私たちは、原稿を読み取るためで公園に感謝します。この作品は、内容はもっぱら著者の責任であり、ないDPとCPLとDTSの国立衛生研究所の助成金を受賞番号K01AR061434と白血病リンパ腫協会フェローシップ賞（5127から09）の下でNIAMSによってサポートされていました必ずしも国立衛生研究所の公式見解を表しています。

1。マウスおよびプレコンディショニング注：すべてのマウスを無菌状態に維持し、すべてのプロトコルがマサチューセッツ総合病院の施設内動物管理使用委員会（IACUC）によって承認された。すべての手術は、オートクレーブし、滅菌装置を使用して、無菌条件下で行われるべきである。MX1〜Creを15、Rosa26遺伝子-のloxPワンストップのloxP-EYFP（ローザ-YFP）、およびRosa26遺伝子-のloxPワンストップのloxP-tdTomato（ローザ·トマト） であったジャクソン研究所から購入した。 オステオカルシン-GFPマウスはヘンリークローネンベルクによって提供された。 インビボで OSPC遊走および増殖の定量分析のために、Mx1の-Creリコンビナーゼ + ローザ-YFP +（単色レポーター）マウスを使用した。 オステオカルシン+成熟骨芽細胞への分化をより詳細に追跡のために、我々は使用三遺伝子MX1〜のCre +ローザ-トマト+ OCN-GFP +マウス。 <ol><li> 生体内でMx1の+細胞を標識するために、無菌のPBS溶液中でポリイノシン-ポリシチジル酸（PIPC、2.5 mg / mlの）を調製し、10日間、腹腔内に一回、一日おきMX1〜のCre +レポーターマウスの20グラム、200μLを注入。 </li><li> Mx1の常駐+造血細胞を排除するために、9.5 Gyでの単回用量をマウスに照射する。照射の24時間後、移植野生型骨髄細胞（1×10 6細胞/マウス）を静脈内16。注意：MX1〜誘導性細胞は造血細胞および造血由来の破骨細胞が含まれるため、Mx1の+造血細胞の排除はMx1の+骨形成細胞の画像品質および定量を向上させます。 </li><li>骨髄移植後に、ドナー骨髄細胞の正常な再増殖を達成するために、4〜6週間のために動物を監視する。 </li></ol> 2。モー準備を使う<ol><li> IACUC承認の手順を使用して、ケタミン/キシラジン（100 mg / kgを、キシラジン12 mg / kg体重）を50μlの腹腔内注射によりマウスを麻酔。注：効果の持続時間は、ケタミン/キシラジンの追加用量で延長することができる。 </li><li>マウスが完全にTOE及び/または尾のピンチに反応しないことによって麻酔をされているかどうか。マウスを麻酔した場合（オプション）、テーピングによる鼻の上イソフルランガスマスクを固定します。動物が完全に鎮静されていることを確認するためにイソフルランのレベルを調整します。 </li><li>電気トリマーや小さなハサミを使って頭髪をクリップ。髪の断片を除去し、70％アルコール綿で露出した皮膚を殺菌。角膜の脱水を防ぐために、涙のゲルを適用します。 </li></ol> 3。微小破壊けが<ol><li>動物が完全に鎮静されていることを確認した後、頭皮を開くために、単一の逆L字​​型の切り込みを入れる。簡単に言えば、第1の横切開（1cm未満）startiを作る別の耳に1耳からngの。 〜60°を回し、離れて鼻から〜2〜3ミリメートルまでの鼻に向かって切開し続けています。注：この切開は画像化される領域の上の瘢痕組織の形成を最小限に抑えます。 </li><li>ピンセットで両側に向かって引いて、皮膚のフラップを分離する。両方前頭骨や矢状および冠状縫合の交点が明確に公開されるべきである。 </li><li>すべての残留毛が除去されるまで滅菌PBSと滅菌ガーゼや綿棒で穏やかにワイピングでフラッシュすることにより開放面を清掃してください。 </li><li>頭蓋冠上の微小破壊を生成するには、片手でマウスの頭と別の手で30 Gの針を保持します。穏やかな圧力で30 Gの針の先端を挿入し、運動をねじることによって矢状および冠状縫合の交差点の近くに左前頭骨上のマイクロ穿刺（&lt;1ミリメートルの深さで巻か〜0.2ミリメートル径）を確認します。注意：これは、それによってbleediを最小限に抑え、脳内に侵入するの針を避けることが重要ですNGと組織の損傷。 </li><li>大きな針（20または25 G）に切り替えて、針（〜0.5ミリメートル径）をひねってパンク穴を広げる。 </li><li>対側前頭骨に対して二の穿刺を生成するために、ステップ3.3から3.5を繰り返します。 </li><li>出血が止まるまでのPBS連続ドロップすることで、負傷したスポットを一掃。 注：骨折部位での出血は、適切な撮像を妨害し、瘢痕形成を誘導する。 </li><li>乾燥を避けるために、頭蓋骨に滅菌生理食塩水または2％のメトセルの低下を適用する。注意：画像の鮮明さを軽減するであろう、頭蓋骨の表面を乾燥させないでください。これは、領域を覆うようにゲルまたは生理食塩水の十分な量を使用することが重要である。 </li></ol> 4。生体内イメージング<ol><li> （任意）イメージングの前に、一部のマウスは、血管を可視化するために、血管色素を注射する。非標的Qtracker 705 80メートルで希釈した（50 mMのホウ酸緩衝液中の2μM溶液）20μlの眼窩注入PBSをL。試薬の無駄を最小限に抑えるためにインスリン注射器を使用してください。信号が十分明るくない場合は、追加の金額が必要になることがあります。 </li><li>スキャナの電源をオンにします。注：ポリゴンベースのレーザスキャナは、ビデオレート（毎秒30フレーム）での同時マルチチャネル画像取得を可能にする。ビデオレートスキャンは、生きている動物のイメージングのために非常に重要である。 </li><li>多光子レーザー（：サファイアレーザフェムト秒チタン）をオンにします。 880 nmの波長を設定し、骨の第二高調波発生イメージング（440 nm）のための電力を調整。 GFP及びtdTomato励起用、491 nmおよび561 nmの固体レーザーをオンにします。 </li><li> （tdTomatoのために第二高調波発生のための435±20 nmのバンドパスフィルタ、GFPについて528±19 nmのバンドパスフィルタ、および590±20）各信号のPMT（光電子増倍管）検出器をオンにします（オプション） Qtracker 705の信号を検出するために638 nmのヘリウム - ネオンレーザーおよび695±27.5 nmのバンドパスフィルタを有するPMTを使用する。 </li><li> PLACXYZ軸電動顕微鏡ステージ上に動物をメール。 （これは、動物の損失のリスクを軽減）、体温を維持するために電気加熱パッドで最も快適な位置にマウスを保管してください。撮像中に動きを最小限に抑え、できるだけ水平に画像化された面積を維持するために、マウスを保持するためにテープを使用しています。 </li><li>乾燥を避けるために、頭蓋骨に暖かい2％メチルセルロースゲルまたは生理食塩水の滴を適用する。撮像面にカバーガラスを置く。頭蓋冠をスキャンするために低倍率レンズ（0.9 NAの30倍の水浸対物レンズ）を使用します。 </li><li> XYZ軸コントローラを使用して、骨からのSHG信号を検出することにより、頭蓋冠の表面を見つけ、そのような矢状縫合および冠状縫合との交点として重要なランドマークの場所を特定する。画像を取得して、XYZ座標を記録します。 </li><li> SHGと蛍光シグナルを観察することにより、損傷の場所を検索し続けています。 </li><li>損傷部位または領域Oするときfは関心が発見された、目的の細胞からのSHG及び蛍光シグナルを含有する最良焦点面の画像を取得する。 XYZ座標と画像の次のラウンドのための彼らの正確な位置を定義するための矢状や冠状縫合の交差点までの距離を保存します。 </li><li>骨折傷害の3D細胞および骨の構造を収集するには、Zスタックによる記録画像 - 骨内膜骨表面から〜100μmの深さを持つ（25μmの間隔）。傷害の片側の撮像が終了した後、次の損傷部位用撮像処理を繰り返す。 </li></ol> 5。ポスト操作手順<ol><li>画像形成後、頭蓋骨からメチルセルロースゲルを除去するために滅菌生理食塩液で撮影部位をすすぐ。滅菌綿棒を使用して、表面上のトリプル抗生物質軟膏を少量を適用します。皮膚フラップをカバーし、外科用縫合で頭皮を再度閉じます。これは低刺激性の縫合糸（吸収性ポリグラクチンSUで行うことができますTURE）と5-0サイズの縫合針。注意：良い縫合技術は、瘢痕形成を最小限に抑えることができます。 </li><li> 0.05〜0.1 mg / kgのブプレノルフィンのIP注射したすべての動物を手術後48時間ごとに12時間の治療。彼らは十分な意識を取り戻すまで、暖かい回収室で動物を保​​管してください。 3〜5日後に、縫合糸を再び開き、骨折治癒中の細胞変化を追跡するために、生体内イメージングの手順を繰り返します。 </li></ol>

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著者は、彼らが競合する経済的利益を持っていないことを宣言します。

骨格筋幹細胞の調節は、骨再生を達成するためのよりよい方法を定義するために非常に重要であり得る。細胞レベルで定量的かつ連続的なイメージングは​​、技術的に困難となっている。マウスの長期骨折モデルが広く使用され、生体力学的研究17に適してきたが、その深部組織の位置、不均一な破断サイズ、軟組織の損傷、および安定化固定具の適用は、順次生体内イメージング...

骨粗しょう症性骨折のリスクが高いにつながる変性骨疾患及び年齢関連骨量減少は、公衆衛生1における大きな課題となっている。骨の維持、骨形成性骨芽細胞と骨吸収破骨細胞によって制御される。骨形成細胞の欠陥は、年齢関連骨量減少および変性骨疾患2,3の主な原因である。大規模な研究は、骨折治癒の改善に焦点を当てているが、変形性骨疾患を治すと骨粗鬆症による骨折の弱点を逆に信頼性の高い医薬品の発見は重要な課題である。したがって、骨の再生および修復における骨形成細胞およびそれらの制御機構の源を研究する骨格再生を促進し、骨量減少疾患を逆転させる新規な洞察を提供する。 骨髄中の多能性間葉細胞の存在は、異なる可能性クローン原集団の同定に基づいて提案されている骨形成にiate、脂肪形成および軟骨形成の系統は、ex vivoで4。最近では、複数の研究は、骨格/間葉系幹細胞（SSCは/ MSCは）骨芽細胞の自然源であり、骨代謝回転、改造、及び骨折修復5,6のために重要であることを報告している。加えて、我々の系譜追跡研究では、成熟骨芽細胞が突然短い半減期（〜60日）があり、継続的に正常な恒常性と骨折修復の条件6の両方で彼らの幹細胞/前駆細胞によって補充されることを明らかにした。しかし、in vivoでの幹細胞のアイデンティティとどのような細胞が傷害やサプライ骨形成細胞を破砕するために反応は不明である。したがって、生理的状況下における内因性のSSC / MSCの遊走、増殖および分化を分析することができる方法を開発することが重要である。 骨折修復複合体の配列によって調節され、多細胞および動的なプロセスであるサイトカインおよび増殖因子7。骨折の研究のための最も一般的なアプローチは、長骨骨折を有する動物モデルを使用し、骨切片および免疫蛍光技法8-10によって骨を分析することである。この修復プロセスは、マイクロCT 11、近赤外蛍光12、および化学発光イメージング13を含む複数の画像化技術によってモニターすることができる。しかし、各技術は、一定の制限があり、in vivoでの細胞レベルでのSSC / MSC機能を監視する有効な方法がなかった。最近では、共焦点/二光子生体顕微鏡が開発され、動物14を生体内にあっても単細胞の解像度でそれらの骨髄微小環境のコンテキスト内で移植された癌細胞及び造血幹細胞を検出するために使用されている。系統トレース一連のモデルでこの技術を組み合わせることで、骨形成幹/前駆細胞が遺伝的に一過交流でマークすることができることを定義することができたミクソウイルス耐性-1（Mx1の ）プロモーターとMX1〜誘起前駆細胞のtivationは、時間の経過とともに成熟骨芽細胞の大部分を維持することができますが、成体マウス6に軟骨細胞の生成に関与しません。加えて、我々はMX1〜ラベルOSPCsが骨折治癒6で新しい骨芽細胞の大半を供給していることを明らかにした。 ここで、骨系統追跡モデルと生体顕微鏡を用いて、プロトコルは、骨折修復におけるMx1の+骨形成性幹/前駆細胞のin vivo動態を定義するために設けられている。このプロトコルは、骨折部位への骨形成幹/前駆細胞の移設と早期修復過程における骨前駆拡大の定量的な測定を追跡するために、順次画像を提供しています。このアプローチは、骨修復を改善するための治療候補の評価を含む複数のコンテキストで有用であり得る。

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			<td height="63" style="height:63px;width:216px;">C57BL/6J (H-2b)</td>
			<td style="width:140px;">Jackson Laboratories (Bar Harbor, ME)</td>
			<td style="width:195px;">000664</td>
			<td style="width:279px;"></td>
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			<td height="42" style="height:42px;width:216px;">Ketamine Hydrochloride Injection</td>
			<td style="width:140px;">Bionichepharma</td>
			<td style="width:195px;">67457-001-10</td>
			<td>&#160;Vial size: 10 ml (50 mg/ml)</td>
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			<td height="21" style="height:21px;width:216px;">Xylazine Sterile Solution</td>
			<td style="width:140px;">Lloyd Inc.</td>
			<td style="width:195px;">NADA# 139-236</td>
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			<td height="21" style="height:21px;width:216px;">Buprenorphine Hl</td>
			<td style="width:140px;">BEDFORD LAB</td>
			<td style="width:195px;">NDC 55390-100-10</td>
			<td>Vial: 0.3 mg/ml, Doses: 0.05-0.1 mg/kg</td>
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			<td style="width:195px;">21-031-CV</td>
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			<td height="42" style="height:42px;width:216px;">Alcohol Prep Pads (70% Isopropyl alcohol)</td>
			<td style="width:140px;">Kendall WEBCOL</td>
			<td style="width:195px;">5110</td>
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			<td height="21" style="height:21px;width:216px;">Fine Surgical Scissor</td>
			<td style="width:140px;">F.S.T</td>
			<td style="width:195px;">14568-09</td>
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			<td height="21" style="height:21px;width:216px;">Extra fine Forceps</td>
			<td style="width:140px;">F.S.T</td>
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			<td height="21" style="height:21px;width:216px;">VICRYL*Plus Suture</td>
			<td style="width:140px;">Ethicon</td>
			<td style="width:195px;">VCP490G</td>
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			<td height="42" style="height:42px;width:216px;">Qtracker 705 non-targeted quantum dot</td>
			<td style="width:140px;">Invitrogen</td>
			<td style="width:195px;">Q21061</td>
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			<td height="21" style="height:21px;width:216px;">Methocel 2%</td>
			<td style="width:140px;">OmmiVision</td>
			<td style="width:195px;"></td>
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			<td height="42" style="height:42px;width:216px;">pIpC (Polyinosinic-polycytidylic acid)&#160;&#160;</td>
			<td style="width:140px;">Sigma</td>
			<td style="width:195px;">P0913-50MG</td>
			<td>100 &#956;l (2.5 mg/ml in PBS) for 10 g of mouse</td>
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			<td height="42" style="height:42px;width:216px;">Mai Tai Tunable Ultrafast Lasers</td>
			<td style="width:140px;">Spectra Physics</td>
			<td style="width:195px;"></td>
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			<td height="42" style="height:42px;width:216px;">Dual Calypso 491 + 532 nm DPSS laser</td>
			<td style="width:140px;">Cobolt AB</td>
			<td style="width:195px;"></td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Radius-635 HeNe laser</td>
			<td style="width:140px;">Coherent</td>
			<td style="width:195px;"></td>
			<td></td>
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sequential in vivo imaging of osteogenic stem/progenitor cells during fracture repair

骨が継続的にオーバーになり、非常に再生損傷後です。骨形成幹/前駆細胞が存在して長いと仮定されているが、そのような細胞のインビボでの実証にごく最近達成された。ここで、内因性の骨形成幹/前駆細胞（OSPCs）および骨修復におけるそれらの子孫の役割を調査するためのインビボイメージング技法が提供される。モデルや頭蓋冠骨における誘導微小破壊の生体内イメージングをトレース骨系細胞を使用して、OSPCs直接の早期修復過程における重要なイベントが発生する損傷後最初の数日間に観察することができる。損傷部位を順次OSPCsが、怪我に移転数が増加し、骨形成骨芽細胞に分化することを明らかに画像化することができる。これらのメソッドは、骨の再生および修復のための幹細胞内因性および外因性の分子制御因子の役割を調査する手段を提供します。

<html>安定化長骨骨折モデルは、骨折の研究で親しまれてきた。しかしながら、長骨骨折または大きなモデルは、複数の組織損傷を引き起こし、従って、骨細胞機能の定量的測定には限界がある。我々は、針の穿孔（ 図1A-1C）で頭蓋冠前頭骨に最小侵襲性損傷（硬膜に最小限または全く浸潤1mm未満の直径）を開発した。この骨は、骨髄と平坦で薄い骨構造を有しているので、我々?...

Watch this Scientific Journal Video about Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair at JoVE.com

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

骨折治癒における骨前駆細胞機能の定量測定は、高分解能シリアルイメージング技術を必要とします。ここで、プロトコルは、画像を順次かつ骨折の修復過程における内因性の骨形成幹細胞/前駆細胞の移動、増殖および分化を定量化する生体顕微鏡と骨系統トラッキングを使用するために提供される。

シーケンシャル<em&gt;インビボ</em&gt;骨折修復中に、骨形成幹細胞/前駆細胞のイメージング

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in ...

sequential-vivo-imaging-osteogenic-stemprogenitor-cells-during

Research

JoVE Journal

Medicine

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

10.4K ビュー.  Harvard Stem Cell Institute. 骨折治癒における骨前駆細胞機能の定量測定は、高分解能シリアルイメージング技術を必要とします。ここで、プロトコルは、画像を順次かつ骨折の修復過程における内因性の骨形成幹細胞/前駆細胞の移動、増殖および分化を定量化する生体顕微鏡と骨系統トラッキングを使用するために提供される。 

ビデオ: Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

In vivo Dual Substrate Bioluminescent Imaging

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate since the advent of in vivo bioluminescent imaging technologies 1-3. Indeed, the ability to locate and quantify tumor growth longitudinally in a single cohort of animals to completion of the study as opposed to sacrificing individual groups of animals at specific assay times has revolutionized how researchers investigate breast cancer metastasis. Unfortunately, current methodologies preclude the real-time assessment of critical changes that transpire in cell signaling systems as breast cancer cells (i) evolve within primary tumors, (ii) disseminate throughout the body, and (iii) reinitiate proliferative programs at sites of a metastatic lesion. However, recent advancements in bioluminescent imaging now make it possible to simultaneously quantify specific spatiotemporal changes in gene expression as a function of tumor development and metastatic progression via the use of dual substrate luminescence reactions. To do so, researchers take advantage for two light-producing luciferase enzymes isolated from the firefly (Photinus pyralis) and sea pansy (Renilla reniformis), both of which react to mutually exclusive substrates that previously facilitated their wide-spread use in in vitro cell-based reporter gene assays 4. Here we demonstrate the in vivo utility of these two enzymes such that one luminescence reaction specifically marks the size and location of a developing tumor, while the second luminescent reaction serves as a means to visualize the activation status of specific signaling systems during distinct stages of tumor and metastasis development. Thus, the objectives of this study are two-fold. First, we will describe the steps necessary to construct dual bioluminescent reporter cell lines, as well as those needed to facilitate their use in visualizing the spatiotemporal regulation of gene expression during specific steps of the metastatic cascade. Using the 4T1 model of breast cancer metastasis, we show that the in vivo activity of a synthetic Smad Binding Element (SBE) promoter was decreased dramatically in pulmonary metastasis as compared to that measured in the primary tumor 4-6. Recently, breast cancer metastasis was shown to be regulated by changes within the primary tumor microenvironment and reactive stroma, including those occurring in fibroblasts and infiltrating immune cells 7-9. Thus, our second objective will be to demonstrate the utility of dual bioluminescent techniques in monitoring the growth and localization of two unique cell populations harbored within a single animal during breast cancer growth and metastasis.

In vivo Dual Substrate Bioluminescent Imaging

Herein we describe the methods to construct, visualize, and quantify the bioluminescent reactions of both firefly and renilla luciferase enzymes expressed in metastatic breast cancer cells during their growth and metastasis in vivo.

生体デュアル基質生物発光イメージングにおける

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate ...

医学

3D-Neuronavigation In Vivo Through a Patient's Brain During a Spontaneous Migraine Headache

A growing body of research, generated primarily from MRI-based studies, shows that migraine appears to occur, and possibly endure, due to the alteration of specific neural processes in the central nervous system. However, information is lacking on the molecular impact of these changes, especially on the endogenous opioid system during migraine headaches, and neuronavigation through these changes has never been done. This study aimed to investigate, using a novel 3D immersive and interactive neuronavigation (3D-IIN) approach, the endogenous &#181;-opioid transmission in the brain during a migraine headache attack in vivo. This is arguably one of the most central neuromechanisms associated with pain regulation, affecting multiple elements of the pain experience and analgesia. A 36 year-old female, who has been suffering with migraine for 10 years, was scanned in the typical headache (ictal) and nonheadache (interictal) migraine phases using Positron Emission Tomography (PET) with the selective radiotracer [11C]carfentanil, which allowed us to measure &#181;-opioid receptor availability in the brain (non-displaceable binding potential - &#181;OR BPND). The short-life radiotracer was produced by a cyclotron and chemical synthesis apparatus on campus located in close proximity to the imaging facility. Both PET scans, interictal and ictal, were scheduled during separate mid-late follicular phases of the patient's menstrual cycle. During the ictal PET session her spontaneous headache attack reached severe intensity levels; progressing to nausea and vomiting at the end of the scan session. There were reductions in &#181;OR BPND in the pain-modulatory regions of the endogenous &#181;-opioid system during the ictal phase, including the cingulate cortex, nucleus accumbens (NAcc), thalamus (Thal), and periaqueductal gray matter (PAG); indicating that &#181;ORs were already occupied by endogenous opioids released in response to the ongoing pain. To our knowledge, this is the first time that changes in &#181;OR BPND during a migraine headache attack have been neuronavigated using a novel 3D approach. This method allows for interactive research and educational exploration of a migraine attack in an actual patient's neuroimaging dataset.

3D-Neuronavigation In Vivo Through a Patient&#039;s Brain During a Spontaneous Migraine Headache

In this study, the authors report for the first time a novel 3D-Immersive &#38; Interactive Neuronavigation (3D-IIN) through the impact of a spontaneous migraine headache attack in the &#956;-opioid system of a patient's brain in vivo.

3D-ニューロ<em&gt;インビボ</em&gt;自発片頭痛時には、患者の脳を通じ

A growing body of research, generated primarily from MRI-based studies, shows that migraine appears to occur, and possibly endure, due to the alteration ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

細胞追跡のためのin vivoでの19 F MRI における

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with distinct functions and gene expression profiles. The roles of microglial activation in health, injury and disease remain incompletely understood due to their dynamic and complex regulation in response to changes in their microenvironment. Thus, it is critical to non-invasively monitor and analyze changes in microglial activation over time in the intact organism. In vivo studies of microglial activation have been delayed by technical limitations to tracking microglial behavior without altering the CNS environment. This has been particularly challenging during chronic neurodegeneration, where long-term changes must be tracked. The retina, a CNS organ amenable to non-invasive live imaging, offers a powerful system to visualize and characterize the dynamics of microglia activation during chronic disorders.
This protocol outlines methods for long-term, in vivo imaging of retinal microglia, using confocal ophthalmoscopy (cSLO) and CX3CR1GFP/+ reporter mice, to visualize microglia with cellular resolution. Also, we describe methods to quantify monthly changes in cell activation and density in large cell subsets (200-300 cells per retina). We confirm the use of somal area as a useful metric for live tracking of microglial activation in the retina by applying automated threshold-based morphometric analysis of in vivo images. We use these live image acquisition and analyses strategies to monitor the dynamic changes in microglial activation and microgliosis during early stages of retinal neurodegeneration in a mouse model of chronic glaucoma. This approach should be useful to investigate the contributions of microglia to neuronal and axonal decline in chronic CNS disorders that affect the retina and optic nerve.

In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

Microglia activation and microgliosis are key responses to chronic neurodegeneration. Here, we present methods for in vivo, long-term visualization of retinal CX3CR1-GFP+ microglial cells by confocal ophthalmoscopy, and for threshold and morphometric analyses to identify and quantify their activation. We monitor microglial changes during early stages of age-related glaucoma.

共焦点検眼鏡イメージングとマウス緑内障における細胞の形態計測：神経変性中に網膜ミクログリアの活性化のインビボダイナミクス

Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with ...

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest improved outcome measures in both experimental and clinical studies. Although the BMMSC are implanted into the tendon lesion in large numbers (usually 10 - 20 million cells), only a relatively small number survive (&#60;10%) although these can persist for up to 5 months after implantation. This appears to be a common observation in other species where BMMSC have been implanted into other tissues and it is important to understand when this loss occurs, how many survive the initial implantation process and whether the cells are cleared into other organs. Tracking the fate of the cells can be achieved by radiolabeling the BMMSC prior to implantation which allows non-invasive in vivo imaging of cell location and quantification of cell numbers.
This protocol describes a cell labeling procedure that uses Technetium-99m (Tc-99m), and tracking of these cells following implantation into injured flexor tendons in horses. Tc-99m is a short-lived (t1/2 of 6.01 hr) isotope that emits gamma rays and can be internalized by cells in the presence of the lipophilic compound hexamethylpropyleneamine oxime (HMPAO). These properties make it ideal for use in nuclear medicine clinics for the diagnosis of many different diseases. The fate of the labeled cells can be followed in the short term (up to 36 hr) by gamma scintigraphy to quantify both the number of cells retained in the lesion and distribution of the cells into lungs, thyroid and other organs. This technique is adapted from the labeling of blood leukocytes and could be utilized to image implanted BMMSC in other organs.

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

This protocol describes the radiolabeling of equine mesenchymal stem cells and their implantation into tendon injuries in the horse in order to determine cell survival and tissue distribution using gamma scintigraphy.

馬腱でテクネチウム-99m標識骨髄間葉系幹細胞のin vivoイメージングと追跡中

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

新しい治療戦略を開発するための膵臓癌幹細胞特性を研究

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

ヒト腫瘍異種移植片からマウス細胞の枯渇は大幅に標的細胞の下流分析を向上させます

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The amount of the different tissues in the callus provides important information on the fracture healing progress. Available in vivo techniques to longitudinally monitor the callus tissue development in preclinical fracture-healing studies using small animals include digital radiography and &#181;CT imaging. However, both techniques are only able to distinguish between mineralized and non-mineralized tissue. Consequently, it is impossible to discriminate cartilage from fibrous tissue. In contrast, magnetic resonance imaging (MRI) visualizes anatomical structures based on their water content and might therefore be able to noninvasively identify soft tissue and cartilage in the fracture callus. Here, we report the use of an MRI-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice. The experiments demonstrated that the fixator and a custom-made mounting device allow repetitive MRI scans, thus enabling longitudinal analysis of fracture-callus tissue development.

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

The evaluation of tissue development in the fracture callus during endochondral bone healing is essential to monitor the healing process. Here, we report the use of a magnetic resonance imaging (MRI)-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice.

生体内で破壊カルス開発マウス大腿骨の骨、MRI 対応のデバイスを用いたマウスの骨治癒過程の評価

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The ...

Middle Cerebral Artery Occlusion Allowing Reperfusion via Common Carotid Artery Repair in Mice

The ischemic stroke is a major cause of adult long-term disability and death worldwide. The current treatments available are limited, with only tissue plasminogen activator (tPA) as an approved drug treatment to target ischemic strokes. Current research in the field of ischemic stroke focuses on better understanding the pathophysiology of stroke, to develop and investigate novel pharmaceutical targets. Reliable experimental stroke models are crucial for the progression of potential treatments. The middle cerebral artery occlusion (MCAO) model is clinically relevant and the most frequently used surgical model of ischemic stroke in rodents. However, the outcomes of this model, such as lesion volume, are associated with high levels of variability, particularly in mice. The alternative MCAO model described here allows the reperfusion of the common carotid artery (CCA) and the increased perfusion of the middle cerebral artery (MCA) territory, using a tissue pad with fibrinogen-based sealant to repair the vessel, and the improved welfare of the mice by avoiding external carotid artery (ECA) ligation. This reduces the reliance on the Circle of Willis, which is known to be highly anatomically variable in mice. Representative data show that using this alternative surgical approach decreases the variability in lesion volumes between the traditional MCAO approach and the alternative approach described here.

Intraluminal filament occlusion of the middle cerebral artery is the most frequently used in vivo model of experimental stroke in rodents. An alternative surgical approach to allow common carotid artery repair is performed here, which allows the reperfusion of the common carotid artery and a full reperfusion to the middle cerebral artery territory.

マウスにおける一般的な頸動脈の修理で再灌流を許可する中大脳動脈閉塞

The ischemic stroke is a major cause of adult long-term disability and death worldwide. The current treatments available are limited, with only tissue ...

A Simplified Stepwise Approach to Echo Guidance during Percutaneous Mitral Valve Repair

Percutaneous transcatheter edge-to-edge reconstruction of the mitral valve is a safe and well-established therapy for severe symptomatic mitral regurgitation in patients with high surgical risk. Echocardiographic guidance in addition to fluoroscopy is the gold standard and should be performed using a standardized technique.
This article lays out our reproducible step by step echocardiographic guide including views, measurements as well as highlighting possible difficulties that may arise during the procedure.
This article provides detailed and chronological echocardiographic views for each step of the procedure, especially preferences between 2D and 3D imaging. If needed, pulse wave, continuous wave and color doppler measurements are described. Furthermore, as there are no official recommendations for the quantification of mitral regurgitation during the percutaneous edge-to-edge-repair procedure, advice is also included for echocardiographic quantification after grasping the mitral leaflets and after device deployment. In addition, the article deals with important echocardiographic views to prevent and deal with possible complications during the procedure.
Echocardiographic guidance during transcatheter mitral valve repair is mandatory. A structured approach improves the collaboration between interventionist and imager and is indispensable for a safe and effective procedure.

This protocol presents in detail how to perform real-time echocardiographic guidance during transcatheter mitral valve repair. The fundamental views and the necessary measurements are described for each stage of the procedure.