この研究は、National Health and Medical Research Council APP1194139/APP1126276(JC)、National Stem Cell Foundation of Australia、New South Wales Government Office of Health and Medical Research(JC)からの助成金によって資金提供されました。DSは、Royal Australasian College of Physicians、Institute of Clinical Pathology and Medical Research、およびAustralian Government Research Training Programの支援を受けました。TDは、Institute of Clinical Pathology and Medical Research、Penfolds Family Scholarship、National Health and Medical Research Council(APP2002783)、National Heart Foundation of Australia(104615)の支援を受けました。

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Dyce, Sack, and Wensing's Textbook of Veterinary Anatomy. , (2018).</li><li>Percie du Sert, 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=Reporting+animal+research:+Explanation+and+elaboration+for+the+ARRIVE+guidelines+2.0.">Reporting animal research: Explanation and elaboration for the ARRIVE guidelines 2.0.</a> PLOS Biology. 18 (7), 3000411 (2020).</li><li>Sch&#252;ttler, 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=A+practical+guide+to+setting+up+pig+models+for+cardiovascular+catheterization,+electrophysiological+assessment+and+heart+disease+research.">A practical guide to setting up pig models for cardiovascular catheterization, electrophysiological assessment and heart disease research.</a> Lab Animals. 51 (2), 46-67 (2022).</li></ol>

著者は何も開示していません。

経心外膜心筋内注射 この手順には、心臓を直接視覚化できるという利点があり、全身投与方法よりも治療薬の局所保持率が高いことが実証されています9,10,14。しかし、胸腔切開術は侵襲的であり、かなりの技術的スキルを必要とし、議論されている他の方法よりも罹患率と死亡率のリスクが高くなります10,15。手順の重要で不安定な段階に関する知識は、この増加したリスクの調停に役立ちます。
不整脈とそれに伴う血行動態の障害のリスクが高いため、心臓の頂点を露出させるために心臓を操作する際には細心の注意を払う必要があります。継続的な侵襲的血圧モニタリングと心電図検査により、低血圧や不安定な不整脈を迅速に特定し、迅速な介入と矯正を容易にします。一過性低血圧は、通常、メタラミノールボーラスで治療できます。.持続的な低血圧は、吸入麻酔薬を減らし(麻酔の深さを注意深く監視)、昇圧剤の注入を開始すると同時に、血行動態の変化の原因を特定することで一時的になる場合があります。.心室頻拍や心室細動などの不安定な不整脈は、静脈内抗不整脈薬の有無にかかわらず、電気除細動によって治療できます。.
動物の生存にとって同様に重要なことは、胸腔を閉じる前に胸膜腔から遊離ガスをうまく除去することです。これを怠ると、気胸を発症し、回復時に人工呼吸器から切り離されると、動物は呼吸障害や死亡の大きなリスクにさらされます。気道陽圧は、気泡が観察されなくなるまで少なくとも30秒間維持する必要があります。.シリコンチューブは、バブリングが止まると速やかに取り外され、胸部は急速に閉じられます。また、胸腔瘻造設チューブを閉鎖時に外科的に留置することも可能であり、次の24〜72時間にわたって手動で空気と炎症性液体を除去することができます。しかし、これは、特に動物が一緒に飼育されている場合、清潔で無傷に保つことは困難です。チューブの損傷や汚染は、膿胸、気胸、または敗血症につながる可能性があります。私たちの経験では、胸部閉鎖前に遊離ガスが適切に除去されていれば、一時的な胸腔ドレーンを挿入する必要はありません。
経皮的経心内膜心筋内注射 この治療的投与方法は、外科的アプローチと比較して侵襲性が低いため、リスクの低い局所組織送達を可能にするという利点があります10,14。この手法は、すでに大動物の研究で使用されており、直接可視化がない場合のガイドとして、透視法と電気機械マッピングの両方を使用しています10,16,17。
心臓が直視されていないことを考えると、処置者は注射部位を選択する際に直交透視図を使用するのが賢明です。さらに、治療薬の投与前および送達前の希釈ヨウ素造影剤の注射は、心筋接触を確認する上で非常に貴重である。適切な接触は、組織採取前の注射成功の唯一のマーカーの1つである可能性のある特徴的な「心筋赤面」を観察することによって確認できます。チャンバー穿孔のリスクがあるため、選択した注射部位の心筋壁の厚さも9mmより大きくすることをお勧めします14,16。
頸静脈浸透圧ミニポンプ 浸透圧ミニポンプは、小動物実験で一般的に使用される一般的な装置です。治療薬を一定期間一定の割合で投与するという独自の利点から、大型動物モデル7,18,19でこのデバイスを使用することへの関心が高まっています。この方法の考えられる制限は、ポンプを交換または取り外さずに薬物の注入速度を変更または停止できないことです。.この方法で治療を試す前に、これを考慮する必要があります。.
この研究は、この方法が豚で高い成功率で、罹患率と死亡率が低いことを実証しました。リンパ節、胸腺、頸動脈など、多くの重要な構造が手術部位に隣接していることに注意する必要があります。これらの構造のいずれかへの不注意な損傷を防ぐために、方法の遵守、および解剖学的テキスト20の相談を強くお勧めします。この方法の最も懸念される合併症は、頸静脈または周囲の構造への不注意な損傷による出血性ショックです。したがって、頸静脈を取り巻く軟部組織を慎重に除去することが重要です。このステップを適切に行わないと、ミニポンプチューブの配置や不注意による出血の制御が困難になる可能性があります。
この記事では、心役活性治療薬を送達するための3つの方法について説明しました。各手法の成功が報告されているにもかかわらず、考慮すべき固有の制限があります。侵襲的処置(経心外膜注射)により、治療送達の精度が向上します。しかし、それらは潜在的に致命的な合併症のリスクを高めます。さらに、侵襲的な分娩では、合併症のリスクを最小限に抑えるための技術的スキルがより必要になります。同様に、透視ガイド下経心内膜注射には、カテーテル挿入とハードウェアの操作にある程度の技術的スキルが必要です。この方法が不適切に行われると、注射の失敗や致命的な合併症を引き起こす可能性があります。
記載された直接注射法は、標的組織への治療薬の1回限りの投与を可能にする。頸静脈浸透圧ミニポンプは、7日間にわたる治療薬の全身投与を可能にします。比較的、この方法はより単純でリスクが少ないですが、心筋への道を見つける全身治療に依存しています。さらに、ポンプが設置されると、動物を再麻酔してポンプを取り外さずに投与を中止したり、線量率を変更したりすることは不可能です。
この記事に記載されているすべての方法は、心筋梗塞の日または2週間後に動物で行われました。したがって、この研究は、健康な動物または代替の心臓病理を受けた動物における上記の方法の成功を報告することはできません。最後に、意図する薬剤の薬理学とバイオテクノロジーは、選択した送達経路の有効性に本質的に関連しているため、慎重に検討する必要があります。これに関する詳細な議論は、この原稿の範囲を超えています。
前臨床法の包括的な描写は、動物福祉とより広い科学コミュニティに利益をもたらします。その結果、手順と結果の再現性が向上し、動物の健康上の合併症が減少し、有意な結果を出すために必要な動物の数が減り、実験結果の信頼性が高まります21,22。この記事では、ブタモデルにおける心筋梗塞の治療のために、新規治療薬の3つの投与方法について説明します。使用される技術を詳細に説明し、それぞれの利点とリスクを明確にすることで、研究者は研究目標に合った一貫性のある信頼性の高い前臨床モデルを快適に作成できるようになることが期待されます。

冠動脈疾患(CAD)および関連するST上昇型心筋梗塞(STEMI)は、世界中で主要な死因です。過去20年間で、経皮的冠動脈インターベンション、線溶療法、および再灌流がタイムリーに達成されるようにするための治療アルゴリズムの標準化の出現により、STEMIを呈する患者の院内死亡率の低下に大きな進歩が見られました1,2,3.それにもかかわらず、STEMIに関連する罹患率は依然として大きな負担であり、したがって、新しい心臓保護および再生療法の開発に大きなニーズが生じています2,3。治療法開発に不可欠な要素は、新規治療法をどのように投与するかを決定することです4。各方法の安全性、有効性、および実現可能性は、治療自体の特性と一致する必要があります。
生理学的に関連性のある大型動物モデルは、さまざまな治療送達戦略のこれらの属性を評価する上で重要です5。心血管生理学、冠動脈血管の解剖学、および心臓の重量と体重の比率がヒトと類似しているため、ブタは心筋梗塞の新しい治療法の前臨床評価において好ましい種の1つです6。我々は以前、ブタSTEMIモデルを用いて組換えタンパク質療法の修復能力を実証しており7、このモデルを用いた新規の薬理学的、細胞的、遺伝子的治療の研究を続けている。ここでは、梗塞作成後の豚モデルで使用される治療投与の3つの技術、すなわち開胸術および経心上膜注射、経皮的経心内膜注射、および頸静脈浸透圧ミニポンプ移植について説明する。最初の2つの方法は、局所組織送達を可能にし、必要な投与量、オフターゲット効果、および肝臓の初回通過代謝を低減します8,9,10。浸透圧ミニポンプは、半減期の短い薬剤の連続送達を可能にし、大型動物モデルでは導入が困難な輸液ポンプや特許の静脈内カニューレへの依存を排除します。
これらの技術を説明することにより、この記事は、トランスレーショナルサイエンティストが大型動物モデルで心筋梗塞後の新しい心臓保護剤または再生剤を研究するのに役立つことが期待されます。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Central line placement</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2-0 sutures</td>
			<td>Ethicon</td>
			<td>JJ9220</td>
			<td></td>
		</tr>
		<tr>
			<td>Arrow&#39; Paediatric Two-Lumen Central Venous Catheterisation Set with Blue FlexTip Catheter (contains 18G cook needle and 0.035&#34; J-tip wire)</td>
			<td>Teleflex</td>
			<td>CS-14502</td>
			<td>Central Line</td>
		</tr>
		<tr>
			<td>Green Fluorsence Protein (GFP)</td>
			<td>Abcam</td>
			<td>ab13970</td>
			<td>1:100 dilution ratio</td>
		</tr>
		<tr>
			<td>Histology antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ku80</td>
			<td>Cell Signalling Technology</td>
			<td>C48E7</td>
			<td>1:500 dilution ratio</td>
		</tr>
		<tr>
			<td>No. 11 scalpel</td>
			<td>Swann-Morton</td>
			<td>203</td>
			<td></td>
		</tr>
		<tr>
			<td>Sparq&#39; Ultrasound System</td>
			<td>Philips</td>
			<td>MP11742 Medpick</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile ultrasound probe cover</td>
			<td>Atris</td>
			<td>28041947</td>
			<td></td>
		</tr>
		<tr>
			<td>Swine Jacket with Pocket, size &#39;Medium&#39;</td>
			<td>Lomir Biomedical</td>
			<td>SS J2YJJET</td>
			<td></td>
		</tr>
		<tr>
			<td>Jugular vein osmotic minipump implantation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Adson Brown Tissue Forceps</td>
			<td>Icon Medical Supplies</td>
			<td>KLINI316012</td>
			<td></td>
		</tr>
		<tr>
			<td>Bellucci Self-Retaining Retractor</td>
			<td>surgicalinstruments.net.au</td>
			<td>group-24.26.02</td>
			<td>Self retaining tissue retractor</td>
		</tr>
		<tr>
			<td>Electrosurgical Pencils with &#39;Edge&#39; Coated Electrodes</td>
			<td>Covidien</td>
			<td>E2450H</td>
			<td>Cautery Pencil</td>
		</tr>
		<tr>
			<td>Metzenbaum Scissors</td>
			<td>Icon Medical Supplies</td>
			<td>ARMO3250</td>
			<td></td>
		</tr>
		<tr>
			<td>No. 22 scalpel blade</td>
			<td>Swann-Morton</td>
			<td>208</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon Suture&#160; (2-0, 3-0)</td>
			<td>Ethicon</td>
			<td>D9635, 663G</td>
			<td></td>
		</tr>
		<tr>
			<td>Osmotic Infusion Minipump</td>
			<td>Alzet</td>
			<td>2ML1, 2ML2, 2ML4</td>
			<td></td>
		</tr>
		<tr>
			<td>Vascular Silicone Ties</td>
			<td>Vecmedical</td>
			<td>95001</td>
			<td></td>
		</tr>
		<tr>
			<td>Vicryl suture (5-0)</td>
			<td>Ethicon</td>
			<td>W9982</td>
			<td></td>
		</tr>
		<tr>
			<td>Percutaneous transedocardial injection</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Artis Zee&#39; C-Arm Fluoroscopy&#160;</td>
			<td>Siemens&#160;</td>
			<td>IR-19-1994</td>
			<td></td>
		</tr>
		<tr>
			<td>CARTO&#39; 3 System&#160;&#160;</td>
			<td>Biosense Webster</td>
			<td></td>
			<td>Electrophysiological Mapping Software &#38; System</td>
		</tr>
		<tr>
			<td>Cook Access Needle</td>
			<td>Cook Medical</td>
			<td>G07174</td>
			<td>Cannulation needle</td>
		</tr>
		<tr>
			<td>Fast-Cath&#39; Introducer (6 French, 8 French)</td>
			<td>Abbott</td>
			<td>406204, 406142</td>
			<td>Vascular sheath with introducer and guidewire</td>
		</tr>
		<tr>
			<td>Myostar&#39; Injection Catheter</td>
			<td>Biosense Webster</td>
			<td>121117S, 121119S, 1211120S</td>
			<td>Intramyocardial injection catheter</td>
		</tr>
		<tr>
			<td>No.11 scalpel</td>
			<td>Swann-Morton</td>
			<td>203</td>
			<td></td>
		</tr>
		<tr>
			<td>Omnipaque&#39; Iohexol Contrast</td>
			<td>GE Healthcare</td>
			<td>AUST R 39861&#160;</td>
			<td>Iodinated contrast agent</td>
		</tr>
		<tr>
			<td>Sparq&#39; Ultrasound System</td>
			<td>Philips</td>
			<td>MP11742 Medpick</td>
			<td></td>
		</tr>
		<tr>
			<td>Sedation &#38; general anaesthesia</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Compound Sodium Lactate Hartmann&#39;s Solution</td>
			<td>Free flex</td>
			<td>894451</td>
			<td></td>
		</tr>
		<tr>
			<td>Fentanyl 50 mcg/mL</td>
			<td>Pfizer</td>
			<td>AUST R 107027.</td>
			<td>Intravenous anaesthesia and analgesia</td>
		</tr>
		<tr>
			<td>Forthane&#39; Isoflurane</td>
			<td>Abbott</td>
			<td>AUST R 29656&#160;</td>
			<td>Inhalant anaesthetic</td>
		</tr>
		<tr>
			<td>GE Aestiva 5 Anaesthesia Machine&#160;</td>
			<td>Datex Ohmeda</td>
			<td>17002-9, 17002A9 Avante Health Solutions</td>
			<td>Anaesthetic Machine&#160;</td>
		</tr>
		<tr>
			<td>Hypnovel&#39; Midazolam 5 mg/mL</td>
			<td>Roche</td>
			<td>AUST R 13726</td>
			<td>Sedative</td>
		</tr>
		<tr>
			<td>Intravenous cannula</td>
			<td>BD Angiocath</td>
			<td>381137</td>
			<td>20 gauge cannula</td>
		</tr>
		<tr>
			<td>Ketamil&#39; Ketamine 10 mg/mL</td>
			<td>Ilium</td>
			<td>APVMA number: 51188c&#160;</td>
			<td>Sedative</td>
		</tr>
		<tr>
			<td>Laryngoscope</td>
			<td>Miller</td>
			<td>VDI-6205</td>
			<td></td>
		</tr>
		<tr>
			<td>Medetomidine 1 mg/mL</td>
			<td>Ilium</td>
			<td>APVMA number 64251; ACVM number A10488&#160;</td>
			<td>Sedative</td>
		</tr>
		<tr>
			<td>Metaraminol 10 mg/mL</td>
			<td>Phebra</td>
			<td>AUST R 284784</td>
			<td>Short-acting vasopressor</td>
		</tr>
		<tr>
			<td>Methadone 10 mg/mL</td>
			<td>Ilium</td>
			<td>APVMA number: 63712&#160;</td>
			<td>Sedative, Restricted drug</td>
		</tr>
		<tr>
			<td>Onsetron&#39; Ondansetron 2 mg/mL</td>
			<td>Accord Healthcare</td>
			<td>AUST R 205593&#160;</td>
			<td>Anti-emetic</td>
		</tr>
		<tr>
			<td>Propofol-Lipuro&#39; Propofol 10 mg/mL</td>
			<td>Braun</td>
			<td>AUST R 142906&#160;</td>
			<td>Intravenous anaesthetic</td>
		</tr>
		<tr>
			<td>Pulse Oximeter&#160;</td>
			<td>Meditech</td>
			<td>GVPMT-M3S</td>
			<td>Portable pulse oximeter</td>
		</tr>
		<tr>
			<td>Shiley&#39; Cuffed Basic Endotracheal Tube (Size 5.5 &#38; 6.0)</td>
			<td>Medtronic</td>
			<td>86108-, 86109-</td>
			<td></td>
		</tr>
		<tr>
			<td>Shiley&#39; Intubating Stylet, 10 Fr</td>
			<td>Medtronic</td>
			<td>85864</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride 0.9%</td>
			<td>Free flex</td>
			<td>FAH1322</td>
			<td></td>
		</tr>
		<tr>
			<td>Thoracotomy and epicardial Cell Injection</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>27 G Insulin needle</td>
			<td>Terumo</td>
			<td>51907</td>
			<td></td>
		</tr>
		<tr>
			<td>Adson Brown Tissue Forceps</td>
			<td>Icon Medical Supplies</td>
			<td>KLINI316012</td>
			<td></td>
		</tr>
		<tr>
			<td>CARTO&#39; 3 System&#160;&#160;</td>
			<td>Biosense Webster</td>
			<td></td>
			<td>Electrophysiological Mapping Software &#38; System</td>
		</tr>
		<tr>
			<td>Cefazolin 1 g Vial</td>
			<td>AFT Pharmaceuticals</td>
			<td>9421900137367 CH2</td>
			<td>Antibiotic Prophylaxis&#160;</td>
		</tr>
		<tr>
			<td>Chest drainage tube</td>
			<td>SurgiVet</td>
			<td>SKU-336</td>
			<td></td>
		</tr>
		<tr>
			<td>Cook Access Needle</td>
			<td>Cook Medical</td>
			<td>G07174</td>
			<td>Cannulation needle</td>
		</tr>
		<tr>
			<td>Cooley Sternotomy Retractor Paediatric</td>
			<td>Millennium Surgical</td>
			<td>9-61287</td>
			<td></td>
		</tr>
		<tr>
			<td>Durogesic&#39; 100 mcg/h Fentanyl Patch</td>
			<td>Janssen</td>
			<td>AUST R 112371&#160;</td>
			<td>Postoperative analgesia</td>
		</tr>
		<tr>
			<td>Electrosurgical Pencils with &#39;Edge&#39; Coated Electrodes</td>
			<td>Covidien</td>
			<td>E2450H</td>
			<td>Cautery Pencil</td>
		</tr>
		<tr>
			<td>Electrosurgical Pencils with &#39;Edge&#39; Coated Electrodes</td>
			<td>Covidien</td>
			<td>E2450H</td>
			<td>Cautery Pencil</td>
		</tr>
		<tr>
			<td>Fast-Cath&#39; Introducer (6 French, 8 French)</td>
			<td>Abbott</td>
			<td>406204, 406142</td>
			<td>Vascular sheath with introducer and guidewire</td>
		</tr>
		<tr>
			<td>Lignocaine 20 mg/mL</td>
			<td>Pfizer</td>
			<td>AUST R 49296, AUST R 49297, AUST R 49293 and AUST R 49295.</td>
			<td>Local anaesthesia, anti-arrhythmic</td>
		</tr>
		<tr>
			<td>Marcaine&#39; Bupivacaine 0.5%</td>
			<td>Pfizer</td>
			<td>AUST R 48328&#160;</td>
			<td>Local anaesthesia.&#160;</td>
		</tr>
		<tr>
			<td>Metzenbaum Scissors</td>
			<td>Icon Medical Supplies</td>
			<td>ARMO3250</td>
			<td></td>
		</tr>
		<tr>
			<td>No. 22 scalpel</td>
			<td>Swann-Morton</td>
			<td>208</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon Suture&#160; (2-0, 3-0)</td>
			<td>Ethicon</td>
			<td>D9635, JJ76264</td>
			<td></td>
		</tr>
		<tr>
			<td>Size 1 PDS suture</td>
			<td>Ethicon</td>
			<td>JJ75414</td>
			<td></td>
		</tr>
		<tr>
			<td>Sparq&#39; Ultrasound System</td>
			<td>Philips</td>
			<td>MP11742 Medpick</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile gauze</td>
			<td>Kerlix</td>
			<td>KE5072</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile laparotomy sponges</td>
			<td>Propax</td>
			<td>2907950</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermocool Smartouch&#39; Catheter</td>
			<td>Biosense Webster</td>
			<td>D133601, D133602, D133603</td>
			<td>Epicardial Mapping Catheter</td>
		</tr>
	</tbody>
</table>

delivery of cardioactive therapeutics in a porcine myocardial infarction model

心筋梗塞は、世界中の主要な死因の1つであり、新しい心臓保護または再生戦略が緊急に必要とされています。医薬品開発に不可欠な要素は、新規治療薬をどのように投与するかを決定することです。生理学的に関連性のある大型動物モデルは、さまざまな治療戦略の実現可能性と有効性を評価する上で非常に重要です。心血管生理学、冠動脈血管の解剖学、および心臓の重量と体重の比率がヒトと類似しているため、豚は心筋梗塞の新しい治療法の前臨床評価において好ましい種の1つです。本プロトコルは、ブタモデルにおいて心活性治療薬を投与する3つの方法を記述する。経皮的に誘発された心筋梗塞の後、雌の在来種豚は、(1)開胸術と経心外膜注射、(2)カテーテルベースの経心内膜注射、または(3)頸静脈浸透圧ミニポンプによる静脈内注入のいずれかによって新規薬剤 による 治療を受けました。各技術に採用されている手順は再現性があり、信頼性の高い心活性薬物送達を実現します。これらのモデルは、個々の研究デザインに合わせて簡単に適応させることができ、これらの提供技術のそれぞれを使用して、さまざまな可能な介入を調査することができます。したがって、これらの方法は、心筋梗塞後の心臓修復における新しい生物学的アプローチを追求するトランスレーショナルサイエンティストにとって有用なツールです。

開胸術と心外膜細胞注射 開胸術と心外膜注射を受けた29匹の動物のうち、26匹が生存した。組織学的分析により、生存しているすべての動物において、この方法によって送達されたヒト細胞の生着が確認されました(図1E)。1匹の動物は細胞注射中に致命的な不整脈を経験し、蘇生できませんでした。もう1人は、閉鎖中に脈拍のない電気的活動を経験し、気道に陽圧が長時間加えられ、回復することができませんでした。3匹目の動物は、抜管時に嘔吐し、呼吸停止に陥った。この動物は蘇生できませんでした。
2匹の動物が大きな合併症を経験しましたが、回復することができました。1匹の動物は心筋内注射中に心室細動を発症し、体内の除細動パドルと心臓マッサージで蘇生することができました。2匹目の動物は抜管時に嘔吐し、短時間の呼吸停止がありましたが、迅速に再挿管することができ、順調に回復しました。これらの事象はすべて初期の実験中に発生し、プロトコルに関するチームの経験が増加するにつれて有害事象は減少しました(表1)。
頸静脈浸透圧ミニポンプ移植術 頸動脈浸透圧ミニポンプの移植に関連する死亡率または重篤な合併症の報告はなかった。7匹の動物のほとんどが、最初の24時間以内に手術部位に軽度の腫脹を経験し、介入なしで治癒した。ポンプ移植後3日目に血清に対してELISAを実施したところ、ポンプの有効性が実証され、対照群と比較して 血小板由来成長因子-ABヒト(PDGF-AB)の血中濃度が有意に向上しました7(図2E)。
経皮的経心内膜注射 合計22匹の動物が心内膜注射を受けました。これらの注射のうち、17 回は「成功」と見なされ、死後検査で標的組織に観察された蛍光またはインク染色によって決定されました(図 3B)。この処置に関連する死亡例はなかった。1匹の動物は、右心室穿孔から少量の心嚢液貯留を発症しました。.これは自己限定的であり、心血管系の障害には至らなかった。この同じ動物は確かに死んだ。しかし、これは心筋内注射後の無関係な追加処置によるものでした。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64177/64177fig01.jpg"> 図1:経心外膜心筋細胞注射は、心臓を直接可視化し、心筋に送達される生細胞の割合を高くします。 (A)心臓の頂点は、心臓の基部の下に導かれた湿らせたガーゼスリングを通して露出します。(B)心外膜マッピングカテーテルは、瘢痕と境界ゾーンを描写し、注射部位に注釈を付けます。(C)31Gの針を使用して、細胞を心筋に経心的に注入します。(D)注射部位のアノテーションを含む心外膜電圧マップ。紫色:正常な電圧、健康な心筋。赤:異常な電圧、病気の心筋;灰色の点:注射部位。犠牲の後、心臓は収集され、下流の組織学的評価のためにホルマリン固定されます。(E)では、ヒト抗核抗体Ku80と抗GFP抗体を免疫染色することにより、生着したヒト細胞を検出します。スケールバー = 200 μm。 <a href="https://www.jove.com/files/ftp_upload/64177/64177fig01large.jpg" target="_blank">この図の拡大版をご覧になるには、ここをクリックしてください。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64177/64177fig02.jpg"> 図2:頸静脈ミニポンプ挿入は、7日間にわたってPDGFを安全かつ確実に送達する方法を提供します 。 (A)右頸静脈が露出し、軟部組織が血管から取り除かれます。(B)14Gの針を使用して穿刺し、ミニポンプチューブに通す間、血管タイが血管を閉塞します。(C)ミニポンプチューブを静脈内に進め、ミニポンプ本体を隣接する軟部組織に固定する。(D)移植前のミニポンプ本体とチューブ。(E)ミニポンプとPDGF-AB を介して 送達された組換えタンパク質の血清濃度は、移植後3日目に各動物からELISAを使用して測定されました。PDGF-ABを投与された動物は、対照動物よりもPDGF-ABの血中濃度が有意に高いことが示され、浸透圧ミニポンプ投与法の有効性が確認されました。**はグループ間の統計的に有意な差を示します(p = 0.005、Mann-Whitney U検定) <a href="https://www.jove.com/files/ftp_upload/64177/64177fig02large.jpg" target="_blank">この図の拡大版をご覧になるには、ここをクリックしてください。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64177/64177fig03.jpg"> 図3:経心内膜心筋内注射は、低侵襲治療薬の投与方法を可能にします。 (A)造影剤(黄色矢印)を心筋に注入する注射カテーテル(白矢印)を示す右前方斜め透視画像。造影剤の注射は、心筋内の針の配置を確認できるように、治療注射の前後に行われます。(B)注入したベクターは緑色蛍光タンパク質(GFP)を発現し、注入された物質が組織採取時に蛍光を発し、注入の成功が確認されました。 <a href="https://www.jove.com/files/ftp_upload/64177/64177fig03large.jpg" target="_blank">この図の拡大版をご覧になるには、ここをクリックしてください。</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td></td>
			<td>経心外膜注射(n = 29)。</td>
			<td>経心内膜注射(n = 22)</td>
			<td>浸透圧ミニポンプ(n = 7)</td>
		</tr><tr><td>死亡率</td>
			<td>3</td>
			<td>0</td>
			<td>0</td>
		</tr><tr><td>術後の嘔吐と呼吸停止</td>
			<td>1</td>
			<td>0</td>
			<td>0</td>
		</tr><tr><td>注射中の心室細動</td>
			<td>1</td>
			<td>0</td>
			<td>0</td>
		</tr><tr><td>胸部閉鎖時のパルスレス電気的活動</td>
			<td>1</td>
			<td>0</td>
			<td>0</td>
		</tr><tr><td>罹患 率</td>
			<td>0</td>
			<td>1</td>
			<td>0</td>
		</tr><tr><td>気胸</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
		</tr><tr><td>胸水</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
		</tr><tr><td>心室穿孔</td>
			<td>0</td>
			<td>1</td>
			<td>0</td>
		</tr><tr><td>出血</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
		</tr></tbody></table>表1:合併症のリスト。

Watch this Scientific Journal Video about Delivery of Cardioactive Therapeutics in a Porcine Myocardial Infarction Model at JoVE.com

Delivery of Cardioactive Therapeutics in a Porcine Myocardial Infarction Model

本プロトコルは、ブタモデルにおいて心活性治療薬を投与する3つの方法を記述する。雌の在来種豚は、(1)開胸術と経心的心内膜注射、(2)カテーテルベースの経心内膜注射、または(3)頸静脈浸透圧ミニポンプ による 静脈内注入のいずれかで治療を受けました。

ブタ心筋梗塞モデルにおける心役活性治療薬の送達

Myocardial infarction is one of the leading causes of death and disability worldwide, and there is an urgent need for novel cardioprotective or ...

delivery-cardioactive-therapeutics-porcine-myocardial-infarction

Research

JoVE Journal

Medicine

1.1K ビュー.  The University of Sydney. 本プロトコルは、ブタモデルにおいて心活性治療薬を投与する3つの方法を記述する。雌の在来種豚は、(1)開胸術と経心的心内膜注射、(2)カテーテルベースの経心内膜注射、または(3)頸静脈浸透圧ミニポンプ による 静脈内注入のいずれかで治療を受けました。

ビデオ: Delivery of Cardioactive Therapeutics in a Porcine Myocardial Infarction Model

Acute Myocardial Infarction in Rats

With heart failure leading the cause of death in the USA (Hunt), biomedical research is fundamental to advance medical treatments for cardiovascular diseases. Animal models that mimic human cardiac disease, such as myocardial infarction (MI) and ischemia-reperfusion (IR) that induces heart failure as well as pressure-overload (transverse aortic constriction) that induces cardiac hypertrophy and heart failure (Goldman and Tarnavski), are useful models to study cardiovascular disease. In particular, myocardial ischemia (MI) is a leading cause for cardiovascular morbidity and mortality despite controlling certain risk factors such as arteriosclerosis and treatments via surgical intervention (Thygesen). Furthermore, an acute loss of the myocardium following myocardial ischemia (MI) results in increased loading conditions that induces ventricular remodeling of the infarcted border zone and the remote non-infarcted myocardium. Myocyte apoptosis, necrosis and the resultant increased hemodynamic load activate multiple biochemical intracellular signaling that initiates LV dilatation, hypertrophy, ventricular shape distortion, and collagen scar formation. This pathological remodeling and failure to normalize the increased wall stresses results in progressive dilatation, recruitment of the border zone myocardium into the scar, and eventually deterioration in myocardial contractile function (i.e. heart failure). The progression of LV dysfunction and heart failure in rats is similar to that observed in patients who sustain a large myocardial infarction, survive and subsequently develops heart failure (Goldman). The acute myocardial infarction (AMI) model in rats has been used to mimic human cardiovascular disease; specifically used to study cardiac signaling mechanisms associated with heart failure as well as to assess the contribution of therapeutic strategies for the treatment of heart failure. The method described in this report is the rat model of acute myocardial infarction (AMI). This model is also referred to as an acute ischemic cardiomyopathy or ischemia followed by reperfusion (IR); which is induced by an acute 30-minute period of ischemia by ligation of the left anterior descending artery (LAD) followed by reperfusion of the tissue by releasing the LAD ligation (Vasilyev and McConnell). This protocol will focus on assessment of the infarct size and the area-at-risk (AAR) by Evan's blue dye and triphenyl tetrazolium chloride (TTC) following 4-hours of reperfusion; additional comments toward the evaluation of cardiac function and remodeling by modifying the duration of reperfusion, is also presented. Overall, this AMI rat animal model is useful for studying the consequence of a myocardial infarction on cardiac pathophysiological and physiological function.

The rat model of acute myocardial infarction (AMI) is useful to study the consequence of a MI on cardiac pathophysiological and physiological function.

ラットにおける急性心筋梗塞

With heart failure leading the cause of death in the USA (Hunt), biomedical research is fundamental to advance medical treatments for cardiovascular ...

医学

MRI and PET in Mouse Models of Myocardial Infarction

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ideal model for testing novel therapeutic strategies.
In vivo magnetic resonance imaging (MRI) gives excellent views of the heart noninvasively with clear anatomical detail, which can be used for accurate functional assessment. Contrast agents can provide basic measures of tissue viability but these are nonspecific. Positron emission tomography (PET) is a complementary technique that is highly specific for molecular imaging, but lacks the anatomical detail of MRI. Used together, these techniques offer a sensitive, specific and quantitative tool for the assessment of the heart in disease and recovery following treatment.
In this paper we explain how these methods are carried out in mouse models of acute myocardial infarction. The procedures described here were designed for the assessment of putative protective drug treatments. We used MRI to measure systolic function and infarct size with late gadolinium enhancement, and PET with fluorodeoxyglucose (FDG) to assess metabolic function in the infarcted region. The paper focuses on practical aspects such as slice planning, accurate gating, drug delivery, segmentation of images, and multimodal coregistration. The methods presented here achieve good repeatability and accuracy maintaining a high throughput.

We describe how to perform MRI and PET imaging of the mouse heart. The protocol is tailored to assess treatment efficacy in models of myocardial infarction and heart failure.

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ...

Myocardial Infarction and Functional Outcome Assessment in Pigs

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One important prerequisite is a good understanding of all safety and efficacy aspects obtained in a large animal model that validly reflect the human scenario of myocardial infarction (MI). Pigs are widely used in this regard since their cardiac size, hemodynamics, and coronary anatomy are close to that of humans. Here, we present an effective protocol for using the porcine MI model using a closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. This approach is based on 90 min of myocardial ischemia, inducing large left ventricle infarction of the anterior, septal and inferoseptal walls. Furthermore, we present protocols for various measures of outcome that provide a wide range of information on the heart, such as cardiac systolic and diastolic function, hemodynamics, coronary flow velocity, microvascular resistance, and infarct size. This protocol can be easily tailored to meet study specific requirements for the validation of novel cardioregenerative biologics at different stages (i.e. directly after the acute ischemic insult, in the subacute setting or even in the chronic MI once scar formation has been completed). This model therefore provides a useful translational tool to study MI, subsequent adverse remodeling, and the potential of novel cardioregenerative agents.

This protocol describes the porcine myocardial infarction (MI) model using a 90 min closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. Furthermore, the protocol for several outcome parameters, such as cardiac function, hemodynamics, microvascular resistance, and infarct size, are also presented.

豚における心筋梗塞および機能的結果の評価

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One ...

A Hydrogel Construct and Fibrin-based Glue Approach to Deliver Therapeutics in a Murine Myocardial Infarction Model.

The murine MI model is widely recognized in the field of cardiovascular disease, and has consistently been used as a first step to test the efficacy of treatments in vivo1. The traditional, established protocol has been further fine-tuned to minimize the damage to the animal. Notably, the pectoral muscle layers are teased away rather than simply cut, and the thoracotomy is approached intercostally as opposed to breaking the ribs in a sternotomy, preserving the integrity of the ribcage. With these changes, the overall stress on the animal is decreased. 
Stem cell therapies aimed to alleviate the damage caused by MIs have shown promise over the years for their pro-angiogenic and anti-apoptotic benefits. Current approaches of delivering cells to the heart surface typically involve the injection of the cells either near the damaged site, within a coronary artery, or into the peripheral blood stream2-4. While the cells have proven to home to the damaged myocardium, functionality is limited by their poor engraftment at the site of injury, resulting in diffusion into the blood stream5. This manuscript highlights a procedure that overcomes this obstacle with the use of a cell-encapsulated hydrogel patch. The patch is fabricated prior to the surgical procedure and is placed on the injured myocardium immediately following the occlusion of the left coronary artery. To adhere the patch in place, biocompatible external fibrin glue is placed directly on top of the patch, allowing for it to dry to both the patch and the heart surface. This approach provides a novel adhesion method for the application of a delicate cell-encapsulating therapeutic construct.

This protocol aims to alleviate the limitation of poor cell engraftment for stem cell treatment of myocardial infarctions through the use of a hydrogel system and a fibrin-based glue. With this approach, cell-to-tissue contact post-infarction can be maintained, increasing the therapeutic potential of beneficial agents at the site of injury.

ヒドロゲルは、構築し、マウス心筋梗塞モデルにおける治療薬を送達するためのフィブリンベースの接着剤のアプローチ。

The murine MI model is widely recognized in the field of cardiovascular disease, and has consistently been used as a first step to test the efficacy of ...

Primary Outcome Assessment in a Pig Model of Acute Myocardial Infarction

Mortality after acute myocardial infarction remains substantial and is associated with significant morbidity, like heart failure. Novel therapeutics are therefore required to confine cardiac damage, promote survival and reduce the disease burden of heart failure. Large animal experiments are an essential part in the translational process from experimental to clinical therapies. To optimize clinical translation, robust and representative outcome measures are mandatory. The present manuscript aims to address this need by describing the assessment of three clinically relevant outcome modalities in a pig acute myocardial infarction (AMI) model: infarct size in relation to area at risk (IS/AAR) staining, 3-dimensional transesophageal echocardiography (TEE) and admittance-based pressure-volume (PV) loops. Infarct size is the main determinant driving the transition from AMI to heart failure and can be quantified by IS/AAR staining. Echocardiography is a reliable and robust tool in the assessment of global and regional cardiac function in clinical cardiology. Here, a method for three-dimensional transesophageal echocardiography (3D-TEE) in pigs is provided. Extensive insight into cardiac performance can be obtained by admittance-based pressure-volume (PV) loops, including intrinsic parameters of myocardial function that are pre- and afterload independent. Combined with a clinically feasible experimental study protocol, these outcome measures provide researchers with essential information to determine whether novel therapeutic strategies could yield promising targets for future testing in clinical studies.

Reliable and accurate outcome assessment is the key for translation of preclinical therapies into clinical treatment. The current paper describes how to assess three clinically relevant primary outcome parameters of cardiac performance and damage in a pig acute myocardial infarction model.

急性心筋梗塞の豚モデルにおける主要アウトカム評価

Mortality after acute myocardial infarction remains substantial and is associated with significant morbidity, like heart failure. Novel therapeutics are ...

Myocardial Infarction in Neonatal Mice, A Model of Cardiac Regeneration

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and regeneration, and to define new targets for therapeutics. For decades, models of complete heart regeneration existed in amphibians and fish, but a mammalian counterpart was not available. The recent discovery of a postnatal window during which mice possess regenerative capabilities has led to the establishment of a mammalian model of cardiac regeneration. A surgical model of mammalian cardiac regeneration in the neonatal mouse is presented herein. Briefly, postnatal day 1 (P1) mice are anesthetized by isoflurane and placed on an ice pad to induce hypothermia. After the chest is opened, and the left anterior descending coronary artery (LAD) is visualized, a suture is placed around the LAD to inflict myocardial ischemia in the left ventricle. The surgical procedure takes 10-15 min. Visualizing the coronary artery is crucial for accurate suture placement and reproducibility. Myocardial infarction and cardiac dysfunction are confirmed by triphenyl-tetrazolium chloride (TTC) staining and echocardiography, respectively. Complete regeneration 21 days post myocardial infarction is verified by histology. This protocol can be used to as a tool to elucidate mechanisms of mammalian cardiac regeneration after myocardial infarction.

This protocol describes a highly reproducible model of cardiac regeneration by surgical induction of myocardial infarction in the left ventricle of postnatal day 1 mice. The method involves induction of hypothermic anesthesia and ligation of the left anterior descending coronary artery.

新生児マウスの心筋梗塞、心臓再生のモデル

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and ...

Improvement of a Closed Chest Porcine Myocardial Infarction Model by Standardization of Tissue and Blood Sampling Procedures

Myocardial ischemia reperfusion (I/R) injury contributes to almost half of the necrotic area after myocardial infarction. To date there is no approved drug to prevent or reduce myocardial I/R injury. The study and understanding of the pathophysiological mechanisms of myocardial I/R injury is essential to develop successful treatments. Large animal experiments are an important step in translational methods. The porcine model of acute myocardial infarction has been established and described by ourselves and others. We aimed to further improve the value of the model by focusing in detail on the sampling techniques for use in future experiments. Furthermore, we emphasize small but important steps that can affect the quality of the final results. To mimic the clinical situation of myocardial I/R injury, a percutaneous coronary intervention (PCI) catheter was inserted into the left anterior descending coronary artery (LAD) of an anesthetized pig. &#176;&#176;&#176;This model mimics acute myocardial infarction and PCI treatment in humans with the possibility of accurately determining the area at risk as well as the necrotic- and viable ischemic tissue. Here the model was used to investigate the effect of a bicyclic peptide inhibitor of FXIIa. The model can also be modified to allow longer reperfusion times to study later effects of myocardial infarction.

Here we demonstrate a protocol to standardize sampling procedures of an established porcine model of acute myocardial infarction in order to increase its translational value in the understanding of the pathophysiology of myocardial ischemia/reperfusion injury and to test novel drug candidates.

組織と血液サンプリング手順の標準化による閉胸ブタ心筋梗塞モデルの改善

Myocardial ischemia reperfusion (I/R) injury contributes to almost half of the necrotic area after myocardial infarction. To date there is no approved ...

A Cryoinjury Model to Study Myocardial Infarction in the Mouse

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we demonstrate a murine cryoinjury infarct model that generates precise infarct sizes with high reproducibility and replicability. In brief, after intubation and sternotomy of the animal, the heart is lifted from the thorax. The probe of a handheld liquid nitrogen delivery system is applied onto the myocardial wall to induce cryoinjury. Impaired ventricular function and electrical conduction can be monitored with echocardiography or optical mapping. Transmural myocardial remodeling of the infarcted area is characterized by collagen deposition and loss of cardiomyocytes. Compared to other models (e.g., LAD-ligation), this model utilizes a handheld liquid nitrogen delivery system to generate more uniform infarct sizes.

This article demonstrates a model to study cardiac remodeling after myocardial cryoinjury in mice.

マウスの心筋梗塞を研究する凍結傷害モデル

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we ...

Establishing a Swine Model of Post-myocardial Infarction Heart Failure for Stem Cell Treatment

Although advances have been achieved in the treatment of heart failure (HF) following myocardial infarction (MI), HF following MI remains one of the major causes of mortality and morbidity around the world. Cell-based therapies for cardiac repair and improvement of left ventricular function after MI have attracted considerable attention. Accordingly, the safety and efficacy of these cell transplantations should be tested in a preclinical large animal model of HF prior to clinical use. Pigs are widely used for cardiovascular disease research due to their similarity to humans in terms of heart size and coronary anatomy. Therefore, we sought to present an effective protocol for the establishment of a porcine chronic HF model using closed-chest coronary balloon occlusion of the left circumflex artery (LCX), followed by rapid ventricular pacing induced with pacemaker implantation. Eight weeks later, the stem cells were administered by intramyocardial injection in the peri-infarct area. Then the infarct size, cell survival, and left ventricular function (including echocardiography, hemodynamic parameters, and electrophysiology) were evaluated. This study helps establish a stable preclinical large animal HF model for stem cell treatment.

We sought to establish a swine model of heart failure induced by left circumflex artery blockage and rapid pacing to test the effect and safety of intramyocardial administration of stem cells for cell-based therapies.

幹細胞治療のための心筋梗塞後心不全の豚モデルの確立

Although advances have been achieved in the treatment of heart failure (HF) following myocardial infarction (MI), HF following MI remains one of the major ...

Intramyocardial Transplantation of MSC-Loading Injectable Hydrogels after Myocardial Infarction in a Murine Model

One of the major issues facing current cardiac stem cell therapies for preventing postinfarct heart failure is the low retention and survival rates of transplanted cells within the injured myocardium, limiting their therapeutic efficacy. Recently, the use of scaffolding biomaterials has gained attention for improving and maximizing stem cell therapy. The objective of this protocol is to introduce a simple and straightforward technique to transplant bone marrow-derived mesenchymal stem cells (MSCs) using injectable hydroxyphenyl propionic acid (GH) hydrogels; the hydrogels are favorable as a cell delivery platform for cardiac tissue engineering applications due to their ability to be cross-linked in situ and high biocompatibility. We present a simple method to fabricate MSC-loading GH hydrogels (MSC/hydrogels) and evaluate their survival and proliferation in three-dimensional (3D) in vitro culture. In addition, we demonstrate a technique for intramyocardial transplantation of MSC/hydrogels in mice, describing a surgical procedure to induce myocardial infarction (MI) via left anterior descending (LAD) coronary artery ligation and subsequent MSC/hydrogels transplantation.

Stem cell-based therapy has emerged as an efficient strategy to repair injured cardiac tissues after myocardial infarction. We provide an optimal in vivo application for stem cell transplantation using gelatin hydrogels that are able to be enzymatically cross-linked.

マウスモデルにおける心筋梗塞後のMSC負荷注入性ヒドロゲルの筋内移植

One of the major issues facing current cardiac stem cell therapies for preventing postinfarct heart failure is the low retention and survival rates of ...