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

המחברים מודים בני הזוג. ג&#39;יימס הוקינס, Zu-Xi יו וואן צ&#39;ו מהלב הלאומי, הריאות ודם מכון (NHLBI) לסיוע שלהם עם ניתוח עכבר והכנה וצביעה של חלקי פרפין. המחברים מודים לועדת עורכי עמיתי NIH למערכת סיוע.

כל ניסויי העכבר אושרו על ידי הטיפול בבעלי חיים ושימוש בתכנית במכון הלאומי לבריאות (NIH) עם מספר פרוטוקול H0083R3. NHLBI IACUC אישר את הפרוטוקול ללא משככי כאבים. 1. היפותרמיה הרדמה בעכברים בילוד <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לעקר ספוגים וציוד כירורגי בחיטוי לפני הניתוח. הכן את כל החומרים כירורגית ולעבור על דקות 15-20 מעקר חרוז חם מראש להגיע 240 מעלות צלזיוס עד 270 מעלות צלזיוס. </li><li style=";text-align:right;direction:rtl"> העבר את כל C57BL הגורים / 6 (P1 גיל) מאם הסיעוד שלהם לכלוב עכבר נקי עם מצעים טריים וחומרי קינון. ברגע שהגורים נלקחים לחדר ניתוח, לבצע את ניתוח הפסגה מייד כדי למזער את זמנם בילה מופרד מאמו וכדי להפחית את הסיכון של קניבליזציה האימהית. </li><li style=";text-align:right;direction:rtl"> שימו ספוגים על מיטת קרח ולאחר מכן למקם גור אחד בספוג ל~ 3 דקות כדי להשיג הרדמה היפותרמיה. לאשר הרדמה על ידי התבוננות APNea ואקינזיה וצובטת את רגל אחורית. בדוק את המצב של ילודים לעתים קרובות בגלל מעט מדי זמן לא יעשה את האקינטי הגור וapneic, ומשך מוגזם של הרדמה יכול להוריד את שיעור הישרדות 12. </li></ol> 2. פתיחת בית החזה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> העבר את הגור מהמיטה הקרח על אזור benchtop כירורגית ולהשתמש בקלטת כדי לשתק את זרועות, רגליים, והזנב שלה במצב שכיבה. </li><li style=";text-align:right;direction:rtl"> לחטא את החזה באמצעות בטאדין ועדינות לנקות אותו באמצעות 70% משטח הכנת אלכוהול. </li><li style=";text-align:right;direction:rtl"> לעשות חתך בעור רוחבי לאורך האזור הלאה הצלע של חלל החזה באמצעות Vannas אביב מספריים, ולאחר מכן להקהות לנתח את השרירים הלאה צלע כדי להקל על גישה ללב. הערה: שיעורי הישרדות לשפר כאשר אובדן דם ממוזער במהלך התהליך כירורגי. </li></ol> 3. קטיעה כירורגית של לב חדרית איפקס <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ביד, בעדינות להפעיל לחצו על הבטן כדי exteriorizeשיאו של הלב. ספוג דם סביב אזור הניתוח עם החלת כותנה שקצה סטרילי להדמיה ברורה. לילודי שליטת דמה המופעל, להמשיך ישירות לשלב 4 (תפרים ושחזור של עכברים). </li><li style=";text-align:right;direction:rtl"> מתחת לפנס מגדלת ושימוש iridectomy מספריים, בעדינות לבצע כריתת טיפין טיפין של החדר השמאלי (LV) עד תא LV חשוף. להיות זהיר כדי למזער את החלקים שעברו כריתה של LV. כ -15% כריתה היא הכרחית כדי להשיג חשיפה אופטימלית של חדר LV. </li><li style=";text-align:right;direction:rtl"> ודא שהלב חוזר לחלל החזה פעם אחת קאמרי LV חשוף. </li></ol> 4. תפר ושחזור של עכברים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לתפור את הצלעות ושרירים יחד כדי לאטום את חלל החזה באמצעות תפרי פרולין 6-0 סטרילי, ולאחר מכן לסגור בקפידה אתר חתך בעור באמצעות דבק עור. </li><li style=";text-align:right;direction:rtl"> לחמם את הילוד תחת מנורת חום ל~ 3 דקות להתאוששות, ולאחר מכן לנקות את עקבות דם ודבק באמצעות 70% משטח הכנת אלכוהול befoמחדש reintroducing להמלטה שלה. לנסות להשלים את כל ההליך כירורגי בתוך 10 דקות, כי צמצום המשך הזמן מופרד מהאם משפר הישרדות גור לאחר כריתת הפסגה. </li><li style=";text-align:right;direction:rtl"> לאחר כל ניתוח, למקם את הכלים כירורגיים במגע עם חרוזים של מעקר חרוז החם לכ -20 שניות לעיקור מלא. לאפשר כלים כירורגיים להתקרר לRT לפני כל ניתוח. </li><li style=";text-align:right;direction:rtl"> לאחר שסיים את הניתוחים לכל הגורים בהמלטה, לערבב את הגורים עם המצעים של האמא וצואה לפני החזרתם לקן של האמא. הערה: באופן כללי, עכברי ICR / CD-1 הן אמהות אומנה טובה יותר מאשר C57BL / 6, אבל גם בקרב אמהות מניקות מאותו הרקע הגנטי, אינסטינקטים טיפוח להשתנות. אם שינוי אם מניקה יש צורך להפחית תמותה על ידי קניבלים אימהיים, להסיר את גורי P0 לאם מניקה ולאחר מכן לבצע כריתת הפסגה בP1. לבצע גם את פעולת דמה או r הפסגה רקesection בהמלטה אחת גורים, כי דמה ערבוב וגורים כירורגית יכולים להפחית את שיעורי ההישרדות של הקבוצה כירורגית של גורים 6. </li></ol> ניתוח 5. לאחר ניתוח <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> יום אחד לאחר הניתוח, לעקוב אחר הגורים, להבטיח שאין הבדל בין זיוף וקבוצות כירורגית, ולספור את מספר הגורים למדוד את שיעור ההישרדות. אם הניתוחים מבוצעים כראוי, שיעורי ההישרדות צריכים להיות דומים ויותר מ -60% בשני קבוצות דמה וניתוחיות. </li><li style=";text-align:right;direction:rtl"> בידוד לב וקיבעון. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> להרדים את הגורים בימים 1 ו -2 לאחר ניתוח באמצעות עריפת ראש ויום 21 לאחר ניתוח על ידי CO2 עם הבטחה של מוות על ידי נקע בצוואר הרחם, ולאחר מכן לנקות את החזה באמצעות הכנת אלכוהול 70%. </li><li style=";text-align:right;direction:rtl"> לחתוך את העור ושרירים קו האמצע של החזה ולאחר מכן פתח את החזה. </li><li style=";text-align:right;direction:rtl"> בלו כל הלב מחלל בית החזה ולתקן את כל הלב של כל דגימה ב 5 מיליליטר של 4% O paraformaldehyde / N ב RT. </li></ol></li><li style=";text-align:right;direction:rtl"> ביום שלמחרת, להעביר את הדגימות לאתנול 70%. דוגמאות ניתן לאחסן עד שבוע לפני הטבעת פרפין. </li><li style=";text-align:right;direction:rtl"> סעיפי Slice 5-מיקרומטר בעובי פרפין דרך כל החדר ולבצע hematoxylin הסטנדרטי וeosin (H &amp; E) וצביעת Trichrome של האסון לבחון את התגובה 5 משובי, 7. באופן ספציפי, כתם H &amp; E משמש כדי לבחון החלפת שריר וכתם Trichrome של האסון משמש כדי לבחון את תגובות fibrotic 5. </li><li style=";text-align:right;direction:rtl"> בחר את האזור של עניין ולהקים לפחות שלושה מוקדים לדגימה באמצעות סורק שקופיות באופן ידני. תמונה ולנתח את השקופיות מכתימה ב 40 × הגדלה פי הוראות היצרן עם פרמטרים של ברירת מחדל. </li></ol>

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	<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. 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עמלות פרסום לכתבה זו מומנו על ידי מתנה מכלי מדע פיין לד&quot;ר ג&#39;יאן הו.

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 mammal...

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

מחלת לב וכלי דם המכות כל העולם בשל שינויים באורח חיים אינטנסיביים. התחדשות לב טומנת בחובו הבטחה גדולה לתיקון ושחזור cardiomyocytes איבד בגלל פציעה ומחלה. בניגוד להתחדשות הלב החזקה של בעלי חוליות התחתונות מסוימים, לב של יונקים בוגרים בדרך כלל להראות יכולת מינימאלית להתחדשות לב ותיקון. עם זאת, מחקרים שנעשו לאחרונה עוררו עניין מדעי רב עם הממצא ש, בין יום הלידה 1 עד 7 (P1 לP7), לב העכבר בילוד שומר פוטנציאל התחדשות משמעותי לאחר כריתת הפסגה (כלומר, קטיעה כירורגית וחשיפה של קודקוד של חדר השמאלי). מחלוקת עיקרית אחת מעל ממצא זה יכול להיות בגלל הנהלים הקשורים לניתוח המגוונים המשמשים במאמצים כדי לשכפל או להרחיב על ממצא חשוב זה. הוראות אלה באופן דינמי להציג את חומרים ומתודולוגיה לכריתת הפסגה במודל של עכברים. הצעדים הבולטים של surviva מכרסם זההניתוח כרוך בהרדמה l היפותרמיה, פתיחת בית החזה, כריתה כירורגית של קודקוד של חדר לב, ותפר והתאוששות של עכברים. הגישה המתוארת יכול להרחיב את היישום של מודל עכבר כריתת הפסגה למחקר לב וכלי דם.

גורי עכבר היו מורדמים כריתת פוסט-פסגת 1, 2, ו -21 ימים, ולבם נאספו עבור H &amp; E וכתם Trichrome של האסון. צבע כחול בכתם Trichrome של האסון מציין בתצהיר של מטריקס epicardial 5. עם כריתת הפסגה מוצלחת, קריש דם נוצר בצורה יעילה כדי לאטום את כריתת פוסט-פסגת יום אחד LV, כפי שמוצג באיור 1 א.</...

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.

דגם פסגת כריתת עכבר כדי ללמוד התחדשות לב יונקים מוקדמת

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

Research

JoVE Journal

Medicine

11.2K Views.  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.

Video: 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.

השתלת מקרופאגים לב של עכבר יילודים בעכברים בוגרים

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

Biochemistry

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.

לכידת תגובת פגיעה לב של אוכלוסיות תאים ממוקדות באמצעות לב נקי תלת מימדי הדמיה

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

Developmental Biology

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.

קשירת קבע של העורק השמאלי קדמי יורד כלילית בעכברים: דגם של Remodelling אוטם שריר לב ופוסט-אי ספיקת לב

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 ...

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.

אוטם שריר לב עכברים ילודים, מודל התחדשות לב

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.

השתלה של תיקון ביו-מודפס בתלת מיתד במודל מורין של אוטם שריר הלב

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

Bioengineering

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.

שחזור מעגל הדם במודל השתלת לב של עכבר בטן

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.

טכניקת שרוול שונה למודל השתלת לב הטרוטופי של עכבר צוואר הרחם

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.

מודל כריתה אפית בלב הבוגר Xenopus tropicalis

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.

הכנת תרחיף תאים שאינו קרדיומיוציטים לריצוף RNA חד-תאי מלב עכבר בוגר לאחר אוטם שריר הלב

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