The authors would like to thank the Lam Lab at Georgia Tech for many helpful discussions in developing the experimental assays. Research reported in this publication was supported by the National Heart, Lung, and Blood Institute of the National Institutes of Health under Award Number K01HL115486 and by New Innovator Grant 1DP2OD007433-01 from the Office of the Director, National Institutes of Health. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

注：以下のプロトコルは、ジョージア工科大学の治験審査委員会（IRB）によって設定されたガイドラインに準拠しています。 1.採血およ​​び赤血球単離法<ol><li> 10ミリリットルのヘパリン化バキュテナーチューブ中にドナーからの血液の40〜120ミリリットルを収集します。コレクションの4時間以内にPBMC（末梢血単核細胞）の分離を開始します。この時間中、RTで血液を保つ。 注：これは下PBMCの収量になりますようにRTまたは4℃における血液のO / Nストレージが推奨されていません 。 </li><li>氷冷（4℃）を滅菌PBSで1：全血の1希釈する。 </li><li>ピペット空、無菌の50mlの円錐チューブに氷のように冷たい親水性多糖類の10ミリリットル。静かに親水性多糖類の上に希釈した全血を移す。 </li><li>非常にゆっくりとチューブの側面に沿ってゆっくり設定やピペット血液中の25ミリリットルピペットを使用してください。血液が前にセントリに糖類と混合されていないことを確認してくださいfugation親水性多糖類は、細胞に対して毒性とそうでない場合であるため、PBMCの収率が低くなります。 </li><li> 4℃で400×gで30分間遠心血液サンプル。可能な場合は、遠心分離後の良好な分離のための最大値の半分に遠心分離機のブレーキ速度を低下させる。 </li><li>遠心分離した血液サンプルの血漿/血小板分画から吸引する。慎重に直接パックRBC層の上に親水性の多糖類の層を吸引除去する。 </li><li>パックされたRBCの8〜10ミリリットルを収集し、1希釈する：非無菌の0.9％NaCl生理食塩水で1を。 </li></ol> 2.共焦点顕微鏡評価糖化の血塊構造（模擬糖尿病血塊） <ol><li> 37℃でのヒトフィブリノーゲンおよび蛍光標識されたヒトフィブリノゲン結合体を解凍。 </li><li>マイクロ遠心チューブを卒業した0.5ミリリットルに次のようにピペット。 <ol><li>健康、通常のフィブリノゲンの塊については、10％の蛍光標識したヒトフィブリノーゲンヒトフィブリノゲンをミックス5 mg / mlの濃度で50mMのトリス/ 100mMのNaCl緩衝液中のコンジュゲート。 </li><li>人工的に糖化フィブリノゲンは、（糖尿病血栓をシミュレートするため）6.8 mg / mlでの得られた濃度50mMのトリス/ 100mMのNaClに溶解したグルコースの5 mM溶液中のヒトフィブリノーゲンおよび10％の蛍光標識されたフィブリノゲン結合体をインキュベートする。 </li></ol></li><li>光への曝露を避けるために、不透明材料を用いたマイクロ遠心管を覆う。 48時間、37℃の水浴中でチューブをインキュベートする。 </li><li>スライドの2側面に薄い接着剤を貼り付けてガラス顕微鏡スライド上室を作る。 </li><li> 48時間のインキュベーション期間の後、RTで活性化XIII因子およびヒトαトロンビンを解凍することによってフィブリン形成のための試薬を調製する。 </li><li> 5のCaCl 2のバッファに80 U / mlに活性化XIII因子を希釈します。 </li><li> 5のCaCl 2のバッファに4 U / mlにトロンビンを希釈します。 <ol><li>通常の血栓のために、フィブリノゲン、活性化XIII因子、およびトロンビ​​ンの最終濃度は2.5メートルであることを確認G / mlで、20 U / mlで、それぞれ50μlの容量で1単位/ ml、。 </li><li>糖化血栓のために、フィブリノゲン、活性化XIII因子、およびトロンビ​​ンの最終濃度がそれぞれ50μlの容量で、3.4 mg / mlの、20 U / mlとし、1単位/ mlであることを確認してください。 </li></ol></li><li>すぐに接着剤によって形成されたチャンバー内にフィブリン溶液を30μlのピペット。 </li><li>接着剤の2層の上に0.15ミリメートルの厚さの22ミリメートル×22 mmのガラスカバースリップを置きます。乾燥からフィブリン塊を防止するための明確な接着剤で開放側をシール。接着剤はフィブリン塊と相互作用しないことを確認してください。 </li><li>フィブリン塊は、共焦点イメージングの前に2時間21〜23℃で重合することができるようにします。 </li><li> 488nmのアルゴンレーザーを40X / 1.30オイルM27レンズを共焦点顕微鏡を用いて血栓の共焦点顕微鏡画像を取る。 </li></ol>鎌状赤血球患者からRBCを含むフィブリン塊の3共焦点顕微鏡評価<ol><li> 37℃でのヒトフィブリノーゲンおよび蛍光標識されたヒトフィブリノゲン結合体を解凍。 </li><li>マイクロ遠心チューブを卒業した0.5ミリリットルに次のようにピペット。 <ol><li>健康な、正常なフィブリン塊のために、5mg / mlの濃度で50mMのトリス/ 100mMのNaCl緩衝液中の10％の蛍光標識されたヒトフィブリノーゲン複合体とヒトフィブリノゲンを混ぜる。 </li><li> SCD RBCを含む塊、ヒトフィブリノーゲン及び50mMトリス/ 100mMのNaClフィブリノーゲンの得られる濃度が10 mg / mlであるような、10％、蛍光標識されたヒトフィブリノゲン結合体を混合する。 </li></ol></li><li>細胞標識溶液を使用して（通常のとRBC分離プロトコルから取得した鎌状赤血球患者からのもの両方）を単離したRBCにラベルを付けます。 <ol><li>任意の選択された無血清培地1ml当たり1×10 6の密度で細胞を懸濁。 </li><li>細胞標識溶液に細胞懸濁液の5μL/ mlのを追加します。優しく穏やかにピペッティングでよく混ぜる。 </li><li&gt;は37℃で20分間インキュベートする。 </li><li> 37℃で5分間1500 rpmで標識されたサスペンションチューブを遠心する。 </li><li>上清を除去し、静かに37℃の培地中の細胞を再懸濁。 </li><li>洗浄手順（3.3.4および3.3.5）をさらに2回繰り返します。 </li></ol></li><li> 5のCaCl 2のバッファに80 U / mlに活性化XIII因子を希釈します。 </li><li> 5のCaCl 2のバッファに4 U / mlにトロンビンを希釈します。 <ol><li>通常の血栓のために、フィブリノゲン、活性化XIII因子、およびトロンビ​​ンの最終濃度がそれぞれ50μlの容量で、2.5 mg / mlの、20 U / mlとし、1単位/ mlであることを確認してください。 </li><li> SCD RBCを含む塊については、フィブリノーゲン、活性化XIII因子、およびトロンビ​​ンの最終濃度は、50μlの容量でそれぞれ5 mg / mlの、20 U / mlであり、1 U / mlであること。確実</li></ol></li><li>フィブリン溶液へのラベルRBCのピペットを10μl。ピペット顕微鏡ガラス上のRBCとフィブリンの30μL（GLのための準備をスライドを参照してくださいycated血栓）。 </li><li>フィブリンは、共焦点イメージングの前に2時間21〜23℃で重合することができるようにします。 </li><li>共焦点顕微鏡を使用して血餅の共焦点画像を撮影し、蛍光標識したフィブリンおよび赤血球を励起して488nmと633nmの波長の励起レーザを利用する。 </li></ol>糖化クロット構造における線維素溶解料金の4共焦点顕微鏡評価<ol><li>糖化血栓における線維素溶解率評価のための糖化血栓（プロトコルステップ2）の共焦点顕微鏡プロトコルを繰り返します。 </li><li>明確な接着剤を用いて、チャンバの両端をシールしないでください。フィブリン塊は、脱水を防ぐために、250mlの水を含む密閉筐体に21〜23℃で1.5時間重合することができるようにします。 </li><li>重合後、共焦点顕微鏡を用いて血栓の画像を得る。 </li><li>チャンバーの開放端にピペットプラスミンおよび毛管作用を介して、血餅を介してプラスミンを分散させる。 <ol><li>のために通常の濃度は、ピペット室の​​開口部にプラスミン200μgの/ mlの25μlを、正常と糖化線維素溶解実験。 </li><li>減少した濃度、ピペット室の​​開口部には50μg/ mlで25μlの糖化血栓のために。 </li></ol></li><li>時系列機能を使用する血餅を溶解するプラスミンのリアルタイムのビデオ映像をキャプチャするために、共焦点顕微鏡ソフトウェア（ 図1参照）。 <ol><li>取得モードをクリックし、選択して &quot;時系列&quot;をサイクル数と記録時間間隔を選択します。 </li></ol></li></ol> 5.フィブリン溶解料金の計算<ol><li>面積（2500ミクロン2）を設定し、血餅の固定領域を溶解するのに必要な経過時間を計算することにより、溶解速度を決定する。以下の式（ 図2）を使用して、溶解率を計算する。 <img alt="式1" fo:content-width="2in" srC = &quot;/ファイル/ ftp_upload / 52019 / 52019eq1.jpg&quot; /&gt; </li></ol>フィブリン溶解料金の6.統計解析<ol><li>統計分析ソフトウェアを使用して溶解データを分析する。一方向ANOVAおよび事後テューキー·クレーマー検定44,45を実行します。 </li></ol>

<ol>
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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrinogen+and+fibrin.">Fibrinogen and fibrin.</a> Advances in protein chemistry. 70, 247-299 (2005).</li><li>Collet, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+Fibrin+Network+Conformation+and+Fibrin+Fiber+Diameter+on+Fibrinolysis+Speed+Dynamic+and+Structural+Approaches+by+Confocal+Microscopy.">Influence of Fibrin Network Conformation and Fibrin Fiber Diameter on Fibrinolysis Speed Dynamic and Structural Approaches by Confocal Microscopy.</a> Arteriosclerosis, thrombosis, and vascular biology. 20, 1354-1361 (2000).</li><li>J&#246;rneskog, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+properties+of+the+fibrin+gel+structure+in+patients+with+IDDM.">Altered properties of the fibrin gel structure in patients with IDDM.</a> Diabetologia. 39, 1519-1523 (1996).</li><li>Svensson, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acetylation+and+glycation+of+fibrinogen+in+vitro+occur+at+specific+lysine+residues+in+a+concentration+dependent+manner:+A+mass+spectrometric+and+isotope+labeling+study.">Acetylation and glycation of fibrinogen in vitro occur at specific lysine residues in a concentration dependent manner: A mass spectrometric and isotope labeling study.</a> Biochemical and Biophysical Research Communications. 421, 335-342 (2012).</li><li>Pieters, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycation+of+fibrinogen+in+uncontrolled+diabetic+patients+and+the+effects+of+glycaemic+control+on+fibrinogen+glycation.">Glycation of fibrinogen in uncontrolled diabetic patients and the effects of glycaemic control on fibrinogen glycation.</a> Thrombosis Research. 120, 439-446 (2007).</li><li>Baradet, T. C., Haselgrove, J. C., Weisel, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+reconstruction+of+fibrin+clot+networks+from+stereoscopic+intermediate+voltage+electron+microscope+images+and+analysis+of+branching.">Three-dimensional reconstruction of fibrin clot networks from stereoscopic intermediate voltage electron microscope images and analysis of branching.</a> Biophysical journal. 68, 1551-1560 (1995).</li><li>Campbell, R. A., Overmyer, K. A., Selzman, C. H., Sheridan, B. C., Wolberg, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contributions+of+extravascular+and+intravascular+cells+to+fibrin+network+formation,+structure,+and+stability.">Contributions of extravascular and intravascular cells to fibrin network formation, structure, and stability.</a> Blood. 114, 4886-4896 (2009).</li><li>Curtis, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+study+of+microstructural+templating+in+fibrin-thrombin+gel+networks+by+spectral+and+viscoelastic+analysis.">A study of microstructural templating in fibrin-thrombin gel networks by spectral and viscoelastic analysis.</a> Soft Matter. 9, 4883-4889 (2013).</li><li>Kim, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+between+fibrin+network+structure+and+mechanical+properties:+an+experimental+and+computational+analysis.">Correlation between fibrin network structure and mechanical properties: an experimental and computational analysis.</a> Soft Matter. 7, 4983-4992 (2011).</li><li>Keegan, P. M., Surapaneni, S., Platt, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sickle+cell+disease+activates+peripheral+blood+mononuclear+cells+to+induce+cathepsins+k+and+v+activity+in+endothelial+cells.">Sickle cell disease activates peripheral blood mononuclear cells to induce cathepsins k and v activity in endothelial cells.</a> Anemia. 2012, 201781 (2012).</li><li>Allison, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+sickle-cell+haemoglobin.">Properties of sickle-cell haemoglobin.</a> Biochemical Journal. 65, 212 (1957).</li><li>Kuehl, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Design of experiments : statistical principles of research design and analysis. , (2000).</li><li>Lindman, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Analysis of variance in experimental designs. , (1992).</li><li>Pan, W., Galkin, O., Filobelo, L., Nagel, R. L., Vekilov, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metastable+Mesoscopic+Clusters+in+Solutions+of+Sickle-Cell+Hemoglobin.">Metastable Mesoscopic Clusters in Solutions of Sickle-Cell Hemoglobin.</a> Biophysical Journal. 92, 267-277 (2007).</li><li>Wootton, D. M., Popel, A. S., Alevriadou, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+experimental+and+theoretical+study+on+the+dissolution+of+mural+fibrin+clots+by+tissue-type+plasminogen+activator.">An experimental and theoretical study on the dissolution of mural fibrin clots by tissue-type plasminogen activator.</a> Biotechnology and bioengineering. 77, 405-419 (2002).</li><li>Sazonova, I. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=127+Mathematic+model+of+fibrin+clot+lysis+by+plasmin.">127 Mathematic model of fibrin clot lysis by plasmin.</a> Fibrinolysis and Proteolysis. 12, 45 (1998).</li></ol>

The authors have nothing to disclose.

疾患状態における凝固機構の構造についての意味のあるデータを得るためには、これらの条件下でのタンパク質および細胞の効果を決定するために、凝固に関与する因子を単離することが重要である。このプロトコルは、in vitroで 、糖尿病およびSCDの状態でフィブリン塊の構造を調査する目的で開発された。 改変された条件は、凝固亢進、アテローム血栓症、お?...

血管の内皮内層に損傷は止血応答、または血栓の形成を通じて修復される。血液は、細胞外マトリックスに浸透すると、組織因子は、凝固カスケードの開始を容易に血流中の血小板を活性化する。この治癒過程の重要な機械的な構成要素は、高弾性であるフィブリン繊維からなるフィブリンマトリックスであり、かつ大きな力1-4を維持することができる。多くの研究が盛んに過去数十5-13フィブリンの形成構造、および機能を研究している。 例えば、糖尿病および鎌状赤血球のような疾患を有する患者は、心筋梗塞、虚血性心疾患、および脳卒中14-19として血栓性合併症を発症するリスクを有する。 200万人以上の人が新たに米国で毎年糖尿病と診断されている。タイプI：糖尿病には2つの種類があります本体は、体がインスリン抵抗性となり、インスリン、およびタイプII、十分な量を生産することができない。糖尿病患者では、心血管疾患（CVD）は、疾患20,21に関連する罹患率および死亡率の80％の原因である。 鎌状赤血球症（SCD）は、米国22で10万人以上の人々に影響を与える遺伝的血液疾患です。 SCDは難しい細胞が血管系23を通過できるようにすること、三日月形になるように、赤血球を引き起こす点突然変異疾患である。これらの疾患状態の両方は、体内のア​​テローム血栓性の状態を発症する可能性を高める。この理由の一つは、疾患状態14,24-26における変化したフィブリン構造と機能の結果である。 糖尿病や鎌状赤血球症の両方において、アテローム血栓症および心血管疾患（Cを誘導し、凝固亢進とhypofibrinolysis活動がありますVD）健康な患者17,27,28と比較した。これはhypofibrinolysisは、アテローム性動脈硬化症の進行を促進し、早期冠動脈疾患を有する患者について29再発虚血イベントを拘禁することが知られている。現在の原稿では、この特定の設定で、フィブリンの物理的特性の役割を調べた。非疾患患者におけるフィブリン血餅の構造は、薄い繊維、より大きな孔、及び一般に低密度の14,24で構成されている。健康な患者における気孔率の増加及び低密度のフィブリン塊は、線溶16を容易にすることが見出されている。糖尿病及び鎌状赤血球症などhyperthrombotic条件で、健康な患者30-33に2.5 mg / mlの正常レベルから増加するフィブリノーゲン濃度を引き起こすフィブリノゲン産生の増加がある。健康な、非直径と比較して、糖尿病患者に形成されたフィブリン塊は、以上の分岐点を有する、より高密度、より剛性の、多孔性の低いことが見出されているbetic患者14,24,33-35。改変されたフィブリン構造が血栓形成に関与するタンパク質中に存在する糖化メカニズムの結果である。グルコース分子が適切に架橋グルタミンおよびリジン残基33,36,37からのヒト第XIIIa因子（活性化XIII因子）を阻害フィブリノゲン分子、上のリジン残基に結合したときに非酵素（不可逆）糖化が起こる。 フィブリンネットワークの構造解析は、最近広く研究されている。特に、研究者は、電子顕微鏡および血管（内皮）細胞および血管外（線維芽細胞および平滑筋）双方の細胞がフィブリン構造40を分析するフィブリン構造39を利用粘弾性スペクトル解析にどのように影響するかを調べたフィブリンネットワーク38の3D再構成などを利用している実験を使用してフィブリン構造と機械的特性との間の相関を開発し、計算は、41に近づく </sup&gt;。現在の研究の焦点は、シミュレートされた糖尿病及び鎌状赤血球症条件下血餅構造を定式化し、疾患状態における血塊の構造および機能の検査のために、共焦点顕微鏡を使用することであった。フィブリン塊は、ヒトフィブリノーゲン、ヒトトロンビン、および活性化XIII因子から形成された。血栓は、プラスミンを用いて溶解した。糖尿病状態をシミュレートするために、フィブリノゲン濃度の増加は、 インビトロでのフィブリノーゲン糖化を誘発するためにグルコース溶液中でインキュベートした。鎌状赤血球症凝固条件をシミュレートするために、増加したフィブリノゲン濃度は、我々のグループ42によって、以前に行われたように患者から採取した鎌状赤血球ヘマトクリットと混合した。これらの方法は、構造および機能病的条件下でのフィブリン塊の形成及びフィブリン溶解に関与するだけでなく、CVDを誘導するメカニズムを調べた。これらの疾患についての現在の情報に基づいて、糖化されたフィブリン塊構造は、少数のAでより高密度であったND小さな孔。鎌状赤血球患者からの赤血球（RBC）とフィブリン塊はまた、より高密度でありRBCおよび凝集フィブリンクラスターの凝集を示した。これは、以前に43決定された十分に確立された現象である。また、線維素溶解率が健全な、正常なフィブリンと比較して減少しプラスミンとない糖化フィブリン塊に有意に低いであろうと仮説を立てた。結果は、糖化フィブリン塊のために、著しく異なる溶解速度の結果だけ減少プラスミン濃度の条件下で観察されたことを示した。細胞およびタンパク質が凝固活性のリアルタイムビデオのキャプチャを可能にするそれらの天然の状態で残っているため、共焦点顕微鏡を使用して、この実験的手法は、他の撮像方法を超える有意な利点を提供する。合成的に誘導凝固のこの方法は、患者のサンプルを取得し、個々のフィルタリングより効率的で安価でより多くの時間であるタンパク質および酵素。サンプル間のばらつきは、プラズマ中の他のタンパク質の結果として存在しなかったように、また、血栓を合成するために分離されたタンパク質および酵素を使用することにより、血栓を標準化した。

<table border="0" cellpadding="0" cellspacing="0" width="887">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Name of Material/ Equipment</td>
			<td style="width:287px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">PBS</td>
			<td style="width:287px;">Life Technologies</td>
			<td style="width:119px;">10010031</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Ficoll-Paque (hydrophilic polysaccharide)</td>
			<td style="width:287px;">GE Healthcare</td>
			<td style="width:119px;">45-001-749</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10 ml heparinized vacutainer tubes</td>
			<td style="width:287px;">BD Biosciences</td>
			<td style="width:119px;">366643</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Human Fibrinogen</td>
			<td style="width:287px;">Enzyme Research Laboratories</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 488 human fibrinogen conjugate</td>
			<td style="width:287px;">Molecular Probes</td>
			<td style="width:119px;">F13191</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.5 mL graduated microcentrifuge tube</td>
			<td style="width:287px;">Fisher Scientific</td>
			<td style="width:119px;">05-408-120</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Glucose powder</td>
			<td style="width:287px;">Life Technologies</td>
			<td style="width:119px;">15023-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">FXIIIa</td>
			<td style="width:287px;">Enzyme Research Laboratories</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">VIS Confocal Microscope</td>
			<td style="width:287px;">Zeiss LSM 510</td>
			<td style="width:119px;">LSM 510</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">50 mM Tris</td>
			<td style="width:287px;">Lonza</td>
			<td style="width:119px;">S50-642</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:315px;">Calcium Chloride (CaCl2)</td>
			<td style="width:287px;">Sigma Aldrich</td>
			<td style="width:119px;">449709-10G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vybrant DiD cell-labeling solution</td>
			<td style="width:287px;">Life Technologies</td>
			<td style="width:119px;">L7781</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Plasmin</td>
			<td style="width:287px;">Enzyme Research Laboratories</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Sodium Chloride (5 M NaCl)</td>
			<td style="width:287px;">Life Technologies</td>
			<td style="width:119px;">AM9759</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:315px;">Statistical Modeling Software</td>
			<td>IBM</td>
			<td style="width:119px;">SPSS Statistics 22</td>
			<td></td>
		</tr>
	</tbody>
</table>

experimental and imaging techniques for examining fibrin clot structures in normal and diseased states

Fibrin is an extracellular matrix protein that is responsible for maintaining the structural integrity of blood clots. Much research has been done on fibrin in the past years to include the investigation of synthesis, structure-function, and lysis of clots. However, there is still much unknown about the morphological and structural features of clots that ensue from patients with disease. In this research study, experimental techniques are presented that allow for the examination of morphological differences of abnormal clot structures due to diseased states such as diabetes and sickle cell anemia. Our study focuses on the preparation and evaluation of fibrin clots in order to assess morphological differences using various experimental assays and confocal microscopy. In addition, a method is also described that allows for continuous, real-time calculation of lysis rates in fibrin clots. The techniques described herein are important for researchers and clinicians seeking to elucidate comorbid thrombotic pathologies such as myocardial infarctions, ischemic heart disease, and strokes in patients with diabetes or sickle cell disease.

糖化フィブリンクロット構造の共焦点顕微鏡分析正常および糖化血栓の共焦点顕微鏡画像を図3に示されている。通常の糖化血栓の共焦点顕微鏡分析は、糖化血餅とし、血餅重合中の活性化XIII因子を添加せずに両方の正常な凝血塊よりも小さい孔を有するより緻密であることが明らかである。 図3Aおよび3Bに、より高いフィブリン濃?...

Watch this Scientific Journal Video about Experimental and Imaging Techniques for Examining Fibrin Clot Structures in Normal and Diseased States at JoVE.com

Experimental and Imaging Techniques for Examining Fibrin Clot Structures in Normal and Diseased States

In this manuscript, experimental techniques, including blood preparation, confocal microscopy, and lysis rate analysis, to examine the morphological differences between normal and abnormal clot structures due to diseased states are presented.

正常および疾患米国でフィブリンクロット構造を調べるための実験とイメージング技術

Fibrin is an extracellular matrix protein that is responsible for maintaining the structural integrity of blood clots. Much research has been done on ...

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11.3K ビュー.  Georgia Institute of Technology & Emory University School of Medicine. In this manuscript, experimental techniques, including blood preparation, confocal microscopy, and lysis rate analysis, to examine the morphological differences between normal and abnormal clot structures due to diseased states are presented.

ビデオ: Experimental and Imaging Techniques for Examining Fibrin Clot Structures in Normal and Diseased States

Treatment of Osteochondral Defects in the Rabbit's Knee Joint by Implantation of Allogeneic Mesenchymal Stem Cells in Fibrin Clots

The treatment of osteochondral articular defects has been challenging physicians for many years. The better understanding of interactions of articular cartilage and subchondral bone in recent years led to increased attention to restoration of the entire osteochondral unit. In comparison to chondral lesions the regeneration of osteochondral defects is much more complex and a far greater surgical and therapeutic challenge. The damaged tissue does not only include the superficial cartilage layer but also the subchondral bone. For deep, osteochondral damage, as it occurs for example with osteochondrosis dissecans, the full thickness of the defect needs to be replaced to restore the joint surface 1. Eligible therapeutic procedures have to consider these two different tissues with their different intrinsic healing potential 2. In the last decades, several surgical treatment options have emerged and have already been clinically established 3-6.
Autologous or allogeneic osteochondral transplants consist of articular cartilage and subchondral bone and allow the replacement of the entire osteochondral unit. The defects are filled with cylindrical osteochondral grafts that aim to provide a congruent hyaline cartilage covered surface 3,7,8. Disadvantages are the limited amount of available grafts, donor site morbidity (for autologous transplants) and the incongruence of the surface; thereby the application of this method is especially limited for large defects.
New approaches in the field of tissue engineering opened up promising possibilities for regenerative osteochondral therapy. The implantation of autologous chondrocytes marked the first cell based biological approach for the treatment of full-thickness cartilage lesions and is now worldwide established with good clinical results even 10 to 20 years after implantation 9,10. However, to date, this technique is not suitable for the treatment of all types of lesions such as deep defects involving the subchondral bone 11.
The sandwich-technique combines bone grafting with current approaches in Tissue Engineering 5,6. This combination seems to be able to overcome the limitations seen in osteochondral grafts alone. After autologous bone grafting to the subchondral defect area, a membrane seeded with autologous chondrocytes is sutured above and facilitates to match the topology of the graft with the injured site. Of course, the previous bone reconstruction needs additional surgical time and often even an additional surgery. Moreover, to date, long-term data is missing 12.
Tissue Engineering without additional bone grafting aims to restore the complex structure and properties of native articular cartilage by chondrogenic and osteogenic potential of the transplanted cells. However, again, it is usually only the cartilage tissue that is more or less regenerated. Additional osteochondral damage needs a specific further treatment. In order to achieve a regeneration of the multilayered structure of osteochondral defects, three-dimensional tissue engineered products seeded with autologous/allogeneic cells might provide a good regeneration capacity 11.
Beside autologous chondrocytes, mesenchymal stem cells (MSC) seem to be an attractive alternative for the development of a full-thickness cartilage tissue. In numerous preclinical in vitro and in vivo studies, mesenchymal stem cells have displayed excellent tissue regeneration potential 13,14. The important advantage of mesenchymal stem cells especially for the treatment of osteochondral defects is that they have the capacity to differentiate in osteocytes as well as chondrocytes. Therefore, they potentially allow a multilayered regeneration of the defect.
In recent years, several scaffolds with osteochondral regenerative potential have therefore been developed and evaluated with promising preliminary results 1,15-18. Furthermore, fibrin glue as a cell carrier became one of the preferred techniques in experimental cartilage repair and has already successfully been used in several animal studies 19-21 and even first human trials 22.
The following protocol will demonstrate an experimental technique for isolating mesenchymal stem cells from a rabbit's bone marrow, for subsequent proliferation in cell culture and for preparing a standardized in vitro-model for fibrin-cell-clots. Finally, a technique for the implantation of pre-established fibrin-cell-clots into artificial osteochondral defects of the rabbit's knee joint will be described.

Treatment of Osteochondral Defects in the Rabbit&#039;s Knee Joint by Implantation of Allogeneic Mesenchymal Stem Cells in Fibrin Clots

An experimental technique for the treatment of osteochondral defects in the rabbit's knee joint is described. The implantation of allogeneic mesenchymal stem cells into osteochondral defects provides a promising development in the field of tissue engineering. The preparation of fibrin-cell-clots in vitro offers a standardized method for implantation.

フィブリン塊における同種間葉系幹細胞の注入によってウサギの膝関節における骨軟骨欠損の治療

The treatment of osteochondral articular defects has been challenging physicians for many years. The better understanding of interactions of articular ...

医学

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

新規高分解能<em生体内で&gt;</em腫瘍増殖と治療に頭蓋内構造物の動的応答を研究する&gt;イメージング技術

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

Implanting Glass Spinal Cord Windows in Adult Mice with Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) in adult rodents is the standard experimental model for studying autonomic demyelinating diseases such as multiple sclerosis. Here we present a low-cost and reproducible glass window implantation protocol that is suitable for intravital microscopy and studying the dynamics of spinal cord cytoarchitecture with subcellular resolution in live adult mice with EAE. Briefly, we surgically expose the vertebrae T12-L2 and construct a chamber around the exposed vertebrae using a combination of cyanoacrylate and dental cement. A laminectomy is performed from T13 to L1, and a thin layer of transparent silicone elastomer is applied to the dorsal surface of the exposed spinal cord. A modified glass cover slip is implanted over the exposed spinal cord taking care that the glass does not directly contact the spinal cord. To reduce the infiltration of inflammatory cells between the window and spinal cord, anti-inflammatory treatment is administered every 2 days (as recommended by ethics committee) for the first 10 days after implantation. EAE is induced only 2-3 weeks after the cessation of anti-inflammatory treatment.
Using this approach we successfully induced EAE in 87% of animals with implanted windows and, using Thy1-CFP-23 mice (blue axons in dorsal spinal cord), quantified axonal loss throughout EAE progression. Taken together, this protocol may be useful for studying the recruitment of various cell populations as well as their interaction dynamics, with subcellular resolution and for extended periods of time. This intravital imaging modality represents a valuable tool for developing therapeutic strategies to treat autoimmune demyelinating diseases such as EAE.

We describe a method for implanting and maintaining glass windows over the exposed spinal cords of adult mice for studying experimental autoimmune encephalomyelitis. These windows provide chronic optical access to the spinal cord for monitoring cell dynamics at the subcellular level in live animals using two-photon microscopy.

Experimental autoimmune encephalomyelitis (EAE) in adult rodents is the standard experimental model for studying autonomic demyelinating diseases such as ...

A Pipeline for 3D Multimodality Image Integration and Computer-assisted Planning in Epilepsy Surgery

Epilepsy surgery is challenging and the use of 3D multimodality image integration (3DMMI) to aid presurgical planning is well-established. Multimodality image integration can be technically demanding, and is underutilised in clinical practice. We have developed a single software platform for image integration, 3D visualization and surgical planning. Here, our pipeline is described in step-by-step fashion, starting with image acquisition, proceeding through image co-registration, manual segmentation, brain and vessel extraction, 3D visualization and manual planning of stereoEEG (SEEG) implantations. With dissemination of the software this pipeline can be reproduced in other centres, allowing other groups to benefit from 3DMMI. We also describe the use of an automated, multi-trajectory planner to generate stereoEEG implantation plans. Preliminary studies suggest this is a rapid, safe and efficacious adjunct for planning SEEG implantations. Finally, a simple solution for the export of plans and models to commercial neuronavigation systems for implementation of plans in the operating theater is described. This software is a valuable tool that can support clinical decision making throughout the epilepsy surgery pathway.

We describe the steps to use our custom designed software for image integration, visualization and planning in epilepsy surgery.

てんかん手術における3Dマルチモダリティ画像の統合とコンピュータ支援計画のためのパイプライン

Epilepsy surgery is challenging and the use of 3D multimodality image integration (3DMMI) to aid presurgical planning is well-established. Multimodality ...

Visualization of Vascular and Parenchymal Regeneration after 70% Partial Hepatectomy in Normal Mice

A modified silicone injection procedure was used for visualization of the hepatic vascular tree. This procedure consisted of in-vivo injection of the silicone compound, via a 26 G catheter, into the portal or hepatic vein. After silicone injection, organs were explanted and prepared for ex-vivo micro-CT (&#181;CT) scanning. The silicone injection procedure is technically challenging. Achieving a successful outcome requires extensive microsurgical experience from the surgeon. One of the challenges of this procedure involves determining the adequate perfusion rate for the silicone compound. The perfusion rate for the silicone compound needs to be defined based on the hemodynamic of the vascular system of interest. Inappropriate perfusion rate can lead to an incomplete perfusion, artificial dilation and rupturing of vascular trees.
The 3D reconstruction of the vascular system was based on CT scans and was achieved using preclinical software such as HepaVision. The quality of the reconstructed vascular tree was directly related to the quality of silicone perfusion. Subsequently computed vascular parameters indicative of vascular growth, such as total vascular volume, were calculated based on the vascular reconstructions. Contrasting the vascular tree with silicone allowed for subsequent histological work-up of the specimen after &#181;CT scanning. The specimen can be subjected to serial sectioning, histological analysis and whole slide scanning, and thereafter to 3D reconstruction of the vascular trees based on histological images. This is the prerequisite for the detection of molecular events and their distribution with respect to the vascular tree. This modified silicone injection procedure can also be used to visualize and reconstruct the vascular systems of other organs. This technique has the potential to be extensively applied to studies concerning vascular anatomy and growth in various animal and disease models.

Tools used for visualizing vascular regeneration require methods for contrasting the vascular trees. This film demonstrated a delicate injection technique used to achieve optimal contrasting of the vascular trees and illustrate the potential benefits resulting from a detailed analysis of the resulting specimen using &#181;CT and histological serial sections.

正常マウスで70％肝切除後の血管と実質再生の可視化

A modified silicone injection procedure was used for visualization of the hepatic vascular tree. This procedure consisted of in-vivo injection of the ...

An Intramedullary Locking Nail for Standardized Fixation of Femur Osteotomies to Analyze Normal and Defective Bone Healing in Mice

Bone healing models are essential to the development of new therapeutic strategies for clinical fracture treatment. Furthermore, mouse models are becoming more commonly used in trauma research. They offer a large number of mutant strains and antibodies for the analysis of the molecular mechanisms behind the highly differentiated process of bone healing. To control the biomechanical environment, standardized and well-characterized osteosynthesis techniques are mandatory in mice. Here, we report on the design and use of an intramedullary nail to stabilize open femur osteotomies in mice. The nail, made of medical-grade stainless steel, provides high axial and rotational stiffness. The implant further allows the creation of defined, constant osteotomy gap sizes from 0.00 mm to 2.00 mm. Intramedullary locking nail stabilization of femur osteotomies with gap sizes of 0.00 mm and 0.25 mm result in adequate bone healing through endochondral and intramembranous ossification. Stabilization of femur osteotomies with a gap size of 2.00 mm results in atrophic non-union. Thus, the intramedullary locking nail can be used in healing and non-healing models. A further advantage of the use of the nail compared to other open bone healing models is the possibility to adequately fix bone substitutes and scaffolds in order to study the process of osseous integration. A disadvantage of the use of the intramedullary nail is the more invasive surgical procedure, inherent to all open procedures compared to closed models. A further disadvantage may be the induction of some damage to the intramedullary cavity, inherent to all intramedullary stabilization techniques compared to extramedullary stabilization procedures.

This protocol describes an osteosynthesis technique using an intramedullary locking nail for standardized fixation of femur osteotomies, which can be used to analyze normal and defective bone healing in mice.

マウスにおける正常および不良骨治癒を分析するために大腿骨骨切り術の標準化された固定用髄内ロッキングネイル

Bone healing models are essential to the development of new therapeutic strategies for clinical fracture treatment. Furthermore, mouse models are becoming ...

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of cerebral malaria can be difficult. We present an experimental mouse model of cerebral malaria that shares multiple features of the human disease, including edema and microvascular pathology. Using magnetic resonance imaging (MRI), we can detect and track the blood-brain barrier disruption, edema development, and subsequent brain swelling. We describe multiple MRI techniques that can visualize these pertinent pathological changes. Thus, we show that MRI represents a valuable tool to visualize and track pathological changes, such as edema, brain swelling, and microvascular pathology, in vivo.

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

We describe a mouse model of experimental cerebral malaria and show how inflammatory and microvascular pathology can be tracked in vivo using magnetic resonance imaging.

磁気共鳴映像法を用いた実験的大脳マラリアモデルにおける浮腫発生と微小血管病変のin vivo追跡

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of ...

Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently labelled structures in large biological specimens, and is down to cellular resolution. Meanwhile, the constant refinement of underlying protocols and the enhanced availability of specialized commercial systems enable us to investigate the microstructure of whole mouse organs and even allow for the characterization of cellular behavior in various live-cell imaging approaches. Here, we describe a protocol for the spatial whole-mount visualization and quantification of the CD45+ leukocyte population in inflamed mouse hearts. The method employs a transgenic mouse strain (CD11c.DTR)that has recently been shown to serve as a robust, inducible model for the study of the development of fulminant fatal myocarditis, characterized by lethal cardiac arrhythmias. This protocol includes myocarditis induction, intravital antibody-mediated cell staining, organ preparation, and LSFM with subsequent computer-assisted image post-processing. Although presented as a highly-adapted method for our particular scientific question, the protocol represents the blueprint of an easily adjustable system that can also target completely different fluorescent structures in other organs and even in other species.

Here, we describe a light-sheet microscopy approach to visualize the cardiac CD45+ leukocyte infiltrate in a murine model of aseptic fulminant myocarditis, which is induced by the intratracheal diphtheria toxin treatment of CD11c.DTR mice.

光シート顕微鏡による劇症心筋炎のマウスモデルにおける白血球浸潤の定量的可視化

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently ...

In Vivo Morphometric Analysis of Human Cranial Nerves Using Magnetic Resonance Imaging in Meni&#232;re's Disease Ears and Normal Hearing Ears

Analysis of neural structures in Meni&#232;re's Disease (MD) is of importance, since a loss of such structures has previously been proposed for this patient group but has yet to be confirmed. This protocol describes a method of in vivo evaluation of neural changes especially well suitable for cranial nerve analysis using magnetic resonance imaging (MRI). MD-patients and normal hearing persons were examined in a 3-T MR-scanner using a scan protocol including strongly T2-weighted 3D gradient-echo-sequence (3D-CISS). In the patient group, MD was additionally confirmed using MRI-based assessment of endolymphatic hydrops. Morphometric analysis was performed using a freeware DICOM viewer. Evaluation of cranial nerves included measurements of cross-sectional areas (CSAs) of the nerves at different levels as well as orthogonal diametric measurements.

&lt;em&gt;In Vivo&lt;/em&gt; Morphometric Analysis of Human Cranial Nerves Using Magnetic Resonance Imaging in Meni&amp;#232;re&#039;s Disease Ears and Normal Hearing Ears

To evaluate morphological changes of cranial nerves such as loss of neural structures or swelling of cranial nerves in Meni&#232;re&#39;s Disease (MD) or in healthy persons in vivo, a protocol of evaluation has been developed using magnetic resonance imaging (MRI). Additional MRI-based confirmation of MD was performed.

生体内でMenière の病気耳と健聴耳の磁気共鳴イメージ投射を使用して人間の脳神経の形態計測的解析

Analysis of neural structures in Meni&#232;re's Disease (MD) is of importance, since a loss of such structures has previously been proposed for this ...

Cardiac Magnetic Resonance for the Evaluation of Suspected Cardiac Thrombus: Conventional and Emerging Techniques

We present the conventional cardiac magnetic resonance (CMR) protocol for evaluating a suspected thrombus and highlight emerging techniques.&#160;The appearance of a mass on certain magnetic resonance (MR) sequences can help differentiate a thrombus from competing diagnoses such as a tumor. T1 and T2 signal characteristics of a thrombus are related to the evolution of hemoglobin properties. A thrombus typically does not enhance following contrast administration, which also helps differentiation from a tumor. We also highlight the emerging role of T1 mapping in the evaluation of a thrombus, which can add another level of support in diagnosis. Prior to any CMR exam, patient screening and interviews are critical to ensure safety and to optimize patient comfort. Effective communication during the exam between the technologist and the patient promotes proper breath holding technique and higher quality images. Volumetric post processing and structured reporting are helpful to ensure that the radiologist answers the ordering services&#39; question and communicates these results effectively. Optimal pre-MR safety evaluation, CMR exam execution, and post exam processing and reporting allow for delivery of high quality radiological service in the evaluation of a suspected cardiac thrombus.

The goal of this article is to describe how cardiac magnetic resonance can be used for the evaluation and diagnosis of a suspected cardiac thrombus. The method presented will describe data acquisition as well as the pre-procedure and post-procedure protocol.

疑わしい心臓血栓の評価のための心臓磁気共鳴:従来技術と新興技術

We present the conventional cardiac magnetic resonance (CMR) protocol for evaluating a suspected thrombus and highlight emerging techniques.&#160;The ...