isolation of rat heart for testing donor heart preservation methods

<meta charset="utf8"></head><body>使用工作大鼠心脏灌注模型优化供体心脏保存

The success of a heart transplant can be greatly influenced by the preservation method used for the donor heart. Donor hearts are subjected to ischemic insults throughout the organ retrieval, transport, and transplantation process. The degree of ischemic damage to the donor organ can be mitigated by selecting an appropriate donor heart preservation strategy. Cold static storage (CSS) remains the most feasible and common method for donor heart preservation; however, CSS may not necessarily be the best option for all heart donation pathways. For example, hearts procured via the donation after circulatory death (DCD) pathway are maintained and assessed for viability using normothermic machine perfusion, whereas hearts procured via the donation after brain death (DBD) pathway can be preserved using CSS or hypothermic machine perfusion. Another important determinant for the preservation strategy used is the amount of either warm ischemia (for DCD) or cold ischemia (for DBD) the heart is subjected to during organ procurement and transportation.
Many centers use additional pharmacological supplementation of the heart preservation solution to increase ischemic tolerance and improve post-transplant heart function. Rodent studies from our laboratory showed that supplementation of cardioplegia with erythropoietin and glyceryl trinitrate was able to significantly improve cardiac functional recovery after prolonged cold storage of the donor heart1,2. These studies then progressed to porcine studies of donor heart preservation and provided the pre-clinical groundwork that led to the incorporation of erythropoietin and glyceryl trinitrate into the cardioplegia solution of hearts procured via a DCD pathway utilizing normothermic machine perfusion3,4,5. Currently, supplementation with erythropoietin and glyceryl trinitrate is also used in Australian transplant units for clinical DBD heart retrievals incorporating CSS. The overall goal of the isolated working rat heart (IWRH) perfusion protocol outlined below is to test donor heart preservation strategies in a laboratory setting and test their efficacy for preserving donor heart function after reperfusion. The isolated working rat heart protocol described has been used for over two decades within our laboratory and has proven extremely useful in screening for potential supplements and/or strategies that can improve donor heart preservation2,7,8,9.

<meta charset="utf8"></head><body>作者聚焦：通过离体大鼠心脏灌注研究增强供体心脏保存

使用分离的工作大鼠心脏模型测试供体心脏保存策略

Our research uses an isolated rat heart perfusion circuit to test supplementation strategies for donor heart preservation. We aim to identify preservation strategies that can improve the functional recovery of donor hearts after either long periods of cold storage or long periods of warm ischemia following donation after circulatory death. The isolated work in rat heart protocol was used to initially test the current clinical donor heart preservation strategy of using glyceryl trinitrate and erythropoietin in the heart preservation solution.This provided the rationale for testing in a porcine heart transplant study before transitioning to the clinic. The advantage of using an isolated working rat heart perfusion protocol compared to Langendorff perfusion techniques is the ability to obtain functional measurements of the heart, such as aortic flow and cardiac output.

分离大鼠心脏以测试供体心脏保存方法

isolation-of-rat-heart-for-testing-donor-heart-preservation-methods

Research

JoVE Journal

Medicine

Isolation of Rat Heart for Testing Donor Heart Preservation Methods

28 次观看. 首先，将麻醉的大鼠置于仰卧位。用胶带固定动物的四肢。然后在动物身上固定一个鼻锥以给氧。将无菌纱布浸泡在含有 Krebs-Henseleit 缓冲液的容器中。将浸泡过的纱布放在冰上。在腹部做一个侧切口后，移动胃和肠以允许进入左肾静脉。注射肝素并对注射部位施加压力，直到出血停止。让肝素循环几分钟后，切开两侧的胸腔并穿过横膈膜...

视频: 分离大鼠心脏以测试供体心脏保存方法

Using an Isolated Working Rat Heart Model to Test Donor Heart Preservation Strategies

Optimization of donor heart preservation solutions has played a key role in reducing ischemia-reperfusion injury in donor hearts during organ retrieval, transportation, and transplantation. Previous work from our laboratory showed that the addition of glyceryl trinitrate and erythropoietin to donor heart preservation solutions could significantly improve cardiac functional recovery after prolonged cold storage and in donation after circulatory death hearts. This supplementation protocol has been implemented in clinical use in transplant units around Australia, Belgium, and the United Kingdom. Here, we outline a protocol for testing supplementation strategies using an ex vivo isolated working rat heart (IWRH) perfusion circuit. Using this methodology, supplementation strategies can be tested in the context of prolonged cold static storage, donation after brain death, and donation after circulatory death donor heart preservation. Cardiac functional recovery, measured by aortic flow, coronary flow, cardiac output, pulse pressure, and heart rate, can be used to determine whether a particular preservation strategy can minimize ischemia-reperfusion injury of the donor heart.

We describe an ex vivo isolated working rat heart protocol to test donor heart preservation strategies. This paper describes the protocol for use in static cold storage of rodent donor hearts; however, the protocol can also be used for donor hearts obtained after donation after circulatory death and brain death.

Optimization of donor heart preservation solutions has played a key role in reducing ischemia-reperfusion injury in donor hearts during organ retrieval, ...

科研

医学

To begin, place an anesthetized rat in a supine position. Secure the limbs of the animal with tape. Then fix a nose cone over the animal to administer oxygen.Soak sterile gauze in a container with Krebs-Henseleit buffer. Place the soaked gauze on ice. After making a lateral incision across the abdomen, move the stomach and intestines to allow access to the left renal vein.Inject heparin and apply pressure to the injection site until the bleeding stops. After letting the heparin circulate for a couple of minutes, cut through the rib cage on either side and across the diaphragm to completely open the chest cavity. Carefully lift the heart-lung block, and cut along the descending aorta, keeping the aortic arch intact.Transfer the heart-lung block to the Krebs-Henseleit buffer ice slush to arrest the heart.

Testing Donor Heart Preservation Strategies on a Rat Heart Using Perfusion Circuits

To begin, place a freshly excised rat heart lung block on a gauze sheet over ice. Dissect the excess fat around the aorta. Then trim the aorta, leaving about three to five millimeters for cannulation.Make a one to two millimeter long incision in the pulmonary artery. Place the trimmed heart lung block on ice directly underneath the cannulas. Next, use a flow control tubing clamp to adjust the flow from the Langendorff side for aorta cannulation.Carefully cannulate the aorta onto the left cannula. Hold it with a black clamp and tie it with a 2/0 silk suture. Open the flow control tubing clamp completely to increase flow from the Langendorff perfusion.Then open the afterload clamp. Start monitoring heart activity with software. Adjust the heart lung block such that the anterior surface of the lungs faces the operator.Tie the left lung lobe with a 2/0 silk suture. Tie the remaining lung lobes in one sweep with a 2/0 silk suture. Then rotate the heart so that the tied lungs are towards the back.With a pair of small vannas scissors. Trim a two to three millimeter section of the left atrial appendage to create a small opening into the atria. Carefully cannulate the left atrial appendage to the left cannula.Hold it with a black clamp and tie it with a 2/0 silk suture. Adjust the heart chamber water jacket so that the heart sits within the center of the chamber. Now open the pressure transducer to air.Select aortic pressure in the software. Then click on bridge amp, followed by zero and OK to zero the pressure transducer. Perfuse the heart in Langendorff mode for 10 minutes.Next, transfer the air stone from the Langendorff Krebs-Henseleit buffer reservoir into the working Krebs-Henseleit buffer reservoir. Open the three-way tap leading from working perfusate. Close the large clamp leading to the aortic cannula and open the flow leading to the left atrial appendage to switch to working mode.Perfuse the heart in working mode for 15 minutes. Fill a cardioplegia chamber with 50 milliliters of the designated cardiac preservation solution. Lower the water jacket and close the three-way tap on the working mode side.Close the clamp to stop the flow towards the left atrial cannula. Then open the flow to the aortic cannula. Now close the clamp from the Langendorff side and on the afterload.Open the clamp from the cardioplegia line. Flush 15 milliliters of the cardioplegia solution out from the pressure line. Flush the heart with cardioplegia via the aortic cannula and collect the coronary effluent.After three minutes, close the cardioplegia clamp and the small blue clamp leading to the aortic cannula. Place a bulldog clamp on the aortic cannula and another bulldog clamp on the left cannula. Disconnect the heart cannula block from the circuit and place it in a beaker containing cardioplegia in an ice box.To reperfuse the heart after cold static storage, carefully secure the heart lung block after cold storage onto the perfusion circuit. Use a blunt end 18 gauge syringe filled with Krebs-Henseleit buffer to remove any air from the cannula. Connect the aortic cannula first.Then open the tubing clamp on the Langendorff side. Reconnect the left cannula and ensure that air has been removed from the cannula before connecting it to the perfusion circuit. Keep the working side clamp closed until it is time to switch from Langendorff to working mode.An aortic flow of 14 to 22 milliliters per minute resulted in a good Langendorff flow. When switched to working mode, an aortic flow of at least 30 milliliters per minute facilitated heart storage. Despite good baseline traces, some hearts did not yield good recovery after prolonged cold static storage.