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.
Background: Continuous and primary in vitro cultures are largely used to study cellular mechanisms occurring in several pathologic-like or pathological conditions. Continuous cell lines allow to perform long-lasting experiments since they do not undergo senescence.
Methods: The immortalized Moorfields/Institute of Ophtalmology-Müller 1 (MIO-M1) cell type represents a valuable model to analyze the mechanistic pathways characterizing Müller glial cells, both in health and in disease. MIO-M1 can be used to dissect the response of these glial cells following treatments which mimic pathological condition. For instance, MIO-M1 are useful to study the response of this cell type to stress condition as the case of oxidative stress (OS) (cultured with hydrogen peroxide), pathological neovascularization (cultured with VEGF), hypoxic or hyperoxic condition (cultured in low or high oxygen chamber). On the other hand, primary cultures allow to specifically analyze cellular responses without the interference of the whole organ, although the experimental treatment is performed in vivo. Primary Müller cells can be used to perform electrophysiological analyses of different cell sites.
Discussion: We describe how to manage MIO-M1 cells and how to analyze their response to different stress conditions; moreover, we report how to isolate and identify primary Müller cells and how to perform patch clamp and single cell recordings on them.
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: The ex vivo model represented by mouse retinal explants in culture is a useful experimental model to investigate the molecular mechanism involved in neurovascular diseases such as diabetic retinopathy (DR). It ensures an experimental overview with more complete respect to isolate cells and reduce problems in terms of accessibility and management with respect to in vivo model. In particular, it allows the evaluation of the relationship between retinal cells in response to the typical stressors involved in DR pathogenesis.
Methods: Ex vivo retinal fragments derived from 3- to 5-week-old C57BL/6J mice. In particular, after dissection, the retina is cut into 4 separate fragments and transferred onto inserts placed with ganglion cells up. Once in culture, the explants could be treated in stress conditions typical of DR. In particular, this study protocol describes the procedure for the preparation and the culture of retinal explants with specific metabolic stressors such as high glucose (HG), advanced glycation end product (AGE), and oxidative stress (OS). In the end, this paper provides the protocols to perform molecular analyses in order to evaluate the response of retinal explants to stress and/or neuroprotective treatments.
Discussion: The cultured retinal explants represent an ex vivo experimental model to investigate the molecular mechanisms involved in neurovascular diseases such as DR. Moreover, they could be useful to test the effect of neuroprotective compounds in response to metabolic stressors in a fewer time respect to an in vivo model. In conclusion, retinal explants in culture represent a valuable experimental model to conduct further studies to better understand the pathophysiology of DR.
Background: Retinal degeneration is a common feature of several retinal diseases, such as retinitis pigmentosa and age-related macular degeneration (AMD). In this respect, experimental models of photo-oxidative damage reproduce faithfully photoreceptor loss and many pathophysiological events involved in the activation of retinal cell degeneration. Therefore, such models represent a useful tool to study the mechanisms related to cell death. Their advantage consists in the possibility of modulating the severity of damage according to the needs of the experimenter. Indeed, bright light exposure could be regulated in both time and intensity to trigger a burst of apoptosis in photoreceptors, allowing the study of degenerative mechanisms in a controlled fashion, compared to the progressive and slower rate of death in other genetic models of photoreceptor degeneration.
Methods: Here, an exemplificative protocol of bright light exposure in albino rat is described, as well as the main outcomes in retinal function, photoreceptor death, oxidative stress, and inflammation, which characterize this model and reproduce the main features of retinal degeneration diseases.
Discussion: Models of photo-oxidative damage represent a useful tool to study the mechanisms responsible for photoreceptor degeneration. In this respect, it is important to adapt the exposure paradigm to the experimental needs, and the wide range of variables and limitations influencing the final outcomes should be considered to achieve proper results.
Trial Registration: None.
