この作品は、スイスの全米科学財団 (SNSF; #320030_156019) の補助金によって支えられました。著者らは、ハイデルベルク工学 GmBH は、ドイツから金融サポートを受け取った。

すべての手順で BALB/c 雌雄成体 Cx3cr1 のプロモーター gfp を表現するいた使用 24。マウスは、眼科と視覚に関する研究における動物の使用の ARVO 声明に従って扱われ、すべてのプロシージャは、動物福祉に関するスイス連邦政府の規則によるとスイス政府から承認されました。マウス塩酸メデトミジン (0.75 mg/kg) とケタミン (45 の mg/kg の皮下注射で麻酔をかけられました。適切な麻酔は呼吸率および動物の監視によって確認された &#39; 尾ピンチに対して s 反射。実験の最後に、マウスは、CO 2 吸入で安楽死された。
 注: 各撮像セッションを (最大 20 分) 可能な限り迅速に実行、白内障形成以来次の麻酔は網膜の可視化 30 を妨げる可能性があります。
 1 ですシステム構成 <ol><li>コンピューターをオンにします。</li>。
	<li>は、電源をオンに。メッセージ &#34; 開始集録モジュール &#34; デバイスのコントロール パネル画面に表示されます</li>。
	<li>ソフトウェアのソフトウェアを動作するようにデスクトップのショートカットをダブルクリックします</li>。
	<li>データベース ビューをクリックして、&#34; 患者を追加 &#34; アイコン</li>。
	<li>ポップアップ ウィンドウにすべての必要な情報を挿入 (姓、名、タイトル、生年月日、性別、患者 ID)] をクリックして &#34; [ok] を &#34;。ポップアップ ウィンドウに角膜の曲率を 2 mm に設定し、クリックして &#34; ok &#34;.</li>
	<li>標準 30 ° 光の前に 78 D 眼科用非接触スリット標準ランプ レンズを置き、テープで固定して安全にします</li>。
	<li>デバイスの左側にフィルターのレバーは徴候に配置されることを確認してください &#34; A &#34; IR + 10 月と AF ( 図 1) をイメージングできるように</li>。
</ol> 2。マウス作製 リン酸緩衝生理食塩水 (PBS、pH 7.4) 麻酔ソリューションを準備する 45 の mg/kg の <ol><li>使用メデトミジン塩酸塩投与量 0.75 mg/kg と、ケタミンの投与量。したがって、20 g マウス 18 μ L の PBS で 50 μ L の最終巻にケタミンと塩酸メデトミジンの 15 μ L をミックスします。1 週間 4 ° C でソリューションを保存します</li>。
	<li>マウスの襟首をつかみ、瞳孔拡張を達成するためにそれぞれの目のトロピカミド 0.5% + フェニレフリン塩酸塩 2.5% の一滴を適用します</li>。
	<li>は、拘束された、マウスを維持し、麻酔液の 50 μ L を皮下注入インスリン注射器 30 G 針に接続と。加熱パッドの上に配置、そのケージに戻るには、マウスを置きます。マウスは完全に鎮静するまで 3-5 分を待ちます。麻酔の深さを評価するために、マウスの尾をピンチします。角膜反射を確認するには、綿棒を使ってマウス角膜に優しく触れます。正反射が観測されない場合はイメージングに進みます 。
	注: 麻酔中に呼吸数を監視することが重要です。マウスは完全に鎮静がない、痛みを伴う刺激に呼吸数が増加します</li>。
	<li>水和物 2% ヒドロキシプロピルメチル セルロースのアプリケーションでマウス角膜をそれぞれの目にドロップします</li>。
</ol> 3。SD 10 月 <ol><li>デバイスのあごの残り ( 図 1) に接続されているカスタム プラットフォームへおよそ 32 ° C の温暖化のパッチを配置します</li>。
	右の目をイメージする <li>は、プラットフォーム ( 図 1) の左側の部分にマウスを配置します。マウスの右の軌道面レンズ、その体はあるプラットフォームの左部分に発生しやすいことを確認します</li>。
	<li>で右目ヒドロキシプロピルメチル セルロースのドロップを適用し、それを +4 ディオプター厳格なガス透過性コンタクト レンズを慎重に移動 (球状の力: +25.00 ジオプトリーに-25.00)。バランスの取れた塩溶液 (BSS) にコンタクト レンズを格納します。レンズをつかむに損傷を防ぐためプラスチックやシリコンの鉗子を使用します</li>。
	<li>は、集録モジュール ( 図 2 a に緑のボックス) を開始するコントロール パネル表示の右に黄色のボックスを押します。黄色のボックスは緑色になりコントロール パネル メニュー画面 ( 図 2 a) に表示されます</li>。
	<li>B-スキャンの買収は、コントロール パネル ( 図 2 a) のオプション IR + 10 月を選択します。選択した設定がコントロール パネルに青色で強調表示されます</li>。
	<li>選択 &#34; 網膜 &#34; 下 &#34; 構造とアプリケーション &#34; ソフトウェアおよび移動マウス眼レンズのデバイス ( 図 1) のマニピュレーターを使用して。網膜上に焦点を当て、前にことを確認、表示 &#34; OD &#34; 画面の左下の部分で選択しました。ソフトウェアは自動的に左 (OS) を識別し、右 (OD) 目が目的の位置に基づきます 。
	注: プラットフォームの中間に位置づけ、エラー メッセージが画面の下部に表示されます。その場合は、再配置マウス少しソフトウェアが正しい目を認識するまで.</li>
	<li>網膜上フォーカス フォーカスのノブで、コンピューター画面の左上の眼底写真で大きな船が見えるまで。左または右、その方向にカメラを移動するまたは時計回りまたは反時計回りに、カメラを上または下に移動する、マイクロマニピュレーターを回してカメラを移動します 。
	注: プラットフォーム上のマウス位置を変更または赤外線映像で視神経乳頭の最寄りのローカリゼーションを達成するためにレンズ上方または下方に移動します</li>。
	<li>ノブの感度を抑える反時計回りまたは時計回りに眼底画像の明度を上げます。最適なフォーカスを達成すると、SD 10 月 B スキャンは画面の右側に表示されます 。
	メモ: B-スキャンは、フォーカスを調整することによって可視化することはできません、する場合は、キーボードの組み合わせ Ctrl + Alt + Shift + O を押します。ポップアップ ウィンドウに B-スキャンは、画面に表示されるまでに参照アームの値を調整します</li>。
	<li>画面の右下にパターン メニュー ( 図 2 b で単一行) から単一のスキャンを選択します</li>。
	<li>デバイス ( 図 1) のマイクロマニピュレーターをオンに左、右、前方、または後方 (その方向にカメラを回す) する B-スキャンは、上部と下部の間あるように SD 10 月の角スキャン ウィンドウ</li>。
	<li>は、高品質の画像を取得する、少なくとも 9 自動リアルタイム (芸術) 値を設定します 。
	注意: 芸術は、いくつかの連続したスキャンを平均することによって画質を向上します。高い、&#34; アート &#34; 値より高い信号対雑音比および従ってイメージの質。ただし、増、&#34; アート &#34; 値、取得時間も増加します</li>。
	<li>プレス、&#34; を取得 &#34; コントロール パネルの画面で ( 図 2) ボタンをクリックし、画像を取得します</li>。
	<li>左目をイメージし、繰り返し手順 3.2 3.12 マウスを再配置</li>。
	<li>プラスチック/シリコン鉗子でコンタクト レンズを外し、BSS にそれを配置します</li>。
	<li>ヒドロキシプロピルメチル セルロースの新鮮なドロップとマウス角膜をメタンハイド レートし、ティッシュ ペーパーと超過分を削除します</li>。
	<li>標準 30 ° 光学を反時計回りに回転させることにより削除します</li>。
	<li>55 ° のレンズをマウントし、目ごとの手順 3.4 3.12 を繰り返します</li>。
</ol> 4。自動蛍光イメージング <ol><li>プラットフォーム上、マウスを動かさずにコントロール パネルで IR を選択します</li>。
	<li>大きな網膜血管に焦点を当てる、フォーカスノブと</li>。
	<li>コントロール パネルで AF を選択します</li>。
	<li>は、ツマミを減らすために反時計回りに回してください。または時計回りに画像の明度を上げます</li>。
	<li>感度つまみを押し、設定、&#34; アート &#34; 67 以上高品質を得るためのイメージする値</li>。
	<li>ときセット &#34; アート &#34; 値に達してを押して &#34; を取得 &#34; 画像が取得できるコントロール パネルの。平均を停止してもう一度ツマミを押してください。網膜の異なるレイヤーを視覚化する、フォーカスを調整します</li>。
	<li>55 ° のレンズを取り外すし、広いフィールド 102 ° のレンズをマウントします。手順 4.1 &#8211; 4.6 各マウスの両方の目とすべてのマウスします</li>。
	<li>をクリックして &#8220; 画像を保存 &#8221; ウィンドウとクリックの左の上部のボーダーに &#8220; 終了 &#8221;。コンピューターに画像を保存するには、マウスの名前をダブルクリックして、画面の右側の部分のリストからマウスを選択します。個別に各画像をダブルクリックし、フロッピー ディスク アイコンをクリックします。.Tif ファイル、.bmp、.jpg、または目的のフォルダーで .png としてイメージを保存します。ソフトウェアのプレスを終了する &#8220; ファイル &#8221; と &#8220; 終了 &#8221;.</li>
</ol> 5。麻酔逆転 <ol><li>ヒドロキシプロピルメチル セルロースの 1 つのドロップとマウスの目を水和物し、加熱パッドでマウスを返します</li>。
	<li>塩酸メデトミジンの鎮静作用を逆に 2.25 mg/kg の線量で準備アチパメゾール ソリューション。20 g のマウスでは、150 μ L の PBS の最終巻の atipamezol の 9 μ L を追加します。1 週間 4 ° C でソリューションを保存します</li>。
	<li>分後に麻酔薬注射 (手順 2.2)、注入アチパメゾール ソリューションの 150 μ L を皮下</li>。
	<li>は、麻酔から回復までマウスを監視します。マウスが完全に回復した (アチパメゾール注射後 5-10 分) あるとき、ケージに戻す。回復は 1 つの側面に置いたとき、その体に動物の能力によって示される歩行活動と環境刺激応答反応を取り戻すします</li>。
</ol> 6。SD OCT 画像から手動の網膜厚測定 <ol><li>OCT スキャンを開くには、動物の名前をダブルクリックします</li>。
	<li>30 ° または 55 ° のレンズで得られた B-スキャンを開く</li>。
	<li>選択 &#34; 厚さプロファイル &#34;.</li>
	<li>プレス、&#34; 編集レイヤーの区切り &#34; アイコン ( 図 2 b)。ソフトウェアが自動的に内部を制限することを識別し、膜を基本します</li>。
	<li>を必要に応じて、手動で内部制限の位置修正膜を基本とします。これを行うには、画面をクリックし、赤い円オプション ( 図 2 b) の左側にあるから変更するレイヤーを選択します。行を変更するのには押されたマウスのボタンが付いている円の移動</li>。
	<li>は、正しく対応するレイヤーを配置する行を変更します。クリックして &#34; を保存して閉じます &#34; ウィンドウを終了します 。
	注: 脈絡膜の反射率によりソフトウェアが正しく識別するない基本膜。したがって、それは網膜の厚さ測定に進む前に手動でそれを定義することをお勧めします</li>。
	<li>選択 &#34; 網膜 &#34; の下で、&#34; 層 &#34; オプション。網膜厚の図は、画面の下部に表示されます</li>。
	<li>選択した位置の異なる位置図または網膜の厚さ (μ m で示されます) を表示する B-スキャンのいずれかをクリックします</li>。
	<li>視神経乳頭から目的の距離で網膜の厚さを測定し、スプレッドシート内の値をコピーします</li>。
	<li>30 ° と 55 ° のレンズの両方のそれぞれについてマウス 5.1 5.9、手順を繰り返します</li>。
	<li>希望のソフトウェアと統計分析を実行します</li>。
</ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+Analysis+of+Mouse+Retinal+Layers+Using+Automated+Segmentation+of+Spectral+Domain+Optical+Coherence+Tomography+Images.">Quantitative Analysis of Mouse Retinal Layers Using Automated Segmentation of Spectral Domain Optical Coherence Tomography Images.</a> Transl Vis Sci Technol. 4 (4), 9 (2015).</li><li>Sim, D. A., 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+simple+method+for+in+vivo+labelling+of+infiltrating+leukocytes+in+the+mouse+retina+using+indocyanine+green+dye.">A simple method for in vivo labelling of infiltrating leukocytes in the mouse retina using indocyanine green dye.</a> Dis Model Mech. 8 (11), 1479-1487 (2015).</li><li>Bosco, A., Romero, C. O., Ambati, B. K., Vetter, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+dynamics+of+retinal+microglial+activation+during+neurodegeneration:+confocal+ophthalmoscopic+imaging+and+cell+morphometry+in+mouse+glaucoma.">In vivo dynamics of retinal microglial activation during neurodegeneration: confocal ophthalmoscopic imaging and cell morphometry in mouse glaucoma.</a> J Vis Exp. (99), e52731 (2015).</li><li>Acton, J. H., Cubbidge, R. P., King, H., Galsworthy, P., Gibson, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drusen+detection+in+retro-mode+imaging+by+a+scanning+laser+ophthalmoscope.">Drusen detection in retro-mode imaging by a scanning laser ophthalmoscope.</a> Acta Ophthalmol. 89 (5), e404-e411 (2011).</li><li>Greenstein, V. C., 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=Structural+and+functional+changes+associated+with+normal+and+abnormal+fundus+autofluorescence+in+patients+with+retinitis+pigmentosa.">Structural and functional changes associated with normal and abnormal fundus autofluorescence in patients with retinitis pigmentosa.</a> Retina. 32 (2), 349-357 (2012).</li><li>Jung, S., 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=Analysis+of+fractalkine+receptor+CX(3)CR1+function+by+targeted+deletion+and+green+fluorescent+protein+reporter+gene+insertion.">Analysis of fractalkine receptor CX(3)CR1 function by targeted deletion and green fluorescent protein reporter gene insertion.</a> Mol Cell Biol. 20 (11), 4106-4114 (2000).</li><li>Wang, X., 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=Requirement+for+Microglia+for+the+Maintenance+of+Synaptic+Function+and+Integrity+in+the+Mature+Retina.">Requirement for Microglia for the Maintenance of Synaptic Function and Integrity in the Mature Retina.</a> J Neurosci. 36 (9), 2827-2842 (2016).</li><li>Ebneter, A., Casson, R. J., Wood, J. P., Chidlow, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglial+activation+in+the+visual+pathway+in+experimental+glaucoma:+spatiotemporal+characterization+and+correlation+with+axonal+injury.">Microglial activation in the visual pathway in experimental glaucoma: spatiotemporal characterization and correlation with axonal injury.</a> Invest Ophthalmol Vis Sci. 51 (12), 6448-6460 (2010).</li><li>Ebneter, A., Kokona, D., Schneider, N., Zinkernagel, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+Activation+and+Recruitment+of+Circulating+Macrophages+During+Ischemic+Experimental+Branch+Retinal+Vein+Occlusion.">Microglia Activation and Recruitment of Circulating Macrophages During Ischemic Experimental Branch Retinal Vein Occlusion.</a> Invest Ophthalmol Vis Sci. 58 (2), 944-953 (2017).</li><li>Lin, Y. L., Potter-Baker, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+theoretical+models+from+adult+stroke+recovery+to+improve+use+of+non-invasive+brain+stimulation+for+children+with+congenital+hemiparesis.">Using theoretical models from adult stroke recovery to improve use of non-invasive brain stimulation for children with congenital hemiparesis.</a> J Neurophysiol. , (2017).</li><li>Combadiere, C., 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=CX3CR1-dependent+subretinal+microglia+cell+accumulation+is+associated+with+cardinal+features+of+age-related+macular+degeneration.">CX3CR1-dependent subretinal microglia cell accumulation is associated with cardinal features of age-related macular degeneration.</a> J Clin Invest. 117 (10), 2920-2928 (2007).</li><li>Bermudez, M. A., 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=Time+course+of+cold+cataract+development+in+anesthetized+mice.">Time course of cold cataract development in anesthetized mice.</a> Curr Eye Res. 36 (3), 278-284 (2011).</li><li>Toth, C. A., 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+comparison+of+retinal+morphology+viewed+by+optical+coherence+tomography+and+by+light+microscopy.">A comparison of retinal morphology viewed by optical coherence tomography and by light microscopy.</a> Arch Ophthalmol. 115 (11), 1425-1428 (1997).</li><li>Ebneter, A., Kokona, D., Jovanovic, J., Zinkernagel, M. 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L., 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=Reproducibility+of+spectral-domain+optical+coherence+tomography+total+retinal+thickness+measurements+in+mice.">Reproducibility of spectral-domain optical coherence tomography total retinal thickness measurements in mice.</a> Invest Ophthalmol Vis Sci. 51 (12), 6519-6523 (2010).</li><li>Nakao, S., 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=Wide-field+laser+ophthalmoscopy+for+mice:+a+novel+evaluation+system+for+retinal/choroidal+angiogenesis+in+mice.">Wide-field laser ophthalmoscopy for mice: a novel evaluation system for retinal/choroidal angiogenesis in mice.</a> Invest Ophthalmol Vis Sci. 54 (8), 5288-5293 (2013).</li><li>Wang, N. K., 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=Origin+of+fundus+hyperautofluorescent+spots+and+their+role+in+retinal+degeneration+in+a+mouse+model+of+Goldmann-Favre+syndrome.">Origin of fundus hyperautofluorescent spots and their role in retinal degeneration in a mouse model of Goldmann-Favre syndrome.</a> Dis Model Mech. 6 (5), 1113-1122 (2013).</li><li>Wang, N. K., 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=Cellular+origin+of+fundus+autofluorescence+in+patients+and+mice+with+a+defective+NR2E3+gene.">Cellular origin of fundus autofluorescence in patients and mice with a defective NR2E3 gene.</a> Br J Ophthalmol. 93 (9), 1234-1240 (2009).</li><li>Thanos, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sick+photoreceptors+attract+activated+microglia+from+the+ganglion+cell+layer:+a+model+to+study+the+inflammatory+cascades+in+rats+with+inherited+retinal+dystrophy.">Sick photoreceptors attract activated microglia from the ganglion cell layer: a model to study the inflammatory cascades in rats with inherited retinal dystrophy.</a> Brain Res. 588 (1), 21-28 (1992).</li><li>Hughes, E. H., 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=Generation+of+activated+sialoadhesin-positive+microglia+during+retinal+degeneration.">Generation of activated sialoadhesin-positive microglia during retinal degeneration.</a> Invest Ophthalmol Vis Sci. 44 (5), 2229-2234 (2003).</li></ol>

著者が明らかに何もありません。

本稿は、網膜 B-スキャンの買収と同じ撮像セッションでマウス網膜における gfp 陽性ミクログリア分布のイメージングのためのプロトコルを示します。SD 10 月と SLO はますます時間10,14,17,18,21に網膜の変化の情報を提供するために網膜疾患の動物モデルで使用されます。?...

実験眼科で網膜の病理検査では組織学的手法を使用して評価、通常。しかし、組織は動物 euthanization を必要とし、組織の実際のプロパティに変更を引き起こす可能性があります。SD 10 月と SLO が定期的に眼科で使用される臨床診断用といくつかの網膜疾患網膜色素変性症11 や前部虚血性視神経症10、糖尿病黄斑浮腫9などの監視のため.SD 10 月 SLO は、非侵襲的手法、さら、介入を使わないで瞳孔を通して可視化、網膜の高解像度画像を生成します。SD-10 月は、SLO 蛍光網膜の立体の高コントラストの画像を生成するデータを収集中に、網膜の断面像を作成する後方散乱データを集めることによって網膜の厚さと網膜の構造の情報を提供します。今日では、両方の技術はますます小さい齧歯動物12,13,14,15 (または) を使用もゼブラフィッシュ16,17実験眼科で使用され、することができます。両方質的・量的情報12,17,18,19,20,21を提供します。
Lipofuscins のような内因性蛍光物質の蓄積や乳頭部のドルーゼン、網膜の形成が自動蛍光信号として SLO で視覚化できます。この機能は、診断と網膜症加齢黄斑変性症や網膜色素変性症22,23などの監視のための貴重な技術を SLO になります。実験眼科でレポーター マウスの行の特定のセル型の検出の自動蛍光 (AF) を使用できます。たとえば、 Cx3cr124のプロモーターの gfp 発現のヘテロ接合体マウスは通常の網膜におけるミクログリア細胞の生体内可視化およびミクログリア/マクロファージの調査のために有利です網膜疾患21のダイナミクス。ミクログリアは、網膜のマクロファージ組織恒常性と損傷1,25,26の時に組織の修復に重要な役割を果たしています。網膜におけるミクログリアの活性化が、網膜の損傷、虚血、変性、網膜疾患2,3,4,5,におけるこれらの細胞の役割を示唆している報告されています。6。
この議定書の目的Cx3cr1gfp/gfpマウス網膜を使って gfp 陽性ミクログリア細胞の可視化、網膜イメージングおよび SD-OCT による網膜の厚さの測定のための比較的簡単なメソッドを記述することです。SLO (ハイデルベルク Spectralis HRA + OCT システム)。このプロトコルはマウスの各種の行の健康または病気の網膜のイメージングや膜厚計測の利用できます。さらに、同定及び定量化のミクログリア数字と SLO21を使用して網膜におけるミクログリアの活性化の形態計測学的解析を実行できます。ミクログリア細胞は網膜27,28,29を含む中枢神経系 (CNS)、変性疾患に関連付けられます。したがって、この議定書で使用される 2 つの方法を組み合わせることによってミクログリア分布と網膜変性の相関を行うことができますは、重症度監視を容易にすることができますをまたは生体内で治療の有効性に近づきます。

<table>
	<tbody>
		<tr>
			<td>Spectralis Imaging system (HRA+OCT)</td>
			<td>Heidelberg Engineering, Germany</td>
			<td>N/A</td>
			<td>ophthalmic imaging platform system</td>
		</tr>
		<tr>
			<td>Heidelberg Eye Explorer</td>
			<td>Heidelberg Engineering, Germany</td>
			<td>N/A</td>
			<td>Version 1.9.13.0</td>
		</tr>
		<tr>
			<td>78D standard ophthalmic non-contact slit lamp lens</td>
			<td>Volk Optical Inc., Ohio, USA</td>
			<td>V78C</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectralis wide angle 55&#176; lens</td>
			<td>Heidelberg Engineering, Germany</td>
			<td>50897-002</td>
			<td></td>
		</tr>
		<tr>
			<td>ultra widefield 102&#176; lens</td>
			<td>Heidelberg Engineering, Germany</td>
			<td>50117-001</td>
			<td></td>
		</tr>
		<tr>
			<td>medetomidine hydrochloride 1 mg/mL (Domitor)</td>
			<td>Provet AG, Lyssach, Switzerland</td>
			<td>Swissmedic Nr. 50&#39;590 - ATCvet: QN05CM91</td>
			<td>anesthetic/analgesic</td>
		</tr>
		<tr>
			<td>ketamine 50mg/ml (Ketalar)</td>
			<td>Parke-Davis, Zurich, Switzerland</td>
			<td>72276388</td>
			<td>anesthetic</td>
		</tr>
		<tr>
			<td>tropicamide 0.5% + phenylephrine HCl 2.5% (Augentropfen mix)</td>
			<td>ISPI, Bern, Switzerland</td>
			<td>N/A</td>
			<td>pupil dilation</td>
		</tr>
		<tr>
			<td>Omnican Insulin-50 0.5 ml G30 0.3 x 12mm</td>
			<td>B. Braun Mesungen AG, Carl-Braun-Stra&#223;e, Germany</td>
			<td>9151125</td>
			<td></td>
		</tr>
		<tr>
			<td>hydroxypropylmethylcellulose (Methocel 2%)</td>
			<td>OmniVision, Neuhausen, Switzerland</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>+4 dpt rigid gas permeable contact lens</td>
			<td>Quantum I, Bausch + Lomb Inc., Rochester, NY</td>
			<td>N/A</td>
			<td>Base Curve: 7.20 to 8.40 mm 
			Diameter: 9.00 / 9.60 / 10.20 mm 
			Power: -25.00 to +25.00 Diopters</td>
		</tr>
		<tr>
			<td>balanced salt solution (BSS)</td>
			<td>Inselspital, Bern, Switzerland</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>silicon forceps</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>atipamezole 5 mg/mL (Antisedan)</td>
			<td>Provet AG, Lyssach, Switzerland</td>
			<td>N/A</td>
			<td>&#945;2 adrenergic receptor antagonist</td>
		</tr>
		<tr>
			<td>GraphPad Prism 7</td>
			<td>GraphPad Software, Inc, San Diego, CA, USA</td>
			<td>N/A</td>
			<td>statistical analysis software</td>
		</tr>
	</tbody>
</table>

in vivo imaging of cx3cr1gfp/gfp reporter mice with spectral-domain optical coherence tomography and scanning laser ophthalmoscopy

スペクトル領域光干渉断層法 (SD OCT) と走査レーザー眼底検査 (SLO) は、実験眼科で幅広く使用されています。現在のプロトコルを発現するマウス緑蛍光蛋白質 (gfp) Cx3cr1のプロモーター (BALB/c-Cx3cr1gfp/gfp) ミクログリア細胞体内の網膜のイメージのために使用されました。ミクログリアは網膜のマクロファージであり、いくつかの網膜疾患1,2,3,4,5,6に関与しています。このプロトコルは、網膜 B-スキャンの SD-10 月、SLO は生体内で、 Cx3cr1gfp/gfpマウスのミクログリア細胞分布のイメージングと眼画像処理プラットフォーム システムを使用して生成の詳細なアプローチを提供します。プロトコルは、レポーター マウスのいくつかの行で使用することができます。ただし、ここで示されるプロトコルをいくつかの制限があります。まず、両方の SLO と SD 10 月では、高分解能モードで使用する場合高軸方向の解像度が横方向の解像度を持つデータを収集が低い (3.5 μ m と 6 μ m、それぞれ)。さらに、SLO のフォーカスと彩度レベルはパラメーターの選択や目の適切な配置に依存です。さらに、マウスの人間の患者はマウス眼の高い合計光学力のために挑戦のために設計されたデバイスを使用してください。 人間の目と比較してください。これはとりわけマウス レンズによって倍率に依存でもある横倍率誤り7、可能性があります。しかし、軸のスキャン位置にもかかわらず、横倍率に依存して、軸の SD 10 月測定は正確な8。

ここで提示されたプロトコルを使用して、SD OCT スキャンし、SLO 画像は、同じ撮像セッションでCx3cr1gfp/gfpマウスから得られました。図 3には、代表者 SD 10 月 gfp 陽性ミクログリア細胞が視覚化される 55 ° または 102 ° レンズで得られる 30 ° または 55 ° レンズ (図 3 a) と代表的な SLO 画像で得られた単一のスキ...

Watch this Scientific Journal Video about In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy at JoVE.com

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

このプロトコルを記述する方法高解像度イメージング スペクトル領域光干渉断層法などと小さい齧歯動物, 眼科画像処理プラットフォーム システムを使用してに関する情報を取得するに活用できるレーザー眼底をスキャン網膜厚とミクログリア細胞の分布、それぞれ。

生体内でスペクトル領域光干渉断層法と走査レーザー眼底Cx3cr1gfp/gfpレポーター マウスの画像

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the ...

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In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

10.5K ビュー.  Bern University Hospital and University of Bern. このプロトコルを記述する方法高解像度イメージング スペクトル領域光干渉断層法などと小さい齧歯動物, 眼科画像処理プラットフォーム システムを使用してに関する情報を取得するに活用できるレーザー眼底をスキャン網膜厚とミクログリア細胞の分布、それぞれ。

ビデオ: In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

Oral Biofilm Analysis of Palatal Expanders by Fluorescence In-Situ Hybridization and Confocal Laser Scanning Microscopy

Confocal laser scanning microscopy (CLSM) of natural heterogeneous biofilm is today facilitated by a comprehensive range of staining techniques, one of them being fluorescence in situ hybridization (FISH).1,2 We performed a pilot study in which oral biofilm samples collected from fixed orthodontic appliances (palatal expanders) were stained by FISH, the objective being to assess the three-dimensional organization of natural biofilm and plaque accumulation.3,4 FISH creates an opportunity to stain cells in their native biofilm environment by the use of fluorescently labeled 16S rRNA-targeting probes.4-7,19 Compared to alternative techniques like immunofluorescent labeling, this is an inexpensive, precise and straightforward labeling technique to investigate different bacterial groups in mixed biofilm consortia.18,20 General probes were used that bind to Eubacteria (EUB338 + EUB338II + EUB338III; hereafter EUBmix),8-10 Firmicutes (LGC354 A-C; hereafter LGCmix),9,10 and Bacteroidetes (Bac303).11 In addition, specific probes binding to Streptococcus mutans (MUT590)12,13 and Porphyromonas gingivalis (POGI)13,14 were used. The extreme hardness of the surface materials involved (stainless steel and acrylic resin) compelled us to find new ways of preparing the biofilm. As these surface materials could not be readily cut with a cryotome, various sampling methods were explored to obtain intact oral biofilm. The most workable of these approaches is presented in this communication. Small flakes of the biofilm-carrying acrylic resin were scraped off with a sterile scalpel, taking care not to damage the biofilm structure. Forceps were used to collect biofilm from the steel surfaces. Once collected, the samples were fixed and placed directly on polysine coated glass slides. FISH was performed directly on these slides with the probes mentioned above. Various FISH protocols were combined and modified to create a new protocol that was easy to handle.5,10,14,15 Subsequently the samples were analyzed by confocal laser scanning microscopy. Well-known configurations3,4,16,17 could be visualized, including mushroom-style formations and clusters of coccoid bacteria pervaded by channels. In addition, the bacterial composition of these typical biofilm structures were analyzed and 2D and 3D images created.

We present a protocol for structural and compositional analysis of natural oral biofilm from orthodontic appliances with in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM). Oral biofilm samples were collected from palatal expanders, scraping acrylic-resin flakes off their surface and referring them for molecular processing.

蛍光in situハイブリダイゼーションおよび共焦点レーザー走査顕微鏡による口蓋パンダの口腔バイオフィルムの解析

Confocal laser scanning microscopy (CLSM) of natural heterogeneous biofilm is today facilitated by a comprehensive range of staining techniques, one of ...

医学

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The cell line was originally induced by intracranial injection of 3-methyl-cholantrene into a C57BL/6 syngeneic mouse strain 1; therefore, immunologically competent C57BL/6 mice can be used. While we use GL261, the following protocol can be used for the implantation and monitoring of any intracranial mouse tumor model. GL261 cells were engineered to stably express firefly luciferase (GL261-luc). We also created the brighter GL261-luc2 cell line by stable transfection of the luc2 gene expressed from the CMV promoter. C57BL/6-cBrd/cBrd/Cr mice (albino variant of C57BL/6) from the National Cancer Institute, Frederick, MD were used to eliminate the light attenuation caused by black skin and fur. With the use of albino C57BL/6 mice; in vivo imaging using the IVIS Spectrum in vivo imaging system is possible from the day of implantation (Caliper Life Sciences, Hopkinton, MA). The GL261-luc and GL261-luc2 cell lines showed the same in vivo behavior as the parental GL261 cells. Some of the shared histological features present in human GBMs and this mouse model include: tumor necrosis, pseudopalisades, neovascularization, invasion, hypercellularity, and inflammation 1.
Prior to implantation animals were anesthetized by an intraperitoneal injection of ketamine (50 mg/kg), xylazine (5 mg/kg) and buprenorphine (0.05 mg/kg), placed in a stereotactic apparatus and an incision was made with a scalpel over the cranial midline. A burrhole was made 0.1mm posterior to the bregma and 2.3mm to the right of the midline. A needle was inserted to a depth of 3mm and withdrawn 0.4mm to a depth of 2.6mm. Two &mu;l of GL261-luc or GL261-luc2 cells (107 cells/ml) were infused over the course of 3 minutes. The burrhole was closed with bonewax and the incision was sutured.
Following stereotactic implantation the bioluminescent cells are detectable from the day of implantation and the tumor can be analyzed using the 3D image reconstruction feature of the IVIS Spectrum instrument. Animals receive a subcutaneous injection of 150&mu;g luciferin /kg body weight 20 min prior to imaging. Tumor burden is quantified using mean tumor bioluminescence over time. Tumor-bearing mice were observed daily to assess morbidity and were euthanized when one or more of the following symptoms are present: lethargy, failure to ambulate, hunched posture, failure to groom, anorexia resulting in &gt;10% loss of weight. Tumors were evident in all of the animals on necropsy.

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

Intracranial implantation of GL261 cells into C57BL/6 mice produces malignant gliomas that recapitulate many of the hallmarks of human glioblastoma multiforme. We used GL261 cells stably expressing luciferase to allow us to use in vivo imaging to follow tumor progression. The surgery and 3D in vivo imaging are demonstrated.

その後の3Dと頭蓋内注入インビボでの</emマウス神経膠腫の>生物発光イメージング

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The ...

Doppler Optical Coherence Tomography of Retinal Circulation

Noncontact retinal blood flow measurements are performed with a Fourier domain optical coherence tomography (OCT) system using a circumpapillary double circular scan (CDCS) that scans around the optic nerve head at 3.40 mm and 3.75 mm diameters. The double concentric circles are performed 6 times consecutively over 2 sec. The CDCS scan is saved with Doppler shift information from which flow can be calculated. The standard clinical protocol calls for 3 CDCS scans made with the OCT beam passing through the superonasal edge of the pupil and 3 CDCS scan through the inferonal pupil. This double-angle protocol ensures that acceptable Doppler angle is obtained on each retinal branch vessel in at least 1 scan. The CDCS scan data, a 3-dimensional volumetric OCT scan of the optic disc scan, and a color photograph of the optic disc are used together to obtain retinal blood flow measurement on an eye. We have developed a blood flow measurement software called "Doppler optical coherence tomography of retinal circulation" (DOCTORC). This semi-automated software is used to measure total retinal blood flow, vessel cross section area, and average blood velocity. The flow of each vessel is calculated from the Doppler shift in the vessel cross-sectional area and the Doppler angle between the vessel and the OCT beam. Total retinal blood flow measurement is summed from the veins around the optic disc. The results obtained at our Doppler OCT reading center showed good reproducibility between graders and methods (&lt;10%). Total retinal blood flow could be useful in the management of glaucoma, other retinal diseases, and retinal diseases. In glaucoma patients, OCT retinal blood flow measurement was highly correlated with visual field loss (R2&gt;0.57 with visual field pattern deviation). Doppler OCT is a new method to perform rapid, noncontact, and repeatable measurement of total retinal blood flow using widely available Fourier-domain OCT instrumentation. This new technology may improve the practicality of making these measurements in clinical studies and routine clinical practice.

Total retinal blood flow is measured by Doppler optical coherence tomography and semi-automated grading software.

網膜循環のドップラー光コヒーレンストモグラフィー

Noncontact retinal blood flow measurements are performed with a Fourier domain optical coherence tomography (OCT) system using a circumpapillary double ...

Segmentation and Measurement of Fat Volumes in Murine Obesity Models Using X-ray Computed Tomography

Obesity is associated with increased morbidity and mortality as well as reduced metrics in quality of life.1 Both environmental and genetic factors are associated with obesity, though the precise underlying mechanisms that contribute to the disease are currently being delineated.2,3 Several small animal models of obesity have been developed and are employed in a variety of studies.4 A critical component to these experiments involves the collection of regional and/or total animal fat content data under varied conditions.
Traditional experimental methods available for measuring fat content in small animal models of obesity include invasive (e.g. ex vivo measurement of fat deposits) and non-invasive (e.g. Dual Energy X-ray Absorptiometry (DEXA), or Magnetic Resonance (MR)) protocols, each of which presents relative trade-offs. Current invasive methods for measuring fat content may provide details for organ and region specific fat distribution, but sacrificing the subjects will preclude longitudinal assessments. Conversely, current non-invasive strategies provide limited details for organ and region specific fat distribution, but enable valuable longitudinal assessment. With the advent of dedicated small animal X-ray computed tomography (CT) systems and customized analytical procedures, both organ and region specific analysis of fat distribution and longitudinal profiling may be possible. Recent reports have validated the use of CT for in vivo longitudinal imaging of adiposity in living mice.5,6 Here we provide a modified method that allows for fat/total volume measurement, analysis and visualization utilizing the Carestream Molecular Imaging Albira CT system in conjunction with PMOD and Volview software packages.

Fat content analysis is routinely conducted in studies utilizing murine obesity models. Emerging methods in small animal CT imaging and analysis are providing for longitudinal detail rich fat content analysis. Here we detail step by step procedures for performing small animal CT imaging, analysis, and visualization.

X線CTを用いたマウス肥満モデルにおけるセグメンテーションおよびFATボリュームの測定

Obesity is associated with increased morbidity and mortality as well as reduced metrics in quality of life.1 Both environmental and genetic factors are ...

Generation and 3-Dimensional Quantitation of Arterial Lesions in Mice Using Optical Projection Tomography

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating important information on the pathogenesis of lesion formation and the action of novel therapies. Use of atherosclerosis-prone mice, surgical methods of lesion induction, and dietary modification has dramatically improved understanding of the mechanisms that contribute to disease development and the potential of new treatments.
Classically, analysis of lesions is performed ex vivo using 2-dimensional histological techniques. This article describes application of optical projection tomography (OPT) to 3-dimensional quantitation of arterial lesions. As this technique is non-destructive, it can be used as an adjunct to standard histological and immunohistochemical analyses.
Neointimal lesions were induced by wire-insertion or ligation of the mouse femoral artery whilst atherosclerotic lesions were generated by administration of an atherogenic diet to apoE-deficient mice.
Lesions were examined using OPT imaging of autofluorescent emission followed by complementary histological and immunohistochemical analysis. OPT clearly distinguished lesions from the underlying vascular wall. Lesion size was calculated in 2-dimensional sections using planimetry, enabling calculation of lesion volume and maximal cross-sectional area. Data generated using OPT were consistent with measurements obtained using histology, confirming the accuracy of the technique and its potential as a complement (rather than alternative) to traditional methods of analysis.
This work demonstrates the potential of OPT for imaging atherosclerotic and neointimal lesions. It provides a rapid, much needed ex vivo technique for the routine 3-dimensional quantification of vascular remodelling.

Ex vivo analysis of arterial lesions from animal models of cardiovascular disease classically relies on histological and immunohistochemical techniques. These provide 2-dimensional measurements in 3-dimensional lesions. This manuscript describes the generation of arterial lesions for quantitative analysis in 3-dimensions using optical projection tomography.

発生と光学投影トモグラフィを用いたマウスにおける動脈病変の3次元定量

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating ...

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10&#176; steps over 380&#176;. A rectangular&#160;region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo quantitation of a fluorescent marker delivered by saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles. Employing mouse models of cancer and arthritis, we demonstrate how the MAROI signal curve analysis can be used for the precise mapping and biological characterization of disease processes.

蛍光SAPC-DOPSナノベシクルを用いて脳腫瘍、関節炎のin vivo光イメージング中

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...

Combined In vivo Optical and &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in vivo optical and &#956;CT imaging allow the visualization of biological phenomena in an anatomical context. These imaging modalities may be especially useful to study conditions that impact bone. In particular, orthopaedic implant infections are an important problem in clinical orthopaedic surgery. These infections are difficult to treat because bacterial biofilms form on the foreign surgically implanted materials, leading to persistent inflammation, osteomyelitis and eventual osteolysis of the bone surrounding the implant, which ultimately results in implant loosening and failure. Here, a mouse model of an infected orthopaedic prosthetic implant was used that involved the surgical placement of a Kirschner-wire implant into an intramedullary canal in the femur in such a way that the end of the implant extended into the knee joint. In this model, LysEGFP mice, a mouse strain that has EGFP-fluorescent neutrophils, were employed in conjunction with a bioluminescent Staphylococcus aureus strain, which naturally emits light. The bacteria were inoculated into the knee joints of the mice prior to closing the surgical site. In vivo bioluminescent and fluorescent imaging was used to quantify the bacterial burden and neutrophil inflammatory response, respectively. In addition, &#956;CT imaging was performed on the same mice so that the 3D location of the bioluminescent and fluorescent optical signals could be co-registered with the anatomical &#956;CT images. To quantify the changes in the bone over time, the outer bone volume of the distal femurs were measured at specific time points using a semi-automated contour based segmentation process. Taken together, the combination of in vivo bioluminescent/fluorescent imaging with &#956;CT imaging may be especially useful for the noninvasive monitoring of the infection, inflammatory response and anatomical changes in bone over time.

Combined In vivo Optical and  &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Combined optical and &#956;CT imaging in a mouse model of orthopaedic implant infection, utilizing a bioluminescent engineered strain of Staphylococcus aureus, provided the capability to noninvasively and longitudinally monitor the dynamics of the bacterial infection, as well as the corresponding inflammatory response and anatomical changes in the bone.

合わせた<em&gt;インビボ</em&gt;光とμCTイメージングマウスにおける整形外科インプラント感染で感染症、炎症、および骨解剖学を監視する

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in ...

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 ...

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.
First we describe how to generate subcutaneous tumors in mice, using antigen-negative cell lines as negative controls and antigen-positive cells as positive controls in the same mice for intraindividual comparison. We outline how to administer intravenously near-infrared fluorophore labelled (AlexaFluor680) antigen-specific nanobodies and conventional antibodies. In vivo imaging was performed with a small-animal NIRF-Imaging system. After the in vivo imaging experiments the mice were sacrificed. We then describe how to prepare the tumors for parallel ex vivo analyses by flow cytometry and fluorescence microscopy to validate in vivo imaging results.
The use of the near-infrared fluorophore labelled nanobodies allows for non-invasive same day imaging in vivo. Our protocols describe the ex vivo quantification of the specific labeling efficiency of tumor cells by flow cytometry and analysis of the distribution of the antibody constructs within the tumors by fluorescence microscopy. Using near-infrared fluorophore labelled probes allows for non-invasive, economical in vivo imaging with the unique ability to exploit the same probe without further secondary labelling for ex vivo validation experiments using flow cytometry and fluorescence microscopy.

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.

ナノボディ及び抗体ベースの検証<em&gt;インビボ</em使用してマウスにおける&gt;腫瘍異種移植NIRFイメージング実験<em&gt;エクスビボ</em&gt;フローサイトメトリーおよび顕微鏡

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice ...

In Vivo, Percutaneous, Needle Based, Optical Coherence Tomography of Renal Masses

Optical coherence tomography (OCT) is the optical equivalent of ultrasound imaging, based on the backscattering of near infrared light. OCT provides real time images with a 15 &#181;m axial resolution at an effective tissue penetration of 2-3 mm. Within the OCT images the loss of signal intensity per millimeter of tissue penetration, the attenuation coefficient, is calculated. The attenuation coefficient is a tissue specific property, providing a quantitative parameter for tissue differentiation. 
Until now, renal mass treatment decisions have been made primarily on the basis of MRI and CT imaging characteristics, age and comorbidity. However these parameters and diagnostic methods lack the finesse to truly detect the malignant potential of a renal mass. A successful core biopsy or fine needle aspiration provides objective tumor differentiation with both sensitivity and specificity in the range of 95-100%. However, a non-diagnostic rate of 10-20% overall, and even up to 30% in SRMs, is to be expected, delaying the diagnostic process due to the frequent necessity for additional biopsy procedures. 
We aim to develop OCT into an optical biopsy, providing real-time imaging combined with on-the-spot tumor differentiation. This publication provides a detailed step-by-step approach for percutaneous, needle based, OCT of renal masses.

In Vivo, Percutaneous, Needle Based, Optical Coherence Tomography of Renal Masses

Optical coherence tomography (OCT) is a high resolution imaging technique that allows analysis of tissue specific optical properties providing the means for tissue differentiation. We developed needle based OCT, providing real-time imaging combined with on-the-spot tumor differentiation. This publication describes a method for percutaneous, needle based OCT of renal masses.

生体内 、経皮、ニードルベース、腎大衆の光コヒーレンストモグラフィーで

Optical coherence tomography (OCT) is the optical equivalent of ultrasound imaging, based on the backscattering of near infrared light. OCT provides real ...

生体内でスペクトル領域光干渉断層法と走査レーザー眼底Cx3cr1gfp/gfpレポーター マウスの画像

生体内でスペクトル領域光干渉断層法と走査レーザー眼底Cx3cr1^gfp/gfpレポーターマウスの画像