Stems cells are continuously investigated as potential treatments for individuals with myocardial damage, however, their decreased viability and retention within injured tissue can impact their long-term efficacy. In this manuscript we describe an alternative method for stem cell delivery in a murine model of ischemia reperfusion injury.
There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, cardiac stem cell therapy is studied by delivering SCs concurrently with the induction of myocardial injury. However, this approach presents two significant limitations: the early hostile pro-inflammatory ischemic environment may affect the survival of transplanted SCs, and it does not represent the subacute infarction scenario where SCs will likely be used. Here we describe a two-part series of surgical procedures for the induction of ischemia-reperfusion injury and delivery of mesenchymal stem cells (MSCs). This method of stem cell administration may allow for the longer viability and retention around damaged tissue by circumventing the initial immune response. A model of ischemia reperfusion injury was induced in mice accompanied by the delivery of mesenchymal stem cells (3.0 x 105), stably expressing the reporter gene firefly luciferase under the constitutively expressed CMV promoter, intramyocardially 7 days later. The animals were imaged via ultrasound and bioluminescent imaging for confirmation of injury and injection of cells, respectively. Importantly, there was no added complication rate when performing this two-procedure approach for SC delivery. This method of stem cell administration, collectively with the utilization of state-of-the-art reporter genes, may allow for the in vivo study of viability and retention of transplanted SCs in a situation of chronic ischemia commonly seen clinically, while also circumventing the initial pro-inflammatory response. In summary, we established a protocol for the delayed delivery of stem cells into the myocardium, which can be used as a potential new approach in promoting regeneration of the damaged tissue.
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 optimization3,4,5. 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 survival7,8. 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 remodeling9,10,11. 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 area10,12. 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
2. Ischemia reperfusion injury
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.
4. Post-operative care following both procedures
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.
EF% | FS% | EDV (µl) | ESV (µl) | SV (µl) | |
Baseline | 74.19±1.2 | 44.67±2 | 23.8±3.6 | 6.14±0.98 | 17.68±2.7 |
Post-IR | 43.9±3.8 | 30.65±3.8 | 33.88±4.4 | 18.11±1.4 | 15.74±3.2 |
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 signaling10,12. 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.
The authors have nothing to disclose.
None.
0.9% NaCl Irrigation, USP | Baxter | 0338-0048-04 | |
11×12" Press n' Seal surgical drape, autoclavable | SAI Infusion Technologies | PSS-SD | |
24G 3/4" IV catheter tube | Jelco | 4053 | |
28G x 1/2" 1mL allergy syringe | BD | 305500 | Injection of analgesic |
30G x 1/2" 3/10cc insulin syringe | Ulticare | 08222.0933.56 | Injection of stem cells |
6-0 S-29, 12" Vicryl suture | Ethicon | J556G | Intercostal, superficial muscle and skin layer incision closure |
9-0 BV100-4, 5" Ethilon suture | Ethicon | 2829G | Ligation of the LAD artery |
Absorbent underpad | Thermo Fischer Scientific | 14-206-64 | For underneath the animal |
Alcohol prep pads, 2 ply, medium | Coviden | 6818 | |
Anti-fog face mask | Halyard | 49235 | |
Bonn Strabismus scissors, curved, blunt | Fine Science Tools | 14085-09 | |
Buprenorphine HCL SR LAB 1mg/ml, 5 ml | ZooPharm Pharmacy | Buprenorphine narcotic analgesic formulated in a polymer that slows absorption extending duration of action (72 hours duration of activity). | |
Castroviejo needle holders, curved | Fine Science Tools | 12061-01 | |
Curity sterile gauze sponges | Coviden | 397310 | |
Delicate suture tying forceps, 45 angle bent | Fine Science Tools | 11063-07 | |
Electric Razor | Wahl | Fur removal | |
Isoflurane 100 ml | Cardinal Health | PI23238 | Anesthetic |
Lab coat | |||
Monoject 1 mL hypodermic syringe | Coviden | 8881501400 | |
Moria iris forceps, curved, serrated (x2) | Fine Science Tools | 11370-31 | |
Moria speculum retractor | Fine Science Tools | 17370-53 | |
Mouse endotracheal intubation kit | Kent Scientific | ||
Nair depilatory cream | Johnson & Johnson | Fur removal | |
Optixcare eye lube plus | Aventix | Sterile ocular lubricant | |
Physiosuite ventilator | Kent Scientific | ||
PolyE Polyethylene tubing | Harvard Apparatus | 72-0191 | Temporary compression of LAD artery |
Povidone-iodine swabs | PDI | S41125 | |
Scalpel, 10-blade | Bard-Parker | 371610 | |
Sterile 3" cotton tipped applicators | Cardinal Health | C15055-003 | |
Sterile 6" tapered cotton tip applicators | Puritan | 25-826-5WC | |
Sterile gloves | Cardinal Health | N8830 | |
Sterilization pouches | Medline | MPP100525GS | |
Surgery cap | |||
Surgical Microscope | Leica | M125 | |
Suture tying forceps, straight (x2) | Fine Science Tools | 10825-10 | |
Transpore surgical tape | 3M | 1527-1 | |
Triple antibiotic ointment | G&W Laboratories | 11-2683ILNC2 | Topical application to prevent infection |
Vannas-Tübingen Spring Scissors, curved | Fine Science Tools | 15004-08 | |
Vetflo vaporizer | Kent Scientific |