Name: Implantation of Engineered Tissue in the Rat Heart (Video) | JoVE Journal
Uploaded: 2009-06-24T00:00:00.000+00:00
Duration: 7 min 59 s
Description: Learn a surgical protocol for implanting engineered tissue constructs into the rat heart's AV groove. This method creates an alternative electrical conduction pathway for cardiovascular tissue engineering research.

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5 minutes

This work is supported by research grants from the National Institutes of Health (HL068915; HL088206) and contributions to the Cardiac Conduction Fund at Children&rsquo;s Hospital Boston.

Part 1: Induction of general anesthesia
<ol>
<li>A 200-250 g Lewis rat is anesthetized with 50 mg/kg Ketamine and 4 mg/kg Xylazine injected with a 30 G needle into the intraperitoneal cavity. The upper abdomen and right chest are clipped to remove hair and the incision site thoroughly cleaned with 70% ethanol and then a povidone-iodine topical antiseptic solution.</li>
<li>The animal is intubated endotracheally using a 16 G intravenous catheter connected to a small animal respirator (Harvard Apparatus). The respirator is connected to a vaporizer to supply 0.5 to 1.0% Isoflurane (Baxter) mixed with 100% oxygen to maintain general anesthesia. Respiration rate and tidal volume is adjusted according to the body weight of the rat. The animal is placed on a heating pad (Hood Thermo-Pad) to maintain body temperature during the surgical procedure.</li>
<li>To monitor heart rate and oxygen saturation throughout the surgical procedure a pulsoximeter device (Nellcor) is clipped onto one of the back limbs.</li>
</ol>
Part 2: Thoracotomy and engineered tissue implantation
<ol>
<li>The surgical site is prepared with sterile surgical draping that is held in place with towel clamps.</li>
<li>An incision is made from the antero-lateral to the lateral wall of the right chest, just above the 5th intercostal space. The interior of the chest is accessed through the 5th intercostal space and a small retractor (Roboz) is positioned to maximize the view.</li>
<li>The pericardium is cut just above the appendix of the right atrium and right ventricle from the base toward the apex of the heart. Afterward, the AV groove is identified and the epicardial layer adjacent to the aorto-atrio-ventricular triangle is gently teased apart.</li>
<li>A 7-0 Prolene suture (Ethicon) is placed through the basal shoulder of the right ventricle. A 2 x 2 x 2 mm piece is dissected from the engineered tissue construct and positioned within the AV groove and gently tied down with the Prolene suture.</li>
<li>The surgical field is checked for bleeding and two 4-0 Vicryl sutures (Ethicon) are placed between the upper and lower intercostal space for closure of the rib-cage.</li>
<li>Any apparent pneumothorax is excavated by expanding the lungs by way of the ventilator.</li>
<li>The chest wall is then closed in layers.</li>
</ol>
Part 3: Recovery
<ol>
<li>While the animal recovers from the surgery, the incision site is infiltrated with 5 mL 0.25% Bupivacaine[2].</li>
<li>The animal is extubated following weaning from the respirator and is returned to a cage with fresh bedding once it is fully awake and sternal. The rat is subsequently administered 0.01 mg/kg Buprenorphine analgesia subcutaneously every 12 hours for 3 days.</li>
</ol>
Part 4: Representative results
If performed properly, the described surgical implantation of engineered tissue in the AV-groove of the heart (Figure 1) results in greater than 90% recovery of the animals.
<img src="/files/ftp_upload/1139/Figure1.jpg" alt="Cardiac implant placement; labeled heart anatomy; surgical procedure; right atrium, ventricle." data-altupdatedat="1761735611000" data-alt="Figure 1"> Figure 1. A photograph of the exposed heart showing the site of implantation of engineered tissue.

<ol><li>Choi, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cardiac+conduction+through+engineered+tissue%5BTitle%5D)+AND+%22Am+J+Pathol%22%5BJournal%5D)">Cardiac conduction through engineered tissue.</a> Am J Pathol. 169, 72-85 (2006).</li>
<li>Shin, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Low-dose+systemic+bupivacaine+prevents+the+development+of+allodynia+after+thoracotomy+in+rats.+Anesth+Analg%5BTitle%5D)+AND+%22%22%5BJournal%5D)">Low-dose systemic bupivacaine prevents the development of allodynia after thoracotomy in rats. Anesth Analg.</a> 107, 1587-1591 (2008).</li>
<li>Horstick, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Surgical+procedure+affects+physiological+parameters+in+rat+myocardial+ischemia%3A+need+for+mechanical+ventilation%5BTitle%5D)+AND+%22Am+J+Physiol%22%5BJournal%5D)">Surgical procedure affects physiological parameters in rat myocardial ischemia: need for mechanical ventilation.</a> Am J Physiol. 276, H472-H479 (1999).</li>
</ol>

Experiments on animals were performed in accordance with the guidelines and regulations set forth by the Institutional Animal Care and Use Committee at Children s Hospital Boston.

For a favorable outcome to the surgical procedure, it is critical to properly intubate and invasively ventilate the animal. If the rat is simply sedated and allowed to spontaneously breathe, both lungs will inevitably collapse while the thorax is opened due to common pleura in rodents. Before closing the chest, any pneumothorax needs to be completely eliminated and the lungs fully expanded to reduce atelectasis. Otherwise, weaning from the respirator and recovery will be unsuccessful and the animal will become hypercapni...

<table><tbody><tr><td>7x4 Thermo Pad 4/box</td><td>Equipment</td><td>Hood Thermo-Pad, Summerland, BC Canada</td><td>MODEL 704R</td><td></td></tr><tr><td>Inspira ASV Ventilator</td><td>Equipment</td><td>Harvard Apparatus</td><td></td><td></td></tr><tr><td>Handheld Pulse Oximeter</td><td>Equipment</td><td>Nellcor</td><td>N20PA</td><td></td></tr><tr><td>Retractor</td><td>Equipment</td><td>Roboz Surgical Instruments Co.</td><td>RS-6514</td><td></td></tr><tr><td>Isoflurane (Forane)</td><td>Reagent</td><td>Baxter Internationl Inc.</td><td>1001936040</td><td></td></tr><tr><td>Ketamine HCl</td><td>Reagent</td><td>Hospira Inc.</td><td>RL-0065</td><td></td></tr><tr><td>Xylazine (AnaSed)</td><td>Reagent</td><td>Lloyd, Inc.</td><td>LB15705A</td><td></td></tr><tr><td>Buprenorphine HCL Inject</td><td>Reagent</td><td>Bedford Laboratories</td><td>Div-BUP-P00</td><td></td></tr><tr><td>Bupivacaine 0.25%</td><td>Reagent</td><td>Hospira Inc.</td><td></td><td></td></tr></tbody></table>

implantation of engineered tissue in the rat heart

Implantation of Engineered Tissue in the Rat Heart

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in common laboratory rats or mice and these quite frequently serve as an intermediate step between bench-top experiments and large animal testing or human trials. Given that rodents provide an established, cost-effective, and physiologically-relevant model system in which to test novel combinations of scaffolding materials and cells, they are particularly well-suited for cardiovascular tissue engineering studies. Presently, we describe an open-heart surgical procedure to implant engineered tissue containing myogenic progenitor cells in the atrioventricular (AV) groove of a rat heart. These implants are intended to create an electrical conduit between the right atrium and right ventricle with the ultimate goal of providing an alternative treatment to conventional pacemaker implantation in pediatric patients with complete heart block[1]. The engineered tissue is implanted in the AV-groove by means of a thoracotomy. For our purposes, Lewis rats are anesthetized and invasively ventilated to maintain positive airway pressure during the sterile surgical procedure. The approach to the heart is performed by a right thoracotomy through an antero-lateral incision at the 5th intercostal space. The tissue construct is fixed in the AV groove using a single 7-0 Prolene suture and positioned between the right ventricle and atrium at the ventral portion of the heart. The epicardium is partially removed to allow direct contact between the recipient myocardial cells and those contained in the engineered tissue. Following implantation, the chest wall is closed in layers, any pneumothorax is evacuated, and the animal is extubated and treated with analgesic.

Here, we describe a cardiac surgical procedure to implant engineered tissue in the atrioventricular (AV)-groove of an adult Lewis rat.

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in ...

Hi, I am Zu from the laboratory of Douglas Cowan in the Department of Anesthesiology at Children's Hospital and Harvard Medical School. Today we will show you a procedure for implanting engineered tissue in the red heart. We use this procedure in our laboratory to create an alternative atrial ventricular electrical conduction pathway.So let's get started. This video describes a surgical implantation of a myoblast containing engineered tissue construct. This proof of principle experimental protocol provides evidence that tissue casts can be fabricated in vitro and transplanted in vivo into recipients requiring reparation of a non-functional organ.And accompanying video describes how cells were cast into the mold that fits the AV groove of the heart of an adult rat. The protocol begins by anesthetizing a 200 to 250 gram Lewis rat with a ketamine and aine cocktail injected through a 30 gauge needle into the intraperitoneal cavity. Once the rat is fully anesthetized and does not respond to a foot pinch, prepare for the surgical procedure by clipping off the hair of the upper abdomen and right chest.Then the incision side is thoroughly cleansed with 70%ethanol, followed by a povidone iodine, topical antiseptic solution. Using a 16 gauge intravenous catheter, the rat is intubated and Dora lead and connected to a small animal respirator. The respirator is connected to a vaporizer set to supply 0.5 to 1%ISO fluorine mixed with 100%oxygen to maintain general anesthesia.The respiration rate and tidal volume is adjusted according to the body weight of the rat. To maintain body temperature, the intubated rat is kept on a heating pad During the surgical procedure as a final precaution to monitor heart rate and oxygen saturation throughout the surgical procedure, a pulse oximeter device is clipped onto one of the back limbs. The surgical site is prepared with sterile surgical draping that is held in place with towel clamps.This surgery begins with an incision from the anterior lateral to the lateral wall of the right chest, just above the fifth intercostal space. The interior of the chest is accessed through the ribs. In a small retractor is position to maximize the view.The pericardium is cut just above the appendix of the right atrium and right ventricle from the base toward the apex of the heart. Afterward, the AV groove is identified in the epicardial layer adjacent to the aorta. Atrial ventricular triangle is gently teased apart.A seven oh prolene suture is placed through the basal shoulder of the right ventricle from the engineered tissue, dissect out a two millimeter cube of tissue. The two millimeter tissue cube is positioned within the AV groove and gently tied down with the proline suture Before the rat is closed up. Any air in the space around the lungs is removed by expanding the lungs with a ventilator.The surgical field is checked for bleeding and two four oh Vicryl sutures are placed between the upper and lower intercostal space for closure of the ribcage. Finally, to close the chest, the muscular plane is brought together with a running suture and the skin is closed. When an intra cutaneous placed running suture while the animal recovers from the surgery, the incision side is infiltrated with five milliliters, a 0.25%bupivocaine.The animal is extubated following weaning from the respirator and is returned to a cage with fresh bedding. Once it is fully awake and ambulatory, the rat is subsequently administered 0.01 milligram per milliliter of buprenorphine and analgesia subcutaneously every 12 hours for the next three days if performed properly. The described surgical implantation of engineered tissue in the AV groove of the heart results in greater than 90%recovery of the animals.Subsequent experiments have shown that donor cells from implanted tissue functionally integrate into the host myocardium. We've just shown you how to implant an engineered tissue construct into our red heart. When doing this procedure, it is important to eliminate any pneumothorax to allow the lungs to fully expand and to reduce atelectasis.So that's it. Thanks for watching and good luck with your experiments.I.

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Research

JoVE Journal

Biology

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Learn a surgical protocol for implanting engineered tissue constructs into the rat heart's AV groove. This method creates an alternative electrical conduction pathway for cardiovascular tissue engineering research.

Implantation of Engineered Tissue in the Rat Heart

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