Ischemic damage induces a hostile pro-inflammatory environment affecting the survival of transplanted cells. Our protocol presents an alternative method for providing biologics by circumventing the initial immune response to injury. The main advantage of this protocol is that it makes it possible to administer therapeutics one week following injury, mimicking treatment in the clinical environment.
Our primary focus is on the ischemia reperfusion injury model and transplantation of STEM cells. However, this approach can also be utilized for models of inflammatory disease that involves the introduction of biological therapies. Begin by autoclaving all surgical instruments.
If multiple surgeries are to be performed in one session, clean the instruments after each animal and re-sterilize them using a hot bead sterilizer. Subcutaneously administer analgesic, weigh the animal, and input the weight into the ventilator. Shave the left side of the chest from the sternum to the level of the shoulder and apply depilatory cream to remove excess fur.
To prevent lung collapse, maintain the positive end expiratory pressure at two centimeters of water for the ischemia reperfusion procedure and change it to three centimeters of water for the delayed injection of cells procedure. After anesthetizing the mouse, intubate it with a 20 gauge endotracheal tube and transfer it to a controlled heating pad to maintain a body temperature of 35 to 37 degrees Celsius. Place the mouse in lateral recumbency with cranial end on the left and caudal end on the right.
Scrub the surgical area alternating between povidone iodine and alcohol swabs three times and apply ophthalmic ointment to both eyes. Use a number 10 blade scalpel to make a vertical incision 2.5 millimeters to the right of the leftmost nipple in the field of view, then use scissors to cut through the superficial muscle layers until the intercostal muscle and ribs are visible. While lifting the ribs and surrounding tissue, cut through the intercostal space between the fourth and fifth ribs, then insert the eyelid retractor into the open space.
Retract the pericardium using curved forceps, moving the lung upwards and out of the view. Pass a 9-0 nylon suture through the myocardium beneath the LAD artery 2.5 millimeters distal to the left auricle maintaining smooth and consistent movement to prevent tearing of the tissue or puncturing a major blood vessel. Tie a loose square knot, then place one centimeter of polyethylene tubing within the loose knot.
Secure the suture around the tubing and confirm ischemia by pallor and ventricular arrhythmia, then release after 35 minutes. After removing the tubing, wait for five minutes to confirm re-perfusion of the myocardium. Place a 24 gauge IV catheter tube into the thoracic cavity one intercostal space to the right of the opening.
Use 6-0 absorbable sutures to close the intercostal incision in a simple interrupted pattern and the muscle layer in a continuous suture pattern. After closing the superficial muscle layer, remove the chest tube while withdrawing the air from the thoracic cavity with a one milliliter tuberculin syringe. Close the skin incision with a 6-0 absorbable suture in a continuous horizontal mattress pattern.
When finished, administer 1.5 milliliters of warm saline subcutaneously and apply triple antibiotic ointment to the incision site to prevent infection. Transfer the mouse to a bedding free cage or a cage with covered bedding on a warm pad and a temperature of 35 to 37 degrees Celsius until it is fully recovered. After preparing and intubating the mouse as previously described, remove the suture from the skin layer with scissors and forceps, then use a number 10 scalpel to make an incision in the same location as the previous surgery.
Continue to cut through the scar tissue until the muscle layer suture is visible, then use the scissors and forceps to remove the suture and cut the muscle layer open. Remove the sutures holding the ribs together and continue cutting through the intercostal muscle from the previous incision. Place the eyelid retractor into the intercostal space and locate the area of the previous ligation.
Load 300, 000 mesenchymal STEM cells suspended in 20 microliters of PBS into a 30 gauge insulin syringe and bend the needle slightly to achieve the proper angle for injection. Moving in the direction from the apex towards the base of the heart, insert the syringe into the peri-infarct region until the needle opening is completely inside the myocardium, then slowly inject the cells into the myocardium, wait three seconds and remove the needle. Observe the heart closely for three minutes to make sure that there is no abnormal reaction to the cells such as ventricular fibrillation.
After three minutes, place a 24 gauge IV catheter tube into the thoracic cavity one intercostal space to the right of the opening. Close the intercostal muscle and skin layers and remove the chest tube as previously described. Administer 1.5 milliliters of warm saline subcutaneously and apply triple antibiotic ointment to the incision site to prevent infection.
Transfer the mouse to a bedding free cage or cage with covered bedding on a warm pad until it is fully recovered. Post-operative echocardiogram and electrocardiogram were performed on the day proceeding STEM cell implantation, confirming infarction and decreased ventricular contractile function. Further examination of the data showed the ejection fraction and fractional shortening were decreased in mice that received ischemic injury, while the end diastolic and systolic volumes increased.
Masson trichrome staining of myocardial tissue seven days post-injury showed increased collagen deposition and thinning of the left ventricular wall in hearts of mice that received ischemic injury. In vivo bioluminescent imaging or BLI was performed on the day after mesenchymal STEM cell implantation. Successful delivery of MSCs is exemplified by the BLI signal compared to mice that had induced ischemia reperfusion injury but did not receive MSCs.
When attempting this protocol, keep in mind that small intentional movements when cutting through the intercostals, ligating the vessel, and injecting cells are vital for preventing undue damage to the heart or the lung in the field of view. These procedures offer the ability to monitor cardiac function after transplantation through ultrasound, cell viability via bioluminescence imaging, or further molecular or biochemical techniques depending on the therapy used.
