Published: March 20th, 2021
We provide a protocol for neonatal cardiac macrophage separation and transplantation into an adult mouse heart, which could be a promising way to promote cardiac repair.
In an injured neonatal myocardium, macrophages facilitate cardiomyocyte proliferation and angiogenesis and promote heart regeneration. The present study reveals that transplantation of neonatal cardiac macrophages recruited by injury promotes adult heart regeneration after myocardial infarction with improvement of cardiac function and cardiomyocyte proliferation. The results indicate that neonatal cardiac macrophage transplantation could be a promising strategy for cardiac injury treatment. Here, we provide the technical details, including the isolation of neonatal cardiac macrophages from apical resection-injured neonatal mouse hearts, the transplantation of macrophages into myocardial-infarcted adult mice, and the estimation of heart regeneration after a macrophage graft.
Heart regeneration is a promising strategy to recover cardiac function after cardiac injury and to protect against heart failure1,2,3. Following myocardial injury, macrophages infiltrate the injured heart and have been explored as the key factors during neonatal heart regeneration4,5,6. Besides clearing necrotic cellular debris and inducing inflammation, macrophages promote angiogenesis5 and cardiomyocyte proliferation7 after neonatal mice myocardial infarction.
Our previous study illustrates that transplantation of neonatal cardiac macrophages isolated from injured neonatal heart enhances adult heart regeneration7, indicating that neonatal cardiac macrophage transplantation could be a promising strategy to treat cardiac injury. Here we provide the technical details, including isolation of neonatal cardiac macrophages from apical resection-injured neonatal mouse hearts, the transplantation of macrophages in to myocardial-infarcted adult mice, and the estimation of heart regeneration after macrophage graft (Figure 1).
All experiments were conducted in accordance with the Guide for the Use and Care of Laboratory Animals. All animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC), Fuwai Hospital, Chinese Academy of Medical Sciences. Aseptic technique is required throughout the procedure to prevent contamination of the surgical site.
1. Apical resection operation in neonatal 1-day-old Cx3cr1 GFP/+ mouse (C57BL/6 background)
2. Preparation of the neonatal cardiac macrophage suspension
NOTE: All these experimental procedures should be carried out in a dark place and under sterile conditions.
3. Neonatal cardiac macrophage transplantation
4. Result evaluation
The protocol described here is summarized in a flow chart (Figure 1). We performed apical resection operation on 1-day-old Cx3cr1GFP/+ mouse. As shown in Figure 2A, the neonatal Cx3cr1GFP/+ mouse was anchored on the operating platform under the stereoscope after anesthesia. We applied pressure alternately on the mouse chest and abdomen with the help of two forceps, which can be set up as a tract to guide the heart popping out of the chest. Any extra mechanical damage to the heart that may affect heart regeneration should be avoided. The heart was immobilized by the surrounding chest tissues, which facilitated the operation on the myocardium. We found that cutting off less than a 1 mm diameter of the resected apex tissue by iridectomy scissors was appropriate when the left ventricular chamber began oozing. Successful induction of apical resection model is necessary for macrophage transplantation8,9.
We sorted the GFP+ macrophages strictly following a neonatal heart dissociation protocol1 (Figure 2).
Neonatal cardiac macrophages were injected into the myocardial infarcted adult mouse heart shortly after the isolation. In order to confirm the efficiency of the macrophage transplantation, we performed immunofluorescence staining at 7 days after the injection. The results showed that GFP+ macrophages could be found in the adult myocardial infarcted mouse heart, indicating the successful transplantation of neonatal cardiac macrophage. We employed co-immunostaining of pH3 with α-actinin. The co-localization was considered to be cardiomyocyte proliferation. The results illustrated that the number of proliferative cardiomyocytes was upregulated in the macrophage-injected group, indicating that adult cardiomyocyte proliferation ability was enhanced after the transplantation (Figure 3).
Adult mouse heart regeneration was evaluated 1 month after the transplantation. We performed echocardiogram on the adult mouse and found that macrophage injection could enhance the cardiac function after myocardial infarction. Masson's staining was performed, and the results illustrated that infarcted area was remarkably reduced after the neonatal cardiac macrophage transplantation (Figure 3). All these results demonstrated that neonatal cardiac macrophage transplantation promotes adult mouse heart regeneration and cardiomyocyte proliferation.
Figure 1: Schematic representation of neonatal cardiac macrophage transplantation. Please click here to view a larger version of this figure.
Figure 2: Images of macrophage isolation. A) The hearts are cut into small pieces. B) The mouse hearts are dissociated. C) The suspension is filtered and transferred to a 15 mL centrifuge tube. D) The number of neonatal cardiac macrophages is calculated. Please click here to view a larger version of this figure.
Figure 3: Neonatal cardiac macrophage transplantation promotes adult heart regeneration. A) (Left) Immunofluorescence images show the proliferative cardiomyocytes (arrow, pH3 green, α-actinin red). (Right) Statistical analysis show that cardiomyocyte proliferation increases after macrophage transplantation. B) (Left) Echocardiogram images show the cardiac function in adult mouse 1-month after myocardial infarction. (Right) Statistical analysis show that cardiac function is enhanced after macrophage transplantation. C) Masson's staining shows the infarcted area in adult mouse 1-month after myocardial infarction. (Right) Statistical analysis show that infarcted area is reduced after Mϕ, macrophage transplantation. Please click here to view a larger version of this figure.
Here, we provide an effective approach to prepare, acquire, and transplant neonatal cardiac macrophages to promote adult mouse heart regeneration.
Apical resection is a simple and effective operation to stimulate heart regeneration. We have optimized the details of apical resection to ensure the maximum survival rate of the animals involved in the operation10. Anesthesia time should not be longer than 3 minutes, which lead to hypothermia-induced death, or shorter than 2 minutes, which would cause excessive bleeding during the operation. The standard apical resection was supposed to amputate about 1.5 mm diameter of the apex tissue. However, the purpose of the operation here was to stimulate abundant macrophage infiltration during heart regeneration process. Resection of less than 1.5 mm diameter was acceptable as it could guarantee a maximum survival rate and effective macrophage recruitment at the same time. Only the skillfulness of the operator influenced the survival rate, and the operated mice could not survive the prolonged operation duration. The operator should complete the whole procedure within 5 minutes.
In our former study, we found that acute inflammation promoted neonatal heart regeneration. Intramyocardial microinjection of immunogenic zymosan A particles into neonatal mouse heart could promote cardiomyocyte proliferation6. Recently, Molkentin et al. claimed that macrophage infiltration that is stimulated by intracardiac injection of zymosan A, cell debris, and freeze/thaw killed cells can promote cardiac repair, confirming that acute inflammation and macrophages are essential in cardiac repair rather than stem cells differentiating into cardiomyocytes11. Sadek et al.5 reported that macrophages could promote neonatal heart regeneration via angiogenesis. Our recent study revealed that neonatal cardiac macrophage injection could promote adult heart regeneration and increase the ability of adult cardiomyocyte proliferation1,7. Neonatal cardiac macrophage transplantation might be a promising strategy to promote adult mouse heart regeneration. Here we present the protocols to help more researchers on the regenerative application development and explore of heart regeneration mechanisms.
No conflicts of interest.
This work was supported by Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences (CIFMS, 2016-I2M-1-015), the National Key Research and Development Project of China (2019YFA0801500), the National Natural Science Foundation of China (NSFC: 81970243, 81770308), Beijing Natural Science Foundation (7172183, 7182140).
|Anti-mouse alpha actinin
|Anti-rabbit Aurora B
|gentleMACS Octo Dissociator
|Miltenyi Bio Tech, Teterow, Germany
|Goat anti-mouse Alexa Fluor 555
|Goat anti-rabbit Alexa Fluor 488
|Neonatal Heart Dissociation Kit
|Miltenyi Bio Tech, Teterow, Germany
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