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 instr...

<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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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 height="17" style="height:17px;width:322px;">0.9% NaCl Irrigation, USP</td>
			<td style="width:191px;">Baxter</td>
			<td style="width:132px;">0338-0048-04</td>
			<td style="width:328px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">11x12&#34; Press n&#39; Seal surgical drape, autoclavable</td>
			<td>SAI Infusion Technologies</td>
			<td>PSS-SD</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">24G 3/4&#34; IV catheter tube</td>
			<td>Jelco</td>
			<td>4053</td>
			<td></td>
		</tr>
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			<td height="17" style="height:17px;">28G x 1/2&#34; 1mL allergy syringe</td>
			<td>BD</td>
			<td>305500</td>
			<td>Injection of analgesic</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">30G x 1/2&#34; 3/10cc insulin syringe</td>
			<td>Ulticare</td>
			<td>08222.0933.56</td>
			<td>Injection of stem cells</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">6-0 S-29, 12&#34; Vicryl suture</td>
			<td>Ethicon</td>
			<td>J556G</td>
			<td>Intercostal, superficial muscle and skin layer incision closure</td>
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		<tr height="17">
			<td height="17" style="height:17px;">9-0 BV100-4, 5&#34; Ethilon suture</td>
			<td>Ethicon</td>
			<td>2829G</td>
			<td>Ligation of the LAD artery</td>
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		<tr height="17">
			<td height="17" style="height:17px;">Absorbent underpad</td>
			<td>Thermo Fischer Scientific</td>
			<td>14-206-64</td>
			<td>For underneath the animal</td>
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			<td height="17" style="height:17px;">Alcohol prep pads, 2 ply, medium</td>
			<td>Coviden</td>
			<td>6818</td>
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			<td height="17" style="height:17px;">Anti-fog face mask</td>
			<td>Halyard</td>
			<td>49235</td>
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		<tr height="17">
			<td height="17" style="height:17px;">Bonn Strabismus scissors, curved, blunt</td>
			<td>Fine Science Tools</td>
			<td>14085-09</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">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 height="17">
			<td height="17" style="height:17px;">Castroviejo needle holders, curved</td>
			<td>Fine Science Tools</td>
			<td>12061-01</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Curity sterile gauze sponges</td>
			<td>Coviden</td>
			<td>397310</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Delicate suture tying forceps, 45 angle bent</td>
			<td>Fine Science Tools</td>
			<td>11063-07</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Electric Razor</td>
			<td>Wahl</td>
			<td></td>
			<td>Fur removal</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Isoflurane 100 ml</td>
			<td>Cardinal Health</td>
			<td>PI23238</td>
			<td>Anesthetic</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Lab coat</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Monoject 1 mL hypodermic syringe</td>
			<td>Coviden</td>
			<td>8881501400</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Moria iris forceps, curved, serrated (x2)</td>
			<td>Fine Science Tools</td>
			<td>11370-31</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Moria speculum retractor</td>
			<td>Fine Science Tools</td>
			<td>17370-53</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Mouse endotracheal intubation kit</td>
			<td>Kent Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Nair depilatory cream</td>
			<td>Johnson &#38; Johnson</td>
			<td></td>
			<td>Fur removal</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Optixcare eye lube plus</td>
			<td>Aventix</td>
			<td></td>
			<td>Sterile ocular lubricant</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Physiosuite ventilator</td>
			<td>Kent Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<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>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Povidone-iodine swabs</td>
			<td>PDI</td>
			<td>S41125</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Scalpel, 10-blade</td>
			<td>Bard-Parker</td>
			<td>371610</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Sterile 3&#34; cotton tipped applicators</td>
			<td>Cardinal Health</td>
			<td>C15055-003</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Sterile 6&#34; tapered cotton tip applicators</td>
			<td>Puritan</td>
			<td>25-826-5WC</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Sterile gloves</td>
			<td>Cardinal Health</td>
			<td>N8830</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Sterilization pouches</td>
			<td>Medline</td>
			<td>MPP100525GS</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Surgery cap</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Surgical Microscope</td>
			<td>Leica</td>
			<td>M125</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Suture tying forceps, straight (x2)</td>
			<td>Fine Science Tools</td>
			<td>10825-10</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Transpore surgical tape</td>
			<td>3M</td>
			<td>1527-1</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Triple antibiotic ointment</td>
			<td>G&#38;W Laboratories</td>
			<td>11-2683ILNC2</td>
			<td>Topical application to prevent infection</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Vannas-T&#252;bingen Spring Scissors, curved</td>
			<td>Fine Science Tools</td>
			<td>15004-08</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">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...

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, ...

Ischemic damage induces a hostile pro-inflammatory environment affecting the survival of transplanted cells. Our protocol presents an alternative method for providing biologics by circumventing the initial immune response to injury. The main advantage of this protocol is that it makes it possible to administer therapeutics one week following injury, mimicking treatment in the clinical environment.Our primary focus is on the ischemia reperfusion injury model and transplantation of STEM cells. However, this approach can also be utilized for models of inflammatory disease that involves the introduction of biological therapies. Begin by autoclaving all surgical instruments.If multiple surgeries are to be performed in one session, clean the instruments after each animal and re-sterilize them using a hot bead sterilizer. Subcutaneously administer analgesic, weigh the animal, and input the weight into the ventilator. Shave the left side of the chest from the sternum to the level of the shoulder and apply depilatory cream to remove excess fur.To prevent lung collapse, maintain the positive end expiratory pressure at two centimeters of water for the ischemia reperfusion procedure and change it to three centimeters of water for the delayed injection of cells procedure. After anesthetizing the mouse, intubate it with a 20 gauge endotracheal tube and transfer it to a controlled heating pad to maintain a body temperature of 35 to 37 degrees Celsius. Place the mouse in lateral recumbency with cranial end on the left and caudal end on the right.Scrub the surgical area alternating between povidone iodine and alcohol swabs three times and apply ophthalmic ointment to both eyes. Use a number 10 blade scalpel to make a vertical incision 2.5 millimeters to the right of the leftmost nipple in the field of view, then use scissors to cut through the superficial muscle layers until the intercostal muscle and ribs are visible. While lifting the ribs and surrounding tissue, cut through the intercostal space between the fourth and fifth ribs, then insert the eyelid retractor into the open space.Retract the pericardium using curved forceps, moving the lung upwards and out of the view. Pass a 9-0 nylon suture through the myocardium beneath the LAD artery 2.5 millimeters distal to the left auricle maintaining smooth and consistent movement to prevent tearing of the tissue or puncturing a major blood vessel. Tie a loose square knot, then place one centimeter of polyethylene tubing within the loose knot.Secure the suture around the tubing and confirm ischemia by pallor and ventricular arrhythmia, then release after 35 minutes. After removing the tubing, wait for five minutes to confirm re-perfusion of the myocardium. Place a 24 gauge IV catheter tube into the thoracic cavity one intercostal space to the right of the opening.Use 6-0 absorbable sutures to close the intercostal incision in a simple interrupted pattern and the muscle layer in a continuous suture pattern. After closing the superficial muscle layer, remove the chest tube while withdrawing the air from the thoracic cavity with a one milliliter tuberculin syringe. Close the skin incision with a 6-0 absorbable suture in a continuous horizontal mattress pattern.When finished, administer 1.5 milliliters of warm saline subcutaneously and apply triple antibiotic ointment to the incision site to prevent infection. Transfer the mouse to a bedding free cage or a cage with covered bedding on a warm pad and a temperature of 35 to 37 degrees Celsius until it is fully recovered. After preparing and intubating the mouse as previously described, remove the suture from the skin layer with scissors and forceps, then use a number 10 scalpel to make an incision in the same location as the previous surgery.Continue to cut through the scar tissue until the muscle layer suture is visible, then use the scissors and forceps to remove the suture and cut the muscle layer open. Remove the sutures holding the ribs together and continue cutting through the intercostal muscle from the previous incision. Place the eyelid retractor into the intercostal space and locate the area of the previous ligation.Load 300, 000 mesenchymal STEM cells suspended in 20 microliters of PBS into a 30 gauge insulin syringe and bend the needle slightly to achieve the proper angle for injection. 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, then slowly inject the cells into the myocardium, wait three seconds and remove the needle. Observe the heart closely for three minutes to make sure that there is no abnormal reaction to the cells such as ventricular fibrillation.After three minutes, place a 24 gauge IV catheter tube into the thoracic cavity one intercostal space to the right of the opening. Close the intercostal muscle and skin layers and remove the chest tube as previously described. Administer 1.5 milliliters of warm saline subcutaneously and apply triple antibiotic ointment to the incision site to prevent infection.Transfer the mouse to a bedding free cage or cage with covered bedding on a warm pad until it is fully recovered. Post-operative echocardiogram and electrocardiogram were performed on the day proceeding STEM cell implantation, confirming infarction and decreased ventricular contractile function. 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.Masson trichrome staining of myocardial tissue seven days post-injury showed increased collagen deposition and thinning of the left ventricular wall in hearts of mice that received ischemic injury. In vivo bioluminescent imaging or BLI was performed on the day after mesenchymal STEM cell implantation. Successful delivery of MSCs is exemplified by the BLI signal compared to mice that had induced ischemia reperfusion injury but did not receive MSCs.When attempting this protocol, keep in mind that small intentional movements when cutting through the intercostals, ligating the vessel, and injecting cells are vital for preventing undue damage to the heart or the lung in the field of view. These procedures offer the ability to monitor cardiac function after transplantation through ultrasound, cell viability via bioluminescence imaging, or further molecular or biochemical techniques depending on the therapy used.

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

Research

JoVE Journal

Medicine

4.5K visualizações.  Mayo Clinic. Danos isquêmicos induzm um ambiente hostil pró-inflamatório que afeta a sobrevivência de células transplantadas. Nosso protocolo apresenta um método alternativo para fornecer biológicos contornando a resposta imune inicial à lesão. A principal vantagem deste protocolo é que ele possibilita a administração terapêutica uma semana após a lesão, imitando o tratamento no ambiente clínico.Nosso foco principal é no modelo de lesão de reperfusão de isquemia e transplante de ...