We thank Katherine Clark for technical assistance. A.D.R.G. is funded in part by 5K01MH097957-03

混合C57BL / 6バックグラウンド上の成体（3-4ヶ月齢）雄マウスは、このプロトコルで使用しました。動物は、12時間の明/暗サイクルで維持し、食物および水に自由にアクセスさせました。このプロトコルで実行されるすべての手順は、ドレクセル大学施設内動物管理使用委員会によって承認されたプロトコルに従って行いました。 1.手術領域の準備<ol><li>吸収パッドで全体の外科ベンチをカバーし、定位に隣接する手術器具を配置し、その後、70％エタノールで手術台を消毒。 </li><li>マニピュレータアームなし定位装置を設定します。定位の加熱パッドを配置し、37℃に設定してください。動物と加熱パッドとの間のペーパータオルの小片または外科パッドを配置することによって、動物の過熱を防ぎます。 </li><li>オートクレーブはさみを使用して、REAまで0.9％滅菌生理食塩水を含む滅菌ペトリ皿にオートクレーブゲルフォームの小片をカット使用するためにDY。 注意：オートクレーブ処理機器および無菌手術用消耗品を使用して滅菌作業環境を維持します。必要に応じて、10〜15秒間ビーズ滅菌器に浸漬することにより、動物の間で処置中または手術器具を再滅菌します。消毒するために、必要に応じて、70％エタノールで手を擦ることによって作業中は清潔な手袋を維持します。 </li></ol>外科2.プレッピングマウス<ol><li>ホームケージからマウスを削除し、（g）を量ります。 </li><li>場所イソフルラン誘導チャンバ内にマウスや麻酔の外科的平面、約3〜5分を誘導するために2〜5 L /分およびイソフルラン気化器に酸素を設定します。緩徐呼吸や固定化のために監視します。マウスが完全につま先ピンチ反射を使用して鎮静されていることを確認してください。 </li><li>マウスが完全に鎮静されると、イソフルランにチューブが取り付けられているノーズコーンに鼻を確保、定位フレームに配置します。外耳道に耳棒を挿入し、締め、頭が安定して確保します。 </li><li>耳にし、目の間から耳の後ろに耳から頭を剃ります。 </li><li> 、イソプロピルアルコール及びベタジンヨウ素溶液のワイプを交互に3回ずつ肌を殺菌。 </li><li>外科手術中に乾燥からそれらを防ぐために、両眼に人工涙液を適用します。 </li></ol> 3.手術手順<ol><li>つま先や尾をつまんで麻酔深度を監視します。応答がないとき、マウスは、適切な外科的な平面であり、呼吸がゆっくりとさえあります。 </li><li>ちょうどほぼ11番手術用メスの刃を使用して、単一の、しっかりした動きで耳の間に、目の後ろから矢状皮膚切開を行います。脇の皮膚を移動し、止血剤で右側をクリップ。 </li><li> 11番メスと綿の鈍い側を使用して、上層膜の明確な頭蓋骨は、アプリケーターをひっくり返しました。必要に応じて、0.9％の食塩水に浸した綿先端のアプリケーターで頭蓋骨を拭きます。完全に乾燥することができます。 </li><li>米国小さ ​​な定規をる、1ミリメートルの冠状縫合の尾、および恒久的なマーカーと矢状縫合（ 図1）に対して横1ミリメートル、で開頭の左端に開頭術の前縁をマーク。そして、矢状および冠状縫合、それぞれ（ 図1）から4ミリメートルで開頭術の権利と尾側の境界をマークします。 </li><li> 0.5ミリメートルドリルビットを使用して、恒久的なマーカーアウトライン次ゆっくり穿孔することによって開頭を作るために開始します。完全に頭蓋骨を突破しないように注意してください。を押して、優しく第5鉗子で頭頂骨の孤立片に、弱さの領域は、圧力に道を譲るだろう。薄くなった骨が周囲全体に十分に弱い場合、骨片を除去するための準備ができています。 注：調査員が困難一体に骨を除去するが発生した場合、これは、骨が十分に掘削時に間引かれなかったことを示唆しています。 facilita以降の動物で、さらに頭蓋骨をドリル考えてみましょう骨片を容易に除去するTE。 </li><li> 23 G針を装着した10mlシリンジを用いて、単離された骨と掘削面積を浸して0.9％生理食塩水を少量を適用します。 </li><li>定位機器にマニピュレータアームを取り付けます。吻方面した刃の鋭い側とプローブホルダに新しい11番手術用メスの刃を取り付けます。 注：吻側および尾側の組織が刃の鋭く尖っていないエッジを経験しているが、それぞれ、貫通損傷によって誘発される機械的な損傷は、病変の程度全体に匹敵します。私たちは、頭側と尾部の間、GFAP発現または増殖のアップレギュレーションを含む反応性神経膠症の主な機能にはかなりの違いを観察していません。 </li><li>邪魔にならないようにマニピュレータアームを保ち、慎重5/45角度のついたピンセットを用いて、単離された骨を持ち上げます。 FUL 1に残さ骨の一部をやってのけるレバレッジを使用して、孤立した骨やリフトの側面に鉗子の先端部を挿入Lの動き。 注：脳を刺すか、頭蓋骨の下に硬膜を乱さないように注意してください。 </li><li>浸した吸収性ゲルフォームの小片を取り、乾燥からそれを防止し、存在する可能性のある血液を吸収するために覆われていない脳に置きます。 </li><li>ゲルフォームが適所に配置されると、所定の位置にマニピュレータアームを揺動し、ゲルフォーム上に開頭の中央にブレードを調整します。ゲルフォームを取り外し、先端が硬膜を穿刺せずに硬膜に接触​​するまで、ブレードを下げます。定位の垂直アームのバーニアスケールを使用してマーク背/腹座標。 </li><li>マニピュレータアームを使用して、ゆっくりと脳にブレード正確に3ミリメートルを下げます。これは、マニピュレータアームにバーニヤ目盛りを使用することによって達成されます。ブレードは5〜10秒のための場所に滞在することができます。ブレードはopposiに移動する前に、開頭の吻側および尾側の境界に到達することを可能にする3回尾側するブレード取付吻側に定位アームを移動TE終了。 注：硬膜は、ブレードを挿入する前に除去されていません。厚さ7〜80μmであるラットの硬膜とは対照的に、マウスの硬膜がかなり薄くなっている（わずか数細胞層の厚さ）、挿入時のメスの刃にかなりの抵抗を生成しません。各動物が一貫性の損傷を受けることを確実にするために、各マウスのための新しい手術用メスの刃を使用してください。 </li><li>ゆっくりと脳からブレードを取り外し、定位アームを持ち上げます。ブレードを除去した後、すぐに余分な血液や体液を吸収するために、脳の表面にゲルフォームの別の部分を配置します。 </li><li>一方、定位アームを取り外し、11番手術用メスの刃を処分。出血が停止した後、ゲルフォームを削除します。 </li><li>このようなethilonまたはプロレンとして、非吸収性縫合糸で皮膚を縫合することによって傷を閉じます。縫合糸は、手術後9-10日に削除する必要があります。 </li><li>そのホームケージにマウスを返し、マウスがheatinにゆっくりと回復することができます苦痛の兆候がないかのGパッドとモニター。イソフルラン誘発性の麻酔からの回復は、典型的には、イソフルランから除去した後、2〜5分以内に発生します。それは胸骨recumbancyを取り戻したまで無人動物を放置しないでください。 </li><li>水和を確実にするために乳酸リンゲル液の皮下0.5〜1 mlで管理します。 </li></ol> 4.手術後のケア<ol><li>密接植民地に戻る前に麻酔から回復するまで、手術後の動物を監視します。 <ol><li> 、術後の痛みや不快感を軽減直ちに手順に従って、腹腔内注射により0.05〜0.1ミリグラム/ kgのブプレノルフィンを投与すること。 </li><li>このような制限された運動、グルーミングの欠如、または体重減少などの苦痛の重症徴候について術後2〜3日間動物を観察します。苦痛のこれらの兆候のいずれかを実証する動物を安楽死させると研究から取り外します。 </li></ol></li><li>損傷組織の病理組織学的検査を検討するために、スタンにより動物を安楽死させます準の心臓内灌流。 <ol><li>簡単に述べると、ケタミン/キシラジンの過剰摂取で動物を麻酔し、心臓内の0.9％NaCl 15〜20ミリリットルで灌流、または肝臓、実験の終了時に4％パラホルムアルデヒドの60ミリリットル、続いて血液のクリアされるまで。 </li></ol></li><li> 30％ショ糖溶液に移す前に、4％パラホルムアルデヒド中で2〜4時間、脳とポストフィックスを分析。セクション40〜60ミクロンで、クライオスタットの脳、および標準的な組織学的または免疫組織化学的手順による処理、またはガルシア8,9に記載されているように。 </li></ol>

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The authors have nothing to disclose.

それは、頭蓋骨や、基礎となる硬膜を掘削中に損傷していないことが重要です。頭蓋骨がパンクされていないことを確認するために掘削しながら、軽い圧力を使用してください。硬膜が骨にリフトオフされていないことを確認するために頭蓋骨片を持ち上げながら加えて、注意が必要です。 前脳刺し損傷はこちらCNSへの浸透傷害モデルを説明しました。このようなFPIやCC...

A major challenge for developing successful therapies to treat the injured CNS is an incomplete understanding of the complex multicellular events that are triggered by the trauma. Reactive astrocytes are gaining increasing recognition as a promising target for novel therapies1. Though historically regarded as hostile to neural repair, reactive astrocytes are now recognized as critical components of a complex, multicellular neuroprotective response that includes attenuation of inflammatory processes and limiting secondary damage and neurodegeneration2-6. Although the neuropathological characteristics of reactive gliosis have long been well defined, the cellular and molecular mechanisms regulating reactive gliosis, and the diverse array of downstream consequences remain poorly understood. Understanding the mechanisms that drive reactive gliosis, as well as the subsequent cellular and molecular events, is an important step towards developing strategies aimed at promoting the neuroprotective properties of reactive gliosis, while attenuating the detrimental effects. 
Here we demonstrate a method to induce severe reactive astrogliosis in the forebrain of adult mice using a stab injury. In contrast to other traumatic brain injury (TBI) models, such as controlled cortical impact (CCI) or fluid percussion injury (FPI), which require specialized equipment to produce an injury, the forebrain stab requires only a stereotaxic device to stabilize the head and a No. 11 scalpel blade. Thus the forebrain stab lesion model is more broadly accessible to a wide range of laboratories that do not have access to the specialized devices necessary for creating an FPI or CCI injury. The method described here enables investigators to reliably and reproducibly trigger a robust gliosis response to investigate subsequent cellular and molecular events. Once recovered from surgery, animals that have received a forebrain stab injury can survive for prolonged periods without the need for specialized care and can be returned to the colony for acute, intermediate, or chronic studies. Though less clinically translatable than FPI or CCI models of TBI, a forebrain lesion produced by a stab injury serves as a simple yet useful experimental model to investigate basic biological mechanisms underlying reactive gliosis and other neuropathological events following trauma to the CNS.

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			<td>Harvard Apparatus</td>
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			<td>High speed micro drill</td>
			<td>Harvard Apparatus</td>
			<td>724950</td>
			<td></td>
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		<tr>
			<td>stainless steel scalpel blade, #11</td>
			<td>MedVet</td>
			<td>JOR581S</td>
			<td></td>
		</tr>
		<tr>
			<td>5/45 angled forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-35</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelfoam sponge 12cmx7mm</td>
			<td>Fisher</td>
			<td>NC9841478</td>
			<td></td>
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			<td>Antibodies and other reagents</td>
			<td>Manufacturer</td>
			<td>Catalog Number</td>
			<td>Dilution (brightfield) 
			Dilution (fluorescence)</td>
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		<tr>
			<td>Rb anti-GFAP</td>
			<td>DAKO</td>
			<td>&#160;Z033429-2</td>
			<td>1:20k 
			1:1k</td>
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			<td>Shp anti-BrdU</td>
			<td>Abcam</td>
			<td>ab1893</td>
			<td>1:2k 
			1:500</td>
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		<tr>
			<td>Biotinylated goat anti-rabbit</td>
			<td>Vector Laboratories</td>
			<td>BA-1000&#160;</td>
			<td>1:400</td>
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		<tr>
			<td>Biotinylated rabbit anti-sheep</td>
			<td>Vector Laboratories</td>
			<td>BA-6000</td>
			<td>1:400</td>
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		<tr>
			<td>Alexafluor 488 goat anti-rabbit</td>
			<td>Life Technologies</td>
			<td>A-11008</td>
			<td>1:400</td>
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		<tr>
			<td>Alexafluor 568 donkey anti-sheep</td>
			<td>Life Technologies</td>
			<td>A-21099</td>
			<td>1:1000</td>
		</tr>
		<tr>
			<td>DAPI Nucleic Acid Stain</td>
			<td>Life Technologies</td>
			<td>D3571</td>
			<td>1:1000</td>
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		<tr>
			<td>Cresyl Violet Acetate</td>
			<td>Sigma Aldrich</td>
			<td>C5042-10G</td>
			<td>1%</td>
		</tr>
	</tbody>
</table>

triggering reactive gliosis in vivo by a forebrain stab injury

Following injury to the CNS, astrocytes undergo a broad range of biochemical, morphological, and molecular changes collectively referred to as reactive astrogliosis. Reactive astrocytes exert both inflammatory and protective effects that inhibit and promote, respectively, neural repair. The mechanisms underlying the diverse functional properties of reactive astrogliosis are not well understood. Achieving a greater understanding of these mechanisms is critical to developing therapeutic strategies to treat the injured CNS. Here we demonstrate a method to trigger reactive astrogliosis in the adult mouse forebrain using a forebrain stab lesion. This lesion model is simple, reliable, and requires only a stereotaxic device and a scalpel blade to produce the injury. The use of stab lesions as an injury model in the forebrain is well established and amenable to studies addressing a broad range of neuropathological outcomes, such as neuronal degeneration, neuroinflammation, and disruptions in the blood brain barrier (BBB). Thus, the forebrain stab injury model serves as a powerful tool that can be applied for a broad range of studies on the CNS response to trauma.

この処置を受けた動物は、専門的な術後のケアを必要としないので、短期または長期の時間の生存期間は容易に損傷後の急性または慢性の病状を調査する必要性に応じて、試験に組み込まれています。このようなソーマのGFAPおよび肥大のアップレギュレーションのような反応性神経膠症の主要な特徴は、早ければ、損傷後2〜3日のように観察することができます。反応性星状細胞のための増?...

Watch this Scientific Journal Video about Triggering Reactive Gliosis In Vivo by a Forebrain Stab Injury at JoVE.com

Triggering Reactive Gliosis In Vivo by a Forebrain Stab Injury

This article describes a detailed protocol to produce a forebrain stab injury in adult mice. The stab injury induces severe reactive gliosis and glial scar formation which can be subsequently examined by standard immunohistochemistry methods.

反応性神経膠症のトリガ<em&gt;インビボ</em&gt;前脳刺し損傷によって

Following injury to the CNS, astrocytes undergo a broad range of biochemical, morphological, and molecular changes collectively referred to as reactive ...

triggering-reactive-gliosis-in-vivo-by-a-forebrain-stab-injury

Research

JoVE Journal

Medicine

Triggering Reactive Gliosis In Vivo by a Forebrain Stab Injury

10.4K ビュー.  Drexel University. This article describes a detailed protocol to produce a forebrain stab injury in adult mice. The stab injury induces severe reactive gliosis and glial scar formation which can be subsequently examined by standard immunohistochemistry methods. 

ビデオ: Triggering Reactive Gliosis In Vivo by a Forebrain Stab Injury

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

Muscle strains are one of the most common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and physical exam alone, however the clinical presentation can vary greatly depending on the extent of injury, the patient's pain tolerance, etc. In patients with muscle injury or muscle disease, assessment of muscle damage is typically limited to clinical signs, such as tenderness, strength, range of motion, and more recently, imaging studies. Biological markers, such as serum creatine kinase levels, are typically elevated with muscle injury, but their levels do not always correlate with the loss of force production. This is even true of histological findings from animals, which provide a "direct measure" of damage, but do not account for all the loss of function. Some have argued that the most comprehensive measure of the overall health of the muscle in contractile force. Because muscle injury is a random event that occurs under a variety of biomechanical conditions, it is difficult to study. Here, we describe an in vivo animal model to measure torque and to produce a reliable muscle injury. We also describe our model for measurement of force from an isolated muscle in situ. Furthermore, we describe our small animal MRI procedure.

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

An in vivo animal model of injury is described. The method takes advantage of the subcutaneous position of the fibular nerve. Velocity, timing of muscle activation, and arc of motion are all pre-determined and synchronized using commercial software. Post injury changes are monitored in vivo using MR imaging/spectroscopy.

アン in vivoで</em収縮で誘起された損傷と回復の非侵襲的モニタリングの>齧歯類モデル

Muscle strains are one of the most  common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and  physical ...

医学

Implantation of a Carotid Cuff for Triggering Shear-stress Induced Atherosclerosis in Mice

It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce atherosclerosis (reviewed in 1 and 2). The role of shear stress has been extensively studied in vitro investigating the influence of flow dynamics on cultured endothelial cells 1,3,4 and in vivo in larger animals and humans 1,5,6,7,8. However, highly reproducible small animal models allowing systematic investigation of the influence of shear stress on plaque development are rare. Recently, Nam et al. 9 introduced a mouse model in which the ligation of branches of the carotid artery creates a region of low and oscillatory flow. Although this model causes endothelial dysfunction and rapid formation of atherosclerotic lesions in hyperlipidemic mice, it cannot be excluded that the observed inflammatory response is, at least in part, a consequence of endothelial and/or vessel damage due to ligation.
In order to avoid such limitations, a shear stress modifying cuff has been developed based upon calculated fluid dynamics, whose cone shaped inner lumen was selected to create defined regions of low, high and oscillatory shear stress within the common carotid artery 10. By applying this model in Apolipoprotein E (ApoE) knockout mice fed a high cholesterol western type diet, vascular lesions develop upstream and downstream from the cuff. Their phenotype is correlated with the regional flow dynamics 11 as confirmed by in vivo Magnetic Resonance Imaging (MRI) 12: Low and laminar shear stress upstream of the cuff causes the formation of extensive plaques of a more vulnerable phenotype, whereas oscillatory shear stress downstream of the cuff induces stable atherosclerotic lesions 11. In those regions of high shear stress and high laminar flow within the cuff, typically no atherosclerotic plaques are observed.
 
In conclusion, the shear stress-modifying cuff procedure is a reliable surgical approach to produce phenotypically different atherosclerotic lesions in ApoE-deficient mice.

The constricting cuff presented in this article is designed to induce atherosclerosis in the murine common carotid artery. Due to the conical shape of its inner lumen the implanted cuff generates well-defined regions of low, high and oscillatory shear stress triggering the development of atherosclerotic lesions of different inflammatory phenotypes.

マウスでのせん断応力誘発アテローム性動脈硬化症をトリガするための頸動脈カフの注入

It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce ...

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. ...

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. ...

Detecting Anastasis In Vivo by CaspaseTracker Biosensor

Anastasis (Greek for &#8220;rising to life&#8221;) is a recently discovered cell recovery phenomenon whereby dying cells can reverse late-stage cell death processes that are generally assumed to be intrinsically irreversible. Promoting anastasis could in principle rescue or preserve injured cells that are difficult to replace such as cardiomyocytes or neurons, thereby facilitating tissue recovery. Conversely, suppressing anastasis in cancer cells, undergoing apoptosis after anti-cancer therapies, may ensure cancer cell death and reduce the chances of recurrence. However, these studies have been hampered by the lack of tools for tracking the fate of cells that undergo anastasis in live animals. The challenge is to identify the cells that have reversed the cell death process despite their morphologically normal appearance after recovery. To overcome this difficulty, we have developed Drosophila and mammalian CaspaseTracker biosensor systems that can identify and permanently track the anastatic cells in vitro or in vivo. Here, we present in vivo protocols for the generation and use of the CaspaseTracker dual biosensor system to detect and track anastasis in Drosophila melanogaster after transient exposure to cell death stimuli. While conventional biosensors and protocols can label cells actively undergoing apoptotic cell death, the CaspaseTracker biosensor can permanently label cells that have recovered after caspase activation - a hallmark of late-stage apoptosis, and simultaneously identify active apoptotic processes. This biosensor can also track the recovery of the cells that attempted other forms of cell death that directly or indirectly involved caspase activity. Therefore, this protocol enables us to continuously track the fate of these cells and their progeny, facilitating future studies of the biological functions, molecular mechanisms, physiological and pathological consequences, and therapeutic implications of anastasis. We also discuss the appropriate controls to distinguish cells that undergo anastasis from those that display non-apoptotic caspase activity in vivo.

Detecting Anastasis In Vivo by CaspaseTracker Biosensor

Anastasis is technically challenging to detect in vivo because the cells that have reversed the cell death process can be morphologically indistinguishable from normal healthy cells. Here we describe protocols for detecting and tracking cells that undergo anastasis in live animals by using our newly developed in vivo CaspaseTracker biosensor system.

アナスタシスはIn Vivo CaspaseTracker バイオ センサーによる検出

Anastasis (Greek for &#8220;rising to life&#8221;) is a recently discovered cell recovery phenomenon whereby dying cells can reverse late-stage cell death ...

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 ...

Open Tracheostomy Gastric Acid Aspiration Murine Model of Acute Lung Injury Results in Maximal Acute Nonlethal Lung Injury

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute respiratory distress syndrome (ARDS), characterized by neutrophilic alveolitis, and injury to both alveolar epithelium and vascular endothelium. Clinically, ARDS is defined by acute onset of hypoxemia, bilateral patchy pulmonary infiltrates and non-cardiogenic pulmonary edema. Human studies have provided us with valuable information about the physiological and inflammatory changes in the lung caused by ARDS, which has led to various hypotheses about the underling mechanisms. Unfortunately, difficulties determining the etiology of ARDS, as well as a wide range of pathophysiology have resulted in a lack of critical information that could be useful in developing therapeutic strategies.
Translational animal models are valuable when their pathogenesis and pathophysiology accurately reproduce a concept proven in both in vitro and clinical settings. Although large animal models (e.g., sheep) share characteristics of the anatomy of human trachea-bronchial tree, murine models provide a host of other advantages including: low cost; short reproductive cycle lending itself to greater data acquisition; a well understood immunologic system; and a well characterized genome leading to the availability of a variety of gene deletion and transgenic strains. A robust model of low pH induced ARDS requires a murine ALI that targets mainly the alveolar epithelium, secondarily the vascular endothelium, as well as the small airways leading to the alveoli. Furthermore, a reproducible injury with wide differences between different injurious and non-injurious insults is important.
The murine gastric acid aspiration model presented here using hydrochloric acid employs an open tracheostomy and recreates a pathogenic scenario that reproduces the low pH pneumonitis injury in humans. Additionally, this model can be used to examine interaction of a low pH insult with other pulmonary injurious entities (e.g., food particles, pathogenic bacteria).

This protocol induces acute lung injury in a mouse that has close fidelity to the pathogenesis of acid pneumonitis observed in humans. We generate a maximal acute nonlethal low pH lung injury and account for differences in rodent-human anatomic respiratory structure using an open tracheostomy coupled with circumferential pressure release.

最大急性非致死肺損傷における急性肺損傷の結果のオープン気管切開胃酸吸引マウスモデル

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute ...

Reproducible Arterial Denudation Injury by Infrarenal Abdominal Aortic Clamping in a Murine Model

Percutaneous vascular interventions uniformly result in arterial denudation injuries that subsequently lead to thrombosis and restenosis. These complications can be attributed to impairments in re-endothelialization within the wound margins. Yet, the cellular and molecular mechanisms of re-endothelialization remain to be defined. While several animal models to study re-endothelialization after arterial denudation are available, few are performed in the mouse because of surgical limitations. This undermines the opportunity to exploit transgenic mouse lines and investigate the contribution of specific genes to the process of re-endothelialization. Here, we present a step-by-step protocol for creating a highly reproducible murine model of arterial denudation injury in the infrarenal abdominal aorta using external vascular clamping. Immunocytochemical staining of injured aortas for fibrinogen and &#946;-catenin demonstrate the exposure of a pro-thrombotic surface and the border of intact endothelium, respectively. The method presented here has the advantages of speed, excellent overall survival rate, and relative technical ease, creating a uniquely practical tool for imposing arterial denudation injury in transgenic mouse models. Using this method, investigators may elucidate the mechanisms of re-endothelialization under normal or pathological conditions.

Understanding the cellular and molecular mechanisms of re-endothelialization following arterial denudation injury is of paramount importance in preventing thrombosis and restenosis of arteries. Here we describe a protocol for reproducible arterial denudation injury of the infrarenal abdominal aorta. The procedure was developed to investigate the underlying mechanisms that regulate endothelial regeneration using mouse models.

マウスモデルにおける下腹部大動脈クランプすることにより、再現性動脈侵食に起因けが

Percutaneous vascular interventions uniformly result in arterial denudation injuries that subsequently lead to thrombosis and restenosis. These ...

A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System

The regrowth capacity of damaged neurons governs neuroregeneration and functional recovery after nervous system trauma. Over the past few decades, various intrinsic and extrinsic inhibitory factors involved in the restriction of axon regeneration have been identified. However, simply removing these inhibitory cues is insufficient for successful regeneration, indicating the existence of additional regulatory machinery. Drosophila melanogaster, the fruit fly, shares evolutionarily conserved genes and signaling pathways with vertebrates, including humans. Combining the powerful genetic toolbox of flies with two-photon laser axotomy/dendriotomy, we describe here the Drosophila sensory neuron &#8211; dendritic arborization (da) neuron injury model as a platform for systematically screening for novel regeneration regulators. Briefly, this paradigm includes a) the preparation of larvae, b) lesion induction to dendrite(s) or axon(s) using a two-photon laser, c) live confocal imaging post-injury and d) data analysis. Our model enables highly reproducible injury of single labeled neurons, axons, and dendrites of well-defined neuronal subtypes, in both the peripheral and central nervous system.

A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System

Here, we present a protocol using the Drosophila sensory neuron - dendritic arborization (da) neuron injury model, which combines in vivo live imaging, two-photon laser axotomy/dendriotomy, and the powerful fly genetic toolbox, as a platform for screening potential promoters and inhibitors of neuroregeneration.

再生に関する末梢および中枢神経系を研究するため生体内でショウジョウバエ損傷モデル

The regrowth capacity of damaged neurons governs neuroregeneration and functional recovery after nervous system trauma. Over the past few decades, various ...

Inducing Acute Lung Injury in Mice by Direct Intratracheal Lipopolysaccharide Instillation

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. Various approaches have been described, such as the inhalation of aerosolized LPS as well as nasal or intratracheal instillation. The presented protocol describes a detailed step-by-step procedure to induce ALI in mice by direct intratracheal LPS instillation and perform FACS analysis of blood samples, bronchoalveolar lavage (BAL) fluid, and lung tissue. After intraperitoneal sedation, the trachea is exposed and LPS is administered via a 22 G venous catheter. A robust and reproducible inflammatory reaction with leukocyte invasion, upregulation of proinflammatory cytokines, and disruption of the alveolo-capillary barrier is induced within hours to days, depending on the LPS dosage used. Collection of blood samples, BAL fluid, and lung harvesting, as well as the processing for FACS analysis, are described in detail in the protocol. Although the use of the sterile LPS is not suitable to study pharmacologic interventions in infectious diseases, the described approach offers minimal invasiveness, simple handling, and good reproducibility to answer mechanistic immunological questions. Furthermore, dose titration as well as the use of alternative LPS preparations or mouse strains allow modulation of the clinical effects, which can exhibit different degrees of ALI severity or early vs. late onset of disease symptoms.

Presented here is a step-by-step procedure to induce acute lung injury in mice by direct intratracheal lipopolysaccharide instillation and to perform FACS analysis of blood samples, bronchoalveolar lavage fluid, and lung tissue. Minimal invasiveness, simple handling, good reproducibility, and titration of disease severity are advantages of this approach.

直接気管内リポ多糖刺激によるマウスの急性肺損傷の誘導

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. ...