Ann Thorac Surg 2005;79:2169-2171
© 2005 The Society of Thoracic Surgeons
How to do it
Independent Ventilation of the Graft and Native Lungs In Vivo After Rat Lung Transplantation
Marc de Perrot, MD,
Syed M. Quadri, MD,
Yumiko Imai, MD,
Shaf Keshavjee, MD*
Thoracic Surgery Research Laboratory, Toronto General Hospital Research Institute, University of Toronto, Toronto, Ontario, Canada
Accepted for publication January 28, 2004.
* Address reprint requests to Dr Keshavjee, Thoracic Surgery Research Laboratory, Toronto General Hospital, 200 Elizabeth St, EN 10-224, Toronto, Ontario M5G 2C4, Canada (E-mail: shaf.keshavjee{at}uhn.on.ca).
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Abstract
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Rat lung transplantation is a proven experimental technique for the study of lung injury following lung transplantation. We have modified the surgical and ventilatory techniques to allow for independent ventilation in vivo of the transplanted graft and native lungs. This will provide additional data on the physiology and function of the transplanted graft and ameliorate the problem of progressive graft lung collapse and thereby allow for an improved model of ischemia-reperfusion injury and ventilator-induced lung injury in the setting of lung transplantation.
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Introduction
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Ischemia-reperfusion injury remains a significant cause of morbidity and mortality after lung transplantation [1]. The development of an orthotopic left single-lung transplant model in rats has enabled investigators to study the mechanisms of ischemia-reperfusion injury with greater ease over the past decade. The development of the total nonsuture cuff technique has reduced the warm ischemic lung implantation time and allows for excellent graft function and long-term survival in a reproducible fashion [2, 3]. Because of its simplicity, many investigators have frequently used this model to study ischemia-reperfusion injury of the lung [1, 4].
However, the use of the present standard dual-lung ventilation technique with a volume-cycled ventilator (Harvard Rodent Ventilator, Model 683, South Natick, MA), makes it difficult to gather broncho-alveolar lavage samples or data, such as flow-volume curves and peak airway pressures, from the transplanted graft lung alone rather than for both the transplanted graft and native lungs together. Furthermore, the model is often hindered by progressive collapse of the transplanted lung during reperfusion, with poor control of the tidal volume (Vt) and positive end-expiratory pressure (PEEP) applied to the newly grafted lung. Finally, for studies specifically examining the effect of ventilator-induced lung injury or prolonged ischemic storage, this technique is not ideal, as an injured lung requires higher airway pressures to prevent atelectasis and collapse compared with the normal native right lung. This is not possible if a standard dual-lung ventilation technique with a volume-cycled ventilator is used to ventilate both lungs. This drawback appears to be even more important as inadequate ventilation of the transplanted lung may increase lung injury that is induced by ischemia-reperfusion [5]. To address these issues, we recently improved the technique by ventilating the donor and recipient rat lung separately with two different ventilators during the reperfusion period.
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Technique
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All animals received humane care in accordance with the National Institutes of Health guidelines [6].
Harvest and Preparation of Organ for Implantation
Lung harvest and preparation was carried out as described previously [4, 7] with the following changes. After the left lung was prepared for transplantation with the placement of two 14-gauge cuffs into the left pulmonary artery (PA) and the left pulmonary vein (PV), the right main bronchus was ligated. The left main bronchus and the trachea were kept intubated with the 14-gauge catheter connected to a three-way stopcock for separate ventilation after the transplantation procedure (Fig 1). The left lung was kept inflated by occluding the three-way stopcock and was then placed into 40 mL of low potassium dextran (LPD) solution at 4°C for cold ischemic preservation.
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Fig 1. (A) Left lung preparation for independent ventilation. Note that the donor trachea remains connected to the left main bronchus; the donor right main bronchus has been ligated. (B) Position of the left lung and donor trachea in order to reperfuse and allow separate ventilation of the transplanted lung. (PA = pulmonary artery; PV = pulmonary vein.)
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Transplantation
Recipient animals were anesthetized in an isoflurane (5%) bell jar, and a tracheostomy was performed as described for the donor animals. The recipient animals were ventilated with a volume-cycled Harvard Rodent Ventilator, with Vt 10 mL/kg; respiratory rate, 80 breaths/min; PEEP, 2 cm H2O; Fio2, 1.0, and anesthesia was maintained with 2.0% isoflurane.
After a left thoracotomy was performed, the left lung was mobilized by dividing the pulmonary ligament, and the hilar structures were dissected free. The left PA, PV, and main bronchus were identified and clamped with microsurgical aneurysm clamps. The left main bronchus was ligated and divided, and the Vt, which was then limited to the right lung only, was reduced to 6 mL/kg. A ventral incision was made in the recipient PA and PV and the cuffs of the donor lung structures were inserted into the corresponding recipient structures. The anastomoses were secured with 6–0 polypropylene ties around the rigid cuffs. The recipient’s native left lung was then explanted. The total implantation time is usually less than 15 minutes.
The trachea of the transplanted lung was connected to a second volume-cycled Harvard Rodent Ventilator, with Vt, 4 mL/kg; respiratory rate, 80 breaths/min; PEEP, 2 cm H2O; Fio2, 1.0; and the lung was reinflated (Fig 1). It must be emphasized that great care needs to be taken to ensure that the trachea and left main bronchus are kept straight and are prevented from becoming twisted and obstructed—the chest wall retractor securing the donor trachea in place greatly facilitates this alignment (Fig 2).
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Fig 2. Independent lung ventilation after transplantation. Note that the chest wall retractor is used to prevent kinking of the graft airway and ensure patency.
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Immediately after reinflation, a recruitment maneuver was performed by occluding the expiratory limb of the ventilator for two successive inspiratory/expiratory cycles. The recipient’s native right lung and the left transplanted lung were thus ventilated independently. Reperfusion was initiated by unclamping the PV (the patency of the venous anastomosis was verified by noting retrograde flow from the atrium to the pulmonary veins), followed by unclamping the PA. The transplanted lung can then be reperfused for several hours without any significant hemodynamic instability, because the animal is supported by the native lung irrespective of the graft function.
In our experience, this model has been used in more than 60 lung transplant procedures with a reperfusion period of 2 to 4 hours, according to the type of experiments performed [5, 7]. We have found this model to be reliable and reproducible.
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Comment
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Progressive collapse of the transplanted lung can be a problem in the single-lung transplant model, as the lung with normal compliance (the native right lung) begins to hyperinflate and the transplanted lung gets successively smaller tidal volumes as the time period of reperfusion increases. This is especially a problem if the graft lung has been subjected to prolonged periods of cold ischemic storage (> 12 hours) and could be a significant source of error when lung function in this model is evaluated.
Along with solving the problem of progressive collapse of the grafted lung, this simple method of independent lung ventilation also allows one to obtain direct samples and measurements from the transplanted lung in order to precisely study the broncho-alveolar lavage fluid or determine physiologic variables, such as elastance, compliance, and airway pressures of the grafted lung, which can then act as independent markers reflecting the degree of lung injury. Ventilator-induced lung injury or the potential for inducing further injury in an already injured ischemia-reperfused transplanted lung is becoming increasingly recognized. This model allows for precise control and variation of the technique of ventilation of the transplanted single lung in the rat.
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References
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- de Perrot M, Liu M, Waddell TK, Keshavjee S. Ischemia-reperfusion-induced lung injury Am J Respir Crit Care Med 2003;167(4):490-511.[Abstract/Free Full Text]
- Mizuta T, Kawaguchi A, Nakahara K, Kawashima Y. Simplified rat lung transplantation using a cuff technique J Thorac Cardiovasc Surg 1989;97(4):578-581.[Abstract]
- Reis A, Giaid A, Serrick C, Shennib H. Improved outcome of rat lung transplantation with modification of the nonsuture external cuff technique J Heart Lung Transplant 1995;14(2):274-279.[Medline]
- Fischer S, Liu M, MacLean AA, et al. In vivo transtracheal adenovirus-mediated transfer of human interleukin-10 gene to donor lungs ameliorates ischemia-reperfusion injury and improves early posttransplant graft function in the rat Hum Gene Ther 2001;12(12):1513-1526.[Medline]
- de Perrot M, Imai Y, Volgyesi GA, et al. Effect of ventilator-induced lung injury on the development of reperfusion injury in a rat lung transplant model J Thorac Cardiovasc Surg 2002;124(6):1137-1144.[Abstract/Free Full Text]
- National Institutes of Health Guide for the Care and Use of Laboratory Animals Publication # 85–23. Washington, DC: US Government Printing Office; 1985.
- de Perrot M, Young K, Imai Y, et al. Recipient T cells mediate reperfusion injury after lung transplantation in the rat J Immunol 2003;171(10):4995-5002.[Abstract/Free Full Text]
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H. Inokawa, M. Sevala, W. K. Funkhouser, and T. M. Egan
Ex-vivo perfusion and ventilation of rat lungs from non-heart-beating donors before transplant.
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October 1, 2006;
82(4):
1219 - 1225.
[Abstract]
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Abstract
Introduction
