We are supported by research grants from the National Natural Science Foundation of China (NO. 81400590), National Institutes of Health (U54 CA156735) and Takeda Pharmaceutical Company Ltd. (MA-NC-D-156).

全ての動物実験は、施設内動物管理使用委員会により承認されています。 1.マウスの準備<ol><li>体重や手術の時代に〜6週間以上〜20グラムの上にマウスを使用してください。 </li><li>手術前に、マウスを実験用固形飼料および水を自由に与え、12時12分時間の明暗サイクルで維持しています。 </li><li>バリカンで手術領域を剃ります。 80ミリグラム/ kgのケタミンおよび12 ​​mgの/ kgのキシラジンの腹腔内注射によりマウスを麻酔。この用量は、数分で眠りにマウスを置くと、次の外科的処置のための十分な麻酔を提供します。 </li><li>フットパッドが挟まれたときに角膜反射と四肢後退の欠如に基づいて、適切な麻酔を確認してください。乾燥を防ぐために、目に眼軟膏を潤滑に適用します。 </li><li>ベタジン溶液で皮膚を滅菌します。 </li></ol> 2.胃逆流モデル（図1B） <ol><li>アッパーを作ります肛門を指して剣状突起から始まる1〜2センチ正中切開。 </li><li>正中線を通って腹腔を開きます。ハサミで露出を高めるために、剣状突起を外します。 </li><li>セパレート、肝臓と胃の間の結合組織をカット。眼底まで完全に解放するために脾臓および眼底間の維管束を連結し、カット。 </li><li>少し左に眼底を回し、フィールドに胃食道接合部の左側を公開します。上皮を公開するために、鋭いハサミを使用して遠位食道に沿って筋肉の5ミリメートル縦切開を行います。 </li><li>同じ方向（切開1、 図1B）に沿って上皮を開いてカットします。シャープな操作ハサミで胃のほとんどを遮断するために胃（切開2、図3、図4、 図1B）の3つの切開を行います。胃が空でない場合は、慎重に胃内容物を除去します。 </li><li> 8-0プロレンスツ語を配置再正確な粘膜とポイントを介して（胃の）（食道上）と​​点A &#39;（テーパ点針で）反対粘膜します。同様に、それぞれ、点Bと点B &#39;を介して縫合糸を配置し、ポイントの他のペア。均等にスペースこれらの縫合糸互いに3〜4ミリメートル。 </li><li>血液や胃内容物をクリーンアップするために生理食塩水で腹腔を洗浄します。絹縫合糸で腹壁と金属製のクリップで皮膚を閉じます。 </li></ol> 3.混合還流モデル（図1C） <ol><li>フォローは胃食道接合部を露出させるために2.4から2.1を繰り返します。 </li><li>ゆっくり食道の後ろの血管から食道の背側を分離します。食道と血管との間の小さな綿棒を渡します。 注：コットンは、部分的にそのサイズを減少させるためにチップから除去することができます。これは綿の先端が食道を持ち上げ、血管を保護し、2つの目的があります。 </li><li> 2 5ミリメートル縦株式会社を作ります胃食道接合部とシャープな操作ハサミで幽門に隣接する十二指腸の近位端にisionsそれぞれ。十二指腸の切開は、血管を避け、抗腸間膜の国境に配置します。 </li><li>中断8-0プロレン縫合糸で反対粘膜ために正確な粘膜に切開を吻合。通常前面に背側の3-4縫合糸および2-3縫合糸を配置します。 </li><li>綿棒を削除します。通常の生理食塩水で腹腔を洗浄し、腹壁と皮膚を閉じます。 </li></ol> 4.十二指腸還流モデル（図1D） <ol><li>混合逆流を生成するために、3.4から3.1のステップに従ってください。 </li><li>慎重にその裏面を露出させるために胃を持ち上げます。肝臓の葉は、肝臓と胃の背面との間に結合組織によって捕捉することができます。慎重に結合組織をカットし、適切に肝臓を保護し、食道の背側の血管を露出させます。 </li><li>で連結され、血管を切りました。胃食道接合部で食道を連結し、カット。 </li><li>幽門で十二指腸を結紮。腸間膜を連結し、カット。胃全体を削除します。 </li><li>通常の生理食塩水で腹腔を洗浄します。腹壁および皮膚を閉じます。 </li></ol> 5.後の外科的治療<ol><li>手術後に加熱パッドで体温を維持。のみ胸骨横臥位を取り戻した後、他の動物とバックマウスを置きます。 </li><li>感染を予防し、痛みを緩和するための抗生物質や鎮痛剤を与えます。鎮痛剤としての感染を予防するバイトリル（3日間の10mg / kg、腹腔内、QD）、及びブプレノルフィン塩酸塩（0.05ミリグラム/ kg、皮下、入札 。2日間）を注入します。断食は、手術後の必要はありません。 液体またはソフト食事を与えられてもよいです。 </li><li>毎日basis.Ifの一般的な健康状態を評価するマウスは、以下の兆候（劇的な体重減少&gt; 15％、アプリの欠如を示しますetite、発声、口、鼻や目からの排出、猫背ジェスチャー、非アクティブ、異常なグルーミング活性）、マウスを安楽死させます。 </li></ol>

<ol>
	<li>Kandulski, A., Malfertheiner, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gastroesophageal+reflux+disease--from+reflux+episodes+to+mucosal+inflammation.">Gastroesophageal reflux disease--from reflux episodes to mucosal inflammation.</a> Nat Rev Gastroenterol Hepatol. 9 (1), 15-22 (2012).</li><li>Orlando, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+integrity+of+the+esophageal+mucosa.+Balance+between+offensive+and+defensive+mechanisms.">The integrity of the esophageal mucosa. Balance between offensive and defensive mechanisms.</a> Best Pract Res Clin Gastroenterol. 24 (6), 873-882 (2010).</li><li>Stairs, D. B., Kong, J., Lynch, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cdx+genes,+inflammation,+and+the+pathogenesis+of+intestinal+metaplasia.">Cdx genes, inflammation, and the pathogenesis of intestinal metaplasia.</a> Prog Mol Biol Transl Sci. 96, 231-270 (2010).</li><li>Spechler, S. J., Fitzgerald, R. C., Prasad, G. A., History Wang, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=molecular+mechanisms,+and+endoscopic+treatment+of+Barrett's+esophagus.">molecular mechanisms, and endoscopic treatment of Barrett's esophagus.</a> Gastroenterology. 138 (3), 854-869 (2010).</li><li>Fang, Y., 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=Cellular+origins+and+molecular+mechanisms+of+Barrett's+esophagus+and+esophageal+adenocarcinoma.">Cellular origins and molecular mechanisms of Barrett's esophagus and esophageal adenocarcinoma.</a> Ann N Y Acad Sci. 1300, 187-199 (2013).</li><li>Garman, K. S., Orlando, R. C., Chen, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review:+Experimental+models+for+Barrett's+esophagus+and+esophageal+adenocarcinoma.">Review: Experimental models for Barrett's esophagus and esophageal adenocarcinoma.</a> Am J Physiol Gastrointest Liver Physiol. 302 (11), G1231-G1243 (2012).</li><li>Baruah, A., 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=Translational+research+on+Barrett's+esophagus.">Translational research on Barrett's esophagus.</a> Ann N Y Acad Sci. 1325, 170-186 (2014).</li><li>Lechpammer, M., 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=Flavopiridol+reduces+malignant+transformation+of+the+esophageal+mucosa+in+p27+knockout+mice.">Flavopiridol reduces malignant transformation of the esophageal mucosa in p27 knockout mice.</a> Oncogene. 24 (10), 1683-1688 (2005).</li><li>Hao, J., Liu, B., Yang, C. S., Chen, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gastroesophageal+reflux+leads+to+esophageal+cancer+in+a+surgical+model+with+mice.">Gastroesophageal reflux leads to esophageal cancer in a surgical model with mice.</a> BMC Gastroenterol. 9, 59 (2009).</li><li>Mari, 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+pSMAD/CDX2+complex+is+essential+for+the+intestinalization+of+epithelial+metaplasia.">A pSMAD/CDX2 complex is essential for the intestinalization of epithelial metaplasia.</a> Cell Rep. 7 (4), 1197-1210 (2014).</li><li>Fang, Y., 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=Gastroesophageal+reflux+activates+the+NF-kappaB+pathway+and+impairs+esophageal+barrier+function+in+mice.">Gastroesophageal reflux activates the NF-kappaB pathway and impairs esophageal barrier function in mice.</a> Am J Physiol Gastrointest Liver Physiol. 305 (1), G58-G65 (2013).</li><li>Chen, H., 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=Nrf2+deficiency+impairs+the+barrier+function+of+mouse+oesophageal+epithelium.">Nrf2 deficiency impairs the barrier function of mouse oesophageal epithelium.</a> Gut. 63 (5), 711-719 (2014).</li></ol>

One of the authors (XC) received funding that was provided by Takeda Pharmaceutical Company Ltd which supports basic research associated with gastroesophageal reflux disease. None of the reagents or instruments used in this article is produced by this company.

様々な外科モデルは、げっ歯類で胃、十二指腸および混合逆流を模倣するために設立されました。ここに述べた3つの手順は、術後生存のリーズナブルな料金との長期的な実験に適しています。手術経験を持つ研究者は練習の短い期間の後に技術を把握することができるはずです。 出血は、手術前に麻酔薬の腹腔内注射から肝臓、胃、および血管の偶発的な損害との間に結合組織の分離の間、肝臓の裂傷を生じる可能性があります。これは可能な限り避けるべきです。バッテリ駆動のミニ焼灼は、適切な場合に出血を止めるために使用されてもよいです。器官および組織が操作されたときに過度の延伸は可能な限り避けるべきです。ピンセットの先端が綿で包まれてもよく、または綿のヒントは、ピンセットを交換するために箸のように使用することができます。マウスの食道は、2層または2＆＃を持っています8220;チューブ」、外側の赤みを帯びた「筋肉チューブ」と内側白っぽい「上皮チューブ」。切開が食道に行われると、両方の層を切開する必要があります。縫合糸は均等に反対粘膜ために正確な粘膜に間隔を置いて配置する必要があります。狭窄は、縫合糸の間の狭い距離に起因し開発することができるのに対し、漏れは、縫合糸の間に大きな距離から生じ得ます。しかし、狭窄は、食物の通過を阻止することができ、わずかな漏れが地元密着性および線維形成反応により固定することができるのに対し、術後死を引き起こし、したがって、通常は生命を脅かすではありません。 プロトコル内従うべきいくつかの批判一般的な原則があります。 （実験室で約20〜30分）、可能であれば手術の所要時間を短縮する必要があります。手術前と後の断食は不要です。実際には、長期の断食（&gt; 1日）は、有害である可能性があります。手術は最良の外科医によって行われています。アシスタントは、マウスを麻酔し、外科を供給助けることができますiCalの材料。外科医が適切にマイクロサージェリーに訓練されていない限り、顕微鏡は必要ありません。そうでなければ手術の持続時間が必要以上に長くなる傾向があり、従って、術後の生存を潜在的に悪化することがあります。 これらの手順は、以前の刊行物に使用されており、良好な結果を生成しました。野生型および遺伝子改変マウスを用いて、NFκBとのNrf2経路は胃食道逆流11,12の間に食道上皮のバリア機能を調節していることを実証しました。しかし、我々はその代わりに、散在粘液細胞と扁平上皮癌が9誘導された、これらのマウスにおける本格バレット食道または腺癌を生成することができませんでした。マウスの食道と人間の食道の間に組織学的な違いは、バレット食道と食道腺癌の研究で、これらの外科手術用のモデルの固有の制限であってもよいです。 我々は、これらのsuを信じてrgicalモデルは逆流性食道炎の研究に有用です。遺伝子および分子経路の機能は、GERDの分子機構を理解し、機構に基づく治療を開発するために明らかにすることができます。バレット食道および食道腺癌を製造するために使用される場合、遺伝子改変は、逆流手術と組み合わせて必要とされます。

Gastroesophageal reflux disease (GERD) is a chronic disorder caused by the prolonged exposure of distal esophagus to gastric or gastroduodenal contents1. Prolonged exposure to these noxious refluxates impairs the intrinsic defenses within the esophageal epithelium and thus results in esophagitis2. Barrett&#8217;s esophagus arises in the setting of chronic reflux, and is a premalignant lesion with increased risk of esophageal adenocarcinoma3,4. Despite the clinical importance, the mechanisms of GERD, Barrett&#8217;s esophagus and adenocarcinoma have not been well understood. 
Animal models are essential for research on etiology, pathology, molecular mechanisms, prevention and treatment of human diseases. Up to date, various animal models of GERD, Barrett&#8217;s esophagus and adenocarcinoma have been developed using model animals5,6. Mouse esophagus is lined with stratified squamous epithelium which is histologically similar to that in human esophagus. Although a mouse esophagus is different from human esophagus in terms of keratinization and the absence of submucosal glands, the mouse is still an appealing model animal because of its relatively low cost of maintenance and its potential of sophisticated genetic modifications. Two approaches are commonly used to model GERD, Barrett&#8217;s esophagus and adenocarcinoma in mice: reflux surgery and genetic modification. Reflux surgery is the best way to induce reflux and genetic modifications mimics molecular alterations5,7. Reflux surgery can be combined with genetic modifications to further understand disease mechanisms8. 
Many surgical procedures have been reported by us and others6,9: (1) gastric reflux: pyloric ligation, pyloric constriction with forestomach ligation, Wendel cardioplasty, and esophagogastric anastomosis; (2) mixed reflux: esophagogastroduodenal anastomosis, esophagoduodenostomy (or esophagojejunostomy); (3) duodenal reflux: esophagogastroduodenal anastomosis plus gastrectomy; (4) reflux of chemical components: bilious reflux, pancreatic reflux, esophageal perfusion; and (5) esophageal transplantation5. Recently a microsurgical mouse model was reported to produce jejunal reflux via an esophagojejunostomy with magnets10. These surgical models have advantages over in vitro cell culture or organotypic culture models. In vitro, esophageal cells cannot tolerate a medium with high acidity or high concentrations of bile acids. Unconjugated bile acids which are commonly used to produce changes in esophageal epithelial cells in vitro are usually not present in the duodenal refluxate in vivo. Thus conclusions drawn from such in vitro studies should be taken with caution.
Surgery on the mouse esophagus remains a technical challenge because of its small size. A low rate of postoperative survival does not allow experiments which require certain sample size to reach statistically sound conclusions. In the past we have successfully developed and characterized surgical models of gastric reflux, mixed reflux, duodenal reflux with mice in long-term experiments9,11,12. We have also provided consultation to several other groups in their mouse surgery. Herein, we describe three surgical procedures in mice in order to help the community to establish these models in their labs.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Instruments</td>
			<td>Source</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #1 Forceps Dumostar Tip&#160;</td>
			<td>Roboz Surgical Instrument Co. (Gaithersburg, MD)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Clip Applying Forceps 5.5&#34;</td>
			<td>Roboz Surgical Instrument Co. (Gaithersburg, MD)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bonn Scissors 3.5&#34; Straight 15mm Sharp/Sharp Tungsten Carbide Blades</td>
			<td>Roboz Surgical Instrument Co. (Gaithersburg, MD)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Operating Scissors 5.5&#34; Straight Sharp/Sharp SureCut</td>
			<td>Roboz Surgical Instrument Co. (Gaithersburg, MD)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4-0 Silk Black Braid 100 Yard Spool</td>
			<td>Roboz Surgical Instrument Co. (Gaithersburg, MD)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Surgeon&#39;s Needle 1/2 Circle Cutting Edge Size 12 (25 mm Chord Length) Pack 12</td>
			<td>Roboz Surgical Instrument Co. (Gaithersburg, MD)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Halsey Needle Holder 5&#34; Smooth</td>
			<td>Roboz Surgical Instrument Co. (Gaithersburg, MD)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Needle Holder 5.125&#34; Curved Lock .6mm</td>
			<td>Roboz Surgical Instrument Co. (Gaithersburg, MD)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reflex 9mm Wound Clip Applier</td>
			<td>Roboz Surgical Instrument Co. (Gaithersburg, MD)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reflex 9mm Wound Clips Box Of 100</td>
			<td>Roboz Surgical Instrument Co. (Gaithersburg, MD)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PRONOVA Poly (hexafluoropropylene-VDF) Suture 8-0</td>
			<td>Ethicon US, LLC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td>Source</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ringer&#39;s solution</td>
			<td>Henry Schein, Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ketamine</td>
			<td>Henry Schein, Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>xylazine</td>
			<td>Henry Schein, Inc.</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

surgical models of gastroesophageal reflux with mice

Multiple surgical procedures have been reported to induce gastroesophageal reflux in animals. Herein, we report three surgical models with mice aiming to induce reflux of gastric contents, duodenal contents or mixed contents. Surgical procedures and general principles have been described in detail. A researcher with surgical experience should be able to grasp the technique after a short period of practice. After surgery, most mice can survive and develop reflux esophagitis similar to that in humans. However, it should be noted that histological differences between mouse and human esophagus are the inherent limitations of these surgical models. If used for research on Barrett&#8217;s esophagus and adenocarcinoma, these procedures may need to be combined with genetic modifications.

ほとんどのマウス（&gt; 95％）が手術に耐えることができます。周術期の期間中、死亡の主要な原因は、麻酔薬の過剰摂取、出血および未知の理由があります。 手術後4週間は、&gt;十二指腸逆流と胃逆流または混合逆流および&gt; 80％のマウスを用いた90％のマウスが生存することができます。この期間中、マウスは主に食道狭窄と食べることができないことで死にます。これらのマウスは、重度のストレス（猫背の姿勢、非アクティブ、嘔吐、くぼんだ目、発声など ）の兆候を示し、安楽死させる必要があります。通常、抗生物質や点滴での医療処置が死んでから、これらのマウスを保存することはできません。マウスは通常、手術後グラムのカップルが十二指腸逆流と特にを自分の体重を失います。それにも関わらず体重が回復すると、後に増加します。 フォーティ週間手術後、十二指腸逆流と胃と混合逆流および&gt; 70％のマウスと&gt; 80％のマウスが生存することができます。長調この期間をる、いくつかのマウスは、栄養不良または未知の理由で死ぬことがあります。私たちは、手術後80週間のマウスを保持しています。一般的に、彼らはまだ非常にアクティブで、9健康です。 手術後4週目に、食道上皮のアーキテクチャを還流することにより、多かれ少なかれを損傷します。顕微鏡下で、上皮が厚くなり、乳頭が長くなります。上皮細胞および炎症性細胞（例えば、好中球、マスト細胞および好酸球）の浸潤の過剰増殖は、（ 図2）食道炎の明らかな兆候です。食道上皮（例えば、IL1β、IL6およびIL8）でのサイトカインは、特に十二指腸逆流11の存在下で上昇しています。透過型電子顕微鏡の下で、拡張間隙は、上皮バリア機能12の障害を反映しています。機能面では、経上皮電気抵抗は特にduodenの存在下で、劇的に減少しますアル逆流。 <img alt="図1" src="/files/ftp_upload/53012/53012fig1.jpg" /> 図1：（A）マウス（正面図と側面図）で、上部消化管の解剖学; （B）胃逆流（側面図）。 （C）混合逆流（正面図）。 （D）十二指腸逆流（正面図）。 <img alt="図2" src="/files/ftp_upload/53012/53012fig2.jpg" /> 図2：還流なしマウス食道上皮の組織学（A）、又は胃逆流（B）、混合還流（C）、十二指腸の逆流（D）を有します。 =50μmのスケールバー。

Watch this Scientific Journal Video about Surgical Models of Gastroesophageal Reflux with Mice at JoVE.com

Surgical Models of Gastroesophageal Reflux with Mice

This article demonstrates surgical procedures of gastroesophageal reflux with mice. These models are useful tools for research on mechanisms and treatment of gastroesophageal reflux disease and potentially Barrett&#8217;s esophagus and esophageal adenocarcinoma.

マウスを用いた胃食道逆流の外科モデル

Multiple surgical procedures have been reported to induce gastroesophageal reflux in animals. Herein, we report three surgical models with mice aiming to ...

surgical-models-of-gastroesophageal-reflux-with-mice

Nanyang Technological University

Research

JoVE Journal

Medicine

10.0K ビュー.  Ningxia Medical University General Hospital. This article demonstrates surgical procedures of gastroesophageal reflux with mice. These models are useful tools for research on mechanisms and treatment of gastroesophageal reflux disease and potentially Barrett’s esophagus and esophageal adenocarcinoma.

ビデオ: Surgical Models of Gastroesophageal Reflux with Mice

Surgical Management of Meatal Stenosis with Meatoplasty

Meatal stenosis is a common urologic complication after circumcision. Children present to their primary care physicians with complaints of deviated urinary stream, difficult-to-aim, painful urination, and urinary frequency. Clinical exam reveals a pinpoint meatus and if the child is asked to urinate, he will usually have an upward, thin, occasionally forceful urinary stream with incomplete bladder emptying. The mainstay of management is meatoplasty (reconstruction of the distal urethra /meatus). This educational video will demonstrate how this is performed.

Meatoplasty, surgical management of meatal stenosis.

管形成術と外尿道口狭窄の外科的管理

Meatal stenosis is a common urologic complication after circumcision.  Children present to their primary care physicians with complaints of deviated ...

医学

Implantation of Radiotelemetry Transmitters Yielding Data on ECG, Heart Rate, Core Body Temperature and Activity in Free-moving Laboratory Mice

The laboratory mouse is the animal species of choice for most biomedical research, in both the academic sphere and the pharmaceutical industry. Mice are a manageable size and relatively easy to house. These factors, together with the availability of a wealth of spontaneous and experimentally induced mutants, make laboratory mice ideally suited to a wide variety of research areas.
In cardiovascular, pharmacological and toxicological research, accurate measurement of parameters relating to the circulatory system of laboratory animals is often required. Determination of heart rate, heart rate variability, and duration of PQ and QT intervals are based on electrocardiogram (ECG) recordings. However, obtaining reliable ECG curves as well as physiological data such as core body temperature in mice can be difficult using conventional measurement techniques, which require connecting sensors and lead wires to a restrained, tethered, or even anaesthetized animal. Data obtained in this fashion must be interpreted with caution, as it is well known that restraining and anesthesia can have a major artifactual influence on physiological parameters1, 2.
Radiotelemetry enables data to be collected from conscious and untethered animals. Measurements can be conducted even in freely moving animals, and without requiring the investigator to be in the proximity of the animal. Thus, known sources of artifacts are avoided, and accurate and reliable measurements are assured. This methodology also reduces interanimal variability, thus reducing the number of animals used, rendering this technology the most humane method of monitoring physiological parameters in laboratory animals3, 4. Constant advancements in data acquisition technology and implant miniaturization mean that it is now possible to record physiological parameters and locomotor activity continuously and in realtime over longer periods such as hours, days or even weeks3, 5.
Here, we describe a surgical technique for implantation of a commercially available telemetry transmitter used for continuous measurements of core body temperature, locomotor activity and biopotential (i.e. onelead ECG), from which heart rate, heart rate variability, and PQ and QT intervals can be established in freeroaming, untethered mice. We also present pre-operative procedures and protocols for post-operative intensive care and pain treatment that improve recovery, well-being and survival rates in implanted mice5, 6.

A surgical technique for implantation of commercially available telemetry transmitters used for continuous measurement of biopotential (one-lead ECG), heart rate, core body temperature and locomotor activity in freely moving mice is shown. Recommendations and protocols for post-operative care and pain relief, improving recovery, well being and survival rate are also presented.

自由に動く実験用マウスの心電図上のデータ、心拍数、体温および活動の要因となるRadiotelemetryトランスミッターの注入

The laboratory mouse is the animal species of choice for most biomedical research, in both the academic sphere and the pharmaceutical industry. Mice are a ...

Protocols for Assessing Radiofrequency Interactions with Gold Nanoparticles and Biological Systems for Non-invasive Hyperthermia Cancer Therapy

Cancer therapies which are less toxic and invasive than their existing counterparts are highly desirable. The use of RF electric-fields that penetrate deep into the body, causing minimal toxicity, are currently being studied as a viable means of non-invasive cancer therapy. It is envisioned that the interactions of RF energy with internalized nanoparticles (NPs) can liberate heat which can then cause overheating (hyperthermia) of the cell, ultimately ending in cell necrosis.
In the case of non-biological systems, we present detailed protocols relating to quantifying the heat liberated by highly-concentrated NP colloids. For biological systems, in the case of in vitro experiments, we describe the techniques and conditions which must be adhered to in order to effectively expose cancer cells to RF energy without bulk media heating artifacts significantly obscuring the data. Finally, we give a detailed methodology for in vivo mouse models with ectopic hepatic cancer tumors.

We describe the protocols used to investigate the interactions of 13.56 MHz radiofrequency (RF) electric-fields with gold nanoparticle colloids in both non-biological and biological systems (in vitro/vivo). These interactions are being investigated for applications in cancer therapy.

非侵襲ハイパーサーミアがん治療のための金ナノ粒子と生体系とラジオ波の相互作用を評価するためのプロトコル

Cancer therapies  which are less toxic and invasive than their existing counterparts are highly  desirable. The use of RF electric-fields that penetrate ...

Inducing Myointimal Hyperplasia Versus Atherosclerosis in Mice: An Introduction of Two Valid Models

Various in vivo laboratory rodent models for the induction of artery stenosis have been established to mimic diseases that include arterial plaque formation and stenosis, as observed for example in ischemic heart disease. Two highly reproducible mouse models&#160;&#8211;&#160;both resulting in artery stenosis but each underlying a different pathway of development&#160;&#8211; are introduced here. The models represent the two most common causes of artery stenosis; namely one mouse model for each myointimal hyperplasia, and atherosclerosis are shown. To induce myointimal hyperplasia, a balloon catheter injury of the abdominal aorta is performed. For the development of atherosclerotic plaque, the ApoE -/- mouse model in combination with western fatty diet is used. Different model-adapted options for the measurement and evaluation of the results are named and described in this manuscript. The introduction and comparison of these two models provides information for scientists to choose the appropriate artery stenosis model in accordance to the scientific question asked.

This video shows two models of intimal plaque development in murine arteries and emphasizes the differences in myointimal hyperplasia and atherosclerosis.

マウスにおけるアテローム性動脈硬化症に対する筋内膜過形成を誘発する。つの有効なモデルの導入

Various in vivo laboratory rodent models for the induction of artery stenosis have been established to mimic diseases that include arterial plaque ...

Minimal Invasive Surgical Procedure of Inducing Myocardial Infarction in Mice

Myocardial infarction still remains the main cause of death in western countries, despite considerable progress in the stent development area in the last decades. For clarification of the underlying mechanisms and the development of new therapeutic strategies, the availability of valid animal models are mandatory. Since we need new insights into pathomechanisms of cardiovascular diseases under in vivo conditions to combat myocardial infarction, the validity of the animal model is a crucial aspect. However, protection of animals are highly relevant in this context. Therefore, we establish a minimally invasive and simple model of myocardial infarction in mice, which assures a high reproducibility and survival rate of animals. Thus, this models fulfils the requirements of the 3R principle (Replacement, Refinement and Reduction) for animal experiments and assure the scientific information needed for further developing of therapeutical strategies for cardiovascular diseases.

A highly reproducible model for myocardial infarction in mice with minimal invasive manipulations is described. The model can be easily performed, resulting in a high reproducibility and survival rate. Thus, the described model will reduce the number of required animals as requested by the 3R principle (Replacement, Refinement and Reduction).

マウスにおける心筋梗塞を誘発する低侵襲手術手順

Myocardial infarction still remains the main cause of death in western countries, despite considerable progress in the stent development area in the last ...

Using Saccadometry with Deep Brain Stimulation to Study Normal and Pathological Brain Function

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including Parkinson's and Huntington's can disrupt it. People with Parkinson's disease, for example, tend to have increased saccadic latencies. Consequently, the quantitative measurement of saccadic eye movements has received considerable attention as a potential biomarker for neurodegenerative conditions. A lot more can be learned about the brain in both health and disease by observing what happens to eye movements when the function of specific brain areas is perturbed. Deep brain stimulation is a surgical intervention used for the management of a range of neurological conditions including Parkinson's disease, in which stimulating electrodes are placed in specific brain areas including several sites in the basal ganglia. Eye movement measurements can then be made with the stimulator systems both off and on and the results compared. With suitable experimental design, this approach can be used to study the pathophysiology of the disease being treated, the mechanism by which DBS exerts it beneficial effects, and even aspects of normal neurophysiology.

This paper describes the use of quantitative measurement of eye movements in conjunction with stimulation of focal areas of the deep brain in order to study physiology, pathophysiology, and the mechanisms of deep brain stimulation.

正常および病的脳機能を研究するために脳深部刺激でSaccadometryを使用します

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including ...

Cutaneous Surgical Denervation:  A Method for Testing the Requirement for Nerves in Mouse Models of Skin Disease

Cutaneous somatosensory nerves function to detect diverse stimuli that act upon the skin. In addition to their established sensory roles, recent studies have suggested that nerves may also modulate skin disorders including atopic dermatitis, psoriasis and cancer. Here, we describe protocols for testing the requirement for nerves in maintaining a cutaneous mechanosensory organ, the touch dome (TD). Specifically, we discuss methods for genetically labeling, harvesting and visualizing TDs by whole-mount staining, and for performing unilateral surgical denervation on mouse dorsal back skin. Together, these approaches can be used to directly compare TD morphology and gene expression in denervated as well as sham-operated skin from the same animal. These methods can also be readily adapted to examine the requirement for nerves in mouse models of skin pathology. Finally, the ability to repeatedly sample the skin provides an opportunity to monitor disease progression at different stages and times after initiation.

This article includes detailed protocols for genetic labeling of mouse skin, surgical denervation, skin biopsy and visualizing labeled epithelia by whole-mount &#946;-galactosidase staining. These methods can be used to test the requirement for nerves in mouse models of normal and pathological skin.

皮膚外科神経切除：皮膚病のマウスモデルにおける神経のための要件をテストする方法

Cutaneous somatosensory nerves function to detect diverse stimuli that act upon the skin. In addition to their established sensory roles, recent studies ...

Surgical Approach for Middle Cerebral Artery Occlusion and Reperfusion Induced Stroke in Mice

Stroke is a leading cause of death worldwide and continues to be one of the major causes of long-term adult disabilities. About 87% of strokes are ischemic in origin and occur in the territory of the middle cerebral artery (MCA). Currently the only Food and Drug Administration (FDA) approved drug for the treatment of this devastating disease is tissue plasminogen activator (tPA). However, tPA has a small therapeutic window for administration (3 - 6 hr), and is only effective in 4% of the patients who actually receive it. Current research focuses on understanding the pathophysiology of stroke in order to find potential therapeutic targets. Thus, reliable models are crucial, and the MCA occlusion (MCAo) model (also termed the intraluminal filament or suture model) is deemed to be the most clinically relevant surgical model of ischemic stroke, and is fairly non-invasive and easily reproducible. Typically the MCAo model is used with rodents, especially with mice due to all the genetic variations available for this species. Here we describe (and present in the video) how to successfully perform the MCAo model (with reperfusion) in mice to generate reliable and reproducible data.

In order to understand the pathophysiology of stroke, it is important to use reliable models. This paper will describe one of the most frequently used stroke models in mice, termed the middle cerebral artery occlusion (MCAo) model (also termed the intraluminal filament or suture model) with reperfusion.

マウスの中大脳動脈閉塞再灌流誘発性の脳卒中のための外科的アプローチ

Stroke is a leading cause of death worldwide and continues to be one of the major causes of long-term adult disabilities. About 87% of strokes are ...

Intravital Microscopy of Tumor-associated Vasculature Using Advanced Dorsal Skinfold Window Chambers on Transgenic Fluorescent Mice

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static information and is often not sufficient for a correct interpretation. Intravital evaluation, in which a process is followed in time, provides extra and often unexpected information. With the creation of transgenic animals expressing cell-specific markers and live cell tracers, improvements to imaging equipment, and the development of several imaging chambers, intravital microscopy has become an important tool to better understand biological processes. This paper describes an experimental design for the investigation of tumor vessel development and of therapeutic effects in a spatial and temporal manner. Using this setup, the stage of vessel development, tip cell and lumen formation, blood flow, extravasation, an established vascular bed, and vascular destruction can be visualized and followed. Furthermore, therapeutic effects, intratumoral fate, and the localization of chemotherapeutic compounds can also be followed.

This protocol describes the intravital imaging of transgenic mice expressing cell-specific fluorescent markers. Intravital imaging provides a non-invasive method for high-resolution observations in living animals using implantable windows through which the microscopic visualization of processes is possible. This method is especially useful to study longitudinal processes.

腫瘍関連血管系を使用しての生体顕微鏡が背高度な蛍光トランスジェニック マウスの皮下脂肪のウィンドウの部屋

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static ...

Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles

Conventional imaging techniques can provide detailed information about cellular processes. However, this information is based on static images in an otherwise dynamic system, and successive phases are easily overlooked or misinterpreted. Live-cell imaging and time-lapse microscopy, in which living cells can be followed for hours or even days in a more or less continuous fashion, are therefore very informative. The protocol described here allows for the investigation of the fate of chemotherapeutic nanoparticles after the delivery of doxorubicin (dox) in living cells. Dox is an intercalating agent that must be released from its nanocarrier to become biologically active. In spite of its clinical registration for more than two decades, its uptake, breakdown, and drug release are still not fully understood. This article explores the hypothesis that lipid-based nanoparticles are taken up by the tumor cells and are slowly degraded. Released dox is then translocated to the nucleus. To prevent fixation artifacts, live-cell imaging and time-lapse microscopy, described in this experimental procedure, can be applied.

Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles

Live-cell imaging is a powerful tool to visualize dynamic processes. The examination of fixed cells provides only static pictures, which can lead to misinterpretation and confusion about the process. This work presents a method to study uptake, drug release, and intracellular localization of liposomal nanoparticles in living cells.

体外配信生細胞イメージング化学療法を探求する脂質ナノ粒子によってを使用してください。

Conventional imaging techniques can provide detailed information about cellular processes. However, this information is based on static images in an ...