著者らは、これらの実験の主要な資金源として、北米小児整形外科学会のバイオメット脊椎助成金と国立衛生研究所の臨床トランスレーショナルサイエンス研究所KL2助成金、およびHH Lee Surgical Research Grantの両方を受け取ったことに感謝したいと思います。

すべての動物は、動物福祉法(AWA)、1996年の実験動物のケアと使用に関するガイド、実験動物の人道的なケアと使用に関するPHSポリシー、および動物の飼育と使用に関するトレーニングマニュアルに記載されている機関の方針と手順に規定されている連邦規制に定義されている適正動物慣行に厳密に従って取り扱われました。 また、すべての動物実験は、カリフォルニア大学ロサンゼルス校の動物研究委員会(ARC)によって承認されました。
1. 黄色ブドウ球菌 の生物発光株の選択
<ol><li>生物発光 黄色ブドウ球菌 Xen36株を目的の接種物として使用します。 注:この株は、敗血症患者からの臨床分離株である親株黄色 ブドウ球菌 ATCC-49525に由来しました。 黄色ブドウ球菌 Xen36は、最適化され、宿主のネイティブプラスミドに組み込まれた ルクスABCDEオペロンを独自に利用しています。19 その結果、Xen36株は、ピーク波長発光が490nmの青緑色の生物発光光を生成することができる。この発光シグナルは、生きている代謝活性細菌によってのみ生成されます。</li>
</ol>2. S.の準備接種のための黄色ブドウ球菌
<ol><li>ルリアブロスに200 μg/mLのカナマイシンと1.5%寒天を加えて、ルクスオペロンに結合したカナマイシン耐性遺伝子を利用して、黄色ブドウ球菌Xen36を潜在的な汚染物質から単離します19。</li>
	<li>黄色 ブドウ球菌 Xen36細菌をトリプシン大豆寒天プレート(トリプシン大豆ブロス[TSB]と1.5%寒天培地)にストリークし、37°Cで12〜16時間インキュベートします。</li>
	<li>黄色ブドウ球菌Xen36の単一コロニーを単離し、振とうインキュベーター(200rpm)で37°Cで12〜16時間TSBで個別に培養します。</li>
	<li>得られた培養液を1:50の比率で希釈します。</li>
	<li>37°Cでさらに2時間培養し、中対数期の細菌を単離します。</li>
	<li>ペレット化、再懸濁、リン酸緩衝生理食塩水(PBS)でのバクテリアの洗浄を3回行います。</li>
	<li>600nmでの吸光度を測定します。理想的なOD600は0.700〜0.750で、これは4x105 CFU/mLに相当します。段階希釈を行い、目的の細菌接種(1 x 103 CFU/2 μL)を達成します。 注:慢性感染症の確立のためのXen36の最適濃度は、1 x 103 CFUであることがわかりました。バクテリアの投与量が少ないと宿主の免疫系によって除去され、投与量が多いと創傷破壊を引き起こしました。創傷の破壊は、深部インプラント感染と表在性創傷感染を区別しないため、このモデルでは回避されます(図1)20。</li>
</ol>3.マウス
<ol><li>12週齢の雄のC57BL/6J野生型マウスを使用。</li>
	<li>ハツカネズミを一度に最大4匹のケージに入れます。</li>
	<li>常に水を利用できるようにしてください。12時間の明暗サイクルを維持し、サイクルの暗期には実験を行わないでください。</li>
	<li>給餌にはアルファルファを含まないチャウを使用し、蛍光シグナル伝達に干渉する可能性があるため、給餌に使用してください。</li>
	<li>研究スタッフまたは獣医スタッフにマウスを毎日評価してもらい、実験全体を通してマウスの健康状態を確保します。</li>
</ol>4. マウスの外科的処置
<ol><li>マウスをイソフルラン(2%)チャンバーに約5分間入れて麻酔を誘発します。呼吸が覚醒時よりもリズミカルでゆっくりと保たれ、有害な刺激(外科的操作、つま先のつまみなど)に反応して変化しないように呼吸を監視することにより、適切な麻酔深度を確認します。</li>
	<li>麻酔をかけたマウスを調製ステーションに移し、げっ歯類のバリカンで仙骨から上部胸椎までの毛を取り除きます。</li>
	<li>ベタジン溶液とイソプロピルアルコールを交互に3回洗浄して皮膚を洗浄および滅菌します。</li>
	<li>腹臥位で麻酔および滅菌したマウスを、鼻錐形を介して吸入イソフルラン(2%)を投与して麻酔を維持した無菌手術用ベッドに移します。</li>
	<li>腰を最大限に曲げ、脊椎の高さで膝の位置を特定して、腰椎4椎体に近づけます。</li>
	<li>15枚刃の外科用メスで皮膚を縦方向に2cm切開します。</li>
	<li>棘突起を触診して正中線を確認し、骨まで切開を続けます。</li>
	<li>L4棘突起の右側を骨膜下的に解剖し、横突起まで横方向に伸ばします。</li>
	<li>吸収性の編組縫合糸サイズ5-0のセファラッドとコーダッドを筋膜を通してL4ボディに通し、将来の閉鎖に備えて開いたままにします。</li>
	<li>25Gの脊椎針を使用して、25Gの脊椎針を使用してL4の棘突起をリームし、長い腕の産卵頭を持つ椎弓に沿って直径0.1mm、長さ1cmの「L字型」の外科用グレードのステンレス鋼インプラントを挿入します。</li>
	<li>1 x 103 CFU/2 μLの生物発光 黄色ブドウ球菌 Xen36をインプラントに接種し、すべての溶液がインプラントに接触するように注意します。</li>
	<li>約10秒待ってから、接種後に以前に通過した吸収性縫合糸を結び、インプラントに接種物を確実に封じ込めます。</li>
	<li>吸収性縫合糸でランニングファッションで肌を閉じます。</li>
	<li>ブプレノルフィン(0.1 mg / kg)の皮下注射により鎮痛剤を直ちに投与し、その後3日間は12時間ごとに投与します。</li>
	<li>加熱パッドでマウスを回収し、通常の活動に戻るのを監視します。</li>
	<li>術後のX線写真を入手して、インプラントの適切な配置を確認します。</li>
</ol>5. 細菌量測定のための縦断的生体内生物発光イメージング
<ol><li>マウスに吸入イソフルラン(2%)を麻酔する。呼吸が覚醒時よりもリズミカルでゆっくりと保たれ、有害な刺激(外科的操作、つま先のつまみなど)に反応して変化しないように呼吸を監視することにより、適切な麻酔深度を確認します。</li>
	<li>げっ歯類のバリカンで仙骨から胸椎の上部まで毛を取り除きます。</li>
	<li>マウスを生物発光イメージングプラットフォームの視野にロードして、in vivo生物発光イメージング(BLI)を実行します19。</li>
	<li>5分間の取得時間で生物発光シグナルを捕捉します。視野角15cm(B)の大きなビニング設定を利用します。</li>
	<li>術後0、1、3、5、7、10、14、18、21、25、28、および35日目(または特定の実験デザインに基づく他の日)に手順5.1〜5.4を繰り返して、細菌負荷を監視します。</li>
	<li>BLIデータをカラースケールで表示し、グレースケール写真に重ね合わせます。BLIソフトウェアを使用して標準的な卵形関心領域(ROI)を分離し、総フラックス(フォトン/秒)または平均最大フラックス(フォトン/秒/センチメートル2/ステラジアン)のBLIを定量化します。</li>
</ol>6. インプラントや周辺組織に付着した細菌の定量化
<ol><li>AVMAガイドラインに従って、POD 35または選択した代替の術後日でマウスを安楽死させます。二次性子宮頸部脱臼を伴う安楽死を確認します。</li>
	<li>ステップ4.3に従って背側皮膚を滅菌し、マウスを無菌手術野にうつ伏せにします。</li>
	<li>15枚刃の外科用メスを使用して、前の切開部を鋭く切開します。</li>
	<li>滅菌ハサミを使用して、L4棘突起を鈍く解剖し、外科的インプラントを特定します。</li>
	<li>ニードルドライバーを使用して、インプラントを静かにひねり、L4棘突起の位置から取り外します。</li>
	<li>滅菌鉗子とハサミを使用して、外科用インプラントのすぐ周囲の約0.1 gの棘突起骨と軟部組織を採取し、4つの鋭い均質化ビーズを備えた小さな円錐形のサイチューブに1 mLのPBSを入れます。</li>
	<li>収穫前と収穫後に円錐形チューブを計量することにより、軟部組織の重量を記録します。</li>
	<li>インプラントを0.5 mLの0.3% Tween-80 in TSBに入れ、15分間超音波処理します。</li>
	<li>得られたインプラント懸濁液を2分間ボルテックスし、12〜16時間一晩培養します。</li>
	<li>ホモジナイザーを使用して、インプラントを囲む 1 ml PBS に以前に配置した軟部組織と有棘突起を均質化します。</li>
	<li>得られた軟部組織懸濁液を5分間ボルテックスし、12〜16時間一晩培養します。</li>
	<li>一晩培養した後、インプラントと周囲の組織からCFUをそれぞれカウントします。軟部組織では収穫された総CFU/g、超音波処理されたインプラントではCFU/mLとして値を表現します。</li>
</ol>

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著者には開示すべき利益相反はありません。

脊椎のインプラント関連の感染症は、患者の転帰不良の前兆です1,2,3,4,5。体内の他の多くの領域とは異なり、脊椎の感染したハードウェアは、不安定性や神経学的障害のリスクがあるため、取り除くことができないことがよくあります。全身抗生物質療法に耐性のある?...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/60560">In vivo Mouse Model of Spinal Implant Infection</a>. The Authors section was updated from:
Benjamin V. Kelley1 
Stephen D. Zoller1 
Danielle Greig1 
Kellyn Hori1 
Nicolas Cevallos1 
Chad Ishmael1 
Peter Hsiue1 
Rishi Trikha1 
Troy Sekimura2 
Thomas Olson2 
Ameen Chaudry2 
Michael M. Le2 
Anthony A. Scaduto1 
Kevin P. Francis1 
Nicholas M. Bernthal1 
1Department of Orthopaedic Surgery, University of California Los Angeles 
2David Geffen School of Medicine, University of California Los Angeles
to:
Benjamin V. Kelley1 
Christopher Hamad1 
Stephen D. Zoller1 
Danielle Greig1 
Zeinab Mamouei1 
Rene Chun1 
Kellyn Hori1 
Nicolas Cevallos1 
Chad Ishmael1 
Peter Hsiue1 
Rishi Trikha1 
Troy Sekimura2 
Brandon Gettleman3 
Autreen Golzar2 
Adrian Lin2 
Thomas Olson2 
Ameen Chaudry2 
Michael M. Le2 
Anthony A. Scaduto1 
Kevin P. Francis1 
Nicholas M. Bernthal1 
1Department of Orthopaedic Surgery, University of California Los Angeles 
2David Geffen School of Medicine, University of California Los Angeles 
3University of South Carolina School of Medicine, University of South Carolina

An erratum was issued for:&#160;<a href="https://www.jove.com/t/60560">In vivo Mouse Model of Spinal Implant Infection</a>. The Authors section was updated.

Erratum: In vivo Mouse Model of Spinal Implant Infection

この方法の目的は、脊椎インプラント感染症(SII)の新しいマウスモデルを記述することです。このモデルは、in vivoで宿主、病原体、および/またはインプラント変数の影響を柔軟に評価するための安価で正確なツールを提供するように設計されています。このモデルで脊椎インプラント感染症の潜在的な治療法と治療戦略をテストすることは、より大きな動物モデルや臨床試験に適用する前に研究開発を導くことを目的としています。
脊椎手術後のインプラント関連の感染症は壊滅的な合併症であり、残念ながら、選択的脊椎手術を受けている患者の約3〜8%に発生します1,2,3,4,5およびマルチレベルまたは再手術を受けている患者の最大65%6。脊椎インプラント感染症の治療には、多くの場合、複数回の入院、複数回の手術、および長期の抗生物質療法が必要です。SIIは、神経学的障害、障害、死亡リスクの増加など、患者の転帰不良の前兆です。SIIの管理は非常に費用がかかり、患者1人あたり90万ドル以上の費用がかかります7。
黄色ブドウ球菌は、SII 8,9,10,11の最も一般的な病原体です。細菌は、手術中に直接、術後の傷口から、または後に血行性の広がりを介してハードウェアに種をまく可能性があります。金属インプラントの存在下で、黄色ブドウ球菌は抗生物質療法や免疫細胞から細菌を保護するバイオフィルムを形成します。感染したハードウェアの除去は、感染を効果的に根絶するのに役立つ可能性がありますが、これは、不安定化を引き起こし、神経学的障害のリスクを冒さずに脊椎で実行できないことがよくあります12。
感染したハードウェアを摘出できない場合、SIIを予防、検出、治療するための新しいアプローチが必要です。歴史的に、新しい治療法の安全性と有効性を効率的に評価するためのSIIの動物モデルは限られていました。SIIの以前の動物モデルでは、多数の動物と、コロニーカウント、組織学、培養など、安楽死を必要とするデータポイントの収集が必要でした13,14,15。これらのモデルは、縦断的なin vivoモニタリングを欠いているため、1匹の動物につき1つのデータポイントしか提供しないため、高価で非効率的です。
人工膝関節置換術感染のマウスモデルを研究した以前の研究では、感染負荷を縦断的に監視するための非侵襲的in vivo光学イメージングの価値と精度が確立されました16。生物発光の検出により、1匹の動物の細菌負荷を縦断的に、人道的、正確、効率的に定量化することができます。さらに、以前の研究では、in vivo生物発光とインプラントに接着したCFUとの間に高い相関関係があることが実証されています17。感染を経時的に追跡する能力は、インプラント関連の感染のより微妙な理解につながりました。さらに、このように縦断的感染を監視することで、抗生物質療法と新規抗菌薬の有効性を正確に評価できるようになりました16,17,18。
これらのツールを活用して、術後の脊椎インプラント感染のモデルを開発し、検証しました。提示された方法では、生物発光黄色ブドウ球菌Xen36の接種物を利用して、細菌負荷を縦断的に監視するためのSIIのin vivoマウスモデルを確立します16,17,18。この新しいモデルは、より大きな動物モデルや臨床試験に適用する前に、SIIの潜在的な検出、予防、および治療戦略を効率的にテストするための貴重なツールを提供します。

<table>
	<tbody>
		<tr>
			<td>Analytical Balance ME104</td>
			<td>Mettler Toledo</td>
			<td>30029067</td>
			<td>120 g capacity, 0.1 mg readability, backlit LCD, internal adjustment, metal base</td>
		</tr>
		<tr>
			<td>BD Bacto Tryptic Soy Broth</td>
			<td>Becton Dickinson (BD)</td>
			<td>BD 211825</td>
			<td>BD Bacto Tryptic Soy Broth (Soybean-Casein Digest Medium)</td>
		</tr>
		<tr>
			<td>Biomate 3S UV-VIS Spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>840-208300</td>
			<td>Spectrophotometer; Thermo Scientific; BioMate 3S; Six-position cell holder; Spectral bandwidth: 1.8nm; Long-life xenon lamp; Store up to 40 test methods; 16L x 13W x 9 in. H; 19 lb.; 100/240V US line cord</td>
		</tr>
		<tr>
			<td>Bioshield 720+ swinging bucket rotor</td>
			<td>Thermo Scientific</td>
			<td>75003183</td>
			<td>Rotor, Swinging bucket; Thermo Scientific; BIOShield 720 high speed; Capacity: 4 x 180mL (0.72L); Angle: 90 deg. ; Max. speed/RCF: 6300rpm/7188 x g; Max. radius: 16.2cm</td>
		</tr>
		<tr>
			<td>Branson Ultrasonics 2510R-MTH (Sonicator)</td>
			<td>Branson Ultrasonics</td>
			<td>CPX952217R</td>
			<td>*similar model, our model is discontinued* Branson Ultrasonics MH Series Heated Ultrasonic Cleaning Bath, 120V, 0.75 gal</td>
		</tr>
		<tr>
			<td>Bullet Blender Storm Homogenizer</td>
			<td>Next Advance</td>
			<td>BBY24M</td>
			<td>The Bullet Blender Storm is the most powerful member of the Bullet Blender family. Homogenize up to 24 of your toughest samples (mouse femur, skin, cartilage, tumor, etc.) in just minutes. Air cooling&#8482; minimizes sample heat up. Uses 1.5ml screw-cap RINO&#174; tubes or snap-cap Eppendorf&#174; Safe-lock&#8482; tubes.</td>
		</tr>
		<tr>
			<td>Germinator 500</td>
			<td>Electron Microscopy Sciences</td>
			<td>66118-10</td>
			<td>The Germinator 500 is designed to decontaminate metal micro-dissecting instruments only. It is to be 
			used exclusively for research purposes. The Germinator 500 should not be used as a substitute for 
			traditional methods of terminal sterilization. Effective sterilization cannot be assured due to lack of routine 
			sterilization-efficacy monitoring methods for glass bead sterilization. The Germinator 500 has been 
			designed and built to pass the Validation of Dry Sterilizer Spore Suspension Test: USP XXIII, Part 1211.</td>
		</tr>
		<tr>
			<td>Heracell 150i CO2 Incubator</td>
			<td>Thermo Scientific</td>
			<td>51026282</td>
			<td>Single 150L</td>
		</tr>
		<tr>
			<td>IVIS Lumina X5 Imaging System</td>
			<td>Perkin Elmer</td>
			<td>CLS148590</td>
			<td>The IVIS Lumina X5 high-throughput 2D optical imaging system combines high-sensitivity bioluminescence and fluorescence with high-resolution x-ray into a compact system that fits on your benchtop. With an expanded 5 mouse field of view for 2D optical imaging plus our unique line of accessories to accelerate setup and labeling, it has never been easier or faster to get robust data&#8212;and answers&#8212;on anatomical and molecular aspects of disease.</td>
		</tr>
		<tr>
			<td>MAXQ 4450 Digtial Incubating Bench Shaker</td>
			<td>Thermo Scientific</td>
			<td>SHKE4450</td>
			<td>Shaker, Incubated; Thermo Scientific; Digital; MaxQ 4450; Speed 15 to 500rpm +/-1rpm; 5 deg. C above ambient to 80 deg. C; 120V 50/60Hz</td>
		</tr>
		<tr>
			<td>PBS, Phosphate Buffered Saline</td>
			<td>Fisher Bioreagents</td>
			<td>BP24384</td>
			<td>PBS, Phosphate Buffered Saline, 1X Solution, pH 7.4</td>
		</tr>
		<tr>
			<td>Sorvall Legend Micro 21 Centrifuge, Ventilated</td>
			<td>Thermo Scientific</td>
			<td>75002436</td>
			<td>24 x 1.5/2.0mL rotor with ClickSeal biocontainment lid</td>
		</tr>
		<tr>
			<td>SORVALL LEGEND X1R 120V Centrifuge</td>
			<td>Thermo Scientific</td>
			<td>75004261</td>
			<td>Centrifuge, Benchtop; Thermo Scientific; Sorvall Legend X1R (Refrigerated), 1L capacity; Max. Speed/RCF 15,200rpm/25,830 x g; CFC-free cooling -10C to +40C; 120V 60Hz</td>
		</tr>
		<tr>
			<td>Staphylococcus aureus - Xen36</td>
			<td>Perkin Elmer</td>
			<td>119243</td>
			<td>Staphylococcus aureus - Xen36 bioluminescent pathogenic bacteria for in vivo and in vitro drug discovery. This product was derived from a parental strain from the American Type Culture Collection, used under license. Staph. aureus-Xen36 possesses a stable copy of the Photorhabdus luminescens lux operon on the native plasmid.</td>
		</tr>
		<tr>
			<td>TUTTNAUER AUTOCLAVE 2540E 120V</td>
			<td>Heidolph Tuttnauer</td>
			<td>23210401</td>
			<td>Sterilizer, Benchtop; Heidolph; Tuttnauer; Model 2540E; Self-contained design with refillable reservoir controls water purity for sterilization; 120V 50/60Hz; 1400w. With electronic controls</td>
		</tr>
		<tr>
			<td>Tween 80</td>
			<td>Fisher Bioreagents</td>
			<td>BP338-500</td>
			<td>Tween 80, Fisher BioReagents, Non-ionic detergent for selective protein extraction</td>
		</tr>
		<tr>
			<td>Vortex mixer VX-200</td>
			<td>Labnet Internation</td>
			<td>S0200</td>
			<td>120V touch or continuous mixer, 230V: 0 - 2,850 rpm,120V: 0 - 3,400 rpm</td>
		</tr>
		<tr>
			<td>0.9% Sodium Chloride</td>
			<td>Pfizer Injectables/Hospira</td>
			<td>00409-4888-10</td>
			<td>0.9% Sodium Chloride Injection, USP</td>
		</tr>
	</tbody>
</table>

in vivo mouse model of spinal implant infection

脊椎インプラント感染症は、診断が困難であり、外科的根絶が機械的脊椎の安定性と相反するため、予後不良の前兆です。この方法の目的は、脊椎インプラント感染症の潜在的な治療法と治療戦略をテストするための安価で迅速、かつ正確なin vivoツールを提供するために作成された脊椎インプラント感染症(SII)の新しいマウスモデルを説明することです。
この方法では、12週齢のC57BL/6J野生型マウスのL4棘突起にステンレス鋼のkワイヤーを固定し、黄色ブドウ球菌Xen36細菌の生物発光株の1 x 103 CFUを接種する後方アプローチ脊椎手術のモデルを提示します。次に、マウスを術後0、1、3、5、7、10、14、18、21、25、28、および35日目のin vivoで生物発光について縦断的に画像化します。標準化された視野からの生物発光イメージング(BLI)信号を定量化して、in vivoの細菌負荷を測定します。
インプラントやインプラント周囲組織に付着した細菌を定量するために、マウスを安楽死させ、インプラントや周囲の軟部組織を採取します。バクテリアは超音波処理によってインプラントから分離され、一晩培養され、次にコロニー形成単位(CFU)がカウントされます。この方法から得られた結果には、in vivo 黄色ブドウ球菌 の生物発光によって測定された縦断的細菌数(平均最大フラックス)と安楽死後のCFU数が含まれます。
これまでの脊椎感染症の動物モデルには、侵襲的なex vivo組織分析が含まれていましたが、この論文で紹介するSIIのマウスモデルは、生物発光細菌の非侵襲的でリアルタイムのin vivo光学イメージングを活用して、静的組織研究に取って代わります。このモデルの用途は多岐にわたり、代替の生物発光細菌株の利用、宿主の免疫応答を同時に研究するための他の種類の遺伝子改変マウスの組み込み、抗生物質やインプラントコーティングなどの新しい診断および治療モダリティの現在または調査が含まれます。

ここで紹介する手順は、SIIのin vivoマウスモデルにおける抗生物質レジメンの有効性を評価するために使用されました。具体的には、バンコマイシンとリファンピンの併用抗生物質療法の有効性を、バンコマイシン単剤療法および未治療の感染対照と比較した。
手術に先立ち、マウスは併用療法、単剤療法、または感染対照のいずれかに無作為に割り付けられた。サンプ?...

Watch this Scientific Journal Video about In Vivo Mouse Model of Spinal Implant Infection at JoVE.com

In Vivo Mouse Model of Spinal Implant Infection

プロトコルはステンレス鋼kワイヤー インプラントが生物発光黄色 ブドウ球 菌Xen36と感染する脊髄インプラント伝染の新しい生体内のマウス モデルを記述する。細菌負荷は、生物発光イメージングで縦断的に監視され、安楽死後のコロニー形成ユニット数で確認されます。

脊椎インプラント感染のin vivoマウスモデル

Spine implant infections portend poor outcomes as diagnosis is challenging and surgical eradication is at odds with mechanical spinal stability. The ...

in-vivo-mouse-model-of-spinal-implant-infection

Research

JoVE Journal

Medicine

2.2K ビュー.  University of California Los Angeles. プロトコルはステンレス鋼kワイヤー インプラントが生物発光黄色 ブドウ球 菌Xen36と感染する脊髄インプラント伝染の新しい生体内のマウス モデルを記述する。細菌負荷は、生物発光イメージングで縦断的に監視され、安楽死後のコロニー形成ユニット数で確認されます。

ビデオ: In Vivo Mouse Model of Spinal Implant Infection

Ex Vivo Infection of Live Tissue with Oncolytic Viruses

Oncolytic Viruses (OVs) are novel therapeutics that selectively replicate in and kill tumor cells1. Several clinical trials evaluating the effectiveness of a variety of oncolytic platforms including HSV, Reovirus, and Vaccinia OVs as treatment for cancer are currently underway2-5. One key characteristic of oncolytic viruses is that they can be genetically modified to express reporter transgenes which makes it possible to visualize the infection of tissues by microscopy or bio-luminescence imaging6,7. This offers a unique advantage since it is possible to infect tissues from patients ex vivo prior to therapy in order to ascertain the likelihood of successful oncolytic virotherapy8. To this end, it is critical to appropriately sample tissue to compensate for tissue heterogeneity and assess tissue viability, particularly prior to infection9. It is also important to follow viral replication using reporter transgenes if expressed by the oncolytic platform as well as by direct titration of tissues following homogenization in order to discriminate between abortive and productive infection. The object of this protocol is to address these issues and herein describes 1. The sampling and preparation of tumor tissue for cell culture 2. The assessment of tissue viability using the metabolic dye alamar blue 3. Ex vivo infection of cultured tissues with vaccinia virus expressing either GFP or firefly luciferase 4. Detection of transgene expression by fluorescence microscopy or using an In Vivo Imaging System (IVIS) 5. Quantification of virus by plaque assay. This comprehensive method presents several advantages including ease of tissue processing, compensation for tissue heterogeneity, control of tissue viability, and discrimination between abortive infection and bone fide viral replication.

Ex Vivo Infection of Live Tissue with Oncolytic Viruses

Oncolytic viruses are promising for cancer therapeutics. The ability to ascertain the infectability of live tissue specimens obtained from patients prior to treatment is a unique advantage of this therapeutic approach. This protocol describes how to process tissues for ex vivo infection with oncolytic virus and subsequent viral quantification.

腫瘍溶解性ウイルスと生きている組織のex vivoでの感染

Oncolytic Viruses (OVs) are novel therapeutics that selectively replicate in and kill tumor cells1. Several clinical trials evaluating the effectiveness ...

医学

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher frequency of fractures 1. As these fractures often occur in regions that have lost normal sensory function, the patient is at a greater risk of fracture-dependent pathologies, including death. SCI-dependent loss in both bone mineral density (BMD, grams/cm2) and bone mineral content (BMC, grams) has been attributed to mechanical disuse 2, aberrant neuronal signaling 3 and hormonal changes 4. The use of rodent models of SCI-induced osteoporosis can provide invaluable information regarding the mechanisms underlying the development of osteoporosis following SCI as well as a test environment for the generation of new therapies 5-7 (and reviewed in 8). Mouse models of SCI are of great interest as they permit a reductionist approach to mechanism-based assessment through the use of null and transgenic mice. While such models have provided important data, there is still a need for minimally-invasive, reliable, reproducible, and quantifiable methods in determining the extent of bone loss following SCI, particularly over time and within the same cohort of experimental animals, to improve diagnosis, treatment methods, and/or prevention of SCI-induced osteoporosis.
An ideal method for measuring bone density in rodents would allow multiple, sequential (over time) exposures to low-levels of X-ray radiation. This study describes the use of a new whole-animal scanner, the IVIS Lumina XR (Caliper Instruments) that can be used to provide low-energy (1-3 milligray (mGy)) high-resolution, high-magnification X-ray images of mouse hind limb bones over time following SCI. Significant bone density loss was seen in the tibiae of mice by 10 days post-spinal transection when compared to uninjured, age-matched control (na&iuml;ve) mice (13% decrease, p&lt;0.0005). Loss of bone density in the distal femur was also detectable by day 10 post-SCI, while a loss of density in the proximal femur was not detectable until 40 days post injury (7% decrease, p&lt;0.05). SCI-dependent loss of mouse femur density was confirmed post-mortem through the use of Dual-energy X-ray Absorptiometry (DXA), the current "gold standard" for bone density measurements. We detect a 12% loss of BMC in the femurs of mice at 40 days post-SCI using the IVIS Lumina XR. This compares favorably with a previously reported BMC loss of 13.5% by Picard and colleagues who used DXA analysis on mouse femurs post-mortem 30 days post-SCI 9. Our results suggest that the IVIS Lumina XR provides a novel, high-resolution/high-magnification method for performing long-term, longitudinal measurements of hind limb bone density in the mouse following SCI.

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

A longitudinal examination of bone loss in the femurs and tibiae of adult mice was performed following spinal cord injury using sequential low-dose X-ray scans. Tibia bone loss was detected throughout the study, while bone loss in the femur was not detected until 40 days post injury.

小説を使用して、脊髄損傷後のマウス後肢の骨の損失の経時的評価、 in vivoで、方法論

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher ...

Clinical Testing and Spinal Cord Removal in a Mouse Model for Amyotrophic Lateral Sclerosis (ALS)

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder resulting in progressive degeneration of motoneurons. Peak of onset is around 60 years for the sporadic disease and around 50 years for the familial disease. Due to its progressive course, 50% of the patients die within 30 months of symptom onset. In order to evaluate novel treatment options for this disease, genetic mouse models of ALS have been generated based on human familial mutations in the SOD gene, such as the SOD1 (G93A) mutation. Most important aspects that have to be evaluated in the model are overall survival, clinical course and motor function. Here, we demonstrate the clinical evaluation, show the conduction of two behavioural motor tests and provide quantitative scoring systems for all parameters. Because an in depth analysis of the ALS mouse model usually requires an immunohistochemical examination of the spinal cord, we demonstrate its preparation in detail applying the dorsal laminectomy method. Exemplary histological findings are demonstrated. The comprehensive application of the depicted examination methods in studies on the mouse model of ALS will enable the researcher to reliably test future therapeutic options which can provide a basis for later human clinical trials.

Clinical Testing and Spinal Cord Removal in a Mouse Model for Amyotrophic Lateral Sclerosis ALS

A mouse model for amyotrophic lateral sclerosis (ALS) is examined clinically and behaviorally. As a prerequisite for an accompanying immunohistological analysis the preparation of the spinal cord is depicted in detail.

臨床試験および筋萎縮性側索硬化症（ALS）のマウスモデルにおける脊髄の除去

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder resulting in progressive degeneration of motoneurons. Peak of onset is around 60 ...

A Contusion Model of Severe Spinal Cord Injury in Rats

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is described to obtain a consistent model of severe SCI. Use of a stereotactic frame and computer controlled impactor allows for creation of reproducible injury. Hypothermia and urinary tract infection pose significant challenges in the post-operative period. Careful monitoring of animals with daily weight recording and bladder expression allows for early detection of post-operative complications. The functional results of this contusion model are equivalent to transection models. The contusion model can be utilized to evaluate the efficacy of both neuroprotective and neuroregenerative approaches.

A contusion model of severe spinal cord injury is described. Detailed pre-operative, operative and post-operative steps are described to obtain a consistent model.

ラットにおける重度の脊髄損傷挫傷モデル

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is ...

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  &amp;#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 ...

Controlled Cortical Impact Model for Traumatic Brain Injury

Every year over a million Americans suffer a traumatic brain injury (TBI). Combined with the incidence of TBIs worldwide, the physical, emotional, social, and economical effects are staggering. Therefore, further research into the effects of TBI and effective treatments is necessary. The controlled cortical impact (CCI) model induces traumatic brain injuries ranging from mild to severe. This method uses a rigid impactor to deliver mechanical energy to an intact dura exposed following a craniectomy. Impact is made under precise parameters at a set velocity to achieve a pre-determined deformation depth. Although other TBI models, such as weight drop and fluid percussion, exist, CCI is more accurate, easier to control, and most importantly, produces traumatic brain injuries similar to those seen in humans. However, no TBI model is currently able to reproduce pathological changes identical to those seen in human patients. The CCI model allows investigation into the short-term and long-term effects of TBI, such as neuronal death, memory deficits, and cerebral edema, as well as potential therapeutic treatments for TBI.

Traumatic brain injuries (TBIs) remain a serious health problem. Using the controlled cortical impact surgery model, research on the effects of TBI and possible treatment methods may be performed.

外傷性脳損傷のための制御皮質衝撃モデル

Every year over a million Americans suffer a traumatic brain injury (TBI).  Combined with the incidence of TBIs worldwide, the physical, emotional, ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

マウス収縮圧力を測定するために子宮内テレメトリ<em&gt;インビボ</em

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...

A Neonatal Mouse Spinal Cord Compression Injury Model

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal cord. A major current area of research is focused on the mechanisms of adaptive plasticity that underlie spontaneous or induced functional recovery following SCI. Spontaneous functional recovery is reported to be greater early in life, raising interesting questions about how adaptive plasticity changes as the spinal cord develops. To facilitate investigation of this dynamic, we have developed a SCI model in the neonatal mouse. The model has relevance for pediatric SCI, which is too little studied. Because neural plasticity in the adult involves some of the same mechanisms as neural plasticity in early life1, this model may potentially have some relevance also for adult SCI. Here we describe the entire procedure for generating a reproducible spinal cord compression (SCC) injury in the neonatal mouse as early as postnatal (P) day 1. SCC is achieved by performing a laminectomy at a given spinal level (here described at thoracic levels 9-11) and then using a modified Yasargil aneurysm mini-clip to rapidly compress and decompress the spinal cord. As previously described, the injured neonatal mice can be tested for behavioral deficits or sacrificed for ex vivo physiological analysis of synaptic connectivity using electrophysiological and high-throughput optical recording techniques1. Earlier and ongoing studies using behavioral and physiological assessment have demonstrated a dramatic, acute impairment of hindlimb motility followed by a complete functional recovery within 2 weeks, and the first evidence of changes in functional circuitry at the level of identified descending synaptic connections1.

This article describes a method for generating a reproducible spinal cord compression injury (SCI) in the neonatal mouse. The model provides an advantageous platform for studying mechanisms of adaptive plasticity that underlie spontaneous functional recovery.

新生児マウス脊髄圧縮損傷モデル

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal ...

Stem-cell Based Engineered Immunity Against HIV Infection in the Humanized Mouse Model

With the rapid development of stem cell-based gene therapies against HIV, there is pressing requirement for an animal model to study the hematopoietic differentiation and immune function of the genetically modified cells. The humanized Bone-marrow/Liver/Thymus (BLT) mouse model allows for full reconstitution of a human immune system in the periphery, which includes T cells, B cells, NK cells and monocytes. The human thymic implant also allows for thymic selection of T cells in autologous thymic tissue. In addition to the study of HIV infection, the model stands as a powerful tool to study differentiation, development and functionality of cells derived from hematopoietic stem cells (HSCs). Here we outline the construction of humanized non-obese diabetic (NOD)-severe combined immunodeficient (SCID)-common gamma chain knockout (c&#947;-/-)-Bone-marrow/Liver/Thymus (NSG-BLT) mice with HSCs transduced with CD4 chimeric antigen receptor (CD4CAR) lentivirus vector. We show that the CD4CAR HSCs can successfully differentiate into multiple lineages and have anti-HIV activity. The goal of the study is to demonstrate the use of NSG-BLT mouse model as an in vivo model for engineered immunity against HIV. It is worth noting that, because lentivirus and human tissue is used, experiments and surgeries should be performed in a Class II biosafety cabinet in a Biosafety Level 2 (BSL2) with special precautions (BSL2+) facility.

This protocol describes the methods in constructing a humanized bone-marrow/liver/thymus mouse model with stem cell-based engineered immunity against HIV infection.

ヒト化マウスモデルにおけるHIV感染に対する幹細胞ベースの設計さイミュニティ

With the rapid development of stem cell-based gene therapies against HIV, there is pressing requirement for an animal model to study the hematopoietic ...

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 ...