Background: Soft drusen and basal linear deposit (BLinD) are two forms of the same extracellular lipid rich material that together make up an Oil Spill on Bruch’s membrane (BrM). Drusen are focal and can be recognized clinically. In contrast BLinD is thin and diffusely distributed, and invisible clinically, even on highest resolution OCT, but has been detected on en face hyperspectral autofluorescence (AF) imaging ex vivo. We sought to optimize histologic hyperspectral AF imaging and image analysis for recognition of drusen and sub-RPE deposits (including BLinD and basal laminar deposit), for potential clinical application.
Methods: Twenty locations specifically with drusen and 12 additional locations specifically from fovea, perifovea and mid-periphery from RPE/BrM flatmounts from 4 AMD donors underwent hyperspectral AF imaging with 4 excitation wavelengths (λex 436, 450, 480 and 505 nm), and the resulting image cubes were simultaneously decomposed with our published non-negative matrix factorization (NMF). Rank 4 recovery of 4 emission spectra was chosen for each excitation wavelength.
Results: A composite emission spectrum, sensitive and specific for drusen and presumed sub-RPE deposits (the SDr spectrum) was recovered with peak at 510–520 nm in all tissues with drusen, with greatest amplitudes at excitations λex 436, 450 and 480 nm. The RPE spectra of combined sources Lipofuscin (LF)/Melanolipofuscin (MLF) were of comparable amplitude and consistently recapitulated the spectra S1, S2 and S3 previously reported from all tissues: tissues with drusen, foveal and extra-foveal locations.
Conclusions: A clinical hyperspectral AF camera, with properly chosen excitation wavelengths in the blue range and a hyperspectral AF detector, should be capable of detecting and quantifying drusen and sub-RPE deposits, the earliest known lesions of AMD, before any other currently available imaging modality.
Background: Cells of the retinal pigment epithelium (RPE) accumulate different kinds of granules (lipofuscin, melanolipofuscin, melanosomes) within their cell bodies, with lipofuscin and melanolipofuscin being autofluorescent after blue light excitation. High amounts of lipofuscin granules within the RPE have been associated with the development of RPE cell death and age-related macular degeneration (AMD); however, this has not been confirmed in histology so far. Here, based on our previous dataset of RPE granule characteristics, we report the characteristics of RPE cells from human donor eyes that show either high or low numbers of intracellular granules or high or low autofluorescence (AF) intensities.
Methods: RPE flatmounts of fifteen human donors were examined using high-resolution structured illumination microscopy (HR-SIM) and laser scanning microscopy (LSM). Autofluorescent granules were analyzed regarding AF phenotype and absolute number of granules. In addition, total AF intensity per cell and granule density (number of granules per cell area) were determined. For the final analysis, RPE cells with total granule number below 5th or above the 95th percentile, or a total AF intensity ± 1.5 standard deviations above or below the mean were included, and compared to the average RPE cell at the same location. Data are presented as mean ± standard deviation.
Results: Within 420 RPE cells examined, 42 cells were further analyzed due to extremes regarding total granule numbers. In addition, 20 RPE cells had AF 1.5 standard deviations below, 28 RPE cells above the mean local AF intensity. Melanolipofuscin granules predominate in RPE cells with low granule content and low AF intensity. RPE cells with high granule content have nearly twice (1.8 times) as many granules as an average RPE cell.
Conclusions: In normal eyes, outliers regarding autofluorescent granule load and AF intensity signals are rare among RPE cells, suggesting that granule deposition and subsequent AF follows intrinsic control mechanisms at a cellular level. The AF of a cell is related to the composition of intracellular granule types. Ongoing studies using AMD donor eyes will examine possible disease related changes in granule distribution and further put lipofuscin′s role in aging and AMD further into perspective.
Objective: In this review, non-transgenic models of age-related macular degeneration (AMD) are discussed, with focuses on murine retinal degeneration induced by sodium iodate and lipid peroxide (HpODE) as preclinical study platforms.
Background: AMD is the most common cause of vision loss in a world with an increasingly aging population. The major phenotypes of early and intermediate AMD are increased drusen and autofluorescence, Müller glia activation, infiltrated subretinal microglia and inward moving retinal pigment epithelium (RPE) cells. Intermediate AMD may progress to advanced AMD, characterized by geography atrophy and/or choroidal neovascularization (CNV). Various transgenic and non-transgenic animal models related to retinal degeneration have been generated to investigate AMD pathogenesis and pathobiology, and have been widely used as potential therapeutic evaluation platforms.
Methods: Two retinal degeneration murine models induced by sodium iodate and HpODE are described. Distinct pathological features and procedures of these two models are compared. In addition, practical protocol and material preparation and assessment methods are elaborated.
Conclusions: Retina degeneration induced by sodium iodate and HpODE in mouse eye resembles many clinical aspects of human AMD and complimentary to the existent other animal models. However, standardization of procedure and assessment protocols is needed for preclinical studies. Further studies of HpODE on different routes, doses and species will be valuable for the future extensive use. Despite many merits of murine studies, differences between murine and human should be always considered.
Abstract: Animal models are crucial for the study of tumorigenesis and therapies in oncology research. Though rare, uveal melanoma (UM) is the most common intraocular tumor and remains one of the most lethal cancers. Given the limitations of studying human UM cells in vitro, animal models have emerged as excellent platforms to investigate disease onset, progression, and metastasis. Since Greene’s initial studies on hamster UM, researchers have dramatically improved the array of animal models. Animals with spontaneous tumors have largely been replaced by engrafted and genetically engineered models. Inoculation techniques continue to be refined and expanded. Newer methods for directed mutagenesis have formed transgenic models to reliably study primary tumorigenesis. Human UM cell lines have been used to generate rapidly growing xenografts. Most recently, patient-derived xenografts have emerged as models that closely mimic the behavior of human UM. Separate animal models to study metastatic UM have also been established. Despite the advancements, the prognosis has only recently improved for UM patients, especially in patients with metastases. There is a need to identify and evaluate new preclinical models. To accomplish this goal, it is important to understand the origin, methods, advantages, and disadvantages of current animal models. In this review, the authors present current and historic animal models for the experimental study of UM. The strengths and shortcomings of each model are discussed and potential future directions are explored.
Background: Sodium iodate (SI) is a chemical widely applied to induce retina degeneration in animal models. SI treatment caused formation of rosettes/folds in the outer nuclear layer (ONL) of the rat retina, but it was previously unclear whether SI also forms rosettes in mice. In addition, SI induced retina degeneration was never addressed in non-separate sclerochoroid/retina pigment epithelium/retina whole mount. Here we displayed features of retina degeneration including rosette formation in mice and developed a morphological analytic assessment using sclerochoroid/retina pigment epithelium/retina whole mounts.
Methods: SI was intraperitoneally injected in Sprague-Dawley (SD) rats and C57BL/6J mice using a single dose (50 mg/kg) or with a dose range (10 to 50 mg/kg) in BALB/C mice. Rat retinas were investigated up to 2-week post-injection by histology and whole mounts, and mouse retinas were investigated up to 3-week post-injection by histology, fluorescent staining of sections and/or sclerochoroid/retina pigment epithelium/retina whole mounts for the morphological evaluations of the SI-induced retina damage.
Results: SI-induced retina damage caused photoreceptor (PR) degeneration and rosettes/folds formation, as well as retina pigment epithelium degeneration and inward migration. It displayed mixed nuclei from choroid to PRs, due to layer disorganization, as shown by single horizontal images in the sclerochoroid/retina pigment epithelium/retina whole mounts. Measurement of the PR rosette area induced by SI provided a quantitative, morphological evaluation of retina degeneration.
Conclusions: The method of non-separate sclerochoroid/retina pigment epithelium/retina whole staining and mount allows us to observe the integral horizontal view of damage from sclera to PR layers, which cannot be addressed by using sectioned and separate whole mount methods. This method is applicable for morphological evaluation of retina damage, especially in the subretinal layer.
Abstract: Submacular haemorrhage (SMH) is a sight threatening complication that can occur in exudative age related macular degeneration (AMD), but has been described to occur more frequently in eyes with polypoidal choroidal vasculopathy (PCV). Left untreated, SMH carries a grave visual prognosis. Thus, expedient diagnosis and effective management of this complication is of paramount importance. The treatment strategies for SMH include (I) displacement of blood from the fovea, usually by injection of an expansile gas; (II) pharmacologic clot lysis such as with recombinant tissue plasminogen activator (rtPA); and (III) treatment of the underlying choroidal neovascularization (CNV) or PCV, such as with anti-vascular endothelial growth factor (anti-VEGF) agents. These three strategies have been employed in isolation or in combination, some concurrently and others in stages. rtPA has demonstrable effect on the liquefaction of submacular clots but there are remaining uncertainties with regards to the dose, safety and the timing of initial and repeat treatments. Potential side effects of rtPA include retinal pigment epithelial toxicity, increased risk of breakthrough vitreous haemorrhage and systemic toxicity. In cases presenting early, pneumatic displacement alone with anti-VEGF may be sufficient. Anti-VEGF monotherapy is a viable treatment option particularly in patients with thinner SMH and those who are unable to posture post pneumatic displacement.
Background: Retinopathy of prematurity (ROP) is considered as the most common reason for blindness in children, particularly in preterm infants. The disease is characterized by the dysregulation of angiogenic mechanisms due to preterm birth, leading ultimately to vascular abnormalities and pathological neovascularization (NV). Retinal detachment and vision loss could represent a concrete risk connected to the most severe forms of ROP, also characterized by inflammation and retinal cell death.
Methods: During the last decades, many animal models of oxygen-induced retinopathy (OIR) have been recognized as useful tools to study the mechanisms of disease, since they reproduce the hallmarks typical of human ROP. Indeed, modulation of retinal vascular development by exposure to different oxygen protocols is possible in these animals, reproducing the main pathological phenotypes of the disease. The easy quantification of abnormal NV and the possibility to perform electrophysiologic, histological and molecular analyses on these models, make OIR animals a fundamental instrument in studying the pathophysiology of ROP and the effects of novel treatments against the disease.
Discussion: Here, the most commonly used OIR protocols in rodents, such as mice and rats, are described as well as the main pathological outcomes typical of these models. Despite their limitations and variables which should be considered whilst using these models, OIR models display several characteristics which have also been confirmed in human patients, validating the usefulness of such animals in the pre-clinical research of ROP.
Background and Objective: Subthreshold laser therapy has emerged as a therapeutic alternative to traditional laser photocoagulation for certain ophthalmic diseases including central serous chorioretinopathy (CSCR), diabetic macular edema (DME), macular edema secondary to branch retinal vein occlusion (BRVO), and age-related macular degeneration (AMD). The objective of this paper is to review and discuss the clinical applications of subthreshold laser and the mechanisms of different subthreshold laser techniques including subthreshold micropulse laser (SMPL), selective retina therapy (SRT), subthreshold nanosecond laser (SNL), endpoint management (EpM), and transpupillary thermotherapy (TTT).
Methods: A narrative review of English literature and publicly available information published before November 2021 from literature databases and computerized texts. We discuss the currently available subthreshold laser systems and the advancements made to perform different subthreshold laser techniques for various ophthalmic diseases. We highlight various clinical studies and therapeutic techniques that have been conducted to further understand the effectiveness of subthreshold laser in the clinical setting. We conclude the article by covering emerging subthreshold laser systems that are currently being developed for future clinical use. The PubMed database was utilized for peer-reviewed articles and pertinent information on subthreshold systems was cited from publicly available online websites covering specific systems.
Key Content and Findings: Various subthreshold laser systems have been developed to treat certain retinal diseases. Several systems are currently in development for future clinical applications.
Conclusions: While conventional laser photocoagulation has been effective in treating various retinal diseases, subthreshold laser systems aim to provide a therapeutic effect without visible signs of damage to the underlying tissue. This technology may be particularly effective in treating macular disorders. Further clinical studies are needed to evaluate their role in the management of retinal diseases.
