Short Communication Experimental Orthotopic Lung Transplantation ...

Surg Today (2009) 39:641–645 DOI 10.1007/s00595-008-3929-x

Short Communication Experimental Orthotopic Lung Transplantation Model in Rats with Cold Storage RYUJIRO SUGIMOTO1,2,3,5, ATSUNORI NAKAO1,2,5, ITARU NAGAHIRO3, JUNICHI KOHMOTO1,2,3, SEIICHIRO SUGIMOTO3, MIKIO OKAZAKI3, MASAOMI YAMANE3, HIDETOSHI INOKAWA3, TAKAHIRO OTO3, KAZUNORI TAHARA4, JIANGHUA ZHAN1,2, YOSHIFUMI SANO3, and KENNETH R. MCCURRY1,2,5 1

Heart, Lung and Esophageal Surgery Institute, Department of Surgery, University of Pittsburgh, Pittsburgh, PA, USA Department of Surgery, University of Pittsburgh, Pittsburgh, PA, USA 3 Department of Thoracic Surgery, Okayama University Hospital, Okayama, Japan 4 Department of Pediatric Surgery, Dokkyo Medical University Koshigaya Hospital, Koshigaya, Japan 5 Thomas E. Starzl Transplantation Institute, Department of Surgery, University of Pittsburgh, E1551 Biomedical Science Tower, 200 Lothrop Street, Pittsburgh, PA 15213, USA 2

Abstract This report describes a new experimental procedure, a rat unilateral, orthotopic lung transplantation with cold storage, and evaluates its relevancy and reliability to study the early events during cold ischemia/reperfusion (I/R) injury. This model, using the cuff technique, does not require extensive training and is relatively easy to be established. The model can induce reproducible degrees of pulmonary graft injury including impaired gas exchange, proinflammatory cytokine upregulation, or inflammatory infiltrates, depending on the preservation time. The results are consistent with the previous clinical evidence, thus suggesting that this model is a valid and reliable animal model of cold I/R injury. Key words Lung transplantation · Experimental animal model · Ischemia/reperfusion · Cold preservation

Introduction The rodent model of lung cold ischemia/reperfusion (I/ R) injury provides several advantages over large animal models with regard to cost, social concerns, and the ability to perform detailed mechanistic studies. Since the first model of rat lung transplantation was reported in 1971,1 several different experimental models have been used.2–4,5 In particular, a technique using cuff anastomosis developed by Mizuta et al. has considerably shortened the surgery time and reduced the rate of complications; thus the cuff technique has become the standard method for lung transplantation in rats.6,7 It is Reprint requests to: A. Nakao (address 5) Received: September 19, 2008 / Accepted: December 2, 2008 A. Nakao and K.R. McCurry contributed equally to this work.

critical to establish experimental intervention programs using reliable animal models of cold I/R injury following lung transplantation to develop therapeutic strategies for the prevention and management of cold I/R injury. In addition, it is also important to clarify the endpoints of the study to evaluate the therapeutic approaches aimed at improving lung graft outcomes. This laboratory has employed a modification of Mizuta’s procedure for investigations of various events during cold I/R injury process and reported several therapeutic strategies to prevent lung cold I/R injury.8–10 These experiences may be informative and valuable, and help other researchers develop an experimental protocol for studies of lung cold I/R injury. This report describes the surgical procedure for rat unilateral orthotopic lung transplantation using the cuff technique, as well as the methodology to elucidate the endpoints of the graft injury. This experimental model permits the reproducible analysis of the various pathological phenomena associated with lung cold I/R injury and is analogous to the events observed in larger animals and the clinical setting of human lung transplantation.

Surgical Procedure Cuff Preparation Cuffs are prepared for the vascular and bronchial anastomoses prior to beginning the donor surgical procedure. Sixteen-gauge intravenous fluorinated ethylene-propylene (FEP) polymer catheters are used to make each cuff for the pulmonary artery (PA), and 14gauge catheters are used for the pulmonary vein (PV) and bronchus, respectively. The cuff head must be smooth and the surface of the cuff is polished with sandpaper or a file to avoid vessels slipping from the

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R. Sugimoto et al.: Rat Lung Transplantation Model

cuff. One or two grooves are made with the back of scalpel to facilitate keeping the position of tie.

The donor rat is placed in a supine position and median laparotomy is performed. Heparin (300 U) is intravenously injected via the inferior vena cava. A median sternotomy is then performed and both cut edges of sternum are held with Kocher’s forceps, and the chest cavity is exposed. Incisions are made on the left and right atrial appendages to decompress the graft, in addition to cutting the inferior vena cava. The catheter for perfusion is inserted to the main PA through the anterior wall of the right ventricle. The lungs are flushed through the main pulmonary artery with 20 ml of flush solution at a pressure of 20 cmH2O for 60–120 s (Fig. 1A). After the perfusion is completed, the lungs are inflated and the cardiopulmonary block is extracted, separating it from the esophagus and sectioning the supra-aortic trunks, aorta, vena cava, and pulmonary ligament. Immediately after the excision of the heart and lungs, the right lung is taken and kept as a control sample.

Donor Surgery General anesthesia is induced with isoflurane (Isoflo, Abbott Laboratories, Abbott Park, IL, USA). A skin incision is made in the fore neck and the trachea is exposed. A tracheotomy is performed and a tracheal tube (14-gauge intravenous catheter) is inserted and fixed with 4-0 silk tie. The tracheal tube is connected to a ventilator (Harvard Rodent Ventilator Model 683; Harvard Apparatus, Holliston, MA, USA), and then the donor is mechanically ventilated with a mixture of 100% oxygen and isoflurane, with a positive endexpiratory pressure (PEEP) of 2 cmH2O, tidal volume of 10 ml/kg, and respiratory rate of 60 breaths/min. The lungs are ventilated with 100% oxygen throughout the donor surgery.

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Fig. 1. A The grafts are perfused through the tube with 20 ml of perfusate at 4°C with 20 cmH2O pressure. The cannula is inserted into the pulmonary artery (PA) via right ventricle wall. Lt. left; Rt., right; IVC, inferior vena cava; SVC, superior vena cava; RV, right ventricle; LV, left ventricle; RA, right atrium. B Cuff attachment to the graft lung. C The hilum of the recipient left lung is dissected, and the PA, the PV, and the left main bronchus are isolated. The picture shows the exposed PA, bronchus (Br), and PV to be used for anastomosis. The head of the recipient is to the right side of the pictures. D The lung graft is orthotopically transplanted by anastomosing the PA, Br, and PV of the recipient using the cuff technique. E A schematic drawing of the completion of the anastomosis. Abbreviations as in A


Back Table (Graft Preparation) The harvested lung grafts are placed in a Petri dish filled with cold flush solution and covered with wet sponges. The adjacent tissue along with the proximal edges of vessels and trachea are carefully removed. Hypothermia must be maintained during cuff placement into the PA (16 gauge), PV (14 gauge), and bronchus (14 gauge). To facilitate the procedure, the length of the vessels must be long enough to attach the cuff. The vessels and bronchus are passed through the cuffs, everted over the cuffs circumferentially, and secured with 7-0 nonabsorbable monofilament ligature (Fig. 1B). It is important that the cuff extension faces the posterior edge of the lung, so that the structures do not become twisted during implantation. Recipient Surgery The recipient animal is anesthetized, orotracheally intubated, and ventilated on the same settings as the donors. The recipient animal is placed in a right decubitus position and a left posterior lateral thoracotomy is performed, as described. A skin incision is made horizontally at 1 inch above the left costal edge, the pectoral and latissimus dorsi muscles are cut using bipolar cautery, and the fourth intercostal space is entered. The left lung is mobilized by dividing the pulmonary ligament, then the lung is exteriorized and maintained outside of the cavity with a metal clip. The hilum of the left lung is dissected and the PA, the PV, and the left main bronchus are isolated. All three recipient structures are clamped by using microvascular clips (Micro-Serrefine, Fine Science Tools, Foster City, CA, USA). A circumferential ligature of 7-0 silk with the first knot is prepared before anastomosis. Before the native left lung is removed, the lung graft is orthotopically transplanted by anastomosing the PA, bronchus, and PV of the recipient using the cuff technique. After the reperfusion, the pleural cavity is cleaned and hemostasis is confirmed. The graft is moved into the thorax using cotton tip swabs. If partial atelectasis is noted, PEEP will help eliminate it. A pleural drainage tube connected to a 20-ml syringe is introduced into the thorax, and the chest is closed. The drainage tube is aspirated to return the pleural cavity to negative pressure. When the recipient starts breathing spontaneously and then adequately recovers from general anesthesia (usually within 10 min), the thoracic drainage tube and tracheal tube are removed. Evaluations of Cold I/R Injury Orthotopic lung transplantations were performed using syngeneic rat combinations of inbred male LEW (RT.1l)

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rats weighing 250–300 g (Harlan Sprague Dawley, Indianapolis, IN, USA). All procedures were performed according to the guidelines of the Institutional Animal Care and Use Committee at the University of Pittsburgh and the National Research Council’s Guide for the Humane Care and Use of Laboratory Animals. The graft functions for gas exchange were assessed by blood gas analysis (iSTAT Portable Clinical Analyzer; iSTAT, East Windsor, NJ, USA). At the time of sacrifice, the animals were reanesthetized and ventilated on the same settings as those used for donors after a tracheotomy. Blood was drawn from the PV of the graft lung to negate any contribution from the recipient native right lung and assessed by blood gas analysis, on a FiO2 of 1.0 for >5 min. The blood oxygenation at 2 h after reperfusion was deteriorated in the grafts stored for 6 h in low potassium dextran (LPD) solution (Perfadex; Vitrolife, Englewood, CO, USA). PaCO2 levels were comparable among the experimental groups (Fig. 2A). The macrophages were stained by immunohistochemistry for CD68 (ED1) as described previously since inflammatory cellular recruitment is critical for determination of lung cold I/R injury.8 Prolonged cold ischemia resulted in increase of ED1 positive macrophage infiltrations to the lung grafts 2 h after reperfusion (Fig. 2B). As with graft gas exchange, the levels of proinflammatory cytokines such as tumor necrosis factor-α and interleukin-6, determined by real time reverse transcription–polymerase chain reaction as previously described,9,10 were elevated 2 h after reperfusion in grafts with minimal cold ischemic time. Prolonging the cold ischemic period to 6 h resulted in further increases in these cytokine mRNA levels (Fig. 2C).

Discussion The rat model of unilateral lung transplantation has been used in various research centers for the assessment of the repercussions resulting from this procedure. The advantages of rodent models include the availability of established inbred strains, which allow various immunological tests using specific monoclonal antibodies. In addition, the costs of purchasing and housing experimental animals are lower when rodent models are employed. The rat model permits reproduction of I/R injury after transplantation, which is analogous to what occurs in larger animals and in human clinical situations. The current series of experiments used mainly syngeneic, isograft transplant models, since the isograft model is considered an ideal experimental model to study I/R injury by isolating factors related to I/R from other factors involved in alloimmune reactions. Although it is an artificial experimental model and it does not happen in the clinical transplantation setting

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(except for the rare case of identical twins), it is widely used as a tool to study the biology of I/R and to evaluate the effect of therapeutic manipulations. To obtain a reliable and reproducible model of cold I/R injury, it is important to reduce the incidence of surgical complications and the technical differences among the investigators. Nonsuture anastomosis using a cuff technique, reported by Kamada and Calne in 1979 for portal vein anastomosis in rat liver transplantation, may be an ideal method to establish feasible models.11 In 1989, Mizuta et al. first described an external nonsuture cuff technique for vascular anastomosis in rat lung transplantation.6 The surgical procedure described above is very similar to Mizuta’s original method, although several things were modified: the thoracotomy procedure in donor surgery, the size of the cuff, and the method of anastomosis. The advantage of the cuff method in comparison to the suture technique includes a decrease of the warm ischemic time and the prevention of bleeding from the vascular anastomosis site. The cuff technique requires special skills including cuff han-

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Fig. 2. A The graft pulmonary vein (PV) PaO2 values in sham-operated animals were 324.5 ± 11.5 mmHg under mechanical ventilation with 100% oxygen. Graft gas exchange was attenuated after lung transplantation (LTx) with even minimal cold storage (2 h) in low potassium dextran (LPD) solution and PaO2 levels were 229 ± 14.1 mmHg at 2 h after reperfusion. Prolonged cold storage for 6 h in LPD resulted in more severe graft impairment and a significant decrease of graft PV PaO2 levels to 157.9 ± 46.1 mmHg at 2 h after reperfusion. PaCO2 levels were comparable among the experimental groups (n = 5, *P < 0.05, Student’s t-test for two-group analysis). B Prolonged cold storage for 6 h resulted in a further increase of the number of ED1+ macrophage infiltration to 13.7 ± 1.0/0.1 mm2 from those in sham-operated grafts (7.3 ± 2.8/0.1 mm2) 2 h after reperfusion. n = 4– 5, *P < 0.05. The positively stained cells were counted in a blinded fashion in 10 high-power fields (400×) per section and expressed as the number of cells per 0.1 mm2. C Proinflammatory cytokine mRNA levels in the lung grafts elevated 2 h after reperfusion. Extended cold ischemia caused a further upregulation of proinflammatory cytokines such as tumor necrosis factor-α and interleukin-6. CIT, cold ischemia time

dling, the use of the microsurgical instruments, and a surgical microscope; however, it does not require extensive training that is otherwise necessary for the development of microvascular surgery skills required for use of the conventional suture technique. Therefore, mastery of the technique is feasible within a reasonable amount of time with proper instruction and practice. In fact, all surgeons trained in this laboratory between April 2005 and April 2008 required