<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

<ol>
	<li>Kwan, J. C., Gao, L., Macdonald, P. S., Hicks, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardio-protective+signalling+by+glyceryl+trinitrate+and+cariporide+in+a+model+of+donor+heart+preservation.">Cardio-protective signalling by glyceryl trinitrate and cariporide in a model of donor heart preservation.</a> Heart Lung Circ. 24 (3), 306-318 (2015).</li><li>Watson, A. 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=Enhanced+preservation+of+the+rat+heart+after+prolonged+hypothermic+ischemia+with+erythropoietin-supplemented+Celsior+solution.">Enhanced preservation of the rat heart after prolonged hypothermic ischemia with erythropoietin-supplemented Celsior solution.</a> J Heart Lung Transplant. 32 (6), 633-640 (2013).</li><li>Chew, H. C., 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=Outcomes+of+donation+after+circulatory+death+heart+transplantation+in+Australia.">Outcomes of donation after circulatory death heart transplantation in Australia.</a> J Am Coll Cardiol. 73 (12), 1447-1459 (2019).</li><li>Dhital, K. 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=Adult+heart+transplantation+with+distant+procurement+and+ex-vivo+preservation+of+donor+hearts+after+circulatory+death:+a+case+series.">Adult heart transplantation with distant procurement and ex-vivo preservation of donor hearts after circulatory death: a case series.</a> Lancet. 385 (9987), 2585-2591 (2015).</li><li>Joshi, Y., 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+transplantation+from+DCD+donors+in+Australia:+Lessons+learned+from+the+first+74+cases.">Heart transplantation from DCD donors in Australia: Lessons learned from the first 74 cases.</a> Transplantation. 107 (2), 361-371 (2023).</li><li>Villanueva, J. E., 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=Functional+recovery+after+dantrolene-supplementation+of+cold+stored+hearts+using+an+ex+vivo+isolated+working+rat+heart+model.">Functional recovery after dantrolene-supplementation of cold stored hearts using an ex vivo isolated working rat heart model.</a> PLoS One. 13 (10), e205850 (2018).</li><li>Villanueva, J. E., 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+cardioprotective+potential+of+the+sodium-glucose+cotransporter+2+inhibitor+empagliflozin+in+donor+heart+preservation.">The cardioprotective potential of the sodium-glucose cotransporter 2 inhibitor empagliflozin in donor heart preservation.</a> J Heart Lung Transplant. 39 (10), 1151-1153 (2020).</li><li>Gao, L., 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=Critical+role+of+the+STAT3+pathway+in+the+cardioprotective+efficacy+of+zoniporide+in+a+model+of+myocardial+preservation+-+the+rat+isolated+working+heart.">Critical role of the STAT3 pathway in the cardioprotective efficacy of zoniporide in a model of myocardial preservation - the rat isolated working heart.</a> Br J Pharmacol. 162 (3), 633-647 (2011).</li><li>Cropper, J. R., Hicks, M., Ryan, J. B., Macdonald, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardioprotection+by+cariporide+after+prolonged+hypothermic+storage+of+the+isolated+working+rat+heart.">Cardioprotection by cariporide after prolonged hypothermic storage of the isolated working rat heart.</a> J Heart Lung Transplant. 22 (8), 929-936 (2003).</li><li>Gao, L., 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=Enhanced+functional+recovery+of+the+heart+donated+after+circulatory+death+determination+with+antemortem+heparin.">Enhanced functional recovery of the heart donated after circulatory death determination with antemortem heparin.</a> J Heart Lung Transplant. 39 (6), 607-609 (2020).</li><li>Villanueva, J. E., 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+increasing+donor+age+on+myocardial+ischemic+tolerance+in+a+rodent+model+of+donation+after+circulatory+death.">The effect of increasing donor age on myocardial ischemic tolerance in a rodent model of donation after circulatory death.</a> Transplant Direct. 7 (6), e699 (2021).</li><li>Kumarasinghe, G., 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=Improved+heart+function+from+older+donors+using+pharmacologic+conditioning+strategies.">Improved heart function from older donors using pharmacologic conditioning strategies.</a> J Heart Lung Transplant. 35 (5), 636-646 (2016).</li></ol>

<table><tbody><tr><td>1.5 mL colorless Eppendorff tube, 1000 per box</td><td>Bio Strategy Pty Ltd</td><td>0030.125.150</td><td></td></tr><tr><td>15 mL cent/tubes (S) Sleeve/25 PP ctn/500</td><td>Sigma-aldrich Pty Ltd</td><td>CLS430791-500ea</td><td></td></tr><tr><td>BP Transducer/Cable kit</td><td>ADInstruments</td><td>MLT1199</td><td>Data Acquisition</td></tr><tr><td>Bridge Amp</td><td>ADInstruments</td><td>FE221</td><td>Data Acquisition</td></tr><tr><td>Carbogen 555G2</td><td>BOC</td><td>BOC002</td><td></td></tr><tr><td>Checktemp1 thermometer</td><td>Hanna Instruments</td><td>HI98509</td><td>Rig Construction</td></tr><tr><td>Clamp Pinch 1/4-7/16 PK 12</td><td>Thomas Scientific</td><td>2848Y40</td><td>Rig Construction</td></tr><tr><td>Clamp W/Extension Stem Med.</td><td>Thomas Scientific</td><td>8847T08</td><td>Rig Construction</td></tr><tr><td>Clamp W/Extension Stem Sm.</td><td>Thomas Scientific</td><td>8847T02</td><td>Rig Construction</td></tr><tr><td>Clips, Vessel, 60 g Pressure</td><td>Coherent Scientific</td><td>14121</td><td>Surgical Equipment</td></tr><tr><td>Closed Connector</td><td>Thomas Scientific</td><td>8847E25</td><td>Rig Construction</td></tr><tr><td>Covidien Sofsilk 2-0 black precut 45 cm box of 24</td><td>Specialist Medical Supplies</td><td>S195</td><td>Surgical consumables</td></tr><tr><td>Custom Made 250 mL Jacketed Degasser</td><td>Custom Blown Glassware Pty Ltd</td><td>N/A</td><td>Rig Construction</td></tr><tr><td>Custom Made 750 mL Reservoir</td><td>Custom Blown Glassware Pty Ltd</td><td>N/A</td><td>Rig Construction</td></tr><tr><td>D-(+)-Glucose Anhydrous SigmaUltra</td><td>Sigma-aldrich Pty Ltd</td><td>G7528-1Kg</td><td>To make Krebs Buffer</td></tr><tr><td>Dual Channel Console</td><td>ADInstruments</td><td>TS402</td><td>Data Acquisition</td></tr><tr><td>Erlenmyer flasks 2 L&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Filter Microfibre type GF/C glass fibre 47 mm, 100</td><td>Bio-strategy Pty Ltd</td><td>1822047</td><td></td></tr><tr><td>Forceps, 15 cm, 0.3 mm, CRVD</td><td>Coherent Scientific</td><td>14114</td><td>Surgical Equipment</td></tr><tr><td>Four Prong Clamps with 9 mm x 115 mm long arm for holding 2-70 mm diam objects. Vinyl coated</td><td>Met-App Australia Pty Ltd</td><td>1352</td><td>Rig Construction</td></tr><tr><td>Heater Circulator. Digital Solid State Control. (1020 Watts/240 Volts)</td><td>Thermoline Scientific</td><td>TU3</td><td>Rig Construction</td></tr><tr><td>Heparin 5000 U/5 mL box 50 Pfizer 02112115</td><td>Clffird Hallam Healthcare Pty Ltd</td><td>1258693</td><td>Drugs</td></tr><tr><td>Ilium Xylazil 20 Inj 50 mL</td><td>Cenvet Australia Pty Ltd</td><td>X5010</td><td>Anaesthetic</td></tr><tr><td>Johns Hopkins Bulldog Clamp</td><td>Coherent Scientific&amp;nbsp;</td><td>CS-WPI-14117</td><td></td></tr><tr><td>Ketamine 100 mg/50 mL</td><td>Provet (NSW) Pty Ltd</td><td>KETAI1</td><td>Anaesthetic</td></tr><tr><td>Magnesium Sulphate heptahydrate AR 500 g Chemsupply</td><td>Bio Strategy Pty Ltd</td><td>MA048-500g</td><td>To make Krebs Buffer</td></tr><tr><td>Male/Female Hinged Adapter</td><td>Thomas Scientific</td><td>8847V08</td><td>Rig Construction</td></tr><tr><td>Masterflex L/S Easy-Load Head for Precision Tubing, PPS, CRS Rotor</td><td>John Morris</td><td>1015164</td><td>Rig Construction</td></tr><tr><td>Metzenbaum scissors, 11.5 cm curved</td><td>Coherent Scientific</td><td>WPI-501748</td><td>Surgical Equipment</td></tr><tr><td>Metzenbaum scissors, 14.5 cm straight</td><td>Coherent Scientific</td><td>WPI-501252</td><td>Surgical Equipment</td></tr><tr><td>Mounting Hardware F/2-HEADS SS</td><td>John Morris</td><td>1014414</td><td>Mounting screws for pump heads</td></tr><tr><td>Open-sided connector</td><td>Thomas Scientific</td><td>8847E05</td><td>Rig Construction</td></tr><tr><td>Paraformadehyde</td><td>Sigma-Aldrich</td><td>P6148-500G</td><td>Sample processing</td></tr><tr><td>Potassium Chloride (AnalaR NORMAPUR) 500 g</td><td>VWR Chemicals&amp;nbsp;</td><td>26764.26</td><td>To make Krebs Buffer</td></tr><tr><td>Potassium phosphate monobasic&amp;nbsp;</td><td>Sigma-Aldrich Pty Ltd</td><td>P5379-500g</td><td>To make Krebs Buffer</td></tr><tr><td>Powerlab 2/26, 2 channel recorder + Labchart software</td><td>ADInstruments</td><td>ML826</td><td>Computer Hardware and Software</td></tr><tr><td>Precision XN Inline Flowsensor, 3.2 mm (1/8&quot;)ID ME4PXN-KR37 XF</td><td>ADInstruments</td><td>ME4PXN</td><td>Rig Construction</td></tr><tr><td>Scalpel with handle disposable #11 pkt/10</td><td>BSN Medical (Aust) Pty Ltd</td><td>73252-36</td><td></td></tr><tr><td>Silicone Gasket for Swinnex 47 mm 5/PK</td><td>Merck Millipore</td><td>SX0004701</td><td>Rig Construction</td></tr><tr><td>Silicone O-Ring 5-329 10/PK</td><td>Merck Millipore</td><td>XX1104707</td><td>Rig Construction</td></tr><tr><td>Single Buret Clamp</td><td>Thomas Scientific</td><td>8847T32</td><td>Rig Construction</td></tr><tr><td>Slip-on inlet Filter pore size 10 &amp;micro;m (bubbler)</td><td>Sigma-aldrich</td><td>59277</td><td>Rig Construction</td></tr><tr><td>Sm. 360 Rotation Connector</td><td>Thomas Scientific</td><td>8847E35</td><td>Rig Construction</td></tr><tr><td>Sodium bicarbonate</td><td>Sigma-Aldrich Pty Ltd</td><td>S6297 - 250g</td><td>To make Krebs Buffer</td></tr><tr><td>Swinnex Filter Holder, 47 mm</td><td>Merck Millipore</td><td>SX0004700</td><td>Rig Construction</td></tr><tr><td>syringes 1 mL box/100</td><td>Becton Dickinson Pty Ltd</td><td>302100</td><td></td></tr><tr><td>Three Prong Clamps with 9 mm diameter x 125 mm long arm and twin screw for holding 5-80 mm</td><td>Met-App Australia Pty Ltd</td><td>1356</td><td>Rig Construction</td></tr><tr><td>Tubing Flowmeter Module TS410</td><td>ADInstruments</td><td>TS410</td><td>Data Acquisition</td></tr><tr><td>Tubing PVC 6.35 mm ID x 9.52 mm OD 50ft Roll 15.24m, clear , DEHP phthalate free, food grade meets REAC</td><td>Thermo Fisher</td><td>NAL 8701-0600</td><td>Rig Construction</td></tr><tr><td>Tubing PVC 7.94mm ID x 11.1mm OD 50ft Roll 15.24 m, clear , DEHP phthalate free, food grade meets REAC</td><td>Thermo Fisher</td><td>NAL 8701-0900</td><td>Rig Construction</td></tr><tr><td>Tubing PVC 9.52 mm ID x 12.7 mm OD 100ft Roll</td><td>Thermo Fisher</td><td>NAL 8701-4120</td><td>Rig Construction</td></tr><tr><td>Vannas scissors, 8.5 cm, Straight, 7 mm Blades</td><td>Coherent Scientific</td><td>WPI-500-086</td><td>Surgical Equipment</td></tr><tr><td>Water Bath 30 Litre with Suspended Tray</td><td>Thermoline Scientific</td><td>TLWB-30</td><td>Rig Construction</td></tr></tbody></table>

using an isolated working rat heart model to test donor heart preservation strategies

<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

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

using-an-isolated-working-rat-heart-model-to-test-donor-heart

Research

JoVE Journal

Medicine

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

357 次观看.  Victor Chang Cardiac Research Institute. 我们的研究使用离体大鼠心脏灌注回路来测试保存供体心脏的补充策略。我们的目标是确定可以改善供体心脏在循环死亡后长期冷藏或长期热缺血后功能恢复的保存策略。大鼠心脏方案中的分离工作用于初步测试当前临床供体心脏保存策略，即在心脏保存溶液中使用硝酸甘油酯和促红细胞生成素。这为在过渡到临床之前进行猪心脏移植研究的检...

视频: 作者聚焦:通过离体大鼠心脏灌注研究增强供体心脏保存

An Isolated Working Heart System for Large Animal Models

Since its introduction in the late 19th century, the Langendorff isolated heart perfusion apparatus, and the subsequent development of the working heart model, have been invaluable tools for studying cardiovascular function and disease1-15. Although the Langendorff heart preparation can be used for any mammalian heart, most studies involving this apparatus use small animal models (e.g., mouse, rat, and rabbit) due to the increased complexity of systems for larger mammals1,3,11. One major difficulty is ensuring a constant coronary perfusion pressure over a range of different heart sizes &#8211; a key component of any experiment utilizing this device1,11. By replacing the classic hydrostatic afterload column with a centrifugal pump, the Langendorff working heart apparatus described below allows for easy adjustment and tight regulation of perfusion pressures, meaning the same set-up can be used for various species or heart sizes. Furthermore, this configuration can also seamlessly switch between constant pressure or constant flow during reperfusion, depending on the user&#8217;s preferences. The open nature of this setup, despite making temperature regulation more difficult than other designs, allows for easy collection of effluent and ventricular pressure-volume data.

Most studies involving the Langendorff apparatus use small animal models due to the increased complexity of systems for larger mammals. We describe a Langendorff system for large animal models that allows for use across a range of species, including humans, and relatively easy data acquisition.

一个孤立的工作心脏系统的大型动物模型

Since its introduction in the late 19th century, the Langendorff isolated heart perfusion apparatus, and the subsequent development of the working heart ...

科研

医学

The Inverted Heart Model for Interstitial Transudate Collection from the Isolated Rat Heart

The present protocol describes a unique approach that enables the collection of cardiac transudate (CT) from the isolated, saline-perfused rat heart. After isolation and retrograde perfusion of the heart according to the Langendorff technique, the heart is inverted into an upside-down position and is mechanically stabilized by a balloon catheter inserted into the left ventricle. Then, a thin latex cap &#8211; previously cast to match the average size of the rat heart &#8211; is placed over the epicardial surface. The outlet of the latex cap is connected to silicon tubing, with the distal opening 10 cm below the base level of the heart, creating slight suction. CT continuously produced on the epicardial surface is collected in ice-cooled vials for further analysis. The rate of CT formation ranged from 17 to 147 &#181;L/min (n = 14) in control and infarcted hearts, which represents 0.1-1% of the coronary venous effluent perfusate. Proteomic analysis and high performance liquid chromatography (HPLC) revealed that the collected CT contains a wide spectrum of proteins and purinergic metabolites.

This protocol describes a method to collect cardiac interstitial fluid from the isolated, perfused rat heart. To physically separate interstitial transudate from coronary venous effluent perfusate, the Langendorff perfused heart is inverted, and the transudate (interstitial fluid) formed on the cardiac surface is collected using a soft latex cap.

从孤立的大鼠心脏的间质渗出物收集的倒置心脏模型

The present protocol describes a unique approach that enables the collection of cardiac transudate (CT) from the isolated, saline-perfused rat heart. ...

An Immunological Model for Heterotopic Heart and Cardiac Muscle Cell Transplantation in Rats

Heterotopic heart transplantation in rats has been a commonly used model for diverse immunological studies for more than 50 years. Several modifications have been reported since the first description in 1964. After 30 years of performing heterotopic heart transplantation in rats, we have developed a simplified surgical approach, which can be easily taught and performed without further surgical training or background.
After dissection of the ascending aorta and the pulmonary artery and ligation of superior and inferior caval and pulmonary veins, the donor heart is harvested and subsequently perfused with ice-cold saline solution supplemented with heparin. After clamping and incising the recipient abdominal vessels, the donor ascending aorta and pulmonary artery are anastomosed to the recipient abdominal aorta and inferior vena cava, respectively, using continuous running sutures.
Depending on different donor-recipient combinations, this model allows analyses of either acute or chronic rejection of allografts. The immunological significance of this model is further enhanced by a novel approach of in-ear injection of vital cardiac muscle cells and subsequent analysis of draining cervical lymphatic tissue.

We describe a model of heterotopic abdominal heart transplantation in rats, implying modifications of current strategies, which lead to a simplified surgical approach. Additionally, we describe a novel rejection model by in-ear injection of vital cardiac muscle cells, allowing further transplant immunological analyses in rats.

大鼠异位心脏和心肌细胞移植的免疫模型

Heterotopic heart transplantation in rats has been a commonly used model for diverse immunological studies for more than 50 years. Several modifications ...

免疫与感染

Pre-clinical Model of Cardiac Donation after Circulatory Death

Cardiac transplantation demand is on the rise; nevertheless, organ availability is limited due to a paucity of suitable donors. Organ donation after circulatory death (DCD) is a solution to address this limited availability, but due to a period of prolonged warm ischemia and the risk of tissue injury, its routine use in cardiac transplantation is seldom seen. In this manuscript we provide a detailed protocol closely mimicking current clinical practices in the context of DCD with continuous monitoring of heart function, allowing for the evaluation of novel cardioprotective strategies and interventions to decrease ischemia-reperfusion injury.
In this model, the DCD protocol is initiated in anesthetized Lewis rats by stopping ventilation to induce circulatory death. When systolic blood pressure drops below 30 mmHg, the warm ischemic time is initiated. After a pre-set warm ischemic period, hearts are flushed with a normothermic cardioplegic solution, procured, and mounted onto a Langendorff ex vivo heart perfusion system. Following 10 min of initial reperfusion and stabilization, cardiac reconditioning is continuously evaluated for 60 min using intraventricular pressure monitoring. A heart injury is assessed by measuring cardiac troponin T and the infarct size is quantified by histological staining. The warm ischemic time can be modulated and tailored to develop the desired amount of structural and functional damage. This simple protocol allows for the evaluation of different cardioprotective conditioning strategies introduced at the moment of cardioplegia, initial reperfusion and/or during ex vivo perfusion. Findings obtained from this protocol can be reproduced in large models, facilitating clinical translation.

This protocol shows a simple and flexible approach for the evaluation of new conditioning agents or strategies to increase the feasibility of cardiac donation after circulatory death.

循环死亡后心脏捐献的临床前模型

Cardiac transplantation demand is on the rise; nevertheless, organ availability is limited due to a paucity of suitable donors. Organ donation after ...

A Simplified Model for Heterotopic Heart Valve Transplantation in Rodents

There is an urgent clinical need for heart valve replacements that can grow in children. Heart valve transplantation is proposed as a new type of transplant with the potential to deliver durable heart valves capable of somatic growth with no requirement for anticoagulation. However, the immunobiology of heart valve transplants remains unexplored, highlighting the need for animal models to study this new type of transplant. Previous rat models for heterotopic aortic valve transplantation into the abdominal aorta have been described, though they are technically challenging and costly. For addressing this challenge, a renal subcapsular transplant model was developed in rodents as a practical and more straightforward method for studying heart valve transplant immunobiology. In this model, a single aortic valve leaflet is harvested and inserted into the renal subcapsular space. The kidney is easily accessible, and the transplanted tissue is securely contained in a subcapsular space that is well vascularized and can accommodate a variety of tissue sizes. Furthermore, because a single rat can provide three donor aortic leaflets and a single kidney can provide multiple sites for transplanted tissue, fewer rats are required for a given study. Here, the transplantation technique is described, providing a significant step forward in studying the transplant immunology of heart valve transplantation.

This protocol describes a simple and efficient method for the transplantation of aortic valve leaflets under the renal capsule to allow for the study of alloreactivity of heart valves.

啮齿动物异位心脏瓣膜移植的简化模型

There is an urgent clinical need for heart valve replacements that can grow in children. Heart valve transplantation is proposed as a new type of ...

Rodent Working Heart Model for the Study of Myocardial Performance and Oxygen Consumption

Isolated working heart models have been used to understand the effects of loading conditions, heart rate and medications on myocardial performance in ways that cannot be accomplished in vivo. For example, inotropic medications commonly also affect preload and afterload, precluding load-independent assessments of their myocardial effects in vivo. Additionally, this model allows for sampling of coronary sinus effluent without contamination from systemic venous return, permitting assessment of myocardial oxygen consumption. Further, the advent of miniaturized pressure-volume catheters has allowed for the precise quantification of markers of both systolic and diastolic performance. We describe a model in which the left ventricle can be studied while performing both volume and pressure work under controlled conditions. 
In this technique, the heart and lungs of a Sprague-Dawley rat (weight 300-500 g) are removed en bloc under general anesthesia. The aorta is dissected free and cannulated for retrograde perfusion with oxygenated Krebs buffer. The pulmonary arteries and veins are ligated and the lungs removed from the preparation. The left atrium is then incised and cannulated using a separate venous cannula, attached to a preload block. Once this is determined to be leak-free, the left heart is loaded and retrograde perfusion stopped, creating the working heart model. The pulmonary artery is incised and cannulated for collection of coronary effluent and determination of myocardial oxygen consumption. A pressure-volume catheter is placed into the left ventricle either retrograde or through apical puncture. If desired, atrial pacing wires can be placed for more precise control of heart rate. This model allows for precise control of preload (using a left atrial pressure block), afterload (using an afterload block), heart rate (using pacing wires) and oxygen tension (using oxygen mixtures within the perfusate).

Isolated working heart models can be used to measure the effect of loading conditions, heart rate, and medications on myocardial performance and oxygen consumption. We describe methods for preparation of a rodent left heart working model that permits study of systolic and diastolic performance and oxygen consumption under various conditions.

灭鼠工作心脏模型心肌的性能和氧耗的研究

Isolated working heart models have been used to understand the effects of loading conditions, heart rate and medications on myocardial performance in ways ...

生物学

A Heterotopic Rat Heart Transplantation Model using Circulatory Death Donor Hearts

The objective of this protocol is to set up a rat heterotopic heart transplantation model with donation after circulatory death (DCD) donor hearts. There are two setups for this protocol: heart donor setup and recipient setup. In the heart donor setup, Sprague Dawley rats are anesthetized, endotracheally intubated, and ventilated. The right carotid artery is cannulated to deliver heparin and the paralytic agent vecuronium-bromide. The DCD process is initiated by terminating the ventilation. After 20 min, the heart is exposed and the aorta distal to the brachiocephalic branch is clamped. At 25 min from terminating the ventilator, ice-cold University of Wisconsin (UW) solution is perfused through the carotid catheter to flush the heart. The heart is procured by dividing the aorta, pulmonary artery, venae cavae, and pulmonary veins and stored in UW solution for implantation. In the recipient setup, the Lewis rat is anesthetized with isoflurane. Slow-release buprenorphine is administered subcutaneously to facilitate a smooth postoperative recovery. Through a midline abdominal incision, the infra-renal aorta and the inferior vena cava are isolated and clamped with an atraumatic vascular clamp. The donor heart aorta and pulmonary artery are sutured to the recipient abdominal aorta and vena cava, respectively, with a running 8-0 Prolene. The vascular clamp is removed to reperfuse the heart. The abdominal wall is closed and the rat is recovered. After a set interval (24 h to 2 weeks), the recipient rat is anesthetized, the transplanted heart is exposed, and a balloon-tip-catheter is inserted into the left ventricle via the apex to record developed pressure and dP/dt using a data acquisition system. The heart tissue is collected for histology, immunology, or molecular analysis. A successful DCD donor rat heart transplantation model will allow further studies on the cardioprotective approaches to improve heart transplantation outcomes from DCD donors.

Here, we describe the preparation and technical details of murine heterotopic heart transplantation utilizing a circulatory death donor heart.

The objective of this protocol is to set up a rat heterotopic heart transplantation model with donation after circulatory death (DCD) donor hearts. There ...

Large-Animal Model of Donation after Circulatory Death and Normothermic Regional Perfusion for Cardiac Assessment

The increase in demand for cardiac transplantation throughout the years has fueled interest in donation after circulatory death (DCD) to expand the organ donor pool. However, the DCD process is associated with the risk of cardiac tissue injury due to the inevitable period of warm ischemia. Normothermic regional perfusion (NRP) allows for an in situ organ assessment, allowing the procurement of hearts determined to be viable. Here, we describe a clinically relevant large-animal model of DCD followed by NRP. Circulatory death is established in anesthetized pigs by stopping mechanical ventilation. After a preset warm ischemia period, an extracorporeal membrane oxygenator (ECMO) is used for a NRP period lasting at least 30 min. During this reperfusion period, the model allows the collection of various myocardial biopsies and blood samples for initial cardiac evaluation. Once NRP is weaned, biochemical, hemodynamic, and echocardiographic assessments of cardiac function and metabolism can be performed before organ procurement. This protocol closely simulates the clinical scenario previously described for DCD and NRP in heart transplantation and has the potential to facilitate studies aimed at decreasing ischemia-reperfusion injury and enhance cardiac functional preservation and recovery.

The protocol describes a large-animal (porcine) model of donation after circulatory death, followed by thoracoabdominal normothermic regional perfusion that closely simulates the clinical scenario in heart transplantation, and has the potential to facilitate therapeutic studies and strategies.

用于心脏评估的循环死亡后捐献和正常区域灌注的大型动物模型

The increase in demand for cardiac transplantation throughout the years has fueled interest in donation after circulatory death (DCD) to expand the organ ...

Simultaneous Isolation of High Quality Cardiomyocytes, Endothelial Cells, and Fibroblasts from an Adult Rat Heart

The rat is an important animal model used in cardiovascular research, and rat cardiac cells are used routinely for in vitro analysis of the molecular mechanisms of cardiovascular disease progression such as cardiac hypertrophy, fibrosis, and atherosclerosis. Although several attempts with variable success have been made to develop immortalized cell lines from the cardiovascular system to understand these cellular mechanisms, primary cells offer a more natural and close to in vivo environment for such studies. Therefore, different laboratories working on a particular cell type have developed protocols to isolate individual types of rat cardiac cells of interest. A protocol that allows the isolation of more than one cell type, however, is missing. Here an optimized protocol is described that allows the isolation of high-quality major cardiac cell types (cardiomyocytes, endothelial cells, and fibroblasts) from a single preparation and enables their use for cellular analyses. This permits the most efficient use of available resources, which may save time and reduce research costs.

Several protocols have been developed and described for the isolation of different cardiac cell types from a rat heart. Here, an optimized protocol is described that allows the isolation of high-quality major cardiac cell types (cardiomyocytes, endothelial cells, and fibroblasts) from a single preparation, reducing the experimental costs.

从成年大鼠心脏同时分离高质量心肌细胞，内皮细胞和成纤维细胞

The rat is an important animal model used in cardiovascular research, and rat cardiac cells are used routinely for in vitro analysis of the molecular ...

Rat Model of Normothermic Ex-Situ Perfused Heterotopic Heart Transplantation

Heart transplantation is the most effective therapy for end-stage heart failure. Despite the improvements in therapeutic approaches and interventions, the number of heart failure patients waiting for transplantation is still increasing. The normothermic ex situ preservation technique has been established as a comparable method to the conventional static cold storage technique. The main advantage of this technique is that donor hearts can be preserved for up to 12 h in a physiologic condition. Moreover, this technique allows resuscitation of the donor hearts after circulatory death and applies required pharmacologic interventions to improve donor function after implantation. Numerous animal models have been established to improve normothermic ex situ preservation techniques and eliminate preservation-related complications. Although large animal models are easy to handle compared to small animal models, it is costly and challenging. We present a rat model of normothermic ex situ donor heart preservation followed by heterotopic abdominal transplantation. This model is relatively cheap and can be accomplished by a single experimenter.

Here, we present an assessment protocol of a heterotopically implanted heart after normothermic ex situ preservation in the rat model.

异位灌注异位心脏移植大鼠模型

Heart transplantation is the most effective therapy for end-stage heart failure. Despite the improvements in therapeutic approaches and interventions, the ...