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

レチナール色素上皮食作用とリポフスチン蓄積の定量化

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.

著者スポットライト:高度に分極したヒト網膜色素上皮培養における改良されたリポフスチンモデルと外側セグメント食作用能力の定量化  

高度に分極されたヒト網膜色素上皮培養におけるリポフスチンモデルの改善と外セグメント食能力の定量化

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  

光酸化アウターセグメントチップおよびフラグメントの調製

まず、ポリテトラフルオロエチレンまたはPTFEコーティングされたスライドを、バイオセーフティキャビネット内の70%エタノールに10分間浸して滅菌します。スライドを滅菌済みの100mm細胞培養皿で風乾します。各スライドを1つの新しい100ミリメートル細胞培養皿に入れ、200マイクロリットルの血清含有細胞培養培地を各長方形に追加します。次に、ハンドヘルドの254ナノメートルのUV光を100ミリメートルの細胞培養皿に置き、電球をスライドに直接向けて20分間置きます。外側セグメント(OS)の凍結アリコートを摂氏37度の水浴で解凍します。次に、OSを2, 400Gで室温で5分間回転させます。直ちにピペットを使用してバイオセーフティキャビネット内の上清を吸引し、OSをPBSに再懸濁します。次に、スライドから培地を吸引し、各スライド長方形に、ミリリットルPBS溶液あたり2 x 10から8番目のOSの2 x 10からミリリットルPBS溶液あたり8番目のOSを最大500マイクロリットル配置します。ハンドヘルドの254ナノメートルのUV光を細胞培養皿の上に置き、電球をOSに直接向けて40分間置きます。OS-PBS溶液を1.5ミリリットルのチューブに集めます。コーティングされたスライドの長方形を200〜500マイクロリットルのPBSで洗浄し、各洗浄物をマイクロ遠心チューブに集めます。次に、OS-PBS溶液を室温で5分間2, 400Gでスピンダウンします。バイオセーフティキャビネット内のPBSをピペッターで吸引し、網膜色素上皮またはRPE培養に使用する500マイクロリットルの標準培地にペレットを再懸濁します。10マイクロリットルの懸濁液を新しいマイクロ遠心チューブに入れます。さらに細胞培養培地で50〜100倍に希釈し、血球計算盤でOSをカウントします。光酸化OS(OxOS懸濁液)を1ミリリットルあたり5.6 x 10から7番目のOS濃度で標準RPE培地に希釈します。OSの取り込みとリポフスチンの蓄積を促進する食作用ブリッジリガンドを追加します。次に、OxOSを使い捨てストックに分注し、液体窒素で急速凍結します。凍結したアリコートを45マイクロリットルの細胞培養培地で希釈してOxOSアリコートを解凍してから、RPEトランスウェルに添加してリポフスチン蓄積を誘導します。次に、共焦点顕微鏡でラムダスキャンを使用してOxOS発光スペクトルを測定することにより、酸化されたOSを特徴付けます。共焦点イメージングは、処理されたOSが未処理のOSと比較して自家蛍光を増加させたことを示した。

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 ...