This method will allow us to evaluate the role of the pericardial cavity that surrounds the heart and its contents in cardiac repair following myocardial infarction. Compared to conventional coronary ligation methods for modeling myocardial infarction, our method maintains an intact pericardial cavity, allowing the determination of contents and tracks their roles following cardiac ischemic injury. Understanding the contribution of the pericardial cavity and the heart's injury response will provide important insights into therapeutic strategies that target the pericardium to improve heart healing.
To begin with post anesthetization, restrain the paws of the mouse and position it on the surgical table. After preparing the mouse for surgery as described in the manuscript, ventilate the mouse with 2%isoflurane and 100%oxygen as a carrier gas using a commercial ventilator. Then set a rate of 110 breaths per minute, a tidal volume of 250 microliters, and a positive end expiratory pressure of four millimeters of mercury.
Roll the mouse 30%on its right side to position the chest's left side for surgery. Then make a two to three centimeter lateral incision in the chest skin to visualize the pectoralis muscles on the left side. Make a one centimeter incision from the midline outward for cutting the pectoralis major and minor.
Avoid excessive bleeding throughout the cauterization of bleeding vessels. Visualize the intercostal muscles between the third and fourth ribs. Make a two centimeter incision in the left intercostal muscle to introduce air into the chest cavity and allow the heart and lungs to fall away from the incision.
To incise the intercostal without bleeding, temporarily stop the ventilator and expand the opening using a cautery device. Using retractors, retract the ribs to expose the heart. Then observe the pericardium and the underlying heart under a stereo microscope.
Gently place the forceps on the pericardium surface to reduce pericardium and underlying heart movement. Visually landmark the LAD coronary artery by tracing its emergence under the left appendage. Using the microneedle driver, guide an appropriate suture through the pericardium under the LAD.
Observe the suture emerging on the other side of the LAD and pericardium. Representing the permanent ligation model, tie the suture to restrict the blood flow through the coronary artery, making blanching of the anterior portion of the left ventricle visible. Then trim the excess suture with the help of scissors.
Provide maintenance injections of analgesic subcutaneously every 12 hours for 72 hours post surgery. After reducing the isoflurane to 1%turn off the isoflurane while maintaining the ventilation with oxygen. Once the animal shows signs of breathing, remove the 23 gauge tracheal tube from the mouth and place the mouse in a recovery cage to monitor the breathing resumption.
Allow the mouse to recover in the cage with a portion of the cage placed on a warming pad to provide an external heat source. Monitor the health status of the mouse daily for seven days by evaluating the incisions and animal discomfort. If necessary, administer antibiotics for a successful recovery following the invasive surgery.
After maintaining the mouse under general anesthesia with oxygen, place the mouse in a supine position on a heated stage platform and attach the paws to the ECG leads. Then chafe the chest. Acquire ECG images using transducer probe and contact gel and analyze with the appropriate software.
After noninvasive ECG assessment, turn off the isoflurane and return the mouse to the cage. Fix the heart in 10%formalin for at least 24 hours. Cut the sample using a straight razor blade through the right ventricle, interventricular septum, and left ventricle, ensuring that the incision goes through the center of the infarct zone.
And then paraffin embed the tissue samples. The cut sections are then placed in mounting cassette for further paraffin embedding and subsequent sectioning into five micrometer thick sections. Following placement of tissue sections on slides, deparaffinize using toluene and graded alcohol washes with deionized water.
Then rehydrate. Next, stain the section with 0.1%sirius red in picric acid for two hours at room temperature. Wash the section with 0.5%acetic acid for three minutes followed by rinsing with 70%ethanol for one minute.
Dehydrate the section using the reverse order of washes with increasing and graded ethanol concentrations prior to mounting for microscopic evaluation. The successful myocardial infarction induction with the modified coronary ligation was assessed by examining the heart's functional parameters and structural features. For function, decreased left ventricle ejection fraction was assessed by echocardiography within three to four weeks of post myocardial infarction.
The histological staining demonstrated that the functional changes were accompanied by significant fibrosis of the left ventricle wall, which were further confirmed by quantification of the picrosirius red stained fibrosis in the cross-sections of the heart at four weeks post infarct with the disrupted or intact pericardial coronary ligation models. Combining the fluorescent bead labeling method with flow cytometry analysis provided an approach to track the resident Gata6 positive pericardial macrophages. Importantly, little to no labeling was detected in the heart or blood.
Following myocardial infarction, the relocation of the cells can be tracked by flow cytometry and imaging. Quantification of the fluorescent bead labeled pericardial myeloid cells with or without myocardial infarction is shown. Care is needed to prevent inadvertently disrupting or tearing the thin pericardium during the procedure.
This method can be combined with lineage tracing cellular or effector blockade, which will allow tracking and determining the pericardial component's impact on the healing response in the infarcted heart. We have identified the pericardial cavity protects the heart after injury, which is partly mediated by resident pericardial macrophages. This approach opens a new research area for cardiovascular disease.
