この研究は、国立神経疾患·脳卒中研究所、国立衛生研究所（VVD）と神経疾患や脳卒中の国立研究所からの補助金、R21 NS064403によって、ARRA（VVD）とR21を介して国立衛生研究所からの助成金R01NS062842によってサポートされていました生物医学イメージングとバイオエンジニアリングEB006301国立研究所、国立衛生研究所（VVD）。

この方法は、ホルムアルデヒド固定、パラフィン包埋組織切片に適しています。技術は、ラットにおける実験的ストロークにここにそのアプリケーションで実証されている。 1。チラミドシグナル増幅（TSA）システムのコンポーネントの準備 TSA系増強がこの反応のために調製し、標識の最終段階で使用されているが1時間以上行うことができる。したがって、標識化を開始する前に、TSA試薬を作るために便利である。 <ol><li>蛍光団のチラ原液を作る。 TSAラベリングキットと個々のバイアルで供給される蛍光体チラミド試薬を使用してください。チラミド試薬バイアルに300μlのDMSOを追加します。 4℃で保存した場合にフルオロフォアチラミドストック溶液は、少なくとも3ヶ月間安定である</li><li> TNTを含む洗浄緩衝液作る：0.1 Mトリス - 塩酸、pH 7.5; 0.15MのNaCl; 0.05％のTween-20。必要な場合、0.3％トリトンX-100、0.05％のTween-20の代わりに用いることができる。 Alternatively PBS洗浄緩衝液として使用してもよい。 </li><li>作るTNB含むバッファー、ブロッキング：0.1 Mトリス塩酸、pH 7.5; 0.15MのNaCl; 0.5％ブロッキング試薬（キットに付属）。攪拌しながら完全にブロッキング試薬を溶解するために、徐々にバッファに少しずつ追加します。連続的に攪拌しながら55℃に徐々にこのソリューションを加熱する。これは30〜60分かかる場合があります。解決策は、乳白色表示されます。使用前に室温に戻します。長期使用のために-20℃で一定分量や店舗。 </li></ol>セクションの2。準備ホルマリン固定組織から切り出したマウント5ミクロン厚の切片をスライドを使用してください。 <ol><li> 15分間キシレンでセクションを扱う。 </li><li>次のようにセクションを再水：5分毎に96％エタノール2倍につかる。 5分、80％エタノールに浸し; 5分間ずつの水2Xで洗浄する。 </li><li> PBS中50μg/ mlのにプロテイナーゼKストック溶液を希釈することにより、プロテアーゼKワーキング溶液を作る。 </li><li>プロテイナーゼを追加セクションへの使用液のK。 23℃で15分間インキュベート溶液の体積と消化の時間は、切片および組織型のサイズに応じて調整する必要があるかもしれない。小さな（直径5mm以下の）セクションのプロテイナーゼ溶液50μlで十分です。平均サイズ（直径〜10ミリメートル）のセクションでは、プロテアーゼK溶液100μlを加える。非常に大規模なセクションの場合、溶液の体積をスケールアップすることができます。全ての場合において、信号の損失を完了するのにつながり、組織形態を破壊する部分の過剰消化しないように注意してください。インキュベーション時間は25分を超えた場合、これが頻繁に発生。 </li><li> 5分ごとに蒸留水を2倍のセクションを洗ってください。のセクションでは、水中にいる間、アクティブプローブを含む標識ミックスを準備します。 </li></ol> 3。VACC TOPOベースの染色<ol><li>次を組み合わせることにより、アクティブ·プローブ·ソリューションを準備します。 水 - 11.75μL 1 Mトリス-HCl、pHが7.4から1.25μL 12塩基オーバーハング自殺オリゴヌクレオチド100ピコモル - 1μL アダプターオリゴヌクレオチドの100ピコモル - 1μL 50 pmolのVACC TOPO - 10μL </li><li>ピペッティングにより穏やかに混合します。プローブの活性化を可能にするために15分間室温（23℃）でインキュベートする。 </li><li>洗浄瓶からのセクションを取り、残りの水を吸引する。次に、ステップ3.1（〜10×10ミリメートルのセクションごとに25μL）で調製したアクティブ·プローブ·ソリューションを適用します。 </li><li>セクションをカバースリップし、23℃で15分間インキュベートディップは優しくカバースリップを除去するために水にスライドする。 5分間ずつ水3Xで洗う。 </li></ol> 4。TSA信号強調<ol><li> TNBは、先に調製し、室温（23℃）で30分間加湿チャンバー内でスライドをインキュベートし、ブロッキング緩衝液とともに組織切片を覆う。 </li><li> TNBはブロッキング緩衝液中のストレプトアビジン - 西洋ワサビペルオキシダーゼ結合体（SA-HRP）の1:100希釈液を準備します。吸引物TNBSA-HRP溶液を緩衝して適用ブロッキング。室温で30分間SA-HRPでスライドをインキュベートする。一般的には組織切片、セクションごとに100μlをカバーするために十分な試薬量を使用してください。 </li><li>洗浄は、室温でのTNT洗浄バッファー（またはPBS）中で5分間ずつ3回スライドさせる。 </li><li>キットから1X増幅希釈液中の蛍光団のチラミドストックの1:50希釈を準備します。各スライドの上に蛍光体チラ作業溶液をピペット。完全に一般的に組織切片、セクションごとに100μlをカバーするのに十分な実用的なソリューションを使用してください。すぐに各ラベルの前にフルオロフォアチラ作業溶液を作る。解決策は再利用なので、標識した後、それの未使用部分を破棄することはできません。 </li><li> 3〜10分間室温でスライドをインキュベートする。構造体の最初のヒントを見ることができるまで、顕微鏡下で蛍光の増加をモニターする。すぐにステップを洗浄するために進んでください。 </li><li>洗浄は室温tのTNT洗浄緩衝液（またはPBS）中で5分間ずつ3回スライドさせる攪拌しながらemperature。 </li><li> DAPIおよびカバーガラスで褪色防止ソリューションを追加します。蛍光顕微鏡で観察します。 DAPI及びFITC蛍光を可視化するために使用するバンドパスフィルターセット： FITC励起D490/40、発光10分の520; DAPI励起D360/40、放射20分の460、または類似。 </li></ol>

<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。

このビデオでは、局所性脳虚血のどの明細胞死アクティブ食細胞を標識する方法を、組織切片の形式で、示しています。提示された技術は、具体的に飲み込まれ、積極的に細胞の死体を消化しているだけの廃棄物管理の細胞を標識する最初のアプローチである。これは、この機能を持っていない貪食細胞の標識化のための他の既存の方法に関して、それが有利になります。 <p class="jove_conte...

虚血性脳卒中は、影響を受けた脳組織の大きな変化を誘導する。これは、ミクログリア由来の常在食細胞の急速な活性化をもたらす大量の細胞死を開始する。また、好中球、マクロファージ、樹状、および肥満細胞1,2を含む血液由来のプロ食細胞の様々なタイプによる虚血性脳の浸潤をオフに設定します。 それはまだ、虚血性傷害に対するこの応答は、正または負の役割を果たしているかどうか議論されている。それは死んだ細胞をクリアし、炎症を抑制するため、脳卒中後の貪食が有益であることができますが、それはまた、ニューロンの生存に影響を与えるし、組織損傷1-5を悪化させる有毒な活性酸素種を生成します。 食細胞にはいくつかの種類が虚血性脳を浸透しながら、それらのすべては、細胞の死体を巻き込むと再生プロセス1-3の道をクリアすることによって、廃棄物管理に参加しています。このCR脳卒中における細胞死の貪食クリアランスを行う廃棄物管理の細胞の選択的同定のための要件をeat​​es。このような積極的な廃棄物管理の細胞を撮像する場合、それは彼らが巻き込ま細胞死体を劣化させる方法を効率的に質問に答えることも重要です。それは、自己免疫の病理学および核物質6のリリースを防止するので、食作用で死んで細胞のDNAの効果的かつ完全な分解が不可欠である。 ここでは、アクティブな食細胞のマーカーとして、特定のDNA切断を使用する新しいプローブを提示する。これらの署名ブレークは、排他的な機能の廃棄物管理の細胞内で包ま核の崩壊の間に生産されています。したがってプローブは、選択的に巻き込むだけ食細胞を標識し、積極的に細胞の死体を消化。彼らはまた、貪食後の強度とDNAの分解が完了したことを観察することが可能になる。プローブは、強度とefficiの評価に役立つ貪食クリアランスency。 新しいプローブは、非タンパク質ベースのマーカーに依存しているため、広範な虚血性損傷が細胞形態を混乱させるか、特にコア虚血性ゾーン内に、タンパク質ベースのマーカーを枯渇することができ、脳卒中の研究に特に有利であり得る。 技術の原理を図1に示す。図は、酵素、ワクシニアトポイソメラーゼ（VACC TOPO）によって連結し、ヘアピン型オリゴヌクレオチドプローブを示している 5&#39;OH DNAにDNA消化7時のリソソームのDNase IIによって生成され終了します。

<table border="0" cellpadding="0" cellspacing="0" width="696">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="34">
			<td height="34" style="height:34px;width:312px;">Name of the reagent or material</td>
			<td style="width:99px;">Company</td>
			<td style="width:157px;">Catalogue number</td>
			<td style="width:129px;">Comments (optional)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Xylene</td>
			<td>JT Baker</td>
			<td>9490-22</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Proteinase K</td>
			<td>Roche</td>
			<td>3115879001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS Buffer</td>
			<td>Invitrogen</td>
			<td>AM9624</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vaccinia Topoisomerase I</td>
			<td colspan="2">Vivid Technologies</td>
			<td>5 pmol/&#956;L stock&#160;</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">12-Base Overhang Suicide Oligonucleotide</td>
			<td>IDT DNA</td>
			<td></td>
			<td>5&#8217;-AAG GGA CCT GCB GCA GGT CCC TTG ATA CGA TTC TA -3&#8217; ; B &#8211; Biotin-dT; 100 pmol/&#956;L stock in ddH2O&#160;&#160;</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">Adapter Oligonucleotide</td>
			<td>IDT DNA</td>
			<td></td>
			<td>5&#8217;-TAG AAT CGT ATC-3&#8217;; 100 pmol/&#956;L stock in ddH2O</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 M Tris-HCl, PH 7.4</td>
			<td>Lonza</td>
			<td>51237</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TSA, Fluorescein System&#160;</td>
			<td>PerkinElmer&#160;</td>
			<td>NEL701A001KT</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 M NaCl</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMSO&#160;</td>
			<td>Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween-20&#160;</td>
			<td>Sigma</td>
			<td>P9416-50ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vectashield with DAPI</td>
			<td>Vector Laboratories</td>
			<td>H-1200</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Coverslips</td>
			<td></td>
			<td></td>
			<td style="width:129px;">22x22mm or 22x40mm glass or plastic coverslips. Plastic coverslips are preferable during the reaction, as they are easier to remove from the section.</td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;">Rat Brain Sections</td>
			<td></td>
			<td></td>
			<td style="width:129px;">Rat brain taken 48 hours after reproduction of ischemic stroke as described in 11. 5-6&#956;m-thick sections cut from paraformaldehyde-fixed, paraffin-embedded tissue blocks. Use charged and precleaned slides.&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td style="width:129px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Equipment</td>
			<td></td>
			<td></td>
			<td style="width:129px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td style="width:129px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Inverted Microscope</td>
			<td>Olympus</td>
			<td></td>
			<td style="width:129px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Digital Video camera and software</td>
			<td>Micromax</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Software</td>
			<td>Metamorph</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

assessing phagocytic clearance of cell death in experimental stroke by ligatable fluorescent probes

我々は、局所性脳虚血に貪食クリアランスの可視化のための新たな組織化学的なアプローチについて説明します。アプローチは、任意の細胞系譜の廃棄物管理の食細胞による行程の死細胞の除去の研究を可能にします。貪食することができる多数の異なる起源の細胞が虚血性脳中に存在するが、それらの一部だけが積極的に飲み込みおよび細胞死体を消化する。このような積極的な食細胞の廃棄物管理の選択的可視化、定量化、および分析は、虚血に対する脳の反応を評価するのに役立ちます。それは、その後の再生プロセスのために道を開くように効率的な細胞死のクリアランスは、虚血性損傷から脳の回復のために重要である。死体をきれいにする失敗は非分解のDNAやタンパク質によって引き起こされる毒性反応をもたらすであろう。説明する手順は、選択的に貪食中にすべての食細胞で生産特性DNA切断にウイルストポイソメラーゼによって連結させた蛍光プローブを使用していますそして不可逆的に虚血により損傷を受けた細胞を消化。この方法は、虚血性傷害に対する脳の反応の調査のための新しいツールです。

活性な廃棄物管理の食細胞を標識するための技術の原理を図1に示されている。概略は3段階で検出進むことを示している。第一工程は、プローブの活性化（ 図1A）を包含し;第二段階で活性化プローブは、組織切片（ 図1C）内の特定のDNA切断に連結している。第三段階は、蛍光シグナル増幅（ 図1D）を含む。以下のようにより詳細な説明では?...

Watch this Scientific Journal Video about Assessing Phagocytic Clearance of Cell Death in Experimental Stroke by Ligatable Fluorescent Probes at JoVE.com

Assessing Phagocytic Clearance of Cell Death in Experimental Stroke by Ligatable Fluorescent Probes

私たちは、積極的な食細胞のin situラベリングにおける選択のための新しい蛍光技術を提示し、その明確なストローク中の細胞死体オフ。虚血性脳内に存在する食細胞のわずかな割合は、細胞死の隙間に参加しているための方法は、虚血に対する脳の反応を評価するために重要である。

連結可能な蛍光プローブによる実験脳卒中における細胞死の貪食クリアランスを評価する

We describe a new histochemical approach for visualization of phagocytic clearance in focal brain ischemia. The approach permits the study of elimination ...

assessing-phagocytic-clearance-cell-death-experimental-stroke

Research

JoVE Journal

Medicine

8.1K ビュー.  Baylor College of Medicine. 私たちは、積極的な食細胞のin situラベリングにおける選択のための新しい蛍光技術を提示し、その明確なストローク中の細胞死体オフ。虚血性脳内に存在する食細胞のわずかな割合は、細胞死の隙間に参加しているための方法は、虚血に対する脳の反応を評価するために重要である。 

ビデオ: Assessing Phagocytic Clearance of Cell Death in Experimental Stroke by Ligatable Fluorescent Probes

Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are very limited. To meet the need for novel therapeutic approaches, experimental stroke research frequently employs rodent models of focal cerebral ischaemia. Most researchers use permanent or transient occlusion of the middle cerebral artery (MCA) in mice or rats.
Proximal occlusion of the middle cerebral artery (MCA) via the intraluminal suture technique (so called filament or suture model) is probably the most frequently used model in experimental stroke research. The intraluminal MCAO model offers the advantage of inducing reproducible transient or permanent ischaemia of the MCA territory in a relatively non-invasive manner. Intraluminal approaches interrupt the blood flow of the entire territory of this artery. Filament occlusion thus arrests flow proximal to the lenticulo-striate arteries, which supply the basal ganglia. Filament occlusion of the MCA results in reproducible lesions in the cortex and striatum and can be either permanent or transient. In contrast, models inducing distal (to the branching of the lenticulo-striate arteries) MCA occlusion typically spare the striatum and primarily involve the neocortex. In addition these models do require craniectomy. In the model demonstrated in this article, a silicon coated filament is introduced into the common carotid artery and advanced along the internal carotid artery into the Circle of Willis, where it blocks the origin of the middle cerebral artery. In patients, occlusions of the middle cerebral artery are among the most common causes of ischaemic stroke. Since varying ischemic intervals can be chosen freely in this model depending on the time point of reperfusion, ischaemic lesions with varying degrees of severity can be produced. Reperfusion by removal of the occluding filament at least partially models the restoration of blood flow after spontaneous or therapeutic (tPA) lysis of a thromboembolic clot in humans.
In this video we will present the basic technique as well as the major pitfalls and confounders which may limit the predictive value of this model.

Filamentous occlusion of the Middle cerebral artery is a common model for studying ischemic stroke in mice.

マウスにおけるモデリングのストローク - フィラメントモデルと中大脳動脈閉塞

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are ...

医学

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and finding new sources for implantable cells are the focus of recent research4,5. Investigating the effectiveness of cell therapies is not an easy task and new tools are needed to investigate the mechanisms involved in the treatment process6. We designed an experimental protocol of ischemia/reperfusion in order to allow the observation of cellular connections and even subcellular mechanisms during ischemia/reperfusion injury and after stem cell transplantation and to evaluate the efficacy of cell therapy. H9c2 cardiomyoblast cells were placed onto cell culture plates7,8. Ischemia was simulated with 150 minutes in a glucose free medium with oxygen level below 0.5%. Then, normal media and oxygen levels were reintroduced to simulate reperfusion. After oxygen glucose deprivation, the damaged cells were treated with transplantation of labeled human bone marrow derived mesenchymal stem cells by adding them to the culture. Mesenchymal stem cells are preferred in clinical trials because they are easily accessible with minimal invasive surgery, easily expandable and autologous. After 24 hours of co-cultivation, cells were stained with calcein and ethidium-homodimer to differentiate between live and dead cells. This setup allowed us to investigate the intercellular connections using confocal fluorescent microscopy and to quantify the survival rate of postischemic cells by flow cytometry. Confocal microscopy showed the interactions of the two cell populations such as cell fusion and formation of intercellular nanotubes. Flow cytometry analysis revealed 3 clusters of damaged cells which can be plotted on a graph and analyzed statistically. These populations can be investigated separately and conclusions can be drawn on these data on the effectiveness of the simulated therapeutical approach.

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

We demonstrate how to set up an in vitro ischemia/reperfusion model and how to evaluate the effect of stem cell therapy on postischemic cardiac cells.

における幹細胞移植 in vitroで模擬虚血/再灌流モデル

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and ...

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

胎児性アルコール暴露のゼブラフィッシュモデルの催奇形性変化を評価する

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 percent of women with ovarian cancer develop new metastases, and the recurrence is often fatal2. It is, therefore, necessary to understand how secondary metastases arise in order to develop better treatments for intermediate and late stage ovarian cancer. Ovarian cancer metastasis occurs when malignant cells detach from the primary tumor site and disseminate throughout the peritoneal cavity. The disseminated cells can form multicellular clusters, or spheroids, that will either remain unattached, or implant onto organs within the peritoneal cavity3 (Figure 1, Movie 1). 
All of the organs within the peritoneal cavity are lined with a single, continuous, layer of mesothelial cells4-6 (Figure 2). However, mesothelial cells are absent from underneath peritoneal tumor masses, as revealed by electron micrograph studies of excised human tumor tissue sections3,5-7 (Figure 2). This suggests that mesothelial cells are excluded from underneath the tumor mass by an unknown process. 
Previous in vitro experiments demonstrated that primary ovarian cancer cells attach more efficiently to extracellular matrix than to mesothelial cells8, and more recent studies showed that primary peritoneal mesothelial cells actually provide a barrier to ovarian cancer cell adhesion and invasion (as compared to adhesion and invasion on substrates that were not covered with mesothelial cells)9,10. This would suggest that mesothelial cells act as a barrier against ovarian cancer metastasis. The cellular and molecular mechanisms by which ovarian cancer cells breach this barrier, and exclude the mesothelium have, until recently, remained unknown. 
Here we describe the methodology for an in vitro assay that models the interaction between ovarian cancer cell spheroids and mesothelial cells in vivo (Figure 3, Movie 2). Our protocol was adapted from previously described methods for analyzing ovarian tumor cell interactions with mesothelial monolayers8-16, and was first described in a report showing that ovarian tumor cells utilize an integrin &#x2013;dependent activation of myosin and traction force to promote the exclusion of the mesothelial cells from under a tumor spheroid17. This model takes advantage of time-lapse fluorescence microscopy to monitor the two cell populations in real time, providing spatial and temporal information on the interaction. The ovarian cancer cells express red fluorescent protein (RFP) while the mesothelial cells express green fluorescent protein (GFP). RFP-expressing ovarian cancer cell spheroids attach to the GFP-expressing mesothelial monolayer. The spheroids spread, invade, and force the mesothelial cells aside creating a hole in the monolayer. This hole is visualized as the negative space (black) in the GFP image. The area of the hole can then be measured to quantitatively analyze differences in clearance activity between control and experimental populations of ovarian cancer and/ or mesothelial cells. This assay requires only a small number of ovarian cancer cells (100 cells per spheroid X 20-30 spheroids per condition), so it is feasible to perform this assay using precious primary tumor cell samples. Furthermore, this assay can be easily adapted for high throughput screening.

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

The mesothelial clearance assay described here takes advantage of fluorescently labeled cells and time-lapse video microscopy to visualize and quantitatively measure the interactions of ovarian cancer multicellular spheroids and mesothelial cell monolayers. This assay models the early steps of ovarian cancer metastasis.

in vitroで中皮クリアランスアッセイでそのモデルの卵巣がんの転移の初期段階を

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 ...

Experimental Human Pneumococcal Carriage

Experimental human pneumococcal carriage (EHPC) is scientifically important because nasopharyngeal carriage of Streptococcus pneumoniae is both the major source of transmission and the prerequisite of invasive disease. A model of carriage will allow accurate determination of the immunological correlates of protection, the immunizing effect of carriage and the effect of host pressure on the pathogen in the nasopharyngeal niche. Further, methods of carriage detection useful in epidemiologic studies, including vaccine studies, can be compared.
Aim
We aim to develop an EHPC platform that is a safe and useful reproducible method that could be used to down-select candidate novel pneumococcal vaccines with prevention of carriage as a surrogate of vaccine induced immunity. It will work towards testing of candidate vaccines and descriptions of the mechanisms underlying EHPC and vaccine protection from carriage1. Current conjugate vaccines against pneumococcus protect children from invasive disease although new vaccines are urgently needed as the current vaccine does not confer optimal protection against non-bacteraemic pneumonia and there has been evidence of serotype replacement with non-vaccine serotypes2-4.
Method
We inoculate with S. pneumoniae suspended in 100 &mu;l of saline. Safety is a major factor in the development of the EHPC model and is achieved through intensive volunteer screening and monitoring. A safety committee consisting of clinicians and scientists that are independent from the study provides objective feedback on a weekly basis.
The bacterial inoculum is standardized and requires that no animal products are inoculated into volunteers (vegetable-based media and saline). The doses required for colonization (104-105) are much lower than those used in animal models (107)5. Detecting pneumococcal carriage is enhanced by a high volume (ideally &gt;10 ml) nasal wash that is relatively mucus free. This protocol will deal with the most important parts of the protocol in turn. These are (a) volunteer selection, (b) pneumococcal inoculum preparation, (c) inoculation, (d) follow-up and (e) carriage detection.
Results
Our current protocol has been safe in over 100 volunteers at a range of doses using two different bacterial serotypes6. A dose ranging study using S. pneumoniae 6B and 23F is currently being conducted to determine the optimal inoculation dose for 50% carriage. A predicted 50% rate of carriage will allow the EHPC model to have high sensitivity for vaccine efficacy with small study numbers.

Experimental human pneumococcal carriage offers a natural model of carriage and a potential model for use in vaccine development. This technique is valuable yet complex and involves clinical risk by introducing a pathogen into a human. We have developed a detailed protocol.

実験的な人間の肺炎球菌キャリッジ

Experimental human pneumococcal carriage (EHPC) is scientifically important because nasopharyngeal carriage of Streptococcus pneumoniae is both the major ...

Cell Death Associated with Abnormal Mitosis Observed by Confocal Imaging in Live Cancer Cells

Phenanthrene derivatives acting as potent PARP1 inhibitors prevented the bi-focal clustering of supernumerary centrosomes in multi-centrosomal human cancer cells in mitosis. The phenanthridine PJ-34 was the most potent molecule. Declustering of extra-centrosomes causes mitotic failure and cell death in multi-centrosomal cells. Most solid human cancers have high occurrence of extra-centrosomes. The activity of PJ-34 was documented in real-time by confocal imaging of live human breast cancer MDA-MB-231 cells transfected with vectors encoding for fluorescent &gamma;-tubulin, which is highly abundant in the centrosomes and for fluorescent histone H2b present in the chromosomes. Aberrant chromosomes arrangements and de-clustered &gamma;-tubulin foci representing declustered centrosomes were detected in the transfected MDA-MB-231 cells after treatment with PJ-34. Un-clustered extra-centrosomes in the two spindle poles preceded their cell death. These results linked for the first time the recently detected exclusive cytotoxic activity of PJ-34 in human cancer cells with extra-centrosomes de-clustering in mitosis, and mitotic failure leading to cell death. According to previous findings observed by confocal imaging of fixed cells, PJ-34 exclusively eradicated cancer cells with multi-centrosomes without impairing normal cells undergoing mitosis with two centrosomes and bi-focal spindles. This cytotoxic activity of PJ-34 was not shared by other potent PARP1 inhibitors, and was observed in PARP1 deficient MEF harboring extracentrosomes, suggesting its independency of PARP1 inhibition. Live confocal imaging offered a useful tool for identifying new molecules eradicating cells during mitosis.

The cytotoxic activity of the phenanthridine PJ-34 in cancer cells undergoing mitosis was documented in real time by live confocal imaging. PJ-34 eradicated human breast cancer MDA-MB-231 cells harboring extra-centrosomes in mitosis. Unlike normal bi-focal mitosis, the extra-centrosomes were not clustered in the two spindle poles in the presence of PJ-34.

ライブ癌細胞で共焦点イメージングで観測異常有糸分裂と関連付けられる細胞死

Phenanthrene derivatives acting as potent PARP1 inhibitors prevented the bi-focal clustering of supernumerary centrosomes in multi-centrosomal human ...

Neural Stem Cell Transplantation in Experimental Contusive Model of Spinal Cord Injury

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can be used both to investigate the biological responses to injury and to test potential therapies. Contusion or compression injury delivered to the surgically exposed spinal cord are the most widely used models of the pathology. In this report the experimental contusion is performed by using the Infinite Horizon (IH) Impactor device, which allows the creation of a reproducible injury animal model through definition of specific injury parameters. Stem cell transplantation is commonly considered a potentially useful strategy for curing this debilitating condition. Numerous studies have evaluated the effects of transplanting a variety of stem cells. Here we demonstrate an adapted method for spinal cord injury followed by tail vein injection of cells in CD1 mice. In short, we provide procedures for: i) cell labeling with a vital tracer, ii) pre-operative care of mice, iii) execution of a contusive spinal cord injury, and iv) intravenous administration of post mortem neural precursors. This contusion model can be utilized to evaluate the efficacy and safety of stem cell transplantation in a regenerative medicine approach.

Spinal cord injury is a traumatic condition that causes severe morbidity and high mortality. In this work we describe in detail a contusion model of spinal cord injury in mice followed by a transplantation of neural stem cells.

脊髄損傷の実験的挫傷モデルにおける神経幹細胞移植

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can ...

Experimental Glaucoma Induced by Ocular Injection of Magnetic Microspheres

Progress in understanding the pathophysiology, and providing novel treatments for glaucoma is dependent on good animal models of the disease. We present here a protocol for elevating intraocular pressure (IOP) in the rat, by injecting magnetic microspheres into the anterior chamber of the eye. The use of magnetic particles allows the user to manipulate the beads into the iridocorneal angle, thus providing a very effective blockade of fluid outflow from the trabecular meshwork. This leads to long-lasting IOP rises, and eventually neuronal death in the ganglion cell layer (GCL) as well as optic nerve pathology, as seen in patients with the disease. This method is simple to perform, as it does not require machinery, specialist surgical skills, or many hours of practice to perfect. Furthermore, the pressure elevations are very robust, and reinjection of the magnetic microspheres is not usually required unlike in some other models using plastic beads. Additionally, we believe this method is suitable for adaptation for the mouse eye.

We present a method for inducing elevated intraocular pressure (IOP), by injecting magnetic microspheres into the rat eye, to model glaucoma. This leads to strong pressure rises, and extensive neuronal death. This protocol is easy to perform, does not require repeat injections, and produces stable long-lasting IOP rises.

磁気マイクロスフェアの眼注射によって誘発実験緑内障

Progress in understanding the pathophysiology, and providing novel treatments for glaucoma is dependent on good animal models of the disease. We present ...

Bile Duct Ligation in Mice: Induction of Inflammatory Liver Injury and Fibrosis by Obstructive Cholestasis

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well as to excrete bilirubin and other metabolic products. In the liver, the experimental obstruction of the extrahepatic biliary system initiates a complex cascade of pathological events that leads to cholestasis and inflammation resulting in a strong fibrotic reaction originating from the periportal fields. Therefore, surgical ligation of the common bile duct has become the most commonly used model to induce obstructive cholestatic injury in rodents and to study the molecular and cellular events that underlie these pathophysiological mechanisms induced by inappropriate bile flow. In recent years, different surgical techniques have been described that either allow reconnection or reanastomosis after bile duct ligation (BDL), e.g., partial BDL, or other microsurgical methods for specific research questions. However, the most frequently used model is the complete obstruction of the common bile duct that induces a strong fibrotic response after 21 to 28 days. The mortality rate can be high due to infectious complications or technical inaccuracies. Here we provide a detailed surgical procedure for the BDL model in mice that induce a highly reproducible fibrotic response in accordance to the 3R rule for animal welfare postulated by Russel and Burch in 1959.

Disruption of bile flow results in severe inflammatory cholestatic liver injury with a characteristic time-dependent sequence of morphological alterations. Here we present a protocol for the surgical ligation of the common bile duct in mice that allows to induce a strong fibrotic response after 21 to 28 days.

マウスにおける胆管結紮：閉塞性胆汁うっ滞による炎症性肝障害と線維症の誘発

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well ...

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo: A Model of Single Vessel Stroke

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to image cellular events in an intact animal. Additionally, the ability to induce physiological disease states such as stroke in vivo increases its utility. The technique described herein allows for physiological assessment of cellular responses within the CNS following a stroke and can be adapted for other pathological conditions being studied. The technique presented uses laser excitation of the photosensitive dye Rose Bengal in vivo to induce a focal ischemic event in a single blood vessel. 
The video protocol demonstrates the preparation of a thin-skulled cranial window over the somatosensory cortex in a mouse for the induction of a Rose Bengal photothrombotic event keeping injury to the underlying dura matter and brain at a minimum. Surgical preparation is initially performed under a dissecting microscope with a custom-made surgical/imaging platform, which is then transferred to a confocal microscope equipped with an inverted objective adaptor. Representative images acquired utilizing this protocol are presented as well as time-lapse sequences of stroke induction. This technique is powerful in that the same area can be imaged repeatedly on subsequent days facilitating longitudinal in vivo studies of pathological processes following stroke.

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo:  A Model of Single Vessel Stroke

Here, we describe a semi-invasive optical microscopy approach for the induction of a Rose Bengal photothrombotic clot in the somatosensory cortex of a mouse in vivo. The technical aspects of the imaging procedure are described from induction of a photothrombotic event to application and data collection.

共焦点光学イメージングによるローズベンガル光血栓<em&gt;インビボ</em&gt;：単一の容器ストロークのモデル

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to ...