Cauterization of the root of the left coronary artery as a straightforward, large and reproducible ischemic injury model in neonatal mice

lab animal Protocol https://doi.org/10.1038/s41684-024-01443-x Cauterization of the root of the left coronary artery as a straightforward, large and reproducible ischemic injury model in neonatal mice Check for updates Tianyuan Hu, Bernd K. Fleischmann    & Mona Malek Mohammadi    Abstract The adult mammalian heart is known to have very limited regenerative capacity, explaining at least in part the frequency of cardiovascular diseases and their impact as the leading cause of death worldwide. By contrast, the neonatal heart has the ability to regenerate upon injury, and the molecular mechanisms underlying this regenerative capacity are intensely investigated to provide novel cues for the repair of the adult heart. However, the existing rodent neonatal injury models—apex resection, left anterior descending artery ligation and cryoinjury—have limitations, such as being technically demanding, yielding a nonphysiological injury type and/or lack of reproducibility. Here we have therefore established a novel ischemic heart injury method in neonatal mice via cauterization of the root of the left coronary artery. This surgical procedure is technically straightforward, requires less than 10 min for completion and yields reproducible, large ischemic lesions (40% of the left ventricle) with low mortality rates (10% of animals). The injury also induces secondary pulmonary hypertension shortly after surgery, allowing to study the response of the right ventricle. Moreover, neonatal mice at postnatal days 1 and 3 display strongly opposing outcomes after the surgery, because of the lack of cardiac regeneration at the later stage. Thus, this new neonatal heart injury model is of great use for mechanistic studies exploring the regeneration of the left ventricle and the adaptation of the right ventricle upon myocardial infarction. Institute of Physiology I, Life and Brain Center, Medical Faculty, University of Bonn, Bonn, Germany. Lab Animal e-mail: ; lab animal Introduction The limited regenerative capacity of the human heart due to low cardiomyocyte (CM) renewal rate, which further deteriorates with age1, poses a substantial challenge contributing to the prevalence of cardiovascular diseases (CVDs) as the leading cause of death worldwide. New therapeutic strategies are therefore needed to unlock the regenerative ability of the adult heart and reduce the burden of CVDs. To identify new therapies, researchers have used model organisms with endogenous regenerative ability such as the zebrafish2 and, more recently, the neonatal mouse3. Studies have shown that neonatal mice at postnatal day (P)1 can regenerate their heart, but this ability is lost 7 days after birth3. The regenerative response of P1 mice is characterized by enhanced CM proliferation, angiogenesis and minimal hypertrophy or fibrosis3. However, at P7 the lack of regeneration coincides with CM cell-cycle arrest. Unraveling the regenerative mechanisms of the heart, which seem to be part of a complex process involving an interplay between different cell types, as well as transcriptome and epigenetic changes4,5, requires further mechanistic studies. For this purpose, a reliable and physiologically relevant injury model is critical. To this aim, different injury models have been developed, each with advantages and disadvantages. While apex resection and cryoinjury are not ischemic lesions, the induction of myocardial infarction (MI) through left anterior descending artery (LAD) ligation can very well mimic the physiological condition of an acute ischemia and an infarct. However, LAD ligation presents challenges regarding its success rate and the reproducibility of injury size due to suture positioning and/or diverse coronary patterning in mice6. Given the technical difficulties of the surgery, the method can only be performed by experts. Here, we describe a new MI model in neonatal mice by cauterizing the entire left coronary artery (LCA). This newly established neonatal MI model is easy and fast to perform, is reproducible and generates a large ischemic injury in the myocardium without damaging the epicardial or endocardial layer of the heart. Our protocol describes all the details necessary to successfully perform the surgery and aims to make it accessible to a broader range of scientists interested in cardiovascular regeneration. The ultimate goal is to uncover the mechanisms of cardiovascular regeneration in neonatal mice with the aim of discovering therapeutic strategies for the human heart and reducing the burden of CVDs in the future. When employing this surgery model, we unveiled the ability of P1 mice to regenerate after such a large ischemic injury, whereas this ability was already impaired in P3 mice7. Despite both P1 and P3 mice experiencing the same initial injury size, P1 mice exhibited enhanced CM proliferation rate, angiogenesis and protective mechanisms against global activation of apoptosis, whereas P3 mice showed low degrees of CM proliferation and angiogenesis and enhanced apoptosis, resulting in left ventricular dilation and heart failure at 7 days post surgery (dps). Furthermore, the large ischemic lesion induced secondary pulmonary hypertension as a consequence of the left ventricle (LV) failure, raising a prominent adaptive response in P1 right ventricle (RV) characterized by enhanced CM proliferation and angiogenesis, without deterioration of RV function. In P3, however, the RV showed a maladaptive response characterized by dilation, reduced wall thickness and CM hypertrophy as well as reduced function together with LV failure as early as 1 dps. Thus, cauterization of the LCA serves not only as a LV MI model but also as a model of secondary pulmonary hypertension, which is the most common form of pulmonary hypertension leading to RV failure in adults7. Likewise, and regardless of the underlying pathology, persistent pulmonary hypertension is an important clinical problem in newborns8. Development of the method Following the discovery of the regenerative ability of the neonatal mouse heart upon apical resection3, numerous studies have aimed to explore the underlying mechanisms of heart regeneration by implementing this surgery technique. Although this method seemed easy to perform, reproducibility was challenging due to variations in scar size9,10. Consequently, cryoinjury employing a defined cryoprobe was established to standardize the method and injury size11,12. However, it soon became evident that this method was not ideal and did not recapitulate Lab Animal Protocol lab animal the typical features of ischemic lesions. With this method, a necrotic scar is generated that does not fully resolve and persists much longer akin to what has been reported in the zebrafish11,13,14. The different sizes of cryoprobes and different durations of cryoprobe application made it possible to investigate the extent of regeneration in the neonatal mouse heart. These studies showed that neonatal mice cann (...truncated)