我々は、FNABの手順のデモをグラフ化する映像の間に援助のために氏ボニーPhetteplace、RNに感謝。

はじめ全身性アミロイドーシスは、総称してアミロイドと呼ばれる様々なタンパク質の細胞外沈着を参照している非常に可変性疾患です。 βプリーツ構成でこれらのアミロイド線維の沈着が関与する臓器に応じて様々な臨床症状につながる。このような心臓、肝臓、および/または腎臓などの重要臓器の関与がある場合に全身性アミロイドーシスの臨床的に最も関連する症状が記載されています。歴史的に、これらの浸潤臓器は、アミロイドの存在を実証するために生検される。これらの適度侵襲的処置は出血を含む重大なリスクを運んだ。脂肪パッドの吸引は、以来、全身性アミロイドーシス1のアミロイドの検出のための信頼性と非侵襲的方法を提供することが示されている。この手順では、小さな血管の壁2に乏しいアミロイドの沈着を評価するための線維脂肪の組織を取得するために局所麻酔下で前腹壁の皮下脂肪の脂肪吸引に本質的に同等です。 組織におけるアミロイドの同定のための最も頻繁に使用方法は、コンゴレッド染色した切片は、偏光顕微鏡3で視覚化するときに見られる特徴的な青リンゴ色の複屈折パターンのまま。脂肪パッドの吸引が行われるときは、コンゴレッド染色は、直接塗抹スライドまたは吸引脂肪組織の細胞ブロック標本のどちらかで行うことができます。しかし、アミロイドーシスの初期段階で患者が大幅に4,5コンゴ赤染色細胞のブロックのセクションの感度を減少させるわずかなアミロイド沈着を、持っている。電子顕微鏡による脂肪パッドの吸引の超微細構造評価は、より良い再現性と感度の向上4とを有している。したがって、セルブロックの両方を調製するため、電子顕微鏡2を実行するためのすべての脂肪パッドの吸引を提出することをお勧めします。 脂肪パッドの吸引は、比較的低コストおよび全身性アミロイドーシスの診断に組織を得るための非侵襲的方法である。この記事では、電子顕微鏡によるコンゴレッド染色と微細構造評価の両方に検体を提出するサンプルの処理についての詳細と一緒に脂肪パッドの吸引の手順を説明します。このビデオでは、我々は最適な診断材料を取得するために、この再現性と簡単な手順を示しています。 1。前歯部脂肪パッドのFNAの実行ローカルエリアのA.の麻酔（図1＆2） <ol><li>アルコール綿棒または特定の機関によって好ま皮膚洗浄剤を使用して、正中線と臍下（図1）の外側腹部の下腹部の領域で皮膚を洗浄。 </li><li>としてマーキングペンで図1に示す2 × 2インチ約菱形形状の領域をマークします。 </li><li> 1％の吸引約10 mLを18G針でリドカイン。 25G 1を取り付けます½シリンジにインチ針を。液体を分配しても気泡が存在しないされるまでプランジャーを押しながら直立シリンジをタップして、任意の閉じ込められた空気を削除します。 </li><li>図2に示すように、既にマーク菱形領域の境界に沿って麻酔する。ちょうどポイントで皮膚の下に25G針を挿入して起動し、慎重にX.を使用すると、容器内にないことを保証するために注射器のプランジャーを後方に引いて指すように、皮下針を押してください。その後徐々に針は、針がポイント（図2A3）で皮膚から出てくるさせることなく参照するように撤退しながらリドカインの2.5mLの（10 mLシリンジのリドカインの¼約）まで潜入するためにプランジャーを押してください。針でも、皮膚の下に点Y（図2A4）に向かって方向を変更するとポイントY（図2B1）に皮下針を押してください。以前と同じようにゆっくりとポイントを介して針を引き出しながら、針が血管内にないことを確認し、リドカインの約2.5 mLを分注する（2B4〜図2B3）。 YにXとB（2D4〜図2C1）に点Bから点Bと調剤リドカイン皮下から始まる同様の手順を繰り返します。呵責からの出血を防ぎ、無菌ゲージピースの会社のアプリケーションによるB。 </li><li>これで、面積は軽く、隣接する無麻酔皮膚と比較することによって、先端/綿のガーゼ片のコーナーで麻酔菱形の中で皮膚に触れることによって麻酔であることを確認することができます。 </li></ol> B.は、脂肪パッドの吸引（図3）の実行 （前の脂肪パッドの吸引のパフォーマンスへの局所麻酔の適用は、地域や個人の好みに応じて、バイパスされる可能性があります。前方脂肪パッドの吸引のための適切に行わFNABの手順が1つの刺し傷で完了することができます。しかし、個々の患者の痛みのしきい値に応じて、皮下脂肪組織に前後に18G針を操縦するのは比較的distresです歌う。ここで説明する麻酔の方法は、25G針で2つだけの呵責に効果を発揮し、手順に耐性が向上して痛みをaverts。） <ol><li> 10mLのシリンジに18G 1 ½インチの針を組み立てます。適切なアプリケーションと真空のリリースのための注射器のグリップ（&quot;FNABグリップ/銃&quot;）で注射針を装着してください。 </li><li>クレンジングと麻酔菱形の領域（図3a）内に皮下脂肪に針の先端を挿入します。 </li><li>完全真空（図3b）を生成するために皮下組織に挿入された針と注射器のプランジャーを引っ込める。 </li><li>真空を維持し、皮下脂肪（3iの〜図3C）への様々な方向に前後に針を操縦。各ストロークは、針が皮膚から出てくるさせることなく、選択された針の長さをできるだけ長くしてください。最大のサンプリングは、各ストローク方向（図3iを）変えることによって実現される。漿膜への接線と腹腔のパンクチャを避けるために皮膚表面に平行な針の方向を維持することが重要です。 </li><li>線維脂肪の組織は、注射器内に蓄積する。一度十分な線維脂肪の組織の断片（フラグメントを豊富に含む血液の混合試料の最大1​​ mL）を取得され、完全に真空を解放し、針（3K図）を取り外します。 </li><li>患者またはアシスタントが血液の溢出を防止するためのガーゼのパッドを持つプロシージャの領域でしっかり圧力を適用しています。 </li></ol> 2。検体処理<ol><li>電子顕微鏡のためのグルタルアルデヒド溶液（図4B）で線維脂肪の組織の少なくとも5から6の断片を配置。 </li><li>制度的なプロトコルに応じて、線維脂肪の組織の断片のいくつかの塗抹標本は、2つのスライド（図4A）との間で、それらを拡散させることによって調製することができる。 </li><li>残りの材料は、シリンジ内の血栓（これが凝固時間に応じて、5〜7分かかる場合があります）（図4C）に許可されています。 </li><li>凝固した線維脂肪の組織の材料が注射器とフリーフローティング（図4C2）の壁から外れているように注射器で10％ホルマリンを吸引除去する。注射器（図4C3）からプランジャーを取り外し、ノズルの端（図4C4）反対側のシリンジの開放端から10％ホルマリン容器にクロテッド線維脂肪の組織を移す。 </li><li>適切な容器にラベルを付け、電子顕微鏡のためのグルタルアルデヒドを提出し、H＆Eセクションとコンゴレッドのためのホルマリンは、偏光顕微鏡下で評価するために染色。塗抹標本が準備されている場合、それらは個々の実験室のプロトコルに従って処理される可能性があります。 </li></ol><img src="/files/ftp_upload/1747/1747fig1.jpg" alt="図1" /> 図1。前脂肪パッドのFNABのために麻酔になるエリア。 <img src="/files/ftp_upload/1747/1747fig2.jpg" alt="図2" /> 図2。ローカルエリアの麻酔。 <img src="/files/ftp_upload/1747/1747fig3.jpg" alt="図3" /> 図3。前脂肪パッドのFNABを実行。 <img src="/files/ftp_upload/1747/1747fig4.jpg" alt="図4" /> 図4。前脂肪パッドの処理は、アミロイド沈着を検出するための実験室に提出する吸引。 3。代表的な結果<img src="/files/ftp_upload/1747/1747fig5.jpg" alt="図5" /> 図5。線維脂肪の組織フラグメントの小血管の壁にアミロイド。線維脂肪の組織はホルマリン加工、パラフィンコンゴレッドで染色し、埋め込 ​​み、および光学顕微鏡を偏光により調べた。小血管の壁にアミロイドは、アップルグリーン複屈折（白い矢印）を示しています。 <img src="/files/ftp_upload/1747/1747fig6.jpg" alt="図6" /> 図6。アミロイドーシスを欠いている組織は。青リンゴ色の複屈折は、アミロイドーシスのない別の患者の組織中に存在しない。コラーゲン線維の青の複屈折は（白矢印）、ほぼすべての標本で、通常は存在する。 <img src="/files/ftp_upload/1747/1747fig7.jpg" alt="図7" /> 図7。血管壁にアミロイドと一致して線維。血管壁にアミロイドが形成されたストレート、非分岐、ランダムに散らばって80〜10 nmの直径の線維の電子顕微鏡写真。

<ol>
	<li>Westermark, P., Stenkvist, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+for+the+diagnosis+of+systemic+amyloidosis.">A new method for the diagnosis of systemic amyloidosis.</a> Arch Intern Med. 132, 522-523 (1973).</li><li>Devata, S., Hari, P., Markelova, N., Li, R., Shidham, V. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detecting+AL+Amyloid+in+Abdominal+fat+pad+aspirates:+Is+Congo+red+sufficient?.">Detecting AL Amyloid in Abdominal fat pad aspirates: Is Congo red sufficient?.</a> Amyloid. , .</li><li>Merlini, G., Bellotti, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Mechanisms+of+Amyloidosis.">Molecular Mechanisms of Amyloidosis.</a> New Engl J Med. 349 (6), 583-596 (2003).</li><li>Shidham, V. B., Kumar, N., Cihlar, K., Varsegi, G., Markelova, N., Li, R., Hari, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fine+needle+aspiration+of+abdominal+fat+pad+for+diagnosis+of+early+amyloidosis:+How+can+the+clinical+role+of+the+test+be+improved?.">Fine needle aspiration of abdominal fat pad for diagnosis of early amyloidosis: How can the clinical role of the test be improved?.</a> Mod Pathol. 20, 1A-380A (2007).</li><li>Halloush, R., Lavrovskaya, E., Mody, D., Lager, D., Truong, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+and+typing+of+systemic+amyloidosis:+The+role+of+abdominal+fat+pad+fine+needle+aspiration.">Diagnosis and typing of systemic amyloidosis: The role of abdominal fat pad fine needle aspiration.</a> Cytojournal. 6, 24-24 (2010).</li><li>Hazenberg, I. I., Rijswijk, C. V. a. n., H, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+Accuracy+of+Subcutaneous+Abdominal+Fat+Tissue+Aspiration+for+Detecting+Systemic+Amyloidosis+and+Its+Utility+in+Clinical+Practice.">Diagnostic Accuracy of Subcutaneous Abdominal Fat Tissue Aspiration for Detecting Systemic Amyloidosis and Its Utility in Clinical Practice.</a> Arthritis Rheum. , 54-546 (2006).</li><li>Guy, C. D., Jones, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abdominal+fat+pad+aspiration+biopsy+for+tissue+confirmation+of+systemic+amyloidosis:+specificity,+positive+predictive+value,+and+diagnostic+pitfalls.">Abdominal fat pad aspiration biopsy for tissue confirmation of systemic amyloidosis: specificity, positive predictive value, and diagnostic pitfalls.</a> Diagn Cytopathol. 24, 181-185 (2001).</li><li>Westermark, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosing+Amyloidosis.">Diagnosing Amyloidosis.</a> Scand J Rheumatol Suppl. 24, 327-329 (1995).</li><li>Linke, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+Sensitive+Diagnosis+of+Amyloid+and+Various+Amyloid+Syndromes+Using+Congo+Red+Fluorescence.">Highly Sensitive Diagnosis of Amyloid and Various Amyloid Syndromes Using Congo Red Fluorescence.</a> Virchow Arch. 436, 439-448 (2000).</li><li>Giorgadze, T. A., Shiina, N., Baloch, Z. W., Tomaszewski, J. E., Gupta, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+detection+of+amyloid+in+fat+pad+aspiration:+an+evaluation+of+Congo+red+stain+by+fluorescent+microscopy.">Improved detection of amyloid in fat pad aspiration: an evaluation of Congo red stain by fluorescent microscopy.</a> Diagn Cytopathol. 31, 300-306 (2004).</li><li>DeMay, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fine+Needle+Aspiration+Biopsy.">Fine Needle Aspiration Biopsy.</a> The Art &#38; Science of Cytopathology. , 465-46 (1996).</li><li>Arbustini, E., Vrga, L., Concardi, M., Pallandini, G., Obici, L., Merlini, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+and+Immuno-electron+Microscopy+of+Abdominal+Fat+Identifies+and+Characterizes+Amyloid+Fibrils+in+Suspected+Cardiac+Amyloidosis.">Electron and Immuno-electron Microscopy of Abdominal Fat Identifies and Characterizes Amyloid Fibrils in Suspected Cardiac Amyloidosis.</a> Amyloid. 9, 108-114 (2002).</li></ol>

アミロイドーシスの診断は、通常、腎臓、肝臓、および/または心臓などの影響を受ける臓器の組織生検によって達成される。このアプローチは、高い診断率を持っている、しかし、侵襲的であり、出血2を含む合併症に関連付けることができます。直腸、歯肉、および骨髄生検は、1960年代6,7,8で、比較的少ない侵襲的なアプローチとして、診断のために好まれた。腹部の脂肪パッドの吸引は、全身性アミロイドーシス1の組織診断のための安全、低侵襲、シンプル、そして安価な手順として、1973年に報告された。しかし、取得された検体の処理に手順とアプローチの詳細はコヒーレント報告されていません。このビデオと記事は、ステップでの手順のステップを説明します。 脂肪パッドの吸引でアミロイドを検出するためのアプローチは、同様に、また、1,5,6,7,8を 、様々なアプローチを比較し、評価するいくつかの研究でも標準化されていません。コンゴレッド染色で前方の脂肪パッドの吸引の評価が特に乏しいのアミロイド沈着5のアミロイドーシスの早期の場合には下の観察者間の再現性と影響を受けにくいです。免疫組織化学では、ホルマリン固定パラフィン包埋細胞のブロックのセクションで行われ、細胞診塗抹標本上で実行コンゴ赤色蛍光は、診断感度9,10を改善することが報告されている。電子顕微鏡では脂肪体吸引4,5の線維脂肪の組織における微小血管の壁にわずかアミロイド線維の検出と同定を向上させます。この情報に基づいて、この記事では、セルブロックを（診断リンゴ緑色の複屈折偏光顕微鏡下でコンゴレッド染色したセルブロックセクションの評価のためとも示されている免疫組織化学としての）と電子顕微鏡を準備する試料の処理について説明します。しかしながら、アミロイドのための試料の評価のために選択されたアプローチによって、それは細胞診塗抹標本作製、セルブロックの準備、および電子顕微鏡のための処理を施すことができる。いくつかの研究室では、吸引線維脂肪の組織の断片から調製し、蛍光10を研究するためにコンゴレッドで染色した塗抹標本上でテストを実行。 一般的に遅くアミロイドーシスでは、コンゴレッド偏光顕微鏡と光学顕微鏡では十分な場合もありますが、セルブロックのセクションの偏光顕微鏡による疾患コンゴレッド染色の初期段階で脂肪パッド吸引4,5,11に対して感応性が低く、信頼性です。 。チオフラビンTを含むその他の特別な汚れは、同様の制限事項を考慮に入れて使用することができます。アミロイドを検出する他のアプローチは、コンゴレッド偏光顕微鏡と蛍光10のアミロイドのための吸引塗抹標本の評価が含まれています。我々の経験ではこの方法の方が再現可能です。電子顕微鏡によるアミロイドのための脂肪パッドの血管の評価は、これらの制限を4,11を克服しています 。アミロイドのさらなる特徴は、免疫電子顕微鏡12で報告されているが、これらのメソッドは非三次医療の設定で広く利用できない場合があります。 吸引の手順を実行するために使用する針は、（例えば、- 14G）、（21 - 25G）ファイン中間（18 - 20G）、および大規模な11にそれらのゲージサイズに応じて分類されることがあります。前方脂肪パッドの願望は、中間（18〜20G）から罰金（21〜22G）2,10まで可変ゲージを使用して実行することが報告されている。腫瘤病変のほとんどは、セルのブロックに対して追加のサンプルを取得するために、広いゲージの針（例えば18Gなど）と追加のパスと一緒に良い細胞診塗抹標本を得るために細かいゲージの針（例えば25Gなど）で吸引されています。 前脂肪パッドの吸引時には、凝集線維脂肪の組織は、細かい針でも吸引していません。セルブロックの準備と電子顕微鏡では脂肪吸引の線維脂肪の組織フラグメントの小血管壁の評価のために重要である。広いゲージの針（例えば18Gなど）の診断に十分な材料2,10を得るために推奨されています。手順は、従来のFNABに匹敵するように、脂肪パッドの吸引は、広いゲージの針が5それを実行するために使用されていてもFNABと呼ばれています。

A correction was made to <a href="http://www.jove.com/index/details.stp?id=1747">Performing and Processing FNA of Anterior Fat Pad for Amyloid</a>. There was an error with an author's name. The author's last name had a typo and was corrected to:
Safwan S. Jaradeh
instead of:
Safwan S. Jardeh

Erratum: Performing and Processing FNA of Anterior Fat Pad for Amyloid

アルコール綿棒、ガーゼパッド、マーキングペン、10 mLの注射器× 2、1％リドカイン（局所麻酔薬、場合リドカインを単独で使用され、それは点眼後、最初の灼熱感を引き起こします。この1％の1:1混合物を使用することによって防ぐことができるリドカインと1％ナトリウム - ビ - 炭酸塩）、18ゲージ（G）1 ½インチの針× 2（FNABを実行するための）、25G 1 ½インチ針（局所麻​​酔薬を注入するため）、FNAのシリンジホルダー（&quot;ガン&quot;）、包帯、グルタルアルデヒドのバイアル（電子顕微鏡用）、および10％ホルマリンの容器を生検。

performing and processing fna of anterior fat pad for amyloid

歴史的に、心臓、肝臓、および腎臓の生検はアミロイドーシスにおけるアミロイド沈着を証明するために行われた。この疾患の臨床症状は非常に可変と非特異的であるため、これらの生検のリスクは診断率のために余りに大きいです。このような皮膚や歯肉などの低生検のリスクを持っている他のサイトでは、、また、比較的侵襲的で高価です。さらに、これらの生検は常に診断を確立するために十分なアミロイド沈着を持っていない可能性があります。脂肪パッドの吸引は、低コストと最小限の罹患率との良好な臨床的相関関係を実証してきました。しかし、この手順を実行したり、変数やnonreproducible結果につながる吸引標本を、処理するための標準プロトコルはありません。組織でアミロイドを検出するための最も頻繁に利用さモダリティは、偏光顕微鏡を使用してコンゴレッド染色した切片上に青リンゴ色の複屈折である。この手法は、吸引材料のセルブロックの準備が必要です。残念なことに、アミロイドーシスの初期段階で受診した患者は大幅に脂肪パッドの吸引のコンゴレッド染色細胞のブロックのセクションの感度を減少させるアミロイドの最小限の量を持っている。そのため、電子顕微鏡による脂肪パッドの吸引の超微細構造の評価は、アミロイドの検出のために感度の向上を考えると、利用されるべきである。この記事では、コンゴレッドのセルブロックのセクションの染色および超微細構造識別の​​ための電子顕微鏡の両方を利用してアミロイドの検出のための前脂肪パッドの吸引を行うためのシンプルで再現性の手順を示しています。

Watch this Scientific Journal Video about Performing and Processing FNA of Anterior Fat Pad for Amyloid at JoVE.com

Performing and Processing FNA of Anterior Fat Pad for Amyloid

脂肪パッドの吸引は、全身性アミロイドーシスの診断にアミロイドを検出するためには、他の方法に比べて優先、低侵襲、低コストのアプローチです。このビデオの記事では、最適な診断結果のための検体の適切な処理によって脂肪パッドの吸引を行うための手続きの概要を示しています。

アミロイドのための前方脂肪パッドのFNAの実行と処理

Historically, heart, liver, and kidney biopsies were performed to demonstrate amyloid deposits in amyloidosis.  Since the clinical presentation of this ...

performing-and-processing-fna-of-anterior-fat-pad-for-amyloid

Research

JoVE Journal

Medicine

54.8K ビュー.  Medical College of Wisconsin. 脂肪パッドの吸引は、全身性アミロイドーシスの診断にアミロイドを検出するためには、他の方法に比べて優先、低侵襲、低コストのアプローチです。このビデオの記事では、最適な診断結果のための検体の適切な処理によって脂肪パッドの吸引を行うための手続きの概要を示しています。

ビデオ: Performing and Processing FNA of Anterior Fat Pad for Amyloid

Purification and Aggregation of the Amyloid Precursor Protein Intracellular Domain

Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer's disease (AD). APP is characterized by a large extracellular domain and a short cytosolic domain termed the APP intracellular domain (AICD). During maturation through the secretory pathway, APP can be cleaved by proteases termed &alpha;, &beta;, and &gamma;-secretases1. Sequential proteolytic cleavage of APP with &beta; and &gamma;-secretases leads to the production of a small proteolytic peptide, termed A&beta;, which is amyloidogenic and the core constituent of senile plaques. The AICD is also liberated from the membrane after secretase processing, and through interactions with Fe65 and Tip60, can translocate to the nucleus to participate in transcription regulation of multiple target genes2,3. Protein-protein interactions involving the AICD may affect trafficking, processing, and cellular functions of holo-APP and its C-terminal fragments. We have recently shown that AICD can aggregate in vitro, and this process is inhibited by the AD-implicated molecular chaperone ubiquilin-14. Consistent with these findings, the AICD has exposed hydrophobic domains and is intrinsically disordered in vitro5,6, however it obtains stable secondary structure when bound to Fe657. We have proposed that ubiquilin-1 prevents inappropriate inter- and intramolecular interactions of AICD, preventing aggregation in vitro and in intact cells4. While most studies focus on the role of APP in the pathogenesis of AD, the role of AICD in this process is not clear. Expression of AICD has been shown to induce apoptosis8, to modulate signaling pathways9, and to regulate calcium signaling10. Over-expression of AICD and Fe65 in a transgenic mouse model induces Alzheimer's like pathology11, and recently AICD has been detected in brain lysates by western blotting when using appropriate antigen retrieval techniques12. To facilitate structural, biochemical, and biophysical studies of the AICD, we have developed a procedure to produce recombinantly large amounts of highly pure AICD protein. We further describe a method for inducing the in vitro thermal aggregation of AICD and analysis by atomic force microscopy. The methods described are useful for biochemical, biophysical, and structural characterization of the AICD and the effects of molecular chaperones on AICD aggregation.

A method for large-scale purification of the APP intracellular domain (AICD) is described. We also describe methodology to induce in vitro AICD aggregation and visualization by atomic force microscopy. The methods described are useful for biochemical/structural characterization of the AICD and the effects of molecular chaperones on its aggregation.

アミロイド前駆体タンパク質細胞内ドメインの精製と集約

Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer's disease (AD). APP is characterized by a ...

医学

Processing of Human Reduction Mammoplasty and Mastectomy Tissues for Cell Culture

Experimental examination of normal human mammary epithelial cell (HMEC) behavior, and how normal cells acquire abnormal properties, can be facilitated by in vitro culture systems that more accurately model in vivo biology. The use of human derived material for studying cellular differentiation, aging, senescence, and immortalization is particularly advantageous given the many significant molecular differences in these properties between human and commonly utilized rodent cells1-2. Mammary cells present a convenient model system because large quantities of normal and abnormal tissues are available due to the frequency of reduction mammoplasty and mastectomy surgeries.
The mammary gland consists of a complex admixture of many distinct cell types, e.g., epithelial, adipose, mesenchymal, endothelial. The epithelial cells are responsible for the differentiated mammary function of lactation, and are also the origin of the vast majority of human breast cancers. We have developed methods to process mammary gland surgical discard tissues into pure epithelial components as well as mesenchymal cells3. The processed material can be stored frozen indefinitely, or initiated into primary culture. Surgical discard material is transported to the laboratory and manually dissected to enrich for epithelial containing tissue. Subsequent digestion of the dissected tissue using collagenase and hyaluronidase strips stromal material from the epithelia at the basement membrane. The resulting small pieces of the epithelial tree (organoids) can be separated from the digested stroma by sequential filtration on membranes of fixed pore size. Depending upon pore size, fractions can be obtained consisting of larger ductal/alveolar pieces, smaller alveolar clusters, or stromal cells. We have observed superior growth when cultures are initiated as organoids rather than as dissociated single cells. Placement of organoids in culture using low-stress inducing media supports long-term growth of normal HMEC with markers of multiple lineage types (myoepithelial, luminal, progenitor)4-5. Sufficient numbers of cells can be obtained from one individual's tissue to allow extensive experimental examination using standardized cell batches, as well as interrogation using high throughput modalities.
Cultured HMEC have been employed in a wide variety of studies examining the normal processes governing growth, differentiation, aging, and senescence, and how these normal processes are altered during immortal and malignant transformation4-15,16. The effects of growth in the presence of extracellular matrix material, other cell types, and/or 3D culture can be compared with growth on plastic5,15. Cultured HMEC, starting with normal cells, provide an experimentally tractable system to examine factors that may propel or prevent human aging and carcinogenesis.

A method to process human mammary surgical discard material is described. Processed tissue, in the form of organoids, can be stored frozen indefinitely or placed in culture for long-term growth. This method enables experimental examination of normal human epithelial cell biology, and the effects of exogenous perturbations.

細胞培養のための人間の削減乳房形成や乳房切除組織の処理

Experimental  examination of normal human mammary epithelial cell (HMEC) behavior, and how  normal cells acquire abnormal properties, can be facilitated ...

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the functions of proteins involved in oncogenic progression or help to discover new tumor suppressors. In addition, injecting cancer cells into mice with different genotypes might provide a better understanding of the importance of the stromal compartment. Many models may be useful to investigate certain aspects of disease progression but do not recapitulate the entire cancerous process. In contrast, breast cancer cells engraftment to the mammary fat pad of mice better recapitulates the location of the disease and presence of the proper stromal compartment and therefore better mimics human cancerous disease. In this article, we describe how to implant breast cancer cells into mice orthotopically and explain how to collect tissues to analyse the tumor milieu and metastasis to distant organs. Using this model, many aspects (growth, angiogenesis, and metastasis) of cancer can be investigated simply by providing a proper environment for tumor cells to grow.

Cancer is a complex disease that is influenced by the tissue surrounding the tumor as well as local pro- and anti-inflammatory mediators. Therefore, orthotropic injection models, rather than subcutaneous models may be useful to study cancer progression in a manner that better mimics human pathology.

腫瘍増殖を研究するためにマウスの乳房脂肪パッド中に乳癌細胞の同所注入。

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

Assessment of Viability of Human Fat Injection into Nude Mice with Micro-Computed Tomography

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft tissue filler as it is readily available, easily obtained, inexpensive, and inherently biocompatible.1 However, despite its burgeoning popularity, fat grafting is hampered by unpredictable results and variable graft survival, with published retention rates ranging anywhere from 10-80%. 1-3
To facilitate investigations on fat grafting, we have therefore developed an animal model that allows for real-time analysis of injected fat volume retention. Briefly, a small cut is made in the scalp of a CD-1 nude mouse and 200-400 &#181;l&#160;of processed lipoaspirate is placed over the skull. The scalp is chosen as the recipient site because of its absence of native subcutaneous fat, and because of the excellent background contrast provided by the calvarium, which aids in the analysis process. Micro-computed tomography (micro-CT) is used to scan the graft at baseline and every two weeks thereafter. The CT images are reconstructed, and an imaging software is used to quantify graft volumes.
Traditionally, techniques to assess fat graft volume have necessitated euthanizing the study animal to provide just a single assessment of graft weight and volume by physical measurement ex vivo. Biochemical and histological comparisons have likewise required the study animal to be euthanized. This described imaging technique offers the advantage of visualizing and objectively quantifying volume at multiple time points after initial grafting without having to sacrifice the study animal. The technique is limited by the size of the graft able to be injected as larger grafts risk skin and fat necrosis. This method has utility for all studies evaluating fat graft viability and volume retention. It is particularly well-suited to providing a visual representation of fat grafts and following changes in volume over time.

Fat grafting is an essential technique for reconstructing soft tissue deficits. However, it remains an unpredictable procedure characterized by variable graft survival. Our goal was to devise a mouse model that utilizes a novel imaging method to compare volume retention between differing techniques of fat graft preparation and delivery.

マイクロコンピュータ断層撮影とヌードマウスへのヒト脂肪注入の生存率の評価

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft ...

Transplantation Into the Mouse Ovarian Fat Pad

Orthotopic transplantation assays in mice are invaluable for studies of cell regeneration and neoplastic transformation. Common approaches for orthotopic transplantation of ovarian surface and tubal epithelia include intraperitoneal and intrabursal administration of cells. The respective limitations of these methods include poorly defined location of injected cells and limited space volume. Furthermore, they are poorly suited for long-term structural preservation of transplanted organs. To address these challenges, we have developed an alternative approach, which is based on the introduction of cells and tissue fragments into the mouse fat pad. The mouse ovarian fat pad is located in the immediate vicinity of the ovary and uterine tube (aka oviduct, fallopian tube), and provides a familiar microenvironment for cells and tissues of these organs. In our approach fluorescence-labeled mouse and human cells, and fragments of the uterine tube are engrafted by using minimally traumatic dorsal incision surgery. Transplanted cells and their outgrowths are easily located in the ovarian fat pad for over 40 days. Long-term transplantation of the entire uterine tube allows correct preservation of all principle tissue components, and does not result in adverse side effects, such as fibrosis and inflammation. Our approach should be uniquely applicable for answering important biological questions such as differentiation, regenerative and neoplastic potential of specific cell populations. Furthermore, it should be suitable for studies of microenvironmental factors in normal development and cancer.

We describe an ovarian fad pad transplantation assay suitable for studies of normal and transformed epithelia of the female reproductive tract. The mouse fat pad allows transplantation of large tissue fragments, is easily accessible for surgery and imaging, and provides the most favorable native environment for tissues of M&#252;llerian origin.

マウスの卵巣脂肪パッドに移植

Orthotopic transplantation assays in mice are invaluable for studies of cell regeneration and neoplastic transformation. Common approaches for orthotopic ...

Confirmation of Myocardial Ischemia and Reperfusion Injury in Mice Using Surface Pad Electrocardiography

Many animal models have been established for the study of myocardial remodeling and heart failure due to its status as the number one cause of mortality worldwide. In humans, a pathologic occlusion forms in a coronary artery and reperfusion of that occluded artery is considered essential to maintain viability of the myocardium at risk. Although essential for myocardial recovery, reperfusion of the ischemic myocardium creates its own tissue injury. The physiologic response and healing of an ischemia/reperfusion injury is different from a chronic occlusion injury. Myocardial ischemia/reperfusion injury is gaining recognition as a clinically relevant model for myocardial infarction studies. For this reason, parallel animal models of ischemia/reperfusion are vital in advancing the knowledge base regarding myocardial injury. Typically, ischemia of the mouse heart after left anterior descending (LAD) coronary artery occlusion is confirmed by visible pallor of the myocardium below the occlusion (ligature). However, this offers only a subjective way of confirming correct or consistent ligature placement, as there are multiple major arteries that could cause pallor in different myocardial regions. A method of recording electrocardiographic changes to assess correct ligature placement and resultant ischemia as well as reperfusion, to supplement observed myocardial pallor, would help yield consistent infarct sizes in mouse models. In turn, this would help decrease the number of mice used. Additionally, electrocardiographic changes can continue to be recorded non-invasively in a time-dependent fashion after the surgery. This article will demonstrate a method of electrocardiographically confirming myocardial ischemia and reperfusion in real time.

During murine myocardial ischemia/reperfusion surgery, correct placement of the occluding ligature is typically confirmed by visible observation of myocardial pallor. Herein, a method of electrocardiographically confirming ischemia and reperfusion, to supplement observed myocardial pallor, is demonstrated in male C57Bl/6 mice.

表面パッド心電図を使用したマウスにおける心筋虚血および再灌流障害の確認

Many animal models have been established for the study of myocardial remodeling and heart failure due to its status as the number one cause of mortality ...

Stromal Vascular Fraction-enriched Fat Grafting for the Treatment of Symptomatic End-neuromata

The purpose of this study was to methodically illustrate and highlight the crucial steps of stromal vascular fraction (SVF)-enriched fat grafting as a novel treatment of symptomatic end-neuromata of peripheral sensory nerves, and in this study, specifically of the superficial branch of the radial nerve (SBRN). Despite a multitude of existing treatments, persistent postoperative pain and common pain relapse are still very common, independent of the procedure assessed. The neuroma is microsurgically excised accordingly to standardized protocol. Instead of the relocation of the regenerating nerve stump in neighboring anatomical structures, such as muscle or bone, a fat graft is applied perifocally and acts as a mechanical barrier. In order to reduce the fat resorption rate and boost the regenerative potential of the graft, the highly concentrated SVF is integrated in the grafting. The SVF is isolated from subcutaneous fat by enzymatic and mechanic separation of the lipoaspirate by a specific commercial isolation system. The SVF-enriched fat graft provides both a mechanical barrier and various biological effects at the cellular level, including improving angiogenesis, inflammation, and fibrosis. Both mechanical and biologic effects help to reduce the disorganized axonal outgrowth of the nerve stump during nerve regeneration and hence prevent the recurrence of painful end-neuromata.

The purpose of the study is to illustrate the technical procedure of stromal vascular fraction (SVF)-enriched fat grafting as a novel treatment of symptomatic end-neuromata. This technique provides advantages of both the mechanical barrier and the biological action at the cellular level by the processed and concentrated SVF.

間質血管分数濃縮脂肪移植症候性の終わり neuromata の治療のため

The purpose of this study was to methodically illustrate and highlight the crucial steps of stromal vascular fraction (SVF)-enriched fat grafting as a ...

Methodology for Sputum Induction and Laboratory Processing

The technique of sputum induction and processing is a recognized non-invasive method allowing the collection and analysis of cells from the airways, which is interesting in various respiratory diseases like asthma, chronic obstructive pulmonary disease (COPD), chronic cough, or idiopathic pulmonary fibrosis. This technique is well tolerated, safe and non-invasive, but is currently limited to research services and specialized centers in clinical practice because it is technically demanding, time-consuming, and requires trained staff. The success rate of sputum induction and analysis is about 80%.
Here, we describe the induction and laboratory processing of sputum samples. Sputum is induced by inhalation of hypertonic or isotonic saline with salbutamol. For the processing, we use the whole sputum technique. Dithiothreitol (DTT) is used to allow mucolysis of sputum samples. The primary aim of sputum processing is to obtain a differential cell count to study the cell types present in the airway lumen. Additional analyses may also be performed on sputum supernatant and sputum cells, which may allow further investigation into inflammatory processes and immune mechanisms. Examples include studying mediators in sputum supernatant and performing a large spectrum of analysis on sputum cells such as flow cytometry, genomics, or proteomics.
Finally, representative results of sputum analysis in healthy controls, asthmatics, and COPD patients are presented.

Here we describe the technique of sputum induction. This protocol also explains the sputum processing to perform a differential cell count and to collect sputum supernatant and cells for further analysis.

喀痰誘発および実験室の処理のための方法論

The technique of sputum induction and processing is a recognized non-invasive method allowing the collection and analysis of cells from the airways, which ...

Sampling Strategies and Processing of Biobank Tissue Samples from Porcine Biomedical Models

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of genetically modified pig models, and the often-limited number of available animals of these models, establishment of (biobank) collections of adequately processed tissue samples suited for a broad spectrum of subsequent analyses methods, including analyses not specified at the time point of sampling, represent meaningful approaches to take full advantage of the translational value of the model. With respect to the peculiarities of porcine anatomy, comprehensive guidelines have recently been established for standardized generation of representative, high-quality samples from different porcine organs and tissues. These guidelines are essential prerequisites for the reproducibility of results and their comparability between different studies and investigators. The recording of basic data, such as organ weights and volumes, the determination of the sampling locations and of the numbers of tissue samples to be generated, as well as their orientation, size, processing and trimming directions, are relevant factors determining the generalizability and usability of the specimen for molecular, qualitative, and quantitative morphological analyses. Here, an illustrative, practical, step-by-step demonstration of the most important techniques for generation of representative, multi-purpose biobank specimen from porcine tissues is presented. The methods described here include determination of organ/tissue volumes and densities, the application of a volume-weighted systematic random sampling procedure for parenchymal organs by point-counting, determination of the extent of tissue shrinkage related to histological embedding of samples, and generation of randomly oriented samples for quantitative stereological analyses, such as isotropic uniform random (IUR) sections generated by the &#34;Orientator&#34; and &#34;Isector&#34; methods, and vertical uniform random (VUR) sections.

The practical application and performance of methods for generation of representative tissue samples of porcine animal models for a broad spectrum of downstream analyses in biobank projects are demonstrated, including volumetry, systematic random sampling, and differential processing of tissue samples for qualitative and quantitative morphologic and molecular analyses types.

サンプリング戦略とブタの生物医学的モデルからバイオバンク組織サンプルの処理

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of ...

A Training and Testing System for Performing Vascular Reconstruction In Vitro

Manual vascular reconstruction training is essential for a beginner surgeon. However, an optimal training system for vascular reconstruction in vitro has yet to be developed. In this study, we introduce an in vitro training and testing system using a magnetic anchoring technique with which a trainee can practice manual vascular reconstruction individually. Additionally, this system can also be used to test the quality of the reconstruction. The described system includes a vascular reconstruction training machine, magnetic tractors, and a magnetic suture puller. In this manuscript, we detail an end-to-end vein anastomosis using porcine right and left iliac veins. To identify the potential damage caused by a magnetic suture puller on the suture, we created three groups with six segments of 4-0 polypropylene sutures each: a control group with no intervention on the polypropylene suture, a group in which the polypropylene suture is manually pulled with sterile gloves 20x, and a magnetic puller group in which the magnetic puller pulled the polypropylene suture 20x. These groups were tested by light microscopy and breaking strength tests, and the effect of reconstruction was assessed. In the light microscopy test, the control group was less likely to be damaged (p &#60; 0.05) and the number of damaged points of the manual group and magnetic puller group were similar (p &#62; 0.05). The results of the breaking strength test were compared across groups and no significant difference was observed (p &#62; 0.05). The end-to-end anastomosis of the porcine iliac veins was successfully performed using this training system, and the reconstructed veins could undergo 2.0 kPa perfusion pressure. Using this training and testing system the trainee can practice manual vascular reconstruction in vitro individually with the aid of magnetic tractors and a magnetic suture puller, and the quality of the reconstruction can be tested.

Here we present a training and testing system where a trainee can complete manual vascular reconstruction in vitro individually using a magnetic anchoring technique. The system can also be used to test the quality of reconstruction.

インビトロにおける血管再建を行うためのトレーニングと検査システム

Manual vascular reconstruction training is essential for a beginner surgeon. However, an optimal training system for vascular reconstruction in vitro has ...