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

망막 색소 상피 식세포작용 및 리포푸신 축적 정량화

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.

저자 스포트라이트: Improved Lipofuscin Models and Quantification of Outer Segment Phagocytosis Capacity in Highly polarized Human Retinal Pigment Epithelial Cultures  

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

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  

광산화된 외부 세그먼트 팁 및 단편의 제조

폴리테트라플루오로에틸렌 또는 PTFE 코팅 슬라이드를 70% 에탄올에 10분 동안 생물 안전 캐비닛에 담가 멸균하는 것으로 시작합니다. 슬라이드를 멸균된 100mm 세포 배양 접시에서 자연 건조시킵니다. 각 슬라이드를 1개의 새로운 100mm 세포 배양 접시에 넣고 200마이크로리터의 혈청 함유 세포 배양 배지를 각 직사각형에 추가합니다.그런 다음 전구가 슬라이드를 직접 향하도록 하여 휴대용 254나노미터 UV 광선을 100mm 세포 배양 접시에 놓고 20분 동안 놓습니다. 섭씨 37도의 수조에서 외부 세그먼트 또는 OS의 얼어붙은 부분 표본을 해동합니다. 그런 다음 실온에서 5 분 동안 2, 400G에서 OS를 회전시킵니다.피펫을 사용하여 생물안전 캐비닛에서 상청액을 즉시 흡인하고 PBS에서 OS를 다시 일시 중단합니다. 이제, 슬라이드로부터 매체를 흡인하고, 각 슬라이드 직사각형 상에 2 x 10 내지 밀리리터당 8번째 OS PBS 용액의 2 x 10 내지 8번째 OS PBS 용액의 최대 500 마이크로리터를 배치한다. 전구가 OS를 직접 향하도록 하여 40분 동안 휴대용 254나노미터 UV 광선을 세포 배양 접시 위에 놓습니다.1.5 밀리리터 튜브에 OS-PBS 용액을 수집합니다. 코팅된 슬라이드의 직사각형을 200 내지 500 마이크로리터의 PBS로 세척하고, 각각의 세척물을 마이크로원심분리 튜브에 수집한다. 그런 다음 OS-PBS 용액을 실온에서 5분 동안 2, 400G로 스핀다운합니다.피펫터를 사용하여 생물안전 캐비닛에서 PBS를 흡인하고 망막 색소 상피 또는 RPE 배양에 사용할 표준 배지 500마이크로리터에 펠릿을 재현탁합니다. 10마이크로리터의 현탁액을 새 미세 원심분리기 튜브에 넣습니다. 더 많은 세포 배양 배지로 50-100 배 희석하고 혈구계로 OS를 계산합니다.광산화된 OS 또는 OxOS 현탁액을 5.6 x 10 내지 밀리리터당 7번째 OS 농도로 표준 RPE 배지로 희석한다. OS 흡수 및 리포푸신 축적을 촉진하는 식균 작용 가교 리간드를 추가합니다. 그런 다음 OxOS를 일회용 스톡으로 분취하고 액체 질소에서 급속 동결합니다.리포푸신 축적을 유도하기 위해 RPE 트랜스웰에 추가하기 전에 냉동된 분취액을 45마이크로리터의 세포 배양 배지에 희석하여 OxOS 분취액을 해동합니다. 그런 다음 컨포칼 현미경에서 Lambda 스캐닝을 사용하여 OxOS 방출 스펙트럼을 측정하여 산화된 OS를 특성화합니다. 공초점 영상은 처리된 OS가 처리되지 않은 OS에 비해 자가형광이 증가했음을 보여주었습니다.

Research

JoVE Journal

Biology

Preparation of Photo-Oxidized Outer Segment Tips and Fragments

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