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 surg...

<ol>
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P., Virmani, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiology+of+acute+myocardial+infarction.">Pathophysiology of acute myocardial infarction.</a> Medical Clinics of North America. 91 (4), 553-572 (2007).</li><li>Sessions, S. K., Bryant, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+that+regenerative+ability+is+an+intrinsic+property+of+limb+cells+in+Xenopus.">Evidence that regenerative ability is an intrinsic property of limb cells in Xenopus.</a> Journal of Experimental Zoology. 247 (1), 39-44 (1988).</li><li>Laflamme, M. A. <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. 473 (7347), 326-335 (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> Nature Protocols. 9 (2), 305-311 (2014).</li><li>Tzahor, E., Poss, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+regeneration+strategies:+Staying+young+at+heart.">Cardiac regeneration strategies: Staying young at heart.</a> Science. 356 (6342), 1035-1039 (2017).</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 (6020), 1078-1080 (2011).</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=Regulation+of+neonatal+and+adult+mammalian+heart+regeneration+by+the+miR-15+family.">Regulation of neonatal and adult mammalian heart regeneration by the miR-15 family.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (1), 187-192 (2013).</li><li>Poss, K. D., Wilson, L. G., Keating, M. T. <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+in+zebrafish.">Heart regeneration in zebrafish.</a> Science. 298 (5601), 2188-2190 (2002).</li><li>Liao, S., 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=Heart+regeneration+in+adult+Xenopus+tropicalis+after+apical+resection.">Heart regeneration in adult Xenopus tropicalis after apical resection.</a> Cell &amp; Bioscience. 7, 70 (2017).</li><li>Marshall, L. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stage-dependent+cardiac+regeneration+in+Xenopus+is+regulated+by+thyroid+hormone+availability.">Stage-dependent cardiac regeneration in Xenopus is regulated by thyroid hormone availability.</a> Proceedings of the National Academy of Sciences of the United States of America. 116 (9), 3614-3623 (2019).</li><li>Witman, N., Murtuza, B., Davis, B., Arner, A., Morrison, J. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recapitulation+of+developmental+cardiogenesis+governs+the+morphological+and+functional+regeneration+of+adult+newt+hearts+following+injury.">Recapitulation of developmental cardiogenesis governs the morphological and functional regeneration of adult newt hearts following injury.</a> Developmental Biology. 354 (1), 67-76 (2011).</li><li>Cano-Martinez, 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=Functional+and+structural+regeneration+in+the+axolotl+heart+(Ambystoma+mexicanum)+after+partial+ventricular+amputation.">Functional and structural regeneration in the axolotl heart (Ambystoma mexicanum) after partial ventricular amputation.</a> Archivos de Cardiolog&#237;a de M&#233;xico. 80 (2), 79-86 (2010).</li><li>Kragl, M., 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=Cells+keep+a+memory+of+their+tissue+origin+during+axolotl+limb+regeneration.">Cells keep a memory of their tissue origin during axolotl limb regeneration.</a> Nature. 460 (7251), 60-65 (2009).</li><li>Oberpriller, J. 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. G., 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=Apex+resection+in+zebrafish+(Danio+rerio)+as+a+model+of+heart+regeneration:+A+video-assisted+guide.">Apex resection in zebrafish (Danio rerio) as a model of heart regeneration: A video-assisted guide.</a> International Journal of Molecular Sciences. 22 (11), 5865 (2021).</li><li>Lee-Liu, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+expression+profile+of+the+response+to+spinal+cord+injury+in+Xenopus+laevis+reveals+extensive+differences+between+regenerative+and+non-regenerative+stages.">Genome-wide expression profile of the response to spinal cord injury in Xenopus laevis reveals extensive differences between regenerative and non-regenerative stages.</a> Neural Development. 9, 12 (2014).</li><li>Wu, H. 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>

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. 
<p...

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.

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			<td>Acid Alcohol Fast Differentiation Solution</td>
			<td>Beyotime</td>
			<td>C0163M</td>
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			<td>aladdin</td>
			<td>A104916</td>
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			<td>Alcohol Soluble Eosin Y Stainin Solution</td>
			<td>Servicebio</td>
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			<td>ST2600-100g</td>
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			<td>Ethanol absolute</td>
			<td>Guangzhou Chemical Reagent Factory</td>
			<td>HB15-GR-0.5L</td>
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			<td>Hematoxylin Stain Solution</td>
			<td>Servicebio</td>
			<td>G1004-500ML</td>
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			<td>Neutral balsam</td>
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			<td>Operating Scissors</td>
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			<td>Paraffins</td>
			<td>Leica</td>
			<td>39601095</td>
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			<td>Para-formaldehyde Fixative</td>
			<td>Servicebio</td>
			<td>G1101-500ML</td>
			<td></td>
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			<td>Phosphate Buffered Saline (PBS) powder</td>
			<td>Servicebio</td>
			<td>G0002-2L</td>
			<td></td>
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			<td>Phosphomolybdic&#160;acid&#160;hydrate</td>
			<td>Macklin</td>
			<td>P815551</td>
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			<td>Stereo microscope</td>
			<td>Leica</td>
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			<td>surgical forceps</td>
			<td>ChangZhou</td>
			<td>zfq-11-btjw</td>
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			<td>Tricaine</td>
			<td>Macklin</td>
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			<td>Xylene</td>
			<td>Guangzhou Chemical Reagent Factory</td>
			<td>IC02-AR-0.5L</td>
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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&#3...

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. ...

Cardiac injury model has significant implication for elucidating the mechanism of cardiac regeneration. This protocol established an apical resection model in adult X.Tropicalis, which can be employed to elucidate the mechanism of adult heart regeneration. Previous studies have stated that heart can regenerate after apical resection in adult X.Tropicalis.And protocols of apex resection have been published in zebrafish and mouse models, but there is no published protocol for apical resection in X.tropicalis. Losing the regenerative capacity in adult mammals makes heart failure one of the leading causes of death worldwide. Illustrating the mechanisms of cardiac regeneration using this technique will have significant implications for therapies for ischemic heart diseases.It is better to practice the technique well before performing the surgery, as it should not affect the operating process time. And it is highly crucial to keep the frog anesthetized. To begin, place the anesthetized Xenopus tropicalis abdomen up, and cover the abdomen with distilled water-soaked gauze to avoid drying of the animal's skin during the operation.Gently press the chest with forceps and find the center of the chest parallel to the lower forelimb. Lift the skin with ophthalmic scissors, and gently make a small incision of approximately 1 centimeter. 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. Gently, clamp the pericardium with forceps and softly break it using ophthalmic scissors using the apex of the heart. Wait for the pericardium to come off due to systolic blood pumping.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, approximately 14%of the ventricle. Place the heart back into the chest using forceps and an absorbent ball.Suture the skin with a 4-0 non-absorbent surgical thread, ensuring no suture into the muscle layer to prevent postoperative mortality. In the sham group, perform the thoracotomy and open the pericardium, as demonstrated earlier, but suture without performing the apex resection. Place the postoperative animal with its abdomen facing up in a Petri dish containing a small quantity of deionized water.Wait about 10 minutes for the animal to regain consciousness, and observe its mobility and wound suture during activity. Transfer the frogs to a container filled with pure water for cultivation and replace it with pure water daily to avoid wound infection. After apical resection of the Xenopus tropicalis heart, a blood clot formed and sealed the wound in the ventricle, as observed by hematoxylin and eosin staining, as well as through Masson's trichrome staining.Morphological analysis of the heart at 0, 7, 14 and 30 days of post-resection revealed that the blood clot caused by the heart injury disappeared at day 30. At the same time, on day 14, significant regeneration of the apex was seen, and by day 30, the resected heart apex had almost entirely regenerated. Histological analysis showed a fibrin mass at the resection site on day 14, and the heart regenerated completely without scars by day 30.Myocardial fibrosis analysis of the heart by Masson's staining showed that within the 14th day of apical resection, the clot disappeared and was replaced by fibrin. From day 14 to 30, the myocardium incrementally replaced the fibrin and repaired the damaged ventricle apex. This technique will allow researchers to explore the cellular mechanism of heart regeneration in Xenopus tropicalis and know the cells that are related to cardiac regeneration in heart of frogs using lineage-tracing analysis after apex resection.

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Medicine

1.2K visualizações.  Jinan University. O modelo de lesão cardíaca tem implicações significativas para a elucidação do mecanismo de regeneração cardíaca. Este protocolo estabeleceu um modelo de ressecção apical em X.Tropicalis adulto, que pode ser empregado para elucidar o mecanismo de regeneração cardíaca do adulto. Estudos anteriores afirmaram que o coração pode se regenerar após ressecção apical em adultos X.Tropicalis.E protocolos de ressecção de ápice foram publicados em modelos de zebrafish e camu...