著者らはこのモデルの開発に支援をVerleneヘンリーとリンジーホームズに感謝します。この研究の一部はジェームズS.マクダネル財団（＃220020173）によって賄われていた。

1。細胞株<ol><li> U87MG神経膠腫の細胞は、5％ウシ胎児血清（FBS）を添加したDMEM - F12を持つ大規模な組織培養フラスコで培養されています。 </li><li>細胞は温かいリン酸で二回フラスコを洗浄することによって収穫されている生理食塩水（PBS）は、バッファ、37でそれらをインキュベート° 0.25％トリプシンおよび0.05％EDTAを含むPBS 10mlで5分間。一度細胞が解除され、それらは培養培地、遠心分離（4分間300 × g）を10 mlを入れた50 mlチューブに配置されます。 </li><li>洗浄後、細胞を10 × 10 6 / 50,000細胞/5μlの接種のために許容される培地のmlの濃度で再懸濁する。 </li><li>細胞は、頭蓋内注射するまで氷上で保存されています。 </li></ol> 2。ボルト頭蓋内ガイドのネジ この手順は、細胞の注入の前に数日間行うことができます。ここで説明するすべての手順は、厳格な無菌条件下で行われている。 <ol><li>マウス（BALB / c NU / NU（メス）、5-6週間、約18グラム）は、ケタミン（100 mg / kg体重）及びキシラジン（5 mgの混合物の腹腔内（IP）の注入で秤量し、anesthetised、識別の目的で番号が付けられています/ kg）を。 </li><li>皮膚は、ヨウ素液でダウン拭いていると、小さな切開（2-3 mm）が正中線と両耳ラインの前方の右側に沿って行われます。これは、頭蓋骨の冠状および矢状縫合糸を公開します。ブレグマは、これら2つの縫合糸の接合部に位置している。 </li><li>ガイド用ネジのエントリポイントは、ポイント横2.5ミリメートルとブレグマの前方を1 mmにマークされています。この点は、直接に尾状核1の上方に位置しています。 </li><li> 1 × 1ミリメートルの深穴は、硬膜に頭蓋骨を介してツイストドリルを保持し、滅菌手で掘られている。 </li><li> 硬膜への頭蓋骨の表面の下に1.6ミリメートルを延長する滅菌ガイド用ネジは、ネジをドライバーで穴にボルトで固定されて頭蓋骨（図1A）と同じ高さになるまで。 </li><li>滅菌スタイレットまたはスクリューのダミーは、開口部（図1B）をクローズするためのガイド用ネジの中心孔に配置されます。 </li><li>鎮痛のために傷がVetbond組織接着剤で閉じられます（n -ブチルシアノアクリレート）およびマウスreversineの腹腔内注射（小動物）（0.1 ml / kgをを）与えられ、カルプロフェン（5 mg/kg/100μL）。マウスはその後、20分かかることが温暖化防止マット（36℃）で回復させる。彼らは完全に意識されるまでマウスを頻繁にモニターし、この回復時間の間に観察されています。 </li></ol> 3。頭蓋内細胞の生着<ol><li>滅菌カフハミルトンシリンジは、ガイド用ネジコンセント（図1B）の下に拡張するために、針のわずか2 mmを可能に針の先端に小さなプラスチック製のリングを適用することによって調製される。最終的な接種のポイントは、したがって、頭蓋骨の表面の下に3.5ミリメートルです。 </li><li>ガイド用ネジの手術後4日間、マウスは再び（2.1）上記のように麻酔し、小さな切開は、スタイレットを除去するためのガイド用ネジを介して行われます。 </li><li>カフハミルトンシリンジは、どのような気泡を作成したり、策定しないように予防策を取ってよく混合した細胞を5μlで満たされている。 </li><li>シリンジは、注入ポンプに固定され、針をガイドねじ（図1C）に挿入されます。次に、細胞を1時間あたり30μlの割合で注入されています。 </li><li>自動化装置（図1D）は、私たちは一定の流速で一度に10匹まで注入することができました。細胞はまた、カフ付きハミルトンシリンジを使用して手動で注入することができるか、あるいは注射器を針の先端が2mmからガイド用ネジを超えて拡張できるように3 mmでトリミングされている200μlのピペットの先端で手錠することができます。注入は、一定の安定したペースで実行する必要があります。 </li><li>注入が完了すると、注射器を注意深く除去され、スタイレットは、ガイドのネジに置き換えられます。傷口が閉じて接着され、マウスが（2.7）以前のように回復薬を与えられ、温暖化のマットの上に回復するために許可されています。 </li></ol> 4。治療の課題<ol><li> U87MG細胞接種4日後、マウスを秤量し、無作為にコントロールし、治療群に分けられます。 </li><li>治療群がAMG102（PBS100μl中100μgの）のIP注射を与えている間に、対照群は、PBS（100μL）の腹腔内（IP）注射が与えられます。 </li><li>注射は、14日間は毎秒6日目注射（図2）の合計を繰り返している。 </li><li>最後の注射後、マウスを毎日モニターし、一日おきに秤量する。 </li><li>マウスでは有意な神経学的機能障害（平衡感覚障害、麻痺）、脱水、または10％以上の体重減少または瀕死を表示するために始めたとき、彼らは人道的に安楽死された。これらの人道的エンドポイントの日数は、Kaplan - Meier生存曲線を記録した。 </li></ol> 5。治療効果の評価</p&gt; <ol><li> Kaplan - Meier生存曲線は、Windows用グラフパッドプリズム4.03を使用して生成され、グラフパッドソフトウェア、サンディエゴカリフォルニア、アメリカ、 <a href="http://www.graphpad.com">www.graphpad.com</a> 3。死亡またはeuthanizationsは、エンドポイントのイベントとしてマークされ、1としてスコア化した。実験終了時に生きて残った動物は、非イベントとして記録され、0としてスコア化した。 </li></ol> 6。代表的な結果： 対照動物は23日、細胞の初期接種後の神経障害や体重減少の兆しを示し始めた。これは、大幅に35日にAMG102投与群では遅れていた。確かに35日で、対照群の77％（N = 7 / 9）が安楽死されていた。 Kaplan - Meier生存曲線は明らかにAMG102の治療（図3）に対応して生存率の有意な増加を示しています。 70日では、対照マウスの88％は、AMG102投与群の37％（N = 3 / 8）と比較して（N = 8 / 9）安楽死されていた。脳の組織学的分析は、対照群では全9動物発達した腫瘍は唯一の3 8 AMG102投与したマウス（図4）のと比較していることを確認した。 <img alt="図1" src="/files/ftp_upload/3157/3157fig1.jpg" /> 図1：画像は（）を直接尾状核の上にあるエリアに位置して頭蓋内のガイド用ネジを示しています。針の2mmのガイド超えると細胞は尾状核の内部に3.5ミリメートルを注入されるように、カフ付きハミルトンシリンジ（細胞との事前ロ​​ードされたが）その後、ガイド用ネジの内側に配置されます。注射器は、後にスタイレット（B）によって除去し、置き換えられます。自動注入装置（C）は、一度に10匹まで注入することができます。ここに示した5つの同時注射は、（D）です。 <img alt="図2" src="/files/ftp_upload/3157/3157fig2.jpg" /> 図2：治験実施計画書の回路図。細胞は、治療は4日目に開始0日目に注入した。治療群ではAMG102（100μg/100μL）IPの注入を受けた一方、コントロール動物では、PBS（100μL）IPの注射を受けた。これらの注射は10日間一日おきに与えられた。 6注射の合計が指定されました。動物は、神経学的および物理的な障害の兆候がない70日間モニターした。 <img alt="図3" src="/files/ftp_upload/3157/3157fig3.jpg" /> 図3：コントロールとAMG102処置マウスを比較したKaplan - Meier生存曲線。有意な生存率はAMG102（P = 0.005）で処理した後得られた。 <img alt="図4" src="/files/ftp_upload/3157/3157fig4.jpg" /> 図4：腫瘍対照動物の開発（）とAMG102処理された動物（B）のない脳腫瘍の開発を実証する冠状脳切片の顕微鏡写真。スケールバーは1mmである。

<ol>
	<li>Lal, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+implantable+guide-screw+system+for+brain+tumor+studies+in+small+animals.">An implantable guide-screw system for brain tumor studies in small animals.</a> J Neurosurg. 92, 326-333 (2000).</li><li>Jun, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AMG+102,+a+fully+human+anti-hepatocyte+growth+factor/scatter+factor+neutralizing+antibody,+enhances+the+efficacy+of+temozolomide+or+docetaxel+in+U-87+MG+cells+and+xenografts.">AMG 102, a fully human anti-hepatocyte growth factor/scatter factor neutralizing antibody, enhances the efficacy of temozolomide or docetaxel in U-87 MG cells and xenografts.</a> Clin Cancer Res. 13, 6735-6742 (2007).</li><li>Pillay, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+plasticity+of+oncogene+addiction:+implications+for+targeted+therapies+directed+to+receptor+tyrosine+kinases.">The plasticity of oncogene addiction: implications for targeted therapies directed to receptor tyrosine kinases.</a> Neoplasia. 11, 448-458 (2009).</li><li>Buchanan, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiosensitization+of+glioma+cells+by+modulation+of+Met+signaling+with+the+hepatocyte+growth+factor+neutralizing+antibody.+AMG102.">Radiosensitization of glioma cells by modulation of Met signaling with the hepatocyte growth factor neutralizing antibody. AMG102.</a> J Cell Mol Med. 15, 1999-2006 (2011).</li><li>Wen, P. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+phase+II+study+evaluating+the+efficacy+and+safety+of+AMG+102+(rilotumumab)+in+patients+with+recurrent+glioblastoma.">A phase II study evaluating the efficacy and safety of AMG 102 (rilotumumab) in patients with recurrent glioblastoma.</a> Neuro Oncol. 13, 437-446 (2011).</li><li>Caretti, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+of+Tumor+Growth+and+Post-Irradiation+Recurrence+in+a+Diffuse+Intrinsic+Pontine+Glioma+Mouse+Model.">Monitoring of Tumor Growth and Post-Irradiation Recurrence in a Diffuse Intrinsic Pontine Glioma Mouse Model.</a> Brain Pathol. 4, 441-451 (2011).</li><li>Jallo, G. I., Volkov, A., Wong, C., Carson, B. S., Penno, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+brainstem+tumor+model:+functional+and+histopathological+characterization.">A novel brainstem tumor model: functional and histopathological characterization.</a> Childs Nerv. Syst. 22, 1519-1525 (2006).</li><li>Johns, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+efficacy+of+epidermal+growth+factor+receptor-specific+antibodies+against+gliomaxenografts+is+influenced+by+receptor+levels,+activation+status,+and+heterodimerization.">The efficacy of epidermal growth factor receptor-specific antibodies against gliomaxenografts is influenced by receptor levels, activation status, and heterodimerization.</a> Clin Cancer Res. 13, 1911-1925 (2007).</li><li>Scott, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+phase+I+clinical+trial+with+monoclonal+antibody+ch806+targeting+transitional+state+and+mutant+epidermal+growth+factor+receptors.">A phase I clinical trial with monoclonal antibody ch806 targeting transitional state and mutant epidermal growth factor receptors.</a> Proc. Natl. Acad. Sci. U. S. A. 104, 4071-4076 (2007).</li><li>Niyazi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+options+for+recurrent+malignant+glioma.">Therapeutic options for recurrent malignant glioma.</a> Radiother. Oncol. 98, 1-14 (2011).</li></ol>

この作品は、Amgen社から研究助成金によって支えられて

ここで紹介する頭蓋内のガイド用ネジの方法は、頭蓋内異種移植片を迅速かつ再現性の確立を可能にし、訓練された動物の技術者の手に、この手順は非常に簡単に定位装置を必要とせずに実行されます。我々の手法は正確にin vivoでの腫瘍の開発に最適な脳の領域、および一度に最大10匹までの同時接種が可能な自動注入ポンプを見つけるために、ガイド用ネジを組み合わせて使用​​します。ガイド用ネジの増大術は、実行するために5分以内かかります。 30μlの/時間における細胞の自動注入は、5μlのボリュームのためにわずか10分かかります。これは一定の速度で正確な投与を保証し、頭蓋内圧亢進に起因する余分-脳神経領域に細胞の追放のリスクを低減します。我々はまた、10μlの容量でこの手順を実行していると携帯逆流に起因する余分-頭蓋腫瘍の成長を確認されていません。自動注入ポンプはまた、いくつかの動物は我々は時間当たり約15の動物に限定されている定位の手順に比べて時間当たり60匹まで注入することができると同時に接種することができますように。 その位置は、腫瘍の大きさの究極の位置を決定するとして、この手順の中で最も重要なステップは、頭蓋骨のガイド用ネジの位置です。ガイド用ネジがあまりにも中心的である場合、細胞はガイドがはるか前方にに配置されている場合では2つの脳半球の間の領域に注入され、細胞は、すべてで脳内に注入されることはありません。したがって、ガイド横2.5ミリメートルとブレグマ1〜前方1 mmの位置で可能な限り正確であることが不可欠である。プラスチック製のガイド用ネジは、このようなMRIやPET 6などの脳イメージングを必要とする研究にも利用できるが、本研究では、鋼のガイド用ネジを使用していました。自動注入ポンプは、いくつかのグループのためのコストがかかりすぎるされている場合でも、細胞はまだ手動でより迅速に定位のアプローチよりも注入することができる。全体的に、この迅速、正確かつ再現性のある方法は神経膠腫、髄芽腫や脳幹の腫瘍7を研究するために使用することができます。この手順は、予想外の麻酔反応（&lt;1％）による死亡者と我々の2つの研究室間、500人以上のマウスで行われている。ガイド用ネジとボルトで固定し、その後、細胞を注入したマウスは、手続きの結果として、神経学的合併症を死亡するか、受けていない。動物はどんな合併症がなく、100日を超えて生存している。我々はヘアレスマウスを使用するのが好ましいが、適切に提供するのと同様に、SCIDのような髪の免疫不全マウスは、頭蓋骨の髪は、サイトを清潔に保つために、ガイド用ネジに干渉する毛を防ぐために手術前に削除されましたれる。 現在、神経膠腫80から10の治療用に開発されているいくつかの新規治療薬があります。最近では、皮下U87MG神経膠腫異種移植片がAMG102 3で処理することにより抑制できることを示した;ヒト化抗体は、現在、神経膠腫5に臨床評価を受けてHGFに指示。我々は、ヌードマウスの脳で頭蓋内U87MG腫瘍を確立する方法がここに記述するために使用。我々の研究は明らかに法の有用性を示し、AMG102が大幅に同所U87MG異種移植片の増殖を阻害することを示しています。我々は、他の治療法が腫瘍の成長をより効果的にinhibitionを取得するにはAMG102と組み合わせて使用​​することができるかを決定するためにこのモデルを使用し続けます。

<table border="1"><tbody><tr><th>試薬の名前</th><th>会社</th><th>カタログ番号</th><th>コメント（省略可能） </th></tr><tr><td> DMEM/F12 </td><td>インビトロジェン</td><td> 12634010 </td><td></td></tr><tr><td> FCS </td><td> Bovogen </td><td> SFBを</td><td></td></tr><tr><td>トリプシン</td><td>インビトロジェン</td><td> 15050065 </td><td></td></tr><tr><td> EDTA </td><td>シグマ</td><td> E6758 </td><td></td></tr><tr><td> BALB / C nu / nuマウス</td><td> ARCパース</td><td></td><td></td></tr><tr><td>スクリューホルダー</td><td>プラスチック一</td><td> SD - 1 </td><td></td></tr><tr><td>ドリルホルダー</td><td>プラスチック一</td><td> DH - 1 </td><td></td></tr><tr><td>ドリルのビット</td><td>プラスチック一</td><td> D＃60 </td><td></td></tr><tr><td>ネジガイド（金属） </td><td>プラスチック一</td><td> C212SG </td><td></td></tr><tr><td>ネジのダミー（スタイラス） </td><td>プラスチック一</td><td> C212SD </td><td></td></tr><tr><td>ハミルトンシリンジ（10μlを26グラム超硬針） </td><td>グレース課発見科学</td><td> 80075 </td><td></td></tr><tr><td> PHD 2000分の22の注入ポンプ</td><td>ハーバード装置</td><td> 70から2003 </td><td>オプション</td></tr><tr><td> Vetbond </td><td> 3M </td><td> 1469SB </td><td></td></tr><tr><td>サーモパッド</td><td>ハーバード装置</td><td> 340390 </td><td></td></tr></tbody></table>

a simple guide screw method for intracranial xenograft studies in mice

免疫抑制マウスの脳にヒト腫瘍細胞の移植は、神経膠芽腫（グリオーマ）と髄芽腫を含む脳腫瘍の研究のための確立された方法です。広く使用されている定位アプローチは一度に一つの動物の注入を可能にする、労働集約的であり、非常に特殊な装置を必要とします。最初はラルら 、1で開発したガイド用ネジの方法は、面倒な定位の手順を排除するために開発されました。我々は現在、迅速かつ非常に安全であると修正されたガイド用ネジのアプローチを説明し、重要な倫理的配慮が、どちらも。特に、私たちの手続きは今同時に腫瘍細胞を注入することが10匹まで可能に注入ポンプを内蔵しています。 HGF /現在臨床評価2-5を受けて散乱係数に向け完全ヒト抗体を、この手順の有用性を実証するために、我々は、AMG102を投与したマウスの頭蓋内異種移植片、のように人間のU87MG神経膠腫細胞を確立した。 AMG102の全身注射は大幅に頭蓋内U87MGの異種移植片ですべてのマウスの生存を延長し、完全な治療の数となりました。 この研究では、ガイド用ネジのメソッドは頭蓋内異種移植片を確立するための安価な、再現性の高いアプローチであることを示しています。さらに、それは脳腫瘍の治療のための新たな治療戦略を検証するための関連する生理学的モデルを提供します。

Watch this Scientific Journal Video about A Simple Guide Screw Method for Intracranial Xenograft Studies in Mice at JoVE.com

A Simple Guide Screw Method for Intracranial Xenograft Studies in Mice

神経膠腫の治療のための新たな治療パラダイムを評価するために、生理学的な関連モデルが不可欠です。我々は、定位のアプローチよりもより迅速かつ安全である頭蓋内移植モデルの確立のための埋め込み型ガイド用ネジの手順を利用する。

マウスにおける頭蓋内異種移植研究のための簡単​​なガイドスクリュー方式

The grafting of human tumor cells into the brain of immunosuppressed mice is an established method for the study of brain cancers including glioblastoma ...

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Medicine

22.9K ビュー.  Monash Institute of Medical Research. 神経膠腫の治療のための新たな治療パラダイムを評価するために、生理学的な関連モデルが不可欠です。我々は、定位のアプローチよりもより迅速かつ安全である頭蓋内移植モデルの確立のための埋め込み型ガイド用ネジの手順を利用する。

ビデオ: A Simple Guide Screw Method for Intracranial Xenograft Studies in Mice

Epidural Intracranial Pressure Measurement in Rats Using a Fiber-optic Pressure Transducer

Elevated intracranial pressure (ICP) is a significant problem in several forms of ischemic brain injury including stroke, traumatic brain injury and cardiac arrest. This elevation may result in further neurological injury, in the form of transtentorial herniation1,2,3,4, midbrain compression, neurological deficit or increased cerebral infarct2,4. Current therapies are often inadequate to control elevated ICP in the clinical setting5,6,7 . Thus there is a need for accurate methods of ICP measurement in animal models to further our understanding of the basic mechanisms and to develop new treatments for elevated ICP.
In both the clinical and experimental setting ICP cannot be estimated without direct measurement. Several methods of ICP catheter insertion currently exist. Of these the intraventricular catheter has become the clinical 'gold standard' of ICP measurement in humans8. This method involves the partial removal of skull and the instrumentation of the catheter through brain tissue. Consequently, intraventricular catheters have an infection rate of 6-11%9. For this reason, subdural and epidural cannulations have become the preferred methods in animal models of ischemic injury. 
Various ICP measurement techniques have been adapted for animal models, and of these, fluid-filled telemetry catheters10 and solid state catheters are the most frequently used11,12,13,14,15. The fluid-filled systems are prone to developing air bubbles in the line, resulting in false ICP readings. Solid state probes avoid this problem (Figure 1). An additional problem is fitting catheters under the skull or into the ventricles without causing any brain injury that might alter the experimental outcomes. Therefore, we have developed a method that places an ICP catheter contiguous with the epidural space, but avoids the need to insert it between skull and brain. 
An optic fibre pressure catheter (420LP, SAMBA Sensors, Sweden) was used to measure ICP at the epidural location because the location of the pressure sensor (at the very tip of the catheter) was found to produce a high fidelity ICP signal in this model. There are other manufacturers of similar optic fibre technologies13 that may be used with our methodology. Alternative solid state catheters, which have the pressure sensor located at the side of the catheter tip, would not be appropriate for this model as the signal would be dampened by the presence of the monitoring screw. 
Here, we present a relatively simple and accurate method to measure ICP. This method can be used across a wide range of ICP related animal models.

A novel technique to record the pressures within the skull is described. The minimally invasive method uses a fibre-optic pressure sensing system to accurately measure intracranial pressure (ICP) in anaesthetized rats without causing significant brain trauma. The technique may be used in a wide range of experimental models.

光ファイバ圧力トランスデューサを使用したラットの硬膜外頭蓋内圧測定

Elevated intracranial pressure (ICP) is a significant problem in several forms of ischemic brain injury including stroke, traumatic brain injury and ...

医学

A Simple Method of Mouse Lung Intubation

A simple procedure to intubate mice for pulmonary function measurements would have several advantages in longitudinal studies with limited numbers or expensive animal. One of the reasons that this is not done more routinely is that it is relatively difficult, despite there being several published studies that describe ways to achieve it. In this paper we demonstrate a procedure that eliminates one of the major hurdles associated with this intubation, that of visualizing the trachea during the entire time of intubation. The approach uses a 0.5 mm fiberoptic light source that serves as an introducer to direct the intubation cannula into the mouse trachea. We show that it is possible to use this procedure to measure lung mechanics in individual mice over a time course of at least several weeks. The technique can be set up with relatively little expense and expertise, and it can be routinely accomplished with relatively little training. This should make it possible for any laboratory to routinely carry out this intubation, thereby allowing longitudinal studies in individual mice, thereby minimizing the number of mice needed and increasing the statistical power by using each mouse as its own control.

This paper describes a striaghforward and efficient method of intubating mice for pulmonary function measurements or pulmonary instillation, that allows the mice to recover and be studied at later times. The procedure involves an inexpensive fiberoptic light source that directly illuminates the trachea.

マウス肺挿管の簡単な方法

A  simple procedure to intubate mice for pulmonary function measurements would  have several advantages in longitudinal studies with limited numbers or  ...

The In ovo CAM-assay as a Xenograft Model for Sarcoma

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for personalized medicine after stratification, explaining the current interest in developing a reproducible and low-cost xenotransplant model for this disease. The chick chorioallantoic membrane is a natural immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition, it is easily accessed, manipulated and imaged using optical and fluorescence stereomicroscopy. Histology further allows detailed analysis of heterotypic cellular interactions.
This protocol describes in detail the in ovo grafting of the chorioallantoic membrane with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines (Saos-2 and SW1353). The chick survival rates are up to 75%. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior. For localized grafting of single cell suspensions, ECM gel provides significant advantages over inert containment materials. The Ki67 proliferation index is related to the distance of the cells from the surface of the CAM and the duration of application on the CAM, the latter determining a time frame for the addition of therapeutic products.

The In ovo CAM-assay as a Xenograft Model for Sarcoma

The in ovo chorioallantoic membrane (CAM) is grafted with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior.

ザ<em&gt;卵で</em肉腫のためXenograftmodelとして&gt; CAMアッセイ

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

マウスにおけるタキサンの薬物動態学的および薬力学的分析のための頸動脈煎茶

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

A Simple Critical-sized Femoral Defect Model in Mice

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all limb bone fractures fail to heal completely due to the extent of the trauma, disease, or age of the patient. Our ability to improve bone regenerative strategies is critically dependent on the ability to mimic serious bone trauma in test animals, but the generation and stabilization of large bone lesions is technically challenging. In most cases, serious long bone trauma is mimicked experimentally by establishing a defect that will not naturally heal. This is achieved by complete removal of a bone segment that is larger than 1.5 times the diameter of the bone cross-section. The bone is then stabilized with a metal implant to maintain proper orientation of the fracture edges and allow for mobility. 
Due to their small size and the fragility of their long bones, establishment of such lesions in mice are beyond the capabilities of most research groups. As such, long bone defect models are confined to rats and larger animals. Nevertheless, mice afford significant research advantages in that they can be genetically modified and bred as immune-compromised strains that do not reject human cells and tissue.
Herein, we demonstrate a technique that facilitates the generation of a segmental defect in mouse femora using standard laboratory and veterinary equipment. With practice, fabrication of the fixation device and surgical implantation is feasible for the majority of trained veterinarians and animal research personnel. Using example data, we also provide methodologies for the quantitative analysis of bone healing for the model.

Animal models are frequently employed to mimic serious bone injury in biomedical research. Due to their small size, establishment of stabilized bone lesions in mice are beyond the capabilities of most research groups. Herein, we describe a simple method for establishing and analyzing experimental femoral defects in mice.

マウスの簡単な重要なサイズの大腿欠陥モデル

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all ...

Utilizing an Orally Dissolving Strip for Pharmacological and Toxicological Studies: A Simple and Humane Alternative to Oral Gavage for Animals

Prior to testing novel therapeutics in humans, short and long term preclinical (i.e., animal), repetitive pharmacological and toxicological testing is required. In most cases, the preferred route of administration is via oral delivery. At the present time, oral delivery is mostly accomplished using an oral gavage procedure, in part, because it can achieve consistent and precise dosing in the animal model. Although this method is well established it does have complications that can result in a high rate of animal attrition. To this end, the procedure introduced here describes an alternative to the oral gavage method in which the desired drug is incorporated into a tastant, orally dissolving strip (ODS) that can simply be presented to the test animal where it is then rapidly taken up with minimal manipulation of the test subject. Herein, we demonstrate that preclinical, oral drug delivery using the ODS method represents a safe, convenient, and humane alternative to oral gavage.

The following protocol describes a novel method for chronic oral drug administration using an orally dissolving strip (ODS) in lieu of the more commonly used oral gavage method. We demonstrate that preclinical, oral drug delivery using the ODS method represents a safe, convenient, and humane alternative to oral gavage.

薬理学的および毒性試験のための経口溶解ストリップを利用：動物のための強制経口投与にシンプルで動物愛護の代替を

Prior to testing novel therapeutics in humans, short and long term preclinical (i.e., animal), repetitive pharmacological and toxicological testing is ...

An Improved Method for Rapid Intubation of the Trachea in Mice

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity is a commonly used model to study both acute lung injury and fibrosis, and multiple methods have been developed for administering bleomycin (and other toxic agents) into the lungs. However, many of these approaches, such as transtracheal instillation, have inherent drawbacks, including the need for strong anesthetics and survival surgery. This paper reports a quick, reproducible method of intratracheal intubation that involves mild inhaled anesthesia, visualization of the trachea, and the use of a surrogate spirometer to confirm exposure. As a proof of concept, 8-12 week old C57BL/6 mice were administered either 2.0 U/kg of bleomycin or an equivalent volume of PBS, and both damage and fibrotic endpoints were measured post-exposure. This procedure allows researchers to treat a large cohort of mice in a relatively short period with little expense and minimal post-procedure care.

This article presents a rapid and simple method for administering bleomycin directly into the mouse trachea via intubation. Key advantages of this method are that it is highly reproducible, easy to master, and does not require specialized equipment or lengthy recovery times.

マウスの気管の迅速な挿管のための改善された方法

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity ...

A Method for Quantifying Upper Limb Performance in Daily Life Using Accelerometers

A key reason for referral to rehabilitation services after stroke and other neurological conditions is to improve one's ability to function in daily life. It has become important to measure a person's activities in daily life, and not just measure their capacity for activity in the structured environment of a clinic or laboratory. A wearable sensor that is now enabling measurement of daily movement is the accelerometer. Accelerometers are commercially-available devices resembling large wrist watches that can be worn throughout the day. Data from accelerometers can quantify how the limbs are engaged to perform activities in peoples' homes and communities. This report describes a methodology to collect accelerometry data and turn it into clinically-relevant information. First, data are collected by having the participant wear two accelerometers (one on each wrist) for 24 h or longer. The accelerometry data are then downloaded and processed to produce four different variables that describe key aspects of upper limb activity in daily life: hours of use, use ratio, magnitude ratio, and the bilateral magnitude. Density plots can be constructed that visually represent the data from the 24 h wearing period. The variables and their resultant density plots are highly consistent in neurologically-intact, community-dwelling adults. This striking consistency makes them a useful tool for determining if upper limb daily performance is different from normal. This methodology is appropriate for research studies investigating upper limb dysfunction and interventions designed to improve upper limb performance in daily life in people with stroke and other patient populations. Because of its relative simplicity, it may not be long before it is also incorporated in clinical neurorehabilitation practice.

This protocol describes a method to quantify upper limb performance in daily life using wrist-worn accelerometers.

加速度計を使用して日常生活に上肢のパフォーマンスを定量化する方法

A key reason for referral to rehabilitation services after stroke and other neurological conditions is to improve one's ability to function in daily life. ...

Observational Study Protocol for Repeated Clinical Examination and Critical Care Ultrasonography Within the Simple Intensive Care Studies

Longitudinal evaluations of critically ill patients by combinations of clinical examination, biochemical analysis and critical care ultrasonography (CCUS) may detect adverse events of interventions such as fluid overload at an early stage. The Simple Intensive Care Studies (SICS) is a research line that focuses on the prognostic and diagnostic value of combinations of clinical variables.
The SICS-I specifically focused on the use of clinical variables obtained within 24 h of acute admission for prediction of cardiac output (CO) and mortality. Its sequel, SICS-II, focuses on repeated evaluations during ICU admission. The first clinical examination by trained researchers is performed within 3 h after admission consisting of physical examination and educated guessing. The second clinical examination is performed within 24 h after admission and includes physical examination and educated guessing, biochemical analysis and CCUS assessments of heart, lungs, inferior vena cava (IVC) and kidney. This evaluation is repeated at days 3 and 5 after admission. CCUS images are validated by an independent expert, and all data is registered in an online secured database. Follow-up at 90 days includes registration of complications and survival status according to patient&#39;s medical charts and the municipal person registry. The primary focus of SICS-II is the association between venous congestion and organ dysfunction.
The purpose of publishing this protocol is to provide details on the structure and methods of this on-going prospective observational cohort study allowing answering multiple research questions. The design of the data collection of combined clinical examination and CCUS assessments in critically ill patients are explicated. The SICS-II is open for other centers to participate and is open for other research questions that can be answered with our data.

Structured protocols are necessary to provide answers on research questions in critically ill patients. The Simple Intensive Care Studies (SICS) provides an infrastructure for repeated measurements in critically ill patients including clinical examination, biochemical analysis and ultrasonography. SICS projects have specific focus but the structure is flexible to other investigations.

頻回の臨床検査と重要な観測的研究プロトコルは気に簡単な集中治療研究内超音波検査

Longitudinal evaluations of critically ill patients by combinations of clinical examination, biochemical analysis and critical care ultrasonography (CCUS) ...

A High-Throughput In Situ Method for Estimation of Hepatocyte Nuclear Ploidy in Mice

When the liver is injured, hepatocyte numbers decrease, while cell size, nuclear size and ploidy increase. The expansion of non-parenchymal cells such as cholangiocytes, myofibroblasts, progenitors and inflammatory cells also indicate chronic liver damage, tissue remodeling and disease progression. In this protocol, we describe a simple high-throughput approach for calculating changes in the cellular composition of the liver that are associated with injury, chronic disease and cancer. We show how information extracted from two-dimensional (2D) tissue sections can be used to quantify and calibrate hepatocyte nuclear ploidy within a sample and enable the user to locate specific ploidy subsets within the liver in situ. Our method requires access to fixed/frozen liver material, basic immunocytochemistry reagents and any standard high-content imaging platform. It serves as a powerful alternative to standard flow cytometry techniques, which require disruption of freshly collected tissue, loss of spatial information and potential disaggregation bias.

We present a robust, cost-effective, and flexible method for measuring changes in hepatocyte number and nuclear ploidy within fixed/cryopreserved tissue samples that does not require flow cytometry. Our approach provides a powerful sample-wide signature of liver cytology ideal for tracking the progression of liver injury and disease.

マウスにおける肝細胞核プロイドの推定に対するSitu法におけるハイスループット

When the liver is injured, hepatocyte numbers decrease, while cell size, nuclear size and ploidy increase. The expansion of non-parenchymal cells such as ...

マウスにおける頭蓋内異種移植研究のための簡単​​なガイドスクリュー方式

マウスにおける頭蓋内異種移植研究のための簡単なガイドスクリュー方式