Name: delivery of cardioactive therapeutics in a porcine myocardial infarction model
Uploaded: 2023-02-10T12:40:00.000+00:00
Duration: 7 min 20 s
Description: Watch this Scientific Journal Video about Delivery of Cardioactive Therapeutics in a Porcine Myocardial Infarction Model at JoVE.com

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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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><tbody><tr><td>&lt;strong&gt;Central line placement&lt;/strong&gt;</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&#039; Paediatric Two-Lumen Central Venous Catheterisation Set with Blue FlexTip Catheter (contains 18G cook needle and 0.035&quot; 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>&lt;strong&gt;Histology antibodies&lt;/strong&gt;</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&#039; 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 &#039;Medium&#039;</td><td>Lomir Biomedical</td><td>SS J2YJJET</td><td></td></tr><tr><td>&lt;strong&gt;Jugular vein osmotic minipump implantation&lt;/strong&gt;</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 &#039;Edge&#039; 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&amp;nbsp; (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>&lt;strong&gt;Percutaneous transedocardial injection&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Artis Zee&#039; C-Arm Fluoroscopy&amp;nbsp;</td><td>Siemens&amp;nbsp;</td><td>IR-19-1994</td><td></td></tr><tr><td>CARTO&#039; 3 System&amp;nbsp;&amp;nbsp;</td><td>Biosense Webster</td><td></td><td>Electrophysiological Mapping Software &amp;amp; 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&#039; 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&#039; 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&#039; Iohexol Contrast</td><td>GE Healthcare</td><td>AUST R 39861&amp;nbsp;</td><td>Iodinated contrast agent</td></tr><tr><td>Sparq&#039; Ultrasound System</td><td>Philips</td><td>MP11742 Medpick</td><td></td></tr><tr><td>&lt;strong&gt;Sedation &amp;amp; general anaesthesia&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Compound Sodium Lactate Hartmann&#039;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&#039; Isoflurane</td><td>Abbott</td><td>AUST R 29656&amp;nbsp;</td><td>Inhalant anaesthetic</td></tr><tr><td>GE Aestiva 5 Anaesthesia Machine&amp;nbsp;</td><td>Datex Ohmeda</td><td>17002-9, 17002A9 Avante Health Solutions</td><td>Anaesthetic Machine&amp;nbsp;</td></tr><tr><td>Hypnovel&#039; 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&#039; Ketamine 10 mg/mL</td><td>Ilium</td><td>APVMA number: 51188c&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</td><td>Sedative, Restricted drug</td></tr><tr><td>Onsetron&#039; Ondansetron 2 mg/mL</td><td>Accord Healthcare</td><td>AUST R 205593&amp;nbsp;</td><td>Anti-emetic</td></tr><tr><td>Propofol-Lipuro&#039; Propofol 10 mg/mL</td><td>Braun</td><td>AUST R 142906&amp;nbsp;</td><td>Intravenous anaesthetic</td></tr><tr><td>Pulse Oximeter&amp;nbsp;</td><td>Meditech</td><td>GVPMT-M3S</td><td>Portable pulse oximeter</td></tr><tr><td>Shiley&#039; Cuffed Basic Endotracheal Tube (Size 5.5 &amp;amp; 6.0)</td><td>Medtronic</td><td>86108-, 86109-</td><td></td></tr><tr><td>Shiley&#039; 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>&lt;strong&gt;Thoracotomy and epicardial Cell Injection&lt;/strong&gt;</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&#039; 3 System&amp;nbsp;&amp;nbsp;</td><td>Biosense Webster</td><td></td><td>Electrophysiological Mapping Software &amp;amp; System</td></tr><tr><td>Cefazolin 1 g Vial</td><td>AFT Pharmaceuticals</td><td>9421900137367 CH2</td><td>Antibiotic Prophylaxis&amp;nbsp;</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&#039; 100 mcg/h Fentanyl Patch</td><td>Janssen</td><td>AUST R 112371&amp;nbsp;</td><td>Postoperative analgesia</td></tr><tr><td>Electrosurgical Pencils with &#039;Edge&#039; Coated Electrodes</td><td>Covidien</td><td>E2450H</td><td>Cautery Pencil</td></tr><tr><td>Electrosurgical Pencils with &#039;Edge&#039; Coated Electrodes</td><td>Covidien</td><td>E2450H</td><td>Cautery Pencil</td></tr><tr><td>Fast-Cath&#039; 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&#039; Bupivacaine 0.5%</td><td>Pfizer</td><td>AUST R 48328&amp;nbsp;</td><td>Local anaesthesia.&amp;nbsp;</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&amp;nbsp; (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&#039; 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&#039; 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://jove.unicartagenaproxy.elogim.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://jove.unicartagenaproxy.elogim.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://jove.unicartagenaproxy.elogim.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

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

1.2K 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

Gene Transfer for Ischemic Heart Failure in a Preclinical Model

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine their efficacy and safety.
Gene therapy using viral vectors is one of the most promising approaches for treating cardiac diseases. Viral delivery of various different genes by changing the carrier gene has immeasurable therapeutic potential.
In this video, the full process of an animal model of heart failure creation followed by gene transfer is presented using a swine model. First, myocardial infarction is created by occluding the proximal left anterior descending coronary artery. Heart remodeling results in chronic heart failure. Unique to our model is a fairly large scar which truly reflects patients with severe heart failure who require aggressive therapy for positive outcomes. After myocardial infarct creation and development of scar tissue, an intracoronary injection of virus is demonstrated with simultaneous nitroglycerine infusion. Our injection method provides simple and efficient gene transfer with enhanced gene expression. This combination of a myocardial infarct swine model with intracoronary virus delivery has proven to be a consistent and reproducible methodology, which helps not only to test the effect of individual gene, but also compare the efficacy of many genes as therapeutic candidates.

A method of gene transfer for the treatment of ischemic heart failure is described using a swine model of myocardial infarction. Our simple and reproducible method enables us to readily evaluate the efficacy of various gene transfers with a very simple and reproducible way.

前臨床モデルにおける虚血性心不全のための遺伝子導入

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine ...

医学

Pluripotent Stem Cell Derived Cardiac Cells for Myocardial Repair

Human induced pluripotent stem cells (hiPSCs) must be fully differentiated into specific cell types before administration, but conventional protocols for differentiating hiPSCs into cardiomyocytes (hiPSC-CMs), endothelial cells (hiPSC-ECs), and smooth muscle cells (SMCs) are often limited by low yield, purity, and/or poor phenotypic stability. Here, we present novel protocols for generating hiPSC-CMs, -ECs, and -SMCs that are substantially more efficient than conventional methods, as well as a method for combining cell injection with a cytokine-containing patch created over the site of administration. The patch improves both the retention of the injected cells, by sealing the needle track to prevent the cells from being squeezed out of the myocardium, and cell survival, by releasing insulin-like growth factor (IGF) over an extended period. In a swine model of myocardial ischemia-reperfusion injury, the rate of engraftment was more than two-fold greater when the cells were administered with the cytokine-containing patch comparing to the cells without patch, and treatment with both the cells and the patch, but not with the cells alone, was associated with significant improvements in cardiac function and infarct size.

We present three novel and more efficient protocols for differentiating human induced pluripotent stem cells into cardiomyocytes, endothelial cells, and smooth muscle cells and a delivery method that improves the engraftment of transplanted cells by combining cell injection with patch-mediated cytokine delivery.

多能性幹細胞は、心筋の修復のための心臓細胞を派生します

Human induced pluripotent stem cells (hiPSCs) must be fully differentiated into specific cell types before administration, but conventional protocols for ...

発生生物学

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 ...

Testing the Efficacy of Pharmacological Agents in a Pericardial Target Delivery Model in the Swine

To date, many pharmacological agents used to treat or prevent arrhythmias in open-heart cases create undesired systemic side effects. For example, antiarrhythmic drugs administered intravenously can produce drops in systemic pressure in the already compromised cardiac patient. While performing open-heart procedures, surgeons will often either create a small port or form a pericardial cradle to create suitable fields for operation. This access yields opportunities for target pharmacological delivery (antiarrhythmic or ischemic preconditioning agents) directly to the myocardial tissue without undesired side effects.
We have developed a swine model for testing pharmacological agents for target delivery within the pericardial fluid. While fully anesthetized, each animal was instrumented with a Swan-Ganz catheter as well as left and right ventricle pressure catheters, and pacing leads were placed in the right atrial appendage and the right ventricle. A medial sternotomy was then performed and a pericardial access cradle was created; a plunge pacing lead was placed in the left atrial appendage and a bipolar pacing lead was placed in the left ventricle. Utilizing a programmer and a cardiac mapping system, the refractory period of the atrioventricular node (AVN), atria and ventricles was determined. In addition, atrial fibrillation (AF) induction was produced utilizing a Grass stimulator and time in AF was observed. These measurements were performed prior to treatment, as well as 30 min&#160;and 60 min&#160;after pericardial treatment. Additional time points were added for selected studies. The heart was then cardiopleged and reanimated in a four chamber working mode. Pressure measurements and function were recorded for 1 hr after reanimation. This treatment strategy model allowed us to observe the effects of pharmacological agents that may decrease the incidence of cardiac arrhythmias and/or ischemic damage, during and after open-heart surgery.

We have developed a swine model for the target delivery of pharmacological agents within the pericardial space/fluid. Using this approach, the relative benefits of administered agents on induced atrial fibrillation, relative refractory periods and/or ischemic protection can be investigated.

豚に心膜ターゲット・デリバリー・モデルでの薬剤の有効性をテストします

To date, many pharmacological agents used to treat or prevent arrhythmias in open-heart cases create undesired systemic side effects. For example, ...

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 ...

Percutaneous Contrast Echocardiography-guided Intramyocardial Injection and Cell Delivery in a Large Preclinical Model

Cell and gene therapy are exciting and promising strategies for the purpose of cardiac regeneration in the setting of heart failure with reduced ejection fraction (HFrEF). Before they can be considered for use, and implemented in humans, extensive preclinical studies are required in large animal models to evaluate the safety, efficacy, and fate of the injectate (e.g., stem cells) once delivered into the myocardium. Small rodent models offer advantages (e.g., cost effectiveness, amenability for genetic manipulation); however, given inherent limitations of these models, the findings in these rarely translate into the clinic. Conversely, large animal models such as rabbits, have advantages (e.g., similar cardiac electrophysiology compared to humans and other large animals), whilst retaining a good cost-effective balance. Here, we demonstrate how to perform a percutaneous contrast echocardiography-guided intramyocardial injection (IMI) technique, which is minimally invasive, safe, well tolerated, and very effective in the targeted delivery of injectates, including cells, into several locations within the myocardium of a rabbit model. For the implementation of this technique, we also have taken advantage of a widely available clinical echocardiography system. After putting in practice the protocol described here, a researcher with basic ultrasound knowledge will become competent in the performance of this versatile and minimally invasive technique for routine use in experiments, aimed at hypothesis testing of the capabilities of cardiac regenerative therapeutics in the rabbit model. Once competency is achieved, the whole procedure can be performed within 25 min after anaesthetizing the rabbit.

Novel therapeutic strategies in cardiac regenerative medicine require extensive and detailed studies in large preclinical animal models before they can be considered for use in humans. Here, we demonstrate a percutaneous contrast echocardiography-guided intramyocardial injection technique in rabbits, which is valuable for hypothesis testing the efficacy of such novel therapies.

経皮的造影エコー ガイド心筋と大規模な前臨床モデルで携帯配信

Cell and gene therapy are exciting and promising strategies for the purpose of cardiac regeneration in the setting of heart failure with reduced ejection ...

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 ...

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 ...

Large Animal Model for Evaluating the Efficacy of the Gene Therapy in Ischemic Heart

Coronary artery disease is one of the significant causes of mortality and morbidity worldwide. Despite the progression of current therapeutics, a considerable proportion of coronary artery disease patients remain symptomatic. Gene therapy-mediated therapeutic angiogenesis offers a novel therapeutic method for improving myocardial perfusion and relieving symptoms. Gene therapy with different angiogenic factors has been studied in few clinical trials. Due to the novelty of the method, the progress of myocardial gene therapy is a continuous path from bench to bedside. Therefore, large animal models are needed for evaluating the safety and efficacy. The more the large animal model identifies the original disease and the endpoints used in clinics, the more predictable outcomes are from clinical trials. Here, we introduce a large animal model for evaluating the efficacy of the gene therapy in the ischemic porcine heart. We use clinically relevant imaging methods such as ultrasound imaging and 15H2O-PET. For targeting the gene transfers into the desired area, electroanatomical mapping is used. The aim of this method is: (1) to mimic chronic coronary artery disease, (2) to induce therapeutic angiogenesis at hypoxic areas of the heart, and (3) to evaluate the safety and efficacy of the gene therapy by using relevant endpoints.

Myocardial gene therapy for ischemic heart disease holds great promise for future therapeutics. Here, we introduce a large animal model for evaluating the efficacy of gene therapy in the ischemic heart.

虚血性心拍における遺伝子治療の有効性を評価するための大型動物モデル

Coronary artery disease is one of the significant causes of mortality and morbidity worldwide. Despite the progression of current therapeutics, a ...

Myocardial Infarction by Percutaneous Embolization Coil Deployment in a Swine Model

Myocardial infarction (MI) is the leading cause of mortality worldwide. Despite the use of evidence-based treatments, including coronary revascularization and cardiovascular drugs, a significant proportion of patients develop pathological left-ventricular remodeling and progressive heart failure following MI. Therefore, new therapeutic options, such as cellular and gene therapies, among others, have been developed to repair and regenerate injured myocardium. In this context, animal models of MI are crucial in exploring the safety and efficacy of these experimental therapies before clinical translation. Large animal models such as swine are preferred over smaller ones due to the high similarity of swine and human hearts in terms of coronary artery anatomy, cardiac kinetics, and the post-MI healing process. Here, we aimed to describe an MI model in pig by permanent coil deployment. Briefly, it comprises a percutaneous selective coronary artery cannulation through retrograde femoral access. Following coronary angiography, the coil is deployed at the target branch under fluoroscopic guidance. Finally, complete occlusion is confirmed by repeated coronary angiography. This approach is feasible, highly reproducible, and emulates the pathogenesis of human non-revascularized MI, avoiding the traditional open-chest surgery and the subsequent postoperative inflammation. Depending on the time of follow-up, the technique is suitable for acute, sub-acute, or chronic MI models.

Myocardial infarction (MI) animal models that emulate the natural process of the disease in humans are crucial to understanding pathophysiological mechanisms and testing the safety and efficacy of new emergent therapies. Here, we describe an MI swine model created by deploying a percutaneous embolization coil.

豚モデルにおける経皮塞栓コイル展開による心筋梗塞

Myocardial infarction (MI) is the leading cause of mortality worldwide. Despite the use of evidence-based treatments, including coronary revascularization ...

ブタ心筋梗塞モデルにおける心役活性治療薬の送達

要約

さらに動画を探す

この動画の章

前臨床モデルにおける虚血性心不全のための遺伝子導入

多能性幹細胞は、心筋の修復のための心臓細胞を派生します

豚における心筋梗塞および機能的結果の評価

豚に心膜ターゲット・デリバリー・モデルでの薬剤の有効性をテストします

急性心筋梗塞の豚モデルにおける主要アウトカム評価

経皮的造影エコーガイド心筋と大規模な前臨床モデルで携帯配信

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

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

虚血性心拍における遺伝子治療の有効性を評価するための大型動物モデル

豚モデルにおける経皮塞栓コイル展開による心筋梗塞

ブタ心筋梗塞モデルにおける心役活性治療薬の送達

要約

さらに動画を探す

この動画の章

前臨床モデルにおける虚血性心不全のための遺伝子導入

多能性幹細胞は、心筋の修復のための心臓細胞を派生します

豚における心筋梗塞および機能的結果の評価

豚に心膜ターゲット・デリバリー・モデルでの薬剤の有効性をテストします

急性心筋梗塞の豚モデルにおける主要アウトカム評価

経皮的造影エコー ガイド心筋と大規模な前臨床モデルで携帯配信

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

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

虚血性心拍における遺伝子治療の有効性を評価するための大型動物モデル

豚モデルにおける経皮塞栓コイル展開による心筋梗塞

経皮的造影エコーガイド心筋と大規模な前臨床モデルで携帯配信