Picture this: A novel model eye for ophthalmoscopy practice.

WHAT WAS THE EDUCATIONAL CHALLENGE?: Ophthalmoscopy is a challenging skill to learn but essential for identifying ophthalmic pathology. Typically, there are few opportunities for early learners to practice this skill in the non-clinical setting. WHAT WAS THE SOLUTION?: We sought to develop a model eye for the practice of direct and indirect ophthalmoscopy, with realistic dimensions and fundus images, using accessible and affordable supplies. HOW WAS THE SOLUTION IMPLEMENTED?: A simple model eye was created with over-the-counter materials. A free software program was used to modify fundus photographs so that they could be molded to the posterior pole of the model eye. WHAT LESSONS WERE LEARNED THAT ARE RELEVANT TO A WIDER GLOBAL AUDIENCE?: This model eye can be implemented globally given the simple and affordable construction process to facilitate practice of the ophthalmoscopy technique. WHAT ARE THE NEXT STEPS?: Formally assessing the efficacy of our model eye teaching tool with learners, using both self-reported and objective methods.

Clinicomolecular Identification of Conserved and Individualized Features of Granulomatous Uveitis.

Objective: To identify molecular features that distinguish individuals with shared clinical features of granulomatous uveitis. Design: Cross-sectional, observational study. Participants: Four eyes from patients with active granulomatous uveitis. Methods: We performed single-cell RNA-sequencing with antigen-receptor sequence analysis to obtain an unbiased gene expression survey of ocular immune cells and identify clonally expanded lymphocytes. Main Outcomes Measures: For each inflamed eye, we measured the proportion of distinct immune cell types, the amount of B or T cell clonal expansion, and the transcriptional profile of T and B cells. Results: Each individual had robust clonal expansion arising from a single T or B cell lineage, suggesting distinct, antigen-driven pathogenic processes in each patient. This variability in clonal expansion was mirrored by individual variability in CD4 T cell populations, whereas ocular CD8 T cells and B cells were more transcriptionally similar between patients. Finally, ocular B cells displayed evidence of class-switching and plasmablast differentiation within the ocular microenvironment, providing additional support for antigen-driven immune responses in granulomatous uveitis. Conclusions: Collectively, our study identified both conserved and individualized features of granulomatous uveitis, illuminating parallel pathophysiologic mechanisms, and suggesting that future personalized therapeutic approaches may be warranted.

Glaucoma and quality of life: fall and driving risk.

PURPOSE OF REVIEW: Numerous population-based studies suggest that glaucoma is an independent risk factor for falling and motor vehicle collisions, particularly for older adults. These adverse events lead to increased healthcare expenditures and decreased quality of life. Current research priorities, therefore, include identifying factors that predispose glaucoma patients to falling and unsafe driving, and developing screening strategies and targeted rehabilitation. The purpose of this article is to review recent studies that address these priorities. RECENT FINDINGS: Studies continue to support that glaucoma patients, particularly those with advanced disease, have an increased risk of falling or unsafe driving. Risk factors, however, remain variable and include severity and location of visual field defects, contrast sensitivity, and performance on divided attention tasks. Such variability is likely because of the multifactorial nature of ambulating and driving and compensatory strategies used by patients. SUMMARY: Falls and unsafe driving remain a serious public health issue for older adults with glaucoma. Ambulation and driving are complex tasks and there is no consensus yet, regarding the best methods for risk stratification and targeted interventions to increase safety. Therefore, comprehensive and individualized assessments are recommended to most effectively evaluate a patient's risk for falling or unsafe driving.

Reprogramming of adult rod photoreceptors prevents retinal degeneration.

A prime goal of regenerative medicine is to direct cell fates in a therapeutically useful manner. Retinitis pigmentosa is one of the most common degenerative diseases of the eye and is associated with early rod photoreceptor death followed by secondary cone degeneration. We hypothesized that converting adult rods into cones, via knockdown of the rod photoreceptor determinant Nrl, could make the cells resistant to the effects of mutations in rod-specific genes, thereby preventing secondary cone loss. To test this idea, we engineered a tamoxifen-inducible allele of Nrl to acutely inactivate the gene in adult rods. This manipulation resulted in reprogramming of rods into cells with a variety of cone-like molecular, histologic, and functional properties. Moreover, reprogramming of adult rods achieved cellular and functional rescue of retinal degeneration in a mouse model of retinitis pigmentosa. These findings suggest that elimination of Nrl in adult rods may represent a unique therapy for retinal degeneration.

Quantifying the activity of cis-regulatory elements in the mouse retina by explant electroporation.

Transcription factors control gene expression by binding to noncoding regions of DNA known as -cis-regulatory elements (CREs; i.e., enhancer/promoters). Traditionally, cis-regulatory analysis has been carried out via mouse transgenesis which is time-consuming and nonquantitative. Electroporation of DNA reporter constructs into living mouse tissue is a rapid and effective alternative to transgenesis which permits quantitative assessment of cis-regulatory activity. Here, we present a simple technique for quantifying the activity of photoreceptor-specific CREs in living explanted mouse retinas.

Transcriptional regulation of neural retina leucine zipper (Nrl), a photoreceptor cell fate determinant.

The transcription factor neural retina leucine zipper (Nrl) is a critical determinant of rod photoreceptor cell fate and a key regulator of rod differentiation. Nrl(-/-) rod precursors fail to turn on rod genes and instead differentiate as cones. Furthermore, NRL mutations in humans cause retinitis pigmentosa. Despite the developmental and clinical significance of this gene, little is known about the transcriptional regulation of Nrl itself. In this study, we sought to define the cis- and trans-acting factors responsible for initiation and maintenance of Nrl transcription in the mouse retina. Utilizing a quantitative mouse retinal explant electroporation assay, we discovered a phylogenetically conserved, 30-base pair region immediately upstream of the transcription start site that is required for Nrl promoter activity. This region contains binding sites for the retinal transcription factors CRX, OTX2, and RORß, and point mutations in these sites completely abolish promoter activity in living retinas. Gel-shift experiments show that CRX, OTX2, and RORß can bind to the critical region in vitro, whereas ChIP experiments demonstrate binding of CRX and OTX2 to the critical region in vivo. Thus, our results indicate that CRX, OTX2, and RORß directly regulate Nrl transcription by binding to critical sites within the Nrl promoter. We propose a model in which Nrl expression is primarily initiated by OTX2 and RORß and later maintained at high levels by CRX and RORß.

Quantifying the activity of cis-regulatory elements in the mouse retina by explant electroporation.

Transcription factors within cellular gene networks control the spatiotemporal pattern and levels of expression of their target genes by binding to cis-regulatory elements (CREs), short (˜300-600 bp) stretches of genomic DNA which can lie upstream, downstream, or within the introns of the genes they control. CREs (i.e., enhancers/promoters) typically consist of multiple clustered binding sites for both transcriptional activators and repressors(1-3). They serve as logical integrators of transcriptional input giving a unitary output in the form of spatiotemporally precise and quantitatively exact promoter activity. Most studies of mammalian cis-regulation to date have relied on mouse transgenesis as a means of assaying the enhancer function of CREs(4-5). This technique is time-consuming, costly and, on account of insertion site effects, largely non-quantitative. On the other hand, quantitative assays for mammalian CRE function have been developed in tissue culture systems (e.g., dual luciferase assays), but the in vivo relevance of these results is often uncertain. Electroporation offers an excellent alternative to traditional mouse transgenesis in that it permits both spatiotemporal and quantitative assessment of cis-regulatory activity in living mammalian tissue. This technique has been particularly useful in the analysis of cis-regulation in the central nervous system, especially in the cerebral cortex and the retina(6-8). While mouse retinal electroporation, both in vivo and ex vivo, has been developed and extensively described by Matsuda and Cepko(6-7,9), we have recently developed a simple approach to quantify the activity of photoreceptor-specific CREs in electroporated mouse retinas(10). Given that the amount of DNA that is introduced into the retina by electroporation can vary from experiment to experiment, it is necessary to include a co-electroporated 'loading control' in all experiments. In this respect, the technique is very similar to the dual luciferase assay used to quantify promoter activity in cultured cells. When assaying photoreceptor cis-regulatory activity, electroporation is usually performed in newborn mice (postnatal day 0, P0) which is the time of peak rod production(11-12). Once retinal cell types become post-mitotic, electroporation is much less efficient. Given the high rate of rod birth in newborn mice and the fact that rods constitute more than 70% of the cells in the adult mouse retina, the majority of cells that are electroporated at P0 are rods. For this reason, rod photoreceptors are the easiest retinal cell type to study via electroporation. The technique we describe here is primarily useful for quantifying the activity of photoreceptor CREs.

Inherited diseases of photoreceptors and prospects for gene therapy.

The photoreceptor cells of the retina are subject to a wide range of genetic diseases. This review summarizes current knowledge regarding an important group of retinal diseases caused by mutations in photoreceptor-enriched genes. In addition, progress toward treatment of a variety of these diseases in animal models via adeno-associated virus gene therapy is described. Although no human trials have yet been initiated to treat diseases caused by mutations in photoreceptor-enriched genes, there is a great deal of optimism regarding the prospects of treating these diseases using adeno-associated virus gene therapy.