私の研究室での研究はWKU研究財団RCAPは-Iは、生物学のスタートアップ資金のWKU省によってサポートされている＃11から8032を付与し、国民の協会の総合医科学研究所から親の許可によって資金を供給KBRINエリア助成金健康の賞番号5P20GM103436-13アンダー。私はまた、その実験室の私が最初に徐実験室で、この手法を確立し、この技術博士レイモンドPagliariniに導入された博士ティアン徐を承認したいと思います。

1。良性非浸潤性腫瘍の誘導<ol><li>良性腫瘍の誘導のために、表2にリストされている銘柄を使用してください。 </li><li>以下の遺伝子型の「テスター」の株式のためのスターター培養を準備します。浴槽-GAL80、FRT40A、Y、EY-FLP1、W Act5C&gt; Y +&gt;のGal4、UAS-GFP </li><li>以下の遺伝子型の「テスト」の株式のためのスターター培養を準備します。W; FRT40A、UAS-RasのV12 / CYO </li><li> 「テスト」在庫から「テスター」株式と10人の男性から10女性の処女を収集します。 </li><li>フライ食品とバイアルの両方を配置することで、「テスター」と「​​テスト」の在庫からオスとメスを渡ります。 </li><li> 25℃のインキュベーターにクロスを置き、男性と女性が交尾し、24〜48時間のために卵を産むことができます。 </li><li>十分な卵の堆積のためと第齢幼虫は文化が乾燥していないことを確認しての存在のためにバイアルをご確認ください。 <ol><li>オートクレーブ処理を数滴を追加します。（文化は乾燥されている場合）には湿った保つために培養液に蒸留水を。 </li><li>文化を監視し、必要に応じてステップ1.7.1を繰り返し続けています。 </li></ol></li><li>セクション3で概説したように、蛍光実体顕微鏡下に3齢幼虫をさまよう観察します。 </li></ol> 2。浸潤性腫瘍の誘発<ol><li> 浸潤性腫瘍の誘導のために、表2にリストされている銘柄を使用してください。 </li><li>以下の遺伝子型の上記のステップ1.2からスターターカルチャーを使用し、Y EY-FLP1、W、浴槽-GAL80、FRT40A; Act5C&gt; Y +&gt;のGal4、UAS-GFP </li><li> LGL 4、FRT40A、UAS-RasのV12 / CYO、W：以下の遺伝子型の「テスト」の株式のためのスターター培養液を準備します</li><li> 「テスト」在庫から「テスター」株式と10人の男性から10女性の処女を収集します。 </li><li>フライFとバイアル中で、それらの両方を配置することによって、「テスター」と「​​テスト」の在庫から男性と女性を渡るOOD。 </li><li> 25℃のインキュベーターにクロスを置き、男性と女性が交尾し、24〜48時間のために卵を産むことができます。 </li><li>十分な卵の堆積のためと第齢幼虫は文化が乾燥していないことを確認しての存在のためにバイアルをご確認ください。 <ol><li> （文化は乾燥されている場合）には湿った保つために培養するためにオートクレーブし、蒸留水を数滴を追加します。 </li><li>乾きのために文化の監視を続け、必要に応じてステップ2.7.1を繰り返します。 </li></ol></li><li>セクション3で概説したように、蛍光実体顕微鏡下に3齢幼虫を観察します。 </li></ol> 3。良性および浸潤性腫瘍の観察<ol><li>良性腫瘍の分離と可視化のための3齢幼虫を使用してください。 <ol><li>蒸留水にペイントブラシを濡らし、そしてこのペイントブラシを使用して培養バイアルの壁から数3齢幼虫をこすり。 </li><li> 1X PBSでうつ病スライドに幼虫を置き、ウェットペイントブラシスクラップを使用する幼虫表皮から任意の食品材料を切っメール。 </li><li>ペトリ皿に幼虫を置き、幼虫を固定化するために30分間-20℃の冷凍庫に出発。 <ol><li> FLYNAPを使用して幼虫を固定する別の方法。 </li><li> 3.1.3.2からバイアルにFLYNAPに浸しFLYNAPの杖を置き、幼虫を、バイアルのlet差し込まバイアルを接続した後に30から40分間放置。 </li></ol></li><li>カバーガラスで覆い、緑色蛍光タンパク質（GFP）を可視化する機能を備えた蛍光実体顕微鏡下で観察し、光ハロカーボン油の滴を追加し、スライドガラス上に固定化された幼虫を置きます。 </li><li>良性腫瘍を有する幼虫は頭部複合体が存在する前方領域に緑色の蛍光を発する。 </li></ol></li><li>浸潤性腫瘍の観察のため、上記のステップ＃2の培養セットから（誘導後）10日目の幼虫を選択します。浸潤性腫瘍を有する幼虫拡張リットルを持っているので、10日目に幼虫が選択される一般アルバルステージとpupariateに失敗します。 </li><li>代わりに3齢幼虫利用日10幼虫のを除いて3.1.4にステップ3.1.1に従ってください。 <ol><li> 10日目浸潤性腫瘍を有する幼虫は頭部複合体が存在する前方領域に緑色の蛍光を発する。 </li></ol></li><li>蛍光顕微鏡デジタルカメラが装備されている場合は、蛍光良性および浸潤性腫瘍を有する幼虫の写真を撮る。 </li></ol> 4。良性および浸潤性腫瘍の頭部のコンプレックスとさらなる観察の解剖幼虫の半透明の表皮が困難腫瘍の浸潤の程度を観察することができる。このように侵入の程度は良く幼虫から頭部の複雑な解剖により可視化されている。次の手順では、頭部の複合体を解剖するのに利用されるべきである。 <ol><li>湿ったペイントブラシを使用して（良性腫瘍のための）3齢幼虫を選択し、（浸潤性腫瘍の場合）10日目の幼虫Fそれぞれの培養バイアルをROMを</li><li>冷たい1X PBSを含む解剖皿の井戸に幼虫を置く。幼虫表皮から任意の食品の粒子を掻き取るペイントブラシを使用してください。 <ol><li>冷たい1X PBSを含む解剖皿の新鮮なウェルにきれいな幼虫を転送します。 </li><li>蛍光を検出することが可能な実体顕微鏡を用いて、GFP蛍光の有無を確認してください。幼虫を有する非GFPを捨てる。 </li></ol></li><li> （解剖プロトコルは腫瘍はこれらの2つのタイプのために同じまま）のどちらか良性または浸潤性腫瘍を有する幼虫を転送解剖皿に新鮮なウェルに冷たい1×PBSの1.0ミリリットルを追加し、ピンセットを使って。 </li><li>前端から3分の2程度ピンセットを使って幼虫を押したままにします。 <ol><li>鉗子の他のペアを使用し、スマートに幼虫の後部3分の1を分離し、それを廃棄します。 </li><li>幼虫の前方3分の2を手放す。幼虫内の圧力の内容が解放される幼虫は、体腔からにじみ出るう。 </li><li>腸、脂肪体、体腔外に染み出してきた幼虫の他の内側の内容を削除するためにピンセットを使用してください。 </li><li>幼虫の口フックを保持し、幼虫表皮にそれをプッシュするピンセットを使用し、完全に幼虫を反転鉗子の他のペアを使用。 </li><li>優しく、慎重に脂肪体、唾液腺、腸、翼ディスク複合体および任意の気管チューブを取り除くために鉗子を使用してください。頭部複合体は、現在、反転幼虫の口フックに取り付ける表示されるはずです。脳半球と腹側神経索（VNC）はまだ、VNCから発せられる神経を通して幼虫表皮に接続される。 </li><li>優しく頭部神経と幼虫の表皮との間の接続を破るためにピンセットを使用してください。 </li><li> VNCと幼虫表皮の間の神経接続が切断さと余分な脂肪本体やその他の組織が除去されると、頭部COmplexがはっきり見えるようになりだろうと口だけのフックで幼虫表皮に添付されます。 </li><li>複合体は、抗体染色のようなダウンストリームアプリケーションのため以降の視覚化のために固定される必要がある場合、頭部複合体は、幼虫の表皮に装着しておくことができます。 </li><li>それ以上のダウンストリームのアプリケーションが実行されないであろう場合には、頭部の複合体は、幼虫の表皮から分離し、さらに、観察および分析のためのメディアのマウントベースグリセロールの低下に配置することができます。封入剤としてVECTASHIELDを使用してください。 </li></ol></li></ol>

<ol>
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A., Xu, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+cell+polarity+drives+tumor+growth+and+invasion+through+JNK+activation+in+Drosophila.">Loss of cell polarity drives tumor growth and invasion through JNK activation in Drosophila.</a> Curr Biol. 16, 1139-1146 (2006).</li><li>Uhlirova, M., Jasper, H., Bohmann, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-cell-autonomous+induction+of+tissue+overgrowth+by+JNK/Ras+cooperation+in+a+Drosophila+tumor+model.">Non-cell-autonomous induction of tissue overgrowth by JNK/Ras cooperation in a Drosophila tumor model.</a> Proc Natl Acad Sci U S A. 102, 13123-13128 (2005).</li><li>Uhlirova, M., Bohmann, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=JNK-+and+Fos-regulated+Mmp1+expression+cooperates+with+Ras+to+induce+invasive+tumors+in+Drosophila.">JNK- and Fos-regulated Mmp1 expression cooperates with Ras to induce invasive tumors in Drosophila.</a> EMBO J. 25, 5294-5304 (2006).</li><li>Menut, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mosaic+genetic+screen+for+Drosophila+neoplastic+tumor+suppressor+genes+based+on+defective+pupation.">A mosaic genetic screen for Drosophila neoplastic tumor suppressor genes based on defective pupation.</a> Genetics. 177, 1667-1677 (2007).</li><li>Wu, M., Pastor-Pareja, J. C., Xu, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+between+Ras(V12)+and+scribbled+clones+induces+tumour+growth+and+invasion.">Interaction between Ras(V12) and scribbled clones induces tumour growth and invasion.</a> Nature. 463, 545-548 (2010).</li></ol>

私は、開示することは何もありません。

癌は過去に比べてはるかに優れた理解、今日と複雑な疾患である。しかし、まだ多くを学んだし、我々は根本的なメカニズムの全体像を持って前に説明される必要がある。ここで紹介する単純なプロトコルは、遺伝的、生物全体に良性および浸潤性腫瘍を誘発し、このモデルでは腫瘍の進行に関連した生物学を研究することが可能となる。 ショウジョウバエにおける既存の技術および...

転移性の状態に良性の腫瘍の進行は、本体8に存在する防御機構の回避することを特徴とする段階的なプロセスです。例えば、体内の腫瘍細胞は、アポトーシスや免疫システムを回避する基底膜と呼ばれる特殊な細胞外マトリックス（ECM）を突破し、周囲の細胞8が課す社会的コントロールを克服できなければなりません。これは、がん細胞が転移と呼ばれるプロセスでの遠隔部位に移行し、コロニーを形成する能力を獲得という段階的進行を介して行われます。腫瘍細胞は、体によって課さ障壁を克服する方法についての我々の理解は、しかし、研究から出てくる写真は、癌細胞による正常な発生過程およびシグナル伝達経路の繰り返し使用11月13日にこれまでのポイントを行って、まだ始まったばかりである。 フルーツ飛ぶキイロショウジョウバエは、私たちのウントに途方もなく貢献してきました過去数十年の14〜17にわたって開発、洗練された遺伝学的技術を用いて正常な発達と病気のerstanding。我々は、様々な癌遺伝子および癌抑制遺伝子18〜22歳のより良い理解に到達した突然変異誘発および過剰発現のツールを使用して。しかしながら、腫瘍転移は、細胞培養モデル23,24ならびに種々の異種移植モデル25-27主に研究されているいくつかの遺伝子病変間の協力の結果である。それらは完全に生体に見られる条件を模倣しないように、これらのモデルは、強力なしかし、それらの限界がある。さらに、マウスでの利用可能なトランスジェニックモデルが面倒で、侵襲性行動28,29の遺伝子解析に資するものではない。いくつかの研究は、ショウジョウバエ 30,31における腫瘍細胞の浸潤を理解しようと試みてきた。これらの技術は、主にホストに原発性腫瘍の移植を利用して、TRAの追跡に依存している隣接組織32,33の侵入のために腫瘍をnsplanted。 MARCM 10と呼ばれる強力な技術は、ショウジョウバエ 9に腫瘍の浸潤をモデル化するためにPagliariniと徐により適応されました。腫瘍の浸潤のエレガントな遺伝的モデル化は、活性化癌遺伝子や細胞極性の喪失との連携を悪用。このモデル化のパワーは浸潤性腫瘍は、したがって組織の移植のための必要性を回避無傷の生物に作成されるという事実にある。発癌の協力をもたらすために、RasのV12のような活性化癌遺伝子は、幼虫の目を触角にある細胞のクローンで表されます。 MARCM技術の結果、これらのクローンはまた、簡単に視覚化のために、緑色蛍光タンパク質（GFP）が付いていますし、細胞極性の走り書き致死巨大な幼虫のような変異体、および大規模なディスクのためにホモ接合作られています。結果がで浸潤性の腫瘍を、GFPをタグ付けされている頭部複雑。本報告では、私誘導し、無傷の幼虫のコンテキストでかつ摘出頭部複合体の両方で、これらの浸潤性腫瘍を可視化する方法を示します。ここで紹介する腫瘍誘導はショウジョウバエの第二の染色体上の試薬 ​​を利用しています。 表2には、私は同じ目的のために利用することができ、Xおよび第 3染色体上の株式のリストを提供する。私はこの単純化されたプロトコルは、腫瘍の進行の分子基盤を理解することに興味を持って研究者にこの技術は容易にアクセスできるようにと考えています。

<table border="1">
	<tbody>
 <tr>
 <td>10X PBS (phosphate buffered saline) pH 7.2 stock solution</td>
 <td>Invitrogen, Sigma Aldrich</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Chilled 1X PBS pH7.2 working solution</td>
 <td>Invitrogen, Sigma Aldrich</td>
 <td></td>
 <td>Make fresh and refrigerate, can be used up to a week</td>
 </tr>
 <tr>
 <td>Flynap</td>
 <td>Carolina Biologicals</td>
 <td></td>
 <td>Fly anesthesia needed to anesthetize larvae</td>
 </tr>
 <tr>
 <td>Fixative</td>
 <td>0.1M PIPES, pH 7.2, 4% Paraformaldehyde</td>
 <td></td>
 <td>Needed to fix the dissected cephalic complex</td>
 </tr>
 <tr>
 <td>Ice Bucket</td>
 <td>Several</td>
 <td></td>
 <td>Maintain solutions on ice. Also, dissect cephalic complex in chilled 1X PBS and then place on ice in an Eppendorf tube</td>
 </tr>
 <tr>
 <td>1.7ml Eppendorf tube</td>
 <td>Various</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Glass slides, cover glass</td>
 <td>Fisher Scientific</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Vectashield Mounting Media or any other mounting media</td>
 <td>Vector Laboratories</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Halocarbon 200 or 700 Oil</td>
 <td>Polysciences Inc. or Halocarbon.com</td>
 <td></td>
 <td>Halocarbon 200 is used to mount the larvae for visualization on a fluorescence stereoscope</td>
 </tr>
 <tr>
 <td>Sally Hansen "Hard as Nails" nail polish</td>
 <td>Can be found at any general merchandise store</td>
 <td></td>
 <td>Needed to seal the edges of Coverslip</td>
 </tr>
 <tr>
 <td>A Leica MZ16.5 fluorescence stereomicroscope or any other fluorescence stereomicroscope</td>
 <td>Leica and others</td>
 <td></td>
 <td>Needed to observe the GFP fluorescence in larvae</td>
 </tr>
 <tr>
 <td>Dumont #5 forceps</td>
 <td>Fine Science Tools</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Pyrex 9 well spot plate or any other dissection dish</td>
 <td>Sigma Aldrich</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Paint Brush</td>
 <td>Can be found at any general merchandise store</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Table 1. Materials needed to perform the experimental protocol presented in this article.</td>
 </tr>
 </tbody>
 </table>

a protocol for genetic induction and visualization of benign and invasive tumors in cephalic complexes of drosophila melanogaster

ショウジョウバエは、過去数十年の間、通常の開発や病気の遺伝的基礎の理解を照らしており、今日では、複雑な疾患1-7の我々の理解に非常に貢献し続けています。転移性の状態に良性の腫瘍の進行は、複雑なイベント8で、私たちはより良いこの病気9の遺伝的基礎を理解するために、ショウジョウバエでモデル化されています。ここで私は、遺伝的に誘導し、観察した後、 ショウジョウバエの幼虫の腫瘍の進行を分析するための単純なプロトコルを提示する。腫瘍誘発技術はMARCMシステム10に基づいており、活性化癌遺伝子、RasのV12及び細胞極性遺伝子の損失（ 落書き 、 ディスク大きく 、 致死巨大幼虫 ）浸潤性腫瘍9を生成する間の連携を利用している。私は、これらの腫瘍は完全な幼虫で可視化することができる方法を紹介し、次に、どのようにこれらは、さらなる分析のために切開することができます。ここで紹介する単純化したプロトコルにより、この技術は腫瘍の浸潤における遺伝子の役割を理解することに興味の研究者によって利用されるのを確認する必要があります。

プロトコルの結果がここに提示され、ユーザは、幼虫の目触角成虫盤で活性化癌遺伝子を過剰発現させることにより、良性腫瘍を誘発することができます。ユーザはまた、細胞極性遺伝子にも変異細胞のクローンで活性化癌遺伝子を過剰発現させることによって眼触角で浸潤性腫瘍を誘発することができるであろう。腫瘍は簡単に全体の幼虫や幼虫の体腔（ 図1）の摘出された頭...

Watch this Scientific Journal Video about A Protocol for Genetic Induction and Visualization of Benign and Invasive Tumors in Cephalic Complexes of Drosophila melanogaster at JoVE.com

A Protocol for Genetic Induction and Visualization of Benign and Invasive Tumors in Cephalic Complexes of Drosophila melanogaster

RAS V12と走り書き 、腫瘍細胞はまた、侵襲性の挙動を表示するショウジョウバエにおける腫瘍増殖をもたらす様細胞極性遺伝子の突然変異のような活性化癌遺伝子間の協力。ここに良性および浸潤性腫瘍の誘導および観察のための単純なプロトコルが提示される。

遺伝的誘導との頭部の複合体における良性および浸潤性腫瘍の可視化のためのプロトコル<em&gt;キイロショウジョウバエ</em

Drosophila has illuminated our  understanding of the genetic basis of normal development and disease for the  past several decades and today it continues ...

a-protocol-for-genetic-induction-visualization-benign-invasive-tumors

Research

JoVE Journal

Medicine

A Protocol for Genetic Induction and Visualization of Benign and Invasive Tumors in Cephalic Complexes of Drosophila melanogaster

9.4K ビュー.  Western Kentucky University.  RAS V12と走り書き 、腫瘍細胞はまた、侵襲性の挙動を表示するショウジョウバエにおける腫瘍増殖をもたらす様細胞極性遺伝子の突然変異のような活性化癌遺伝子間の協力。ここに良性および浸潤性腫瘍の誘導および観察のための単純なプロトコルが提示される。 

ビデオ: A Protocol for Genetic Induction and Visualization of Benign and Invasive Tumors in Cephalic Complexes of Drosophila melanogaster

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor. 
Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation. 
Wesley S. Bond and Lalita Wadhwa are co-first authors.

A method is described to propagate human retinoblastoma tumors in mice. Tumor cells are directly injected into the eyes of immune deficient mice. Secondary tumors have been successfully established using both cells directly harvested from human tumors and cultured tumorspheres.

免疫不全マウスにおけるヒト網膜芽細胞腫の腫瘍の確立と伝播

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

医学

Non-invasive Optical Measurement of Cerebral Metabolism and Hemodynamics in Infants

Perinatal brain injury remains a significant cause of infant mortality and morbidity, but there is not yet an effective bedside tool that can accurately screen for brain injury, monitor injury evolution, or assess response to therapy. The energy used by neurons is derived largely from tissue oxidative metabolism, and neural hyperactivity and cell death are reflected by corresponding changes in cerebral oxygen metabolism (CMRO2). Thus, measures of CMRO2 are reflective of neuronal viability and provide critical diagnostic information, making CMRO2 an ideal target for bedside measurement of brain health.
Brain-imaging techniques such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT) yield measures of cerebral glucose and oxygen metabolism, but these techniques require the administration of radionucleotides, so they are used in only the most acute cases.
Continuous-wave near-infrared spectroscopy (CWNIRS) provides non-invasive and non-ionizing radiation measures of hemoglobin oxygen saturation (SO2) as a surrogate for cerebral oxygen consumption. However, SO2 is less than ideal as a surrogate for cerebral oxygen metabolism as it is influenced by both oxygen delivery and consumption. Furthermore, measurements of SO2 are not sensitive enough to detect brain injury hours after the insult 1,2, because oxygen consumption and delivery reach equilibrium after acute transients 3. We investigated the possibility of using more sophisticated NIRS optical methods to quantify cerebral oxygen metabolism at the bedside in healthy and brain-injured newborns. More specifically, we combined the frequency-domain NIRS (FDNIRS) measure of SO2 with the diffuse correlation spectroscopy (DCS) measure of blood flow index (CBFi) to yield an index of CMRO2 (CMRO2i) 4,5.
With the combined FDNIRS/DCS system we are able to quantify cerebral metabolism and hemodynamics. This represents an improvement over CWNIRS for detecting brain health, brain development, and response to therapy in neonates. Moreover, this method adheres to all neonatal intensive care unit (NICU) policies on infection control and institutional policies on laser safety. Future work will seek to integrate the two instruments to reduce acquisition time at the bedside and to implement real-time feedback on data quality to reduce the rate of data rejection.

We combined frequency-domain near-infrared spectroscopy measures of cerebral hemoglobin oxygenation with diffuse correlation spectroscopy measures of cerebral blood flow index to estimate an index of oxygen metabolism. We tested the utility of this measure as a bedside screening tool to evaluate the health and development of the newborn brain.

乳幼児の脳代謝と血行動態の非侵襲的光計測

Perinatal brain injury remains a significant cause of infant mortality  and morbidity, but there is not yet an effective bedside tool that can  accurately ...

Renal Capsule Xenografting and Subcutaneous Pellet Implantation for the Evaluation of Prostate Carcinogenesis and Benign Prostatic Hyperplasia

New therapies for two common prostate diseases, prostate cancer (PrCa) and benign prostatic hyperplasia (BPH), depend critically on experiments evaluating their hormonal regulation. Sex steroid hormones (notably androgens and estrogens) are important in PrCa and BPH; we probe their respective roles in inducing prostate growth and carcinogenesis in mice with experiments using compressed hormone pellets. Hormone and/or drug pellets are easily manufactured with a pellet press, and surgically implanted into the subcutaneous tissue of the male mouse host. We also describe a protocol for the evaluation of hormonal carcinogenesis by combining subcutaneous hormone pellet implantation with xenografting of prostate cell recombinants under the renal capsule of immunocompromised mice. Moreover, subcutaneous hormone pellet implantation, in combination with renal capsule xenografting of BPH tissue, is useful to better understand hormonal regulation of benign prostate growth, and to test new therapies targeting sex steroid hormone pathways.

We describe the manufacture of compressed hormone pellets, as well as subcutaneous surgical implantation into mice. This strategy can be combined with the growth of cell and tissue xenografts under the renal capsule of athymic nude mice to evaluate hormonal carcinogenesis and regulation of benign prostate growth.

前立腺発がんおよび良性前立腺過形成の評価のための腎皮膜異種移植および皮下ペレット移植

New therapies for two common prostate diseases,  prostate cancer (PrCa) and benign prostatic hyperplasia (BPH), depend  critically on experiments ...

Induction of Invasive Transitional Cell Bladder Carcinoma in Immune Intact Human MUC1 Transgenic Mice:  A Model for Immunotherapy Development

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy and/or cytotoxic chemotherapy. To induce bladder cancer, C57BL/6 mice (MUC1.Tg and wild type) were treated orally with the carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (OH-BBN) at 3.0 mg/day, 5 days/week for 12 weeks. To assess the effects of OH-BBN on serum cytokine profile during tumor development, whole blood was collected via submandibular bleeds prior to treatment and every four weeks. In addition, a MUC1-targeted peptide vaccine and placebo were administered to groups of mice weekly for eight weeks. Multiplex fluorometric microbead immunoanalyses of serum cytokines during tumor development and following vaccination were performed. At termination, interferon gamma (IFN-&#947;)/interleukin-4 (IL-4) ELISpot analysis for MUC1 specific T-cell immune response and histopathological evaluations of tumor type and grade were performed. The results showed that: (1) the incidence of bladder cancer in both MUC1.Tg and wild type mice was 67%; (2) transitional cell carcinomas (TCC) developed at a 2:1 ratio compared to squamous cell carcinomas (SCC); (3) inflammatory cytokines increased with time during tumor development; and (4) administration of the peptide vaccine induces a Th1-polarized serum cytokine profile and a MUC1 specific T-cell response. All tumors in MUC1.Tg mice were positive for MUC1 expression, and half of all tumors in MUC1.Tg and wild type mice were invasive. In conclusion, using a team approach through the coordination of the efforts of pharmacologists, immunologists, pathologists and molecular biologists, we have developed an immune intact transgenic mouse model of bladder cancer that expresses hMUC1.

An N-butyl-N-(4-hydroxybutyl)nitrosamine-induced bladder cancer model was developed in human mucin 1 (MUC1) transgenic mice for the purpose of testing MUC1-directed immunotherapy. After administering a MUC1-targeted peptide vaccine, a cytotoxic T lymphocyte response to MUC1 was confirmed by measuring serum cytokine levels and T-cell specific activity.

免疫無傷のヒトMUC1トランスジェニックマウスにおける侵襲移行細胞膀胱癌の誘導：免疫療法の開発のためのモデル

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

Induction of Accelerated Atherosclerosis in Mice: The &quot;Wire-Injury&quot; Model

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

マウスにおける加速アテローム性動脈硬化症の誘導:ワイヤー傷害モデル

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

Intravital Microscopy and Thrombus Induction in the Earlobe of a Hairless Mouse

Thrombotic complications of vascular diseases are one leading cause of morbidity and mortality in industrial nations. Due to the complex interactions between cellular and non-cellular blood components during thrombus formation, reliable studies of the physiology and pathophysiology of thrombosis can only be performed in vivo. Therefore, this article presents an ear model in hairless mice and focuses on the in vivo analysis of microcirculation, thrombus formation, and thrombus evolution. By using intravital fluorescence microscopy and the intravenous (iv) application of the respective fluorescent dyes, a repetitive analysis of microcirculation in the auricle can easily be performed, without the need for surgical preparation. Furthermore, this model can be adapted for in vivo studies of different issues, including wound healing, reperfusion injury, or angiogenesis. In summary, the ear of hairless mice is an ideal model for the in vivo study of skin microcirculation in physiological or pathophysiological conditions and for the evaluation of its reaction to different systemic or topical treatments.

The ear model of the hairless SKH1-Hrhr mouse enables intravital fluorescence microscopy of microcirculation and phototoxic thrombus induction without prior surgical preparation in the examined microvascular bed. Therefore, the ear of the hairless mouse is an excellent in vivo model to study the complex interactions during microvascular thrombus formation, thrombus evolution, and thrombolysis.

ヘアレスマウスの耳たぶにおける生体顕微鏡および血栓誘導

Thrombotic complications of vascular diseases are one leading cause of morbidity and mortality in industrial nations. Due to the complex interactions ...

Technique of Minimally Invasive Transverse Aortic Constriction in Mice for Induction of Left Ventricular Hypertrophy

Transverse aortic constriction (TAC) in mice is one of the most commonly used surgical techniques for experimental investigation of pressure overload-induced left ventricular hypertrophy (LVH) and its progression to heart failure. In the majority of the reported investigations, this procedure is performed with intubation and ventilation of the animal which renders it demanding and time-consuming and adds to the surgical burden to the animal. The aim of this protocol is to describe a simplified technique of minimally invasive TAC without intubation and ventilation of mice. Critical steps of the technique are emphasized in order to achieve low mortality and high efficiency in inducing LVH.
Male C57BL/6 mice (10-week-old, 25-30 g, n=60) were anesthetized with a single intraperitoneal injection of a mixture of ketamine and xylazine. In a spontaneously breathing animal following a 3-4 mm upper partial sternotomy, a segment of 6/0 silk suture threaded through the eye of a ligation aid was passed under the aortic arch and tied over a blunted 27-gauge needle. Sham-operated animals underwent the same surgical preparation but without aortic constriction. The efficacy of the procedure in inducing LVH is attested by a significant increase in the heart/body weight ratio. This ratio is obtained at days 3, 7, 14 and 28 after surgery (n = 6 - 10 in each group and each time point). Using our technique, LVH is observed in TAC compared to sham animals from day 7 through day 28. Operative and late (over 28 days) mortalities are both very low at 1.7%.
In conclusion, our cost-effective technique of minimally invasive TAC in mice carries very low operative and post-operative mortalities and is highly efficient in inducing LVH. It simplifies the operative procedure and reduces the strain put on the animal. It can be easily performed by following the critical steps described in this protocol.

The aim of this protocol is to describe step-by-step the technique of minimally invasive transverse aortic constriction (TAC) in mice. By elimination of intubation and ventilation which are mandatory for the commonly used standard procedure, minimally invasive TAC simplifies the operative procedure and reduces the strain put on the animal.

左室肥大の誘導マウスにおける低侵襲の横大動脈狭窄のテクニック

Transverse aortic constriction (TAC) in mice is one of the most commonly used surgical techniques for experimental investigation of pressure ...

Non-invasive Assessment of Dorsiflexor Muscle Function in Mice

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively affect skeletal muscle. This can result in a loss of muscle mass (atrophy) and/or loss of muscle quality (reduced force per unit of muscle mass), both of which are prevalent in chronic disease, muscle-specific disease, immobilization, and aging (sarcopenia). Skeletal muscle function in animals can be evaluated by a range of different tests. All tests have limitations related to the physiological testing environment, and the selection of a specific test often depends on the nature of the experiments. Here, we describe an in vivo, non-invasive technique involving a helpful and easy assessment of force frequency-curve (FFC) in mice that can be performed on the same animal over time. This permits monitoring of disease progression and/or efficacy&#160;of a potential therapeutic treatment.

Measurement of rodent skeletal muscle contractile function is a useful tool that can be used to track disease progression as well as efficacy of therapeutic intervention. We describe here the non-invasive, in vivo assessment of the dorsiflexor muscles that can be repeated over time in the same mouse.

マウスで内がえし筋機能の非侵襲的評価

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively ...

Visualization of Endogenous Mitophagy Complexes In Situ in Human Pancreatic Beta Cells Utilizing Proximity Ligation Assay

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated insulin release. Assessment of mitophagy is challenging and often requires genetic reporters or multiple complementary techniques not easily utilized in tissue samples, such as primary human pancreatic islets. Here we demonstrate a robust approach to visualize and quantify formation of key endogenous mitophagy complexes in primary human pancreatic islets. Utilizing the sensitive proximity ligation assay technique to detect interaction of the mitophagy regulators NRDP1 and USP8, we are able to specifically quantify formation of essential mitophagy complexes in situ. By coupling this approach to counterstaining for the transcription factor PDX1, we can quantify mitophagy complexes, and the factors that can impair mitophagy, specifically within beta cells. The methodology we describe overcomes the need for large quantities of cellular extracts required for other protein-protein interaction studies, such as immunoprecipitation (IP) or mass spectrometry, and is ideal for precious human islet&#160;samples generally not available in sufficient quantities for these approaches. Further, this methodology obviates the need for flow sorting techniques to purify beta cells from a heterogeneous islet population for downstream protein applications. Thus, we describe a valuable protocol for visualization of mitophagy highly compatible for use in heterogeneous and limited cell populations.

This protocol outlines a method for quantitative analysis of mitophagy protein complex formation specifically in beta cells from primary human islet samples. This technique thus allows analysis of mitophagy from limited biological material, which are crucial in precious human pancreatic beta cell samples.

近接リゲーションアッセイを利用したヒト膵臓ベータ細胞における内因性ミトファジー複合体の可視化

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated ...

A Pre-clinical Rat Model for the Study of Ischemia-reperfusion Injury in Reconstructive Microsurgery

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many areas of biomedical research due to its cost-effectiveness and its translation to humans. This protocol describes a method to create a preclinical free skin flap model in rats with ischemia-reperfusion injury. The described 3 cm x 6 cm rat free skin flap model is easily obtained after the placement of several vascular ligatures and the section of the vascular pedicle. Then, 8 h after the ischemic insult and completion of the microsurgical anastomosis, the free skin flap develops the tissue damage. These ischemia-reperfusion injury-related damages can be studied in this model, making it a suitable model for evaluating therapeutic agents to address this pathophysiological process. Furthermore, two main monitoring techniques are described in the protocol for the assessment of this animal model: transit-time ultrasound technology and laser speckle contrast analysis.

Here, we describe a pre-clinical animal model for studying the pathophysiology of ischemia-reperfusion injury in reconstructive microsurgery. This free skin flap model based on the superficial caudal epigastric vessels in the rat may also allow for the evaluation of different therapies and compounds to counteract ischemia-reperfusion injury-related damage.

再建微小外科における虚血再灌流損傷の研究のための前臨床ラットモデル

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many ...