Name: apical resection mouse model to study early mammalian heart regeneration
Uploaded: 2016-01-23T15:00:00.000+00:00
Duration: 6 min 8 s
Description: Watch this Scientific Journal Video about Apical Resection Mouse Model to Study Early Mammalian Heart Regeneration at JoVE.com

著者は、博士に感謝します。マウスの手術と準備し、パラフィン切片の染色との支援のための国立心肺血液研究所（NHLBI）からジェームズ・ホーキンス、祖-XIゆうと玄屈原。著者は、編集支援のためのNIHフェロー編集委員会に感謝しています。

すべてのマウスの実験では、プロトコル番号H0083R3と国立衛生研究所（NIH）での動物管理使用プログラムによって承認されました。 NHLBI IACUCは鎮痛剤なしでプロトコルを承認しました。 新生児マウスの1の低体温麻酔<ol><li>手術前にオートクレーブにスポンジや手術用機器を滅菌します。すべての外科的材料を準備し、270℃まで240℃に到達するために、事前にホットビーズ滅菌器で15〜20分を切り替えます。 </li><li>新鮮な寝具、ネスティング材料ときれいなマウスのケージへの看護母からすべてのC57BL / 6仔（年齢P1）を転送します。子犬が手術室に取り込まれると、母親から分離され、母体の共食いの危険性を低減するために費やされた自分の時間を最小限にするために速やかに頂端手術を行います。 </li><li>氷のベッドの上にスポンジを入れた後、低体温麻酔を達成するために〜3分間スポンジに1子犬を置きます。 APを観察することによって麻酔を確認NEAと無動とリアの足をつまんで。あまりにも少しの時間が子犬の無動や無呼吸をすることはありませんし、麻酔の過剰な期間は生存率12を低下させることができるので、頻繁に新生児の状態を確認してください。 </li></ol> 2.開胸<ol><li>外科的なベンチトップ型領域に氷床から子犬を転送し、仰臥位でその腕、脚、尾を固定するテープを使用しています。 </li><li>ベタジンを使用して胸を消毒し、ゆっくりと70％アルコールの準備パッドを使用して清掃してください。 </li><li> Vannas春シザーを用いて、胸腔の第肋間領域に沿って横方向の皮膚切開を行い、その後、心臓へのアクセスを容易にするために記載肋間筋を解剖鈍ら。 注：血液損失は、外科的処理中に最小化されたときに生存率が向上します。 </li></ol>ハート心室アペックスの3外科切断術<ol><li>手で、優しく体外に出すために、腹部に圧力を適用します心尖​​。明確な可視化のための滅菌綿棒アプリケーターで手術領域の周りに血液を吸収します。偽手術対照新生児の場合は、4（縫合糸とマウスの回復）をステップに進んで。 </li><li> LV室が露出するまで拡大ランプの下や虹彩切除鋏を使用して、静かに左心室（LV）の断片的な切除を行います。 LVの切除部分を最小限にするように注意してください。約15％の切除は、LV室の最適な露出を達成するために必要です。 </li><li> LV室が露出されると、心臓が胸腔に返すことを確認してください。 </li></ol> 4.縫合およびマウスの回復<ol><li>無菌プロリン6-0縫合糸を使用して胸腔を密封するために一緒に肋骨や筋肉を縫合した後、慎重に皮膚接着剤を用いた皮膚切開部位を閉じます。 </li><li>回復のために〜3分間加熱ランプの下で新生児を温め、その後、70％のアルコールプレップパッドbefoを用いて血液や接着剤の痕跡をきれいにその同腹子に再導入する再。母親から分離かかる時間を最小限に抑えること頂端切除後の仔の生存率を改善するため、10分以内に全体の外科的手順を完了してみてください。 </li><li>各手術後、完全な殺菌のため約20秒間ホットビーズ滅菌器のビーズと接触した手術器具を配置します。手術器具がそれぞれの外科手術の前に室温まで冷却してください。 </li><li>ごみのすべての子犬のための手術を完了した後、母親の巣に戻す前に、母親の寝具や排泄物との子犬を混ぜます。 注：一般的に、ICR / CD-1マウスは、C57BL / 6よりも優れ養母であるが、同じであっても遺伝的背景の授乳中の母親の間で、育成本能が異なります。看護母を変更すると、母体の共食いによって死亡率を減少させる看護母にP0仔を削除して、P1で頂端切除を行う必要がある場合。のみ偽手術または頂端Rのいずれかを実行します子犬の1リターのesection、混合偽手術子犬は子犬6の手術群の生存率を低下させる可能性があるため。 </li></ol> 5.術後の分析<ol><li>ある日手術後、子犬を監視し、偽手術群の間に差がないことを確認し、生存率を測定するために、仔の数を数えます。手術が適切に行われている場合、生存率は、偽手術群の両方に類似しており、60％以上でなければなりません。 </li><li>ハートの分離と固定。 <ol><li> 70％アルコールの準備を使用して胸をきれいその後、頚椎脱臼により死亡の保証とCO2によって断頭し、21日目、手術後経由で1日目および2日目、手術後に子犬を安楽死させます。 </li><li>胸の正中線の皮膚や筋肉を切開して、胸を開きます。 </li><li>消費税​​胸腔から全体の心と室温で4％パラホルムアルデヒド、O / N 5mlに、各サンプルの心臓全体を固定します。 </li></ol></li><li>翌日、70％エタノールにサンプルを転送します。サンプルは、パラフィン包埋の前に一週間まで保存することができます。 </li><li>スライス5ミクロンの厚さのパラフィン切片全体の心室を通って、再生応答5,7を調べるために 、標準的なヘマトキシリンおよびエオシン（H＆E）およびマッソントリクローム染色を行う。具体的には、H＆E染色は、筋肉の交換を検討するために使用され、マッソントリクローム染色を使用しています線維性応答5を検討します。 </li><li>関心のある領域を選択し、スライドスキャナを使用して手動でサンプル当たり少なくとも三つの焦点を設定します。デフォルトのパラメータで、製造元の指示に従って40倍の倍率で染色スライドをイメージし、分析します。 </li></ol>

<ol>
	<li>Lin, Z., Pu, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+for+cardiac+regeneration+and+repair.">Strategies for cardiac regeneration and repair.</a> Sci Transl Med. 6, 231-239 (2014).</li><li>Xin, M., Olson, E. N., Bassel-Duby, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mending+broken+hearts:+cardiac+development+as+a+basis+for+adult+heart+regeneration+and+repair.">Mending broken hearts: cardiac development as a basis for adult heart regeneration and repair.</a> Nat Rev Mol Cell Biol. 14, 529-541 (2013).</li><li>Mahmoud, A. I., Porrello, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Turning+back+the+cardiac+regenerative+clock:+lessons+from+the+neonate.">Turning back the cardiac regenerative clock: lessons from the neonate.</a> Trends Cardiovasc Med. 22, 128-133 (2012).</li><li>Porrello, E. R., Olson, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+neonatal+blueprint+for+cardiac+regeneration.">A neonatal blueprint for cardiac regeneration.</a> Stem Cell Res. 13, 556-570 (2014).</li><li>Porrello, E. R., 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=Transient+regenerative+potential+of+the+neonatal+mouse+heart.">Transient regenerative potential of the neonatal mouse heart.</a> Science. 331, 1078-1080 (2011).</li><li>Mahmoud, A. I., Porrello, E. R., Kimura, W., Olson, E. N., Sadek, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+models+for+cardiac+regeneration+in+neonatal+mice.">Surgical models for cardiac regeneration in neonatal mice.</a> Nat Protoc. 9, 305-311 (2014).</li><li>Allen, T. C., Prophet, E. B., Mills, B., Arrington, J. B., Sobin, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematoxylin+and+eosin.">Hematoxylin and eosin.</a> Laboratory methods in Histotechnology. , 53-58 (1992).</li><li>Andersen, D. C., Ganesalingam, S., Jensen, C. H., Sheikh, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Do+neonatal+mouse+hearts+regenerate+following+heart+apex+resection?.">Do neonatal mouse hearts regenerate following heart apex resection?.</a> Stem cell reports. 2, 406-413 (2014).</li><li>Kotlikoff, M. I., Hesse, M., Fleischmann, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comment+on+'Do+neonatal+mouse+hearts+regenerate+following+heart+apex+resection?'.">Comment on 'Do neonatal mouse hearts regenerate following heart apex resection?'.</a> Stem cell reports. 3, 2 (2014).</li><li>Sadek, H. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-investigator+letter+on+reproducibility+of+neonatal+heart+regeneration+following+apical+resection.">Multi-investigator letter on reproducibility of neonatal heart regeneration following apical resection.</a> Stem cell reports. 3, 1 (2014).</li><li>Andersen, D. C., Jensen, C. H., Sheikh, S. P., 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=Response+to+Sadek+et+al.+and+Kotlikoff+et+al.">Response to Sadek et al. and Kotlikoff et al.</a> Stem cell reports. 3, 3-4 (2014).</li><li>Phifer, C. B., Terry, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+hypothermia+for+general+anesthesia+in+preweanling+rodents.">Use of hypothermia for general anesthesia in preweanling rodents.</a> Physiol Behav. 38, 887-890 (1986).</li><li>Laflamme, M. A., Murry, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerating+the+heart.">Regenerating the heart.</a> Nat Biotechnol. 23, 845-856 (2005).</li><li>Laflamme, M. A., Murry, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+regeneration.">Heart regeneration.</a> Nature. , 326-335 (2011).</li><li>Jesty, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=c-kit++precursors+support+postinfarction+myogenesis+in+the+neonatal,+but+not+adult,+heart.">c-kit+ precursors support postinfarction myogenesis in the neonatal, but not adult, heart.</a> Proc Natl Acad Sci U S A. 109, 13380-13385 (2012).</li><li>Gonzalez-Rosa, J. M., Martin, V., Peralta, M., Torres, M., Mercader, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extensive+scar+formation+and+regression+during+heart+regeneration+after+cryoinjury+in+zebrafish.">Extensive scar formation and regression during heart regeneration after cryoinjury in zebrafish.</a> Development. 138, 1663-1674 (2011).</li></ol>

この記事の発行手数料は博士建侯にファイン科学ツールからの贈り物が主催しました。

Cardiac regeneration shows potential for the treatment and prevention of heart failure1, 2. Animal models are indispensable and play a critical role in understanding how cardiac regeneration occurs3-6. Many amphibians and fish regenerate heart tissue in response to injury, providing insight into our understanding of human cardiovascular disease13, 14. In terms of evolution, however, the pathophysiology should be more conserved between a mouse model and humans. Heart regeneration in mammalian animal models is a relatively new but burgeoning research field for the therapeutics of heart failure3-6. 
Recently, two surgical approaches, apical resection and myocardial infarction, have been established in the study of heart regeneration in neonatal mice6. Although apical resection provides a relatively easy and direct model for loss and/or injury of cardiomyocytes, the main limitation of apical resection compared to myocardial infarction is that this surgical method cannot be performed beyond P7 due to high mortality6. There are, however, a number of ways to reduce mortality with this method. For apical surgery on P7 pups, the pups are cooled to 16&#176;C for optimum hypothermia anesthesia. Generally, sterile conditions and surgical proficiency are critical for successful apical resection surgery. To improve survival, the surgery should be performed as quickly and gently as possible and a minimally invasive operation will reduce bleeding. Additionally, ICR/CD-1 mice are thought as good foster mothers and can effectively avoid of severe maternal cannibalization. For example, placing the P0 C57BL/6 pups with an ICR/CD-1 mother increases the survival rates of P1 pups with apical resection approximately from 60% to 80%ref.6. In addition, cryoinjury is an alternative procedure for study of cardiac regeneration. Compared to apical resection, cryoinjury model represents a different regenerative response and may be a more reproducible model15, 16. Thus far, a comparison of apical resection with other models warrants further investigations. 
Using phospho-histone H3 and 5-bromo-2-deoxyuridine staining, Porrello and colleagues showed that cardiomyocyte proliferation contributes to the heart regeneration of neonatal P1 mice after apical resection response. Moreover, no significant cardiac hypertrophy and increase of cell size have been observed in mice with apical resection relative to sham-operated controls. After apical resection of 7-day-old mice, there is no regenerative response for myocardium5. A more detailed time course analysis of morphology and cardiac fibrosis after apical resection has been demonstrated5. Of note, the usefulness of apical resection in neonatal mouse models for heart regeneration research has been questioned by Anderson and colleagues8. Although many independent groups have demonstrated the reproducibility of apical resection, variations in the details of surgical procedures and different amounts of resected myocardium might be responsible for confusing results10. Quantification of the genomic DNA in resected heart is helpful for control of minimal chamber exposition and reproducibility of this technique. Additional research will be needed to fully understand neomyogenesis and complete vs. incomplete heart regeneration in the apical resection model8-11.
The main aim of this study is to present the procedures of apical resection in a visualized way. This video of apical resection in the neonatal mouse will be helpful to increase the use of this method and teach it to heart regeneration researchers. In addition, it may promote the pursuit of better approaches for mammalian heart regeneration in animal models and facilitate the discovery and translation of the molecular and cellular mechanisms responsible for heart regeneration in humans.

Prolonged human life span leads to various aging- and lifestyle-related diseases, including heart failure, a leading cause of mortality. However, without replacement of lost or dysfunctional cardiac muscle cells, current therapeutics can only transiently improve cardiac function1, 2. Thus, it is necessary to discover and develop innovative strategies for cardiac regeneration and repair. The adult mammalian heart has limited regenerative potential. Studies from lower vertebrates, such as urodele amphibians and teleost fish, have provided unprecedented insights into the molecular and cellular mechanisms underlying heart regeneration3, 4. Recently, a neonatal mouse model of heart regeneration has emerged that might enable identification and characterization of more evolutionarily conserved pathophysiological events required for human heart regeneration5. 
Apical resection refers to the surgical removal of left ventricular apex. This procedure is restricted to 1- to 7-day-old (P1 to P7) mice due to its high lethality in older mice6. The cardiac regeneration process in neonatal mice after apical resection is expected to be as follows: (I) rapid and effective formation of a hematoma to seal the apex and prevent exsanguination; (II) cardiomyocyte regeneration and restoration of systolic function5, 7. Recent work has stimulated debate on the significance and efficiency of this model8-11. Thus, it is important to present the apical resection clearly and in detail. To this end, this protocol vividly and specifically describes a video of how I did apical resection based on a previous protocol6. 
Understanding the molecular mechanisms underlying cardiac regeneration is of importance for treatment of heart disease characterized by loss and/or injured cardiomyocytes, such as heart failure1, 2. Given the current and promising progress of apical resection in the research of cardiac regeneration, this study could promote the use of this technique and its uses in cardiac regeneration research.

<table><tbody><tr><td>Olsen-Hegar Needle Holders with Scissors, 1.5 mm</td><td>Fine Science Tools</td><td>12002-12</td><td></td></tr><tr><td>Vannas Spring Scissors - 2 mm Cutting Edge</td><td>Fine Science Tools</td><td>15000-03</td><td>Iridectomy scissors</td></tr><tr><td>Hot Bead Sterilizer</td><td>Fine Science Tools</td><td>18000-45</td><td></td></tr><tr><td>Iris Forceps, Straight, Serrated</td><td>Fine Science Tools</td><td>11064-07</td><td></td></tr><tr><td>Iris Forceps, Curved, Serrated</td><td>Fine Science Tools</td><td>11065-07</td><td></td></tr><tr><td>Shea Scissors - Curved/Blunt-Blunt/12cm</td><td>Fine Science Tools</td><td>14105-12</td><td></td></tr><tr><td>Round Handled Suture Tying Forceps, Straight</td><td>Fine Science Tools</td><td>18025-12</td><td></td></tr><tr><td>Round Handled Vannas Spring Scissors</td><td>Fine Science Tools</td><td>15400-12</td><td></td></tr><tr><td>Fine Hemostat</td><td>Fine Science Tools</td><td>13007-12</td><td></td></tr><tr><td>Magnifying Lamp</td><td>Luxolamp Corp</td><td>IM120&amp;nbsp;</td><td></td></tr><tr><td>Heating lamp</td><td>Brandt Equipment llc</td><td>51152/3</td><td></td></tr><tr><td>6-0 Prolene sutures</td><td>Ethicon</td><td>8889H</td><td></td></tr><tr><td>Skin glue-Vetbond Tissue Adhesive&amp;nbsp;</td><td>3M&amp;nbsp;</td><td>1469</td><td></td></tr><tr><td>Sterile Cotton Tipped Applicators</td><td>Dynarex</td><td>4305</td><td></td></tr><tr><td>WEBCOL Alcohol Prep Pad</td><td>Covidien</td><td>6818</td><td>Medium 2 PLY, 200/BOX, Satured with 70% Isopropyl Alcohol</td></tr><tr><td>Curity All Purpose Sponges</td><td>Covidien</td><td>9024</td><td>Non-woven 4 PLY, 4&quot;x4&quot; (10.2cm&amp;times;10.2cm)</td></tr><tr><td>Bench top protector sheet</td><td>KIMTECH SCIENCE</td><td>7546</td><td>18&quot; x 19.5&quot; (45.72cm x 49.53cm) x 50</td></tr><tr><td>0.9% Sodium Chloride, 250ml</td><td>Hospira Inc.</td><td>NDC 0409-6138-22</td><td></td></tr><tr><td>Betadine Solution Swabsticks</td><td>Purdue Products L.P.</td><td>NDC 67618-153-03</td><td></td></tr><tr><td>Autoclave</td><td>TOMY Digital Biology</td><td>SX-700</td><td>HIGH-PRESSURE STEAM STERILIZER</td></tr><tr><td>Slide scanner</td><td>HAMAMATSU</td><td>NanoZoomer 2.0-RS</td><td></td></tr></tbody></table>

apical resection mouse model to study early mammalian heart regeneration

心血管疾患は集中的なライフスタイルの変化による全世界を悩ませています。ハートの再生には、負傷や病気に負けた心筋細胞を修復し、復元するための大きな可能性を秘めています。一定の下等脊​​椎動物の強固な心臓再生とは対照的に、大人の哺乳類の心臓は、通常、心臓の再生および修復のための最小限の容量を示しています。しかし、最近の研究では、（P7へP1）1〜7生後日の間、新生児マウスの心臓が心尖切除（ すなわち 、外科的切断および左心室の頂点の曝露）後の重要な再生能力を保持し、知見とかなりの科学的関心を巻き起こしています。この発見を介して1つの主要な論争は、この重要な知見に複製または拡大するための努力で使用される多様な手術に関連した手順に起因する可能性があります。これらの命令は、動的にマウスモデルにおいて、頂端切除するための材料および方法論を提示します。このげっ歯類survivaの顕著なステップL手術は低体温麻酔、開胸、心臓心室尖部の外科的切断、縫合糸およびマウスの回復を伴います。記載されているアプローチは、心血管研究のための頂端切除マウスモデルの適用を拡大することができます。

仔マウスは1、2、および21日後に根尖切除術を安楽死させ、彼らの心は、H＆Eおよびマッソントリクローム染色のために回収しました。マッソントリクローム染色で青色は心外膜の細胞外マトリックス5の堆積を示しています。 図1Aに示すように、成功した頂端切除と、血餅を効果的に、LV一日後、頂端切除を封止するように形成されています。血液凝固および早期の心臓線維症の段階的な吸収が観測され、心筋の完全な再生は21日、頂端切除術（ 図1BおよびC）の後に行われるまで心筋組織による置換が発生します。形態素解析は、手術後21日目に切除および偽手術心（ 図1CおよびD）の間に差を示さありません。 <img alt="図1" src="/files/ftp_upload/53488/53488fig1.jpg" /> 図1.代表的H＆Eとマッソントリクローム染色。心室新生仔マウス心臓1（A）の心筋、 図2（B）、または21（C）日頂端切除後21日偽手術（D）の後 。各副図の場合は、左側のパネルのスケールバーは1mmで、右側のパネルのスケールバーは100μmである。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53488/53488fig1large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a>

Watch this Scientific Journal Video about Apical Resection Mouse Model to Study Early Mammalian Heart Regeneration at JoVE.com

Apical Resection Mouse Model to Study Early Mammalian Heart Regeneration

A step-by-step video protocol of apical resection is demonstrated in this study. Apical resection is a recently highlighted surgical approach in mammalian heart regeneration research. This study may promote the application of apical resection as a standard methodology in research into the mechanism underlying heart regeneration.

アピカル切除マウスモデルは、初期の哺乳類の心臓の再生を研究するために

Cardiovascular disease plagues the whole world due to intensive lifestyle changes. Heart regeneration holds great promise for repairing and restoring ...

apical-resection-mouse-model-to-study-early-mammalian-heart

Universidad San Sebastian

Research

JoVE Journal

Medicine

11.2K ビュー.  National Institutes of Health. A step-by-step video protocol of apical resection is demonstrated in this study. Apical resection is a recently highlighted surgical approach in mammalian heart regeneration research. This study may promote the application of apical resection as a standard methodology in research into the mechanism underlying heart regeneration.

ビデオ: Apical Resection Mouse Model to Study Early Mammalian Heart Regeneration

Transplantation of Neonatal Mouse Cardiac Macrophages into Adult Mice

In an injured neonatal myocardium, macrophages facilitate cardiomyocyte proliferation and angiogenesis and promote heart regeneration. The present study reveals that transplantation of neonatal cardiac macrophages recruited by injury promotes adult heart regeneration after myocardial infarction with improvement of cardiac function and cardiomyocyte proliferation. The results indicate that neonatal cardiac macrophage transplantation could be a promising strategy for cardiac injury treatment. Here, we provide the technical details, including the isolation of neonatal cardiac macrophages from apical resection-injured neonatal mouse hearts, the transplantation of macrophages into myocardial-infarcted adult mice, and the estimation of heart regeneration after a macrophage graft.

We provide a protocol for neonatal cardiac macrophage separation and transplantation into an adult mouse heart, which could be a promising way to promote cardiac repair.

成体マウスへの新生児マウス心臓マクロファージ移植

In an injured neonatal myocardium, macrophages facilitate cardiomyocyte proliferation and angiogenesis and promote heart regeneration. The present study ...

生化学

Capturing the Cardiac Injury Response of Targeted Cell Populations via Cleared Heart Three-Dimensional Imaging

Cardiovascular disease outranks all other causes of death and is responsible for a staggering 31% of mortalities worldwide. This disease manifests in cardiac injury, primarily in the form of an acute myocardial infarction. With little resilience following injury, the once healthy cardiac tissue will be replaced by fibrous, non-contractile scar tissue and often be a prelude to heart failure. To identify novel treatment options in regenerative medicine, research has focused on vertebrates with innate regenerative capabilities. One such model organism is the neonatal mouse, which responds to cardiac injury with robust myocardial regeneration. In order to induce an injury in the neonatal mouse that is clinically relevant, we have developed a surgery to occlude the left anterior descending artery (LAD), mirroring a myocardial infarction triggered by atherosclerosis in the human heart. When matched with the technology to track changes both within cardiomyocytes and non-myocyte populations, this model provides us with a platform to identify the mechanisms that guide heart regeneration. Gaining insight into changes in cardiac cell populations following injury once relied heavily on methods such as tissue sectioning and histological examination, which are limited to two-dimensional analysis and often damage the tissue in the process. Moreover, these methods lack the ability to trace changes in cell lineages, instead providing merely a snapshot of the injury response. Here, we describe how technologically advanced methods in lineage tracing models, whole organ clearing, and three-dimensional (3D) whole-mount microscopy can be used to elucidate mechanisms of cardiac repair. With our protocol for neonatal mouse myocardial infarction surgery, tissue clearing, and 3D whole organ imaging, the complex pathways that induce cardiomyocyte proliferation can be unraveled, revealing novel therapeutic targets for cardiac regeneration.

Cardiomyocyte proliferation following injury is a dynamic process that requires a symphony of extracellular cues from non-myocyte cell populations. Utilizing lineage tracing, passive CLARITY, and three-dimensional whole-mount confocal microscopy techniques, we can analyze the influence of a variety of cell types on cardiac repair and regeneration.

クリアされた心臓3次元イメージングによる標的細胞集団の心臓損傷応答のキャプチャ

Cardiovascular disease outranks all other causes of death and is responsible for a staggering 31% of mortalities worldwide. This disease manifests in ...

発生生物学

Permanent Ligation of the Left Anterior Descending Coronary Artery in Mice: A Model of Post-myocardial Infarction Remodelling and Heart Failure

Heart failure is a syndrome in which the heart fails to pump blood at a rate commensurate with cellular oxygen requirements at rest or during stress. It is characterized by fluid retention, shortness of breath, and fatigue, in particular on exertion. Heart failure is a growing public health problem, the leading cause of hospitalization, and a major cause of mortality. Ischemic heart disease is the main cause of heart failure.
Ventricular remodelling refers to changes in structure, size, and shape of the left ventricle. This architectural remodelling of the left ventricle is induced by injury (e.g., myocardial infarction), by pressure overload (e.g., systemic arterial hypertension or aortic stenosis), or by volume overload. Since ventricular remodelling affects wall stress, it has a profound impact on cardiac function and on the development of heart failure. A model of permanent ligation of the left anterior descending coronary artery in mice is used to investigate ventricular remodelling and cardiac function post-myocardial infarction. This model is fundamentally different in terms of objectives and pathophysiological relevance compared to the model of transient ligation of the left anterior descending coronary artery. In this latter model of ischemia/reperfusion injury, the initial extent of the infarct may be modulated by factors that affect myocardial salvage following reperfusion. In contrast, the infarct area at 24 hr after permanent ligation of the left anterior descending coronary artery is fixed. Cardiac function in this model will be affected by 1) the process of infarct expansion, infarct healing, and scar formation; and 2) the concomitant development of left ventricular dilatation, cardiac hypertrophy, and ventricular remodelling.
Besides the model of permanent ligation of the left anterior descending coronary artery, the technique of invasive hemodynamic measurements in mice is presented in detail.

Heart failure is the leading cause of hospitalization and a major cause of mortality. A model of permanent ligation of the left anterior descending coronary artery in mice is applied to investigate ventricular remodelling and cardiac dysfunction post-myocardial infarction. The technique of invasive hemodynamic measurements in mice is presented.

マウスの左前下冠動脈の動脈の常設ライゲーション：心筋梗塞後のリモデリングおよび心不全のモデル

Heart failure is a syndrome in which the heart fails to pump blood at a rate commensurate with cellular oxygen requirements at rest or during stress. It ...

医学

Clonal Analysis of the Neonatal Mouse Heart using Nearest Neighbor Modeling

By replacing lost or dysfunctional myocardium, tissue regeneration is a promising approach to treat heart failure. However, the challenge of detecting bona fide heart regeneration limits the validation of potential regenerative factors. One method to detect new cardiomyocytes is multicolor lineage tracing with clonal analysis. Clonal analysis experiments can be difficult to undertake, because labeling conditions that are too sparse lack sensitivity for rare events such as cardiomyocyte proliferation, and diffuse labeling limits the ability to resolve clones. Presented here is a protocol to undertake clonal analysis of the neonatal mouse heart by using statistical modeling of nearest neighbor distributions to resolve cardiomyocyte clones. This approach enables resolution of clones over a range of labeling conditions and provides a robust analytical approach for quantifying cardiomyocyte proliferation and regeneration. This protocol can be adapted to other tissues and can be broadly used to study tissue regeneration.

Presented here is a protocol for using multicolor lineage tracing and nearest-neighbor modeling to identify clonally derived cardiomyocytes during growth and regeneration in mice. This approach is objective, works across different labeling conditions, and can be adapted to incorporate a variety of image analysis pipelines.

By replacing lost or dysfunctional myocardium, tissue regeneration is a promising approach to treat heart failure. However, the challenge of detecting ...

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 ...

Transplantation of a 3D Bioprinted Patch in a Murine Model of Myocardial Infarction

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ligation is a challenging procedure and has a high mortality rate due to its nature. We developed a method to consistently transplant bioprinted patches of cells and hydrogels onto the epicardium of an infarcted mouse heart to test their regenerative properties in a robust and feasible way. First, a deeply anesthetized mouse is carefully intubated and ventilated. Following left lateral thoracotomy (surgical opening of the chest), the exposed LAD is permanently ligated and the bioprinted patch transplanted onto the epicardium. The mouse quickly recovers from the procedure after chest closure. The advantages of this robust and quick approach include a predicted 28-day mortality rate of up to 30% (lower than the 44% reported by other studies using a similar model of permanent LAD ligation in mice). Moreover, the approach described in this protocol is versatile and could be adapted to test bioprinted patches using different cell types or hydrogels where high numbers of animals are needed to optimally power studies. Overall, we present this as an advantageous approach which may change preclinical testing in future studies for the field of cardiac regeneration and tissue engineering.

This protocol aims to transplant a 3D bioprinted patch onto the epicardium of infarcted mice modeling heart failure. It includes details regarding anesthesia, the surgical chest opening, permanent ligation of the left anterior descending (LAD) coronary artery and application of a bioprinted patch onto the infarcted area of the heart.

心筋梗塞のマウスモデルにおける3次元バイオプリントパッチの移植

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ...

生物工学

Blood Circuit Reconstruction in an Abdominal Mouse Heart Transplantation Model

The surgical technique of heterotopic abdominal heart transplantation in mice is a standard model for research in transplantation immunology. Here, the established technique for a modified blood circuit reconstruction in a heterotopic abdominal heart transplantation model is presented. This method uses the intrathoracic inferior vena cava (IIVC) instead of the pulmonary artery of the donor heart for the anastomosis to the inferior vena cava of the recipient. It is facilitating and improving success rates for abdominal heart transplantation in mice.

A novel technique for blood circuit reconstruction in a heterotopic abdominal mouse heart transplantation model is demonstrated.

腹部マウス心臓移植モデルにおける血液回路再建

The surgical technique of heterotopic abdominal heart transplantation in mice is a standard model for research in transplantation immunology. Here, the ...

A Modified Cuff Technique for Mouse Cervical Heterotopic Heart Transplantation Model

Cardiac allograft rejection limits the long-term survival of patients after heart transplantation. A mouse heart transplantation model is ideal for investigating the mechanism of cardiac allograft rejection in preclinical studies because of their high homology with human genes. This understanding would help develop unique approaches to improving patients&#39; long-term survival treated with cardiac allografts. In a mouse model, abdominal donor heart implantation is commonly performed with an end-to-end&#160;anastomosis to the recipient&#39;s aorta and inferior vena cava using stitches. In this model, the donor&#39;s heart is implanted by end-to-end anastomosis to the recipient&#39;s carotid artery and jugular vein by the modified-Cuff technique. The transplantation surgery is performed without stitching and thus may increase the survival of the recipient since there is no interference with the blood supply and venous reflux of the lower body. This mouse model would help investigate the mechanisms underlying the immunological and pathological (acute/chronic) rejection of cardiac allografts.

In the present protocol, a mouse heart transplantation model is used for investigating the mechanism of cardiac allograft rejection. In this heterotopic heart transplantation model, operation efficiency is improved, and the survival of cardiac grafts is ensured by a cervical end-to-end anastomosis of heart implantation using a modified Cuff technique.

マウス頸部異所性心臓移植モデルのための修正カフ法

Cardiac allograft rejection limits the long-term survival of patients after heart transplantation. A mouse heart transplantation model is ideal for ...

An Apical Resection Model in the Adult Xenopus tropicalis Heart

It is known that in adult mammals, the heart has lost its regenerative capacity, making heart failure one of the leading causes of death worldwide. Previous research has demonstrated the regenerative ability of the heart of the adult Xenopus tropicalis, an anuran amphibian with a diploid genome and a close evolutionary relationship with mammals. Additionally, studies have shown that following ventricular apex resection, the heart can regenerate without scarring in X. tropicalis. Consequently, these previous results suggest that X. tropicalis is an appropriate alternative vertebrate model for the study of adult heart regeneration. A surgical model of cardiac regeneration in the adult X. tropicalis is presented herein. Briefly, the frogs were anesthetized and fixed; then, a small incision was made with iridectomy scissors, penetrating the skin and pericardium. Gentle pressure was applied to the ventricle, and the apex of the ventricle was then cut out with scissors. Cardiac injury and regeneration were confirmed by histology at 7-30 days post resection (dpr). This protocol established an apical resection model in adult X. tropicalis, which&#160;can be employed to elucidate the mechanisms of adult heart regeneration.

An Apical Resection Model in the Adult Xenopus tropicalis Heart

Xenopus tropicalis is an ideal model for regenerative research as many of its organs possess a remarkable regenerative capacity. Here, we present a method for constructing a heart injury model in X. tropicalis via apex resection.

成人 アフリカツメガ エル心臓の頂端切除モデル

It is known that in adult mammals, the heart has lost its regenerative capacity, making heart failure one of the leading causes of death worldwide. ...

Preparation of a Non-Cardiomyocyte Cell Suspension for Single-Cell RNA Sequencing from a Post-Myocardial Infarction Adult Mouse Heart

Myocardial infarction (MI) is one of the most common cardiovascular diseases, with increasing mortality worldwide. Non-cardiomyocytes account for more than half of the total cardiac cell population, and they contribute to adaptive compensations upon myocardial injury, including inflammatory responses, tissue repair, and scar formation. To study the post-MI cardiac microenvironment, single-cell RNA sequencing (scRNA-seq) is widely used to identify different cardiac cell types and intercellular communications. Among the procedures of scRNA-seq sample preparation, preparing the cell suspension is one of the most critical steps, because the cell viability can affect the quality of the scRNA-seq results. Therefore, we designed an experimental protocol for preparing a non-cardiomyocyte cell suspension from post-MI mouse hearts with an extra focus on improving the cell viability by choosing mild digestive enzymes, controlling the digestion time, and applying fluorescence-activated cell sorting (FACS). Finally, we isolated CD45+ cells from the non-cardiomyocyte cell suspension obtained through this protocol, and then we performed scRNA-seq.

Here, we describe a protocol to isolate a sufficient amount of single non-cardiomyocytes with high viability from a post-myocardial infarction (MI) mouse heart. This can be used for subsequent single-cell sequencing, flow cytometry analysis, and primary cell culture.

心筋梗塞後の成体マウス心臓からのシングルセルRNAシーケンシングのための非心筋細胞懸濁液の調製

Myocardial infarction (MI) is one of the most common cardiovascular diseases, with increasing mortality worldwide. Non-cardiomyocytes account for more ...

アピカル切除マウスモデルは、初期の哺乳類の心臓の再生を研究するために

要約

さらに動画を探す

この動画の章

成体マウスへの新生児マウス心臓マクロファージ移植

クリアされた心臓3次元イメージングによる標的細胞集団の心臓損傷応答のキャプチャ

マウスの左前下冠動脈の動脈の常設ライゲーション：心筋梗塞後のリモデリングおよび心不全のモデル

Clonal Analysis of the Neonatal Mouse Heart using Nearest Neighbor Modeling

新生児マウスの心筋梗塞、心臓再生のモデル

心筋梗塞のマウスモデルにおける3次元バイオプリントパッチの移植

腹部マウス心臓移植モデルにおける血液回路再建

マウス頸部異所性心臓移植モデルのための修正カフ法

成人アフリカツメガエル心臓の頂端切除モデル

心筋梗塞後の成体マウス心臓からのシングルセルRNAシーケンシングのための非心筋細胞懸濁液の調製

アピカル切除マウスモデルは、初期の哺乳類の心臓の再生を研究するために

要約

さらに動画を探す

この動画の章

成体マウスへの新生児マウス心臓マクロファージ移植

クリアされた心臓3次元イメージングによる標的細胞集団の心臓損傷応答のキャプチャ

マウスの左前下冠動脈の動脈の常設ライゲーション：心筋梗塞後のリモデリングおよび心不全のモデル

Clonal Analysis of the Neonatal Mouse Heart using Nearest Neighbor Modeling

新生児マウスの心筋梗塞、心臓再生のモデル

心筋梗塞のマウスモデルにおける3次元バイオプリントパッチの移植

腹部マウス心臓移植モデルにおける血液回路再建

マウス頸部異所性心臓移植モデルのための修正カフ法

成人 アフリカツメガ エル心臓の頂端切除モデル

心筋梗塞後の成体マウス心臓からのシングルセルRNAシーケンシングのための非心筋細胞懸濁液の調製

成人アフリカツメガエル心臓の頂端切除モデル