彼女のサポートのためグンドラ ・ Hertl を認識したいと思います。

このプロトコルのすべてのプロシージャは、ドイツ動物福祉法及びガイドラインで承認されています。10-12 週齢当所ヌードマウスと c57bl/6 マウスの使用を検討 (ドナー: 古い女性; 受信者: 男性) 実験中。膵島分離に当所ヌードマウスを使用します。ローカル動物施設の規則に従って定義された条件の下でマウスを維持します。
1. 誘導によるストレプトゾトシン糖尿病の
<ol><li>-4 日 12 週齢当所ヌードマウスと c57bl/6 マウスの計量機を使用して 10 の体重を測定します。</li>
	<li>測定体重に従ってマウスあたり生理食塩水を 0.1 mL のストレプトゾトシンの 180 mg/kg の線量を準備します。 注: ストレプトゾトシン新鮮な使用前に、の準備、それは光に敏感なアルミ箔で覆われています。</li>
	<li>ストレプトゾトシン (180 mg/kg) を 0.1 mL の単回投与と腹腔内 26 G 針を使用してそれぞれの同系と同種のマウスを挿入します。</li>
	<li>-3 日-2 と-1 は、非絶食マウスの尾静脈を穿刺によって 2-4 μ L の血液を収集します。</li>
	<li>当所ヌードマウスと c57bl/6 マウスの血糖値を測定するのに血液サンプル テスト ストリップあたりの 2-4 μ L を使用します。 注: この時点で、マウスの 80% が糖尿病に (20 mmol/L)。<ol>
<li>血糖値が 20 ミリ モル/l. に達したら、膵島移植のマウスを使用します。</li>
	</ol>
</li>
</ol>2. マウスの膵島の分離
<ol><li>26 G 針で腹腔内投与によるケタミン (100 mg/kg 体重) とキシラジン (20 mg/kg 体重) マウスを麻酔します。 注: 動物 (0.10 mL/10 g) の体重によるとケタミン ・ キシラジンのボリュームを求めた。</li>
	<li>仰臥位でマウスを維持し、ポビドン ヨード液で消毒します。ペダルの反射をチェックして適切な安楽死を確認します。 注: C57Bl/6 の場合、腹部領域を剃る。</li>
	<li>微細鉗子で肌を保持し、腹腔内を公開するハサミで正中切開。滅菌ガーゼ湿布に腹部キャビティの外側と左腸の内臓を配置します。</li>
	<li>膵臓および他の関連を検索する別の方向で胃を調整する組織。</li>
	<li>大動脈に到達し、血液を排出する小さなカットをします。血液吸収に滅菌ガーゼを使用します。</li>
	<li>腸を慎重に取り外します。胃と脾臓から膵臓を区切ります。</li>
	<li>膵臓を切除、新鮮なシャーレ内に置きます。鉗子を使用して脂肪など、すべての不要な物質を削除します。</li>
	<li>コラゲナーゼの 4 mL と 5 mL シリンジを記入し、26 G 針と膵臓に挿入 (貯蔵液: ハンクの溶液 12 mL 中のコラゲナーゼの 30 mg)。</li>
	<li>消化力の改善と 12 mL 赤キャップ チューブへの転送のための表面積を高めるためハサミで膵臓をミンチします。 注: ハンクの平衡塩溶液: HBSS 10 (100 mL)、x (10 mL) のペニシリン-ストレプトマイシン、ゲンタマイシン (1 mL)、シプロフロキサシン (10 mL)、HEPES (35 mL)、蒸留 H2O (900 mL)</li>
	<li>振動水バースの 37 ° C で 10 分間の消化を実行します。渦 15 の定期的な間隔で三度サンプル s。</li>
	<li>3 分間の手によって積極的に消化膵臓をミックスし、10 の氷の上に置きます s。</li>
	<li>消化反応と 560 x g で室温で 3 分間遠心を停止する冷たいハンクの溶液 6 mL を追加します。</li>
	<li>上澄みを除去し、室温でパーカー/胎児の子牛血清 (P/FCS) 培地 10 mL にペレットを溶解します。 注: P/FCS 中: TCM 199 10 (100 mL) x FCS (50 mL)、ペニシリン ・ ストレプトマイシン (10 mL)、HEPES (20 mL)、蒸留 H2O (900 mL)</li>
	<li>光学顕微鏡下でその形態 (回転楕円体、金茶色色) に基づく小島を識別します。中心部が暗い島は避けてください。純度、光学顕微鏡による染色ジチゾンを実行することで生存率をチェックします。 注: ジチゾン亜鉛イオンに結合し、小島に赤い染みを提供します。</li>
	<li>1 mL ピペットを使用して、カウントし光学顕微鏡の下で小島を手で摘むし、P/FCS 培地 4 mL を含んでいる新鮮なペトリ皿 (60 mm × 15 mm) にそれらを転送します。 注: のボリュームP/FCS ペトリ皿中は酸素の可用性を確保するために 4 mL を超えない。</li>
	<li>インキュベーターの 37 ° C で膵島の P/FCS 中一晩 4 mL を維持します。</li>
</ol>3. 動物の膵島移植のための準備
<ol><li>37 ° C の定温器から膵島を削除し、流れベンチ下に配置。ペトリ皿の揺れ円運動と小島を中心します。</li>
	<li>P/FCS 中の 0.1 mL を 1 mL の注射器 (青 23 G 針) を入力します。その後、ハンクの溶液 0.3 mL で 350 島を追加します。解決する島を許可する垂直方向の位置に注射をしてください。</li>
	<li>0.05 mL に音量を下げるし、茶色の 26 G 針に青 23 G 針を置き換えます。同じ注射器に注射器で 0.2 mL の最終的なボリュームに到達する上に 0.15 mL Ficoll (密度勾配媒体) を追加します。空気の泡を避けるため。</li>
	<li>垂直位置に注射器を保つため、小島の凝集を避けるために少し回します。小島は 2-3 分以内で注射器のコーンで解決されます。</li>
	<li>受信者のネズミのボディ重量および血ブドウ糖のレベルを測定します。血糖値が約 20 ミリ モル/l. をする必要があります。</li>
	<li>26 G 針で腹腔内投与によるケタミン (100 mg/kg 体重) とキシラジン (20 mg/kg 体重) マウスを麻酔します。</li>
	<li>暖かいサーマル プレート (37 ° C) に仰臥位にマウスを配置します。つま先をつまんで麻酔の深さを確認します。</li>
	<li>ポビドン ヨード液 (皮膚の消毒剤) と腹部をクリーンアップします。目の乾燥を防ぐためにそれらを保つ眼軟膏で湿潤。 注: C57Bl/6 の場合、腹部領域を剃る。</li>
</ol>4. 門脈経由膵島移植
<ol><li>開腹した、0 日に始めるにはピンセットで皮膚を押しながら 2-3 cm を作る-ハサミで正中切開。場所滅菌濡れたガーゼを傷周囲にそれを湿った保つに圧縮します。</li>
	<li>滅菌ガーゼ湿布に腹部キャビティの外左に腸の内臓を配置します。 注: は、腸に予め温めておいた 0.9 %nacl 水溶液や食塩に浸した滅菌ガーゼを使ってプロシージャ全体で潤いを保ちます。</li>
	<li>十二指腸に移動し、膵臓を探します。膵臓を上向きに押すことで腹側のサイトに位置して門脈を探します。指と親指を使用して門脈を公開して、公開、一度穴を肝臓の近くに。</li>
	<li>1 分以内ゆっくりと着実には、島の漏洩を避けるために拡大鏡の検査で門脈に小島 (3.4 の手順から 0.2 mL) を挿入します。 メモ: 注入後常にシリンジの残りの島のために確認します。新鮮なペトリ皿と顕微鏡下でカウントにフラッシュの注射器 1 mL P/FCS メディアを取る。</li>
	<li>穿刺部位に滅菌ゲージを置き、人差し指で圧力を適用 (浸透深さ: 0.5-1 cm)、出血を止めるため 6 分間。 注: 注入量は 0.2 mL を超えない。過度な量と圧力は、せん断応力による島に損傷を与えます。</li>
	<li>慎重に腸の内臓、腹膜、腹部筋層、筋膜を元の位置に配置します。</li>
	<li>鉗子を使用して、皮膚を持ち上げて、シルク縫合糸と筋切開と 2-3 クリップ上部の皮膚層を閉じます。</li>
	<li>皮膚の消毒剤で表面をきれいに傷の治療師を適用し、個々 のケージにマウスを置きます。傷の治療師は耐水性アクリル層を提供し、浸軟、二次感染を防ぐ。 注: 十分な意識を取り戻しているまでマウスを観察、流体と共にトラマドール (マウスあたり 0.2 mg) を提供します。このプロトコルではすぐに麻酔から回復したマウスとトラマドール管理が苦痛を減少します。7 日後、傷を癒す必要があります。</li>
	<li>通常の餌と水でマウスを供給します。ストレプトゾトシン投与後食料と水の消費量と尿中排泄が増加します。</li>
	<li>最初の 1 週間と同系マウスの週後に毎日血糖値を測定します。同種のマウスで最大 15 日間の週 3 回血糖値を測定します。</li>
</ol>

<ol>
	<li>McCall, M., Shapiro, 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=Update+on+islet+transplantation.">Update on islet transplantation.</a> Cold Spring Harbor Perspectives in Medicine. 2 (7), a007823 (2012).</li><li>Shapiro, A. 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=Islet+transplantation+in+seven+patients+with+type+1+diabetes+mellitus+using+a+glucocorticoid-free+immunosuppressive+regimen.">Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen.</a> New England Journal of Medicine. 343 (4), 230-238 (2000).</li><li>Cantarelli, 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=Bone+marrow+as+an+alternative+site+for+islet+transplantation.">Bone marrow as an alternative site for islet transplantation.</a> Blood. 114 (20), 4566-4574 (2009).</li><li>Echeverri, G. J., 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=Endoscopic+gastric+submucosal+transplantation+of+islets+(ENDO-STI):+technique+and+initial+results+in+diabetic+pigs.">Endoscopic gastric submucosal transplantation of islets (ENDO-STI): technique and initial results in diabetic pigs.</a> American Journal of Transplantation. 9 (11), 2485-2496 (2009).</li><li>Korsgren, O., 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=Optimising+islet+engraftment+is+critical+for+successful+clinical+islet+transplantation.">Optimising islet engraftment is critical for successful clinical islet transplantation.</a> Diabetologia. 51 (2), 227-232 (2008).</li><li>Merani, S., Toso, C., Emamaullee, J., Shapiro, 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=Optimal+implantation+site+for+pancreatic+islet+transplantation.">Optimal implantation site for pancreatic islet transplantation.</a> British Journal of Surgery. 95 (12), 1449-1461 (2008).</li><li>Lingwal, N., 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=Inhibition+of+gelatinase+B+(matrix+metalloprotease-9)+activity+reduces+cellular+inflammation+and+restores+function+of+transplanted+pancreatic+islets.">Inhibition of gelatinase B (matrix metalloprotease-9) activity reduces cellular inflammation and restores function of transplanted pancreatic islets.</a> Diabetes. 61 (8), 2045-2053 (2012).</li><li>Chen, C., 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=Improved+intraportal+islet+transplantation+outcome+by+systemic+IKK-beta+inhibition:+NF-kappaB+activity+in+pancreatic+islets+depends+on+oxygen+availability.">Improved intraportal islet transplantation outcome by systemic IKK-beta inhibition: NF-kappaB activity in pancreatic islets depends on oxygen availability.</a> American Journal of Transplantation. 11 (2), 215-224 (2011).</li><li>Wang, Y., Wang, T., Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three+aspects+are+critical+for+carrying+out+intraportal+islet+transplants+successfully+in+a+diabetes+mouse+model.">Three aspects are critical for carrying out intraportal islet transplants successfully in a diabetes mouse model.</a> Experimental and Clinical Transplantation. 10 (3), 302-304 (2012).</li><li>Neuman, J. C., Truchan, N. A., Joseph, J. W., Kimple, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+mouse+pancreatic+islet+isolation+and+intracellular+cAMP+determination.">A method for mouse pancreatic islet isolation and intracellular cAMP determination.</a> Journal of Visualized Experiments. (88), e50374 (2014).</li><li>Zmuda, E. J., Powell, C. A., Hai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+murine+islet+isolation+and+subcapsular+kidney+transplantation.">A method for murine islet isolation and subcapsular kidney transplantation.</a> Journal of Visualized Experiments. (50), (2011).</li><li>Rother, K. I., Harlan, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Challenges+facing+islet+transplantation+for+the+treatment+of+type+1+diabetes+mellitus.">Challenges facing islet transplantation for the treatment of type 1 diabetes mellitus.</a> Journal of Clinical Investigation. 114 (7), 877-883 (2004).</li><li>Su, Z., 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=Small+islets+are+essential+for+successful+intraportal+transplantation+in+a+diabetes+mouse+model.">Small islets are essential for successful intraportal transplantation in a diabetes mouse model.</a> Scandinavian Journal of Immunology. 72 (6), 504-510 (2010).</li><li>Rekittke, N. E., Ang, M., Rawat, D., Khatri, R., Linn, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerative+Therapy+of+Type+1+Diabetes+Mellitus:+From+Pancreatic+Islet+Transplantation+to+Mesenchymal+Stem+Cells.">Regenerative Therapy of Type 1 Diabetes Mellitus: From Pancreatic Islet Transplantation to Mesenchymal Stem Cells.</a> Stem Cells International. 2016, 22 (2016).</li></ol>

すべての著者は、利害の衝突があるないことを宣言します。

本研究では、膵島移植の門脈内のルートが検討されます。このプロトコルは糖尿病のストレスの緩和に非常に効率的な膵島移植の研究を探索する詳細な機会を提供しています。このプロトコルは、約 350 マウス島が高血糖状態を逆転させることができることを確認します。また、プロシージャの間に死んだマウスのどれも、血糖コントロールは、すべての受信者で達成されました。本体重量および血ブドウ糖のレベルは、能力、標準、および高血糖を逆転させることで移植膵島の機能を反映するように定期的に記録されました。Syngeneically 移植マウスの血糖値は、毎日最初の週と週に一度その後を測定しました。膵島移植、低酸素条件の後の要因は 14 日後 VEGF などプロ血管新生因子を分泌して、図 3に示すように、肝臓の血管の生着を高めるに内皮細胞をトリガーします。同種のマウスの血糖値は最大 15 日間の週 3 回を測定しました。15 日後免疫抑制剤を投与しない限りは、免疫の攻撃による同種のモデルで移植膵島の拒否されました。
文献10,11で公開されていくつかの膵島隔離のプロトコルがあります。コラゲナーゼの消化力の後のサイズと形態に基づいて手動で小島をピックアップする最も簡単です。これは膵島だけを選択する最も重要な手順の 1 つと外分泌細胞、破片など選択した島を避けるためには、消化中に発生する機械的ストレスから回復することを許可するインキュベーターで一晩 37 ° C で維持されるべきプロシージャ。ストレプトゾトシン投与量および門脈内膵島移植の選択もこのプロトコルに重要であります。体温は規制されるべきし、手術中のサーマル プレートを使用して維持されます。滅菌ガーゼは、出血を避けるために穿刺のサイトで 6 分間の最小限の圧力を適用しなければなりません。彼らは十分な意識を取り戻すまで、観察の下でマウスを保管すべき。
このプロトコルの特定のステップを変更できます。たとえば、消化時間の最適化とコラゲナーゼのボリュームは、異なるマウス系統の島の収量を増加可能性があります。糖尿病 (ストレプトゾトシン 180 mg/kg) の急速な誘導、代わりにゆっくりと高血糖の増加を誘起すること時間の長期間にわたって低用量を使用して (例えば、 40 mg/kg/日 5 日間連続のストレプトゾトシン)。これは膵島移植より生着を提供可能性があります。門脈静脈に代わるものとして骨髄、大網ポーチ、筋肉内の地域、胃粘膜、脾臓、腎臓などの他のサイトを探索できます。
移植膵島の生着不全はまだ臨床試験で主要な関心事です。他には、不一致移植、免疫抑制療法、血液を介した反応12,13前後後の血糖値を維持するために、ドナーの不足が含まれます。適切な生着を評価する手法の欠如は、別の障害です。磁気共鳴または生物発光イメージングによる移植膵島を追跡可能性があるブドウ糖負荷試験や c-ペプチドなど古典的な方法を置き換えることができます。今後、島とともに移植された幹細胞は栄養あります、免疫サポート (14)。

膵島移植は、タイプ 1 の糖尿病1の治療に有望なアプローチです。糖尿病患者に羊膵フラグメントを転送する最初の試みは 1893 年にワトソン ウィリアムズが行った。臨床膵島移植で画期的だったエドモントンの議定書を達成し、その後、国家プログラムのシリーズが開発された2。過去には、前臨床モデルで骨髄、大網ポーチ、筋肉内地域、胃粘膜、脾臓、腎臓のカプセルなど、いくつかのサイトを検討したが、門脈系は適切かつ効果的なサイトの 1 つとして考慮されます。臨床プログラム3,4,5,6。
インスリンの外因性投与は、アイレット門脈内注入によって置き換えることができます。これは酸素の供給がポータル動脈と静脈の合流点の下流の場所のためのネイティブの膵臓に匹敵するので最寄りのサイトと見なされます。また、小島の立体構造を保持することがありますので、大きい表面積があるし、血管新生促進5ができます。糖尿病マウスの受信者、2,000 の人間や豚の膵島同等または 350 マウス島高血糖状態7、8を逆に適切です。外因のレベルは、同系マウスに移植に 120 日以上の異種膵島と同種のマウス-マウス モデルにおけるマウスの受信者モデルで 15 日間の報告されました。
門脈を介して膵島移植の有効性に関連する要因は、適切な麻酔穿刺・止血9法です。麻酔は、吸入 (5% イソフルラン) または (ケタミン、キシラジン、またはペントバルビ タール) の注入によって誘起することし、物質は頻繁に結合されます。深さの制御と麻酔の時間は、マウス、色の粘膜、目の蓋、角膜反射、呼吸数、体温などの状態に留意する必要があります。動物の苦労はないとそれが操作を存続を確保することが重要です。赤電球に、加熱パッドなどの異なる暖房機器で温度を維持できるなどの暖かいサーマル プレートはマウスとの発生を防止する操作テーブルの間 25-30 ° C の温度を維持するために使用されています低体温症。麻酔薬の投与量が重要で、それらのすべては、肝臓で代謝され、肝機能は一過性島ペンション.の注入によって乱れることがあります。門脈の理想的な穿刺のポイントは、最初と 2 番目の支流静脈間位置です。
小島と意図した注入量の数は、過剰なボリュームは、せん断応力を高める可能性がありますとも、移植の結果に影響を与えます。0.2 mL ボリュームは門脈に膵島移植に適したと見なされます。貫通傷 (26 g 針) は、タイムリーかつ効果的な方法で停止する必要がある門脈からの出血を誘発します。約 6 分、出血を止めるための穿刺のサイトで最低限の圧力で滅菌ガーゼまたは指を適用できます。撮影一緒に、ポータルの膵島の注入は効果的な化学物質誘発糖尿病マウスの血糖値を規制を提供しています。

<table border="0" cellpadding="0" cellspacing="0" width="600">
	<tbody>
		<tr height="20">
			<td>C57Bl/6 mice&#160;</td>
			<td>Charles River (Sulzfeld, Germany)</td>
			<td></td>
			<td>(10-12 weeks)</td>
		</tr>
		<tr height="20">
			<td>BALB/c mice</td>
			<td>Charles River (Sulzfeld, Germany)</td>
			<td></td>
			<td>(10-12 weeks)</td>
		</tr>
		<tr height="20">
			<td>NMRI nude mice</td>
			<td>Charles River (Sulzfeld, Germany)</td>
			<td></td>
			<td>(10-12 weeks)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ketamine</td>
			<td>Bela-pharm</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Xylazine</td>
			<td>CEVA TIERGESUNDHEIT GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Syringe, 23G and 26G needle</td>
			<td>BD</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Petri Dish</td>
			<td>Falcon</td>
			<td>303800</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaCl</td>
			<td>Carl Roth</td>
			<td>351007</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sterile Gauze</td>
			<td>Fuhrmann</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Shaking Water bath</td>
			<td>Kotterman</td>
			<td>1501</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Scissors</td>
			<td>Braun</td>
			<td>1501</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glucose Meter&#160;</td>
			<td>One Touch</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Warm Thermal Plate</td>
			<td>Thermo Fisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Braunol (povidone-iodide solution)</td>
			<td>B.Braun Melsungen AG</td>
			<td>3864154</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Liposic Edo - Sprinkled Ointment (eye ointment)</td>
			<td colspan="2">Dr. G Mann Chem.-Pharm.&#160; GmbH</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Incubator</td>
			<td>Fa. Roth</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Flow Bench</td>
			<td>Herdus</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ficoll</td>
			<td>Sigma-Aldrich</td>
			<td>10771</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5-0 Silk Suture Vicryl</td>
			<td>Braun</td>
			<td></td>
			<td>(7.5 *1.75mm)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Michel Clamps (clips)</td>
			<td>Aesculap&#160;</td>
			<td>BN507R</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">P/FCS Solution</td>
			<td>Gibco</td>
			<td>21180-021</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TCM-199 (10x)</td>
			<td>Gibco</td>
			<td>21180-021</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FCS</td>
			<td>Biowest</td>
			<td>S1810-500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin-streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES (1 M)</td>
			<td>Biochrom</td>
			<td>L 1613</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gentamycin</td>
			<td>Ratiopharm</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ciprofloxacin</td>
			<td>Fresenius Kabi</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hank&#39;s Solution</td>
			<td>Gibco</td>
			<td>14060-040</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Collagenase</td>
			<td>Roche</td>
			<td>11213865001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Streptozotocin</td>
			<td>Calbiochem</td>
			<td>572204</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">OPSITE-spray (wound healer)</td>
			<td>Smith and nephew</td>
			<td>66004978</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Reugular food</td>
			<td>Altromin</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Head light LED 16042 (magnifying glass)</td>
			<td>Eschenbach</td>
			<td>16042</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rat anti-mouse PECAM-1(CD31) monoclonal primary antibody</td>
			<td>Millipore, Chemicon</td>
			<td>CBL1337</td>
			<td>Dilution (1:100)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Donkey anti-rat secondary antibody</td>
			<td>Dianova</td>
			<td>712095153</td>
			<td>Dilution (1:400)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Polyclonal guinea pig anti-insulin primay antibody</td>
			<td>Dako</td>
			<td>A0564</td>
			<td>Dilution (1:500)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Donkey anti-guinea pig secondary antibody</td>
			<td>Dianova</td>
			<td>706-259-148</td>
			<td>Dilution (1:400)</td>
		</tr>
	</tbody>
</table>

intraportal transplantation of pancreatic islets in mouse model

高血糖を抑える膵島移植は化学的に誘発性糖尿病を持つ齧歯類で非常に成功しました。実験的膵島移植における最も一般的な移植サイトは腎被膜です。ただし、以下は膵島の血液成分との相互作用について知られている、また理にかなって実験的膵島移植における門脈アプローチを利用します。
このプロトコルは、当所ヌードマウスにおける門脈内の膵島移植技術を示しています。ストレプトゾトシン (180 mg/kg) を受信者のマウスにおける高血糖を誘発する腹腔内注入します。20 ミリ モル/l. より大きい非空腹時血糖で糖尿病と見なされます1 日、移植前にマウスの膵島がドナーの膵臓由来のコラゲナーゼの消化力; によって350 小島の最小値は、糖尿病の受信者ごと利用されています。膵島分離歩留まりによりますが、2 つ以上のドナー マウス受信者ごとに活用されます。37 ° C で一晩培養後の小島は門脈を介して受信者の肝臓に管理されています。マウスの赤い「マクロロン」家で保護されているとまで観察の手術後、目を覚ましています。このプロトコルは、同系マウスの 120 日間と同種のマウスで 15 日間の血糖コントロールを維持します。

化学物質誘発糖尿病マウスにおける移植膵島の能力を調べた。小島は糖尿病マウスにおける門脈経由で移植されました。ドナーと受信者の比 2:1 であった。同系マウス モデルにおける 2 つのドナー マウスが活かされて 350 島を取得します。小島それから糖尿病マウスに移植。血糖値を減らすことで膵ランゲルハンス島移植の役割を示唆して、移植後 1 日目に血糖値の低下を認めた.ストレプトゾトシンが約 22 ミリ モル/l. まで血糖値のレベルを増加しました。4.6 モル/L (1 日目) に血糖値の低下は、膵島移植 (図 1 a) 後に観察されました。な場合は、図 1 aに示すように、121 日間維持されました。ボディの重量および血ブドウ糖のレベルほぼ同じパターンに従ってください。ストレプトゾトシン投与、手術後、体重が減少。2 日目から以降、図 1 bに示すように普通 29.7 g (121 日)、25.3 g (4 日目) から上昇傾向にあった。
主要組織適合抗原 (MHC) の場合一致しないマウス、マウス二、C57Bl/6 (H2d) から 350 島は糖尿病 (H2b) BALB/c マウスに移植されました。ストレプトゾトシン投与後血糖値増加最大 26.6 ミリ モル/l.膵島移植後血糖値を落とした 4.4 mmol/l (日 1;図 2 a)。10 日間、15 日間 (20.1 ミリ モル/L) 後ではなく、な場合 (4.44 7.2 mmol/l) が観察されました。糖尿病マウスの体重は 23 g (0 日) に、21.5 g (1 日目) 26 g (日-3) から落ちた移植後は、体重は、25 g (10 日目) に徐々 にで 23 g (2 日目) から回復。MHC の不一致により血中グルコース濃度の増加し、体重減少 (図 2 b) 膵島移植後 10 日。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57559/57559fig1.jpg"> 図 1: 同系膵島移植の効果。ストレプトゾトシン誘発糖尿病マウスで異なる時点で (A) 血糖値のレベルを測定した (n = 3)。ストレプトゾトシン 180 mg/kg の単回投与は、それぞれの受信者 (0 日) に 350 の小島の移植前に注入しました。121 日前まで測定した血糖値 (mmol/L) (日-3: 17 ± 2.3; 0 日: 22.6 ± 1; 日 1: 4.6 ± 0.3; 日 5: 7.5 ± 0.4; と日 121: 5.3 ± 0.09)。(B) 膵島移植後体重増加。上記のデータは、平均 ± 標準偏差を表します。<a href="/files/ftp_upload/57559/57559fig1large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57559/57559fig2.jpg"> 図 2: 同種膵島移植の効果。ストレプトゾトシン誘発糖尿病マウスで異なる時点で (A) 血糖値のレベルを測定した (n = 2)。ストレプトゾトシン 180 mg/kg の単回投与は、350 の膵島 (0 日目) の移植前に注入しました。15 日間測定した血糖値 (mmol/L) (日-1: 25.5 ± 5.3; 0 日: 26.6 ± 1.4; 日 2:8 ± 0.9; 日 10: 6.2 ± 0.08; と一日 15: 20.1 ± 1.6)。Normoglycemic 状態は、血中グルコース濃度の増加とその後 10 日間を維持しました。(B) 体重前に、と膵島移植後のフォロー アップ。データは、平均 ± 標準偏差を表します。<a href="/files/ftp_upload/57559/57559fig2large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57559/57559fig3.jpg"> 図 3:肝臓に移植膵島の代表染色画像: 10% ロバ血清インスリンの一次抗体 (1: 500) と血小板の 4 ° C で一晩インキュベート続いてブロック、pbs 肝セクション (5 μ m) を洗う血管内皮細胞接着分子 1 PECAM 一次抗体 (1: 100)。次の日、1 時間、二次抗体で染色カウンター ヘキスト染色 PBS で洗浄し、ライカ蛍光顕微鏡でイメージをキャプチャーします。<a href="/files/ftp_upload/57559/57559fig3large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。

Watch this Scientific Journal Video about Intraportal Transplantation of Pancreatic Islets in Mouse Model at JoVE.com

Intraportal Transplantation of Pancreatic Islets in Mouse Model

膵島移植で 1 型糖尿病な場合を達成するために道はあります。糖尿病マウスの通常のブドウ糖のレベルを維持するために門脈内のルートを通じて移植技術について説明します。

マウス モデルにおける膵島の門脈内移植

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation ...

intraportal-transplantation-of-pancreatic-islets-in-mouse-model

Nanyang Technological University

Research

JoVE Journal

Medicine

9.4K ビュー.  Clinical Research Lab. 膵島移植で 1 型糖尿病な場合を達成するために道はあります。糖尿病マウスの通常のブドウ糖のレベルを維持するために門脈内のルートを通じて移植技術について説明します。

ビデオ: Intraportal Transplantation of Pancreatic Islets in Mouse Model

A Mouse Model of in Utero Transplantation

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus. For example, in utero transplantation (IUT) of hematopoietic stem cells has been used to successfully treat patients with severe combined immunodeficiency.1,2 In several other conditions, however, IUT has been attempted without success.3 Given these mixed results, the availability of an efficient non-human model to study the biological sequelae of stem cell transplantation and gene therapy is critical to advance this field. We and others have used the mouse model of IUT to study factors affecting successful engraftment of in utero transplanted hematopoietic stem cells in both wild-type mice4-7 and those with genetic diseases.8,9 The fetal environment also offers considerable advantages for the success of in utero gene therapy. For example, the delivery of adenoviral10, adeno-associated viral10, retroviral11, and lentiviral vectors12,13 into the fetus has resulted in the transduction of multiple organs distant from the site of injection with long-term gene expression. in utero gene therapy may therefore be considered as a possible treatment strategy for single gene disorders such as muscular dystrophy or cystic fibrosis. Another potential advantage of IUT is the ability to induce immune tolerance to a specific antigen. As seen in mice with hemophilia, the introduction of Factor IX early in development results in tolerance to this protein.14 
In addition to its use in investigating potential human therapies, the mouse model of IUT can be a powerful tool to study basic questions in developmental and stem cell biology. For example, one can deliver various small molecules to induce or inhibit specific gene expression at defined gestational stages and manipulate developmental pathways. The impact of these alterations can be assessed at various timepoints after the initial transplantation. Furthermore, one can transplant pluripotent or lineage specific progenitor cells into the fetal environment to study stem cell differentiation in a non-irradiated and unperturbed host environment.
The mouse model of IUT has already provided numerous insights within the fields of immunology, and developmental and stem cell biology. In this video-based protocol, we describe a step-by-step approach to performing IUT in mouse fetuses and outline the critical steps and potential pitfalls of this technique.

A Mouse Model of in Utero Transplantation

The mouse model of in utero transplantation is a versatile tool that can be used to study the potential clinical applications of stem cell transplantation and gene therapy in the fetus. In this protocol, we present a general approach to performing this technique

のマウスモデル子宮内で移植

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus.  For example, in ...

医学

Mouse Model of Surgically-induced Endometriosis by Auto-transplantation of Uterine Tissue

Endometriosis is a chronic, painful disease whose etiology remains unknown. Furthermore, treatment of endometriosis can require laparoscopic removal of lesions, and/or chronic pharmaceutical management of pain and infertility symptoms. The cost associated with endometriosis has been estimated at 22 billion dollars per year in the United States1. To further our understanding of mechanisms underlying this enigmatic disease, animal models have been employed. Primates spontaneously develop endometriosis and therefore primate models most closely resemble the disease in women. Rodent models, however, are more cost effective and readily available2. The model that we describe here involves an autologous transfer of uterine tissue to the intestinal mesentery (Figure 1) and was first developed in the rat3 and later transferred to the mouse4. The goal of the autologous rodent model of surgically-induced endometriosis is to mimic the disease in women. We and others have previously shown that the altered gene expression pattern observed in endometriotic lesions from mice or rats mirrors that observed in women with the disease5,6. One advantage of performing the surgery in the mouse is that the abundance of transgenic mouse strains available can aid researchers in determining the role of specific components important in the establishment and growth of endometriosis. An alternative model in which excised human endometrial fragments are introduced to the peritoneum of immunocompromised mice is also widely used but is limited by the lack of a normal immune system which is thought to be important in endometriosis2,7. Importantly, the mouse model of surgically induced endometriosis is a versatile model that has been used to study how the immune system8, hormones9,10 and environmental factors11,12 affect endometriosis as well as the effects of endometriosis on fertility13 and pain14.

A description of the surgical induction of endometriosis in mice and rats by auto-transplantation of uterine tissue to the arterial cascade of the intestinal mesentery.

子宮組織の自動移植による外科的に誘発される子宮内膜症のマウスモデル

Endometriosis is a chronic, painful disease whose etiology remains unknown. Furthermore, treatment of endometriosis can require laparoscopic removal of ...

Staining Protocols for Human Pancreatic Islets

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and beta mass ranges from 500 to 1500 mg 1-3. With this known heterogeneity, a standard processing and staining procedure was developed so that pancreatic regions were clearly defined and islets characterized using rigorous histopathology and immunolocalization examinations. 
Standardized procedures for processing human pancreas recovered from organ donors are described in part 1 of this series. The pancreas is processed into 3 main regions (head, body, tail) followed by transverse sections. Transverse sections from the pancreas head are further divided, as indicated based on size, and numbered alphabetically to denote subsections. This standardization allows for a complete cross sectional analysis of the head region including the uncinate region which contains islets composed primarily of pancreatic polypeptide cells to the tail region.
The current report comprises part 2 of this series and describes the procedures used for serial sectioning and histopathological characterization of the pancreatic paraffin sections with an emphasis on islet endocrine cells, replication, and T-cell infiltrates. Pathology of pancreatic sections is intended to characterize both exocrine, ductular, and endocrine components. The exocrine compartment is evaluated for the presence of pancreatitis (active or chronic), atrophy, fibrosis, and fat, as well as the duct system, particularly in relationship to the presence of pancreatic intraductal neoplasia4. Islets are evaluated for morphology, size, and density, endocrine cells, inflammation, fibrosis, amyloid, and the presence of replicating or apoptotic cells using H&amp;E and IHC stains. 
The final component described in part 2 is the provision of the stained slides as digitized whole slide images. The digitized slides are organized by case and pancreas region in an online pathology database creating a virtual biobank. Access to this online collection is currently provided to over 200 clinicians and scientists involved in type 1 diabetes research. The online database provides a means for rapid and complete data sharing and for investigators to select blocks for paraffin or frozen serial sections.

This video demonstrates procedures for characterization of human pancreatic islets using hematoxylin and eosin (H&E) and immunohistochemistry (IHC). Pancreatic sections from head, body, and tail regions are stained by both H&amp;E and IHC to determine islet endocrine composition (insulin, glucagon, and pancreatic polypeptide), cell replication (Ki67), and inflammatory infiltrates (H&E, CD3). The uncinate region is localized using IHC for pancreatic polypeptide.

ヒト膵島の染色プロトコル

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and ...

Surgical Procedures for a Rat Model of Partial Orthotopic Liver Transplantation with Hepatic Arterial Reconstruction

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as studies on graft preservation and ischemia-reperfusion injury 1,2, immunological responses 3,4, hemodynamics 5,6, and small-for-size syndrome 7. The rat OLT is among the most difficult animal models in experimental surgery and demands advanced microsurgical skills that take a long time to learn. Consequently, the use of this model has been limited. Since the reliability and reproducibility of results are key components of the experiments in which such complex animal models are used, it is essential for surgeons who are involved in rat OLT to be trained in well-standardized and sophisticated procedures for this model.
While various techniques and modifications of OLT in rats have been reported 8 since the first model was described by Lee et al. 9 in 1973, the elimination of the hepatic arterial reconstruction 10 and the introduction of the cuff anastomosis technique by Kamada et al. 11 were a major advancement in this model, because they simplified the reconstruction procedures to a great degree. In the model by Kamada et al., the hepatic rearterialization was also eliminated. Since rats could survive without hepatic arterial flow after liver transplantation, there was considerable controversy over the value of hepatic arterialization. However, the physiological superiority of the arterialized model has been increasingly acknowledged, especially in terms of preserving the bile duct system 8,12 and the liver integrity 8,13,14.
In this article, we present detailed surgical procedures for a rat model of OLT with hepatic arterial reconstruction using a 50% partial graft after ex vivo liver resection. The reconstruction procedures for each vessel and the bile duct are performed by the following methods: a 7-0 polypropylene continuous suture for the supra- and infrahepatic vena cava; a cuff technique for the portal vein; and a stent technique for the hepatic artery and the bile duct.

Orthotopic liver transplantation in rats is an indispensable experimental model for biomedical research. Here we present our surgical procedures for orthotopic rat liver transplantation with hepatic arterial reconstruction using a 50% partial graft.

肝動脈再建を用いた部分同所性肝移植のラットモデルのための外科的処置

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as ...

Improved Protocol For Laser Microdissection Of Human Pancreatic Islets From Surgical Specimens

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected cells can be used for a variety of investigations, such as transcriptomic or proteomic studies, DNA assessment or chromosomal analysis 2,3. An especially challenging application of LMD is transcriptome analysis, which, due to the lability of RNA 4, can be particularly prominent when cells are dissected from tissues that are rich of RNases, such as the pancreas. A microdissection protocol that enables fast identification and collection of target cells is essential in this setting in order to shorten the tissue handling time and, consequently, to ensure RNA preservation. 
Here we describe a protocol for acquiring human pancreatic beta cells from surgical specimens to be used for transcriptomic studies 5. Small pieces of pancreas of about 0.5-1 cm3 were cut from the healthy appearing margins of resected pancreas specimens, embedded in Tissue-Tek O.C.T. Compound, immediately frozen in chilled 2-Methylbutane, and stored at -80 &deg;C until sectioning. Forty serial sections of 10 &mu;m thickness were cut on a cryostat under a -20 &deg;C setting, transferred individually to glass slides, dried inside the cryostat for 1-2 min, and stored at -80 &deg;C. 
Immediately before the laser microdissection procedure, sections were fixed in ice cold, freshly prepared 70% ethanol for 30 sec, washed by 5-6 dips in ice cold DEPC-treated water, and dehydrated by two one-minute incubations in ice cold 100% ethanol followed by xylene (which is used for tissue dehydration) for 4 min; tissue sections were then air-dried afterwards for 3-5 min. Importantly, all steps, except the incubation in xylene, were performed using ice-cold reagents - a modification over a previously described protocol 6. utilization of ice cold reagents resulted in a pronounced increase of the intrinsic autofluorescence of beta cells, and facilitated their recognition. For microdissection, four sections were dehydrated each time: two were placed into a foil-wrapped 50 ml tube, to protect the tissue from moisture and bleaching; the remaining two were immediately microdissected. This procedure was performed using a PALM MicroBeam instrument (Zeiss) employing the Auto Laser Pressure Catapulting (AutoLPC) mode. The completion of beta cell/islet dissection from four cryosections required no longer than 40-60 min. Cells were collected into one AdhesiveCap and lysed with 10 &mu;l lysis buffer. Each single RNA specimen for transcriptomic analysis was obtained by combining 10 cell microdissected samples, followed by RNA extraction using the Pico Pure RNA Isolation Kit (Arcturus). This protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their rapid and accurate recognition and collection. Further improvement of this procedure could enable the dissection of phenotypically different beta cells, with possible implications for better understanding the changes associated with type 2 diabetes.

Laser microdissection is a technique that allows the recovery of selected cells from minute amounts of parenchyma. Here we describe a protocol for acquiring human pancreatic islets from surgical specimens to be used for transcriptomic studies. Our protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their collection.

手術標本からヒト膵島のレーザーマイクロダイセクションのために改良されたプロトコル

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected ...

Transplantation of Pulmonary Valve Using a Mouse Model of Heterotopic Heart Transplantation

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed results. However, the cellular and molecular mechanisms of the neotissue development, valve thickening, and stenosis development are not researched extensively. To answer the above questions, we developed a murine heterotopic heart valve transplantation model. A heart valve was harvested from a valve donor mouse and transplanted to a heart donor mouse. The heart with a new valve was transplanted heterotopically to a recipient mouse. The transplanted heart showed its own heartbeat, independent of the recipient&#8217;s heartbeat. The blood flow was quantified using a high frequency ultrasound system with a pulsed wave Doppler. The flow through the implanted pulmonary valve showed forward flow with minimal regurgitation and the peak flow was close to 100 mm/sec. This murine model of heart valve transplantation is highly versatile, so it can be modified and adapted to provide different hemodynamic environments and/or can be used with various transgenic mice to study neotissue development in a tissue engineered heart valve.

In order to understand the cellular and molecular mechanisms underlying neotissue formation and stenosis development in tissue engineered heart valves, a murine model of heterotopic heart valve transplantation was developed. A pulmonary heart valve was transplanted to recipient using the heterotopic heart transplantation technique.

異心臓移植のマウスモデルを用いた肺動脈弁の移植

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed ...

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, the role of beta-cell dysfunction in type 2 diabetes has been clearly established 2. Research progress has required methods for the isolation of pancreatic islets. The protocol of the islet isolation presented here shares many common steps with protocols from other groups, with some modifications to improve the yield and quality of isolated islets from both the wild type and diabetic Leprdb (db/db) mice. A live-cell 2-photon imaging method is then presented that can be used to investigate the control of insulin secretion within islets.

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

We present here a protocol for the isolation of islets from the mouse model of type 2 diabetes, Leprdb and details of a live-cell assay for measurement of insulin secretion from intact islets that utilizes 2 photon microscopy.

LEPR<sup&gt; DB</sup&gt; 2型糖尿病のマウスモデル：無傷の膵島の膵島単離およびライブセル2光子イメージング

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, ...

Orthotopic Hind Limb Transplantation in the Mouse

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and translational research in the field of transplantation medicine. A vast array of reagents is available exclusively in this setting, including mono- and polyclonal antibodies for both diagnostic and interventional applications. In addition, a vast number of genotyped, inbred, transgenic, and knock out strains allow detailed investigation of the individual contributions of humoral and cellular components to the complex interplay of an immune response and make the mouse the gold standard for immunological research. 
Vascularized Composite Allotransplantation (VCA) delineates a novel field of transplantation using allografts to replace "like with like" in patients suffering traumatic or congenital tissue loss. This surgical methodological protocol shows the use of a non-suture cuff technique for super-microvascular anastomosis in an orthotopic mouse hind limb transplantation model. The model specifically allows for comparison between established paradigms in solid organ transplantation with a novel form of transplants consisting of various different tissue components. Uniquely, this model allows for the transplantation of a viable vascularized bone marrow compartment and niche that have the potential to exert a beneficial effect on the balance of immune acceptance and rejection. This technique provides a tool to investigate alloantigen recognition and allograft rejection and acceptance, as well as enables the pursuit of functional nerve regeneration studies to further advance this novel field of transplantation.

This novel model for orthotopic hind limb transplantation in the mouse, applying a non-suture cuff technique for super-microvascular anastomosis, provides a powerful tool for in&#160;vivo mechanistic immunological research related to vascularized composite allotransplantation (VCA).

マウスで同所後肢移植

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and ...

Using the Sleeve Technique in a Mouse Model of Aortic Transplantation - An Instructional Video

Orthotopic aortic transplantation using the sleeve technique reduces injury to the aorta with failure rate of only 10-20%. The time to anastomose the aorta in mice using the sleeve method was short and easy averaging 20 min, permitting studies of iso/allo grafts. The following article describes the aortic transplantation procedure used in our laboratory. The mice were anesthetized with a mixture of 1.5% volume isoflurane and 100% oxygen through a face mask. At this point, the segment of the aorta between the renal arteries and its bifurcation was separated from the vena cava, freely prepared and clampedat the proximal and distal segments with a single silk suture. Prior to the removal of the aorta, a saline solution containing heparin was injected into the inferior vena cava. Then the aorta was cut between the clamps and a saline heparin solution was used to flush the lumen. The sleeve technique with monofilament sutures was used in order to transplant the abdominal aorta in the orthotopic position.

We present an orthotopic aortic transplantation model using the sleeve technique in mice. It is a very rapid anastomosis method, which can be employed in studies of vascular disease.

大動脈弁移植 - 教則ビデオのマウスモデルでスリーブを用いた

Orthotopic aortic transplantation using the sleeve technique reduces injury to the aorta with failure rate of only 10-20%. The time to anastomose the ...

Mouse Model for Pancreas Transplantation Using a Modified Cuff Technique

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the availability of the widest range of molecular probes and reagents to perform in vivo as well as in vitro studies. Based on our experience with various murine transplantation models, we developed a heterotopic pancreas transplantation model in mice with the intent to analyze mechanisms underlying severe ischemia reperfusion injury-associated early graft damage. In contrast to previously described techniques using suture techniques, herein we describe a new procedure using a non-suture cuff technique. 
In recent years, we have performed more than 300 pancreas transplantations in mice with an overall success rate of &gt;90%, a success rate never described before in mouse pancreas transplantation. The backbone of this non-suture cuff technique for graft revascularization consists of two major steps: (I) pulling the recipient vessel over a polyethylene/polyamide cuff and fixing it with a circumferential ligature, and (II) placing the donor vessel over the everted recipient vessel and fixing it with a second circumferential ligature. The resultant continuity of the endothelial layer results in less thrombogenic lesions with high patency rates and, finally, high success rates.
In this model, arterial anastomosis is achieved by pulling the abdominal aorta of the donor graft over the everted common carotid artery of the recipient animal. Venous drainage of the graft is achieved by pulling the portal vein of the graft over the everted external jugular vein of the recipient. This manuscript provides details and crucial steps of the organ recovery and organ implantation procedures, which will allow researchers with microsurgical skills to perform the transplantation successfully in their laboratories.

Among abdominal solid organ transplantation, pancreatic grafts are prone to develop severe ischemia reperfusion injury-associated graft damage, leading eventually to early graft loss. This protocol describes a model of murine pancreas transplantation using a non-suture cuff technique, ideally suited for analyzing these early, deleterious damages.

マウス モデルにおける膵島の門脈内移植

マウスモデルにおける膵島の門脈内移植