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 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.
Ischemic heart disease is a leading cause of fatalities globally1,2. Ischemia induced myocardial infarction results in widespread cell death in cardiomyocytes3. Although rapid medical intervention can reduce the risk of immediate demise, the ensuing fibrotic response that humans share with traditional mammalian model animals (mouse, rat, rabbit, pig, etc.) results in scar tissue formation that can ultimately lead to cardiac hypertrophy, arrhythmias, and heart failure4. Contrary to mammals, cardiac regeneration is well established in some non-amnionic animal models such as zebrafish5 and salamanders6. Traditionally, cardiac regeneration has been studied in these species after partial ventricular resection or pinching5,6,7,8,9,10. However, in 2011, three groups independently developed a cryoinjury-induced myocardial infarction technique in zebrafish11,12,13. The cryoinjury technique results in necrosis and apoptosis in a major part of the zebrafish heart ventricle and an initial accumulation of fibrotic tissue that more closely models the pathological development of the mammalian heart following ischemic infarction compared to ventricular resection11,12,13. Additionally, methodological comparisons of cryoinjury-induced myocardial infarction to ischemia-induced myocardial infarction by coronary artery ligation in the mouse and the pig have proven the cryoinjury technique to be a useful alternative in mammalian animal models14,15. Inspired by the methods involved in the zebrafish cryoinjury model16,17 we have developed a similar model in the axolotl18, an amphibian renowned for its regenerative capabilities19, that allows investigation of the mechanisms involved in cardiac repair in this tetrapod after tissue damage rather than tissue removal.
Here we present a detailed protocol on how to perform cryoinjury induced myocardial infarction in the axolotl. We place special emphasis on rapid and minimally invasive crucial steps that increase survival, recovery, and experimental reproducibility. Additionally, we provide instructions for appropriate techniques for evaluating anatomical regeneration in vivo using echocardiography and ex vivo using unbiased stereology based quantitative histology.
Cryoinjury-induced myocardial infarction in the axolotl can be applied to investigate basic mechanisms involved in myocardial regeneration in this tetrapod. The axolotl is tolerant of cryoinjury-induced myocardial infarction, which affects at least 45% of the ventricle, resulting in a decrease in stroke volume and cardiac output without any behavioral changes in its relatively languid lifestyle, however, more severe injuries will potentially lead to decreased animal health.
In short, to induce cryoinjury, a ventral incision is made on the thorax of an anaesthetized axolotl. The ventricle is accessed using blunt dissection and a precooled cryoprobe is applied for 10 s to induce cryoinjury. The ventral incision is sutured and the animal quickly recovers with no signs of behavioral changes after consciousness is regained. Control/sham animals receive similar treatment but experience a non-cooled cryoprobe. Cardiac performance can be monitored using echocardiography (high frequency ultrasound systems necessary, ≥20 MHz) as exhaustively described on healthy axolotl hearts previously20, and infarction fraction can be estimated non-invasively and repeatedly during the regenerative process. Infarcted hearts can be harvested at any time during the 3-month regenerative process, cryosectioned for histology, and stained using standard procedures (e.g., eosin & hematoxylin or Masson’s trichrome staining). In particular, Masson’s trichrome staining allows a clear distinction between infarcted and healthy parts of the ventricle. The infarction fraction of the ventricle is determined using stereological techniques previously described for cardiac studies21.
To minimize experimental variation, the surgical procedure of the cryoinjury should follow sterile procedures and surgical training should be conducted on a number of animals before attempting to use animals for specific regenerative experiments. With training, the cryoinjury procedure can be conducted on a large range of axolotl sizes and ages, from juveniles (5 g, 7 cm) to large adults (100 g, 25 cm). It is critical that the cryoprobe has a sufficient size and is cooled adequately to provide a robust and repeatable cryoinjury to the axolotl ventricle. For very small animals (5-8 g, 7-9 cm), the cryoprobe can be constructed with a smaller diameter. While it is imperative that the cryoinjury protocol is conducted using an anesthetic with analgesic properties like benzocaine and MS-222 (or with the additional application of a secondary analgesic agent), the follow up echocardiography can be conducted with other anesthetics with only limited analgesic properties, such as propofol, which has been described to affect cardiac function less than benzocaine and MS-222 in the axolotl22.
The cryoinjury procedure is limited in the sense that it does not produce an ischemia-induced myocardial infarction by coronary artery occlusion in the same manner as coronary artery ligation procedures that more closely resemble clinical cases of myocardial infarction in humans. However, the ligation-based method is not applicable in the trabeculated amphibian heart with very little coronary vasculature and a mostly luminal oxygen supply. Also, cryoinjury-induced myocardial infarction has been described to recapitulate most of the pathological consequences of ischemia-induced myocardial infarctions14,16. The injury zone generated by cryoinjury is highly localized to the tissue in proximity to the cryoprobe and, though this does not resemble a complex and branchlike infarction resulting from coronary blockage, it is advantageous in an experimental setting as the border zone between healthy and infarcted tissue can easily be recognized, and the progression of newly formed cardiomyocytes can be studied.
The axolotl is a considerably larger animal than the zebrafish with a more complicated cardiovascular system, including a heart that consists of three chambers (two atria, one ventricle) and a functional, though not anatomical, separation of blood flow in oxygenated and deoxygenated currents23 compared to the two chambered heart and serial flow system found in teleosts. The cryoinjury procedure previously described in the zebrafish does not involve post-operative suturing of the incised ventral surface16,17. This is necessary in the axolotl to avoid unnecessary exposure of the heart to the non-sterile aquatic environment in the animal’s laboratory habitat.
Unbiased stereology-based quantitative histology is currently underreported in the regenerative field in which most quantitative measurements of infarction fraction relies on area drawing on histological section at the mid infarction zone and in some cases two neighboring sections24. Since the concept of stereology can be applied in histological examinations in any model species to provide more robust and unbiased measurements, we propose that this freely available method should be incorporated in quantitative evaluations of heart regeneration not only in the axolotl, but in all regenerative species.
The authors have nothing to disclose.
We wish to acknowledge Casper Bindzus Foldager, Asger Andersen, and Michael Pedersen (all at the Department of Clinical Medicine, Aarhus University) and David Gardiner (Department of Developmental and Cell Biology, University of California, Irvine) for help in the initial development of the axolotl cryoinjury model and ultrasound examination.
S&T Scissor SAS-15 | S&T AG – Microsurgical Instruments | Iridectomy scissors | |
Vevo 2100 | Fujifilm, VisualSonics | High frequency ultrasound scanner |
.