Ann Thorac Surg 1997;63:796-799
© 1997 The Society of Thoracic Surgeons
Original Article: General Thoracic
Sequential Bilateral Isolated Lung Perfusion in the Rat: An Experimental Model
Sumihiko Nawata, MD,
Amir Abolhoda, MD,
Howard M. Ross, MD,
Ari Brooks, MD,
Michael E. Burt, MD, PhD
Thoracic Oncology Laboratory, Department of Surgery, Memorial Sloan-Kettering Cancer Center, New York, New York
Accepted for publication October 25, 1996.
 |
Abstract
|
|---|
Background. A model of isolated single-lung perfusion in the rat has been established in our laboratory to study the chemotherapeutic treatment of pulmonary metastases. A sequential bilateral isolated lung perfusion model was designed to investigate the feasibility of staged perfusions in the rat.
Methods. Twenty-four Fischer rats were randomized into three experimental groups of 8 rats each. All rats underwent left isolated lung perfusion. One, 2, or 3 weeks later, the rats in groups I, II, and III, respectively, underwent contralateral (right) perfusion. Five control animals (group IV) underwent sequential bilateral sham thoracotomies 1 week apart. Arterial blood gas analysis was performed 1 week after the second operation in the rats in groups I and IV.
Results. All animals survived the first operation, with 100% (8/8), 75% (6/8), and 100% (8/8) of the animals in perfusion groups I, II, and III, respectively, surviving the second operation. All control animals (group IV) survived the second sham thoracotomy. Arterial blood gas analysis did not show a significant difference in the oxygen or carbon dioxide partial pressure or the pH between group I and IV (p = 0.32, 0.96, and 0.76, respectively).
Conclusions. Our experiments demonstrate that sequential bilateral isolated lung perfusion is safe in and well tolerated by the rat. This model can be used to investigate the safety and efficacy of staged perfusions with chemotherapeutic agents in the treatment of bilateral pulmonary metastases in the rat.
|
Introduction
|
|---|
See also page 799a.
Soft-tissue sarcoma frequently and almost exclusively metastasizes to the lungs [1]. Surgical removal remains the mainstay of treatment for pulmonary sarcoma metastases, but resection yields only a 25% overall 5-year survival [2–5]. We have previously reported a rodent model of left isolated lung perfusion (ILP) as a method for delivering organ specific chemotherapy [6]. This technique allows the exposure of pulmonary metastases to high concentrations of chemotherapeutic agents while minimizing systemic toxicities. Efficacy studies in which doxorubicin was delivered by ILP against experimental pulmonary sarcoma metastases in the rat have shown significant tumor response [7] and established the technical feasibility of ILP. However, our current rodent model is of limited value for the evaluation of long-term toxicity or the durability of remission after isolated single-lung chemoperfusions, because the perfused animal dies approximately 2 to 3 weeks after the treatment as the result of the uncontrolled sarcoma metastases in the untreated right lung. Because most human pulmonary metastases are bilateral, it is also therefore imperative that a bilateral perfusion protocol be developed. Prior investigations in large animals have involved simultaneous bilateral perfusions using cardiopulmonary bypass [8]. The objectives of the present study were to develop a surgical model to examine the feasibility of staged perfusions of both lungs and to evaluate the influence of sequential bilateral perfusions on pulmonary gas exchange. Such a model would not only constitute a prototype for the future clinical trials of ILP but also provide a means for assessing the long-term effects of the chemoperfusion on the normal lung and the pulmonary metastases in the rat.
|
Material and Methods
|
|---|
Animal Care
Male Fischer 344 rats (Charles River Laboratories, Kingston, NY) were used in all experiments. Animals were treated in accordance with the Animal Welfare Act and the "Guide for the Care and Use of Laboratory Animals" (NIH Publication 86-23, Revised 1985). All experiments were approved by the Institutional Animal Care and Use Committee of Memorial Sloan-Kettering Cancer Center. Animals were allowed access to standard laboratory rat food (Purina Rat Chow; Ralston Purina, St. Louis, MO) and water ad libitum. Housing was temperature controlled and provided a 12-hour light-dark cycle.
Surgical Anatomy of Fischer Rat Lung
The left lung of Fischer rats consists of one large lobe with one pulmonary artery and one vein. The right lung is composed of four lobes with a short common pulmonary artery and two dominant veins draining into a short common pulmonary vein (Fig 1
).
View larger version (34K):
[in this window]
[in a new window]
|
Fig 1. . Anatomy of Fischer 344 rat lungs. The right lung is composed of four lobes, and the left lung is a unilobar structure.
|
|
Operative Technique
All procedures were performed under clean operative conditions. Animals were anesthetized with pentobarbital sodium (50 mg/kg) delivered intraperitoneally and intubated endotracheally with a 16-gauge intravenous catheter under direct visualization through an otoscope [9]. A Harvard Apparatus rodent volume ventilator (model 683; Harvard Apparatus, South Natick, MA) was used to ventilate the animals with room air at a tidal volume of 10 mL/kg and a respiratory rate of 80 strokes/min.
LEFT ISOLATED-LUNG PERFUSION.
Left ILP was performed using the method previously established in this laboratory [6]. The left chest was shaved and prepared with a 10% povidone-iodine solution, and a left thoracotomy through the fourth intercostal space was performed. The left pulmonary artery and vein were visualized under an operative microscope (OpMi-1, 16x; Carl Zeiss, Wotan, Germany). Microvascular clamps were placed proximally on the pulmonary artery and vein. A 3-0 silk tie was passed around the artery. An arteriotomy was performed, and the artery was cannulated with a PE-10 catheter (Beckton Dickinson & Co, Parsippany, NJ). The silk tie was then tightened around the catheter and tied once to secure the catheter in place. Perfusate was instilled through this catheter. A pulmonary venotomy was created, and the effluent was collected by a suction catheter placed near the venotomy (Fig 2). At the completion of the perfusion, the arteriotomy was repaired with a single 9-0 Ethilon suture (Ethicon, Somerville, NJ). The microvascular clamps were removed, and the left lung was returned to its anatomic position. Point pressure was applied using a moist gauze over the lung to tamponade the bleeding from the venotomy. A 16-gauge catheter connected to a 5-mL syringe was introduced into the left chest cavity through a separate puncture wound to facilitate lung reexpansion. The thoracotomy incision was closed in three layers with 4-0 silk. When the animals were alert and breathing spontaneously, their chest and endotracheal tubes were removed.
RIGHT ISOLATED LUNG PERFUSION.
Right ILP was performed using a slight modification to accommodate the anatomic differences between the right and left lungs in the Fischer rat. The right chest was shaved and prepared with a 10% povidone-iodine solution, and a right thoracotomy through the fourth intercostal space was performed. The right superior and middle lobes were retracted from the right thoracic cavity. The mediastinal pleura was incised longitudinally along the superior vena cava to identify the right main pulmonary artery. The right pulmonary artery, coursing behind the superior vena cava, was dissected free along its entire length. Subsequently, the right inferior and postcaval lobes were retracted from the right thoracic cavity and all lobes were wrapped with wet gauze and exteriorized to facilitate hilar dissection. Next, the right main pulmonary vein was dissected. A microvascular clamp was placed proximally on the right main pulmonary artery, and a 3-0 silk tie was passed around the artery. A 3-0 silk tie was used to occlude the main pulmonary vein. An arteriotomy was performed, and the artery was cannulated with a PE-10 catheter. Two separate pulmonary venotomies were created in the two venous tributaries of the main pulmonary vein, and the perfusion was begun (Fig 3). After completion of the perfusion, the pulmonary artery cannula and the encircling silk tie were removed and the arteriotomy was repaired with a single 9-0 Ethilon suture. The microvascular clamp on the artery and the occluding silk tie on the main pulmonary vein were then removed, and the right lung was returned to the chest cavity. As in left ILP, gentle pressure was used to tamponade the bleeding from the venotomies. The thoracotomy incision was closed after placement of a 16-gauge catheter as a chest tube.
Experimental Design
Twenty-nine male Fischer 344 rats were divided into four groups. On day 0, 24 rats in groups I to III (n = 8 in each group) underwent left ILP for 15 minutes with buffered hetastarch (BHE, 6% hetastarch in 0.9% sodium chloride; Dupont Pharma, Wilmington, DE) at a rate of 0.5 mL/min. One, two, or three weeks later, the rats in groups I, II, and III, respectively, underwent contralateral (right) ILP. The control group (group IV; n = 5) underwent sequential bilateral sham thoracotomies 1 week apart. The animals were allowed to survive after each operation and were monitored in the postoperative period with daily weights and close observations of their behavioral and respiratory patterns.
Pulmonary Gas Exchange Evaluation
One week after the second thoracotomy, arterial blood gas analysis was performed in the rats in groups I and IV. Anesthetized rats were intubated endotracheally and supported on the rodent volume ventilator on room air to ensure uniform minute ventilation before arterial blood sampling. A midline laparotomy was then performed, and the abdominal aorta was directly punctured to collect arterial blood samples in a preheparinized syringe. After this, the animals were euthanized with carbon dioxide inhalation.
Statistical Analysis
A two-tailed unpaired t test was used for statistical comparison of the arterial blood gas values; a p value of less than 0.05 was considered significant.
|
Results
|
|---|
All rats survived left ILP and left sham thoracotomy. Survival after contralateral ILP or sham thoracotomy was 100% (8/8) in group I, 75% (6/8) in group II, 100% (8/8) in group III, and 100% (5/5) in group IV. The weight curves of the perfused animals in group I were similar to those of the control group (Fig 4). The rats lost weight for the first 2 days after each perfusion, but they recovered their original weight within 1 week. The two deaths in group II occurred during the second thoracotomy and were clearly a result of a technical error made during the dissection that led to fatal hemorrhage.
View larger version (24K):
[in this window]
[in a new window]
|
Fig 4. . Postoperative weight curves for the group receiving the bilateral isolated lung perfusion (group I) and the group undergoing bilateral sham thoracotomy (group IV).
|
|
Arterial blood gas analysis did not show a significant difference in the pH or oxygen or carbon dioxide partial pressure between groups I and IV (p = 0.76, 0.32, and 0.96, respectively) (Table 1).
|
Comment
|
|---|
Isolated lung perfusion is a unique model for the delivery of high-dose chemotherapy with minimal systemic toxicity. When we began our investigations in this area, we elected to use Fischer rats for the following three reasons: (1) the left lung of this strain of rats is unilobar with a long segment of extrapericardial pulmonary artery and vein, which allows greater ease in hilar dissection and pulmonary artery cannulation; (2) Fischer rats can survive on one functioning lung, as repeatedly shown by animal survival after unilateral pneumonectomy; and (3) multiple transplantable cell lines are available that can reproducibly establish bilateral pulmonary metastases after intravenous injection. We faced some difficulties in our initial attempts with right-sided perfusions stemming from the more complicated hilar anatomy of the right lung. The surgically accessible segment of the right main pulmonary artery is relatively short because of its retrocaval position and the early take-off of the right upper lobe branch. This anatomic constraint necessitated a more detailed hilar dissection before pulmonary artery cannulation to ensure uniform perfusion of the entire right lung. Furthermore, there are two distinct pulmonary venous tributaries that enter a short common vein before draining into the left atrium. This necessitated occlusion of the common trunk and the creation of two separate venotomies to ensure unobstructed drainage of the effluent and prevent pulmonary congestion. After these technical modifications were made, we were able to perform right-sided perfusions with equal success.
There are several potential applications of bilateral staged perfusions. As noted previously, evaluation of the long-term results of left ILP with chemotherapeutic agents is limited by progression of the metastatic disease in the untreated right lung. Using our bilateral perfusion model, we believe we can prolong animal survival and thus extend the period of observation for the assessment of the durability of response to the chemoperfusions. Additionally, the staged bilateral delivery of chemotherapeutic agents in a tumor-free model would allow us to evaluate the effects of such treatment on pulmonary physiology. Finally, the results of these preclinical studies will contribute to the development of future treatment protocols for human subjects.
In summary, our experiments demonstrate that sequential bilateral ILP is safe and well tolerated in the rat. This model can be used to investigate the safety and efficacy of staged perfusions with chemotherapeutic agents in the treatment of bilateral pulmonary metastases.
|
Footnotes
|
|---|
Address reprint requests to Dr Burt, Department of Surgery, Memorial Sloan-Kettering Cancer Center, 1275 York Ave, New York, NY 10021.
|
References
|
|---|
- Potter DA, Glenn S, Kinsella T. Patterns of recurrence in patients with high grade soft-tissue sarcomas. J Clin Oncol 1985;3:353–66.
- Casson AG, Putnam JB Jr, Natarajan G, et al. Five-year survival after pulmonary metastasectomy for adult soft tissue sarcoma. Cancer 1992;69:662–8.[Medline]
- Huth JF, Holmes EC, Vernon SE, Callery CD, Ramming KP, Morton DL. Pulmonary resection for metastatic sarcoma. Am J Surg 1980;140:9–16.[Medline]
- Jablons D, Steinberg SM, Roth JA, Pittaluga S, Rosenberg SA, Pass HI. Metastasectomy for soft tissue sarcoma. J Thorac Cardiovasc Surg 1989;97:695–705.[Abstract]
- Lanza LA, Putnam JB Jr, Benjamin RS, Roth JA. Response to chemotherapy does not predict survival after resection of sarcomatous pulmonary metastases. Ann Thorac Surg 1991;51:219–24.[Abstract]
- Weksler BA, Schneider B, Ng B, Burt ME. Isolated single lung perfusion in the rat: an experimental model. J Appl Physiol 1993;74:2736–9.[Abstract/Free Full Text]
- Weksler BJ, Lenert J, Ng B, Burt M. Isolated single lung perfusion with doxorubicin is effective in eradicating soft tissue sarcoma lung metastases in a rat model. J Thorac Cardiovasc Surg 1994;107:50–4.[Abstract/Free Full Text]
- Johnston MR, Christensen CW, Minchin RF, et al. Isolated total lung perfusion as a means to deliver organ-specific chemotherapy: long-term studies in animals. Surgery 1985;98:35–44.[Medline]
- Weksler B, Ng B, Lenert J, Burt M. A simplified method for endotracheal intubation in the rat. J Appl Physiol 1994;76:1823–5.[Abstract/Free Full Text]
Related Article
-
- Michael R. Johnston
Ann. Thorac. Surg. 1997 63: 799.
[Extract]
[Full Text]
Top
Abstract
Introduction
