Isolation and Preparation of Embryonic Zebrafish Retinal Cells for Single-Cell RNA Sequencing.

Recent technological advances in single-cell RNA sequencing (scRNA-Seq) have enabled scientists to answer novel questions in biology with unparalleled precision. Indeed, in the field of ocular development and regeneration, scRNA-Seq studies have resulted in a number of exciting discoveries that have begun to revolutionize the way we think about these processes. Despite the widespread success of scRNA-Seq, many scientists are wary to perform scRNA-Seq experiments due to the uncertainty of obtaining high-quality viable cell populations that are necessary for the generation of usable data that enable rigorous computational analyses. Here, we describe methodology to reproducibility generate high-quality single-cell suspensions from embryonic zebrafish eyes. These single-cell suspensions served as inputs to the 10× Genomics v3.1 system and yielded high-quality scRNA-Seq data in proof-of-principle studies. In describing methodology to quantitatively assess cell yields, cell viability, and other critical quality control parameters, this protocol can serve as a useful starting point for others in designing their scRNA-Seq experiments in the zebrafish eye and in other developing or regenerating tissues in zebrafish or other model systems.

Experimental Framework for Assessing Mouse Retinal Regeneration Through Single-Cell RNA-Sequencing.

Retinal degenerative diseases including age-related macular degeneration and glaucoma are estimated to currently affect more than 14 million people in the United States, with an increased prevalence of retinal degenerations in aged individuals. An expanding aged population who are living longer forecasts an increased prevalence and economic burden of visual impairments. Improvements to visual health and treatment paradigms for progressive retinal degenerations slow vision loss. However, current treatments fail to remedy the root cause of visual impairments caused by retinal degenerations-loss of retinal neurons. Stimulation of retinal regeneration from endogenous cellular sources presents an exciting treatment avenue for replacement of lost retinal cells. In multiple species including zebrafish and Xenopus, Müller glial cells maintain a highly efficient regenerative ability to reconstitute lost cells throughout the organism's lifespan, highlighting potential therapeutic avenues for stimulation of retinal regeneration in humans. Here, we describe how the application of single-cell RNA-sequencing (scRNA-seq) has enhanced our understanding of Müller glial cell-derived retinal regeneration, including the characterization of gene regulatory networks that facilitate/inhibit regenerative responses. Additionally, we provide a validated experimental framework for cellular preparation of mouse retinal cells as input into scRNA-seq experiments, including insights into experimental design and analyses of resulting data.

Models of Photoreceptor Degeneration in Adult Zebrafish.

Zebrafish maintain a remarkable ability to regenerate their neural retina following rapid and extensive loss of retinal neurons. This is mediated by Müller glial cells (MG), which re-enter the cell cycle to produce amplifying progenitor cells that eventually differentiate into the lost retinal neurons. For example, exposing adult albino zebrafish to intense light destroys large numbers of rod and cone photoreceptors, which are then restored by MG-mediated regeneration. Here, we describe an updated method for performing these acute phototoxic lesions to adult zebrafish retinas. Next, we contrast this method to a chronic, low light lesion model that results in a more muted and sustained damage to photoreceptors and does not trigger a MG-mediated regeneration response. Thus, these two methods can be used to compare and contrast the genetic and morphological changes associated with acute and chronic methods of photoreceptor degeneration.

In Vivo Imaging of Rodent Retina in Retinal Disease.

High-quality imaging of the retina is crucial to the diagnosis and monitoring of disease, as well as for evaluating the success of therapeutics in human patients and in preclinical animal models. Here, we describe the basic principles and methods for in vivo retinal imaging in rodents, including fundus imaging, fluorescein angiography, optical coherence tomography, fundus autofluorescence, and infrared imaging. After providing a concise overview of each method and detailing the retinal diseases and conditions that can be visualized through them, we will proceed to discuss the advantages and disadvantages of each approach. These protocols will facilitate the acquisition of optimal images for subsequent quantification and analysis. Additionally, a brief explanation will be given regarding the potential results and the clinical significance of the detected abnormalities.

Retinal Explant Culture from Mouse, Human, and Nonhuman Primates and Its Applications in Vision Research.

The retinal explant culture system is a valuable tool for studying the pharmacological, toxicological, and developmental aspects of the retina. It is also used for translational studies such as gene therapy. While no photoreceptor-like cell lines are available for in vitro studies of photoreceptor cell biology, the retinal explant culture maintains the laminated retinal structure ex vivo for as long as a month. Human and nonhuman primate (NHP) postmortem retinal explants cut into small pieces offer the possibility of testing multiple conditions for safety and adeno-associated viral (AAV) vector optimization. In addition, the cone-enriched foveal area can be studied using the retinal explants. Here, we present a detailed working protocol for retinal explant isolation and culture from mouse, human, and NHP for testing drug efficacy and AAV transduction. Future applications of this protocol include combining live imaging and multiwell retinal explant culture for high-throughput drug screening systems in rodent and human retinal explants to identify new drugs against retinal degeneration.

Isolation and Characterization of Extracellular Vesicles to Activate Retina Regeneration.

Mammals do not possess the ability to spontaneously repair or regenerate damaged retinal tissue. In contrast to teleost fish which are capable of retina regeneration through the action of Müller glia, mammals undergo a process of reactive gliosis and scarring that inhibits replacement of lost neurons. Thus, it is important to discover novel methods for stimulating mammalian Müller glia to dedifferentiate and produce progenitor cells that can replace lost retinal neurons. Inducing an endogenous regenerative pathway mediated by Müller glia would provide an attractive alternative to stem cell injections or gene therapy approaches. Extracellular vesicles (EVs) are now recognized to serve as a novel form of cell-cell communication through the transfer of cargo from donor to recipient cells or by the activation of signaling cascades in recipient cells. EVs have been shown to promote proliferation and regeneration raising the possibility that delivery of EVs could be a viable treatment for visual disorders. Here, we provide protocols to isolate EVs for use in retina regeneration experiments.

Automated In Vivo Phenotypic Screening Platform for Identifying Factors that Affect Cell Regeneration Kinetics.

Various strategies for replacing retinal neurons lost in degenerative diseases are under investigation, including stimulating the endogenous regenerative capacity of Müller Glia (MG) as injury-inducible retinal stem cells. Inherently regenerative species, such as zebrafish, have provided key insights into mechanisms regulating MG dedifferentiation to a stem-like state and the proliferation of MG and MG-derived progenitor cells (MGPCs). Interestingly, promoting MG/MGPC proliferation is not sufficient for regeneration, yet mechanistic studies are often focused on this measure. To fully account for the regenerative process, and facilitate screens for factors regulating cell regeneration, an assay for quantifying cell replacement is required. Accordingly, we adapted an automated reporter-assisted phenotypic screening platform to quantify the pace of cellular regeneration kinetics following selective cell ablation in larval zebrafish. Here, we detail a method for using this approach to identify chemicals and genes that control the rate of retinal cell regeneration following selective retinal cell ablation.

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.

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.

Evaluation of optical coherence tomography angiography and pattern and flash electroretinography in diabetes mellitus without retinopathy.

PURPOSE: To evaluate the findings and the correlation of optical coherence tomography angiography and pattern and flash electroretinography in diabetes mellitus without retinopathy. METHODS: Seventy-six eyes of 38 diabetic patients and age- and gender-matched control subjects were included in the study. The foveal avascular zone (FAZ), whole, foveal, parafoveal and perifoveal vascular densities of the superficial capillary plexus (SCP), deep capillary plexus (DCP) and choriocapillary plexus (CCP) layers were analyzed using optical coherence tomography angiography (OCTA). The amplitudes and implicit times of P50 and N95 waves of the pattern ERG (pERG) and the amplitudes and implicit times of the scotopic and photopic b-waves and oscillatory potentials (OP) of the flash ERG (fERG) tests were evaluated using the Metrovision brand monpack model device. RESULTS: The mean age of the patients was 59.7 ± 7.9 [range 43-79] years. Eighteen (47%) of the patients were female and 20 (53%) were male. The mean duration of diabetes was 7.45 ± 6.2 [range 1-20] years. No significant difference in FAZ area was found between study subjects and controls. Vascular density (VD) values of the superficial capillary plexus (SCP) layer were significantly lower (whole VD, 44.7 ± 3.3 vs. 46.6 ± 3.2%, p = 0.01, foveal VD 16.8 ± 6.4 vs. 24.9 ± 6.1%, p < 0.01, parafoveal VD 45.6 ± 4.5 vs. 47.1 ± 4.4%, p = 0.27 and perifoveal VD 45.5 ± 3.3 vs. 47.3 ± 3.1%, p = 0.01, respectively) in the diabetic group except the parafoveal area. VD measurements in deep and choriocapillary plexuses did not significantly differ between the groups (p > 0.05). ERG tests revealed significantly lower scotopic b-wave amplitudes (130.2 ± 39.3 µV vs.163.3 ± 47.8 µV, p < 0.01) and photopic b-wave amplitudes (83.2 ± 20.7 µV vs. 99.6 ± 29.4 µV, p < 0.01) in the diabetic patients. The implicit time of the photopic responses was significantly prolonged (28.9 ± 1.3 ms vs. 27.8 ± 2.1 ms, p = 0.01) in the patients. Oscillatory potentials in all components consisting of O1 to O4 and the sum of the OP potentials were lower in the diabetic group than the control subjects (p < 0.001). The P50 and N95 amplitudes and implicit times were comparable between the groups (p > 0.05). Correlation analysis showed a positive correlation between N95 amplitudes in pERG and the superficial vessel densities in OCTA (r = 0.26, p = 0.04). A negative correlation was found between photopic implicit times in fERG and the choriocapillary vessel densities (r=-0.27, p = 0.03). CONCLUSION: OCTA revealed decreased superficial vascular densities with the onset of the metabolic process of diabetes mellitus. As a result of these structural changes, lower scotopic and photopic amplitudes, decreased OP amplitudes, and prolonged implicit times in flash ERG were obtained.

Bio-spectroscopic investigation linking changes of retinal structure with short-term administration of Amiodarone and revealing the ameliorative effect of vitamin E supplementation.

Long term use of Amiodarone (AMIO) is associated with the development of ocular adverse effects. This study investigates the short term effects, and the ameliorative consequence of vitamin E on retinal changes that were associated with administration of AMIO. This is accomplished by investigating both retinal structural and conformational characteristics using Fourier transform infrared spectroscopy (FTIR) and Fundus examination. Three groups of healthy rabbits of both sexes were used; the first group served as control. The second group was orally treated with AMIO (160 mg /kg body weight) in a daily basis for two weeks. The last group orally received AMIO as the second group for two weeks then, oral administration of vitamin E (100 mg/kg body weight) for another two weeks as well. FTIR results revealed significant structural and conformational changes in retinal tissue constituents that include lipids and proteins due to AMIO administration. AMIO treatment was associated with fluctuated changes (increased/decreased) in the band position and bandwidth of NH, OH, and CH bonds. This was concomitant with changes in the percentage of retinal protein constituents in particularly α-helix and Turns. AMIO facilitates the formation of intra-molecular hydrogen bonding and turned retinal lipids to be more disordered structure. In conclusion, the obtained FTIR data together with principal component analysis provide evidence that administration of vitamin E following the treatment with AMIO can ameliorate these retinal changes and, these biophysical changes are too early to be detected by Fundus examination.

Orchestrating Blood Flow in the Retina: Interpericyte Tunnelling Nanotube Communication.

The retina transforms light into electrical signals, which are sent to the brain via the optic nerve to form our visual perception. This complex signal processing is performed by the retinal neuron and requires a significant amount of energy. Since neurons are unable to store energy, they must obtain glucose and oxygen from the bloodstream to produce energy to match metabolic needs. This process is called neurovascular coupling (NVC), and it is based on a precise mechanism that is not totally understood. The discovery of fine tubular processes termed tunnelling nanotubes (TNTs) set a new type of cell-to-cell communication. TNTs are extensions of the cellular membrane that allow the transfer of material between connected cells. Recently, they have been reported in the brain and retina of living mice, where they connect pericytes, which are vascular mural cells that regulate vessel diameter. Accordingly, these TNTs were termed interpericyte tunnelling nanotubes (IPTNTs), which showed a vital role in blood delivery and NVC. In this chapter, we review the involvement of TNTs in NVC and discuss their implications in retinal neurodegeneration.

Preparation and Analysis of Histological Slides of Rat and Mouse Eyeballs to Evaluate the Retina.

A rodent eyeball is a powerful tool for researching the pathomechanisms of many ophthalmic diseases, such as glaucoma, hypertensive retinopathy, and many more. Preclinical experiments enable researchers to examine the efficacy of novel drugs, develop new methods of treatment, or seek new pathomechanisms involved in the disease's onset or progression. A histological examination provides a lot of information necessary to assess the effects of the conducted experiments and can reveal degeneration, tissue remodeling, infiltration, and many other pathologies. In clinical research, there is rarely any chance of obtaining eye tissue suitable for a histological examination, which is why researchers should take advantage of the opportunity offered by the examination of eyeballs from rodents. This manuscript presents a protocol for the histological preparation of rodent eyeballs' sections. The procedure is presented for the eyeballs of mice and rats and has the following steps: (i) harvesting the eyeball, (ii) preserving the eyeball for further analysis, (iii) processing the tissue in paraffin, (iv) preparing slides, (v) staining with hematoxylin and eosin, (vi) assessing the tissue under a light microscope. With the proposed method, the retina can be easily visualized and assessed in detail.

First Application of Whole Genome Sequencing in Myelinated Retinal Nerve Fibers (MRNF).

Genetic features are currently unknown in myelinated retinal nerve fibers (MRNF). For a 20-year-old asymptomatic female with unilateral MRNF, we performed whole genome sequencing (WGS) by standard workflow protocol to produce contiguous long-read sequences with Illumina DNA PCR-Free Prep. After tagmentation, libraries were sequenced on separate runs via NovaSeq 6000 platform at 2 x 150bp read length. Gene variants included rs2248799, rs2672589, rs7555070, rs247616_T and rs2043085_C all associated with an increased macular degeneration risk, and seven novel variants of uncertain significance. For optic disc enlargement, variants rs9988687_A, rs11079419_T, rs6787363 and rs10862708_A suggested an increased risk for this condition. In contrast, modeling revealed retinal detachment risk was reduced by variants identified at rs9651980_T, rs4373767_T, and rs7940691_T which were among five other previously unreported variants. WGS data placed proband at the 66th and 64th percentiles for disc anomaly and retinal detachment risk, respectively. Additionally, risk determined from 16 loci associated with age-related macular degeneration found the patient to be at the 18th percentile for this diagnosis (i.e., below average genetic predisposition). Fundoscopic findings showed mean RNFL thickness was lower with MRNF (77 OS vs. 96?m OD) and RNFL symmetry was impaired (43 %) but stable between 2020 and 2023. Rim area and cup volume were also substantially different (2.33 OS vs. 1.34mm2 OD, and 0.001 OS vs. 0.151mm3 OD, respectively). As the first known evaluation of MRNF via WGS, these data reveal a mixed picture with variants associated with different risks for potentially related ocular pathologies. In addition, we identify multiple new variants of unknown significance. Factors affecting gene expression in MRNF require further study. Key words: Whole genome sequencing, Retina, Myelination, Anatomy, Gene variants.

Loss of ON-Pathway Function in Mice Lacking Lrit3 Decreases Recovery From Lens-Induced Myopia.

Purpose: To determine whether the Lrit3-/- mouse model of complete congenital stationary night blindness with an ON-pathway defect harbors myopic features and whether the genetic defect influences the recovery from lens-induced myopia. Methods: Retinal levels of dopamine (DA) and 3,4 dihydroxyphenylacetic acid (DOPAC) from adult isolated Lrit3-/- retinas were quantified using ultra performance liquid chromatography after light adaptation. Natural refractive development of Lrit3-/- mice was measured from three weeks to nine weeks of age using an infrared photorefractometer. Susceptibility to myopia induction was assessed using a lens-induced myopia protocol with -25 D lenses placed in front of the right eye of the animals for three weeks; the mean interocular shift was measured with an infrared photorefractometer after two and three weeks of goggling and after one and two weeks after removal of goggles. Results: Compared to wild-type littermates (Lrit3+/+), both DA and DOPAC were drastically reduced in Lrit3-/- retinas. Natural refractive development was normal but Lrit3-/- mice showed a higher myopic shift and a lower ability to recover from induced myopia. Conclusions: Our data consolidate the link between ON pathway defect altered dopaminergic signaling and myopia. We document for the first time the role of ON pathway on the recovery from myopia induction.

Oculomic stratification of COVID-19 patients' intensive therapy unit admission status and mortality by retinal morphological findings.

To investigate if retinal thickness has predictive utility in COVID-19 outcomes by evaluating the statistical association between retinal thickness using OCT and of COVID-19-related mortality. Secondary outcomes included associations between retinal thickness and length of stay (LoS) in hospital. In this retrospective cohort study, OCT scans from 230 COVID-19 patients admitted to the Intensive Care Unit (ITU) were compared with age and gender-matched patients with pneumonia from before March 2020. Total retinal, GCL + IPL, and RNFL thicknesses were recorded, and analysed with systemic measures collected at the time of admission and mortality outcomes, using linear regression models, Pearson's R correlation, and Principal Component Analysis. Retinal thickness was significantly associated with all-time mortality on follow up in the COVID-19 group (p = 0.015), but not 28-day mortality (p = 0.151). Retinal and GCL + IPL layer thicknesses were both significantly associated with LoS in hospital for COVID-19 patients (p = 0.006 for both), but not for patients with pneumonia (p = 0.706 and 0.989 respectively). RNFL thickness was not associated with LoS in either group (COVID-19 p = 0.097, pneumonia p = 0.692). Retinal thickness associated with LoS in hospital and long-term mortality in COVID-19 patients, suggesting that retinal structure could be a surrogate marker for frailty and predictor of disease severity in this group of patients, but not in patients with pneumonia from other causes.

An attentional mechanism model for segmenting multiple lesion regions in the diabetic retina.

Diabetic retinopathy (DR), a leading cause of blindness in diabetic patients, necessitates the precise segmentation of lesions for the effective grading of lesions. DR multi-lesion segmentation faces the main concerns as follows. On the one hand, retinal lesions vary in location, shape, and size. On the other hand, the currently available multi-lesion region segmentation models are insufficient in their extraction of minute features and are prone to overlooking microaneurysms. To solve the above problems, we propose a novel deep learning method: the Multi-Scale Spatial Attention Gate (MSAG) mechanism network. The model inputs images of varying scales in order to extract a range of semantic information. Our innovative Spatial Attention Gate merges low-level spatial details with high-level semantic content, assigning hierarchical attention weights for accurate segmentation. The incorporation of the modified spatial attention gate in the inference stage enhances precision by combining prediction scales hierarchically, thereby improving segmentation accuracy without increasing the associated training costs. We conduct the experiments on the public datasets IDRiD and DDR, and the experimental results show that the proposed method achieves better performance than other methods.

Simultaneous profiling of RNA isoforms and chromatin accessibility of single cells of human retinal organoids.

Single-cell multi-omics sequencing is a powerful approach to analyze complex mechanisms underlying neuronal development and regeneration. However, current methods lack the ability to simultaneously profile RNA alternative splicing and chromatin accessibility at the single-cell level. We develop a technique, single-cell RNA isoform and chromatin accessibility sequencing (scRICA-seq), which demonstrates higher sensitivity and cost-effectiveness compared to existing methods. scRICA-seq can profile both isoforms and chromatin accessibility for up to 10,000 single cells in a single run. Applying this method to human retinal organoids, we construct a multi-omic cell atlas and reveal associations between chromatin accessibility, isoform expression of fate-determining factors, and alternative splicing events in their binding sites. This study provides insights into integrating epigenetics, transcription, and RNA splicing to elucidate the mechanisms underlying retinal neuronal development and fate determination.

Pericyte-to-Endothelial Cell Communication via Tunneling Nanotubes Is Disrupted by a Diol of Docosahexaenoic Acid.

The pericyte coverage of microvessels is altered in metabolic diseases, but the mechanisms regulating pericyte-endothelial cell communication remain unclear. This study investigated the formation and function of pericyte tunneling nanotubes (TNTs) and their impact on endothelial cell metabolism. TNTs were analyzed in vitro in retinas and co-cultures of pericytes and endothelial cells. Using mass spectrometry, the influence of pericytes on endothelial cell metabolism was examined. TNTs were present in the murine retina, and although diabetes was associated with a decrease in pericyte coverage, TNTs were longer. In vitro, pericytes formed TNTs in the presence of PDGF, extending toward endothelial cells and facilitating mitochondrial transport from pericytes to endothelial cells. In experiments with mitochondria-depleted endothelial cells displaying defective TCA cycle metabolism, pericytes restored the mitochondrial network and metabolism. 19,20-Dihydroxydocosapentaenoic acid (19,20-DHDP), known to disrupt pericyte-endothelial cell junctions, prevented TNT formation and metabolic rescue in mitochondria-depleted endothelial cells. 19,20-DHDP also caused significant changes in the protein composition of pericyte-endothelial cell junctions and involved pathways related to phosphatidylinositol 3-kinase, PDGF receptor, and RhoA signaling. Pericyte TNTs contact endothelial cells and support mitochondrial transfer, influencing metabolism. This protective mechanism is disrupted by 19,20-DHDP, a fatty acid mediator linked to diabetic retinopathy.

Gold Nanoparticles for Retinal Molecular Optical Imaging.

The incorporation of gold nanoparticles (GNPs) into retinal imaging signifies a notable advancement in ophthalmology, offering improved accuracy in diagnosis and patient outcomes. This review explores the synthesis and unique properties of GNPs, highlighting their adjustable surface plasmon resonance, biocompatibility, and excellent optical absorption and scattering abilities. These features make GNPs advantageous contrast agents, enhancing the precision and quality of various imaging modalities, including photoacoustic imaging, optical coherence tomography, and fluorescence imaging. This paper analyzes the unique properties and corresponding mechanisms based on the morphological features of GNPs, highlighting the potential of GNPs in retinal disease diagnosis and management. Given the limitations currently encountered in clinical applications of GNPs, the approaches and strategies to overcome these limitations are also discussed. These findings suggest that the properties and efficacy of GNPs have innovative applications in retinal disease imaging.