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
<ol>
	<li>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.</li>
	<li>Anesthetize the mice with 3.5-4% isoflurane at 1 L/min O2 in an induction chamber.</li>
	<li>Administer Buprenorphine SR 1 mg/kg (analgesic) subcutaneously, weigh the animal, and input the weight into the ventilator.</li>
	<li>Shave the left side of the chest from the sternum to the level of the shoulder and apply depilatory cream to remove excess fur.</li>
	<li>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.</li>
	<li>Intubate the animal using a 20 G endotracheal tube, transfer to a controlled heating pad to maintain a body temperature of 35-37 &#176;C.</li>
	<li>Place the mouse on a ventilator in lateral recumbency with cranial end on the left and caudal end on the right.</li>
	<li>Maintain anesthesia at 2-2.5% isoflurane at 1 L/min O2 for the remainder of the procedure.</li>
	<li>Scrub the surgical area alternating between povidone-iodine and alcohol swabs three times and apply ophthalmic ointment to both eyes.</li>
</ol>
2. Ischemia reperfusion injury
<ol>
	<li>Using a #10 blade scalpel make a vertical incision 2.5 mm to right of the leftmost nipple in the field of view.</li>
	<li>Using scissors cut through the superficial muscle layers until the intercostal muscles and ribs are visible.</li>
	<li>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.</li>
	<li>Retract the pericardium using curved forceps, moving the lung upwards and out of view.</li>
	<li>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.</li>
	<li>Cut 1 cm of polyethylene tubing and place it within the loose knot.</li>
	<li>Secure the suture around the tubing, confirm ischemia, then release after 35 min. 
	NOTE: Confirm ischemia by pallor and ventricular arrhythmia.</li>
	<li>After releasing the ligation and removing the tubing, wait for 5 min to confirm reperfusion of the myocardium.</li>
	<li>Place a 24 G I.V. catheter tube into the thoracic cavity one intercostal space to right of the opening.</li>
	<li>Close the intercostal incision with a 6-0 absorbable suture in a simple interrupted pattern.</li>
	<li>Close the muscle layer with a 6-0 absorbable suture in a continuous suture pattern.</li>
	<li>After closing the superficial muscle layer, remove the chest tube while withdrawing the air from thoracic cavity using a 1 mL tuberculin syringe.</li>
	<li>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.</li>
	<li>Administer 1.5 mL of warm saline subcutaneously and apply triple-antibiotic ointment to the incision site to prevent infection.</li>
	<li>Turn off isoflurane and allow the animal to breathe through the ventilator on 100% O2 until it can breathe continuously without aid.</li>
	<li>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 &#176;C until fully recovered.</li>
</ol>
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.
<ol>
	<li>Complete the preparation and intubation steps as done previously for the first procedure.</li>
	<li>Remove the suture from the skin layer using scissors and forceps.</li>
	<li>With a #10 scalpel, make an incision in the same location as the previous surgery.</li>
	<li>Continue to use the scalpel to cut through scar tissue until muscle layer suture is visible</li>
	<li>Using the scissors and forceps remove the suture and cut the muscle layer open.</li>
	<li>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.</li>
	<li>Place the eyelid retractor into the intercostal space and locate the area of the previous ligation.</li>
	<li>Load the mesenchymal stem cells (3.0 x 105), suspended in 20 &#181;L 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.</li>
	<li>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.</li>
	<li>Once inside slowly inject the cells into the myocardium, wait 3 s, then remove the needle.</li>
	<li>Observe the heart closely for 3 min to be sure of no abnormal reactions to the cells such as ventricular fibrillation.</li>
	<li>Place a 24 G IV catheter tube into the thoracic cavity one intercostal space to right of the opening.</li>
	<li>Close the intercostal, muscle, and skin layers and remove the chest tube in the same method as the first procedure.</li>
	<li>Administer 1.5 mL of warm saline subcutaneously and apply triple-antibiotic ointment to the incision site to prevent infection.</li>
	<li>Turn off isoflurane and allow animal to breathe through the ventilator on 100% O2 until it is able to breathe continuously without aid.</li>
	<li>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 &#176;C until fully recovered.</li>
</ol>
4. Post-operative care following both procedures
<ol>
	<li>Observe the animal continuously until spontaneous breathing, sternal recumbency and normal movement is established.</li>
	<li>Continue observation every 15-30 min for at least 3 h on the day of the surgery.</li>
	<li>Check the mice for wound dehiscence or abnormal pain once daily for 5 days, then 2-3 times weekly.</li>
	<li>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.</li>
</ol>

<ol>
	<li>Franchi, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Myocardial+Microenvironment+Modulates+the+Biology+of+Transplanted+Mesenchymal+Stem+Cells.">The Myocardial Microenvironment Modulates the Biology of Transplanted Mesenchymal Stem Cells.</a> Molecular Imaging Biology. , (2020).</li><li>Bergmann, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+cardiomyocyte+renewal+in+humans.">Evidence for cardiomyocyte renewal in humans.</a> Science. 324 (5923), 98-102 (2009).</li><li>Writing Group, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+Disease+and+Stroke+Statistics-2016+Update:+A+Report+From+the+American+Heart+Association.">Heart Disease and Stroke Statistics-2016 Update: A Report From the American Heart Association.</a> Circulation. 133 (4), 38 (2016).</li><li>Gersh, B. J., Simari, R. D., Behfar, A., Terzic, C. M., Terzic, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+cell+repair+therapy:+a+clinical+perspective.">Cardiac cell repair therapy: a clinical perspective.</a> Mayo Clinic Protocol. 84 (10), 876-892 (2009).</li><li>Terzic, A., Behfar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerative+heart+failure+therapy+headed+for+optimization.">Regenerative heart failure therapy headed for optimization.</a> European Heart Journal. 35 (19), 1231-1234 (2014).</li><li>Beegle, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxic+preconditioning+of+mesenchymal+stromal+cells+induces+metabolic+changes,+enhances+survival,+and+promotes+cell+retention+in+vivo.">Hypoxic preconditioning of mesenchymal stromal cells induces metabolic changes, enhances survival, and promotes cell retention in vivo.</a> Stem Cells. 33 (6), 1818-1828 (2015).</li><li>Kubli, D. A., Gustafsson, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria+and+mitophagy:+the+yin+and+yang+of+cell+death+control.">Mitochondria and mitophagy: the yin and yang of cell death control.</a> Circulation Research. 111 (9), 1208-1221 (2012).</li><li>Psaltis, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+monitoring+of+oxidative+stress+in+transplanted+mesenchymal+stromal+cells.">Noninvasive monitoring of oxidative stress in transplanted mesenchymal stromal cells.</a> JACC Cardiovascular Imaging. 6 (7), 795-802 (2013).</li><li>Peet, C., Ivetic, A., Bromage, D. I., Shah, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+monocytes+and+macrophages+after+myocardial+infarction.">Cardiac monocytes and macrophages after myocardial infarction.</a> Cardiovasc Research. 16 (6), 1101-1112 (2020).</li><li>Swirski, F. K., Nahrendorf, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardioimmunology:+the+immune+system+in+cardiac+homeostasis+and+disease.">Cardioimmunology: the immune system in cardiac homeostasis and disease.</a> Nature Reviews Immunology. 18 (12), 733-744 (2018).</li><li>Zhang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+Stem+Cells+Promote+the+Resolution+of+Cardiac+Inflammation+After+Ischemia+Reperfusion+Via+Enhancing+Efferocytosis+of+Neutrophils.">Mesenchymal Stem Cells Promote the Resolution of Cardiac Inflammation After Ischemia Reperfusion Via Enhancing Efferocytosis of Neutrophils.</a> Journal of the American Heart Association. 9 (5), 014397 (2020).</li><li>Saxena, A., Russo, I., Frangogiannis, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation+as+a+therapeutic+target+in+myocardial+infarction:+learning+from+past+failures+to+meet+future+challenges.">Inflammation as a therapeutic target in myocardial infarction: learning from past failures to meet future challenges.</a> Translational Research. 167 (1), 152-166 (2016).</li><li>Prabhu, S. D., Frangogiannis, N. 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The authors have nothing to disclose.

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&#160;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&#8217;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.

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.

<table><tbody><tr><td>0.9% NaCl Irrigation, USP</td><td>Baxter</td><td>0338-0048-04</td><td></td></tr><tr><td>11x12&quot; Press n&#039; Seal surgical drape, autoclavable</td><td>SAI Infusion Technologies</td><td>PSS-SD</td><td></td></tr><tr><td>24G 3/4&quot; IV catheter tube</td><td>Jelco</td><td>4053</td><td></td></tr><tr><td>28G x 1/2&quot; 1mL allergy syringe</td><td>BD</td><td>305500</td><td>Injection of analgesic</td></tr><tr><td>30G x 1/2&quot; 3/10cc insulin syringe</td><td>Ulticare</td><td>08222.0933.56</td><td>Injection of stem cells</td></tr><tr><td>6-0 S-29, 12&quot; Vicryl suture</td><td>Ethicon</td><td>J556G</td><td>Intercostal, superficial muscle and skin layer incision closure</td></tr><tr><td>9-0 BV100-4, 5&quot; Ethilon suture</td><td>Ethicon</td><td>2829G</td><td>Ligation of the LAD artery</td></tr><tr><td>Absorbent underpad</td><td>Thermo Fischer Scientific</td><td>14-206-64</td><td>For underneath the animal</td></tr><tr><td>Alcohol prep pads, 2 ply, medium</td><td>Coviden</td><td>6818</td><td></td></tr><tr><td>Anti-fog face mask</td><td>Halyard</td><td>49235</td><td></td></tr><tr><td>Bonn Strabismus scissors, curved, blunt</td><td>Fine Science Tools</td><td>14085-09</td><td></td></tr><tr><td>Buprenorphine HCL SR LAB 1mg/ml, 5 ml</td><td>ZooPharm Pharmacy</td><td></td><td>Buprenorphine narcotic analgesic formulated in a polymer that slows absorption extending duration of action (72 hours duration of activity).</td></tr><tr><td>Castroviejo needle holders, curved</td><td>Fine Science Tools</td><td>12061-01</td><td></td></tr><tr><td>Curity sterile gauze sponges</td><td>Coviden</td><td>397310</td><td></td></tr><tr><td>Delicate suture tying forceps, 45 angle bent</td><td>Fine Science Tools</td><td>11063-07</td><td></td></tr><tr><td>Electric Razor</td><td>Wahl</td><td></td><td>Fur removal</td></tr><tr><td>Isoflurane 100 ml</td><td>Cardinal Health</td><td>PI23238</td><td>Anesthetic</td></tr><tr><td>Lab coat</td><td></td><td></td><td></td></tr><tr><td>Monoject 1 mL hypodermic syringe</td><td>Coviden</td><td>8881501400</td><td></td></tr><tr><td>Moria iris forceps, curved, serrated (x2)</td><td>Fine Science Tools</td><td>11370-31</td><td></td></tr><tr><td>Moria speculum retractor</td><td>Fine Science Tools</td><td>17370-53</td><td></td></tr><tr><td>Mouse endotracheal intubation kit</td><td>Kent Scientific</td><td></td><td></td></tr><tr><td>Nair depilatory cream</td><td>Johnson &amp;amp; Johnson</td><td></td><td>Fur removal</td></tr><tr><td>Optixcare eye lube plus</td><td>Aventix</td><td></td><td>Sterile ocular lubricant</td></tr><tr><td>Physiosuite ventilator</td><td>Kent Scientific</td><td></td><td></td></tr><tr><td>PolyE Polyethylene tubing</td><td>Harvard Apparatus</td><td>72-0191</td><td>Temporary compression of LAD artery</td></tr><tr><td>Povidone-iodine swabs</td><td>PDI</td><td>S41125</td><td></td></tr><tr><td>Scalpel, 10-blade</td><td>Bard-Parker</td><td>371610</td><td></td></tr><tr><td>Sterile 3&quot; cotton tipped applicators</td><td>Cardinal Health</td><td>C15055-003</td><td></td></tr><tr><td>Sterile 6&quot; tapered cotton tip applicators</td><td>Puritan</td><td>25-826-5WC</td><td></td></tr><tr><td>Sterile gloves</td><td>Cardinal Health</td><td>N8830</td><td></td></tr><tr><td>Sterilization pouches</td><td>Medline</td><td>MPP100525GS</td><td></td></tr><tr><td>Surgery cap</td><td></td><td></td><td></td></tr><tr><td>Surgical Microscope</td><td>Leica</td><td>M125</td><td></td></tr><tr><td>Suture tying forceps, straight (x2)</td><td>Fine Science Tools</td><td>10825-10</td><td></td></tr><tr><td>Transpore surgical tape</td><td>3M</td><td>1527-1</td><td></td></tr><tr><td>Triple antibiotic ointment</td><td>G&amp;amp;W Laboratories</td><td>11-2683ILNC2</td><td>Topical application to prevent infection</td></tr><tr><td>Vannas-T&amp;uuml;bingen Spring Scissors, curved</td><td>Fine Science Tools</td><td>15004-08</td><td></td></tr><tr><td>Vetflo vaporizer</td><td>Kent Scientific</td><td></td><td></td></tr></tbody></table>

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 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 &#181;L 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.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61546/61546fig1.jpg" /> 
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. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61546/61546fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td></td>
			<td>EF%</td>
			<td>FS%</td>
			<td>EDV (&#181;l)</td>
			<td>ESV (&#181;l)</td>
			<td>SV (&#181;l)</td>
		</tr>
		<tr>
			<td>Baseline</td>
			<td>74.19&#177;1.2</td>
			<td>44.67&#177;2</td>
			<td>23.8&#177;3.6</td>
			<td>6.14&#177;0.98</td>
			<td>17.68&#177;2.7</td>
		</tr>
		<tr>
			<td>Post-IR</td>
			<td>43.9&#177;3.8</td>
			<td>30.65&#177;3.8</td>
			<td>33.88&#177;4.4</td>
			<td>18.11&#177;1.4</td>
			<td>15.74&#177;3.2</td>
		</tr>
	</tbody>
</table>
Table 1: Echocardiography analysis. Variables are expressed as Mean &#177; Standard Error of the Mean. EF: Ejection Fraction, FS: Fractional Shortening, EDV: End-Diastolic Volume, ESV: End-Systolic Volume, SV: Stroke Volume.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61546/61546fig2.jpg" /> 
Figure 2: Histological Staining of Heart Tissue. Masson&#8217;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. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61546/61546fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61546/61546fig3.jpg" /> 
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). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61546/61546fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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, ...

delayed-intramyocardial-delivery-stem-cells-after-ischemia

Universidad San Sebastian

Research

JoVE Journal

Medicine

4.6K Views.  Mayo Clinic. 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.

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

Coronary Artery Ligation and Intramyocardial Injection in a Murine Model of Infarction

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo. With the advent of genetic modifications to the whole organism or the myocardium and an array of biological and/or synthetic materials, there is great potential for any combination of these to assuage the extent of acute ischemic injury and impede the onset of heart failure pursuant to myocardial remodeling. 
Here we present the methods and materials used to reliably perform this microsurgery and the modifications involved for temporary (with reperfusion) or permanent coronary artery occlusion studies as well as intramyocardial injections. The effects on the heart that can be seen during the procedure and at the termination of the experiment in addition to histological evaluation will verify efficacy. 
Briefly, surgical preparation involves anesthetizing the mice, removing the fur on the chest, and then disinfecting the surgical area. Intratracheal intubation is achieved by transesophageal illumination using a fiber optic light. The tubing is then connected to a ventilator. An incision made on the chest exposes the pectoral muscles which will be cut to view the ribs. For ischemia/reperfusion studies, a 1 cm piece of PE tubing placed over the heart is used to tie the ligature to so that occlusion/reperfusion can be customized. For intramyocardial injections, a Hamilton syringe with sterile 30gauge beveled needle is used. When the myocardial manipulations are complete, the rib cage, the pectoral muscles, and the skin are closed sequentially. Line block analgesia is effected by 0.25% marcaine in sterile saline which is applied to muscle layer prior to closure of the skin. The mice are given a subcutaneous injection of saline and placed in a warming chamber until they are sternally recumbent. They are then returned to the vivarium and housed under standard conditions until the time of tissue collection. At the time of sacrifice, the mice are anesthetized, the heart is arrested in diastole with KCl or BDM, rinsed with saline, and immersed in fixative. Subsequently, routine procedures for processing, embedding, sectioning, and histological staining are performed. 
Nonsurgical intubation of a mouse and the microsurgical manipulations described make this a technically challenging model to learn and achieve reproducibility. These procedures, combined with the difficulty in performing consistent manipulations of the ligature for timed occlusion(s) and reperfusion or intramyocardial injections, can also affect the survival rate so optimization and consistency are critical.

Numerous genetic manipulations and/or intramyocardial injections of genes, proteins, cells, and/or biomaterials are superimposed upon the dimension of time in studies of acute ischemia/ reperfusion injury and chronic remodeling in mice. This video illustrates the microsurgical procedures for ischemia/reperfusion, permanent coronary artery ligation, and intramyocardial injection studies.

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo.  With the advent of genetic modifications to ...

A Murine Closed-chest Model of Myocardial Ischemia and Reperfusion

Surgical trauma by thoracotomy in open-chest models of coronary ligation induces an immune response which modifies different mechanisms involved in ischemia and reperfusion. Immune response includes cytokine expression and release or secretion of endogenous ligands of innate immune receptors. Activation of innate immunity can potentially modulate infarct size. We have modified an existing murine closed-chest model using hanging weights which could be useful for studying myocardial pre- and postconditioning and the role of innate immunity in myocardial ischemia and reperfusion. This model allows animals to recover from surgical trauma before onset of myocardial ischemia.
Volatile anesthetics have been intensely studied and their preconditioning effect for the ischemic heart is well known. However, this protective effect precludes its use in open chest models of coronary artery ligation. Thus, another advantage could be the use of the well controllable volatile anesthetics for instrumentation in a chronic closed-chest model, since their preconditioning effect lasts up to 72 hours. Chronic heart diseases with intermittent ischemia and multiple hit models are other possible applications of this model.
For the chronic closed-chest model, intubated and ventilated mice undergo a lateral blunt thoracotomy via the 4th intercostal space. Following identification of the left anterior descending a ligature is passed underneath the vessel and both suture ends are threaded through an occluder. Then, both suture ends are passed through the chest wall, knotted to form a loop and left in the subcutaneous tissue. After chest closure and recovery for 5 days, mice are anesthetized again, chest skin is reopened and hanging weights are hooked up to the loop under ECG control.
At the end of the ischemia/reperfusion protocol, hearts can be stained with TTC for infarct size assessment or undergo perfusion fixation to allow morphometric studies in addition to histology and immunohistochemistry.

Surgical trauma induces an inflammatory response. Cytokines and endogenous ligands are known to modulate myocardial infarct size following ischemia and reperfusion. We present a modified closed-chest model of murine ischemia and reperfusion using hanging weights to minimize effects of thoracotomy.

Surgical trauma by thoracotomy in open-chest models of coronary ligation induces an immune response which modifies different mechanisms involved in ...

Pluripotent Stem Cell Derived Cardiac Cells for Myocardial Repair

Human induced pluripotent stem cells (hiPSCs) must be fully differentiated into specific cell types before administration, but conventional protocols for differentiating hiPSCs into cardiomyocytes (hiPSC-CMs), endothelial cells (hiPSC-ECs), and smooth muscle cells (SMCs) are often limited by low yield, purity, and/or poor phenotypic stability. Here, we present novel protocols for generating hiPSC-CMs, -ECs, and -SMCs that are substantially more efficient than conventional methods, as well as a method for combining cell injection with a cytokine-containing patch created over the site of administration. The patch improves both the retention of the injected cells, by sealing the needle track to prevent the cells from being squeezed out of the myocardium, and cell survival, by releasing insulin-like growth factor (IGF) over an extended period. In a swine model of myocardial ischemia-reperfusion injury, the rate of engraftment was more than two-fold greater when the cells were administered with the cytokine-containing patch comparing to the cells without patch, and treatment with both the cells and the patch, but not with the cells alone, was associated with significant improvements in cardiac function and infarct size.

We present three novel and more efficient protocols for differentiating human induced pluripotent stem cells into cardiomyocytes, endothelial cells, and smooth muscle cells and a delivery method that improves the engraftment of transplanted cells by combining cell injection with patch-mediated cytokine delivery.

Human induced pluripotent stem cells (hiPSCs) must be fully differentiated into specific cell types before administration, but conventional protocols for ...

Developmental Biology

Cell-based Therapy for Heart Failure in Rat: Double Thoracotomy for Myocardial Infarction and Epicardial Implantation of Cells and Biomatrix

Cardiac cell therapy has gained increasing interest and implantation of biomaterials associated with cells has become a major issue to optimize myocardial cell delivery. Rodent model of myocardial infarction (MI) consisting of Left Anterior Descending Artery (LAD) ligation has commonly been performed via a thoracotomy; a second open-heart surgery via a sternotomy has traditionally been performed for epicardial application of the treatment. Since the description of LAD ligation model, post-surgery mortality rate has dropped from 35-13%, however the second surgery has remained critical. In order to improve post-surgery recovery and reduce pain and infection, minimally invasive surgical procedures are presented.&#160;Two thoracotomies were performed, the initial one for LAD ligation and the second one for treatment epicardial administration. Biografts consisting of cells associated with solid or gel type matrices were applied onto the infarcted area.&#160;LAD ligation resulted in loss of heart function as confirmed by echocardiography performed after 2 and 6 weeks. Goldner trichrome staining performed on heart sections confirmed transmural scar formation. First and second surgeries resulted in less that 10%&#160;post-operative mortality.&#160;

Implantation of a biograft to treat myocardial infarction induced by LAD ligation in a rodent model has conventionally required two open-heart surgeries. In order to reduce mortality and provide optimal conditions for fixation of solid and gelatinous biomatrices associated with cells, minimally invasive procedures have been developed.

Cardiac cell therapy has gained increasing interest and implantation of biomaterials associated with cells has become a major issue to optimize myocardial ...

Bioengineering

Intramyocardial Cell Delivery: Observations in Murine Hearts

Previous studies showed that cell delivery promotes cardiac function amelioration by release of cytokines and factors that increase cardiac tissue revascularization and cell survival. In addition, further observations revealed that specific stem cells, such as cardiac stem cells, mesenchymal stem cells and cardiospheres have the ability to integrate within the surrounding myocardium by differentiating into cardiomyocytes, smooth muscle cells and endothelial cells.
Here, we present the materials and methods to reliably deliver noncontractile cells into the left ventricular wall of immunodepleted mice. The salient steps of this microsurgical procedure involve anesthesia and analgesia injection, intratracheal intubation, incision to open the chest and expose the heart and delivery of cells by a sterile 30-gauge needle and a precision microliter syringe.
Tissue processing consisting of heart harvesting, embedding, sectioning and histological staining showed that intramyocardial cell injection produced a small damage in the epicardial area, as well as in the ventricular wall. Noncontractile cells were retained into the myocardial wall of immunocompromised mice and were surrounded by a layer of fibrotic tissue, likely to protect from cardiac pressure and mechanical load.

Intramyocardial cell delivery in murine models of cardiovascular diseases, such as hypertension or myocardial infarction, is widely used to test the therapeutic potential of different cell types in regenerative studies. Therefore, a detailed description and a clear visualization of this surgical procedure will help to define the limits and advantages of cardiovascular cell therapeutic analyses in small rodents.

Previous studies showed that cell delivery promotes cardiac function amelioration by release of cytokines and factors that increase cardiac tissue ...

LAD-Ligation: A Murine Model of Myocardial Infarction

Research models of infarction and myocardial ischemia are essential to investigate the acute and chronic pathobiological and pathophysiological processes in myocardial ischemia and to develop and optimize future treatment.
Two different methods of creating myocardial ischemia are performed in laboratory rodents. The first method is to create cryo infarction, a fast but inaccurate technique, where a cryo-pen is applied on the surface of the heart (1-3). Using this method the scientist can not guarantee that the cryo-scar leads to ischemia, also a vast myocardial injury is created that shows pathophysiological side effects that are not related to myocardial infarction. The second method is the permanent ligation of the left anterior descending artery (LAD). Here the LAD is ligated with one single stitch, forming an ischemia that can be seen almost immediately. By closing the LAD, no further blood flow is permitted in that area, while the surrounding myocardial tissue is nearly not affected. This surgical procedure imitates the pathobiological and pathophysiological aspects occurring in infarction-related myocardial ischemia.
The method introduced in this video demonstrates the surgical procedure of a mouse infarction model by ligating the LAD. This model is convenient for pathobiological and pathophysiological as well as immunobiological studies on cardiac infarction. The shown technique provides high accuracy and correlates well with histological sections.

This video demonstrates how to use a fast and reliable model to study pathobiological and pathophysiological processes of myocardial ischemia.

Research models of infarction and myocardial ischemia are essential to investigate the acute and chronic pathobiological and pathophysiological processes ...

Biology

Ultrasound-guided Transthoracic Intramyocardial Injection in Mice

Murine models of cardiovascular disease are important for investigating pathophysiological mechanisms and exploring potential regenerative therapies. Experiments involving myocardial injection are currently performed by direct surgical access through a thoracotomy. While convenient when performed at the time of another experimental manipulation such as coronary artery ligation, the need for an invasive procedure for intramyocardial delivery limits potential experimental designs. With ever improving ultrasound resolution and advanced noninvasive imaging modalities, it is now feasible to routinely perform ultrasound-guided, percutaneous intramyocardial injection. This modality efficiently and reliably delivers agents to a targeted region of myocardium. Advantages of this technique include the avoidance of surgical morbidity, the facility to target regions of myocardium selectively under ultrasound guidance, and the opportunity to deliver injectate to the myocardium at multiple, predetermined time intervals. With practiced technique, complications from intramyocardial injection are rare, and mice quickly return to normal activity on recovery from anesthetic. Following the steps outlined in this protocol, the operator with basic echocardiography experience can quickly become competent in this versatile, minimally invasive technique.

Echocardiography-guided percutaneous intramyocardial injection represents an efficient, reliable, and targetable modality for the delivery of gene transfer agents or cells into the murine heart. Following the steps outlined in this protocol, the operator can quickly become competent in this versatile, minimally invasive technique.

Murine models of cardiovascular disease are important for investigating pathophysiological mechanisms and exploring potential regenerative therapies. ...

Percutaneous Contrast Echocardiography-guided Intramyocardial Injection and Cell Delivery in a Large Preclinical Model

Cell and gene therapy are exciting and promising strategies for the purpose of cardiac regeneration in the setting of heart failure with reduced ejection fraction (HFrEF). Before they can be considered for use, and implemented in humans, extensive preclinical studies are required in large animal models to evaluate the safety, efficacy, and fate of the injectate (e.g., stem cells) once delivered into the myocardium. Small rodent models offer advantages (e.g., cost effectiveness, amenability for genetic manipulation); however, given inherent limitations of these models, the findings in these rarely translate into the clinic. Conversely, large animal models such as rabbits, have advantages (e.g., similar cardiac electrophysiology compared to humans and other large animals), whilst retaining a good cost-effective balance. Here, we demonstrate how to perform a percutaneous contrast echocardiography-guided intramyocardial injection (IMI) technique, which is minimally invasive, safe, well tolerated, and very effective in the targeted delivery of injectates, including cells, into several locations within the myocardium of a rabbit model. For the implementation of this technique, we also have taken advantage of a widely available clinical echocardiography system. After putting in practice the protocol described here, a researcher with basic ultrasound knowledge will become competent in the performance of this versatile and minimally invasive technique for routine use in experiments, aimed at hypothesis testing of the capabilities of cardiac regenerative therapeutics in the rabbit model. Once competency is achieved, the whole procedure can be performed within 25 min after anaesthetizing the rabbit.

Novel therapeutic strategies in cardiac regenerative medicine require extensive and detailed studies in large preclinical animal models before they can be considered for use in humans. Here, we demonstrate a percutaneous contrast echocardiography-guided intramyocardial injection technique in rabbits, which is valuable for hypothesis testing the efficacy of such novel therapies.

Cell and gene therapy are exciting and promising strategies for the purpose of cardiac regeneration in the setting of heart failure with reduced ejection ...

Establishing a Swine Model of Post-myocardial Infarction Heart Failure for Stem Cell Treatment

Although advances have been achieved in the treatment of heart failure (HF) following myocardial infarction (MI), HF following MI remains one of the major causes of mortality and morbidity around the world. Cell-based therapies for cardiac repair and improvement of left ventricular function after MI have attracted considerable attention. Accordingly, the safety and efficacy of these cell transplantations should be tested in a preclinical large animal model of HF prior to clinical use. Pigs are widely used for cardiovascular disease research due to their similarity to humans in terms of heart size and coronary anatomy. Therefore, we sought to present an effective protocol for the establishment of a porcine chronic HF model using closed-chest coronary balloon occlusion of the left circumflex artery (LCX), followed by rapid ventricular pacing induced with pacemaker implantation. Eight weeks later, the stem cells were administered by intramyocardial injection in the peri-infarct area. Then the infarct size, cell survival, and left ventricular function (including echocardiography, hemodynamic parameters, and electrophysiology) were evaluated. This study helps establish a stable preclinical large animal HF model for stem cell treatment.

We sought to establish a swine model of heart failure induced by left circumflex artery blockage and rapid pacing to test the effect and safety of intramyocardial administration of stem cells for cell-based therapies.

Although advances have been achieved in the treatment of heart failure (HF) following myocardial infarction (MI), HF following MI remains one of the major ...

Intramyocardial Transplantation of MSC-Loading Injectable Hydrogels after Myocardial Infarction in a Murine Model

One of the major issues facing current cardiac stem cell therapies for preventing postinfarct heart failure is the low retention and survival rates of transplanted cells within the injured myocardium, limiting their therapeutic efficacy. Recently, the use of scaffolding biomaterials has gained attention for improving and maximizing stem cell therapy. The objective of this protocol is to introduce a simple and straightforward technique to transplant bone marrow-derived mesenchymal stem cells (MSCs) using injectable hydroxyphenyl propionic acid (GH) hydrogels; the hydrogels are favorable as a cell delivery platform for cardiac tissue engineering applications due to their ability to be cross-linked in situ and high biocompatibility. We present a simple method to fabricate MSC-loading GH hydrogels (MSC/hydrogels) and evaluate their survival and proliferation in three-dimensional (3D) in vitro culture. In addition, we demonstrate a technique for intramyocardial transplantation of MSC/hydrogels in mice, describing a surgical procedure to induce myocardial infarction (MI) via left anterior descending (LAD) coronary artery ligation and subsequent MSC/hydrogels transplantation.

Stem cell-based therapy has emerged as an efficient strategy to repair injured cardiac tissues after myocardial infarction. We provide an optimal in vivo application for stem cell transplantation using gelatin hydrogels that are able to be enzymatically cross-linked.

One of the major issues facing current cardiac stem cell therapies for preventing postinfarct heart failure is the low retention and survival rates of ...