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.

Nuclei Isolation from Ocular Tissues of the Embryonic Chicken for Single-Nucleus Profiling.

The generation of quality data from a single-nucleus profiling experiment requires nuclei to be isolated from tissues in a gentle and efficient manner. Nuclei isolation must be carefully optimized across tissue types to preserve nuclear architecture, prevent nucleic acid degradation, and remove unwanted contaminants. Here, we present an optimized workflow for generating a single-nucleus suspension from ocular tissues of the embryonic chicken that is compatible with various downstream workflows. The described protocol enables the rapid isolation of a high yield of aggregate-free nuclei from the embryonic chicken eye without compromising nucleic acid integrity, and the nuclei suspension is compatible with single-nucleus RNA and ATAC sequencing. We detail several stopping points, either via cryopreservation or fixation, to enhance workflow adaptability. Further, we provide a guide through multiple QC points and demonstrate proof-of-principle using two commercially available kits. Finally, we demonstrate that existing in silico genotyping methods can be adopted to computationally derive biological replicates from a single pool of chicken nuclei, greatly reducing the cost of biological replication and allowing researchers to consider sex as a variable during analysis. Together, this tutorial represents a cost-effective, simple, and effective approach to single-nucleus profiling of embryonic chicken eye tissues and is likely to be easily modified to be compatible with similar tissue types.

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.

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.

The toxic effects of polystyrene microplastic/nanoplastic particles on retinal pigment epithelial cells and retinal tissue.

The increasing use of contact lenses, artificial tears, and anti-vascular endothelial growth factor (anti-VEGF) drug injections for age-related macular degeneration has heightened the likelihood of eye exposure to microplastic particles. Extensive research has established that microplastic particles can induce oxidative stress on the ocular surface, resulting in damage. However, the impact of these particles on the retina remains unclear. Therefore, this study investigated whether microplastics/nanoplastics (MPs/NPs) cause retinal damage. In vitro human retinal pigment epithelial (RPE) cells were exposed to polystyrene MPs and NPs for 48 h. Assessment of cell viability using WST-8; evaluation of TNF-α and IL-1ß expression; observation of cell morphology and particle invasion via TEM; measurement of ROS levels using the DCFDA reagent; and western blot analysis of SOD2, FIS1, Drp1, and LC3B expression were conducted. In vivo experiments involved intravitreal injection of MPs/NPs in rats, followed by retinal H&E staining 24 h later and evaluation of TNF-α and IL-1ß expression. Results indicated that exposure to MPs did not significantly alter RPE cell viability, whereas exposure to NPs led to a noticeable decrease. TEM images revealed NPs' penetration into cells, causing increased oxidative stress (SOD2), mitochondrial fission (FIS1, Drp1), and mitochondrial autophagy (LC3B). In vivo experiments demonstrated an increase in inflammatory cells in retinal tissues exposed to NPs, along with elevated levels of TNF-α and IL-1ß. Conclusively, both MPs and NPs impact the retina, with NPs displaying greater toxicity. NPs significantly elevate ROS levels in the retina and induce mitochondrial fission and mitophagy in RPE cells compared to MPs.

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.

Predicting the impact of retinal vessel density on retinal vessel and tissue oxygenation using a theoretical model.

Vascular impairments, including compromised flow regulation, have been identified as significant contributors to glaucomatous disease. Recent studies have shown glaucoma patients with significantly reduced peripapillary, macular, and optic nerve head vessel densities occurring with early glaucomatous structural changes prior to detectable visual field loss. This study aims to quantify the potential impact of decreased vessel densities on retinal perfusion and oxygen metabolism. In our clinical observations, pre-perimetric glaucoma patients exhibited a 10-13% reduction in vessel density compared to healthy individuals. Our theoretical model of the retinal vasculature is adapted in this study to assess the potential impact of this reduction in vessel density on retinal oxygenation. The model predicts a 1% and 38% decrease in mean oxygen saturation in retinal vessels immediately downstream of the capillaries when vessel density is decreased from its reference value by 10% and 50%, respectively. The impact of capillary loss on oxygen extraction fraction and the partial pressure of oxygen in retinal tissue is also predicted. Reductions in vessel density are simulated in combination with impaired flow regulation, and the resulting effects on saturation and flow are predicted. The model results showed a nonlinear relationship between vessel density and downstream saturation, indicating that larger decreases in the density of capillaries have a disproportionate impact on oxygenation. The model further demonstrates that the detrimental effects of minor vessel density reductions are exacerbated when combined with other vascular impairments.

Autologous Serum for the Treatment of Macular Holes: A Systematic Review and Meta-Analysis.

This systematic review and meta-analysis evaluated the efficacy and safety of autologous human serum as an adjuvant agent in pars plana vitrectomy (PPV) for macular holes (MH). Thus, a comprehensive search was conducted across PubMed, Web of Science, Embase, and the Cochrane Library databases up to August 20th, 2023. The inclusion criteria targeted randomized clinical trials (RCTs) or non-RCTs that compared the use of autologous serum in vitrectomy for MH with the same procedure without the serum. The outcomes were MH closure rates and postoperative complications such as retinal detachment and cataracts. Odds ratios (OR) and mean differences (MDs) were calculated using a random-effects model. Review Manager 5.3 (The Cochrane Collaboration, Oxford, UK) was used for statistical analysis. Four studies, comprising two RCTs and two non-randomized cohort studies with 373 eyes of 372 patients, were included. The pooled analysis showed no significant difference in MH closure rates (OR 1.28; 95% confidence interval (CI): 0.48 to 3.43; P=0.62) and no difference concerning the incidence of adverse events (OR 0.97; 95% CI: 0.30-3.09; P=0.96). Leave-one-out sensitivity analysis excluding the study by Lauritzen et al. revealed a significant difference in anatomical closure, favoring the serum arm, and demonstrated a reduction in the level of heterogeneity. Our meta-analysis demonstrated no difference between groups in the pooled analysis of all studies. However, considering the quality assessment of one of the included studies, and observing the divergent result in sensitivity analysis following its exclusion, there are indications that might suggest the superiority of the serum in terms of the analyzed endpoints. This finding highlights the existing research gaps and the imperative need for additional high-quality randomized trials to further investigate this treatment.

Assessment of Retinal Volume in Individuals Without Ocular Disorders Based on Wide-Field Swept-Source OCT.

Purpose: To evaluate retinal volume (RV) in eyes without retinal disease using wide-field swept-source OCT (SS-OCT). Design: Observational, cross-sectional design. Participants: A total of 332 eyes of 166 healthy participants. Methods: The eyes were imaged with OCT-S1 (Canon) using a protocol centered on the fovea cube scans (20 × 23 mm) of SS-OCT images. Retinal volume (6-mm circle, 6-20-mm ring) and various parameters were evaluated in a multivariate analysis using a generalized estimating equation model. Each quadrant of the macula except for the fovea (1-6 mm in diameter) and peripheral ring (6-20 mm in diameter) was also evaluated. Main Outcome Measures: Retinal volume. Results: In the multivariate analysis, older age and longer axial length were associated with smaller macular RV, whereas older age and left eye were associated with smaller peripheral RV. The temporal area was significantly smaller than all other areas in the macula (1-6 mm), whereas the inferior area was significantly smaller than all other areas in the peripheral retina (6-20 mm). Conclusions: In wide-field SS-OCT images, age and left eye are negatively correlated with peripheral RV. The thinnest part of the retinal quadrant differs between the macular and peripheral retinas. Financial Disclosures: Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Clinical spectrum and possible pathogenesis of progressive outer retinal necrosis.

OBJECTIVE: Progressive outer retinal necrosis (PORN) is an alphaherpesvirus-caused panuveitis with devastating consequences for the eye. Our study aims to describe new findings in the clinical spectrum and propose a mechanism for the pathogenesis of PORN. METHODS: Observational, consecutive case series. Seven eyes from five patients diagnosed with PORN were enrolled. Detailed case histories, ocular examination findings and multimodal images of retina were collected. Optic nerve and brain imaging were obtained by MRI. RESULTS: All eyes were confirmed human alphaherpesviruses positive in ocular fluid by qPCR. Optic nerve oedema was observed on MRI in all eyes. A relative afferent pupillary defect was recorded in the affected eye for the unilateral cases. Two patients with unilateral involvement had a history of viral encephalitis and focal encephalomalacia found in the temporal lobe on brain MRI. The affected eyes were characterised by sensory retinal necrosis sparing retinal pigment epithelium, starting at the end of the retinal nerve fibre (horizontal raphe or peripheral area of the retina) and progressing rapidly along the nerve fibre. The wall of the retinal artery and vein was destroyed, resulting in blood flow interruption on fluorescein angiography and retinal haemorrhages along the large vessels. CONCLUSIONS: Combination the neurotropic characteristics of alphaherpesviruses and the signs of PORN, we hypothesised that the reactivated PORN virus originated from the lateral geniculate nucleus, then propagated along the optic nerve and was released at the terminals, causing necrosis of the entire sensory retina rather than just affecting the outer segment.

Minocycline-induced retinal pigment epithelium hyperpigmentation masquerading as age-related macular degeneration: Case presentation and proposed mechanism.

Purpose: We describe the case of an 80-year-old man with bilateral minocycline-induced retinal pigment epithelium (RPE) hyperpigmentation, which initially masqueraded as AMD. Secondarily, using multimodal imaging features, we propose a mechanism for the development of minocycline-induced RPE hyperpigmentation. Observations: The patient was referred with concern for AMD given the presence of macular drusenoid deposits on optical coherence tomography. However, funduscopic evaluation showed dense granular parafoveal hyperpigmentation, with a diffuse slate-colored hyperpigmentation throughout the peripheral fundus. Short-wavelength fundus autofluorescence of the macula disclosed no irregularities (as would be expected with drusen) while on near-infrared reflectance (NIR) imaging, numerous hyperreflective foci were noted corresponding to the hyperpigmented granules observed clinically (as would instead be seen with melanin deposits). Clinical examination was notable for blue-gray hyperpigmentation of the lower and upper extremities, as well as of the face, periorbital skin, and sclera. Upon further questioning, the patient disclosed daily oral minocycline use for 15 years for acne rosacea, confirming a diagnosis of minocycline-induced hyperpigmentation of the RPE. Conclusions: Multimodal imaging can be useful for differentiating minocycline-induced RPE hyperpigmentation from similar masquerade entities. Timely diagnosis can prevent progressive vision loss.

An Approximate Bayesian Computation Approach for Embryonic Astrocyte Migration Model Reduction.

During embryonic development of the retina of the eye, astrocytes, a type of glial cell, migrate over the retinal surface and form a dynamic mesh. This mesh then serves as scaffolding for blood vessels to form the retinal vasculature network that supplies oxygen and nutrients to the inner portion of the retina. Astrocyte spreading proceeds in a radially symmetric manner over the retinal surface. Additionally, astrocytes mature from astrocyte precursor cells (APCs) to immature perinatal astrocytes (IPAs) during this embryonic stage. We extend a previously-developed continuum model that describes tension-driven migration and oxygen and growth factor influenced proliferation and differentiation. Comparing numerical simulations to experimental data, we identify model equation components that can be removed via model reduction using approximate Bayesian computation (ABC). Our results verify experimental studies indicating that the choroid oxygen supply plays a negligible role in promoting differentiation of APCs into IPAs and in promoting IPA proliferation, and the hyaloid artery oxygen supply and APC apoptosis play negligible roles in astrocyte spreading and differentiation.

Neuronal and glial cell alterations involved in the retinal degeneration of the familial dysautonomia optic neuropathy.

Familial dysautonomia (FD) is a rare genetic neurodevelopmental and neurodegenerative disorder. In addition to the autonomic and peripheral sensory neuropathies that challenge patient survival, one of the most debilitating symptoms affecting patients' quality of life is progressive blindness resulting from the steady loss of retinal ganglion cells (RGCs). Within the FD community, there is a concerted effort to develop treatments to prevent the loss of RGCs. However, the mechanisms underlying the death of RGCs are not well understood. To study the mechanisms underlying RGC death, Pax6-cre;Elp1loxp/loxp male and female mice and postmortem retinal tissue from an FD patient were used to explore the neuronal and non-neuronal cellular pathology associated with the FD optic neuropathy. Neurons, astrocytes, microglia, Müller glia, and endothelial cells were investigated using a combination of histological analyses. We identified a novel disruption of cellular homeostasis and gliosis in the FD retina. Beginning shortly after birth and progressing with age, the FD retina is marked by astrogliosis and perturbations in microglia, which coincide with vascular remodeling. These changes begin before the onset of RGC death, suggesting alterations in the retinal neurovascular unit may contribute to and exacerbate RGC death. We reveal for the first time that the FD retina pathology includes reactive gliosis, increased microglial recruitment to the ganglion cell layer (GCL), disruptions in the deep and superficial vascular plexuses, and alterations in signaling pathways. These studies implicate the neurovascular unit as a disease-modifying target for therapeutic interventions in FD.

Decreased sialylation elicits complement-related microglia response and bipolar cell loss in the mouse retina.

Sialylation plays an important role in self-recognition, as well as keeping the complement and innate immune systems in check. It is unclear whether the reduced sialylation seen during aging and in mice heterozygous for the null mutant of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (Gne+/-), an essential enzyme for sialic acid biosynthesis, contributes to retinal inflammation and degeneration. We found a reduction of polysialic acid and trisialic acid expression in several retinal layers in Gne+/- mice at 9 months of age compared to Gne+/+ wildtype (WT) mice, which was associated with a higher microglial expression of the lysosomal marker CD68. Furthermore, the total number of rod bipolar cells was reduced in 12 months old Gne+/- mice in comparison to WT mice, demonstrating loss of these retinal interneurons. Transcriptome analysis showed up-regulation of complement, inflammation, and apoptosis-related pathways in the retinas of Gne+/- mice. Particularly, increased gene transcript levels of the complement factors C3 and C4 and the pro-inflammatory cytokine Il-1ß were observed by semi-quantitative real-time polymerase chain reaction (sqRT-PCR) in 9 months old Gne+/- mice compared to WT mice. The increased expression of CD68, loss of rod bipolar cells, and increased gene transcription of complement factor C4, were all prevented after crossing Gne+/- mice with complement factor C3-deficient animals. In conclusion, our data show that retinal hyposialylation in 9 and 12 months old Gne+/- mice was associated with complement-related inflammation and lysosomal microglia response, as well as rod bipolar cells loss, which was absent after genetic deletion of complement factor C3.

Self-Assembly Hypoxic and ROS Dual Response Nano Prodrug as a New Therapeutic Approach for Glaucoma Treatments.

Glaucoma is an irreversible blinding eye disease characterized by retinal ganglion cell (RGC) death.Previous studies have demonstrated that protecting mitochondria and activating the CaMKII/CREB signaling pathway can effectively protect RGC and axon. However, currently treatments are often unsatisfactory, and the pathogenesis of glaucoma requires further elucidation. In this study, a ROS-responsive dual drug conjugate (OLN monomer) is first designed that simultaneously bonds nicotinamide and oleic acid. The conjugate self-assembled into nanoparticles (uhOLN-NPs) through the aggregation of multiple micelles and possesses ROS scavenging capability. Then, a polymer with a hypoxic response function is designed, which encapsulates uhOLN-NPs to form nanoparticles with hypoxic and ROS responses (HOLN-NPs). Under hypoxia in RGCs, the azo bond of HOLN-NPs breaks and releases uhOLN-NPs. Meanwhile, under high ROS conditions, the thioketone bond broke, leading to the dissociation of nano-prodrug. The released nicotinamide and oleic acid co-scavenge ROS and activate the CaMKII/CREB pathway, protecting mitochondria in RGCs. HOLN-NPs exhibit a significantly superior protective effect on R28 cells in glutamate models of glaucoma. The accumulation of HOLN-NPs in retinal RGCs lead to significant inhibition of RGC apoptosis and axonal damage in vivo. Notably, HOLN-NPs provide a new therapeutic approach for patients with neurodegenerative disease.

Liquid Biopsy for Proliferative Diabetic Retinopathy: Single-Cell Transcriptomics of Human Vitreous Reveals Inflammatory T-Cell Signature.

Purpose: Current therapies for proliferative diabetic retinopathy (PDR) do not specifically target VEGF-independent, cell-type-specific processes that lead to vision loss, such as inflammatory pathways. This study aimed to identify targetable cell types and corresponding signaling pathways by elucidating the single-cell landscape of the vitreous of patients with PDR. Design: Case series. Subjects: Vitreous and peripheral blood obtained from 5 adult patients (6 eyes) undergoing pars plana vitrectomy for vision-threatening PDR. Methods: Single-cell RNA sequencing (scRNA-seq) was performed on vitreous cells obtained from diluted cassette washings during vitrectomy from 6 eyes and peripheral blood mononuclear cells (PBMCs, n = 5). Droplet-based scRNA-seq was performed using the Chromium 10x platform to obtain single-cell transcriptomes. Differences in tissue compartments were analyzed with gene ontology enrichment of differentially expressed genes and an unbiased ligand-receptor interaction analysis. Main Outcome Measures: Single-cell transcriptomic profiles of vitreous and peripheral blood. Results: Transcriptomes from 13 675 surgically harvested vitreous cells and 22 636 PBMCs were included. Clustering revealed 4 cell states consistently across all eyes with representative transcripts for T cells (CD2, CD3D, CD3E, and GZMA), B cells (CD79A, IGHM, MS4A1 (CD20), and HLA-DRA), myeloid cells (LYZ, CST3, AIF1, and IFI30), and neutrophils (BASP1, CXCR2, S100A8, and S100A9). Most vitreous cells were T cells (91.6%), unlike the peripheral blood (46.2%), whereas neutrophils in the vitreous were essentially absent. The full repertoire of adaptive T cells including CD4+, CD8+ and T regulatory cells (Treg) and innate immune system effectors (i.e., natural killer T cells) was present in the vitreous. Pathway analysis also demonstrated activation of CD4+ and CD8+ memory T cells and ligand-receptor interactions unique to the vitreous. Conclusions: In the first single-cell transcriptomic characterization of human vitreous in a disease state, we show PDR vitreous is primarily composed of T cells, a critical component of adaptive immunity, with activity and proportions distinct from T cells within the peripheral blood, and neutrophils are essentially absent. These results demonstrate the feasibility of liquid vitreous biopsies via collection of otherwise discarded, diluted cassette washings during vitrectomy to gain mechanistic and therapeutic insights into human vitreoretinal disease. Financial Disclosures: Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Interocular Symmetry and Intermachine Reproducibility of Optic Disc and Macular Parameters Measured by Two Different Models of Optical Coherence Tomography.

Purpose: To compare the interocular symmetry and investigate the intermachine reproducibility of optic disc and macular data measured by spectral-domain high-definition optical coherence tomography (HD-OCT) Cirrus HD-OCT 4000 and HD-OCT 5000 from healthy subjects. Patients and Methods: Forty-three volunteers were examined with both HD-OCT 4000 and HD-OCT 5000 at the same visit. Optic nerve head (ONH) and macular data were acquired using ONH Cube 200×200 scans and macular volume cube 512×128 scans, respectively. Results: The average age of the participants was 33 ± 8.6 years. Interocular OCT parameters of ONH and macula showed a high correlation between the right and left eyes regardless of HD-OCT models, displaying a low coefficient of variation (CV). However, the average retinal nerve fiber layer (RNFL) was thicker (96.67±11.19µm vs 95.3±10.89µm, p<0.01), and the average central subfield thickness (261.51±17.45µm vs 262.51±17.39 µm, p<0.01) and cube average thickness (283.91± 13.59µm vs 286.55±13.09µm, p<0.05) were thinner when measured by Cirrus 4000 compared to 5000. Intermachine reproducibility and reliability of RNFL and macular parameters exhibited a high intraclass correlation coefficient (ICC) (0.985) and low CV (2.4%). Ganglion cell-inner plexiform layer (GCIPL) measured by two OCT models showed similar values with an average thickness of 85 µm and had high intermachine reproducibility with high ICC (0.993) and low CV (1.2%). Conclusion: High interocular symmetry was observed across both HD-OCT models. Intermachine reproducibility for RNFL and all macular parameters was also high. GCIPL showed minimal intermachine differences with high reproducibility and reliability. Thus, the results imply that GCIPL values measured by two Cirrus OCT models may be used interchangeably.

Deriving the cone fundamentals: a subspace intersection method.

Two ideas, proposed by Thomas Young and James Clerk Maxwell, form the foundations of colour science: (i) three types of retinal receptors encode light under daytime conditions, and (ii) colour matching experiments establish the critical spectral properties of this encoding. Experimental quantification of these ideas is used in international colour standards. However, for many years, the field did not reach consensus on the spectral properties of the biological substrate of colour matching: the spectral sensitivity of the cone fundamentals. By combining auxiliary data (thresholds, inert pigment analyses), complex calculations, and colour matching from genetically analysed dichromats, the human cone fundamentals have now been standardized. Here, we describe a new computational method to estimate the cone fundamentals using only colour matching from the three types of dichromatic observers. We show that it is not necessary to include data from trichromatic observers in the analysis or to know the primary lights used in the matching experiments. Remarkably, it is even possible to estimate the fundamentals by combining data from experiments using different, unknown primaries. We then suggest how the new method may be applied to colour management in modern image systems.

Evaluation of carotid intima-media thickness measurements in patients with central serous chorioretinopathy.

BACKGROUND: Central serous chorioretinopathy (CSC) is a systemic disease more than a disease localized to the eye, and there may be vascular involvement in its pathogenesis. OBJECTIVE: This study aimed to evaluate the carotid intima-media thickness (IMT) of patients with CSC, to compare it with that of healthy individuals, and to explore whether there might be an association between CSC and subclinical carotid atherosclerotic disease. MATERIALS AND METHODS: Adult patients with CSC (n = 30) and healthy individuals (n = 30) were included in this prospective study. All participants underwent complete ophthalmologic imaging and were then referred to the radiology department. Carotid IMT measurements were performed using ultrasound imaging. Measurements of the two groups were obtained and compared. RESULTS: There was no statistically significant difference between patients with CSC and the control group with respect to age, gender, and smoking habits. The mean (±standard deviation, SD) carotid IMT values obtained by ultrasound measurements for the right and left sides in the patient group were 0.71 mm (±â€¯0.19) and 0.71 mm (±â€¯0.21), respectively. The mean (±SD) carotid IMT values for the right and left sides in the control group were 0.61 mm (±â€¯0.15) and 0.60 mm (±â€¯0.15), respectively. The mean carotid IMT values in the patient group were significantly higher than those in the control group for the right and left sides (p = 0.02 and p = 0.03, respectively). CONCLUSION: Carotid IMT is increased in patients with CSC compared to healthy individuals. This outcome might reinforce the benefit of carotid artery screening following diagnosis of CSC by ophthalmologists for early detection of subclinical carotid atherosclerotic disease.

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.