Delayed Intramyocardial Delivery of Stem Cells after Ischemia Reperfusion Injury in a Murine Model

Cardiovascular disease remains the most common cause of morbidity and mortality worldwide. Cardiac ischemic events have been found to be detrimental to the overall function of the myocardium and surrounding cells¹. Only   ̴0.45-1.0% of cardiomyocytes will regenerate every year after myocardial damage occurs². Despite the growing demand and inherent focus on developing treatments, therapies aiding in the regeneration of injured tissue have been difficult to establish and still require further optimization^3,4,5. Stem cell therapies have been introduced as an alternative path to rejuvenate damaged tissue after an ischemic event; however, advancement of these therapies has been challenged by the limited survival and retention of the cells to an injured area⁶.

The microenvironment of the heart after an ischemic event can be characterized as hypoxic, pro-oxidant, and pro-inflammatory, presenting hostile conditions for therapeutic stem cells to adapt to for survival^7,8. As an immune response is triggered following injury, naïve lymphocytes, macrophages, neutrophils and mast cells attempt to repair the damage by removing dying cells and modulating the process for tissue remodeling^9,10,11. Within the first 3 days post-ischemia, inflammation is at its peak with the release of pro-inflammatory cytokines with high numbers of neutrophils and monocytes in the area^10,12. After 7 days, much of the inflammation has subsided and the transition to reparative cells begins, continuing until the remodeling cascade is complete, approximately 14 days in mice¹³. Our surgical method is a potential alternative approach to the introduction of biologics into the myocardium to bypass the peak innate immune response after ischemia reperfusion injury. At the same time, it will allow for the study of any treatments in a condition of subacute/chronic ischemia where there may be different variables to consider compared to acute myocardial infarction.

The experiments were performed on female C57BL/6 mice, age 10-12 weeks and 20-25 g body weight. All animal procedures complied with the standards stated in the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Academy of Sciences, Bethesda, MD, USA) and were approved by the Mayo Clinic College of Medicine Institutional Animal Care and Use Committee (IACUC).

1. Preparation and intubation

1. Autoclave all surgical instruments before surgery. If multiple surgeries are to be performed in one session, clean the instruments after each animal and re-sterilize using a hot bead sterilizer.
2. Anesthetize the mice with 3.5-4% isoflurane at 1 L/min O₂ in an induction chamber.
3. Administer Buprenorphine SR 1 mg/kg (analgesic) subcutaneously, weigh the animal, and input the weight into the ventilator.
4. Shave the left side of the chest from the sternum to the level of the shoulder and apply depilatory cream to remove excess fur.
5. For the ischemia reperfusion procedure maintain the positive end-expiratory pressure (PEEP) on the ventilator at 2 cmH₂O. For the delayed injection of cells procedure change the PEEP to 3 cmH₂O to prevent lung collapse.
6. Intubate the animal using a 20 G endotracheal tube, transfer to a controlled heating pad to maintain a body temperature of 35-37 °C.
7. Place the mouse on a ventilator in lateral recumbency with cranial end on the left and caudal end on the right.
8. Maintain anesthesia at 2-2.5% isoflurane at 1 L/min O₂ for the remainder of the procedure.
9. Scrub the surgical area alternating between povidone-iodine and alcohol swabs three times and apply ophthalmic ointment to both eyes.

2. Ischemia reperfusion injury

1. Using a #10 blade scalpel make a vertical incision 2.5 mm to right of the leftmost nipple in the field of view.
2. Using scissors cut through the superficial muscle layers until the intercostal muscles and ribs are visible.
3. While lifting the ribs and surrounding tissue, cut through the intercostal space between the 4th and 5th ribs, then insert the eyelid retractor into the open space.
4. Retract the pericardium using curved forceps, moving the lung upwards and out of view.
5. Visualize LAD artery and, using a 9-0 nylon suture, pass through the myocardium beneath the artery 2.5 mm distal to the left auricle and tie a loose square knot.
6. Cut 1 cm of polyethylene tubing and place it within the loose knot.
7. Secure the suture around the tubing, confirm ischemia, then release after 35 min.
   NOTE: Confirm ischemia by pallor and ventricular arrhythmia.
8. After releasing the ligation and removing the tubing, wait for 5 min to confirm reperfusion of the myocardium.
9. Place a 24 G I.V. catheter tube into the thoracic cavity one intercostal space to right of the opening.
10. Close the intercostal incision with a 6-0 absorbable suture in a simple interrupted pattern.
11. Close the muscle layer with a 6-0 absorbable suture in a continuous suture pattern.
12. After closing the superficial muscle layer, remove the chest tube while withdrawing the air from thoracic cavity using a 1 mL tuberculin syringe.
13. Close the skin incision with a 6-0 absorbable suture in a continuous horizontal mattress pattern
    NOTE: Nylon sutures and a discontinuous suture pattern may also be used for the skin layer.
14. Administer 1.5 mL of warm saline subcutaneously and apply triple-antibiotic ointment to the incision site to prevent infection.
15. Turn off isoflurane and allow the animal to breathe through the ventilator on 100% O₂ until it can breathe continuously without aid.
16. Transfer the mouse to a bedding-free cage or a cage with covered bedding (paper towel or drape) on a warm pad with a temperature of 35-37 °C until fully recovered.

3. Mouse mesenchymal stem cell delivery

NOTE: The strain of mice used for the procedure are an inbred line and are deemed genetically identical. The mesenchymal stem cells were obtained from animals of the same strain and, by protocol design, immunosuppression was not induced¹.

1. Complete the preparation and intubation steps as done previously for the first procedure.
2. Remove the suture from the skin layer using scissors and forceps.
3. With a #10 scalpel, make an incision in the same location as the previous surgery.
4. Continue to use the scalpel to cut through scar tissue until muscle layer suture is visible
5. Using the scissors and forceps remove the suture and cut the muscle layer open.
6. Visualize and remove the sutures holding the ribs together and continue cutting through the intercostal muscle from the previous incision.
   NOTE: The lungs may have adhered to the chest wall, if this occurs, use blunt or curved forceps to carefully separate and release them.
7. Place the eyelid retractor into the intercostal space and locate the area of the previous ligation.
8. Load the mesenchymal stem cells (3.0 x 10⁵), suspended in 20 µL PBS, into a 30 G insulin syringe, bend the needle slightly as needed for the proper angle to inject.
   NOTE: Mesenchymal stem cells (MSCs) were isolated from the adipose tissue of 4-6-week-old C56BL/6 mice. Early passage cells (p3) were transduced with a vector expressing the firefly luciferase gene under the CMV promoter to allow in vivo cell viability monitoring. Adipose-derived mouse MSC were characterized by flow cytometry and the cells were positive for CD44, CD29, CD90 and CD105 but negative for the hematopoietic marker CD45¹⁴. Prior to the injection, MSCs were cultured for at least one passage to avoid the loss of cells from the thawing process.
9. 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.
10. Once inside slowly inject the cells into the myocardium, wait 3 s, then remove the needle.
11. Observe the heart closely for 3 min to be sure of no abnormal reactions to the cells such as ventricular fibrillation.
12. Place a 24 G IV catheter tube into the thoracic cavity one intercostal space to right of the opening.
13. Close the intercostal, muscle, and skin layers and remove the chest tube in the same method as the first procedure.
14. Administer 1.5 mL of warm saline subcutaneously and apply triple-antibiotic ointment to the incision site to prevent infection.
15. Turn off isoflurane and allow animal to breathe through the ventilator on 100% O₂ until it is able to breathe continuously without aid.
16. Transfer the mouse to a bedding-free cage or a cage with covered bedding (paper towel or drape) on a warm pad with a temperature of 35-37 °C until fully recovered.

4. Post-operative care following both procedures

1. Observe the animal continuously until spontaneous breathing, sternal recumbency and normal movement is established.
2. Continue observation every 15-30 min for at least 3 h on the day of the surgery.
3. Check the mice for wound dehiscence or abnormal pain once daily for 5 days, then 2-3 times weekly.
4. If the animal shows signs of pain (i.e. arched back, minimal movement, grimacing, or scruffy fur) after 72 h post-op, provide an additional dose of the Buprenorphine SR analgesic.

Ischemia reperfusion injury was induced in mice on day 0, followed by a post-operative echocardiogram and electrocardiogram on the day preceding stem cell implantation. Ultrasound and electrocardiogram analysis confirmed infarction and decreased ventricular contractile function (Figure 1A-D). 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 (Table 1). Compared to a normal mouse heart (Figure 2A), Masson Trichrome staining of myocardial tissue 7 days post-injury (Figure 2B) showed increased collagen deposition and thinning of the left ventricular wall. The second procedure was performed 7 days after injury; mice were given an intramyocardial injection of mesenchymal stem cells (3.0 x 10⁵ in 20 µL PBS) stably expressing the reporter gene firefly luciferase under the constitutively expressed CMV promoter. In vivo bioluminescent imaging (BLI) of these mice was completed the day after stem cell implantation for confirmation of a successful injection. The successful delivery of MSCs is exemplified by the BLI signal, compared to mice that had induced ischemia reperfusion injury but did not receive MSCs (Figures 3A,B). This dual interventional procedure had an attrition rate of 22%, similar to that observed in animals that received MSCs in the acute scenario.

Figure 1: Imaging of mice heart function. Ultrasound analysis of mouse at baseline (A) shows uniform contraction of left ventricle myocardium compared to a mouse after ischemia reperfusion injury (B), which shows decreased ventricular movement. When compared to the baseline electrocardiogram of a normal mouse (C), there are significant shifts in the ST segment of a mouse with ischemia reperfusion injury (D), indicating a decrease in ventricular function. Please click here to view a larger version of this figure.

Table 1: Echocardiography analysis. Variables are expressed as Mean ± Standard Error of the Mean. EF: Ejection Fraction, FS: Fractional Shortening, EDV: End-Diastolic Volume, ESV: End-Systolic Volume, SV: Stroke Volume.

Figure 2: Histological Staining of Heart Tissue. Masson’s Trichrome staining of the myocardium in normal mouse (A) shows no injury to the cardiac tissue, whereas the mouse with ischemia reperfusion injury (B) shows increased collagen deposition and thinning in the myocardium of left ventricle, supporting the determination of a successful infarction. Please click here to view a larger version of this figure.

Figure 3: In vivo bioluminescent imaging. A mouse with ischemia reperfusion injury that did not receive intramyocardial injection of stem cells showed no bioluminescent signal (A). A mouse with ischemia reperfusion injury which received a delayed injection of mesenchymal stem cells (CMV-FLUC) showed a significant amount of signal (B). Please click here to view a larger version of this figure.

Over 85 million people worldwide are affected by cardiovascular disease³. The high prevalence of these ischemic events warrants further development and expansion of alternative therapies for promoting the regeneration of damaged tissue. Traditional methods utilize the ischemia reperfusion procedure in an acute setting with subsequent administration of therapeutics¹. Inflammatory reactions are at its peak between 3-4 days postdating a cardiac ischemic event, with infiltration of neutrophils, macrophages, and increased cytokine signaling^10,12. After this period of dead cell degregation, the primary immune response begins to subside and transition towards remodeling phases¹³. Furthermore, it is important that treatments are investigated within the same scenario as presented in the clinical setting. In this manuscript, we are showing representative results obtained from ischemic mice to demonstrate the feasibility and the safety of the double surgical procedure, with delayed injection of MSCs. We believe that this approach can be used not only for myocardial ischemia animal models, but also for animal models of disease where inflammation may play a critical role, altering the success of therapeutic strategies that involve biologics, such as cell or drug therapies.

Therefore, in this manuscript we describe a surgical method for delivering stem cells into a subacute infarction, 7-10 days after inducing ischemia reperfusion injury in mice. This technique will be useful in studying stem cell viability and biology in connection to different stages of the immune response and in the subacute/chronic phase of the ischemic disease process. Murine models are ideal subjects for this method of study in terms of reproducibility and convenience, however, they may bear some disadvantages. The size of the animal warrants a certain degree of surgical skill although, with practice, these procedures can be completed successfully.

To perform the procedures presented in this manuscript, it is important to note some key steps and observations essential to the successful completion of these surgeries. A critical step of the first procedure is the ligation of the left anterior descending coronary artery (LAD) and placement of polyethylene tubing to achieve temporary ischemia of the myocardium. Use of sterile tapered tip cotton swabs to place pressure on the cardiac tissue distal to the atrium allows for enhanced delineation of the LAD. Once the tubing is in place and the suture tightly secured, observation of arrhythmia and pallor of the tissue is essential to determining successful induction of ischemia. The period of ischemia and the subsequent reperfusion, once the suture is released, is important for consistency of injury across multiple animals. Additionally, during the second described procedure, the injection of mesenchymal stem cells must be performed with horizontal movements in the distal to proximal direction. Due to resulting fibrosis from the first procedure, significant but steady pressure is required to insert the needle followed by a slow consistent injection of the cells to prevent shock. Finally, providing continuous heat and supplemental subcutaneous fluids before waking mice from anesthesia, will prevent heat loss and aid in the replacement of any blood lost during the procedures, as well as the animal’s overall recovery.

In this manuscript, we provide a protocol for completing multiple procedures as a method of administering stem cells as a therapeutic treatment in a murine model of chronic ischemia reperfusion injury. Utilization of these surgical procedures offers a new approach for the delivery of stem cells into the hostile ischemic environment after injury to enhance their viability over time. Use of this approach for the study of stem cell therapy will significantly complement other studies focusing on the use of SCs in the acute setting. In conclusion, the described protocol is successful in inducing ischemic injury and the ensuing delayed implantation of stem cells for use as a model in preclinical studies.