Name: an apical resection model in the adult xenopus tropicalis heart
Uploaded: 2022-11-18T14:30:00.000+00:00
Duration: 5 min 45 s
Description: Watch this Scientific Journal Video about An Apical Resection Model in the Adult Xenopus tropicalis Heart at JoVE.com

This work was supported by grants from the National Key R&#38;D Program of China (2016YFE0204700), the National Natural Science Foundation of China (82070257, 81770240), and the Research Grant of Key Laboratory of Regenerative Medicine, Ministry of Education, Jinan University (ZSYXM202004 and ZSYXM202104), China.

All the experimental protocols related to X. tropicalis were approved by the Jinan University Animal Care Committee.
1. Surgery
<ol>
	<li>Preoperative preparation: Keep ready ophthalmic scissors, ophthalmic forceps, needle forceps, absorbent balls, filter paper, and surgical sutures/needles for apex resection in the hearts of X. tropicalis. Refer to the Table of Materials for detailed information. Before use, sterilize all the surgical instruments by autoclaving, and prepare an adequate quantity of ice for future use.</li>
	<li>Anesthetize an adult X. tropicalis with tricaine by placing it&#160;in 500 mL of tricaine solution (1 mg/mL) at room temperature for 4 min19, and then put it on a surface of ice on the operating table to ensure the frog does not wake up during the operating process. 
	&#8203;CAUTION: The whole procedure requires 5-10 min; the time of anesthesia should not be too long, otherwise the X. tropicalis may not be able to wake up after the operation.</li>
	<li>Thoracotomy
	<ol>
		<li>Place the anesthetized X. tropicalis abdomen up, and cover the abdomen of the frog with gauze presoaked in distilled water to avoid the drying of the animal&#39;s skin during the operation.</li>
		<li>Gently press the chest with forceps, find the center of the chest parallel to the lower forelimb, lift the skin with ophthalmic scissors, and gently make a small incision of ~1 cm. Using ophthalmic scissors, pick up the muscle layer under the skin, and create a wound in the central chest muscle. As the heart is located in the upper position of the wound site, gently press the chest with the ophthalmic forceps to squeeze the heart out from the wound. 
		CAUTION: As the skin of X. tropicalis can produce antimicrobial peptides, it is unnecessary to employ conventional disinfection procedures on the surgical site of the X. tropicalis. Any disinfectant will damage the skin of the X. tropicalis.</li>
	</ol>
	</li>
	<li>Ventricular apex resection
	<ol>
		<li>Gently clamp the pericardium with forceps, and gently break it using ophthalmic scissors near the apex of the heart. Wait for the pericardium to come off due to systolic blood pumping (Figure 1A, B).</li>
		<li>Hold the tip of the heart with forceps in the non-dominant hand, and lift the heart slightly according to the cardiac contraction rhythm. When the heart contracts to recirculate blood through the blood vessels, quickly cut off the apex of the heart (~14% of the ventricle) (Figure 1C).</li>
		<li>To ensure the amount of apex resection is approximately 14% of the whole heart, analyze the heart weight (HW) and surface area with or without resection20. Place the heart into the chest using forceps and an absorbent ball. 
		NOTE: Do not press the heart directly with the forceps; otherwise, other parts of the heart will be punctured.</li>
	</ol>
	</li>
	<li>Suture the skin with a 4-0 non-absorbent non-silk surgical thread suture (Figure 1D). Be careful to avoid suturing into the muscle layer to prevent postoperative mortality. Wait for the skin wound to naturally heal within 1 week following surgery.</li>
	<li>In the sham operation group, perform the thoracotomy, open the pericardium, and suture, without performing the apex resection.</li>
</ol>
2. Surgical recovery
<ol>
	<li>Place the postoperative X. tropicalis with its abdomen facing up in a Petri dish containing a small quantity of deionized water (do not completely flood the animal). Wait for the X. tropicalis to wake up within ~10 min.</li>
	<li>Upon regaining consciousness, observe the animal&#39;s mobility and activity, as well as the wound suture during activity. Transfer the frogs that have regained their balance to a container filled with pure water for cultivation. Replace the water with pure water every day to avoid wound infection. 
	&#8203;NOTE: With these measures, the survival rate could reach ~90%. A prolonged time of anesthesia and excessive bleeding during surgery both lead to death, which typically occurs on the day of surgery.</li>
</ol>
3. Detection of the repair condition after cardiac injury
<ol>
	<li>Collect the hearts of X. tropicalis at multiple time points after surgery.
	<ol>
		<li>After anesthetizing the X. tropicalis with tricaine, open the abdomen, and use forceps to remove the other internal organs and tissues to find the location of the heart. 
		NOTE: Due to the apex resection, the heart is likely to develop alongside other tissues and organs during the repair process. Sometimes, it sticks to the muscle wall and is not easy to separate, so it needs to be handled carefully during collection.</li>
		<li>After finding the heart, use forceps to gently tear the other organs from the heart. Gently peel away the other tissues surrounding the heart to expose the heart. Use forceps to gently lift the heart, and cut it out using scissors. Place the heart immediately in PBS to remove any remaining blood, and photograph it with a stereoscope for documentation (Figure 2).</li>
	</ol>
	</li>
	<li>Gradient dehydration
	<ol>
		<li>Blot the hearts with filter paper to dry the excess PBS residues, and place them in a 24-well cell culture dish. Use 1-2 ml of 4% paraformaldehyde to fix the heart tissues overnight. The following day, perform ethanol dehydration. First, use 70% ethanol overnight, followed by 80% ethanol, 90% ethanol, and 100% ethanol for gradient dehydration (1 h each time). Repeat the use of 100% ethanol three times.</li>
	</ol>
	</li>
	<li>Paraffin embedding
	<ol>
		<li>Treat the dehydrated heart tissue with xylene for 6-8 min. Place the xylene-treated tissue in a glass container filled with paraffin wax at 65 &#176;C for 2-3 h. 
		&#8203;NOTE: It is essential to avoid air bubbles as they affect the section during the embedding process.</li>
	</ol>
	</li>
	<li>Freeze the embedded tissue at &#8722;20 &#176;C for 1 h, and section it.</li>
	<li>After sectioning the heart, perform standard hematoxylin and eosin (H&#38;E) and Masson&#39;s trichrome staining techniques on the sections11.</li>
</ol>

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O., Oberpriller, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+of+the+adult+newt+ventricle+to+injury.">Response of the adult newt ventricle to injury.</a> Journal of Experimental Zoology. 187 (2), 249-253 (1974).</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 (9), 1663-1674 (2011).</li><li>Ellman, D. 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Y., 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=Fosl1+is+vital+to+heart+regeneration+upon+apex+resection+in+adult+Xenopus+tropicalis.">Fosl1 is vital to heart regeneration upon apex resection in adult Xenopus tropicalis.</a> npj Regenerative Medicine. 6 (1), 36 (2021).</li><li>Chablais, F., Jazwinska, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+myocardial+infarction+in+adult+zebrafish+using+cryoinjury.">Induction of myocardial infarction in adult zebrafish using cryoinjury.</a> Journal of Visualized Experiments. (62), e3666 (2012).</li></ol>

The authors have no conflicts of interest to declare.

Apical resection, which involves the surgical amputation of the apex of the heart, has been described in zebrafish and mice6,18; however, this has not been described in X. tropicalis. This report describes a credible model of cardiac injury and demonstrates that the heart of adult X. tropicalis can fully regenerate after apical resection without scarring. However, some shortcomings need to be improved, and certain details need attention. 
Although apical resection can establish a heart injury model, ensuring the same extent of excision of the heart is challenging due to the diversity of the frogs, which have different heart sizes. To ensure that hearts with apexes of the same size are resected, it is necessary to select frogs of the same size, and the operator must practice extensively prior to the experiment. 
Second, the ability to accurately and quickly locate and expose the heart is critical to the procedure's success, as the process of finding the heart can result in the puncture of the adjacent blood vessels and cause bleeding, thus impeding the procedure's progress or even causing failure. The duration of anesthesia should not be too short or too long. If it is too short, the frog may wake up before the operation is complete, and if this process is too long, it is likely to lead to the death of the frog. As mentioned in protocol sections 2-5, the suturing must be done cautiously. To ensure that the ratio of the amount of apex resection compared to the whole heart is about the same across frogs, the heart weight (HW) and surface area with or without resection are analyzed20, and a small portion of the heart apex is resected to ensure chamber exposure6. 
Cryoinjury, MI, and apical resection are the three most widely used methods to induce cardiac injury in zebrafish and mice6,18,21. The limitation of this article is that only the third method was used, meaning this work does not provide any information about the different regenerative responses after injury with the other two methods6. Cryoinjury results in the death of a large number of cells in ~20% of the ventricular wall, similar to mammalian infarcts21. MI, which is also called coronary artery occlusion, has higher survival rates in X. tropicalis in comparison to apical resection in mice6 but is more difficult to achieve in X. tropicalis because the hearts of tropical clawed frogs are smaller than those of mice. The X. tropicalis apical resection method established in this study is simpler to perform and has a high postoperative survival rate. 
The apical resection developed in this report will be a significant method for investigating the molecular mechanisms of regeneration in the hearts of X. tropicalis. A previous study also demonstrated that Fosl1 is indispensable for cardiac regeneration in X. tropicalis following apical resection20. This model of apical resection heart injury in X. tropicalis can be used for learning more about the molecular mechanisms underlying cardiac regeneration to promote the study of the genes and molecular mechanisms related to human cardiac regeneration.

Heart failure has been a leading cause of mortality worldwide in recent years. Since 2000, the number of deaths due to heart failure has been increasing over time. More than 9 million people died from cardiomyopathy in 2019, which accounted for 16% of the total number of mortalities globally1. Due to the loss of the regenerative capacity of the heart in adult mammals, in some cases, there are not enough cardiomyocytes to maintain the contraction functions in the heart, which affects heart function and contributes to abnormal ventricular remodeling and heart failure2,3,4. Indeed, in mammals, the heart has the poorest regenerative capacity compared to the other organs, such as the liver, lungs, intestines, bladder, bone, and skin. As the aging of the world&#39;s population becomes a global megatrend, the challenges we face with heart disease will intensify5.
Elucidating the mechanisms of cardiac regeneration may have significant implications for therapies for ischemic heart disease. Reports have revealed that the hearts of neonatal mice have a regenerative capacity after apex resection6. Nevertheless, this regenerative capacity is lost after 7 days of age7. Studies have demonstrated that adult mammalian hearts are unable to regenerate because their capacity for cardiomyocyte proliferation has diminished8,9. However, the hearts of lower vertebrates possess a powerful regenerative capacity after injury. For instance, zebrafish10, X. tropicalis11, Xenopus laevis12, newt13, and axolotl14 are capable of complete regeneration after apex resection. Additionally, the other parts of the bodies of some lower vertebrates can also undergo complete regeneration, such as the limbs of newts and the tails, lenses, and arms of tropical clawed frogs4,15,16.
Establishing cardiac injury models is the first step to elucidating the mechanisms underlying cardiac regeneration and has great significance in regenerative research. Researchers have developed various methods to build cardiac injury models, including stabbing, contusing, genetic ablation, cryoinjury, and infarction5,6.
Cryoinjury, myocardial infarction (MI), and apex resection are widely used for inducing cardiac injury, and the type of injury may have substantial effects on the following regeneration of cardiomyocytes6. Depending on the surgical technique, the heart&#39;s response to regeneration could vary. Cryoinjury causes massive cell death and produces fibrotic scars in the hearts of zebrafish17, thus creating a model that resembles mammalian infarction. Apical resection is performed by cutting away a part of the ventricular tissues, which has been done in zebrafish10 and X. tropicalis11, without causing permanent scars. This study performed apical resection, which is a simpler operation and requires fewer surgical instruments than cryoinjury. Using lineage-tracing analysis, a previous study demonstrated that cardiac regeneration is related to the proliferation of cardiomyocytes that pre-exist in the hearts of the mouse6 and zebrafish18, but no reports exist for amphibians. Hence, the model of apex resection in X. tropicalis plays an important role in elucidating the mechanisms underlying regenerative responses.

<table><tbody><tr><td>Acetic acid</td><td>GHTECH</td><td>64-19-7-500ml</td><td></td></tr><tr><td>Acid Alcohol Fast Differentiation Solution</td><td>Beyotime</td><td>C0163M</td><td></td></tr><tr><td>Acid Fuchsin</td><td>aladdin</td><td>A104916</td><td></td></tr><tr><td>Alcohol Soluble Eosin Y Stainin Solution</td><td>Servicebio</td><td>G1001-500ML</td><td></td></tr><tr><td>BioReagent</td><td>Beyotime</td><td>ST2600-100g</td><td></td></tr><tr><td>Ethanol absolute</td><td>Guangzhou Chemical Reagent Factory</td><td>HB15-GR-0.5L</td><td></td></tr><tr><td>Hematoxylin Stain Solution</td><td>Servicebio</td><td>G1004-500ML</td><td></td></tr><tr><td>Neutral balsam</td><td>Solarbio</td><td>G8590</td><td></td></tr><tr><td>Operating Scissors</td><td>Prosperich</td><td>HC-JZ-YK-Z-10cm</td><td></td></tr><tr><td>Paraffins</td><td>Leica</td><td>39601095</td><td></td></tr><tr><td>Para-formaldehyde Fixative</td><td>Servicebio</td><td>G1101-500ML</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS) powder</td><td>Servicebio</td><td>G0002-2L</td><td></td></tr><tr><td>Phosphomolybdic&amp;nbsp;acid&amp;nbsp;hydrate</td><td>Macklin</td><td>P815551</td><td></td></tr><tr><td>Stereo microscope</td><td>Leica</td><td></td><td></td></tr><tr><td>surgical forceps</td><td>ChangZhou</td><td>zfq-11-btjw</td><td></td></tr><tr><td>Surgical Suture</td><td>HUAYON</td><td>18-5140</td><td></td></tr><tr><td>Tricaine</td><td>Macklin</td><td></td><td></td></tr><tr><td>Xylene</td><td>Guangzhou Chemical Reagent Factory</td><td>IC02-AR-0.5L</td><td></td></tr></tbody></table>

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.

The hearts were collected at 0 dpr, 7 dpr, 14 dpr, and 30 dpr. The morphological analysis revealed that the blood clot caused by the heart injury disappeared at 30 dpr (Figure 2). At the same time, the appearance of the hearts at 30 dpr in the resection group was similar to that of the hearts in the sham operation group; there were no obvious wounds (Figure 2). After apical resection, a blood clot formed and sealed the wound in the ventricle, as observed by H&#38;E (Figure 3) and Masson&#39;s trichrome staining (Figure 4). Within 14 dpr, the clot gradually disappeared and was replaced by fibrin (Figure 4). From 14 dpr to 30 dpr, the myocardium incrementally replaced the fibrin and repaired the damaged ventricle apex (Figure 4). The histological analysis showed no difference between the sham and resected hearts at 30 dpr (Figure 3 and Figure 4). This morphological and histological analysis revealed that adult X. tropicalis could repair and regenerate the injured ventricular apex by 30 dpr.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64719/64719fig01.jpg" /> 
Figure 1: Surgical procedure. (A) Representative image of an exposed heart covered by the pericardium. (B) Representative image of a heart in the systolic phase, during which the pericardium was peeled off; the dashed line indicates the site of the apical resection of the heart. (C) Representative image of the apex resection of the heart. (D) Representative image of the skin suture after surgery. Scale bars = 1 mm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64719/64719fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64719/64719fig02.jpg" /> 
Figure 2: Morphological analysis of X. tropicalis hearts from 0 dpr to 30 dpr. (A) Representative images of hearts from the sham (left) and apical resection groups (right) at 0 dpr, with the apex of the heart significantly absent in the resection group. (B,C) Representative images of hearts from the sham (left) versus resection groups (right) at 7-14 dpr, with many blood clots appearing in the hearts of the resection group at 7 dpr. At 14 dpr, significant regeneration is seen at the apical resection site, but the edges of the apex of the heart are not even. (D) Representative image of hearts from the sham (left) versus resection groups (right) at 30 dpr; the apex of the resected heart has almost entirely regenerated. Scale bars = 1 mm. Abbreviation: dpr = days post resection. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64719/64719fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64719/64719fig03.jpg" /> 
Figure 3: Histological analysis of Xenopus tropicalis hearts from 0 dpr to 30 dpr. Representative images of H&#38;E staining using hearts from the sham and resection groups at 0 dpr to 30 dpr. (A) Representative image of H&#38;E staining using hearts from the sham group, with dashed lines indicating the approximate amputation planes. (B) Representative image of H&#38;E staining using hearts from the resection group at 0 dpr. (C-E) Representative images of H&#38;E staining using hearts from the resection group at 7-30 dpr, with large amounts of red blood cells accumulated within the wound at 7 dpr (arrows). A mass of fibrin has appeared in the resection site at 14 dpr (arrowheads). The hearts have regenerated completely without scars at 30 dpr. Scale bar = 500 &#181;m. Abbreviation: dpr = days post resection. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64719/64719fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64719/64719fig04.jpg" /> 
Figure 4: Myocardial fibrosis analysis of X. tropicalis hearts from 0 dpr to 30 dpr. Representative images of Masson&#39;s staining of hearts from the sham and resection groups at 0 dpr to 30 dpr. (A) Representative image of Masson&#39;s staining of hearts from the sham group, with dashed lines indicating the approximate amputation planes. (B) Representative image of Masson&#39;s staining of hearts from the resection group at 0 dpr. (C-E) Representative images of Masson&#39;s staining of hearts from the resection group at 7-30 dpr, with large amounts of red blood cells accumulated within the wound at 7 dpr (arrows). A mass of fibrin has appeared in the resection site at 14 dpr (arrowheads). The hearts have regenerated completely without scars at 30 dpr. Scale bar = 500 &#181;m. Abbreviation: dpr = days post resection. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64719/64719fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about An Apical Resection Model in the Adult Xenopus tropicalis Heart at JoVE.com

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.

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. ...

an-apical-resection-model-in-the-adult-xenopus-tropicalis-heart

Nanyang Technological University

Research

JoVE Journal

Medicine

1.3K Views.  Jinan University. Het hartletselmodel heeft een belangrijke implicatie voor het ophelderen van het mechanisme van hartregeneratie. Dit protocol stelde een apicale resectiemodel vast bij volwassen X.Tropicalis, dat kan worden gebruikt om het mechanisme van volwassen hartregeneratie op te helderen. Eerdere studies hebben verklaard dat het hart kan regenereren na apicale resectie bij volwassen X.Tropicalis.En protocollen van apische resectie zijn gepubliceerd in zebravis- en muismodellen, maar er is geen g...

Video: An Apical Resection Model in the Adult Xenopus tropicalis Heart

Imaging Cleared Embryonic and Postnatal Hearts at Single-cell Resolution

Single clonal tracing and analysis at the whole-heart level can determine cardiac progenitor cell behavior and differentiation during cardiac development, and allow for the study of the cellular and molecular basis of normal and abnormal cardiac morphogenesis. Recent emerging technologies of retrospective single clonal analyses make the study of cardiac morphogenesis at single cell resolution feasible. However, tissue opacity and light scattering of the heart as imaging depth is increased hinder whole-heart imaging at single cell resolution. To overcome these obstacles, a whole-embryo clearing system that can render the heart highly transparent for both illumination and detection must be developed. Fortunately, in the last several years, many methodologies for whole-organism clearing systems such as CLARITY, Scale, SeeDB, ClearT, 3DISCO, CUBIC, DBE, BABB and PACT have been reported. This lab is interested in the cellular and molecular mechanisms of cardiac morphogenesis. Recently, we established single cell lineage tracing via the ROSA26-CreERT2; ROSA26-Confetti system to sparsely label cells during cardiac development. We adapted several whole embryo-clearing methodologies including Scale and CUBIC (clear, unobstructed brain imaging cocktails and computational analysis) to clear the embryo in combination with whole mount staining to image single clones inside the heart. The heart was successfully imaged at single cell resolution. We found that Scale can clear the embryonic heart, but cannot effectively clear the postnatal heart, while CUBIC can clear the postnatal heart, but damages the embryonic heart by dissolving the tissue. The methods described here will permit the study of gene function at a single clone resolution during cardiac morphogenesis, which, in turn, can reveal the cellular and molecular basis of congenital heart defects.

We describe a protocol to volumetrically image fluorescent protein labeled cells deep inside intact embryonic and postnatal hearts. Utilizing tissue-clearing methods in combination with whole mount staining, single fluorescent protein-labeled cells inside an embryonic or postnatal heart can be imaged clearly and accurately.

Single clonal tracing and analysis at the whole-heart level can determine cardiac progenitor cell behavior and differentiation during cardiac development, ...

Developmental Biology

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The efficient in vivo visualization of blood vessels and the reliable quantification of their complexity are key to understanding the biology and disease of the vascular network. Here, we provide a detailed method to visualize blood vessels with a commercially available fluorescent dye, human plasma acetylated low density lipoprotein DiI complex (DiI-AcLDL), and to quantify their complexity in Xenopus tropicalis. Blood vessels can be labeled by a simple injection of DiI-AcLDL into the beating heart of an embryo, and blood vessels in the entire embryo can be imaged in live or fixed embryos. Combined with gene perturbation by the targeted microinjection of nucleic acids and/or the bath application of pharmacological reagents, the roles of a gene or of a signaling pathway on vascular development can be investigated within one week without resorting to sophisticated genetically engineered animals. Because of the well-defined venous system of Xenopus and its stereotypic angiogenesis, the sprouting of pre-existing vessels, vessel complexity can be quantified efficiently after perturbation experiments. This relatively simple protocol should serve as an easily accessible tool in diverse fields of cardiovascular research.

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

This protocol demonstrates a fluorescence-based method to visualize the vasculature and to quantify its complexity in Xenopus tropicalis. Blood vessels can be imaged minutes after the injection of a fluorescent dye into the beating heart of an embryo after genetic and/or pharmacological manipulations to study cardiovascular development in vivo.

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The ...

Cryo-injury Induced Heart Regeneration in the Axolotl and Echocardiography and Unbiased Quantitative Histology to Evaluate Regenerative Progression

The urodele amphibians, salamanders and newts, represent the phylogenetic group closest to mammals capable of performing complete myocardial regeneration following ventricular resection. The resection model has generated a basic knowledge of the processes involved in cardiac repair. However, the model does not relate well to clinical situations in which tissue damage, apoptosis, necrosis, fibrosis, and hypertrophy are all key detrimental consequences of ischemia-induced myocardial infarctions rather than tissue removal. On the other hand, cryoinjury-induced myocardial infarction resembles ischemia-induced myocardial infarction more closely. Here we provide a detailed description of the cryoinjury procedure in the axolotl (Ambystoma mexicanum), which provides a tool for investigating basic mechanisms in cardiac repair in a tetrapod model. Additionally, we provide quantitative methods for estimating infarction size non-invasively in vivo with echocardiography and for measuring infarction size precisely with unbiased quantitative histology ex vivo.

Here we present a surgery protocol to induce cryoinjury to the ventricular myocardium of the axolotl. Additionally, we present a protocol to non-invasively estimate infarction fraction during the regenerative process with echocardiography and a protocol to precisely measure infarction fraction in the excised heart with unbiased quantitative histology.

The urodele amphibians, salamanders and newts, represent the phylogenetic group closest to mammals capable of performing complete myocardial regeneration ...

Apical Resection Mouse Model to Study Early Mammalian Heart Regeneration

Cardiovascular disease plagues the whole world due to intensive lifestyle changes. Heart regeneration holds great promise for repairing and restoring cardiomyocytes lost due to injury and disease. In contrast to the robust cardiac regeneration of certain lower vertebrates, adult mammalian hearts typically show minimal capacity for heart regeneration and repair. However, recent studies have sparked considerable scientific interest with the finding that, between postnatal day 1 to 7 (P1 to P7), the neonatal mouse heart retains significant regenerative potential after apical resection (i.e., surgical amputation and exposure of left ventricular apex). One major controversy over this finding might be due to the diverse surgery-related procedures used in efforts to replicate or expand upon this important finding. These instructions dynamically present the materials and methodology for apical resection in a mouse model. The salient steps of this rodent survival surgery involve hypothermia anesthesia, thoracotomy, surgical amputation of heart ventricular apex, and suture and recovery of mice. The approach described could expand the application of the apical resection mouse model for cardiovascular research.

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.

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

2D and 3D Echocardiography in the Axolotl (Ambystoma Mexicanum)

Cardiac malfunction as a result of ischemic heart disease is a major challenge, and regenerative therapies to the heart are in high demand. A few model species such as zebrafish and salamanders that are capable of intrinsic heart regeneration hold promise for future regenerative therapies for human patients. To evaluate the outcome of cardioregenerative experiments it is imperative that heart function can be monitored. The axolotl salamander (A. mexicanum) represents a well-established model species in regenerative biology attaining sizes that allows for evaluation of cardiac function. The purpose of this protocol is to establish methods to reproducibly measure cardiac function in the axolotl using echocardiography. The application of different anesthetics (benzocaine, MS-222, and propofol) is demonstrated, and the acquisition of two-dimensional (2D) echocardiographic data in both anesthetized and unanesthetized axolotls is described. 2D echocardiography of the three-dimensional (3D) heart can suffer from imprecision and subjectivity of measurements, and to alleviate this phenomenon a solid method, namely intra/inter-operator/observer analysis, to measure and minimize this bias is demonstrated. Finally, a method to acquire 3D echocardiographic data of the beating axolotl heart at a very high spatiotemporal resolution and with pronounced blood-to-tissue contrast is described. Overall, this protocol should provide the necessary methods to evaluate cardiac function and model anatomy, and flow dynamics in the axolotl using ultrasound imaging with applications in both regenerative biology and general physiological experiments.

2D and 3D Echocardiography in the Axolotl Ambystoma Mexicanum

Here we present echocardiography protocols for two-dimensional and three-dimensional image acquisition of the beating heart of the axolotl salamander (Ambystoma mexicanum), a model species in heart regeneration. These methods allow for longitudinal evaluation of cardiac function at a high spatiotemporal resolution.

Cardiac malfunction as a result of ischemic heart disease is a major challenge, and regenerative therapies to the heart are in high demand. A few model ...

Facial Transplants in Xenopus laevis Embryos

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. To identify where signaling pathways and tissues act during patterning of the developing face, a 'face transplant' technique has been developed in embryos of the frog Xenopus laevis. A region of presumptive facial tissue (the "Extreme Anterior Domain" (EAD)) is removed from a donor embryo at tailbud stage, and transplanted to a host embryo of the same stage, from which the equivalent region has been removed. This can be used to generate a chimeric face where the host or donor tissue has a loss or gain of function in a gene, and/or includes a lineage label. After healing, the outcome of development is monitored, and indicates roles of the signaling pathway within the donor or surrounding host tissues. Xenopus is a valuable model for face development, as the facial region is large and readily accessible for micromanipulation. Many embryos can be assayed, over a short time period since development occurs rapidly. Findings in the frog are relevant to human development, since craniofacial processes appear conserved between Xenopus and mammals.

Facial Transplants in Xenopus laevis Embryos

A technique for transplanting "Extreme Anterior Domain" facial tissue between Xenopus laevis embryos has been developed. Tissue can be moved from one gene expression background into another, allowing the study of local requirements for craniofacial development and for signaling interactions between facial regions.

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. ...

Biology

Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation

The neural crest (NC) is a transient dorsal neural tube cell population that undergoes an epithelium-to-mesenchyme transition (EMT) at the end of neurulation, migrates extensively towards various organs, and differentiates into many types of derivatives (neurons, glia, cartilage and bone, pigmented and endocrine cells). In this protocol, we describe how to dissect the premigratory cranial NC from Xenopus laevis embryos, in order to study NC development in vivo and in vitro. The frog model offers many advantages to study early development; abundant batches are available, embryos develop rapidly, in vivo gain and loss of function strategies allow manipulation of gene expression prior to NC dissection in donor and/or host embryos. The NC explants can be plated on fibronectin and used for in vitro studies. They can be cultured for several days in a serum-free defined medium. We also describe how to graft NC explants back into host embryos for studying NC migration and differentiation in vivo.

Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation

This protocol describes how to dissect premigratory cranial neural crest (NC) from Xenopus laevis neurulas. These explants can be plated on fibronectin and cultured in vitro, or grafted back into host embryos. This technique allows studying the mechanisms of NC epithelium-to-mesenchyme transition, migration, and differentiation.

The neural crest (NC) is a transient dorsal neural tube cell population that undergoes an epithelium-to-mesenchyme transition (EMT) at the end of ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

Standardized Dissection and Sampling Protocol for Adult Xenopus Organs

Techniques for Rapidly Sampling Six Crucial Organs in Adult Xenopus

Xenopus has been a powerful model organism for understanding vertebrate development and disease for over a hundred years. While experimental analysis and dissection techniques of the embryo have been well documented, descriptions of adult Xenopus structures and organs, together with techniques for working with adults, have not been updated to take into consideration the requirements of such modern approaches as quantitative proteomics and single-cell transcriptomics. The cell-type and gene-centric perspectives require contrasting observations in embryonic stages to those in adult tissues. The organs of the larva undergo significant changes in their overall structure, morphology, and anatomical location all along the larval to adult transition, most notably during massive metamorphosis remodeling. Establishing robust standards for organ identification and dissection is crucial to ensure datasets resulting from studies performed at different laboratories can be consistent. The present protocol identifies six of the organs in the adult Xenopus, demonstrating methods for dissection and sampling of the heart ventricle, liver, fat body, pancreas, paired kidney, and skin of the adult Xenopus. Depending on the preservation methods, the dissected organs can be used for quantitative proteomics, single cell/nuclei transcriptomics, in situ hybridization, immunohistochemistry, histology, etc. This protocol aims to standardize tissue sampling and facilitate multi-lab investigations of the adult organ systems.

Author Spotlight: Standardizing Tissue Sampling in Proteomics and Immunochemistry Research

This article presents a guide for sampling six significant and diverse organs in adult Xenopus that can be rapidly and easily accessed: the heart ventricle, liver lobe, pancreas, fat bodies, paired kidneys, and skin.

Xenopus has been a powerful model organism for understanding vertebrate development and disease for over a hundred years. While experimental analysis and ...

Apical Resection Neonatal Mouse Model: A Surgical Procedure to Amputate the Ventricular Apex to Study Regenerative Potential in a Neonate Mouse Heart

Source: Li, Y. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/62108">Transplantation of Neonatal Mouse Cardiac Macrophages into Adult Mice.</a> J. Vis. Exp. (2021).
In this video, we demonstrate the procedure to surgically remove the left ventricular apex of a neonatal mouse heart.

Source: Li, Y. et al. Transplantation of Neonatal Mouse Cardiac Macrophages into Adult Mice. J. Vis. Exp. (2021).
In this video, we demonstrate the ...

An Apical Resection Model in the Adult Xenopus tropicalis Heart

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