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Ann Thorac Surg 1995;60:958-962
© 1995 The Society of Thoracic Surgeons

Original Articles: General Thoracic

Lung Preservation Threshold in a Compromised Septic Lung Injury Model

Shigeyuki Sasaki, MD, PhD, James D. McCully, PhD, John D. Palombo, ScD, R. Armour Forse, MD, PhD, Joseph LoCicero, III, MD

Section of General Thoracic Surgery, Department of Surgery, Harvard Medical School, New England Deaconess Hospital, Boston, Massachusetts

Accepted for publication May 12, 1995.


   Abstract
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 Footnotes
 Abstract
Introduction
 Material and Methods
 Results
 Comment
 References
 
Background. Discrepancy between clinical and research works in lung transplantation could be due to differences between compromised clinical donor lungs and intact research lungs. The purpose of this laboratory study was to produce compromised lungs to compare with normal ones.

Methods. Sprague-Dawley rats were continuously infused with lipopolysaccharide (5 mg/kg) for 24 hours before organ harvest. Lungs were stored in University of Wisconsin solution at 4°C for the following period: group 1: intact lungs, no storage (n = 12); group 2: septic lungs, no storage (n = 6); group 3: septic lungs for 6 hours (n = 5); and group 4: septic lungs for 12 hours (n = 5). All lungs were reperfused for 2 hours with venous blood using an isolated, pulsatile perfused lung system.

Results. Experimental variables were comparable between groups 1, 2, and 3. All septic lungs stored for 12 hours (group 4) failed within 1 hour of perfusion.

Conclusions. These results indicate that compromised lungs with septic injury functioned at near control levels after 6 hours of preservation. Six hours may be a safe limit for human donor lungs, all of which are compromised in some way by the time of harvest.


   Introduction
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 Abstract
 Introduction
Material and Methods
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 Comment
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See also page 962.

Currently accepted usual lung storage intervals for transplantation remain up to 6 hours, which may restrict the pulmonary donor pool available for clinical lung transplantation [1, 2]. Experimental studies have shown that lungs were successfully preserved for at least 12 hours, maintaining lung function after storage. However, extended storage intervals longer than 6 hours (8 hours in bilateral lung transplantation) are not often used in clinical work [2–6]. One of the major reasons for this discrepancy may be the lack of a standard experimental model.

However, a more compelling reason is the difference in donor material between lungs in clinical situations and those used for research. Most experimental studies have used intact lungs; however, most clinical lung grafts are compromised to some degree by a wide variety of conditions including aspiration, neurogenic pulmonary edema, trauma, overhydration, or sepsis before harvest [1, 2]. The purpose of this study, therefore, was to develop a compromised rat lung preservation model allowing for mimicking of the clinical situation. In these experiments, we modified our standard model by using compromised lungs with early septic injury as donor lungs for preservation.


   Material and Methods
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 Abstract
 Introduction
 Material and Methods
Results
 Comment
 References
 
Male Sprague-Dawley rats (325 to 350 g; 4 to 6 months) were obtained from Harlan Sprague Dawley Inc, Indianapolis, IN. All experiments were approved by the New England Deaconess Hospital Animal Care and Use Committee, and conformed to the US National Institutes of Health guidelines regulating the care and use of laboratory animals. Unless otherwise stated, all reagents were purchased from Sigma Chemical (St. Louis, MO).

Experimental Protocol
Animals used in this study were divided into four groups. In the first group, lungs isolated from healthy normal rats were reperfused without storage (group 1: control; n = 12). In the second group, lungs pretreated with lipopolysaccharide (LPS) were reperfused without storage (group 2; n = 6). In the third and fourth groups, lungs pretreated with LPS were stored for 6 hours (group 3; n = 5) or 12 hours (group 4; n = 5) in University of Wisconsin solution (composition of the University of Wisconsin solution: 50 g/L hydroxyethyl starch, 100 mmol/L lactobionic acid, 25 mmol/L KH2PO4, 5 mmol/L MgSO4, 30 mmol/L raffinose, 5 mmol/L adenosine, 1 mmol/L allopurinol, 3 mmol/L glutathione; total Na, 20 mmol/L; K, 140 mmol/L; pH, 7.4; 320 mOsm; ViaSpan, Du Pont Pharmaceuticals, Wilmington, DE).

Lipopolysaccharide Infusion Before Storage
For continuous infusion with LPS, rats in groups 2, 3, and 4 (total n = 16) underwent internal jugular venous catheterization under general anesthesia with ether. Silicone venous catheters (Dow Corning Corp, Midland, MI) were tunneled subcutaneously to the interscapular region and threaded through a metal sheath attached to a swivel device (Instech Laboratories, Horsham, PA) for free movement of the rats during the infusion period. The catheter was filled with heparin and clamped until the infusion commenced. The next day, rats were infused with LPS (5 mg/kg; dissolved in 12 mL of physiologic saline solution) for 24 hours at a constant flow of 0.5 mL/h through the jugular catheter using a constant-infusion pump (Harvard Apparatus, Natick, MA).

Lung Isolation and Storage
After infusion of LPS for 24 hours (groups 2, 3, and 4) or without pretreatment (group 1), rats were anesthetized by an intraperitoneal injection of sodium pentobarbital (50 mg/kg) and underwent tracheostomy. Rats were ventilated with 38°C humidified room air at a respiratory rate of 30 breaths per minute. The tidal volume was set at 10 mL/kg body weight with a positive end-expiratory pressure of 2.5 cm H2O (Harvard Rodent Ventilator, Harvard Apparatus, Natick, MA). Heparin (300 IU) was injected through the inferior vena cava, and the lungs were then exposed through a median sternotomy. Lungs for immediate reperfusion (groups 1 and 2) were flushed in situ with 100 mL/kg body weight of physiologic saline solution. Lungs for storage (groups 3 and 4) were flushed in situ first with physiologic saline solution then with 100 mL/kg body weight of cold (4°C) University of Wisconsin solution at a constant pressure of 30 cm of water. The lungs and heart were harvested en bloc after flushing, and ventilation was held at the half inflated point of inspiration. Lungs in groups 3 and 4 were stored in University of Wisconsin solution at 4°C for 6 hours (group 3) or 12 hours (group 4).

Isolated, Pulsatile Blood Perfusion
After storage in University of Wisconsin solution (groups 3 and 4) or immediately after harvest (groups 1 and 2), lungs were reperfused with fresh venous blood exsanguinated from donor rats for the assessment of lung function using the isolated, pulsatile blood-perfused working lung model. This system was previously reported from our laboratory [7]. Briefly, blood perfusate was delivered by a pulsatile pump (Medical Engineering Consultants, Los Angeles, CA) at a pulmonary flow of 10 mL•100 g body weight-1•min-1 and at a pulse rate of 150/min. Ventilation was with an inspired oxygen fraction of 0.21, 2.5 cm H2O positive end-expiratory pressure, and a tidal volume of 10 mL/kg body weight. The ventilation rate was adjusted to provide normocarbia. The pulmonary flow was monitored using an electromagnetic flowmeter (Carolina Medical Electronics Inc, King, NC), and the pulmonary arterial pressure and the pulmonary airway pressure were measured using Mikro-tip catheter transducers (model SPR-249A; Millar Instruments, Inc, Houston, TX). Airway flow velocity and integrated flow volume were measured using a Fleisch pressure pneumotach (Oem Medical, Inc, Richmond, VA) connected to a Validyne differential pressure transducer (Validyne Engineering Corp, Northridge, CA). Effluent from the pulmonary vein was continuously deoxygenated during the perfusion period with a gas mixture of 95% nitrogen (N2) and 5% carbon dioxide (CO2) and recirculated. The pulmonary arterial pressure, the pulmonary arterial flow, the airway pressure, and the airway flow velocity were continuously monitored and recorded. Pulmonary arterial and venous blood gas tensions were measured using a Corning 238 pH/blood gas analyzer (Essex, UK). Values for hemoglobin and hematocrit in perfusate were measured with Corning 270 Co-oximeter (Essex, UK).

Measurements
Lungs were perfused for 2 hours or until lung failure occurred, determined by the appearance of bronchial fluid in the tracheal cannula. The following variables were calculated and assessed during the perfusion period: blood gas exchange, total pulmonary vascular resistance, and lung airway resistance were measured as routine parameters. The following three variables were calculated by the airway volume--pressure loop to examine elasticity of the lungs [8]: dynamic lung compliance, elastic work, and flow resistive work. Shunt fractions were calculated from blood gas tensions for evaluating the ventilation-perfusion relationship [9].

Statistical Analysis
All results were expressed as means ± standard error of the mean, and differences were considered significant when the p value was less than 0.05. Data from all groups were compared by one-way analysis of variance with post-hoc pairwise comparisons (Tukey-Kramer).


   Results
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 Footnotes
 Abstract
 Introduction
 Material and Methods
 Results
Comment
 References
 
Lung Survival
Lungs stored for up to 6 hours (groups 1, 2, and 3) survived the 2-hour perfusion period. Lipopolysaccharide-pretreated lungs stored for 12 hours (group 4) all failed within 1 hour of the reperfusion period (32.0 ± 2.0 min), which was significantly (p < 0.001) shorter than those of groups 1, 2, and 3.

Blood Gas Analysis and Shunt Fraction
Figure 1Go shows the changes in oxygen tension during reperfusion after storage. Lungs in group 4 showed significantly lower values relative to those in the other groups. Normal lungs (group 1) and LPS-pretreated lungs stored for up to 6 hours (groups 2 and 3) maintained stable oxygen tension values during the perfusion period. The values for shunt fraction indicated the same trend as those for oxygen tension. Shunt fractions at 30 minutes after the onset of reperfusion for groups 1, 2, and 3 were 0.17 ± 0.03, 0.16 ± 0.03, and 0.20 ± 0.07, respectively, which were significantly better than that for group 4 (0.52 ± 0.07; p < 0.01). In addition, lungs in groups 1, 2, and 3 maintained stable shunt fraction values for 2 hours.



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  		Fig 1. . Changes in oxygen tension (pO2) for each group during the perfusion period. (LPS = lipopolysaccharide; **p < 0.01 versus groups 1, 2, and 3.)

 
Aerodynamic and Hemodynamic Parameters
Figure 2Go shows changes in lung airway resistance. Values for groups 1, 2, and 3 at 30 minutes of perfusion were 2.65 ± 0.07, 3.01 ± 0.16, and 3.36 ± 0.17 cm H2O•mL-1 s-1), respectively, which were significantly better than that for group 4 (5.94 ± 0.13 cm H2O•mL-1•s-1; p < 0.01). Furthermore, lungs stored for up to 6 hours maintained stable airway resistance values throughout the 2-hour perfusion period.



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  		Fig 2. . Changes in lung airway resistance (RA) for each group during the perfusion period. (LPS = lipopolysaccharide; **p < 0.01 versus groups 1, 2, and 3.)

 
Figure 3Go shows changes in flow resistive work, which is a marker for lung mechanics indicating the work done to overcome the nonelastic forces of the lungs. Values of flow resistive work indicated the same trend as the lung airway resistance.



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  		Fig 3. . Changes in flow resistive work (Wres) for each group during the perfusion period. (LPS = lipopolysaccharide; **p < 0.01 versus groups 1, 2, and 3.)

 
Values for the dynamic lung compliance, the elastic work, and the total pulmonary vascular resistance are shown in Table 1Go. The dynamic lung compliance and the elastic work indicated the same trend as other aerodynamic parameters. However, alterations in total pulmonary vascular resistance values were independent of those for aerodynamic variables in this model. No significant differences were found in total pulmonary vascular resistance values among experimental groups.


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  		Table 1. . Changes in Dynamic Lung Compliance, Elastic Work, and Total Pulmonary Vascular Resistance for Each Group During the Perfusion Period
 

   Comment
Top
 Footnotes
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
References
 
There have been two major reasons for the discrepancy between clinical and research work regarding storage intervals for lung preservation. One is the large variety of experimental models, and the other is the difference of materials between compromised clinical lungs and intact research lungs [1, 2]. Numerous studies show extended storage intervals to allow availability of cadaver lungs to increase the size of the pulmonary donor pool for transplantation [2–4, 6, 10–13]. However, successful research results have not been realized in clinical situations.

All donor lungs in clinical situations are compromised by a diverse range of pulmonary insults. These insults may be categorized into preexisting damage, acute traumatic insult, and ischemia-reperfusion injury. Human lungs are injured, often chronically, by environmental factors such as urban living and first-hand or second-hand smoke. Age has its own effect, with some diminution of vital capacity. More importantly, donor lungs frequently sustain major insult either directly from trauma such as pulmonary contusion and hemorrhage or secondarily from intubation and ventilation, aspiration, overhydration, sepsis during the trauma recovery phase, or secondary insult from ischemia reperfusion injury to other organs affecting the alveolar-capillary membrane. Finally, donor lungs receive additional insult from the obligatory ischemia and reperfusion injury of harvest, preservation, and reimplantation.

To induce a lung injury that might mimic a clinical situation, we used a septic lung injury model with a continuous infusion of LPS. This produces a response indistinguishable from early adult respiratory distress syndrome (ARDS). Sepsis is a common reason for pulmonary insults leading to lung injury in both ARDS [14] and potential donor lungs in the intensive care unit setting [2]. The LPS-infusion model used in this study is a traditional model inducing sepsis [15]. Previous work has shown significant lung injury with atelectasis, leukocyte infiltration, and patchy loss of integrity of the alveolar-capillary membrane [16]. These lungs show histologic evidence of damage with thickened alveolar septae, proteinaceous alveolar exudates, and platelet aggregation in capillaries [17].

In the present study, prestorage of lungs after the infusion of endotoxin (LPS) for 24 hours is thought to mimic pre-ARDS conditions because those lungs without storage (group 2) showed no deterioration in lung function during the subsequent reperfusion period when compared with control lungs (group 1). However, the safe storage interval for the LPS-pretreated lungs was 6 hours, whereas those for normal lungs was at least 18 hours in previous studies [10–13]. Our previous work showed that undamaged normal animal lungs preserved for 6, 12, and even 18 hours performed as well as normal lungs reperfused immediately. These results on storage intervals in the present study are more similar to those in clinical situations than in the studies with the use of intact donor lungs.

The reasons for the differences between storage intervals for intact lungs and those for compromised lungs are not well defined. It is most likely a multifactorial problem similar to ARDS [15]. Neutrophils are thought to contribute to lung injury in ARDS by release of oxygen radicals and enzymes such as elastase [14]. Lung injury induced by elastase is most striking after exposure to oxidants but causes no damage in isolated lungs not subjected to oxidants [18]. Release of chemotactic agents due to airway insults, complement components, or endotoxin (LPS) may initiate neutrophil priming, which may be an important prerequisite for the development of lung damage [14].

Previously, we evaluated leukosequestration by measurement of myeloperoxidase activity in the perfused lung [19]. Lipopolysaccharide-pretreated lungs before storage had a significantly elevated myeloperoxidase activity relative to normal control lungs. Primed neutrophils may play an important role during preservation and at the onset of reperfusion for further impairment of lung function [20, 21].

Despite the myriad of laboratory claims of extended preservation in animals with normal lungs, there has been little success in extended human lung preservation. This is probably due to the fact that most human donors have predamaged lungs. Yet, in our laboratory, predamaged lungs such as the ones used in these experiments and stored for 6 hours seem to function at near-normal (control) levels. After further evaluation and confirmation in other animal models, storage times of 6 hours and less may well be the best safe limit for human donor lungs that have received a variety of known and unknown insults.


   Footnotes
Top
 Footnotes
Abstract
 Introduction
 Material and Methods
 Results
 Comment
 References
 
Address reprint requests to Dr LoCicero, Division of General Thoracic Surgery, Harvard Medical School, New England Deaconess Hospital, 110 Francis St, Suite 2C, Boston, MA 02215


   References
Top
 Footnotes
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 References
 

  1. Kaiser L, Cooper J. The current status of lung transplantation. Adv Surg 1992;25:259–307.[Medline]
  2. Novick R, Menkis A, McKenzie F. New trends in lung preservation: a collective review. J Heart Lung Transplant 1992;11:377–92.[Medline]
  3. Miyoshi S, Shimokawa S, Schreinemakers H, et al. Comparison of the University of Wisconsin preservation solution and other crystalloid perfusates in a 30-hour rabbit lung preservation model. J Thorac Cardiovasc Surg 1992;103: 27–32.[Abstract]
  4. Mayer E, Puskas JD, Cardoso PFG, et al. Reliable eighteen-hour lung preservation at 4 and 10°C by pulmonary artery flush after high-dose prostaglandin E1 administration. J Thorac Cardiovasc Surg 1992;103:1136–42.[Abstract]
  5. Bonser R, Fragomeni L, Jamieson S, et al. Effects of prostaglandin E1 in twelve-hour lung preservation. J Heart Lung Transplant 1991;10:310–6.[Medline]
  6. Kawahara K, Ikari H, Hisano H, et al. Twenty-four-hour canine lung preservation using UW solution. Transplantation 1991;51:584–7.[Medline]
  7. Sasaki S, McCully JD, LoCicero J. Development of an isolated blood-perfused working rat lung model. Surg Forum 1993;44:312–4.
  8. Berne R, Levy M. Physiology. 2nd ed. St. Louis: Mosby, 1988;575--97.
  9. Mountcastle V. Medical physiology. 14th ed. St. Louis: Mosby, 1980:1715--7.
  10. Breda M, Hall T, Stuart R, et al. Twenty-four hour lung preservation by hypothermia and leucocyte depletion. Heart Transplant 1985;4:325–9.
  11. Puskas JD, Cardoso PF, Mayer E, et al. Equivalent eighteen-hour lung preservation with low-potassium dextran or Euro-Collins solution after prostaglandin E1 infusion. J Thorac Cardiovasc Surg 1992;104:83–9.[Abstract]
  12. Bresticker M, LoCicero J, Oba J, Greene R. Successful extended lung preservation with UW solution. Transplantation 1992;54:780–4.[Medline]
  13. Ulicny K, Egan T, Lambert C, Reddick R, Wilcox B. Cadaver lung donors: effect of preharvest ventilation on graft function. Ann Thorac Surg 1993;55:1185–91.[Abstract]
  14. Repine JE. Scientific perspectives on adult respiratory distress syndrome. Lancet 1992;339:466–9.[Medline]
  15. Simons R, Maier R, Chi E. Pulmonary effects of continuous endotoxin infusion in the rat. Circ Shock 1991;33:233–43.[Medline]
  16. Swails WS, Bell SJ, Bistrian BR, et al. Fish-oil-containing diet and platelet aggregation. Nutrition 1993;9:211–7.[Medline]
  17. Macnaughton PD, Evans TW. Management of adult respiratory distress syndrome. Lancet 1992;339:469–72.[Medline]
  18. Baird B, Cheronis J, Sandhaus R, Berger E, White C, Repine J. O2 metabolites and neutrophil elastase synergistically cause edematous injury in isolated rat lungs. J Appl Physiol 1986;61:2224–9.[Abstract/Free Full Text]
  19. Sasaki S, McCully JD, LoCicero J, Levitsky S, Forse RA, Palombo JD. Effects of endotoxin on pulmonary gas exchange, aerodynamic and hemodynamic function in an isolated blood-perfused rat lung model. Surg Forum 1994;45:286–8.
  20. McCord J. Human disease, free radicals, and the oxidant/antioxidant balance. Clin Biochem 1993;26:351–7.[Medline]
  21. Kilgore KS, Lucchesi BR. Reperfusion injury after myocardial infarction: the role of free radicals and the inflammatory response. Clin Biochem 1993;26:359–70.[Medline]

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