Name: delayed intramyocardial delivery of stem cells after ischemia reperfusion injury in a murine model
Uploaded: 2020-09-03T13:00:00
Description: Watch this Scientific Journal Video about Delayed Intramyocardial Delivery of Stem Cells after Ischemia Reperfusion Injury in a Murine Model at JoVE.com

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
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	<li>Autoclave all surgical instr...

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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...

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&#160;&#160; &#820;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, na&#239;ve 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.

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			<td>ZooPharm Pharmacy</td>
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			<td>Buprenorphine narcotic analgesic formulated in a polymer that slows absorption extending duration of action (72 hours duration of activity).</td>
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			<td height="17" style="height:17px;">Delicate suture tying forceps, 45 angle bent</td>
			<td>Fine Science Tools</td>
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			<td>Cardinal Health</td>
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			<td>Anesthetic</td>
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			<td></td>
			<td></td>
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			<td height="17" style="height:17px;">Monoject 1 mL hypodermic syringe</td>
			<td>Coviden</td>
			<td>8881501400</td>
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			<td height="17" style="height:17px;">Moria iris forceps, curved, serrated (x2)</td>
			<td>Fine Science Tools</td>
			<td>11370-31</td>
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			<td height="17" style="height:17px;">Moria speculum retractor</td>
			<td>Fine Science Tools</td>
			<td>17370-53</td>
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			<td height="17" style="height:17px;">Mouse endotracheal intubation kit</td>
			<td>Kent Scientific</td>
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			<td height="17" style="height:17px;">Nair depilatory cream</td>
			<td>Johnson &#38; Johnson</td>
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			<td>Fur removal</td>
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			<td height="17" style="height:17px;">Optixcare eye lube plus</td>
			<td>Aventix</td>
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			<td>Kent Scientific</td>
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			<td height="17" style="height:17px;">PolyE Polyethylene tubing</td>
			<td>Harvard Apparatus</td>
			<td>72-0191</td>
			<td>Temporary compression of LAD artery</td>
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			<td>PDI</td>
			<td>S41125</td>
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			<td>Bard-Parker</td>
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			<td>Cardinal Health</td>
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			<td>Cardinal Health</td>
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			<td height="17" style="height:17px;">Suture tying forceps, straight (x2)</td>
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			<td>Fine Science Tools</td>
			<td>15004-08</td>
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			<td>Kent Scientific</td>
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delayed intramyocardial delivery of stem cells after ischemia reperfusion injury in a murine model

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.

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...

Watch this Scientific Journal Video about Delayed Intramyocardial Delivery of Stem Cells after Ischemia Reperfusion Injury in a Murine Model at JoVE.com

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

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, ...

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