The authors would like to acknowledge the Netherlands Organization for Scientific Research (NWO, grant 17.106.329)

動物実験は、ライデン大学医療センター（LUMC）の動物福祉委員会によって承認された。 細胞、楽器やマウスの作製<ol><li>操作前日は、正中線に第四の乳首からNOD / SCIDガンマ（NSG）マウスを剃ると、すべてのマウスがほぼ同程度の重みを持っていることを確認するために、マウスの重量を量る。 </li><li>はさみ、2ピンセットと2ストレートモスキート鉗子（ 図1A）をオートクレーブ。 </li><li>操作の日に、リン酸緩衝生理食塩水（PBS）で一回注射するヒト乳腺癌（MDA-MB-231）細胞を洗浄し、pH7.4で、細胞をトリプシン処理。細胞の上の10mlの血清含有DMEM培地を添加することによりトリプシンをクエンチする。血清を含まないPBS中またはメディアのいずれかで細胞を再懸濁することによって血清を除去するために、室温で7分間1200 rpmで細胞を遠心します。完全に血清の痕跡を除去するためにそれらを再度遠心します。 PBSに細胞を再懸濁またはメディア。 </li><li>血球計数器を用いて細胞をカウントし、細胞の量を計算する。しないより150以上μlの500,000細胞/マウスを使用しています。 PBSまたはメディアへのオプション、マトリゲルで細胞を懸濁します。 注：使用する細胞の種類に応じて、細胞の量を決定します。 </li><li>滅菌マイクロチューブに細胞を配置し、氷の上に保管してください。 </li><li> 35 PBS pHを7.4 mlの70％エタノールを有する別の50mlの円錐形遠心分離管と、ワン50mlコニカル遠心チューブを埋める。各チューブに綿棒を浸す。 </li></ol> 2.同所インジェクション<ol><li>無菌雰囲気を維持するために、無菌フード内で手術を行う。それぞれの10mg / kgを、100 mg / kg体重の用量でキシラジン/ケタミン混合物を皮下注射することによってマウスを麻酔。加熱パッド上でマウスを修正。つま先のピンチに反応の欠如によって麻酔の成功を確認してください。 NOTE：一つは外科手術手順に精通していない場合は、手術が長くかかる場合があります。アレンジ麻酔はそれに応じて投与量。 </li><li>乾燥から目を防ぐために、マウスの眼に眼軟膏を適用します。 </li><li>ステップ1.6で作成エタノールに浸漬綿棒を使って、剃らエリアを清掃してください。離れて計画された切開部位から回転する円形の方法でベタジンとアルコール3回を使用して交互にスクラブを実行する必要があります。また、消毒剤としてイオノフォアを使用しています。 </li><li>はさみで四ニップルと正中線の間に小さな切開を行い、PBSのpHは7.4で湿らせた綿棒を挿入してポケットを作る。 </li><li>乳腺脂肪体を露出させるためにピンセットを使用してください。その白い色で脂肪パッドを観察します。 </li><li>そのベースから他のピンセットで脂肪パッドを絞る。これを行うことで、完全に簡単に注入を実行するために脂肪パッド（ 図1B）を公開します。 </li><li>ピペッティングにより細胞混合物を均質化する。静かにインスリン注射器に細胞懸濁液50μlを吸引しに注入水平に針を保持することによって乳腺脂肪パッド。脂肪パッドの腫れのためにチェックすることで成功​​した注射を確認します。 </li><li>優しく脂肪パッドを離します。 </li><li>針ドライバを使用して切開部を縫合する。 3時計回り、反時計回りおよび時計回りに針ドライバの周りに縫合糸を回すことによりスロー行う。縫合ごとに2つの結び目を使用する必要があります。 注：ノットが締まっているそうでマウスが縫合糸を開くことができていることを確認してください。 </li><li>手術後、痛みを緩和するために、0.05〜0.1ミリグラム/ kg体重、皮下にかかるtemgesicとして、鎮痛剤を注入する。 </li><li>彼らが意識を獲得し、胸骨横臥を維持するまで無人の動物に放置しないでください。彼らは完全に回復するまで、別のケージにすべての動物を置きます。 </li><li>無菌性を維持するために、オートクレーブケージと水を使用する。清浄な雰囲気を保つために3日ごとにケージを変更します。 </li></ol>分析のための3.収穫臓器<ol><li>アット収穫の日は、8週間の細胞の移植後、各マウスのための3ミリリットルブアン液で15ミリリットルコニカル遠心管を埋める。また、マウス当たり5ミリリットルホルマリン溶液で充填された2つの15ミリリットルチューブを使用。 </li><li> 10ミリグラムの用量で、キシラジン/ケタミンを注射することによって動物を麻酔/ 100 mg / kg体重の皮下キロ。動物が足指ピンチ引っ込め反射を失うまで待ってください。 </li><li>ベース上の針を動物に固定してください。はさみとの長い、垂直正中切開を行います。右の前脚の下と後脚の上に二つの水平切開を行います。ベースに皮膚を固定することによって腫瘍を公開します。キャリパーを用いて腫瘍体積を測定する。 </li><li>ハサミを用いて皮膚からの腫瘍を解離。スナップは、RNA単離のために液体窒素中での腫瘍の一部を凍結する。パラフィン包埋以下の免疫組織化学を実行するためにホルマリンで満たされたコニカル遠心チューブに、他の部分を配置します。 </li><li>ゆっくりと肺を取り出します。置きブアン溶液中に肺を残した。 3日間溶液中の肺にしてください。肉眼ではっきりと表面的な転移巣を観察します。必要に応じて、ヘマトキシリン​​およびエオシン（H＆E）染色を使用して微小転移を確認するためにホルマリンに右肺を置く。 注：肺転移が乳癌で頻繁に見られ、が、1にも転移を分析するために骨、肝臓、脳および脾臓を収集する場合があります。転移領域における細胞はより高密度と形態学的に異なっており、したがって、肺組織から容易に区別することができる。 </li><li>日は収穫後、15ミリリットル管からのホルマリン溶液を吸引し、70％エタノールで交換してください。パラフィンで組織を埋め込み、免疫組織化学研究を行う。 </li><li>血液分析のために、はさみで胸腔を開く。心臓穿刺を介して450μlの血液を撤回。 3.2％クエン酸ナトリウムを50μl含むマイクロ遠心チューブに血液試料を置く。血液が収集された後、10マイルのために2000×gで、それをスピンN。さらに使用するまで-20℃で血漿サンプルを保管してください。 注：一般的慣行20と実験科学者の選択に依存して使用する必要がある技術で使用されるいくつかの他の血液採取技術がある。血液サンプルを必要としない場合は、動物プロトコルや機関のガイドラインに従って応じて動物を安楽死させる。 </li></ol>

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The authors declare that they have no competing financial interests.

乳癌細胞の同所注射は、癌の成長のすべての側面を研究するための強力なモデルである。マウスの乳腺脂肪体におけるこれらの細胞の注入は、慎重に腫瘍成長の変化を防止するために行われるべきである。最も重要なことは、各マウスへの細胞の同量を注入することは重要である。これを行うためには、細胞の生存に影響を与えることなく、厳密に細胞をトリプシン処理すべきである。非生?...

がんは何世紀にもわたってのための研究の対象となっている非常に複雑な疾患である。乳がんは、最も一般的な癌型です。それは女性で主に発生するが、散発的にも、男性27内に発生する可能性があります。この疾患は、主に次に体内の細胞の無限増殖をもたらす細胞分裂を支配する制御機構の損失によって引き起こされる。これらの不具合がいくつかのメカニズムによって引き起こされる可能性がありますまず、正常細胞は、癌細胞が自分自身の成長因子を行うため、より高い増殖率1を得る成長因子受容体の発現を増加させるのに対して、増殖するために、周囲のセルからの増殖シグナルを必要とする。第二に、癌細胞は、抗増殖シグナル8を受けにくい。第三の、また必要とされる体細胞死細胞数のバランスをとる。しかし、癌細胞は、プログラム細胞死から脱出アポトーシス14と呼ばれる。第四に、細胞は、細胞外マトリックスに付着生き残るために、腫瘍細胞は付着を必要とせずに成長し、アノイキス19に対する抵抗性を示すことができるために。第五、テロメラーゼの活性化は、テロメアの短縮を回避し、複製老化21を防ぎます。最後のではなく、少なくとも、変更された遺伝的内容15,16における有糸分裂の結果、以下のDNAの品質管理のスキップ。この調節解除された増殖に役割を果たす癌遺伝子または腫瘍抑制因子を同定するために、マウスにおける腫瘍増殖実験は重要である。 原発腫瘍の成長は、一般的に死の主な理由ではありません。セカンダリサイトへのプライマリサイトから癌細胞の移動、転移と呼ばれるが、ほとんどの癌患者の死亡の主要な原因で22です。転移はセカンダリサイトでの腫瘍細胞の浸潤、血管内、循環を通過する、免疫攻撃を回避し、血管外漏出と成長を伴う。間葉移行（EMT）の上皮は、キープロセスである転移および転移性細胞12のための前提条件である、より高い運動性と侵襲性で細胞をもたらす遺伝子発現プロファイル、スイッチを伴う。癌処理としては、がん細胞、ストローマ細胞とプロと抗炎症性細胞との間の相互の相互作用を含む様々なアクションの組み合わせの結果として、癌にin vitroでのアプローチは、多くの場合、癌のプロセスに完全な洞察を提供していませんされている。同様に、腫瘍血管系に影響を与える抗癌治療は、しばしば、したがって、インビボでの使用が避けられない近づき、 インビトロで研究することができない。 乳癌の進行を研究するために、異なる実験的方法が開発されている。最も広く使用されるモデルは、マウス5に乳癌細胞の皮下注射である。この実験では、研究者は、in vitroで選択した細胞株の変化の広い範囲を導入することができる（ すなわちアップレギュレーション、タンパク質のダウンレギュレーション）と皮膚の下に細胞を注入する。この方法は簡単で、注入プロセスがマウスの手術を実行する必要なしに単純であるが、腫瘍が注入される部位は、ローカル乳腺腫瘍環境を表していないと、この環境の欠如は、乳癌の発達をもたらし得るヒト病態において観察されるものとは異なる。第二に、遺伝子操作されたマウスは、乳癌の進行を研究するためのin vivoでのツールとして頻繁に使用される。このモデルでは、癌遺伝子（ すなわち PyMT、ノイ）の発現は、自発乳癌sの形成をもたらす乳房組織特異的プロモーターによって駆動される。この実験は、時間3における腫瘍の大きさを確認しながら、薬物または抗体を注入することによって、疾患の治療の側面を研究するのに有用である。しかし、欠損またはまたインを与えるかもしれない、目的の遺伝子に変異した他のマウス系統とこれらのマウスの繁殖乳房腫瘍増殖24の異なるタンパク質の役割にights。このモデルの欠点は、腫瘍の大きさおよび数の変動を受けやすいということである。さらに、導入遺伝子発現のレベルは、ゲノム中の組込み部位に依存し、別の4一つのマウス株から変更することができる。ヒト疾患において、細胞の亜集団のみが癌遺伝子を発現するかまたは腫瘍抑制レベル26を下方のに対し、このモデルでは、導入遺伝子の発現は、上皮起源のすべての細胞によって達成することができる。転移を研究するために、乳癌細胞を静脈内注射することができる25（モデルは、実験的転移と呼ばれる）。しかし、このアプローチは、部分的にしか転移プロセスを再現する。それは、腫瘍細胞が侵入しintravasateするための要件を回避し、腫瘍細胞が容易に循環中に存在するところから始まる。 私たちの研究では、同所注入を使用乳癌進行13における目的の遺伝子の関与を研究するためのイオンモデル。本発明者らは、ヒト乳癌細胞においてタンパク質を過剰発現し、NOD / SCIDガンマ（NSG）マウスの乳房脂肪パッドにそれらを注入する。この方法は、多くの点で有利である：それは、注入された細胞株において非常に急速な多様な遺伝的変化を可能にし、それは病理学的に関連する部位に転移する原発性腫瘍成長の乳癌進行のプロセス全体をカバーし、それはまた、良好な実験モデルを提供疾患の初期または後期段階での治療処置の影響を研究するため。また、このモデルを用いたものは、遺伝的に改変されたマウスまたは細胞を用いて疾患の進行中の癌細胞由来のタンパク質対間質の役割を調べることができる。皮下注射は実行が容易であるが、同所性モデルは、より腫瘍形成、より転移性癌細胞集団を生じる。したがって、結果は、皮下注射により得られるSは、偽陰性または腫瘍増殖を研究するための同所モデルの使用を奨励6,17偽陽性のいずれかである可能性があります。

<table border="0" cellpadding="0" cellspacing="0" width="1093">
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:185px;">Name of material/equipment</td>
			<td style="width:172px;">Company</td>
			<td style="width:95px;">Catalog number</td>
			<td style="width:641px;">Comments/Description</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Bouin&#39;s solution</td>
			<td>Sigma-Aldrich</td>
			<td>HT10132</td>
			<td>Used for investigating the metastasis on lungs</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Formalin solution</td>
			<td>Sigma-Aldrich</td>
			<td>HT501128</td>
			<td>Used to fix the tissues</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Matrigel, growth factor reduced</td>
			<td>Corning</td>
			<td>356230</td>
			<td>Cells can be resuspended in matrigel for injection</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Mosquito forceps</td>
			<td>Fine Science Tools</td>
			<td>13008-12</td>
			<td>Used for stiching</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Angled forceps</td>
			<td>Electron microscopy sciences</td>
			<td>72991-4c</td>
			<td>These make the exposure of mammary fat pad easier</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Scissors</td>
			<td>B Braun Medicals</td>
			<td>BC056R</td>
			<td>Used to cut open the mice</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Straight forceps</td>
			<td>B Braun Medicals</td>
			<td>BD025R</td>
			<td>This is used to open up the skin to expose mammary fat pad</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">NOD scid gamma mice</td>
			<td>Charles River</td>
			<td>005557</td>
			<td>Experimental animal used for experiment</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">MDA-MB-231</td>
			<td>Sigma-Aldrich</td>
			<td>92020424</td>
			<td>Experimental cells used for injections</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Oculentum simplex</td>
			<td>Teva Pharmachemie</td>
			<td></td>
			<td>Opthalmic ointment used to prevent drying out of eyes</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Betadine</td>
			<td>Fischer Scientific</td>
			<td>19-898-859</td>
			<td>Ionophore, used to disinfect the surgical area</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Xylazin/Ketamine</td>
			<td>Sigma-Aldrich</td>
			<td>X1251, K2753</td>
			<td>Use injected anesthesia as 10mg/kg and 100mg/kg body weight respectively</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Temgesic</td>
			<td>Schering-Plough</td>
			<td></td>
			<td>Use the painkiller as 0,05-0,1mg/kg body weight</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">DMEM</td>
			<td>Life sciences</td>
			<td>11995</td>
			<td>For trypsin neutralization,use media with serum(FBS:media 1:10 volume); for injection, use media with no serum</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Buffered sodium citrate</td>
			<td>Aniara</td>
			<td>A12-8480-10</td>
			<td>Use the volume ratio as citrate:blood; 1:9</td>
		</tr>
	</tbody>
</table>

orthotopic injection of breast cancer cells into the mammary fat pad of mice to study tumor growth.

<html>乳癌増殖は、モデルの過多を使用してマウスで研究することができる。乳癌細胞の遺伝子操作は、発癌の進行に関与するタンパク質の機能への洞察を提供するか、または新たな腫瘍抑制因子を発見するのを助けることができる。さらに、異なる遺伝子型を有するマウスに癌細胞を注入する間質区画の重要性のよりよい理解を提供するかもしれない。多くのモデルは、疾患の進行の特定の局面を調査するために有用であり得るが、全体の癌のプロセスを反復しない。対照的に、マウスの乳房脂肪パッドに乳癌細胞の生着が、より適切な間質区画、従ってより良い模倣するヒト癌性疾患の疾患および存在の位置を再現する。この記事では、同所マウスに乳癌細胞を移植し、分析するために、組織を収集する方法を説明する方法を説明します：14px;行の高さ：28px; &quot;&gt;腫瘍環境および転移遠隔臓器にこのモデルを用いて、癌の多くの側面（成長、血管新生、および転移）は、腫瘍細胞が成長するための適切な環境を提供することにより、簡単に調べることができる。

「同所乳癌モデル」の成功したアプリケーションは、乳房脂肪パッドへの細胞の適切な注入に基づいています。このような細胞または漏れの不正確接種の実験誤差は、腫瘍の大きさまたは対照緩衝液（ 図2A）を注射した乳房脂肪パッドに似て見て構造が形成される腫瘍であっても非存在下でのばらつきにつながるかもしれない。腫瘍の増殖速度は、注入された細胞株の性質に依?...

Watch this Scientific Journal Video about Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth. at JoVE.com

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

癌は、腫瘍の周囲の組織だけでなく、ローカルプロ - および抗炎症性メディエーターによって影響される複雑な疾患である。したがって、むしろ皮下モデルより異方性注入モデルは、方法をより良く模倣するヒト病理における癌の進行を研究するために有用であり得る。

腫瘍増殖を研究するためにマウスの乳房脂肪パッド中に乳癌細胞の同所注入。

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

orthotopic-injection-breast-cancer-cells-into-mammary-fat-pad-mice-to

Research

JoVE Journal

Medicine

78.4K ビュー.  Leiden University Medical Center. 癌は、腫瘍の周囲の組織だけでなく、ローカルプロ - および抗炎症性メディエーターによって影響される複雑な疾患である。したがって、むしろ皮下モデルより異方性注入モデルは、方法をより良く模倣するヒト病理における癌の進行を研究するために有用であり得る。 

ビデオ: Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Performing and Processing FNA of Anterior Fat Pad for Amyloid

Historically, heart, liver, and kidney biopsies were performed to demonstrate amyloid deposits in amyloidosis. Since the clinical presentation of this disease is so variable and non-specific, the associated risks of these biopsies are too great for the diagnostic yield. Other sites that have a lower biopsy risk, such as skin or gingival, are also relatively invasive and expensive. In addition, these biopsies may not always have sufficient amyloid deposits to establish a diagnosis. Fat pad aspiration has demonstrated good clinical correlation with low cost and minimal morbidity. However, there are no standardized protocols for performing this procedure or processing the aspirated specimen, which leads to variable and nonreproducible results. The most frequently utilized modality for detecting amyloid in tissue is an apple-green birefringence on Congo red stained sections using a polarizing microscope. This technique requires cell block preparation of aspirated material. Unfortunately, patients presenting in early stage of amyloidosis have minimal amounts of amyloid which greatly reduces the sensitivity of Congo red stained cell block sections of fat pad aspirates. Therefore, ultrastructural evaluation of fat pad aspirates by electron microscopy should be utilized, given its increased sensitivity for amyloid detection. This article demonstrates a simple and reproducible procedure for performing anterior fat pad aspiration for the detection of amyloid utilizing both Congo red staining of cell block sections and electron microscopy for ultrastructural identification.

Fat pad aspiration is a preferred, minimally invasive, and low cost approach as compared to other methods to detect amyloid for diagnosis of systemic amyloidosis. This video article demonstrates a procedural outline for performing fat pad aspiration with appropriate processing of the specimen for the optimal diagnostic outcome.

アミロイドのための前方脂肪パッドのFNAの実行と処理

Historically, heart, liver, and kidney biopsies were performed to demonstrate amyloid deposits in amyloidosis.  Since the clinical presentation of this ...

医学

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent until many years after removal of the primary tumor and adjuvant therapy. A likely explanation of this phenomenon is that tumor cells have seeded metastatic sites, are resistant to conventional therapies, and remain dormant for long periods of time 1-4.
The existence of dormant cancer cells at secondary sites has been described previously as quiescent solitary cells that neither proliferate nor undergo apoptosis 5-7. Moreover, these solitary cells has been shown to disseminate from the primary tumor at an early stage of disease progression 8-10 and reside growth-arrested in the patients' bone marrow, blood and lymph nodes 1,4,11. Therefore, understanding mechanisms that regulate dormancy or the switch to a proliferative state is critical for discovering novel targets and interventions to prevent disease recurrence. However, unraveling the mechanisms regulating the switch from tumor dormancy to metastatic growth has been hampered by the lack of available model systems.
in vivo and ex vivo model systems to study metastatic progression of tumor cells have been described previously 1,12-14. However these model systems have not provided in real time and in a high throughput manner mechanistic insights into what triggers the emergence of solitary dormant tumor cells to proliferate as metastatic disease. We have recently developed a 3D in vitro system to model the in vivo growth characteristics of cells that exhibit either dormant (D2.OR, MCF7, K7M2-AS.46) or proliferative (D2A1, MDA-MB-231, K7M2) metastatic behavior in vivo . We demonstrated that tumor cells that exhibit dormancy in vivo at a metastatic site remain quiescent when cultured in a 3-dimension (3D) basement membrane extract (BME), whereas cells highly metastatic in vivo readily proliferate in 3D culture after variable, but relatively short periods of quiescence. Importantly by utilizing the 3D in vitro model system we demonstrated for the first time that the ECM composition plays an important role in regulating whether dormant tumor cells will switch to a proliferative state and have confirmed this in in vivo studies15-17. Hence, the model system described in this report provides an in vitro method to model tumor dormancy and study the transition to proliferative growth induced by the microenvironment.

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

A modified 3-D in vitro system is presented in which growth characteristics of several tumor cell lines in reconstituted basement membrane correlate with the dormant or proliferative behavior of the tumor cells at a metastatic secondary site in vivo.

アンインビトロ</em転移性増殖に腫瘍の休眠とスイッチを研究する>システム

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent ...

An Orthotopic Bladder Tumor Model and the Evaluation of Intravesical saRNA Treatment

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder tumor model. The mouse model is established by urethral catheterization under inhaled general anesthetic. Chemical burn is then introduced to the bladder mucosa using intravesical silver nitrate solution to disrupt the bladder glycosaminoglycan layer and allows cells to attach. Following several washes with sterile water, human bladder cancer KU-7-luc2-GFP cells are instilled through the catheter into the bladder to dwell for 2 hours. Subsequent growth of bladder tumors is confirmed and monitored by in vivo bladder ultrasound and bioluminescent imaging. The tumors are then treated intravesically with saRNA formulated in lipid nanoparticles (LNPs). Tumor growth is monitored with ultrasound and bioluminescence. All steps of this procedure are demonstrated in the accompanying video.

Establishing an orthotopic bladder tumor model to evaluate antitumor effects of intravesically delivered saRNA and monitoring tumor growth by ultrasound and bioluminescent imaging.

同所性膀胱腫瘍モデルと膀胱内Sarnaの治療の評価

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder ...

Orthotopic Aortic Transplantation in Mice for the Study of Vascular Disease

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. This is largely true, but there are a number of important principles that can reduce the difficulty of these procedures and enhance reproducibility. Orthotopic aortic transplantation is an excellent procedure in which to learn these principles because it involves only two end-to-end anastomoses, but requires good suturing technique and handling of the vessels for consistent success. This procedure begins with the procurement of a length of abdominal aorta from a donor animal, followed by division of the native aorta in the recipient. The procured aorta is then placed between the divided ends of the recipient aorta and sutured into place using end-to-end anastomoses. To accomplish this objective successfully requires a high degree of concentration, good tools, a steady hand, and an appreciation of how easily the vasculature of a mouse can be damaged, resulting in thrombosis. Learning these important principles is what occupies most of the beginner's time when learning microsurgery in small rodents. Throughout this protocol, we refer to these important points. This model can be used to study vascular disease in a variety of different experimental systems1-8. In the context shown here, it is most often used for the study of post-transplant vascular disease, a common long-term complication of solid organ transplantation in which intimal hyperplasia occurs within the allograft. The primary advantage of the model is that it facilitates quantitative morphometric analyses and the transplanted vessel lies contiguous to the endogenous vessel, which can serve as an additional control9. The technique shown here is most often used for mice weighing 18-25 grams. We have accumulated most of our experience using the C57BL/6J, BALB/cJ, and C3H/HeJ strains.

We describe a technique in which a section of the abdominal aorta from a mouse is transplanted orthotopically to just below the renal arteries in an allogeneic or syngeneic recipient. This technique can be useful in studies in which transplantation of large arteries of uniform size is deemed advantageous.

血管疾患の研究のためのマウスにおける同所性大動脈移植

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. ...

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 &mu;m x 700 &mu;m fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.

乳がんの遺伝子改変マウスモデル由来の初代前癌乳腺上皮細胞のタイムラプスイメージング

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

新規高分解能<em生体内で&gt;</em腫瘍増殖と治療に頭蓋内構造物の動的応答を研究する&gt;イメージング技術

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The interplay of signals between cancer cells and their microenvironment exerts a powerful influence on breast cancer growth and biological behavior1. However, most of these signals from the extracellular matrix are lost or their relevance is understudied when cells are grown in two dimensional culture (2D) as a monolayer. In recent years, three dimensional (3D) culture on a reconstituted basement membrane has emerged as a method of choice to recapitulate the tissue architecture of benign and malignant breast cells. Cells grown in 3D retain the important cues from the extracellular matrix and provide a physiologically relevant ex&#160;vivo system2,3. Of note, there is growing evidence suggesting that cells behave differently when grown in 3D as compared to 2D4. 3D culture can be effectively used as a means to differentiate the malignant phenotype from the benign breast phenotype and for underpinning the cellular and molecular signaling involved3. One of the distinguishing characteristics of benign epithelial cells is that they are polarized so that the apical cytoplasm is towards the lumen and the basal cytoplasm rests on the basement membrane. This apico-basal polarity is lost in invasive breast carcinomas, which are characterized by cellular disorganization and formation of anastomosing and branching tubules that haphazardly infiltrates the surrounding stroma. These histopathological differences between benign gland and invasive carcinoma can be reproduced in 3D6,7. Using the appropriate read-outs like the quantitation of single round acinar structures, or differential expression of validated molecular markers for cell proliferation, polarity and apoptosis in combination with other molecular and cell biology techniques, 3D culture can provide an important tool to better understand the cellular changes during malignant transformation and for delineating the responsible signaling.

Three dimensional culture of mammary epithelial cells on a reconstituted basement membrane is a useful method to recapitulate the in vivo architecture of the benign breast, and to differentiate the malignant phenotype from the benign breast phenotype. Importantly, this system can be applied to study invasive carcinomas in other tissues.

三次元文化：乳細胞の悪性形質転換正常対腺房アーキテクチャを研究するためのツール

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The ...

Tracking the Mammary Architectural Features and Detecting Breast Cancer with Magnetic Resonance Diffusion Tensor Imaging

Breast cancer is the most common cause of cancer among women worldwide. Early detection of breast cancer has a critical role in improving the quality of life and survival of breast cancer patients. In this paper a new approach for the detection of breast cancer is described, based on tracking the mammary architectural elements using diffusion tensor imaging (DTI). 
The paper focuses on the scanning protocols and image processing algorithms and software that were designed to fit the diffusion properties of the mammary fibroglandular tissue and its changes during malignant transformation. The final output yields pixel by pixel vector maps that track the architecture of the entire mammary ductal glandular trees and parametric maps of the diffusion tensor coefficients and anisotropy indices. 
The efficiency of the method to detect breast cancer was tested by scanning women volunteers including 68 patients with breast cancer confirmed by histopathology findings. Regions with cancer cells exhibited a marked reduction in the diffusion coefficients and in the maximal anisotropy index as compared to the normal breast tissue, providing an intrinsic contrast for delineating the boundaries of malignant growth. Overall, the sensitivity of the DTI parameters to detect breast cancer was found to be high, particularly in dense breasts, and comparable to the current standard breast MRI method that requires injection of a contrast agent. Thus, this method offers a completely non-invasive, safe and sensitive tool for breast cancer detection.

We describe how to obtain parametric and vector maps of the diffusion tensor of the breast using magnetic resonance imaging. The protocol and final output following imaging processing are tailored for tracking breast architectural features and detecting breast malignancy.

乳腺アーキテクチャの特長を追跡し、磁気共鳴拡散テンソルイメージングと乳がんの検出

Breast cancer is the most common cause of cancer among women worldwide. Early detection of breast cancer has a critical role in improving the quality of ...

Assessment of Viability of Human Fat Injection into Nude Mice with Micro-Computed Tomography

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft tissue filler as it is readily available, easily obtained, inexpensive, and inherently biocompatible.1 However, despite its burgeoning popularity, fat grafting is hampered by unpredictable results and variable graft survival, with published retention rates ranging anywhere from 10-80%. 1-3
To facilitate investigations on fat grafting, we have therefore developed an animal model that allows for real-time analysis of injected fat volume retention. Briefly, a small cut is made in the scalp of a CD-1 nude mouse and 200-400 &#181;l&#160;of processed lipoaspirate is placed over the skull. The scalp is chosen as the recipient site because of its absence of native subcutaneous fat, and because of the excellent background contrast provided by the calvarium, which aids in the analysis process. Micro-computed tomography (micro-CT) is used to scan the graft at baseline and every two weeks thereafter. The CT images are reconstructed, and an imaging software is used to quantify graft volumes.
Traditionally, techniques to assess fat graft volume have necessitated euthanizing the study animal to provide just a single assessment of graft weight and volume by physical measurement ex vivo. Biochemical and histological comparisons have likewise required the study animal to be euthanized. This described imaging technique offers the advantage of visualizing and objectively quantifying volume at multiple time points after initial grafting without having to sacrifice the study animal. The technique is limited by the size of the graft able to be injected as larger grafts risk skin and fat necrosis. This method has utility for all studies evaluating fat graft viability and volume retention. It is particularly well-suited to providing a visual representation of fat grafts and following changes in volume over time.

Fat grafting is an essential technique for reconstructing soft tissue deficits. However, it remains an unpredictable procedure characterized by variable graft survival. Our goal was to devise a mouse model that utilizes a novel imaging method to compare volume retention between differing techniques of fat graft preparation and delivery.

マイクロコンピュータ断層撮影とヌードマウスへのヒト脂肪注入の生存率の評価

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft ...

An Ex vivo Model to Study Hormone Action in the Human Breast

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.

An Ex vivo Model to Study Hormone Action in the Human Breast

We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.

アン<em&gt;エクスビボ</emヒト乳癌でホルモンの作用を研究するために&gt;モデル

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...