astrocyte phenotypes and their effects on central nervous system degeneration and repair remain poorly understood. Here we show that injured white matter astrocytes differentiate into two distinct C3-positive and C3-negative reactive populations, previously simplified as neurotoxic (A1) and neuroprotective (A2)1,2, which can be further subdivided into unique subpopulations defined by proliferation and differential gene expression signatures. We find the balance of neurotoxic versus neuroprotective astrocytes is regulated by discrete pools of compartmented cyclic adenosine monophosphate derived from soluble adenylyl cyclase and show that proliferating neuroprotective astrocytes inhibit microglial activation and downstream neurotoxic astrocyte differentiation to promote retinal ganglion cell survival. Finally, we report a new, therapeutically tractable viral vector to specifically target optic nerve head astrocytes and show that raising nuclear or depleting cytoplasmic cyclic AMP in reactive astrocytes inhibits deleterious microglial or macrophage cell activation and promotes retinal ganglion cell survival after optic nerve injury. Thus, soluble adenylyl cyclase and compartmented, nuclear- and cytoplasmic-localized cyclic adenosine monophosphate in reactive astrocytes act as a molecular switch for neuroprotective astrocyte reactivity that can be targeted to inhibit microglial activation and neurotoxic astrocyte differentiation to therapeutic effect. These data expand on and define new reactive astrocyte subtypes and represent a step towards the development of gliotherapeutics for the treatment of glaucoma and other optic neuropathies.

An ON-type direction-selective ganglion cell in primate retina.

To maintain a stable and clear image of the world, our eyes reflexively follow the direction in which a visual scene is moving. Such gaze-stabilization mechanisms reduce image blur as we move in the environment. In non-primate mammals, this behaviour is initiated by retinal output neurons called ON-type direction-selective ganglion cells (ON-DSGCs), which detect the direction of image motion and transmit signals to brainstem nuclei that drive compensatory eye movements1. However, ON-DSGCs have not yet been identified in the retina of primates, raising the possibility that this reflex is mediated by cortical visual areas. Here we mined single-cell RNA transcriptomic data from primate retina to identify a candidate ON-DSGC. We then combined two-photon calcium imaging, molecular identification and morphological analysis to reveal a population of ON-DSGCs in the macaque retina. The morphology, molecular signature and GABA (γ-aminobutyric acid)-dependent mechanisms that underlie direction selectivity in primate ON-DSGCs are highly conserved with those in other mammals. We further identify a candidate ON-DSGC in human retina. The presence of ON-DSGCs in primates highlights the need to examine the contribution of subcortical retinal mechanisms to normal and aberrant gaze stabilization in the developing and mature visual system.

CD73 controls ocular adenosine levels and protects retina from light-induced phototoxicity.

ATP and adenosine have emerged as important signaling molecules involved in vascular remodeling, retinal functioning and neurovascular coupling in the mammalian eye. However, little is known about the regulatory mechanisms of purinergic signaling in the eye. Here, we used three-dimensional multiplexed imaging, in situ enzyme histochemistry, flow cytometric analysis, and single cell transcriptomics to characterize the whole pattern of purine metabolism in mouse and human eyes. This study identified ecto-nucleoside triphosphate diphosphohydrolase-1 (NTPDase1/CD39), NTPDase2, and ecto-5'-nucleotidase/CD73 as major ocular ecto-nucleotidases, which are selectively expressed in the photoreceptor layer (CD73), optic nerve head, retinal vasculature and microglia (CD39), as well as in neuronal processes and cornea (CD39, NTPDase2). Specifically, microglial cells can create a spatially arranged network in the retinal parenchyma by extending and retracting their branched CD39high/CD73low processes and forming local "purinergic junctions" with CD39low/CD73- neuronal cell bodies and CD39high/CD73- retinal blood vessels. The relevance of the CD73-adenosine pathway was confirmed by flash electroretinography showing that pharmacological inhibition of adenosine production by injection of highly selective CD73 inhibitor PSB-12489 in the vitreous cavity of dark-adapted mouse eyes rendered the animals hypersensitive to prolonged bright light, manifested as decreased a-wave and b-wave amplitudes. The impaired electrical responses of retinal cells in PSB-12489-treated mice were not accompanied by decrease in total thickness of the retina or death of photoreceptors and retinal ganglion cells. Our study thus defines ocular adenosine metabolism as a complex and spatially integrated network and further characterizes the critical role of CD73 in maintaining the functional activity of retinal cells.

Retinal ganglion cell topography and spatial resolving power of eye in the native chicken of Bangladesh / Topografía de células ganglionares de la retina y poder de resolución espacial del ojo en el pollo nativo de Bangladesh

SUMMARY: The distribution of retinal ganglion cells (RGCs) was observed in the retinal wholemount of native chicken (Gallus gallus domestricus) of Bangladesh by using light microscopy. We considered five different anatomic regions (central, nasal, temporal, dorsal, and ventral) of Nissl stained wholemount, and the RGCs were counted, plotted, and measured accordingly. The average area of the retina was 431.75 mm2 while the total number of ganglion cells was 2124431 on average. Only the central area of the retina was the peak density (10400 cells/mm2) area, signifying the acute visual area, whilst the maximum spatial resolving power was 11 cycles/degree. The overall concentration of RGCs gradually declined towards the periphery but the size of cells generally decreased towards centrally. The size of ganglion cell was not uniform (12 to 180 µm2), specifically the central retina, just above the optic disc was packed with tiny-sized cells. The number, topographic distribution, and size of RGCs in native chicken signified their domesticated or terrestrial characters, uneven visual acuteness, and possibly only the central retina was the area for fine vision as the function of RGCs.

RESUMEN: En este studio se observó la distribución de las células ganglionares en la retina (CGR) de pollo nativo (Gallus gallus domesticus) de Bangladesh mediante el uso de microscopía óptica. Consideramos cinco regiones anatómicas diferentes (central, nasal, temporal, dorsal y ventral). Las muestras de CGR se tiñeron con Nissl, posteriormente, se midieron y contó el número de células totales. El área promedio de la retina fue de 431,75 mm2, mientras que el promedio del número total de células ganglionares fue de 2124431. El área central de la retina fue el área de densidad máxima (10400 células / mm2), señalando el área visual aguda, mientras que el poder de resolución espacial máximo fue de 11 ciclos / grado. La concentración general de CGR disminuyó gradualmente hacia la periferia, sin embargo, el tamaño de las células disminuyó hacia el centro. El tamaño de las CGR no fue uniforme (12 a 180 mm2), específicamente en la retina central, por encima del disco óptico, aumentaron significativamente las células pequeñas. El número, la distribución topográfica y el tamaño de las CGR en pollos nativos determinaron las características domésticas o terrestres, agudeza visual desigual y, posiblemente, la función de las CGR, en la retina central era el área de visión fina.

Polydopamine nanoparticles attenuate retina ganglion cell degeneration and restore visual function after optic nerve injury.

BACKGROUND: Oxidative stress contributes to retina ganglion cells (RGCs) loss in variety of ocular diseases, including ocular trauma, ocular vein occlusion, and glaucoma. Scavenging the excessed reactive oxygen species (ROS) in retinal neurovascular unit could be beneficial to RGCs survival. In this study, a polydopamine (PDA)-based nanoplatform is developed to protect RGCs. RESULTS: The PDA nanoparticles efficiently eliminate multi-types of ROS, protect endothelia and neuronal cells from oxidative damage, and inhibit microglia activation in retinas. In an optic nerve crush (ONC) model, single intravitreal injection of PDA nanoparticles could significantly attenuate RGCs loss via eliminating ROS in retinas, reducing the inflammatory response and maintaining barrier function of retinal vascular endothelia. Comparative transcriptome analysis of the retina implied that PDA nanoparticles improve RGCs survival probably by altering the expression of genes involved in inflammation and ROS production. Importantly, as a versatile drug carrier, PDA nanoparticles could deliver brimonidine (a neuroprotection drug) to synergistically attenuate RGCs loss and promote axon regeneration, thus restore visual function. CONCLUSIONS: The PDA nanoparticle-based therapeutic nanoplatform displayed excellent performance in ROS elimination, providing a promising probability for treating retinal degeneration diseases.

Single Cell Sequencing of Induced Pluripotent Stem Cell Derived Retinal Ganglion Cells (iPSC-RGC) Reveals Distinct Molecular Signatures and RGC Subtypes.

We intend to identify marker genes with differential gene expression (DEG) and RGC subtypes in cultures of human-induced pluripotent stem cell (iPSC)-derived retinal ganglion cells. Single-cell sequencing was performed on mature and functional iPSC-RGCs at day 40 using Chromium Single Cell 3' V3 protocols (10X Genomics). Sequencing libraries were run on Illumina Novaseq to generate 150 PE reads. Demultiplexed FASTQ files were mapped to the hg38 reference genome using the STAR package, and cluster analyses were performed using a cell ranger and BBrowser2 software. QC analysis was performed by removing the reads corresponding to ribosomal and mitochondrial genes, as well as cells that had less than 1X mean absolute deviation (MAD), resulting in 4705 cells that were used for further analyses. Cells were separated into clusters based on the gene expression normalization via PCA and TSNE analyses using the Seurat tool and/or Louvain clustering when using BBrowser2 software. DEG analysis identified subsets of RGCs with markers like MAP2, RBPMS, TUJ1, BRN3A, SOX4, TUBB3, SNCG, PAX6 and NRN1 in iPSC-RGCs. Differential expression analysis between separate clusters identified significant DEG transcripts associated with cell cycle, neuron regulatory networks, protein kinases, calcium signaling, growth factor hormones, and homeobox transcription factors. Further cluster refinement identified RGC diversity and subtype specification within iPSC-RGCs. DEGs can be used as biomarkers for RGC subtype classification, which will allow screening model systems that represent a spectrum of diseases with RGC pathology.

Protocol for evaluating the role of a gene in protecting mouse retinal ganglion cells.

The degeneration of retinal ganglion cells (RGCs) leads to irreversible vision loss in a variety of pathological states. Here, we describe a protocol to evaluate the role of a gene in protecting mouse RGCs when they sustain injuries from excitotoxicity or axonal damage. This protocol includes the procedures for gene transfer through AAV intravitreal injection, induction of RGC injuries by NMDA-induced excitotoxicity or optic nerve crush, and retina immunohistochemistry to assess RGC survival. For complete details on the use and execution of this protocol, please refer to Guo et al. (2021).

Serotonin transporter-mediated molecular axis regulates regional retinal ganglion cell vulnerability and axon regeneration after nerve injury.

Molecular insights into the selective vulnerability of retinal ganglion cells (RGCs) in optic neuropathies and after ocular trauma can lead to the development of novel therapeutic strategies aimed at preserving RGCs. However, little is known about what molecular contexts determine RGC susceptibility. In this study, we show the molecular mechanisms underlying the regional differential vulnerability of RGCs after optic nerve injury. We identified RGCs in the mouse peripheral ventrotemporal (VT) retina as the earliest population of RGCs susceptible to optic nerve injury. Mechanistically, the serotonin transporter (SERT) is upregulated on VT axons after injury. Utilizing SERT-deficient mice, loss of SERT attenuated VT RGC death and led to robust retinal axon regeneration. Integrin ß3, a factor mediating SERT-induced functions in other systems, is also upregulated in RGCs and axons after injury, and loss of integrin ß3 led to VT RGC protection and axon regeneration. Finally, RNA sequencing analyses revealed that loss of SERT significantly altered molecular signatures in the VT retina after optic nerve injury, including expression of the transmembrane protein, Gpnmb. GPNMB is rapidly downregulated in wild-type, but not SERT- or integrin ß3-deficient VT RGCs after injury, and maintaining expression of GPNMB in RGCs via AAV2 viruses even after injury promoted VT RGC survival and axon regeneration. Taken together, our findings demonstrate that the SERT-integrin ß3-GPNMB molecular axis mediates selective RGC vulnerability and axon regeneration after optic nerve injury.

Transplanted Mouse Embryonic Stem Cell-Derived Retinal Ganglion Cells Integrate and Form Synapses in a Retinal Ganglion Cell-Depleted Mouse Model.

Purpose: Retinal ganglion cell (RGC) transplantation is a therapeutic approach to replace irreversibly degenerated RGCs in diseases such as glaucoma. However, the application of primary RGCs is limited by the availability of tissues. The goal of this study was to evaluate whether transplanted mouse embryonic stem cell (mESC)-derived RGCs can integrate into the host retina and form cell connectivity with host cells. Methods: In this study, we prepared small retinal fragments containing RGC as THY1-enhanced green fluorescent protein (EGFP)+ cells from mESCs and placed them near the retinal surface in the air-injected mouse eyes with or without N-methyl-d-aspartate (NMDA)-induced RGC depletion. After transplantation, THY1-EGFP+ cell integration was observed in whole-mounts and with immunostaining for synaptic markers. Results: Transplanted THY1-EGFP+ cells survived for 12 weeks and extended neurites into the inner plexiform layer (IPL) of the host retina. Presumptive synapse formation was identified between grafted RGCs and host bipolar cells. The ratio of transplanted eyes with integration of THY1-EGFP+ neurites in the host IPL was higher in RGC-injured mice compared with healthy controls. Conclusions: This report shows the potential for therapeutic use of pluripotent cell-derived RGCs by grafting the cells in healthy conditions and with an appropriate technical approach.

Morphometric and Microstructural Changes During Murine Retinal Development Characterized Using In Vivo Optical Coherence Tomography.

Purpose: The purpose of this study was to develop an in vivo optical coherence tomography (OCT) system capable of imaging the developing mouse retina and its associated morphometric and microstructural changes. Methods: Thirty-four wild-type mice (129S1/SvlmJ) were anesthetized and imaged between postnatal (P) day 7 and P21. OCT instrumentation was developed to optimize signal intensity and image quality. Semi-automatic segmentation tools were developed to quantify the retinal thickness of the nerve fiber layer (NFL), inner plexiform layer (IPL), inner nuclear layer (INL), and the outer retinal layers (ORL), in addition to the total retina. The retinal maturation was characterized by comparing layer thicknesses between consecutive time points. Results: From P7 to P10, the IPL increased significantly, consistent with retinal synaptogenesis. From P10 to P12, the IPL and ORL also increased, which is coherent with synaptic connectivity and photoreceptor maturation. In contrast, during these periods, the INL decreased significantly, consistent with cellular densification and selective apoptotic "pruning" of the tissue during nuclear migration. Thereafter from P12 to P21, the INL continued to thin (significantly from P17 to P21) whereas the other layers remained unchanged. No time-dependent changes were observed in the NFL. Overall, changes in the total retina were attributed to those in the IPL, INL, and ORL. Regions of the retina adjacent to the optic nerve head were thinner than distal regions during maturation. Conclusions: Changes in retinal layer thickness are consistent with retinal developmental mechanisms. Accordingly, this report opens new horizons in using our system in the mouse to characterize longitudinally developmental digressions in models of human diseases.

Dim light in the evening causes coordinated realignment of circadian rhythms, sleep, and short-term memory.

Light provides the primary signal for entraining circadian rhythms to the day/night cycle. In addition to rods and cones, the retina contains a small population of photosensitive retinal ganglion cells (pRGCs) expressing the photopigment melanopsin (OPN4). Concerns have been raised that exposure to dim artificial lighting in the evening (DLE) may perturb circadian rhythms and sleep patterns, and OPN4 is presumed to mediate these effects. Here, we examine the effects of 4-h, 20-lux DLE on circadian physiology and behavior in mice and the role of OPN4 in these responses. We show that 2 wk of DLE induces a phase delay of ∼2 to 3 h in mice, comparable to that reported in humans. DLE-induced phase shifts are unaffected in Opn4-/- mice, indicating that rods and cones are capable of driving these responses in the absence of melanopsin. DLE delays molecular clock rhythms in the heart, liver, adrenal gland, and dorsal hippocampus. It also reverses short-term recognition memory performance, which is associated with changes in preceding sleep history. In addition, DLE modifies patterns of hypothalamic and cortical cFos signals, a molecular correlate of recent neuronal activity. Together, our data show that DLE causes coordinated realignment of circadian rhythms, sleep patterns, and short-term memory process in mice. These effects are particularly relevant as DLE conditions-due to artificial light exposure-are experienced by the majority of the populace on a daily basis.

Red Light Irradiation In Vivo Upregulates DJ-1 in the Retinal Ganglion Cell Layer and Protects against Axotomy-Related Dendritic Pruning.

Retinal ganglion cells (RGCs) undergo dendritic pruning in a variety of neurodegenerative diseases, including glaucoma and autosomal dominant optic atrophy (ADOA). Axotomising RGCs by severing the optic nerve generates an acute model of RGC dendropathy, which can be utilized to assess the therapeutic potential of treatments for RGC degeneration. Photobiomodulation (PBM) with red light provided neuroprotection to RGCs when administered ex vivo to wild-type retinal explants. In the current study, we used aged (13-15-month-old) wild-type and heterozygous B6;C3-Opa1Q285STOP (Opa1+/-) mice, a model of ADOA exhibiting RGC dendropathy. These mice were pre-treated with 4 J/cm2 of 670 nm light for five consecutive days before the eyes were enucleated and the retinas flat-mounted into explant cultures for 0-, 8- or 16-h ex vivo. RGCs were imaged by confocal microscopy, and their dendritic architecture was quantified by Sholl analysis. In vivo 670 nm light pretreatment inhibited the RGC dendropathy observed in untreated wild-type retinas over 16 h ex vivo and inhibited dendropathy in ON-center RGCs in wild-type but not Opa1+/- retinas. Immunohistochemistry revealed that aged Opa1+/- RGCs exhibited increased nitrosative damage alongside significantly lower activation of NF-κB and upregulation of DJ-1. PBM restored NF-κB activation in Opa1+/- RGCs and enhanced DJ-1 expression in both genotypes, indicating a potential molecular mechanism priming the retina to resist future oxidative insult. These data support the potential of PBM as a treatment for diseases involving RGC degeneration.

Neuroprotective effects of glial mediators in interactions between retinal neurons and Müller cells.

Progressive retinal ganglion cell (RGC) loss underlies a number of retinal neurodegenerative disorders, which may lead to permanent vision loss. However, secreted neuroprotective factors, such as PEDF, VEGF and IL-6, which are produced by Müller cells, have been shown to promote RGC survival. Assuming that the communication of RGCs with Müller cells involves a release of glioactive substances we sought to determine whether retinal neurons are able to modulate expression levels of Müller cell-derived PEDF, VEGF and IL-6. We demonstrate elevated mRNA levels of these factors in Müller cells in co-cultures with RGCs or R28 cells when compared to homotypic Müller cell cultures. Furthermore, R28 cells were more protected from apoptosis when co-cultured with Müller cells. IL-6 and VEGF were upregulated in Müller cells under hypoxia. Both cytokines, as well as PEDF, induced an altered neuronal expression of members of the Bcl-2 family, which are central molecules in the regulation of apoptosis. These results suggest that in retinal ischemia, via own secreted mediators, RGCs can resist a potential demise by stimulating Müller cells to increase production of neuroprotective factors, which counteract RGC apoptosis.

Retinal Ganglion Cell Transplantation: Approaches for Overcoming Challenges to Functional Integration.

As part of the central nervous system, mammalian retinal ganglion cells (RGCs) lack significant regenerative capacity. Glaucoma causes progressive and irreversible vision loss by damaging RGCs and their axons, which compose the optic nerve. To functionally restore vision, lost RGCs must be replaced. Despite tremendous advancements in experimental models of optic neuropathy that have elucidated pathways to induce endogenous RGC neuroprotection and axon regeneration, obstacles to achieving functional visual recovery through exogenous RGC transplantation remain. Key challenges include poor graft survival, low donor neuron localization to the host retina, and inadequate dendritogenesis and synaptogenesis with afferent amacrine and bipolar cells. In this review, we summarize the current state of experimental RGC transplantation, and we propose a set of standard approaches to quantifying and reporting experimental outcomes in order to guide a collective effort to advance the field toward functional RGC replacement and optic nerve regeneration.

Establishing the ground squirrel as a superb model for retinal ganglion cell disorders and optic neuropathies.

Retinal ganglion cell (RGC) death occurs after optic nerve injury due to acute trauma or chronic degenerative conditions such as optic neuropathies (e.g., glaucoma). Currently, there are no effective therapies to prevent permanent vision loss resulting from RGC death, underlining the need for research on the pathogenesis of RGC disorders. Modeling human RGC/optic nerve diseases in non-human primates is ideal because of their similarity to humans, but has practical limitations including high cost and ethical considerations. In addition, many retinal degenerative disorders are age-related making the study in primate models prohibitively slow. For these reasons, mice and rats are commonly used to model RGC injuries. However, as nocturnal mammals, these rodents have retinal structures that differ from primates - possessing less than one-tenth of the RGCs found in the primate retina. Here we report the diurnal thirteen-lined ground squirrel (TLGS) as an alternative model. Compared to other rodent models, the number and distribution of RGCs in the TLGS retina are closer to primates. The TLGS retina possesses ~600,000 RGCs with the highest density along the equatorial retina matching the location of the highest cone density (visual streak). TLGS and primate retinas also share a similar interlocking pattern between RGC axons and astrocyte processes in the retina nerve fiber layer (RNFL). In addition, using TLGS we establish a new partial optic nerve injury model that precisely controls the extent of injury while sparing a portion of the retina as an ideal internal control for investigating the pathophysiology of axon degeneration and RGC death. Moreover, in vivo optical coherence tomography (OCT) imaging and ex vivo microscopic examinations of the retina in optic nerve injured TLGS confirm RGC loss precedes proximal axon degeneration, recapitulating human pathology. Thus, the TLGS retina is an excellent model, for translational research in neurodegeneration and therapeutic neuroprotection.