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

Quantifizierung der retinalen Pigmentepithel-Phagozytose und der Lipofuszin-Akkumulation

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 im Rampenlicht: Verbesserte Lipofuszinmodelle und Quantifizierung der Phagozytosekapazität des äußeren Segments in hochpolarisierten humanen retinalen Pigmentepithelkulturen  

Verbesserte Lipofuszin-Modelle und Quantifizierung der Phagozytosekapazität des äußeren Segments in hochpolarisierten humanen retinalen Pigmentepithelkulturen

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  

Präparation von photooxidierten äußeren Segmentspitzen und -fragmenten

Beginnen Sie mit der Sterilisation von Objektträgern mit Polytetrafluorethylen oder PTFE-Beschichtung, indem Sie sie 10 Minuten lang in einer Biosicherheitswerkbank in 70%iges Ethanol tauchen. Lassen Sie die Objektträger in einer sterilen 100-Millimeter-Zellkulturschale an der Luft trocknen. Legen Sie jeden Objektträger in 1 neue 100-Millimeter-Zellkulturschale und geben Sie 200 Mikroliter serumhaltiges Zellkulturmedium in jedes Rechteck.Legen Sie dann 20 Minuten lang ein tragbares 254-Nanometer-UV-Licht auf die 100-Millimeter-Zellkulturschale, wobei die Glühbirne direkt auf den Objektträger zeigt. Tauen Sie die gefrorenen Aliquots der äußeren Segmente (OS) in einem 37 Grad Celsius warmen Wasserbad auf. Drehen Sie dann das Betriebssystem fünf Minuten lang bei Raumtemperatur bei 2.400 G.Saugen Sie den Überstand in der Biosicherheitswerkbank sofort mit einer Pipette ab und resuspendieren Sie das OS in PBS. Saugen Sie nun das Medium aus den Objektträgern ab und geben Sie bis zu 500 Mikroliter der 2 x 10 bis 8. OS pro Milliliter PBS-Lösung der 2 x 10 bis 8. OS pro Milliliter PBS-Lösung auf jedes Objektträgerrechteck. Platzieren Sie 40 Minuten lang ein tragbares 254-Nanometer-UV-Licht über der Zellkulturschale, wobei die Glühbirne direkt zum Betriebssystem zeigt.Sammeln Sie die OS-PBS-Lösung in einem 1,5-Milliliter-Röhrchen. Waschen Sie das Rechteck des beschichteten Objektträgers mit 200 bis 500 Mikrolitern PBS und sammeln Sie jede Wäsche im Mikrozentrifugenröhrchen. Drehen Sie dann die OS-PBS-Lösung bei 2.400 G für fünf Minuten bei Raumtemperatur herunter.Saugen Sie das PBS in der Biosicherheitswerkbank mit einem Pipettiergerät ab und resuspendieren Sie das Pellet in 500 Mikrolitern Standardmedium, das für das retinale Pigmentepithel oder die RPE-Kulturen verwendet wird. Geben Sie 10 Mikroliter der Suspension in ein neues Mikrozentrifugenröhrchen. 50 bis 100 Mal mit mehr Zellkulturmedium verdünnen und das OS mit einem Hämozytometer zählen.Verdünnen Sie das photooxidierte OS oder die OxOS-Suspension bei einer Konzentration von 5,6 x 10 bis 7 OS pro Milliliter in ein Standard-RPE-Medium. Fügen Sie Phagozytose-überbrückende Liganden hinzu, die die Aufnahme des Betriebssystems und die Akkumulation von Lipofuszin erleichtern. Dann aliquotieren Sie OxOS in Einwegvorräte und frieren Sie es in flüssigem Stickstoff ein.Tauen Sie OxOS-Aliquots auf, indem Sie das gefrorene Aliquot in 45 Mikrolitern Zellkulturmedien verdünnen, bevor Sie es zu RPE-Transwellen hinzufügen, um eine Lipofuszin-Akkumulation zu induzieren. Charakterisieren Sie dann das oxidierte OS durch Messung des OxOS-Emissionsspektrums mittels Lambda-Scanning auf einem konfokalen Mikroskop. Die konfokale Bildgebung zeigte, dass das behandelte OS im Vergleich zum unbehandelten OS eine erhöhte Autofluoreszenz aufwies.

Research

JoVE Journal

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 ...