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.
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Method Article
Delayed Intramyocardial Delivery of Stem Cells after Ischemia Reperfusion Injury in a Murine Model
In This Article
Summary
Abstract
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.
Introduction
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. Ste....
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Protocol
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
- 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 ....
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Results
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.......
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Discussion
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 infiltratio.......
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Disclosures
The authors have nothing to disclose.
Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| 0.9% NaCl Irrigation, USP | Baxter | 0338-0048-04 | |
| 11x12" 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 |
References
- Franchi, F., et al. The Myocardial Microenvironment Modulates the Biology of Transplanted Mesenchymal Stem Cells. Molecular Imaging Biology. , (2020).
- Bergmann, O., et al. Evidence for cardiomyocyte renewal in humans. Science. 324 (5923), 98-....
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