Name: a murine model of vertical sleeve gastrectomy
Uploaded: 2017-12-18T13:00:00.000+00:00
Duration: 6 min 47 s
Description: Watch this Scientific Journal Video about A Murine Model of Vertical Sleeve Gastrectomy at JoVE.com

この研究は、NIH/NCI R21CA195002 01A1、コーネル大学女性とニューヨーク州立大学大学院多様性フェローシップの大統領の議会によって支えられました。カミングス博士の研究所も受けたコーネル大学比較がん生物学のトレーニング プログラムとイーライリリー社からプロジェクト期間中に資金調達。

すべての実験のプロトコルは、コーネル大学機関動物ケアおよび使用委員会によって承認されています。
1. 術前準備
注: 研究マウス、通常並進人間の肥満とインスリン抵抗性に関連する研究をさせるため C57BL 食餌誘導性肥満背景です。オスとメスのマウスは、以降の手順で説明するように学ぶことができます。
<ol>
	<li>2 ヶ月の頃に 2 ヶ月間 (材料の表を参照してください) 45% または 60% の高脂肪食のマウスを配置します。 注: マウスは肥満とインスリン抵抗性の表現型を作成する高脂肪食を供給されています。高脂肪食給餌の短いまたは長い期間は、研究の目的によって使用されるかもしれません。</li>
	<li>マウスを供給し続ける手術まで自由。ベースライン測定が必要な 7 週間高脂肪飼料飼育後場合、6 時間マウスを高速または一晩し、関心 (例えばブドウ糖) の代謝物の測定のベースラインの血液サンプルを取る。</li>
	<li>マウス高脂肪の食事し、偽手術または VSG 前に採血後回復することマウスは一週間続けます。液体にマウスを置く (材料の表を参照) を手術前に 4 日間のダイエットです。 注: マウスは流動食に維持した治癒反応を損なう可能性のある粒子状物質の消化管を取り除く手術の前後に。</li>
	<li>手術前に次の項目を滅菌: 箔、ガーゼ、綿の先端アプリケータ (CTAs) および手術器具 (シザー、アイリス シザー、デュモン鉗子、止血鋸歯状ブラウン アドソン鉗子、強制経口投与針、マイクロニードル ドライバー、春シザー).</li>
</ol>
2. 垂直スリーブ胃切除術と偽の手順
<ol>
	<li>手術の日に基準体重の測定値を取得するためにマウスの重量を量る。</li>
	<li>麻酔システム、温水浴加熱パッドと手術室を装備します。37 ° C に風呂の水を設定し、0.9% 食塩水灌漑を暖めるために使用します。70% エタノール手術野をきれいにし無菌のオートクレーブ手術パックを開き、ツールを使ってください。 注: 加熱パッドは、手術と麻酔からの回復の間に動物を暖かく保つために使用されます。</li>
	<li>5% イソフルランと O2流量 1 L/min マウスはつま先のピンチを実行することによって麻酔の適切な平面で確認のマウスを麻酔誘導室を用いたします。麻酔の一貫性のある平面が達成されたら、1-3% イソフルランと、O2 0.6 L/分の流量でマウスを維持します。</li>
	<li>マウスを麻酔すると、一度配置眼軟膏 (材料の表を参照してください) その目は、手術中に湿潤を保つために。この時点でまた、メロキシカムなど、鎮痛剤を管理 (2 mg/kg 皮下)。臍から張力を提供するために、優しく皮膚を押し腋窩に髪をクリップします。
	<ol>
		<li>マウスの皮膚は薄くデリケートな部分、髪の毛をクリッピング皮膚病変が発生しないように世話をします。ポビドン ヨードとアルコールで皮膚をきれい。</li>
		<li>滅菌手袋と滅菌の手術野や楽器を準備します。生殖不能の技術を使用してプロシージャの残りの部分を実行します。滅菌の手術野を準備するには、マウスの両側に滅菌外科用ドレープを配置します。オートクレーブのスズ箔を使用すると、マウスの手術用覆布を作成します。</li>
		<li>マウスの腹部へのアクセスを許可するようにスズ箔に小さな穴をカットします。</li>
	</ol>
	</li>
	<li>アイリス シザーを使用して剣軟骨のレベルに中間腹部 (臍) から皮膚の切開を行います。アルバと使用のアイリスが白線に沿って体壁をカットするはさみリネアを識別するアルバ。腹部から胃を優しく昇格し、胃の大弯から大網をにべもなく解剖 CTAs を使用します。</li>
	<li>7-0 モノフィラメント縫合糸を使用して 2 つの合字を配置することによって胃の底と脾臓を結んで短胃動脈を結紮します。2 つの合字の間をカットする春はさみを使用します。動脈を切断すると、一度完全に腹腔内から胃を外在します。[胃と組織の潤いを保つために生理食塩水で湿ガーゼを配置します。</li>
	<li>偽の手順
	<ol>
		<li>偽の手順 6-0 モノフィラメント縫合糸を用いたテーパ針縫合の緩やかな単純な連続パターンを配置します。食道の右側 (外科医の右) に 2 mm を開始します。胃の腹側の壁に沿って縫合線を配置し、背側の胃の壁に沿って進みます。 注: すべての胃の操作、感染のリスクを減らすためだけモノフィラメント縫合糸を使用します。さらに、切削針の縫合"プルから「胃組織損傷つながると創傷裂開リスクを増大するとすべて胃操作用テーパー針付き縫合のみを使用します。</li>
		<li>縫合線を配置しながら 1 つの胃の壁を完全に針を渡します。縫合はフラット、横になっているが、胃を制限しないことを確認します。軽く 3-4 スローを使用して縫合糸を結ぶ、2.9 の手順に進みます。</li>
		<li>VSG プロシージャの手順 2.7 および 2.8 の手順に進みます。</li>
	</ol>
	</li>
	<li>VSG の手順
	<ol>
		<li>VSG プロシージャの胃動脈、7-0 のモノフィラメント縫合糸テーパー針 3 を使用して各ノットのスローと静脈の顕著な枝を縛る。断裂の意図している行 (すなわち胃の小彎側の方) のすぐ下の合字を配置します。 注: 断裂の意図している行開始 〜 2 mm 上 (胃の大弯に向かって意味) で胃とマーゴ pilcatus 以下、少なくとも 2 ミリメートルの心臓ノッチと膵右葉の近位端に伸びる。通常、結紮する; する必要があります。 胃の壁あたり 4 隻があります。ただし、これはマウスによって異なります。</li>
		<li>胃の中に胃内容物の流出を防ぐため、テーパー針と 6-0 モノフィラメント縫合糸を使用して断裂の意図している行のすぐ下の両方の胃の壁を通過縫合の単純な連続ラインを配置します。外科医の右食道とマーゴ pilcatus と膵臓のすぐ上の端の下に縫合線を開始します。</li>
		<li>薄い先端止血縫合線の上を置き、縫合線と止血剤をカットする春はさみを使用します。滅菌の手術野から切断の胃の組織を削除します。</li></ol></li></ol>血や胃のめん羊をきれいに CTAs を使用します。
		<li>簡単な不連続パターンを使用してテーパー針と 6-0 モノフィラメント縫合糸を使用して胃の壁の同格を強化します。 注: これは簡単な連続的な縫合パターンよりもより安全な閉鎖を提供しますので、簡単な不連続な縫合パターンは推奨します。</li>
		<li>しっとりときれいな組織を保つためにプロシージャ全体で生理食塩水と残胃をフラッシュします。強制経口投与の 20 G 針 20 mL の注射器に接続されているを使用すると、生理食塩水で胃洗浄を実行できます。過度の圧力から胃の組織の損傷や正規の先の尖った針の使用からの偶発的な胃組織の損傷を危険にさらすことがなく十分な圧力を提供するのにには、このサイズを使用します。</li>
		<li>20 ノットの最小値を使用して安全に胃を閉じるこちら側はアクセスが難しく、したがって、しばしば裂のサイトとしては、食道側に沿って閉鎖に注意を払います。</li>
		<li>CTAs と胃に優しく押すことによってリークがないことを確認します。リークが特定される場合は、漏れの分野でさらに簡単な不連続ノットを置くし、テストを再び漏れます。リークテストまで漏れが検出されません。</li>
		<li>伝染性の粒子状物質を残されているはないように、少なくとも 60 mL の生理食塩水を用いた胃の最終的な徹底的な洗浄を行います。</li>
	
	
	<li>アテは CTA を使用して肝臓の下で腹腔内に胃を配置します。すべての余分な水分を吸収する腹腔の背側面に沿って CTAs を配置します。閉鎖直前に、腹腔内に直接抗生物質 (20 mg/kg エンロフロキサシン ± 0.5 mL LRS) の有無 (LRS) リンゲル液を注入鈍 18 G の針を使用して。 注: これは手術中に経験した流体損失を置き換え、回復を支援する手術部位に抗生物質の直接アプリケーションのためのメソッドを提供します。</li>
	<li>簡単な不連続パターンでテーパー針と 6-0 モノフィラメント縫合糸を使用して腹部の筋肉層を閉じます。単純な連続的なパターンで 6-0 モノフィラメント吸収性縫合糸で皮膚の層を閉じます。</li>
	<li>皮膚の組織接着剤を置き、皮膚を折り重ね、マウスは術後創傷閉鎖を中断できないように縫合を埋めるため縫合線。</li>
	<li>イソフルランを切り、マウスをその家のケージに戻る 10-15 minbefore の加熱パッドで回復をしましょう。意識を取り戻したが、移動することができるまで、無人すぎる動物は放置しないでください。</li>
	<li>手術から完全に回復するまで単独で収容動物を飼います。 注: 正確な食品摂取量測定を研究を通して取得する場合は、マウス続けなければならない研究を通して単独で収容されます。ホーム床敷を食べてから異物の閉塞の危険性があります。動物がケージの濃縮を提供する材料をネスト以外の寝具の無料に収納する必要があります (表の資料を参照してください)。</li>

3. 術後マウス ケアと測定
<ol>
	<li>その家のケージに戻すには、加熱パッドのケージを配置します。手術後少なくとも 7 日間の流動食にマウスを維持します。鎮痛剤、メロキシカムなどを管理 (2 mg/kg 皮下)、手術後 2 日間。 注: 手術後 7 日間、エンロフロキサシン (20 mg/kg) などの抗生物質が与えられます。抗生物質は手術からの感染のリスクを最小限に抑えるために与えられていると、プロシージャ全体で維持される不妊のレベルに応じて必要とされない可能性があります。ただし、抗生物質を使用するが一貫性を維持するために同じ調査ですべてのマウスに投与する必要があります。</li>
	<li>術後の期間中に、体重、食事摂取、排便、活動レベル、および適切な治癒と回復を確保するため、少なくとも毎日処分を評価します。術後後 14 日目の術後のモニタリングを実施します。 注: 外科的障害は通常見られる手術直後後や 3 日後以内作動。食べることや手術後 2 日目に飲んでいない動物は、手術の合併症のため密接に監視する必要があります。</li>
	<li>術後の期間が正常に完了後、体の重量と食品の摂取量を適切な表現型の維持するため研究を通じて定期的に測定します。</li>
	<li>グルコース刺激によるインスリン分泌、耐糖能を評価するために経口ブドウ糖負荷試験 (OGTT) を行い、消化管ホルモンの分泌。
	<ol>
		<li>断食後のマウスで、OGTT を実行 6 h とし、50% ブドウ糖液 1 g/kg の用量で経口マウスのそれら。</li>
		<li>強制経口投与後 120 分で 0、2.5、5、15、30、60、尾血液サンプルを収集します。これらの時点で glucometer して血糖値を測定します。インスリンと GLP-1 などの様々 なホルモンの測定のための各時点での血清サンプルを収集します。</li>
		<li>安楽死の前に 6 時間高速マウス。最終的な血液サンプルおよびグルコース測定を取る。ベースライン測定値を最終的なグルコース測定を比較します。200 mg/kg のペントバルビ タールの腹腔内注入マウスを安楽死させます。</li>
		<li>追加検体を測定し、手術前および手術後の値を比較する基準と最後の血清を使用します。</li>
	</ol>
	</li>
</ol>

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D., 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=Health+benefits+of+gastric+bypass+surgery+after+6+years.">Health benefits of gastric bypass surgery after 6 years.</a> Jama. 308 (11), 1122-1131 (2012).</li><li>McGavigan, A. 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K., 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=FXR+is+a+molecular+target+for+the+effects+of+vertical+sleeve+gastrectomy.">FXR is a molecular target for the effects of vertical sleeve gastrectomy.</a> Nature. 509 (7499), 183-188 (2014).</li><li>Wilson-Perez, H. E., 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=Vertical+sleeve+gastrectomy+is+effective+in+two+genetic+mouse+models+of+glucagon-like+Peptide+1+receptor+deficiency.">Vertical sleeve gastrectomy is effective in two genetic mouse models of glucagon-like Peptide 1 receptor deficiency.</a> Diabetes. 62 (7), 2380-2385 (2013).</li><li>Pressler, J. W., 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=Vertical+sleeve+gastrectomy+restores+glucose+homeostasis+in+apolipoprotein+A-IV+KO+mice.">Vertical sleeve gastrectomy restores glucose homeostasis in apolipoprotein A-IV KO mice.</a> Diabetes. 64 (2), 498-507 (2015).</li></ol>

著者は博士カミングス イーライリリー社から資金を受けている場合を除き、利害の対立を宣言しません。

肥満手術は、肥満症の最も効果的な長期的な治療で 2 型糖尿病および高血圧症寛解1,9,15の高レートなどその他健康上の利点の結果します。モデルマウスの肥満手術は、肥満症治療手術が肥満合併症の迅速かつ顕著な改善を引き起こすメカニズムを識別するための強力なツールを提供します。さらに、肥満症治療手術のマウ...

肥満の伝染病が世界的な成長を続けている肥満手術は、肥満1の最も効果的な長期的な治療として人気を集めています。残念ながら、食事と運動による減量は長期2,3以上に達成するために困難で、比較的効果が。垂直方向のスリーブ胃切除術 (VSG) などの肥満症治療手術は、重量損失1,4の目的のため消化管の操作として定義されます。減量は肥満手術の顕著な結果が、肥満手術は、肥満の合併症を改善し、寿命5の拡張などその他健康上の利点を提供します。たとえば、肥満症治療手術結果癌1,6、7の特定の種類の開発の寿命の危険の減少と高血圧、2 型糖尿病の寛解率が高い。注記のうち、手術後すぐにおよび重量損失8,9の前に、2 型糖尿病と高血圧の赦しを引き起こしている肥満手術の効果がよく見られます。これは手術後に観察された健康上の利点に貢献する体重の独立したメカニズムがあること概念を強調表示します。肥満症治療手術の動物モデルが開発されており、これらの健康上の利点は、10、11,12を発生するメカニズムを研究に役立てられています。
我々 は、肥満手術は肥満合併 2 型糖尿病、高血圧、大腸がん10などを向上させる機構を研究する様々 な遺伝子組み換えマウス モデルに適用されている VSG のマウスモデルを検証しました,11,12. 齧歯動物モデルはより実験的な制御と特定の遺伝子の役割や関心のシグナル伝達経路を定義する遺伝的または医薬品の操作を実行する能力を許可します。VSG はアメリカ合衆国13でクリニックで最も一般的に実行の肥満の手順、VSG で主に注力しています。また、VSG は Roux en Y 胃バイパスまたは胆道膵転用など他のプロシージャと比較して少ない解剖学的修正と簡単な手術モデルです。
VSG のマウス モデルを繰り返す人間にみられる肥満手術の次の効果: 減量、減らされた滋養、改良された血糖調節、膵島機能を改善、食後グルカゴン様ペプチド 1 (GLP-1) 分泌を増加動脈血圧、および高められた循環胆汁酸濃度10,11,12,13,14,15を削減しました。したがって、本体重量従属と独立機構または VSG が改善、肥満合併症を解決を研究する理想的なモデルです。また、さまざまな疾患をより追究12下 VSG の影響の調査を可能にする、他の手術と組み合わせることができます信頼性の高いモデルです。

<table><tbody><tr><td>45% high fat diet</td><td>Research Diets</td><td>D12451</td><td></td></tr><tr><td>60% high fat diet</td><td>Research Diets</td><td>D12492</td><td></td></tr><tr><td>Boost</td><td>Nestle</td><td>160-67538</td><td>rich chocolate flavor</td></tr><tr><td>6-0 Suture</td><td>Ethicon</td><td>Z432</td><td>monofilament absorbable/taper</td></tr><tr><td>7-0 Suture</td><td>Covidien</td><td>8866127-01</td><td>monofilament absorbable/taper</td></tr><tr><td>Cotton Swabs</td><td>Fisherbrand</td><td>23-400-118</td><td>small</td></tr><tr><td>Cotton Swabs</td><td>Fisherbrand</td><td>233-400-101</td><td>large</td></tr><tr><td>Gauze</td><td>Various</td><td></td><td>4x4 4 ply and 2x2 4 ply</td></tr><tr><td>Foil</td><td>Various</td><td></td><td></td></tr><tr><td>Surgery drape</td><td>Various</td><td></td><td></td></tr><tr><td>0.9% saline solution</td><td>Various</td><td></td><td></td></tr><tr><td>LRS</td><td>Hospira</td><td>170RX</td><td></td></tr><tr><td>Betadine</td><td>Various</td><td></td><td></td></tr><tr><td>Alcohol</td><td>Various</td><td></td><td></td></tr><tr><td>Eye Ointment</td><td>Paralube&amp;reg; Vet Ointment</td><td>17033-211-38</td><td></td></tr><tr><td>Tissue Adhesive</td><td>Vetbond</td><td>1469SB</td><td></td></tr><tr><td>Meloxicam (Metacam)</td><td>Boehringer Ingelheim</td><td>141-213</td><td>5 mg/ml</td></tr><tr><td>Enrofloxacin</td><td>Baytril</td><td>08713254-186599</td><td>22.7 mg/ml</td></tr><tr><td>Thin tipped hemostats</td><td>Fine Science Tools</td><td>13021-12</td><td></td></tr><tr><td>Metzenbaum Scissor</td><td>Fine Science Tools</td><td>14018-18</td><td></td></tr><tr><td>Iris Scissors</td><td>Fine Science Tools</td><td>14058-09</td><td></td></tr><tr><td>Dumont Forcep</td><td>Fine Science Tools</td><td>11251-20</td><td></td></tr><tr><td>Serrated Forcep</td><td>Fine Science Tools</td><td>11020-12</td><td></td></tr><tr><td>Gavage needle</td><td>Fine Science Tools</td><td>18060-20</td><td></td></tr><tr><td>Microneedle driver</td><td>Fine Science Tools</td><td>12075-14</td><td></td></tr><tr><td>Spring Scissor</td><td>Fine Science Tools</td><td>15396-00</td><td></td></tr><tr><td>Insulin syringe</td><td>Various</td><td></td><td></td></tr><tr><td>1mL syringe</td><td>Various</td><td></td><td></td></tr><tr><td>20mL syringe</td><td>Various</td><td></td><td></td></tr><tr><td>Glucometer (one touch ultra mini)</td><td>Lifescan</td><td>70021208</td><td></td></tr><tr><td>Multiplex insulin and GLP-1 kit</td><td>Meso Scale Discovery</td><td>K15171C-1</td><td></td></tr><tr><td>GraphPad Prism 6.00</td><td>GraphPad Software</td><td></td><td></td></tr><tr><td>Nestlets</td><td>Ancare</td><td>NES3600</td><td></td></tr></tbody></table>

a murine model of vertical sleeve gastrectomy

垂直方向のスリーブ胃切除術 (VSG) などの肥満症治療手術、減量を目的として行われる消化管の手術です。肥満手術は、肥満症の最も効果的な長期的な治療では現在です。体重減少に加えて肥満手術は、2 型糖尿病、高血圧症の寛解の赦しなどその他健康上の利点を生成し、特定のタイプの癌を開発する危険の減少します。これらの利点の重量損失を超えてメカニズムの不完全に定義されたままです。したがって、肥満症治療手術の動物モデルである開発および消化生理学の理解し、新しい治療上のターゲットを識別の向上を目的に、これらの利点につながるメカニズムを識別するために検証します。体重減少や代謝改善、生産に高い効果があるのでアメリカ合衆国でクリニックで最も一般的に実行の肥満の手順となっているし、他の肥満の手順よりも実行する方が簡単です。したがって、我々 は開発し、VSG のマウスモデルを検証します。このマウスモデル VSG VSG 改善血糖、血圧の調節など、人間での効果の多くを繰り返します。メソッドは、胃の大弯に沿って transecting によって胃の分離、胃の血管の結紮と胃の 70% が除去に基づいています。正常に VSG の利点機構の貢献者を定義する様々 な遺伝子組み換えマウス ラインにこの外科手術を行った.さらに、この VSG マウスモデルを深く追究を達成するために、他の手術手技と結合されています。したがって、消化管生理学、肥満手術の健康上の利点を研究するためのシンプルで汎用性の高いモデルです。

シャムと VSG の手順は、図 1に描かれています。図 1 aは、偽の手順中の胃の壁に沿って縫合線を配置する場所を示しています。これと同じエリアは、VSG 手術中に胃をカットする場所です。図 1 bは、VSG のパフォーマンスの後胃の左の管状の名残を示しています。
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Watch this Scientific Journal Video about A Murine Model of Vertical Sleeve Gastrectomy at JoVE.com

A Murine Model of Vertical Sleeve Gastrectomy

次に、マウスの垂直方向のスリーブ胃切除のパフォーマンスについて説明します。これは胃の約 70% の除去は、減量手術の一種です。

垂直方向のスリーブ胃切除術のマウスモデル

Bariatric surgery, such as vertical sleeve gastrectomy (VSG), is a surgery of the gastrointestinal tract that is performed for the purpose of weight loss. ...

a-murine-model-of-vertical-sleeve-gastrectomy

Research

JoVE Journal

Biology

11.1K ビュー.  Cornell University. 次に、マウスの垂直方向のスリーブ胃切除のパフォーマンスについて説明します。これは胃の約 70% の除去は、減量手術の一種です。

ビデオ: A Murine Model of Vertical Sleeve Gastrectomy

Murine Pancreatic Islet Isolation

Hi, I am Greg Zott. I'm with the UCSF Diabetes Center in the Bluestone Laboratory, and today I'm gonna show you the process for isolating mouse eyelets. The procedure involves the injection of a collagenase solution into the pancreas of a C3 H donor mouse, where we will then remove that pancreas, digest it away, purify the eyelets on a fial gradient, and then handpick them for ultimate transplant into the kidney capsule of a diabetic NOD mouse, Which will be our recipient.Before we start this procedure, we diluted our ATE solution in a modified Hanks buffer and we have adjusted the concentration specific for the strain of mouse that we're using the C3 H.We found digests better at a 0.8 mgs per mil collagenase solution, and we will use that to inject into the common bile duct to distend the pancreas.Performing. The procedure today Will be Pavo codre of the diabetes center. He will inject, open up the mouse, expose the the intestines, and then inject into the common bile duct.We will spray down the mouse with ethanol to keep the hair down, and then we will also verify that the mouse has been sacrificed properly with a toe pinch. Pavo will now open up the mouse and exposing the intestine and the pancreas with a midline incision. The diaphragm will also be cut to verify that the mouse will be sacrificed.Before we get started, I'd like to point out some of the important structures. The stomach is lying right here with the pancreas attached underneath into the duodenum and above. This is the liver where we'll find the gallbladder and the common bile duct, which you can see right here.So what Pavo will do next is he will isolate the small intestine, find where the common bile duct enters the small intestine. Once the intestine is exposed, we clamp on either side of the common bile duct with mosquito clamps. We find that this allows more collagenates to enter the pancreas while other groups try to direct the clamp on top of the common bile duct.We find that this is a better technique in allowing enzyme to enter the pancreas. This is a gentle procedure. You don't want to tear the bowel.You wanna allow the clamps to lie gently on either side of the common bile duct. Now we will find a gallbladder, and once the gallbladder's exposed, we will use that as a guide to injecting the collagenase into the common bile duct. Powell's using a 30 gauge needle with a five cc syringe containing three mills of collagenase solution.As he's entering the common bile duct, he's distending that duct with collagenase solution and then he travels down the common bile duct till he reaches near the intestine. And as he's going down, the enzyme solutions being pumped into the injected into the pancreas. Now that the pancreas is inflated with collagenase solution, Powell's gonna remove the hemostats and then remove the pancreas from the the mouse carefully tearing it away from the intestine.He removes it from the stomach. Okay, and then using the spleen as a handle, he lifts up the pancreas and then slowly snips it away in the common bile duct, and that's it. Pancreas is laid out and then the spleen is removed, and there he's removing the meso enteric tissue, and then the pancreas is placed in a two mill collagenase solution and a sterilized glass vial and it's placed back on Ice.The isolated pancreas that we put into the glass vials will now be placed into a 37 degree water bath for approximately 17 minutes. We placed them into a 50 mil conical rack so that they don't float up. Once during the bath, the timer starts and the digestion begins.Once the digestion time is completed, vials are removed, and then they are shaken and disrupted gently. And once the tissue starts falling apart, the vial is then placed on ice. A sterile screen is placed over a 400 mil beaker that contains washing buffer.The pancreas digest is then carefully poured over the screen into the wash buffer. For demonstration purposes, the hood has been turned off and the glass of the hood has been raised so we could see what we're doing. Once the pancreas digest is poured into the beaker, the vials are rinsed with the same washing buffer and the wash buffer placed over the screen.We have a syringe filled with washing buffer. We will use that to forcefully wash the tissue through the screen. The idea is to release any loose eyelets that are unattached to the matrix, but leaving the membranes and the undigested tissue on the screen.And when you're finished washing a tissue, this is what you usually See at the end of the procedure. The Pancreas tissue that's in the bottom of the tube will now be transferred to two 50 mil conical, and the tissue will be washed, the tissue's mixed evenly, and then distributed evenly so that we have a equal pellet volume in each tube. The beaker is now rinsed with more washing buffer.The sides are rinse so that you get all tissue that is entered the beaker again. Then it's equally distributed between each two, both 50 mil. Conical then are topped off with wash buffer.The tissue is inverted several times and then placed into a centrifuge. This is a quick spin. The centrifuge is allowed to reach a thousand RPM and then is turned off.The tissue at this time is very S eyelets are very sensitive, and you don't want to over compact them. The tubes are removed and we will do this wash step three more times. This is the end of the third wash.The components of the 50 mil conical tube are transferred to the 15 conical tube and centrifuge for a thousand RP M and then stopped. The supernat will be removed and the first part of the FI Call will be started. We are at the step where we Have our pelleted di pancreas digest.We'll resuspend that tissue so it's nice and loose to the pellets. We'll add the first density gradient, which is 1 1 0 8. We'll add five mils to each tube.The pellet is then mixed thoroughly in each of the gradients. It's important that you get good tissue distribution in your heaviest gradient. Now we will layer two mils of 1 0 9 6 density.We will then on top of that, layer two mils of 1 0 6 9 density, and then on top of that 1 0 3 7 density. It's important while you're layering the densities that you don't mix. Then all the layers have been added to the tube.And you can see this is the top of 1 1 0 8, the top of 9 1 0 9 6, the top of 1 0 6 9 and the top of 1 0 3 7. And you could see the tissue is already starting to move to its proper density even without centri fusion. The fial gradients are brought up to speed at 1800 RPM for 15 minutes, and remember to keep the brake off after the tubes are removed from the centrifuge.You can see between the 0 0 9 6 and 0 0 7 layer, there's a nice band of eyelet tissue Pablo's. Now removing the 0 3 7 layer to remove any tissue debris and DNA that might have been released during the processing. Using a sterile plastic pasture pipette, he will now remove the islet layer and transfer them to a 50 mil conical containing 25 mils of wash buffer.You want do this step quickly because fol is toxic to eyelets. We actually removed the entire 0 6 9 and 0 9 6 layer. Pavo cuts the top of the plastic pasture pipette and rinses it with wash buffer.The eyelets are then centrifuge like we centrifuge previously. Centrifuge is brought up to a Thousand RPM and then we stop it and We aspirate off. The supernatant eyelets will be washed three times with wash media.So we just completed the eyelid Isolation process we took from the digest to the fol gradient to the purified eyelets after our third wash with wash buffer, we resuspended the eyelets in our RPMI media and we played it in a tissue culture plate. These eyelets are now ready for in vitro assays or in vivo preps like eyelet Transplant.

マウス膵島単離

生物学

Murine Model of Hindlimb Ischemia 

In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide. Medical therapies for symptomatic relief are limited. Surgical or endovascular interventions are useful for some individuals, but long-term results are often disappointing. As a result, there is a need for developing new therapies to treat PAD. The murine hindlimb ischemia preparation is a model of PAD, and is useful for testing new therapies. When compared to other models of tissue ischemia such as coronary or cerebral artery ligation, femoral artery ligation provides for a simpler model of ischemic tissue. Other advantages of this model are the ease of access to the femoral artery and low mortality rate. 
In this video, we demonstrate the methodology for the murine model of unilateral hindimb ischemia. The specific materials and procedures for creating and evaluating the model will be described, including the assessment of limb perfusion by laser Doppler imaging. This protocol can also be utilized for the transplantation and non-invasive tracking of cells, which is demonstrated by Huang et al.1.

Murine Model of Hindlimb Ischemia

The surgical procedure for induction of unilateral hindlimb ischemia is demonstrated, with confirmation of ischemia by laser Doppler perfusion imaging.

後脚虚血症のマウスモデル

In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide.  Medical therapies for ...

Murine Renal Transplantation Procedure

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell et al over 30 years ago (1) and refined by Zhang et al years later (2), few people in the world have mastered this procedure. In our laboratory we have successfully performed 1200 orthotopic kidney transplantations with &gt; 90% survival rate. The key points for success include stringent control of reperfusion injury, bleeding and thrombosis, both during the procedure and post-transplantation, and use of 10-0 instead of 11-0 suture for anastomoses.
Post-operative care and treatment of the recipient is extremely important to transplant success and evaluation. All renal graft recipients receive antibiotics in the form of an injection of penicillin immediately post-transplant and sulfatrim in the drinking water continually. Overall animal health is evaluated daily and whole blood creatinine analyses are performed routinely with a portable I-STAT machine to assess graft function.

Renal transplantation in mice is a technically challenging procedure that requires careful post-operative care and treatment for success.

マウス腎移植の手順

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell ...

Murine Model for Parkinson's Disease: from 6-OH Dopamine Lesion to Behavioral Test

Parkinson's disease (PD) affects at least 6.5 million people worldwide, irrespective of gender, social, ethnic, economic, or geographic boundaries. Key symptoms, such as tremor, rigidity and bradikinesia, develop when about 3/4 of dopaminergic cells are lost in the substantia nigra, and fail to provide for the smooth, coordinated regulation of striatal motor circuits. Depression and hallucinations are common, and dementia eventually occurs in 20% of patients. At this time, there is no treatment to delay or stop the progression of PD. Rather, the medications currently available aim more towards the alleviation of these symptoms. New surgical strategies may reversibly switch on the functionally damaged circuits through the electrical stimulation of deep brain structures, but although deep brain stimulation is a major advance, it is not suitable for all patients. It remains therefore necessary to test new cell therapy approaches in preclinical models.
Selective neurotoxic disruption of dopaminergic pathways can be reproduced by injection of 6-hydroxydopamine (6-OHDA) or MPTP (1-methyl-4-phenyl-1,2,3,6-tertahydropyridine) whereas depleting drugs and oxidative-damaging chemicals may also reproduce specific features of PD in rodents. Unlike MPTP, 6-OHDA lesions cause massive irreversible neuronal loss, and can be uni- or bilateral. The 6-OHDA lesion model is reliable, leads to robust motor deficits, and is the most widely used after 40 years of research in rats1. As interactions between grafted cells and host can now be studied more thoroughly in mice rather than in rats, the model has been transposed to mice2,3, where it has been recently characterized4.
In this video, we demonstrate how to lesion the left nigro-striatal pathway of anesthetized mice by slowly delivering 2.0 &mu;L of 6-OHDA through a stereotaxically inserted micro-syringe needle. The loss of dopaminergic input occurs within days, and the functional impairments can be monitored over post-operative weeks and months by rating animal rotations induced by dopaminergic agents5. Here, we show full-body contralateral rotations occurring 10 minutes after a single subcutaneous administration of apomorphine, measured one month after the lesion. Outcomes and drawbacks are discussed below.

Parkinson disease is caused by loss of dopaminergic innervation to the striatum, which can be experimentally induced by 6-OH-dopamine. We describe how to perform a stereotaxic lesion and to monitor apomorphine-induced rotational behavior in mice. This model is useful and reliable for testing new therapies for Parkinson disease.

パーキンソン病モデルマウス：6 - OHドーパミンの病変から行動試験に

Parkinson's disease (PD) affects at least 6.5 million people worldwide, irrespective of gender, social, ethnic, economic, or geographic boundaries. Key ...

LAD-Ligation: A Murine Model of Myocardial Infarction

Research models of infarction and myocardial ischemia are essential to investigate the acute and chronic pathobiological and pathophysiological processes in myocardial ischemia and to develop and optimize future treatment.
Two different methods of creating myocardial ischemia are performed in laboratory rodents. The first method is to create cryo infarction, a fast but inaccurate technique, where a cryo-pen is applied on the surface of the heart (1-3). Using this method the scientist can not guarantee that the cryo-scar leads to ischemia, also a vast myocardial injury is created that shows pathophysiological side effects that are not related to myocardial infarction. The second method is the permanent ligation of the left anterior descending artery (LAD). Here the LAD is ligated with one single stitch, forming an ischemia that can be seen almost immediately. By closing the LAD, no further blood flow is permitted in that area, while the surrounding myocardial tissue is nearly not affected. This surgical procedure imitates the pathobiological and pathophysiological aspects occurring in infarction-related myocardial ischemia.
The method introduced in this video demonstrates the surgical procedure of a mouse infarction model by ligating the LAD. This model is convenient for pathobiological and pathophysiological as well as immunobiological studies on cardiac infarction. The shown technique provides high accuracy and correlates well with histological sections.

This video demonstrates how to use a fast and reliable model to study pathobiological and pathophysiological processes of myocardial ischemia.

LAD -ライゲーション：心筋梗塞のマウスモデル

Research models of infarction and myocardial ischemia are essential to investigate the acute and chronic pathobiological and pathophysiological processes ...

Techniques of Sleeve Gastrectomy and Modified Roux-en-Y Gastric Bypass in Mice

Obesity is a major public health issue, with a prevalence of 4 to 28% for men and 6.2 to 36.5% for women in Europe (from 2003 to 2008). Morbid obesity is frequently associated with metabolic complications, such as type 2 diabetes, hypertension, and dyslipidemia, reducing life expectancy and quality. In the absence of any effective noninvasive treatments, bariatric surgery is a valuable therapeutic option for patients with morbid obesity (body mass index (BMI) &#62;40 kg/m2), leading to long-term, sustained weight loss and improvements in metabolic complications. However, the underlying cellular and molecular mechanisms sustaining the beneficial effects of bariatric surgery are not yet fully understood. Due to the numerous genetically-modified strains available, the mouse model is the most convenient animal model to explore the molecular mechanisms behind the pleiotropic beneficial effects of bariatric surgeries. Here, we detailed the optimized healthcare methods and surgical protocols in mice for the two most widely-used bariatric surgeries: the sleeve gastrectomy and the modified Roux-en-Y gastric bypass. Deciphering the molecular mechanisms underlying the therapeutic effects of bariatric surgeries offers the promise of identifying new therapeutics targets.

Bariatric surgery is the most efficient way to reduce body weight and the deadly metabolic complications (diabetes, obesity, and dyslipidemia) frequently associated with morbid obesity. Mouse models of bariatric surgery represent a unique asset for deciphering molecular mechanisms behind the beneficial effects of these surgeries on diabetes, hypertension, and dyslipidemia.

スリーブ胃切除およびマウスにおける変更されたルーワイ胃バイパスのテクニック

Obesity is a major public health issue, with a prevalence of 4 to 28% for men and 6.2 to 36.5% for women in Europe (from 2003 to 2008). Morbid obesity is ...

医学

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

生物物理学研究のための昆虫細胞からの効率的な生産と組換えマウスKindlin-3の精製

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

Sleeve Gastrectomy in Mice using Surgical Clips

The number of people who are overweight and obese is continually increasing both in the adult and adolescent populations. This coincides with the increased universal phenomenon of type 2 diabetes (T2D) and other metabolic problems. Bariatric surgery, such as SG, is currently one of the most effective and commonly used long-term treatment for obesity and T2D, but the association between them is not completely explored yet. The mechanisms underlying the outcomes seen after bariatric surgery in humans can be investigated based on preclinical animal studies. The SG reduces body weight, glucose levels and many metabolic parameters, and is easy to perform with a low incidence of complications. The goal of this work is to provide a simple method and an uncomplicated preclinical model of bariatric surgery in animals for researchers.

The prevalence of diabetes and obesity is continuously increasing worldwide. The mechanisms between diabetes, obesity and their associated mortality and co-morbidities need to be further investigated. Here, we present a protocol for sleeve gastrectomy (SG) in animals as an uncomplicated preclinical model of bariatric surgery.

外科用クリップを用いたマウスにおけるスリーブ胃切れ術

The number of people who are overweight and obese is continually increasing both in the adult and adolescent populations. This coincides with the ...

Single-Anastomosis Duodeno-Ileal Bypass with Sleeve Gastrectomy Model in Mice

Obesity is a major health issue worldwide. As a response, bariatric surgeries have emerged to treat obesity and its related comorbidities (e.g., diabetes mellitus, dyslipidemia, non-alcoholic steatohepatitis, cardiovascular events, and cancers) through restrictive and malabsorptive mechanisms. Understanding the mechanisms by which these procedures allow such improvements often require their transposition into animals, especially in mice, because of the ease of generating genetically modified animals. Recently, the single-anastomosis duodeno-ileal bypass with sleeve gastrectomy (SADI-S) has emerged as a procedure that uses both restrictive and malabsorptive effects, which is being used as an alternative to gastric bypass in case of major obesity. Thus far, this procedure has been associated with strong metabolic improvements, which has led to a marked increase in its use in daily clinical practice. However, the mechanisms underlying these metabolic effects have been poorly studied as a result of a lack of animal models. In this article, we present a reliable and reproducible model of SADI-S in mice, with a special focus on perioperative management. The description and use of this new rodent model will be helpful for the scientific community to better understand the molecular, metabolic, and structural changes induced by the SADI-S and to better define the surgical indications for clinical practice.

Single-anastomosis duodeno-ileal bypass (SADI-S) is an emerging bariatric procedure with important metabolic effects. In this article, we present a reliable and reproducible model of SADI-S in mice.

マウスにおけるスリーブ状胃切除モデルを用いた単一吻合十二指腸回腸バイパス術

Obesity is a major health issue worldwide. As a response, bariatric surgeries have emerged to treat obesity and its related comorbidities (e.g., diabetes ...

Murine Vertical Sleeve Gastrectomy: A Surgical Procedure to Downsize Stomach in Experimental Mouse Models

Source: Garibay, D., Cummings, B. P. <a href="https://www.jove.com/video/56534" target="_blank">A Murine Model of Vertical Sleeve Gastrectomy.</a> J. Vis. Exp. (2017)
In this video, a vertical sleeve gastrectomy procedure is performed on an experimental mouse model. This procedure helps remove a significant portion of the stomach along its greater curvature.

マウス垂直スリーブ胃切除術:実験マウスモデルにおける胃のサイズを縮小するための外科的処置

Source: Garibay, D., Cummings, B. P. A Murine Model of Vertical Sleeve Gastrectomy. J. Vis. Exp. (2017)
In this video, a vertical sleeve gastrectomy ...