An Apical Resection Model in the Adult Xenopus tropicalis Heart

Podsumowanie

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.

Streszczenie

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 can be employed to elucidate the mechanisms of adult heart regeneration.

Wprowadzenie

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 failure^2,3,4. 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'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 diminished^8,9. 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 frogs^4,15,16.

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 infarction^5,6.

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'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.

Protokół

All the experimental protocols related to X. tropicalis were approved by the Jinan University Animal Care Committee.

1. Surgery

1. 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.
2. Anesthetize an adult X. tropicalis with tricaine by placing it 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.
   ​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.
3. Thoracotomy
   1. 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's skin during the operation.
   2. 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.
4. Ventricular apex resection
   1. 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).
   2. 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).
   3. 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.
5. 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.
6. In the sham operation group, perform the thoracotomy, open the pericardium, and suture, without performing the apex resection.

2. Surgical recovery

1. 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.
2. Upon regaining consciousness, observe the animal'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.
   ​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.

3. Detection of the repair condition after cardiac injury

1. Collect the hearts of X. tropicalis at multiple time points after surgery.
   1. 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.
   2. 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).
2. Gradient dehydration
   1. 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.
3. Paraffin embedding
   1. 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 °C for 2-3 h.
      ​NOTE: It is essential to avoid air bubbles as they affect the section during the embedding process.
4. Freeze the embedded tissue at −20 °C for 1 h, and section it.
5. After sectioning the heart, perform standard hematoxylin and eosin (H&E) and Masson's trichrome staining techniques on the sections¹¹.

Wyniki

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...

Dyskusje

Apical resection, which involves the surgical amputation of the apex of the heart, has been described in zebrafish and mice^6,18; 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.

Materiały

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