Name: Preparation of Photo-Oxidized Outer Segment Tips and Fragments
Uploaded: 2023-04-14T14:30:00
Description: Watch this Scientific Journal Video about Preparation of Photo-Oxidized Outer Segment Tips and Fragments at JoVE.com

The video outlines a detailed protocol for preparing and characterizing photo-oxidized outer segments (OS) from retinal cells. It covers the sterilization of slides, treatment with UV light, the resuspension and dilution of OS in culture medium, and assessing lipofuscin accumulation using confocal microscopy.

preparation of photo-oxidized outer segment tips and fragments

Cuantificación de la fagocitosis epitelial pigmentaria de la retina y acumulación de lipofuscina

The retinal pigment epithelium (RPE) provides critical support for overlying photoreceptors, including the daily uptake and degradation of photoreceptor outer segment tips or fragments (throughout this protocol, the abbreviation OS stands for OS tips or fragments rather than whole outer segments). This daily uptake in the post-mitotic RPE eventually overloads phagolysosomal capacity and leads to the buildup of undigestible, autofluorescent intracellular material, termed lipofuscin. Interestingly, several studies have also demonstrated that RPE lipofuscin can accumulate without OS phagocytosis1,2. Lipofuscin has many components, including cross-linked adducts derived from visual cycle retinoids, and can occupy nearly 20% of RPE cell volume for those over the age of 803.
Whether lipofuscin is toxic has been hotly debated. Stargardt&#39;s disease is an autosomal recessive degeneration of the photoreceptors and RPE in which a mutation in ABCA4 triggers improper processing of visual cycle retinoids contained within photoreceptor outer segments. Improper retinoid processing leads to aberrant cross-linking and formation of bis-retinoid species, including the bis-retinoid N-retinylidene-N-retinylethanolamine (A2E). Studies have demonstrated multiple mechanisms for A2E toxicity4,5. Lipofuscin contributes to fundus autofluorescence signals during clinical imaging, and both Stargardt&#39;s patients and animal models display increased fundus autofluorescence prior to retinal degeneration, suggesting a correlation between lipofuscin levels and toxicity6,7. However, with age, lipofuscin accumulates in all humans without triggering an RPE degeneration. Further, in age-related macular degeneration (AMD), where RPE degeneration occurs only in elderly patients, those with early and intermediate forms of the disease have less fundus autofluorescence signals than age-matched non-diseased humans8. These clinical findings have been verified at the histologic level as well9,10.
Animal models of RPE lipofuscin accumulation have also left some ambiguity about lipofuscin toxicity. The ABCA4 knockout mouse does not display retinal degeneration on a pigmented background, whereas it does on an albino background or when exposed to blue light11,12. Further, the toxicity of lipofuscin derived via ABCA4 knockout likely differs from the more slowly accumulating lipofuscin that occurs with natural aging, as seen in AMD13.
In vitro models of lipofuscin accumulation provide an alternative to studying the effects of lipofuscin accumulation on RPE health. Such models allow for manipulating lipofuscin components, from feeding single retinoid components to feeding OS, and allow study in human rather than animal RPE. In the last couple of decades, multiple methods have been developed to model RPE lipofuscin in culture. Along with other groups, Dr. Boulton&#39;s group fed bovine OS daily for up to three months on passage 4 to 7 human primary RPE cells from donors aged 4 to 85 years old14. Alternatively, inhibition of autophagy has also led to lipofuscin accumulation in passages 3 to 7 primary human RPE cultures15. However, sub-lethal lysosomal inhibition in highly differentiated, passage 1, primary human pre-natal RPE (hfRPE) cultures failed to induce lipofuscin, even with the repeated addition of OS on a daily basis16.
As a more reductionist approach, others have fed single lipofuscin components to cultures, especially the bis-retinoid A2E4,17. Such studies are valuable in that they define potential direct mechanisms of toxicity for individual lipofuscin components, implicating, for example, lysosomal cholesterol and ceramide homeostasis18. At the same time, there is debate about the toxicity of A2E19, and feeding it directly to cells circumvents the typical pathway for lipofuscin accumulation, which involves phagocytosis of photoreceptor OS. In an attempt to deliver all components of lipofuscin to RPE cultures, Boulton and Marshall purified lipofuscin from human eyes and fed this to passage 4 to 7 human primary RPE cultures derived from both fetal and elderly human donors20. While innovative, this method represents a limited lipofuscin source for repeated experiments.
While repeated feedings of OS to RPE cultures produce lipofuscin in many systems, it fails to do so in highly differentiated primary RPE cultures16. Photo-oxidizing OS induces cross-linking reactions like bis-retinoid formation that naturally occurs during lipofuscin formation in vivo. This can accelerate lipofuscin-like granule formation in RPE culture systems, even those that are highly differentiated and resistant to lipofuscin accumulation16. Here, a method to induce lipofuscin-like granule accumulation in highly differentiated hfRPE and human iPSC-RPE is introduced, modified from Wihlmark&#39;s published protocol21. This method has the advantage of inducing lipofuscin-like granules employing the same source (photoreceptor OS) and pathway (phagolysosomal OS uptake) as occurs for lipofuscinogenesis in vivo. Further, it is done on human RPE cultures that are highly differentiated and validated in multiple studies to replicate human RPE in vivo22,23,24. These lipofuscin-like granules are termed undigestible autofluorescent material (UAM), and provide data and discussion in this protocol comparing UAM to in vivo lipofuscin. Along with methods for building and evaluating UAM-laden cultures in highly differentiated human RPE, an updated method to assess RPE OS phagocytosis is also introduced. Multiple excellent pulse-chase methods for quantifying OS phagocytosis have been introduced, including Western blotting, immunocytochemistry, and FACS25,26,27. However, early in the OS pulse-chase, conditions that lead to poor OS uptake can be conflated with conditions that promote rapid degradation of internalized OS. The method presented here measures the total amount of introduced OS that is fully consumed/degraded by the RPE (&#34;Total Consumptive Capacity&#34;), helping eliminate this ambiguity. It is anticipated that insights about lipofuscin toxicity utilizing these protocols, including effects on OS phagocytosis rates utilizing the &#34;Total Consumptive Capacity&#34; method, will be used to shed light on the toxicity of lipofuscin in vivo.

Autor destacado: Modelos mejorados de lipofuscina y cuantificación de la capacidad de fagocitosis del segmento externo en cultivos epiteliales de pigmento de retina humano altamente polarizados  

Modelos mejorados de lipofuscina y cuantificación de la capacidad de fagocitosis del segmento externo en cultivos epiteliales de pigmento retiniano humano altamente polarizados

While lipofuscin universally accumulates in the RPE with aging, discerning its toxicity has been difficult. Here, we develop protocols that allow us to develop lipofuscin-like accumulation in highly polarized and mature RPE cultures to help discern lipofuscin's effects on RPE physiology. Studying lipofuscin toxicity in humans is confounded by the fact that lipofuscin decreases as the RPE dies.Animal models of lipofuscin toxicity have had widely disparate results. We have carefully created in vitro models of lipofuscin-like material accumulation to facilitate the study of lipofuscin toxicity. The key to the success of our models is maintaining highly differentiated cultures while inducing lipofuscin genesis via the same process that triggers lipofuscin in vivo, namely phagocytosis of photoreceptor outer segments.Our in vitro protocol is unique in that lipofuscin-like material accumulates in RPE cultures that are highly differentiated to faithfully mimic RPE in vivo. In these circumstances, we found that RPE culture are highly resistant to the lipofuscin accumulation and to toxic effect from lipofuscin. Additionally, in the process of assaying for phenotypes induced by lipofuscin-like accumulation in our RPE cultures, we created a new type of phagocytosis assay called total consumptive capacity.This assay allows us to determine the entire capacity of the RPE to phagocytose outer segments while avoiding some of the confounding interpretations that come with classical pulse-chase phagocytosis assays. We are excited to utilize this model of lipofuscin-like material accumulation to better understand what RPE stressors may take normally inert lipofuscin into a more pathological role in RPE cell deaths.

Watch this Scientific Journal Video about Preparation of Photo-Oxidized Outer Segment Tips and Fragments at JoVE.com

Preparation of Photo-Oxidized Outer Segment Tips and Fragments  

Preparación de puntas y fragmentos de segmentos externos fotooxidados

Comience esterilizando los portaobjetos recubiertos de politetrafluoroetileno o PTFE sumergiéndolos en etanol al 70% durante 10 minutos en un gabinete de bioseguridad. Deje que los portaobjetos se sequen al aire en una placa estéril de cultivo celular de 100 milímetros. Coloque cada portaobjetos en 1 nueva placa de cultivo celular de 100 milímetros y agregue 200 microlitros de medio de cultivo celular que contenga suero en cada rectángulo.Luego coloque una luz UV portátil de 254 nanómetros en el plato de cultivo celular de 100 milímetros con la bombilla directamente hacia el portaobjetos durante 20 minutos. Descongele las alícuotas congeladas de los segmentos externos, o OS, en un baño de agua de 37 grados centígrados. Luego gire el sistema operativo a 2, 400G durante cinco minutos a temperatura ambiente.Aspirar inmediatamente el sobrenadante en el gabinete de bioseguridad con una pipeta y volver a suspender el sistema operativo en PBS. Ahora, aspire el medio de los portaobjetos y coloque hasta 500 microlitros de la solución PBS de 2 x 10 a 8º OS por mililitro de la solución PBS de 2 x 10 a 8º OS por mililitro en cada rectángulo de diapositiva. Coloque una luz UV portátil de 254 nanómetros sobre el plato de cultivo celular con la bombilla directamente hacia el sistema operativo durante 40 minutos.Recoja la solución OS-PBS en un tubo de 1,5 mililitros. Lave el rectángulo del portaobjetos recubierto con 200 a 500 microlitros de PBS y recoja cada lavado en el tubo de microcentrífuga. Luego gire la solución OS-PBS a 2, 400G durante cinco minutos a temperatura ambiente.Aspirar el PBS en el gabinete de bioseguridad con un pipeteo y resuspender el pellet en 500 microlitros de medio estándar para ser utilizado para el epitelio pigmentario de la retina, o cultivos de EPR. Coloque 10 microlitros de la suspensión en un nuevo tubo de microcentrífuga. Diluir de 50 a 100 veces con más medio de cultivo celular y contar la SG con un hemocitómetro.Diluir la OS fotooxidada, o suspensión OxOS, a una concentración de 5.6 x 10 a la 7ª OS por mililitro en un medio RPE estándar. Agregue ligandos puente de fagocitosis, que facilitan la absorción de SG y la acumulación de lipofuscina. Luego alícuota OxOS en existencias de un solo uso y congelar a presión en nitrógeno líquido.Descongelar las alícuotas de OxOS diluyendo la alícuota congelada en 45 microlitros de medios de cultivo celular antes de agregarla a los pozos de RPE para inducir la acumulación de lipofuscina. Luego caracterice el sistema operativo oxidado midiendo el espectro de emisión de OxOS utilizando el escaneo Lambda en un microscopio confocal. Las imágenes confocales mostraron que la SG tratada había aumentado la autofluorescencia en comparación con la SG no tratada.

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Biology

Preparation of Photo-Oxidized Outer Segment Tips and Fragments

Improved Lipofuscin Models and Quantification of Outer Segment Phagocytosis Capacity in Highly Polarized Human Retinal Pigment Epithelial Cultures

The daily phagocytosis of photoreceptor outer segments by the retinal pigment epithelium (RPE) contributes to the accumulation of an intracellular aging ...