This work was funded by US Army Grant (W81XWH-08-1-0701) and a Fellowship from Carle Foundation Hospital.

Procedure is in full compatibility with IACUC protocol number 13302 and has been approved by the division of animal resources.
1. Instrument Preparation
<ol>
	<li>Autoclave all non-disposable instruments used prior to the surgical procedure to ensure sterility.</li>
	<li>Sterilize instruments used multiple times in a session with a glass bead sterilizer between uses.</li>
</ol>
2. Hydrogel Preparation
<ol>
	<li>Thaw and plate stem cells at least 24 hr prior to creation of the hydrogel construct.</li>
	<li>Prepare patches one day prior to the surgical procedure using a stereolithography apparatus (or similar instrument) which has been described in full detail in previous publications8,18.</li>
	<li>To create the hydrogel, first generate a computer aided design (CAD) model of the desired patch and export the design to stereolithographic format (STL).</li>
	<li>Create a pre-gel solution by dissolving poly(ethylene glycol) dimethacrylate (PEGDMA) in sterile 1x PBS to obtain a 20% w/v solution. Concurrently, dissolve the photoinitiator 1-[4-(2-hydroxy- ethoxy)-phenyl]-2-hydroxy-2-methyl-1-propane-1-one in DMSO. Add the photoinitiator to the PEGDMA solution at a final w/v of 0.5%. Directly before crosslinking, add 100 ml of the desired density of cells. Note: A cell density between 2.0 x 105-2.0 x 106 cells/ml&#160;is recommended.</li>
	<li>Pipette the pre-gel solution into a dish at the center of the SLA platform and run the SLA software with the previously uploaded STL formatted design.</li>
	<li>Remove the patches from the apparatus and incubate the hydrogels O/N in Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM) supplemented with 5% fetal bovine serum (FBS), 100 U/ml&#160;penicillin, and 100 mg/ml&#160;streptomycin to allow for cells to adjust to the environment within the patch.</li>
</ol>
3. Animal Preparation and Oral intubation
<ol>
	<li>Evaluate each mouse prior to the procedure to ensure that the animal is in good health, as determined from their grooming habits and interactions with their cage mates. Note: Female 8-10&#160;week&#160;old C57BL/6J mice were used for this procedure, however, any mouse age or type can be used.</li>
	<li>Place animals into an anesthetic chamber and expose to 5% isofluorane with a 1 L/min O2 support. Monitor the level of anesthesia by toe pinch reflex.</li>
	<li>Once the animal is properly anesthetized, weigh and place them on the intubation stand.</li>
	<li>Direct a light source towards the animal&#8217;s chest cavity and use forceps to retract the laryngeal surface and expose the vocal cords. Carefully guide a 20 G angiocath between the vocal cords, and smoothly insert into the trachea. Detect proper insertion by a mechanical movement of the chest cavity once the catheter is connected to the small animal ventilator.</li>
	<li>Adjust ventilation settings to the weight of the animal based on the manufacturer&#8217;s guidelines.</li>
	<li>Once intubated, place the animal in the supine position on a heating pad to prevent hypothermia. Ensure that the heating pad does not exceed 40 &#176;C. If necessary, place a barrier between the animal and the heating pad to minimize the chance of burns.</li>
	<li>Remove hair from the surgical site through the use of a depilatory cream. Sterilize the site by 3 alternating scrubs of betadine and 75% ethanol to ensure proper sterilization. Place a drape to expose just the surgical site, and place vet ointment on the eyes to prevent dryness during the procedure.</li>
</ol>
4. Surgical Procedure
<ol>
	<li>The thoracotomy is performed on the left side of the animal, therefore, make a skin incision approximately 1 cm to the left of the sternum, that runs the length of the sternum.</li>
	<li>Use forceps to separate the muscle layer from the underlying ribcage in two steps. Observe a delineating line that represents the pectoralis major muscles of the animal. Lift this muscle slightly, separate it from the underlying external oblique muscle and retract medially. Then, free the external oblique muscle from the underlying ribcage in the same manner, and retract laterally, providing an unobscured view of the second, third, and fourth ribs.</li>
	<li>Perform the thoracotomy between the third and fourth ribs. Gently lift the fourth rib and use a cauterizer to open the chest cavity between the third and fourth ribs. Place retractors to further open the cavity and expose the heart.</li>
	<li>Use forceps to rupture the thin pericardium of the heart.</li>
	<li>Ligate the left coronary artery with an 8-0 monofilament nylon suture. Place the suture approximately 4 mm from the apex of the heart, directly below the bottom tip of the left atrium. Determine proper suture placement by a blanching of the ventricular myocardium and an increase in the size of the left atrium after the suture has been tied.</li>
</ol>
5.&#160;Patch Placement and Glue
<ol>
	<li>Keep patches at 37 &#176;C and 5% CO2 conditions until use.</li>
	<li>Gently lift the patch using a flat ended spatula and gently place it on the surface of the heart, at the site of infarction. 
	Note: To prevent slipping off the left ventricle, the patch can be held in place by lightly maintaining contact using either the spatula or the tip of a pair of forceps.</li>
	<li>Prepare the glue by mixing a 4:1 ratio of fibrinogen to thrombin. Mix the solution by repeated pipetting until it begins to thicken, usually within 1 min after the beginning of the preparation.</li>
	<li>Once the solution viscosity reaches the desired level, quickly transfer approximately 10 ml&#160;to the patch surface. Note: The clotting time of the fibrin glue is rapid, providing a small window of opportunity for efficient transfer of the glue.</li>
</ol>
6. Suturing
<ol>
	<li>Perform all closing sutures using a 6-0 monofilament nylon suture.</li>
	<li>Close the rib layer with three to four individual interrupted sutures.</li>
	<li>Before full closure of the intercostal layer, insert a PE-10 cannula into the incision to evacuate the chest cavity after closure is complete and reestablish proper intrapleural pressure.</li>
	<li>Close the pectoral muscles with three to four individual interrupted sutures.</li>
	<li>Seal the skin layer with a continuous suture.</li>
	<li>Following full closure, attach a 1.0 ml syringe to the end of the cannula and use it to evacuate the chest cavity.</li>
	<li>Apply a tissue adhesive to the incision site to reinforce the suture site.</li>
</ol>
7. Post-surgical Treatment
<ol>
	<li>Give mice an injection of buprenorphine (0.05-1.0 mg/kg) and carprofen (2.2 mg/kg) subcutaneously immediately following the coronary ligation and least 20 min&#160;prior to revival. Administer both drugs 6-8 hr after surgery, and then give twice daily for up to three days to control pain and distress.</li>
	<li>Continuously monitor the animals until conscious and every hour for the first 4 hr following surgery. Perform a second checkup the night of the surgery (2-4 hr later) to administer a second dose of analgesic.</li>
	<li>Monitor the animals daily after the second day following the procedure, until animals appear stable.</li>
</ol>
8. Analysis of Heart Function and Histology
<ol>
	<li>Perform echocardiographs on the mice 4 weeks following the procedure to determine the extent of damage following the MI.</li>
	<li>Anesthetize mice with 4-5% isoflurane within an anesthesia chamber for induction of inhalation anesthesia, and then 2% isoflurane via face mask for maintenance anesthesia.</li>
	<li>Clear the imaging area with a depilatory cream and use a small animal ultrasound to obtain a 2D M-mode echocardiography scan of the beating heart and record the functional parameters associated with it.</li>
	<li>Euthanize the mice in a carbon dioxide chamber, then collect the hearts from the animals and place in a 10% formalin solution for 24 hr. After 24 hr, transfer the hearts to 70% ethanol and keep at 4 &#176;C until tissue processing.</li>
	<li>Process the hearts and embed them in paraffin blocks.</li>
	<li>Cut 5 mm sections through the ventricular region of the heart, starting at the apex and ending at the atrium.</li>
	<li>Stain sectioned slices with both Masson&#8217;s trichrome and hematoxylin and eosin stain using standard protocols.</li>
</ol>

<ol>
	<li>Kolk, M. V., 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=LAD-ligation:+a+murine+model+of+myocardial+infarction.">LAD-ligation: a murine model of myocardial infarction.</a> Journal of visualized experiments : JoVE. , (2009).</li><li>Li, Y., Yao, Y., Sheng, Z., Yang, Y., Ma, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual-modal+tracking+of+transplanted+mesenchymal+stem+cells+after+myocardial+infarction.">Dual-modal tracking of transplanted mesenchymal stem cells after myocardial infarction.</a> International journal of nanomedicine. 6, 815-823 (2011).</li><li>Nagaya, N., 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=Intravenous+administration+of+mesenchymal+stem+cells+improves+cardiac+function+in+rats+with+acute+myocardial+infarction+through+angiogenesis+and+myogenesis.">Intravenous administration of mesenchymal stem cells improves cardiac function in rats with acute myocardial infarction through angiogenesis and myogenesis.</a> American journal of physiology. Heart and circulatory physiology. 287, H2670-H2676 (2004).</li><li>Wang, J. S., Shum-Tim, D., Chedrawy, E., Chiu, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+coronary+delivery+of+marrow+stromal+cells+for+myocardial+regeneration:+pathophysiologic+and+therapeutic+implications.">The coronary delivery of marrow stromal cells for myocardial regeneration: pathophysiologic and therapeutic implications.</a> The Journal of thoracic and cardiovascular surgery. 122, 699-705 (2001).</li><li>Cashman, T. J., Gouon-Evans, V., Costa, K. D. <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+for+cardiac+therapy:+practical+challenges+and+potential+mechanisms.">Mesenchymal stem cells for cardiac therapy: practical challenges and potential mechanisms.</a> Stem cell reviews. 9, 254-265 (2013).</li><li>Sutton, M. G., Sharpe, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Left+ventricular+remodeling+after+myocardial+infarction:+pathophysiology+and+therapy.">Left ventricular remodeling after myocardial infarction: pathophysiology and therapy.</a> Circulation. 101, 2981-2988 (2000).</li><li>Smolina, K., Wright, F. L., Rayner, M., Goldacre, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+survival+and+recurrence+after+acute+myocardial+infarction+in+England,+2004+to+2010.">Long-term survival and recurrence after acute myocardial infarction in England, 2004 to 2010.</a> Circulation. Cardiovascular quality and outcomes. 5, 532-540 (2012).</li><li>Chan, V., Zorlutuna, P., Jeong, J. H., Kong, H., Bashir, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+photopatterning+of+hydrogels+using+stereolithography+for+long-term+cell+encapsulation.">Three-dimensional photopatterning of hydrogels using stereolithography for long-term cell encapsulation.</a> Lab on a chip. 10, 2062-2070 (2010).</li><li>Kai, D., 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=Stem+cell-loaded+nanofibrous+patch+promotes+the+regeneration+of+infarcted+myocardium+with+functional+improvement+in+rat+model.">Stem cell-loaded nanofibrous patch promotes the regeneration of infarcted myocardium with functional improvement in rat model.</a> Acta biomaterialia. 10, 2727-2738 (2014).</li><li>Serpooshan, V., 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+effect+of+bioengineered+acellular+collagen+patch+on+cardiac+remodeling+and+ventricular+function+post+myocardial+infarction.">The effect of bioengineered acellular collagen patch on cardiac remodeling and ventricular function post myocardial infarction.</a> Biomaterials. 34, 9048-9055 (2013).</li><li>Cavallo, L. M., Solari, D., Somma, T., Savic, D., Cappabianca, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Awake+Endoscope-Guided+Sealant+Technique+with+Fibrin+Glue+in+the+Treatment+of+Postoperative+Cerebrospinal+Fluid+Leak+After+Extended+Transsphenoidal+Surgery:+Technical+Note.">The Awake Endoscope-Guided Sealant Technique with Fibrin Glue in the Treatment of Postoperative Cerebrospinal Fluid Leak After Extended Transsphenoidal Surgery: Technical Note.</a> World neurosurgery. , (2013).</li><li>Chung, H. W., Mehta, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrin+glue+for+Gundersen+flap+surgery.">Fibrin glue for Gundersen flap surgery.</a> Clinical Ophthalmology. 7, 479-484 (2013).</li><li>Dunn, C. J., Goa, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrin+sealant:+a+review+of+its+use+in+surgery+and+endoscopy.">Fibrin sealant: a review of its use in surgery and endoscopy.</a> Drugs. 58, 863-886 (1999).</li><li>Chi, N. H., 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=Cardiac+repair+achieved+by+bone+marrow+mesenchymal+stem+cells/silk+fibroin/hyaluronic+acid+patches+in+a+rat+of+myocardial+infarction+model.">Cardiac repair achieved by bone marrow mesenchymal stem cells/silk fibroin/hyaluronic acid patches in a rat of myocardial infarction model.</a> Biomaterials. 33, 5541-5551 (2012).</li><li>Erb, M. A., Claus, T., Hartrumpf, M., Bachmann, S., Albes, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+Tachosil+surgical+patch+or+fibrin+glue+in+coronary+artery+surgery+does+not+affect+quality+of+anastomosis+or+provoke+postoperative+adhesions+in+pigs.">The use of Tachosil surgical patch or fibrin glue in coronary artery surgery does not affect quality of anastomosis or provoke postoperative adhesions in pigs.</a> European journal of cardio-thoracic surgery : official journal of the European Association for Cardio-thoracic Surgery. 36, 703-707 (2009).</li><li>Okada, K., 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=Surgical+treatment+for+rupture+of+left+ventricular+free+wall+after+acute+myocardial+infarction.">Surgical treatment for rupture of left ventricular free wall after acute myocardial infarction.</a> Interactive cardiovascular and thoracic surgery. 4, 203-206 (2005).</li><li>Simpson, D., Liu, H., Fan, T. H., Nerem, R., Dudley, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+tissue+engineering+approach+to+progenitor+cell+delivery+results+in+significant+cell+engraftment+and+improved+myocardial+remodeling.">A tissue engineering approach to progenitor cell delivery results in significant cell engraftment and improved myocardial remodeling.</a> Stem Cells. 25, 2350-2357 (2007).</li><li>Jeong, J. H., 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=Living'+microvascular+stamp+for+patterning+of+functional+neovessels;+orchestrated+control+of+matrix+property+and+geometry.">Living' microvascular stamp for patterning of functional neovessels; orchestrated control of matrix property and geometry.</a> Advanced Materials. 24, 58-63 (2012).</li><li>Wu, X., Ren, J., Li, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrin+glue+as+the+cell-delivery+vehicle+for+mesenchymal+stromal+cells+in+regenerative+medicine.">Fibrin glue as the cell-delivery vehicle for mesenchymal stromal cells in regenerative medicine.</a> Cytotherapy. 14, 555-562 (2012).</li></ol>

The authors declare that they have no competing financial interests.

With this approach to the murine MI model, we have developed a system that minimizes the damage to non-myocardium areas that are associated with other murine MI techniques. These areas include damage caused by a tracheostomy, the cutting of the muscular layer, and the breakage of ribs to expose the chest cavity. We believe that these changes improve the overall surgical outcome thanks to the care taken to keep as much of the major structures, including the ribs an...

A myocardial infarction (MI) is defined as the interruption of blood to a region of the heart caused by the occlusion of a major coronary artery. The damage resulting from an MI is due to the remodeling of the viable heart tissue into non-functional scar tissue, which decreases the ability of the heart or, more specifically, the left ventricle, to beat properly. This results in a decrease in the volume of blood that can be delivered to the body with every heartbeat, known as the stroke volume, and the percentage of blood that is pumped out of the heart with each heartbeat, known as the ejection fraction6. These, along with other diminished functions, increases the strain on the rest of the heart to maintain adequate function. Often, this increased strain can become so severe that it causes a second heart attack, a phenomenon seen in approximately 10% of individuals7.
While medical practices have evolved to treat the immediate aftermath of an MI, no technique has been developed to halt, slow, or reverse the negative side effects of tissue remodeling. Stem cell therapies have emerged as a possible avenue for such a treatment, however, despite their promising potential, stem cells have not proven successful in the clinical setting. One theory for their shortcomings is the inability to ensure the beneficial cells remain at the site of infarction long enough to generate favorable results5. It has been shown that no more than 24% of cells that are simply injected into the site of infarction survived and remained at the damaged site 1 day post-delivery2. A possible prospect for addressing this issue of cell retention is to develop biocompatible hydrogel systems that encapsulate either cells or therapeutics, which can be delivered to the damaged site. The hydrogel of choice in this protocol is a poly(ethylene glycol) dimethacrylate due to its previous use in cell encapsulation procedures, however, any hydrogel capable of encapsulation may be used8. The delivery of the patch directly to the site of injury ensures cell-to-tissue contact over an extended period of time, increasing the length of time the cells can provide beneficial factors to the underlying myocardium.
A bottleneck to the patch approach is the difficulty of adhering the patch to the heart surface. Many groups have overcome this through a variety of techniques, the most prevalent being a simple suture to tie the construct to the heart surface9,10. This has proven successful in a number of cases in which the construct is made of a stiffer material, but fails when attempted on a hydrogel system, due to the high water concentration and delicate nature of the patch construct. To overcome this, we have utilized a fibrin glue external adhesive system that mimics the chemistry of clot formation. Fibrin glue has been used in numerous medical surgeries, including dura tears, bronchial fistulas, and corneal transplantation, highlighting the biocompatibility of the product as a wound sealant11-13. Additionally, fibrin has been used for a variety of cardiac purposes, including surgical treatment of left ventricular ruptures and coronary artery bypass surgeries, however, its use as an adhesion glue for a cardiac patch is not commonly used14-17. A simple formulation of thrombin and fibrinogen results in a biocompatible glue that can be placed directly on the outside of an external cardiac patch, providing a viable adhesion system to ensure patch to heart interaction.

<table border="0" cellpadding="0" cellspacing="0">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr>
			<td>Harvard Model 687 Mouse Ventilator</td>
			<td>Harvard Apparatus</td>
			<td>55-0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Inintech Biosciences LLC Dry Glass Bead Sterilizer</td>
			<td>Fisher Scientific</td>
			<td>NC9531961</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica MZ6 surgical microscope</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cautery Kit</td>
			<td>Gemini</td>
			<td>GEM 5917</td>
			<td></td>
		</tr>
		<tr>
			<td>Delicate Forceps - 0.4mm Tips Angled</td>
			<td>Fine Science Tools</td>
			<td>11063-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Agricola Retractor - 3.5cm Spread</td>
			<td>Fine Science Tools</td>
			<td>17005-04</td>
			<td></td>
		</tr>
		<tr>
			<td>Spring Scissors - 2.5mm Blades Straight</td>
			<td>Fine Science Tools</td>
			<td>15000-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Castroviejo Needle Holder - w/Lock Tungsten Carbide 14cm</td>
			<td>Fine Science Tools</td>
			<td>12565-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Iris Scissors - Delicate Straight 10.5 cm</td>
			<td>Fine Science Tools</td>
			<td>14060-10</td>
			<td></td>
		</tr>
		<tr>
			<td>8-0 monofilament suture</td>
			<td>Ethicon</td>
			<td>8730P</td>
			<td></td>
		</tr>
		<tr>
			<td>6-0 Silk suture</td>
			<td>Ethicon</td>
			<td>639G</td>
			<td></td>
		</tr>
		<tr>
			<td>Thrombin</td>
			<td>Sigma</td>
			<td>T7009</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibrinogen</td>
			<td>Sigma</td>
			<td>F3879</td>
			<td></td>
		</tr>
		<tr>
			<td>Vetbond Tissue Adhesive</td>
			<td>3M</td>
			<td>1469SB</td>
			<td></td>
		</tr>
	</tbody>
</table>

a hydrogel construct and fibrin-based glue approach to deliver therapeutics in a murine myocardial infarction model.

The murine MI model is widely recognized in the field of cardiovascular disease, and has consistently been used as a first step to test the efficacy of treatments in vivo1. The traditional, established protocol has been further fine-tuned to minimize the damage to the animal. Notably, the pectoral muscle layers are teased away rather than simply cut, and the thoracotomy is approached intercostally as opposed to breaking the ribs in a sternotomy, preserving the integrity of the ribcage. With these changes, the overall stress on the animal is decreased. 
Stem cell therapies aimed to alleviate the damage caused by MIs have shown promise over the years for their pro-angiogenic and anti-apoptotic benefits. Current approaches of delivering cells to the heart surface typically involve the injection of the cells either near the damaged site, within a coronary artery, or into the peripheral blood stream2-4. While the cells have proven to home to the damaged myocardium, functionality is limited by their poor engraftment at the site of injury, resulting in diffusion into the blood stream5. This manuscript highlights a procedure that overcomes this obstacle with the use of a cell-encapsulated hydrogel patch. The patch is fabricated prior to the surgical procedure and is placed on the injured myocardium immediately following the occlusion of the left coronary artery. To adhere the patch in place, biocompatible external fibrin glue is placed directly on top of the patch, allowing for it to dry to both the patch and the heart surface. This approach provides a novel adhesion method for the application of a delicate cell-encapsulating therapeutic construct.

During the surgical procedure, ligation of the left coronary artery can be identified by a marked blanching downstream of the occluded artery. As a test before tying the knot, the suture can be tightened briefly to check if it is in the appropriate place. Additionally, since occlusion of the artery results in almost instantaneous decrease in the ability of the left atrium to properly beat, the left atrium will enlarge in response to a backflow of blood in the system.
M-mode echocardiography me...

Watch this Scientific Journal Video about A Hydrogel Construct and Fibrin-based Glue Approach to Deliver Therapeutics in a Murine Myocardial Infarction Model. at JoVE.com

A Hydrogel Construct and Fibrin-based Glue Approach to Deliver Therapeutics in a Murine Myocardial Infarction Model.

This protocol aims to alleviate the limitation of poor cell engraftment for stem cell treatment of myocardial infarctions through the use of a hydrogel system and a fibrin-based glue. With this approach, cell-to-tissue contact post-infarction can be maintained, increasing the therapeutic potential of beneficial agents at the site of injury.

The murine MI model is widely recognized in the field of cardiovascular disease, and has consistently been used as a first step to test the efficacy of ...

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8.6K Views.  University of Illinois at Urbana-Champaign. This protocol aims to alleviate the limitation of poor cell engraftment for stem cell treatment of myocardial infarctions through the use of a hydrogel system and a fibrin-based glue. With this approach, cell-to-tissue contact post-infarction can be maintained, increasing the therapeutic potential of beneficial agents at the site of injury.

Video: A Hydrogel Construct and Fibrin-based Glue Approach to Deliver Therapeutics in a Murine Myocardial Infarction Model.

Acute Myocardial Infarction in Rats

With heart failure leading the cause of death in the USA (Hunt), biomedical research is fundamental to advance medical treatments for cardiovascular diseases. Animal models that mimic human cardiac disease, such as myocardial infarction (MI) and ischemia-reperfusion (IR) that induces heart failure as well as pressure-overload (transverse aortic constriction) that induces cardiac hypertrophy and heart failure (Goldman and Tarnavski), are useful models to study cardiovascular disease. In particular, myocardial ischemia (MI) is a leading cause for cardiovascular morbidity and mortality despite controlling certain risk factors such as arteriosclerosis and treatments via surgical intervention (Thygesen). Furthermore, an acute loss of the myocardium following myocardial ischemia (MI) results in increased loading conditions that induces ventricular remodeling of the infarcted border zone and the remote non-infarcted myocardium. Myocyte apoptosis, necrosis and the resultant increased hemodynamic load activate multiple biochemical intracellular signaling that initiates LV dilatation, hypertrophy, ventricular shape distortion, and collagen scar formation. This pathological remodeling and failure to normalize the increased wall stresses results in progressive dilatation, recruitment of the border zone myocardium into the scar, and eventually deterioration in myocardial contractile function (i.e. heart failure). The progression of LV dysfunction and heart failure in rats is similar to that observed in patients who sustain a large myocardial infarction, survive and subsequently develops heart failure (Goldman). The acute myocardial infarction (AMI) model in rats has been used to mimic human cardiovascular disease; specifically used to study cardiac signaling mechanisms associated with heart failure as well as to assess the contribution of therapeutic strategies for the treatment of heart failure. The method described in this report is the rat model of acute myocardial infarction (AMI). This model is also referred to as an acute ischemic cardiomyopathy or ischemia followed by reperfusion (IR); which is induced by an acute 30-minute period of ischemia by ligation of the left anterior descending artery (LAD) followed by reperfusion of the tissue by releasing the LAD ligation (Vasilyev and McConnell). This protocol will focus on assessment of the infarct size and the area-at-risk (AAR) by Evan's blue dye and triphenyl tetrazolium chloride (TTC) following 4-hours of reperfusion; additional comments toward the evaluation of cardiac function and remodeling by modifying the duration of reperfusion, is also presented. Overall, this AMI rat animal model is useful for studying the consequence of a myocardial infarction on cardiac pathophysiological and physiological function.

The rat model of acute myocardial infarction (AMI) is useful to study the consequence of a MI on cardiac pathophysiological and physiological function.

With heart failure leading the cause of death in the USA (Hunt), biomedical research is fundamental to advance medical treatments for cardiovascular ...

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 ...

Myocardial Infarction and Functional Outcome Assessment in Pigs

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One important prerequisite is a good understanding of all safety and efficacy aspects obtained in a large animal model that validly reflect the human scenario of myocardial infarction (MI). Pigs are widely used in this regard since their cardiac size, hemodynamics, and coronary anatomy are close to that of humans. Here, we present an effective protocol for using the porcine MI model using a closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. This approach is based on 90 min of myocardial ischemia, inducing large left ventricle infarction of the anterior, septal and inferoseptal walls. Furthermore, we present protocols for various measures of outcome that provide a wide range of information on the heart, such as cardiac systolic and diastolic function, hemodynamics, coronary flow velocity, microvascular resistance, and infarct size. This protocol can be easily tailored to meet study specific requirements for the validation of novel cardioregenerative biologics at different stages (i.e. directly after the acute ischemic insult, in the subacute setting or even in the chronic MI once scar formation has been completed). This model therefore provides a useful translational tool to study MI, subsequent adverse remodeling, and the potential of novel cardioregenerative agents.

This protocol describes the porcine myocardial infarction (MI) model using a 90 min closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. Furthermore, the protocol for several outcome parameters, such as cardiac function, hemodynamics, microvascular resistance, and infarct size, are also presented.

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One ...

Primary Outcome Assessment in a Pig Model of Acute Myocardial Infarction

Mortality after acute myocardial infarction remains substantial and is associated with significant morbidity, like heart failure. Novel therapeutics are therefore required to confine cardiac damage, promote survival and reduce the disease burden of heart failure. Large animal experiments are an essential part in the translational process from experimental to clinical therapies. To optimize clinical translation, robust and representative outcome measures are mandatory. The present manuscript aims to address this need by describing the assessment of three clinically relevant outcome modalities in a pig acute myocardial infarction (AMI) model: infarct size in relation to area at risk (IS/AAR) staining, 3-dimensional transesophageal echocardiography (TEE) and admittance-based pressure-volume (PV) loops. Infarct size is the main determinant driving the transition from AMI to heart failure and can be quantified by IS/AAR staining. Echocardiography is a reliable and robust tool in the assessment of global and regional cardiac function in clinical cardiology. Here, a method for three-dimensional transesophageal echocardiography (3D-TEE) in pigs is provided. Extensive insight into cardiac performance can be obtained by admittance-based pressure-volume (PV) loops, including intrinsic parameters of myocardial function that are pre- and afterload independent. Combined with a clinically feasible experimental study protocol, these outcome measures provide researchers with essential information to determine whether novel therapeutic strategies could yield promising targets for future testing in clinical studies.

Reliable and accurate outcome assessment is the key for translation of preclinical therapies into clinical treatment. The current paper describes how to assess three clinically relevant primary outcome parameters of cardiac performance and damage in a pig acute myocardial infarction model.

Mortality after acute myocardial infarction remains substantial and is associated with significant morbidity, like heart failure. Novel therapeutics are ...

Myocardial Infarction in Neonatal Mice, A Model of Cardiac Regeneration

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and regeneration, and to define new targets for therapeutics. For decades, models of complete heart regeneration existed in amphibians and fish, but a mammalian counterpart was not available. The recent discovery of a postnatal window during which mice possess regenerative capabilities has led to the establishment of a mammalian model of cardiac regeneration. A surgical model of mammalian cardiac regeneration in the neonatal mouse is presented herein. Briefly, postnatal day 1 (P1) mice are anesthetized by isoflurane and placed on an ice pad to induce hypothermia. After the chest is opened, and the left anterior descending coronary artery (LAD) is visualized, a suture is placed around the LAD to inflict myocardial ischemia in the left ventricle. The surgical procedure takes 10-15 min. Visualizing the coronary artery is crucial for accurate suture placement and reproducibility. Myocardial infarction and cardiac dysfunction are confirmed by triphenyl-tetrazolium chloride (TTC) staining and echocardiography, respectively. Complete regeneration 21 days post myocardial infarction is verified by histology. This protocol can be used to as a tool to elucidate mechanisms of mammalian cardiac regeneration after myocardial infarction.

This protocol describes a highly reproducible model of cardiac regeneration by surgical induction of myocardial infarction in the left ventricle of postnatal day 1 mice. The method involves induction of hypothermic anesthesia and ligation of the left anterior descending coronary artery.

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and ...

Murine Myocardial Infarction Model using Permanent Ligation of Left Anterior Descending Coronary Artery

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine models of MI with permanent ligation of left-anterior descending (LAD) coronary artery closely mimics MI in humans. Murine models benefit from the extensive genetic engineering available nowadays. Here we propose a reproducible murine surgical model of myocardial infarction by permanent LAD coronary ligation. Our technique comprises anesthesia with ketamine/xylazine that can be rapidly reversed by administration of an antagonist, intubation without tracheotomy for mechanical-assisted ventilation, ventilation with application of extrinsic positive end-expiratory pressure (PEEP) to avoid alveolar collapse, a thoracotomy method limiting to the minimum surgical lesions made to skeletal muscles, and lung inflation without thoracentesis. This method is sparsely invasive, reproducible and reduces post-surgery mortality and complications.

Herein we describe a surgical procedure showing how to achieve permanent ligation of the left-anterior descending coronary artery in mice. This model is of high relevance to investigate the pathophysiology of myocardial infarction and the concomitant biological processes.

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine ...

A Cryoinjury Model to Study Myocardial Infarction in the Mouse

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we demonstrate a murine cryoinjury infarct model that generates precise infarct sizes with high reproducibility and replicability. In brief, after intubation and sternotomy of the animal, the heart is lifted from the thorax. The probe of a handheld liquid nitrogen delivery system is applied onto the myocardial wall to induce cryoinjury. Impaired ventricular function and electrical conduction can be monitored with echocardiography or optical mapping. Transmural myocardial remodeling of the infarcted area is characterized by collagen deposition and loss of cardiomyocytes. Compared to other models (e.g., LAD-ligation), this model utilizes a handheld liquid nitrogen delivery system to generate more uniform infarct sizes.

This article demonstrates a model to study cardiac remodeling after myocardial cryoinjury in mice.

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we ...

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 ...