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Extended Preservation of Ischemic Pulmonary Graft by Postmortem Alveolar Expansion
Extended Preservation of Ischemic Pulmonary Graft by Postmortem Alveolar Expansion Dirk E. M. Van Raemdonck, MD, Nicole C. P. Jannis, Filip R. L. Rega, Paul R. J. De Leyn, MD, PhD, Willem J. Flameng, MD, PhD, and Toni E. Lerut, MD, PhD Center for Experimental Surgery and Anesthesiology, Katholieke Universiteit Leuven, Leuven, Belgium
Background. If lungs could be retrieved for transplantation from non– heart-beating cadavers, the shortage of donors might be significantly alleviated. Methods. Peak airway pressure, mean pulmonary artery pressure, pulmonary vascular resistance, and wet to dry weight ratio were measured during delayed hypothermic crystalloid flush in rabbit lungs (n 5 6) at successive intervals after death comparing cadavers with lungs left deflated (group 1), inflated with room air (group 2) or 100% oxygen (group 4), or ventilated with room air (group 3), or 100% nitrogen (group 5), or 100% oxygen (group 6). Results. There was a gradual increase in mean pulmonary artery pressure and pulmonary vascular resistance with longer postmortem intervals in all study groups (p 5 not significant, group 1 versus group 2 versus group 3). There was also a gradual increase in peak airway pressure and wet-to-dry weight ratio over time in all groups, which reflected edema formation during flush (airway pressure, from 14.5 6 1.0 cm H2O to 53.7 6 12.2 cm H2O, and wet-to-dry weight ratio, from 3.6 6 0.1
to 11.5 6 1.2, in group 1 at 0 and 6 hours postmortem, respectively; p < 0.05). Compared with group 1, however, the increase in groups 2 and 3 was much slower (airway pressure, 20.9 6 0.5 cm H2O and 18.8 6 1.2 cm H2O, and wet-to-dry weight ratio, 5.2 6 0.3 and 4.6 6 0.4 at 6 hours postmortem, respectively; p < 0.05 versus group 1 and p 5 not significant, group 2 versus group 3). Airway pressure and wet-to-dry weight ratio did not differ between groups 2 and 4 or between groups 3, 5, and 6. Conclusions. These data suggest that (1) pulmonary edema will develop in atelectatic lungs if hypothermic flush is delayed for 2 hours after death, (2) postmortem inflation is as good as ventilation in prolonging warm ischemic tolerance, (3) inflation with oxygen or ventilation with nitrogen or oxygen is no different from that with room air, and (4) therefore, prevention of alveolar collapse appears to be the critical factor in protecting the lung from warm ischemic damage independent of continued oxygen delivery. (Ann Thorac Surg 1997;64:801– 8) © 1997 by The Society of Thoracic Surgeons
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however, there will always be a certain delay between (unexpected) circulatory arrest and the start of cold in situ flush of the organs. In previous rabbit animal studies from our laboratory, we have investigated the effect of postmortem lung inflation, ventilation, and cooling on catabolism of adenine nucleotides [5] and pulmonary cell viability [6, 7]. We [8] also have looked at the effect of external cadaver cooling on pulmonary temperatures at intervals after death. The current study was undertaken to investigate pulmonary hemodynamic and aerodynamic changes during hypothermic flush at intervals after cardiac arrest. It was our aim to determine the effect of postmortem cadaver lung stretching by inflation and ventilation and to evaluate the effect of different gas mixtures during lung expansion in a rabbit NHBD model.
ung transplantation, like other forms of solid-organ transplantation, is limited by a scarcity of good donor organs. It is estimated that less than 10% of all available multiorgan donors have lungs suitable for transplantation [1]. With continued progress in organ transplantation, the demand for transplants and thus the need for organs have increased markedly. The result is a growing waiting time for a suitable organ and an augmented risk of premature death of patients listed for lung transplantation. To alleviate this critical organ shortage, there is growing interest in increasing the potential donor pool by turning to alternative sources such as the use of lobar or split transplants [2, 3], living-related donors [4], or organs from circulation-arrested cadavers, so called non– heartbeating donors (NHBDs). Rapid cooling of perfused organs by in situ flush with a cold crystalloid solution forms the basis of any solidorgan preservation before transplantation. In the NHBD,
Presented at the Thirty-third Annual Meeting of The Society of Thoracic Surgeons, San Diego, CA, Feb 3–5, 1997. Address reprint requests to Dr Van Raemdonck, Department of Thoracic Surgery, University Hospital Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium (e-mail: [email protected]).
© 1997 by The Society of Thoracic Surgeons Published by Elsevier Science Inc
Material and Methods Experimental Groups One hundred fifty New Zealand white rabbits (mean weight, 2,632 6 20 g) were sacrificed and left at room temperature (624°C). Cadavers were assigned to one of six groups of animals. Postmortem condition of the lungs inside the cadaver differed between groups. In group 1 0003-4975/97/$17.00 PII S0003-4975(97)00627-9
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Table 1. Time Intervals Between Cardiac Arrest and Pulmonary Flush in the Six Study Groups Postmortem Interval (min) Group 0: 1: 2: 3: 4: 5: 6: a
a
Immediate flush Deflatedb Inflated room air Ventilated room air Inflated 100% O2 Ventilated 100% N2 Ventilated 100% O2
Control heart-beating donor.
b
0
30
60
90
120
180
240
360
480
x ... ... ... ... ... ...
... x x x ... ... ...
... x x x ... ... ...
... x ... ... ... ... ...
... x x x ... ... ...
... x ... ... ... ... ...
... x x x x x x
... x x x ... ... ...
... ... x x x x x
Control non– heart-beating donor.
x 5 pulmonary flush.
(control NHBD), cadavers were left with lungs deflated (Defl) by disconnecting the endotracheal cannula from the ventilator, thus resulting in progressive atelectasis. In group 2, lungs were fully inflated with room air (Infl-RA) immediately after cardiac arrest by clamping the endotracheal cannula at end-tidal volume with end-expiratory pressure higher than 30 cm H2O. In group 3, the lungs were continuously ventilated with room air (Vent-RA) at a respiratory rate of 30 breaths/min, a tidal volume of 10 mL/kg of body weight, and a positive end-expiratory pressure of 2 cm H2O. In group 4, lungs were inflated with 100% oxygen (Infl-O2). In groups 5 and 6, lungs were ventilated with 100% nitrogen (Vent-N2) and 100% oxygen (Vent-O2), respectively. Cadaver lungs (n 5 6 at each time interval) were flushed after 0.5, 1, 1.5, 2, 3, 4, and 6 hours in group 1, after 0.5, 1, 2, 4, 6, and 8 hours in groups 2 and 3, and after 4 and 8 hours in groups 4, 5, and 6. Finally, in nonischemic animals (n 5 6), lungs were flushed immediately on insertion of the pulmonary artery catheter reflecting the situation in the heart-beating donor to derive control values at 0 hour (Table 1).
The pH of the flush solution was adjusted to 7.4 in all groups. Weight of the animals and temperature of the flush solution in all groups are listed in Table 2. Except for the weight in group 4 (Infl-O2) at 8 hours and the temperature of the flush solution at 4 hours in group 3 (Vent-RA) and at 6 hours in group 2 (Infl-RA), there were no significant differences between control and ischemic values at successive intervals after death within a group or between groups at any interval.
Animal Preparation All animals received humane care in compliance with the “Guide for the Care and Use of Laboratory Animals” published by the National Institutes of Health (NIH publication 85-23, revised 1985). Rabbits were premedicated and anesthetized by intramuscular injection with 0.25 mL/kg of Imalge`ne (50 mg/mL of ketamine hydrochloride; Rhoˆne Me´rieux, Lyon, France) and 0.15 mL/kg of Domitor (1 mg/mL of medetomidin-chlorhydrate 1 1 mg/mL of paramethylhydroxybenzoate 1 0.2 mg/mL of parapropylhydroxyben-
Table 2. Animal Weight and Temperature of Flush Solution in All Study Groupsa Postmortem Interval (min) Group
30
60
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360
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2,568 6 115 2,544 6 31 2,478 6 43 ... ... ...
2,577 6 131 2,493 6 46 2,447 6 64 ... ... ...
2,541 6 94 2,574 6 108 2,395 6 63 ... ... ...
2,598 6 140 2,558 6 56 2,770 6 95 2,820 6 103 2,699 6 109 2,586 6 11
2,560 6 89 2,650 6 78 2,779 6 122 ... ... ...
... 2,728 6 148 2,865 6 67 2,846 6 98c 2,661 6 133 2,712 6 71
5.0 6 0.3 4.0 6 0.4 4.1 6 0.3 ... ... ...
3.6 6 0.2 3.9 6 1.1 3.0 6 0.3 ... ... ...
4.2 6 0.4 4.4 6 0.1 3.6 6 0.3 ... ... ...
3.5 6 0.2 3.6 6 0.2 2.2 6 0.4c 3.6 6 0.2 3.8 6 0.3 4.6 6 1.3
3.5 6 0.2 2.7 6 0.1e 3.8 6 0.1 ... ... ...
... 3.7 6 0.3 4.2 6 0.4 3.7 6 0.1 3.6 6 0.4 4.2 6 1.1
b
Weight (g) 1: Deflated 2: Inflated room air 3: Ventilated room air 4: Inflated 100% O2 5: Ventilated 100% N2 6: Ventilated 100% O2 Temperature (°C)d 1: Deflated 2: Inflated room air 3: Ventilated room air 4: Inflated 100% O2 5: Ventilated 100% N2 6: Ventilated 100% O2
b Data are shown as the mean 6 the standard error of the mean from six experiments. The control (preischemic) value is 2,491 6 c d Significance: p , 0.05 versus control value (analysis of variance with repeated measurements). The control (preischemic) value is 4.0° 6 85 g. e Significance: p , 0.05 versus other groups (analysis of variance with factorial analysis). 0.4°C. a
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zoate; Orion Corporation, Farmos, Espoo, Finland). The animals were intubated using a cannula with a 3.5-mm inner diameter (Mallinckrodt Medical, Athlone, Ireland) through a cervical tracheostomy, and the lungs were ventilated using a Harvard rodent ventilator (model 683; Harvard Apparatus, Inc, South Natick, MA) with room air (respiratory rate, 30 breaths/min; tidal volume, 10 mL/kg of body weight; positive end-expiratory pressure, 2 cm H2O). The chest was opened through a median sternotomy. Thymic tissue was excised. Pleural cavities were opened. Both superior caval veins, the inferior caval vein, the ascending aorta, and the main pulmonary artery were encircled by individual ligatures. Heparin Novo, 700 IU/kg (sodium heparin, 5,000 IU/mL; Novo Nordisk, Bagsvaerd, Denmark), was administered through a marginal ear vein. The main pulmonary artery was cannulated through the right ventricular outflow tract using a 10-gauge catheter (Angiocath; Becton Dickinson Vascular Access, Sandy, UT). The pulmonary artery was isolated from the right ventricle by a ligature around the tip of the catheter just distal to the pulmonary valve, creating pulmonary ischemia. The animal was then sacrificed by ligating the ascending aorta and caval veins, which resulted in cardiac arrest. Both the endotracheal cannula and the pulmonary artery catheter remained in place until pulmonary flush. The cadaver was left at room temperature with sternal edges reapproximated using towel clips. The whole procedure was carried out under clean but not sterile conditions. In preliminary experiments, rabbits were sacrificed by intravenous injection through a marginal ear vein with 100 mg/kg of Nembutal (60 mg/mL of sodium pentobarbital; Abbott Laboratories, North Chicago, IL) inducing immediate cardiac arrest. This resulted in massive edema formation during pulmonary flush, even in control animals without ischemia (unpublished results). The edema was attributed to a direct pulmonotoxic effect of pentobarbital on the pulmonary vasculature. This method to sacrifice the animal was therefore abandoned.
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Bonaduz, Switzerland) connected to a pH meter (WTW pH-91, Weilheim, Germany), respectively. At the end of the flush, the heart-lung block was excised, and the pulmonary artery catheter and endotracheal cannula were removed, collecting the endotracheal edema fluid.
Assessment of Graft Function Immediately before the flush, the endotracheal cannula and the pulmonary artery catheter were connected to pressure transducers (Uniflow type 43-600F; Baxter, Uden, the Netherlands) and zeroed at this level. During the flush, peak airway pressure (AwP) and mean pulmonary artery pressure (mPAP) were continuously measured and recorded with an amplifier (Carrier amplifier AP-601G; Nihon Kohden, Tokyo, Japan) on a fourchannel recorder (heat writing recorder model WT-645G; Nihon Kohden). Flushing time (306 6 7 seconds) was recorded using a stopwatch (Hanhart Profile 1; Germany). Pulmonary vascular resistance (PVR) was calculated using the following formula: PVR 5 mean pulmonary artery flush pressure/(flush volume/flush time). At the end of the flush, the heart-lung block was weighed and dried in an oven (model HT 600; Heraeus, Hanau, Germany) at 150°C overnight to constant weight. Wet-to-dry weight ratio (W/D) was calculated as an estimate of the extent of lung edema.
Statistical Analysis Data are presented as the mean 6 the standard error of the mean. Differences within a group between control and ischemic values at successive intervals after death were calculated using one-way analysis of variance with repeated measurements followed by Scheffe´’s multiple comparison test [9]. Differences between groups at the same postmortem interval were compared using analysis of variance with factorial analysis (StatView SE1 Graphics [Abacus Concepts Inc, Berkeley, CA] on a Macintosh Performa 630 computer). Values of p less than 0.05 were accepted as significant.
Pulmonary Flush
Results
Both lungs were flushed through the pulmonary artery catheter by gravitational pressure at 30 cm H2O with 200 mL (680 mL/kg) of cold (3.7° 6 0.1°C) modified Krebs-Henseleit bicarbonate buffer (KHBB) solution (composition in millimoles per liter: NaCl, 118; NaHCO3, 25; KCl, 5.6; CaCl2, 2.9; MgCl2, 0.6; NaH2PO4, 1.2; and d-glucose, 11; pH, 7.4; osmolarity, 321 mOsm/L). The tip of the left atrial appendage was transected to allow free drainage of the flush solution prior to the start of the flush. During the flush, the lungs were ventilated with room air (respiratory rate, 30 breaths/min; tidal volume, 10 mL/kg of body weight; positive end-expiratory pressure, 2 cm H2O). The temperature and the pH of the KHBB solution were noted at the start of the flush using a rectal probe (type AR 1; Ellab Rødovre, Denmark) connected to a five-channel digital thermometer (Ellab, Copenhagen, Denmark) and a pH electrode (Hamilton Liq-Glass,
Values for mPAP, PVR, AwP, and W/D in all study groups are presented in Table 3. There was a gradual increase in both mPAP and PVR with longer ischemic intervals in all groups (mPAP, from 9.5 6 0.6 mm Hg to 15.2 6 1.7 mm Hg, and PVR, from 16.7 6 1.2 mm Hg z mL21 z s21 to 25.2 6 5.4 mm Hg z mL21 z s21, in group 1 [Defl] at 0 and 6 hours postmortem, respectively; p , 0.05). No significant differences were observed at any interval between groups 1 (Defl), 2 (Infl-RA), and 3 (Vent-RA), between groups 2 and 4 (Infl-O2), or between groups 3, 5 (Vent-N2), and 6 (Vent-O2) (see Table 3). Figure 1 shows AwP in groups 1 (Defl), 2 (Infl-RA), and 3 (Vent-RA). There was a sharp and constant increase in AwP in group 1 starting 2 hours after circulatory arrest (from 14.5 6 1.0 cm H2O [control value] to 53.7 6 12.2 cm H2O at 6 hours postmortem; p , 0.05). In contrast, the increase in AwP in groups 2 and 3 was much slower (20.9 6 0.5 cm H2O and 18.8 6 1.2 cm H2O at 6 hours after
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Table 3. Graft Variables at End of Cold Flush in All Study Groupsa Postmortem Interval (min) Group
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60
120
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10.8 6 0.3 12.0 6 0.5 10.8 6 0.3 ... ... ...
11.5 6 0.7 11.0 6 0.4 11.5 6 0.3 ... ... ...
10.5 6 0.6 12.0 6 1.1 11.8 6 0.3 ... ... ...
12.3 6 1.2 13.7 6 0.2c 13.0 6 0.6 12.7 6 0.5c,d 13.5 6 0.7c,d 12.7 6 1.1c,d
15.2 6 1.7c 14.2 6 0.7c 14.8 6 0.8c ... ... ...
... 15.3 6 0.5c 16.5 6 1.4c 14.3 6 0.3c,d 16.5 6 1.0c,d 16.5 6 0.8c,d
14.0 6 0.6 14.7 6 0.9 13.2 6 0.8 ... ... ...
17.3 6 1.1 13.5 6 0.8 14.3 6 0.6 ... ... ...
15.4 6 1.2 17.8 6 3.3 14.5 6 0.4 ... ... ...
17.6 6 1.6 19.8 6 0.6 17.6 6 1.3 17.4 6 1.2d 19.4 6 2.1d 20.4 6 3.8d
25.2 6 5.4c 23.5 6 1.9 24.6 6 2.8 ... ... ...
... 26.9 6 2.3c 36.5 6 7.2c 24.0 6 0.8c,d 34.7 6 4.1c,d 50.7 6 8.7c,d
15.0 6 0.8 15.2 6 0.6 16.1 6 0.4 ... ... ...
18.6 6 1.4 17.0 6 0.6 14.5 6 0.6g ... ... ...
21.8 6 2.5 15.0 6 0.6g 14.3 6 0.8g ... ... ...
36.0 6 8.3 18.8 6 0.8g 18.8 6 0.6g 16.3 6 0.5g 17.7 6 0.9d,g 18.6 6 0.9d,g
53.7 6 12.2c 20.9 6 0.5c,g 18.8 6 1.2g ... ... ...
... 21.1 6 1.6c 32.9 6 12.0c 21.5 6 1.1c,d 38.5 6 11.1c,d 26.3 6 5.7c,d
4.2 6 0.1 4.2 6 0.1 4.5 6 0.1 ... ... ...
3.7 6 0.1 4.3 6 0.1 4.7 6 0.1 ... ... ...
5.4 6 0.8 3.9 6 0.2 4.2 6 0.2 ... ... ...
6.9 6 0.7 4.1 6 0.1g 4.5 6 0.2g 4.4 6 0.4d,g 4.1 6 0.1d,g 4.8 6 0.4d,g
11.5 6 1.2c 5.2 6 0.3c,g 4.6 6 0.4g ... ... ...
... 5.6 6 0.4c 6.2 6 1.1c 5.2 6 0.5c,d 7.7 6 1.5c,d 7.2 6 1.5c,d
b
mPAP (mm Hg) 1: Deflated 2: Inflated room air 3: Ventilated room air 4: Inflated 100% O2 5: Ventilated 100% N2 6: Ventilated 100% O2 PVR (mm Hg z mL21 z s21)e 1: Deflated 2: Inflated room air 3: Ventilated room air 4: Inflated 100% O2 5: Ventilated 100% N2 6: Ventilated 100% O2 AwP (cm H2O)f 1: Deflated 2: Inflated room air 3: Ventilated room air 4: Inflated 100% O2 5: Ventilated 100% N2 6: Ventilated 100% O2 W/Dh 1: Deflated 2: Inflated room air 3: Ventilated room air 4: Inflated 100% O2 5: Ventilated 100% N2 6: Ventilated 100% O2
b Data are shown as the mean 6 the standard error of the mean from six experiments. The control (preischemic) value is 9.5 6 c d Significance: p , 0.05 versus control value (analysis of variance with repeated measurements). Significance: p 5 not significant, 0.6 mm Hg. e The control (preischemic) value is 16.7 6 group 2 versus group 4 and group 3 versus groups 5 and 6 (analysis of variance with factorial analysis). 21 21 f g h z s . The control (preischemic) value is 14.5 6 1.0 cm H2O. Significance: p , 0.05 versus group 1. The control 1.2 mm Hg z mL (preischemic) value is 3.6 6 0.1. a
AwP 5 peak airway pressure; ratio.
mPAP 5 mean pulmonary artery pressure;
death, respectively; p , 0.01 for group 2 and p 5 not significant for group 3 versus control value). The difference between groups 1 and 3 was significant starting at 1 hour and between groups 1 and 2, at 2 hours (see Fig 1). No significant differences were observed between groups 2 and 3 at any interval. In group 4 (Infl-O2), AwP was lower at 4 hours compared with group 2 (p , 0.05), but no significant difference was seen between groups 2 and 4 at 8 hours or between groups 3, 5 (Vent-N2), and 6 (VentO2) at 4 and 8 hours (Fig 2). Figure 3 shows the W/D in groups 1 (Defl), 2 (Infl-RA), and 3 (Vent-RA). There was a sharp and constant increase in W/D in group 1 starting 2 hours after cessation of circulation (from 3.6 6 0.1 [control value] to 11.5 6 1.2 at 6 hours postmortem; p , 0.001). In contrast, the increase in W/D in groups 2 and 3 was much slower (5.2 6 0.3 and 4.6 6 0.4 at 6 hours postmortem, respectively; p , 0.01 for group 2 and p 5 not significant for group 3 versus control value). The differences between group 1 and groups 2 and 3 were highly significant (p ,
PVR 5 pulmonary vascular resistance;
W/D 5 wet-to-dry weight
0.01 at 4 hours and p , 0.001 at 6 hours) (see Fig 3). No significant differences were observed between groups 2 and 3 at any interval, between groups 2 and 4 (Infl-O2), or between groups 3, 5 (Vent-N2), and 6 (Vent-O2) at 4 and 8 hours (Fig 4).
Comment Rapid cooling of perfused organs by in situ flush with a cold crystalloid solution forms the basis of any solidorgan preservation prior to transplantation. Lungs preserved in this way can be transplanted safely after up to 6 to 8 hours of cold ischemia. In the NHBD, however, there will always be a certain delay between (unexpected) circulatory arrest and the start of cold in situ flush of the organs. This critical interval, so-called warm ischemia, will rapidly lead to tissue and cellular damage [10, 11]. This might result in organ dysfunction jeopardizing the transplant recipient and remains the most difficult factor to overcome, especially after lung transplantation.
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Fig 1. Peak airway pressure (mean 6 standard error of the mean) in rabbit lungs (n 5 6) at end of cold (64°C) crystalloid flush at intervals after death comparing lungs left deflated (Defl) versus lungs inflated with room air (Infl-RA), versus lungs ventilated with room air (Vent-RA). (* 5 p , 0.05, Infl-RA [except 60 minutes] and Vent-RA versus Defl by analysis of variance; ∧ 5 p , 0.05 versus control value; ∧∧ 5 p , 0.01 versus control value.)
Indeed, besides extracorporeal membrane oxygenation, no valid alternative organ replacement therapy is available today in case of primary nonfunction of the pulmonary allograft. The period of inevitable warm ischemia in the NHBD, therefore, should be kept as short as possible. However, organizing organ retrieval and obtaining family consent for organ donation consume precious time. During this interval, organs have to be protected against cellular autolysis by preservation inside the dead body. The clinical use of lungs from NHBDs is still anecdotal [12, 13]. Nevertheless, transplantation of lungs retrieved from cadavers after cardiac arrest has been investigated in an
Fig 2. Peak airway pressure (mean 6 standard error of the mean) in rabbit lungs (n 5 6) at end of cold (64°C) crystalloid flush 4 and 8 hours after death comparing lungs inflated with room air (Infl-RA) versus lungs inflated with 100% oxygen (Infl-O2) and lungs ventilated with room air (Vent-RA), versus lungs ventilated with 100% nitrogen (Vent-N2), versus lungs ventilated with 100% oxygen (Vent-O2). (N.S. 5 not significant by analysis of variance; p 5 p , 0.05.)
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Fig 3. Wet-to-dry weight ratio (mean 6 standard error of the mean) in rabbit lungs (n 5 6) after cold (64°C) crystalloid flush at intervals after death comparing lungs left deflated (Defl) versus lungs inflated with room air (Infl-RA) versus lungs ventilated with room air (Vent-RA). (pp 5 p , 0.01, Infl-RA and Vent-RA versus Defl by analysis of variance; ppp 5 p , 0.001, Infl-RA and Vent-RA versus Defl by analysis of variance; ∧ 5 p , 0.05 versus control value; ∧∧ 5 p , 0.01 versus control value; ∧∧∧ 5 p , 0.001 versus control value.)
increasing number of animal transplant experiments during recent years [14 –19]. In the present study, we wanted to investigate graft function during delayed hypothermic crystalloid pulmonary flush at intervals after death. A gradual increase in mPAP and PVR over time was seen in all study groups. No intergroup differences were observed. It is well known that PVR is increased on reperfusion of the
Fig 4. Wet-to-dry weight ratio (mean 6 standard error of the mean) in rabbit lungs (n 5 6) after cold (64°C) crystalloid flush 4 and 8 hours after death comparing lungs inflated with room air (Infl-RA) versus lungs inflated with 100% oxygen (Infl-O2) and lungs ventilated with room air (Vent-RA) versus lungs ventilated with 100% nitrogen (Vent-N2) versus lungs ventilated with 100% oxygen (Vent-O2). (N.S. 5 not significant by analysis of variance.)
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allograft after a period of pulmonary ischemia [20]. The exact mechanism remains unclear. Hypoxic vasoconstriction, mediator-induced vasospasm, and microvascular plugging of blood elements have all been recognized as possible causes of increased resistance. Increased microvascular hydrostatic pressure and postischemic endothelial damage may then lead to permeability pulmonary edema and clinically apparent acute graft dysfunction after reperfusion [21]. In fact, these delayed flush experiments after a period of warm ischemia could be seen as blood-free reperfusion experiments, and therefore the findings could be interpreted in the same way. We observed a significant difference in AwP and W/D after hypothermic flush of lungs already subjected to a period of warm ischemia when comparing postmortem expanded versus nonexpanded lungs. The increase in group 1 (Defl) became evident as early as 2 hours after circulatory arrest. This suggests that warm ischemic tolerance in the atelectatic lung is limited to 690 minutes. Tolerance to warm ischemia could be extended to 4 hours if the lungs remained inflated or ventilated. No differences in W/D were found at 4 and 8 hours after death between postmortem inflation with room air (group 2 [Infl-RA]) versus oxygen (group 4 [Infl-O2]) and ventilation with room air (group 3 [Vent-RA]) versus nitrogen (group 5 [Vent-N2]) and oxygen (group 6 [VentO2]). In a study conducted by Ulicny and co-workers [15], no difference in early gas exchange was seen after canine single-lung transplantation and hilar occlusion of the native lung after a 4-hour period of postmortem donor ventilation with either 100% oxygen or 100% nitrogen. The authors therefore concluded, as we did in the present study, that the mechanics of ventilation after cessation of circulation appear to confer a functional advantage independent of a continued supply of oxygen. However, in a subsequent identical transplant study [19], postmortem ventilation with alveolar gas was inferior to ventilation with 100% oxygen. Further rat experiments by the same group showed that adenine nucleotides were well preserved after postmortem oxygen but not nitrogen ventilation [22], and this correlated well with light microscopy pulmonary cell viability quantified by pulmonary artery infusion with trypan blue vital dye [10]. This latter study suggested that a continued oxygen supply, not ventilation itself, is important to maintain aerobic metabolism and prevent cellular damage. We [6] also looked at rabbit pulmonary cell viability using trypan blue dye exclusion and came to the same conclusions. On the other hand, Koyama and colleagues [23] compared the effect of postmortem ventilation with room air versus oxygen versus nitrogen in a canine isolated reperfusion model and reported superior function in lungs ventilated with nitrogen. The authors concluded that reperfusion injury seen in oxygenventilated animals was mediated by oxygen free radicals. Briefly, conflicting data have been reported from metabolic, morphologic, and functional studies. The ideal gas mixture for delivery to ischemic pulmonary grafts during cadaveric storage remains an open question. We prefer to use KHBB as flush solution, a crystalloid
Ann Thorac Surg 1997;64:801– 8
with electrolytes of extracellular composition, without the addition of a prostanoid as vasodilator to examine solely the effect of increasing periods of warm ischemia on vascular resistance during pulmonary flush. It is well known that the high potassium concentration of an intracellular type of flush solution such as Euro-Collins [24] or University of Wisconsin [25] induces pulmonary vasoconstriction. In preliminary experiments, we also observed higher PVR in control animals (n 5 8) during flush with Euro-Collins solution compared with KHBB solution (52.2 6 7.6 mm Hg z mL21 z s21 versus 13.2 6 1.0 mm Hg z mL21 z s21, respectively; p , 0.0001). The absence of a high-molecular-weight osmotic-impermeant component in KHBB solution might have aggravated pulmonary edema in ischemic lungs. However, no significant difference in W/D was observed in an additional group of cadavers (n 5 7) with deflated lungs flushed 4 hours postmortem with low-potassium– dextran solution, a crystalloid with similar electrolyte composition as KHBB but with dextran 40 as an impermeant, compared with KHBB (6.0 6 0.6 versus 6.9 6 0.6, respectively; p 5 not significant). D’Armini and co-workers [26] investigated W/D in rat lungs ventilated postmortem with 100% oxygen for 4 hours followed by flush with modified Euro-Collins, University of Wisconsin, or Carolina rinse solution. The lungs were then fully expanded and stored in the same cold solution for another 4 hours. The W/D in lungs flushed with and stored in University of Wisconsin solution was significantly lower, with a value similar to that of fresh tissue. Another potential criticism of this NHBD model is the use of heparin in the donors before sacrifice [17]. We elected to heparinize the animals to exclude intravascular thrombosis as a cause of poor lung function during delayed hypothermic flush. In a supplemental group of nonheparinized animals (n 5 6), deflated lungs were flushed in an identical manner 4 hours after death. The PVR at the end of flush was significantly higher compared with that in heparinized animals (23.4 6 0.7 mm Hg z mL21 z s21 versus 17.6 6 1.6 mm Hg z mL21 z s21; p , 0.05). No significant differences, however, were observed in mPAP, AwP, and W/D between both groups. Finally, we also compared the effect of cold (3.5° 6 0.2°C) versus warm (22.5° 6 0.1°C) flush with KHBB solution in deflated lungs (n 5 6) 4 hours postmortem. No significant differences in mPAP, AwP, and W/D were found between groups. However, PVR was significantly higher after hypothermic flush (17.6 6 1.6 mm Hg z mL21 z s21 versus 12.1 6 1.2 mm Hg z mL21 z s21; p , 0.05). In this study, we measured relatively gross estimates of lung function during a very short period (65 minutes). Assessment of gas exchange at reperfusion with deoxygenated blood is probably the most reliable variable to evaluate the quality of lung preservation and permits clear differentiation between well-preserved and poorly preserved lungs. We have used these flush experiments as a rapid screening method to define the length of tolerable warm ischemia in all study groups. In later experiments (unpublished results) comparing the same study groups, we have investigated the effect of 4 hours’
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in situ warm ischemia in a rabbit isolated, pressurelimited, homologous blood reperfusion and room air– ventilated model (n 5 4 in each group). Arteriovenous oxygen pressure gradient after 1 hour of reperfusion was only 9 6 5 mm Hg in cadaver lungs that were left deflated during the ischemic interval versus 95 6 13 mm Hg in lungs that were inflated with room air, 96 6 7 mm Hg in lungs that were ventilated with room air, and 96 6 4 mm Hg in lungs ventilated with 100% nitrogen (p , 0.05, Defl versus all other groups; p 5 not significant, Infl-RA versus Vent-RA versus Vent-N2). This study therefore validates the conclusion of the present study that the prevention of postmortem alveolar collapse itself will confer a functional advantage independent of continued supply of oxygen. The exact mechanism of extended warm ischemic tolerance of the lung by preventing the alveolar space to collapse remains unclear. Release of surfactant from type II pneumocytes is known to be stimulated by inflation [27] and ventilation of lungs [28] and by mechanical stretch of isolated pneumocytes in culture [29]. Alveolar surfactant activity is reduced in the atelectatic lung [30]. Although no measurements of surfactant or surfactant activity were made in the present study, it is reasonable to speculate that repetitive or continuous alveolar expansion during warm ischemia in our study groups may have stimulated the release of pulmonary surfactant, thereby decreasing the alveolar surface tension, preventing damage to the alveolar-capillary membrane, and protecting against permeability pulmonary edema during flush [21]. From this study, we can conclude that in the NHBD (1) pulmonary edema will develop in atelectatic lungs if hypothermic flush is delayed for 2 hours after death, (2) postmortem inflation is as good as ventilation in protecting the lung against edema formation, thereby prolonging warm ischemic tolerance up to 4 hours, (3) postmortem inflation with oxygen or ventilation with nitrogen or oxygen is no different from that with room air, and (4) therefore, prevention of alveolar collapse appears to be the critical factor in protecting the lung from warm ischemic damage independent of continued oxygen delivery. Further studies are necessary to investigate whether lungs from human NHBDs will become a realistic alternative to expand the pulmonary donor pool. This work is supported by grant 7.0036.94 from the Nationaal Fonds voor Wetenschappelijk onderzoek—Levenslijn 1994. We thank Peter Lemmens, Magda Mathys, Kanigula Mubagwa, MD, and Anne Vancauwenbergh for expert technical and secretarial assistance.
References 1. Van Raemdonck D, Roels L, Verleden G, et al. Whence the lungs? Assessment of the use of lungs for transplantation from 156 consecutive donors. In: Cooper JD, Weder W, eds. Proceedings of the third international lung transplant symposium (Zurich, Switzerland, June 24 –25, 1993). 1993:40. 2. Starnes VA, Barr ML, Cohen RG. Lobar transplantation. Indications, technique, and outcome. J Thorac Cardiovasc Surg 1994;108:403–11.
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3. Couetil JPA, Tolan MJ, Loulmet DF, et al. Pulmonary bipartitioning and lobar transplantation. A new approach to donor organ shortage. J Thorac Cardiovasc Surg 1997;113: 529–37. 4. Starnes VA, Barr ML, Cohen RG, et al. Living-donor lobar lung transplantation experience. Intermediate results. J Thorac Cardiovasc Surg 1996;112:1284–91. 5. Van Raemdonck DEM, Jannis NCP, Rega FRL, De Leyn PRJ, Flameng WJ, Lerut TE. Delay of adenosine triphosphate depletion and hypoxanthine formation in rabbit lung after death. Ann Thorac Surg 1996;62:233– 41. 6. Kuang JQ, Van Raemdonck DEM, Jannis NCP, et al. Pulmonary cell death in warm ischemic rabbit lung is related to the postmortem alveolar oxygen reserve [Abstract]. J Heart Lung Transplant 1997;16:99. 7. Kuang JQ, Van Raemdonck DEM, Jannis NCP, et al. Pulmonary grafts should be inflated with 100% oxygen prior to hypothermic storage [Abstract]. J Heart Lung Transplant 1997;16:99. 8. Van Raemdonck DEM, Jannis NCP, Rega FRL, De Leyn PRJ, Flameng WJ, Lerut TE. External cooling of warm ischemic rabbit lungs after death. Ann Thorac Surg 1996;62:331–7. 9. Scheffe´ HA. A method for judging all contrast in the analysis of variance. Biometrika 1953;40:87–104. 10. D’Armini AM, Roberts CS, Griffith PK, Lemasters JJ, Egan TM. When does the lung die? I. Histochemical evidence of pulmonary viability after “death.” J Heart Lung Transplant 1994;13:741–7. 11. Alessandrini F, D’Armini AM, Roberts CS, Reddick RL, Egan TM. When does the lung die? II. Ultrastructural evidence of pulmonary viability after “death.” J Heart Lung Transplant 1994;13:748–57. 12. D’Alessandro AM, Hoffmann RM, Knechtle SJ, et al. Successful extrarenal transplantation from non– heart-beating donors. Transplantation 1995;59:977– 82. 13. Love RB, Stringham JC, Chomiak PN, Warner T, Pellett JR, Mentzer RM. Successful lung transplantation using a non– heart-beating donor [Abstract]. J Heart Lung Transplant 1995;14:S88. 14. Egan TM, Lambert CJ Jr, Reddick R, Ulicny KS Jr, Keagy BA, Wilcox BR. A strategy to increase the donor pool: use of cadaver lungs for transplantation. Ann Thorac Surg 1991;52: 1113–21. 15. Ulicny KS Jr, Egan TM, Lambert CJ Jr, Reddick RL, Wilcox BR. Cadaver lung donors: effect of preharvest ventilation on graft function. Ann Thorac Surg 1993;55:1185–91. 16. Buchanan SA, DeLima NF, Binns OAR, et al. Pulmonary function after non– heart-beating lung donation in a survival model. Ann Thorac Surg 1995;60:38– 46. 17. Umemori Y, Date H, Uno K, Aoe M, Ando A, Shimizu N. Improved lung function by urokinase infusion in canine lung transplantation using non– heart-beating donors. Ann Thorac Surg 1995;59:1513– 8. 18. Roberts CS, D’Armini AM, Egan TM. Canine double-lung transplantation with cadaveric donors. J Thorac Cardiovasc Surg 1996;112:577– 83. 19. Hennington MH, D’Armini AM, Lemasters JJ, Egan TM. Cadaver lungs for transplantation. Effect of ventilation with alveolar gas. Transplantation 1996;61:1009–14. 20. Allison RC, Kyle J, Adkins WK, Prasad VR, McCord JM, Taylor AE. Effect of ischemia reperfusion or hypoxia reoxygenation on lung vascular permeability and resistance. J Appl Physiol 1990;69:597– 603. 21. Novick RJ, Gehman KE, Ali IS, Lee J. Lung preservation: the importance of endothelial and alveolar type II cell integrity. Ann Thorac Surg 1996;62:302–14. 22. D’Armini AM, Tom EJ, Roberts CS, Henke DC, Lemasters JJ, Egan TM. When does the lung die? Time course of high energy phosphate depletion and relationship to lung viability after death. J Surg Res 1995;59:468–74. 23. Koyama I, Toung TJK, Rogers MC, Gurtner GH, Traystman RJ. O2 radicals mediate reperfusion lung injury in ischemic O2ventilated canine pulmonary lobe. J Appl Physiol 1987;63:111–5.
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24. Yamazaki F, Yokomise H, Keshavjee SH, et al. The superiority of an extracellular fluid solution over Euro-Collins’ solution for pulmonary preservation. Transplantation 1990; 49:690– 4. 25. 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. 26. D’Armini AM, Lemasters JJ, Egan TM. Studies of rat lung viability and adenine nucleotide metabolism after death. Ann Thorac Surg 1996;62:1448–53.
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27. Faridy EE. Effect of distension on release of surfactant in excised dogs’ lungs. Respir Physiol 1976;27:99 –114. 28. Oyarzun MJ, Clements JA. Control of lung surfactant by ventilation, adrenergic mediators, and prostaglandins in the rabbit. Am Rev Respir Dis 1978;117:879–91. 29. Wirtz HRW, Dobbs LG. Calcium mobilization and exocytosis after one mechanical stretch of lung epithelial cells. Science 1990;250:1266–9. 30. Oyarzun MJ, Stevens P, Clements JA. Effect of lung collapse on alveolar surfactant in rabbits subjected to unilateral pneumothorax. Exp Lung Res 1989;15:909–24.
DISCUSSION DR THOMAS M. EGAN (Chapel Hill, NC): That was a very nice study, and it adds to the body of literature supporting the hypothesis that lungs may be useful for transplantation even at intervals after death. I am a little curious about the group of lungs that were fully deflated. I gather from your manuscript that these lungs were deflated because, as part of the preparation, you opened both pleural spaces. I wonder if another control group might have been useful as well, that is, lungs maintained at functional residual capacity, because a fully deflated lung does not really mimic the clinical scenario of a cadaver unless the body dies of bilateral stab wounds to the chest. Do you think there would have been any difference had you studied lungs in a noninflated state, but lungs that were allowed to maintain volume at functional residual capacity? As I have said, this was an interesting study. It does suffer from one serious limitation, and that is that the duration of your perfusion was relatively short, and no gas exchange data could be accrued. DR VAN RAEMDONCK: Thank you, Dr Egan, for your kind remarks. It is well appreciated that your group has revived interest in the use of lungs from non– heart-beating donors. In regard to your first question about the group with deflated lungs, it is true that the lungs were deflated because we opened both pleural spaces after sternotomy. We started doing these experiments by killing the animals with an overdose of pentobarbital, and we observed that even in the control animals without any ischemia, we had a massive amount of lung edema. That is why we started to look for another method to kill the rabbits. Indeed, these lungs were not at functional residual capacity but below functional residual capacity. Before sacrifice, the lungs were ventilated with room air; as you know, room air is a gas mixture of 21% oxygen and 79% nitrogen. These lungs will not become fully deflated to the complete zero level as nitrogen
will not be absorbed. But indeed, as you suggested, we should study another group of animals with lungs at functional residual capacity. In regard to your second comment, we studied a relatively gross estimate of lung function during a very short period; the Flush was only 5 minutes. Gas exchange after reperfusion with deoxygenated blood is probably the most reliable variable to study the quality of lung preservation and can distinguish between well-preserved and poorly preserved lungs. We first used these flush experiments as a screening model to rapidly define the length of tolerable warm ischemia in all study groups. We have now extended these rabbit experiments in an isolated, pressure-limited, and homologous deoxygenated blood reperfusion and room air–ventilated model using the same study groups. After an ischemic period of 4 hours, we compared lungs that were deflated versus lungs that were inflated with room air and lungs ventilated with room air, and we looked at oxygenation capacity, defined as the arteriovenous oxygen pressure gradient. During a reperfusion period of 4 hours, there was a highly significant difference in oxygenation capacity between the lungs that were inflated with room air and ventilated with room air versus the deflated lungs. Also, the wet to dry weight ratio in the deflated lungs after 4 hours of reperfusion with homologous blood was significantly higher than in the lungs inflated with room air and those ventilated with room air. However, there was no significant difference in oxygenation capacity between the group ventilated with room air and the group ventilated with nitrogen, nor was there a difference in wet to dry weight ratio between these same two groups. We think this study confirms the conclusion of our present study that alveolar expansion itself, not continued oxygen supply, represents a functional advantage in postmortem lung preservation.
Background. If lungs could be retrieved for transplantation from non– heart-beating cadavers, the shortage of donors might be significantly alleviated. Methods. Peak airway pressure, mean pulmonary artery pressure, pulmonary vascular resistance, and wet to dry weight ratio were measured during delayed hypothermic crystalloid flush in rabbit lungs (n 5 6) at successive intervals after death comparing cadavers with lungs left deflated (group 1), inflated with room air (group 2) or 100% oxygen (group 4), or ventilated with room air (group 3), or 100% nitrogen (group 5), or 100% oxygen (group 6). Results. There was a gradual increase in mean pulmonary artery pressure and pulmonary vascular resistance with longer postmortem intervals in all study groups (p 5 not significant, group 1 versus group 2 versus group 3). There was also a gradual increase in peak airway pressure and wet-to-dry weight ratio over time in all groups, which reflected edema formation during flush (airway pressure, from 14.5 6 1.0 cm H2O to 53.7 6 12.2 cm H2O, and wet-to-dry weight ratio, from 3.6 6 0.1
to 11.5 6 1.2, in group 1 at 0 and 6 hours postmortem, respectively; p < 0.05). Compared with group 1, however, the increase in groups 2 and 3 was much slower (airway pressure, 20.9 6 0.5 cm H2O and 18.8 6 1.2 cm H2O, and wet-to-dry weight ratio, 5.2 6 0.3 and 4.6 6 0.4 at 6 hours postmortem, respectively; p < 0.05 versus group 1 and p 5 not significant, group 2 versus group 3). Airway pressure and wet-to-dry weight ratio did not differ between groups 2 and 4 or between groups 3, 5, and 6. Conclusions. These data suggest that (1) pulmonary edema will develop in atelectatic lungs if hypothermic flush is delayed for 2 hours after death, (2) postmortem inflation is as good as ventilation in prolonging warm ischemic tolerance, (3) inflation with oxygen or ventilation with nitrogen or oxygen is no different from that with room air, and (4) therefore, prevention of alveolar collapse appears to be the critical factor in protecting the lung from warm ischemic damage independent of continued oxygen delivery. (Ann Thorac Surg 1997;64:801– 8) © 1997 by The Society of Thoracic Surgeons
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however, there will always be a certain delay between (unexpected) circulatory arrest and the start of cold in situ flush of the organs. In previous rabbit animal studies from our laboratory, we have investigated the effect of postmortem lung inflation, ventilation, and cooling on catabolism of adenine nucleotides [5] and pulmonary cell viability [6, 7]. We [8] also have looked at the effect of external cadaver cooling on pulmonary temperatures at intervals after death. The current study was undertaken to investigate pulmonary hemodynamic and aerodynamic changes during hypothermic flush at intervals after cardiac arrest. It was our aim to determine the effect of postmortem cadaver lung stretching by inflation and ventilation and to evaluate the effect of different gas mixtures during lung expansion in a rabbit NHBD model.
ung transplantation, like other forms of solid-organ transplantation, is limited by a scarcity of good donor organs. It is estimated that less than 10% of all available multiorgan donors have lungs suitable for transplantation [1]. With continued progress in organ transplantation, the demand for transplants and thus the need for organs have increased markedly. The result is a growing waiting time for a suitable organ and an augmented risk of premature death of patients listed for lung transplantation. To alleviate this critical organ shortage, there is growing interest in increasing the potential donor pool by turning to alternative sources such as the use of lobar or split transplants [2, 3], living-related donors [4], or organs from circulation-arrested cadavers, so called non– heartbeating donors (NHBDs). Rapid cooling of perfused organs by in situ flush with a cold crystalloid solution forms the basis of any solidorgan preservation before transplantation. In the NHBD,
Presented at the Thirty-third Annual Meeting of The Society of Thoracic Surgeons, San Diego, CA, Feb 3–5, 1997. Address reprint requests to Dr Van Raemdonck, Department of Thoracic Surgery, University Hospital Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium (e-mail: [email protected]).
© 1997 by The Society of Thoracic Surgeons Published by Elsevier Science Inc
Material and Methods Experimental Groups One hundred fifty New Zealand white rabbits (mean weight, 2,632 6 20 g) were sacrificed and left at room temperature (624°C). Cadavers were assigned to one of six groups of animals. Postmortem condition of the lungs inside the cadaver differed between groups. In group 1 0003-4975/97/$17.00 PII S0003-4975(97)00627-9
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Table 1. Time Intervals Between Cardiac Arrest and Pulmonary Flush in the Six Study Groups Postmortem Interval (min) Group 0: 1: 2: 3: 4: 5: 6: a
a
Immediate flush Deflatedb Inflated room air Ventilated room air Inflated 100% O2 Ventilated 100% N2 Ventilated 100% O2
Control heart-beating donor.
b
0
30
60
90
120
180
240
360
480
x ... ... ... ... ... ...
... x x x ... ... ...
... x x x ... ... ...
... x ... ... ... ... ...
... x x x ... ... ...
... x ... ... ... ... ...
... x x x x x x
... x x x ... ... ...
... ... x x x x x
Control non– heart-beating donor.
x 5 pulmonary flush.
(control NHBD), cadavers were left with lungs deflated (Defl) by disconnecting the endotracheal cannula from the ventilator, thus resulting in progressive atelectasis. In group 2, lungs were fully inflated with room air (Infl-RA) immediately after cardiac arrest by clamping the endotracheal cannula at end-tidal volume with end-expiratory pressure higher than 30 cm H2O. In group 3, the lungs were continuously ventilated with room air (Vent-RA) at a respiratory rate of 30 breaths/min, a tidal volume of 10 mL/kg of body weight, and a positive end-expiratory pressure of 2 cm H2O. In group 4, lungs were inflated with 100% oxygen (Infl-O2). In groups 5 and 6, lungs were ventilated with 100% nitrogen (Vent-N2) and 100% oxygen (Vent-O2), respectively. Cadaver lungs (n 5 6 at each time interval) were flushed after 0.5, 1, 1.5, 2, 3, 4, and 6 hours in group 1, after 0.5, 1, 2, 4, 6, and 8 hours in groups 2 and 3, and after 4 and 8 hours in groups 4, 5, and 6. Finally, in nonischemic animals (n 5 6), lungs were flushed immediately on insertion of the pulmonary artery catheter reflecting the situation in the heart-beating donor to derive control values at 0 hour (Table 1).
The pH of the flush solution was adjusted to 7.4 in all groups. Weight of the animals and temperature of the flush solution in all groups are listed in Table 2. Except for the weight in group 4 (Infl-O2) at 8 hours and the temperature of the flush solution at 4 hours in group 3 (Vent-RA) and at 6 hours in group 2 (Infl-RA), there were no significant differences between control and ischemic values at successive intervals after death within a group or between groups at any interval.
Animal Preparation All animals received humane care in compliance with the “Guide for the Care and Use of Laboratory Animals” published by the National Institutes of Health (NIH publication 85-23, revised 1985). Rabbits were premedicated and anesthetized by intramuscular injection with 0.25 mL/kg of Imalge`ne (50 mg/mL of ketamine hydrochloride; Rhoˆne Me´rieux, Lyon, France) and 0.15 mL/kg of Domitor (1 mg/mL of medetomidin-chlorhydrate 1 1 mg/mL of paramethylhydroxybenzoate 1 0.2 mg/mL of parapropylhydroxyben-
Table 2. Animal Weight and Temperature of Flush Solution in All Study Groupsa Postmortem Interval (min) Group
30
60
120
240
360
480
2,568 6 115 2,544 6 31 2,478 6 43 ... ... ...
2,577 6 131 2,493 6 46 2,447 6 64 ... ... ...
2,541 6 94 2,574 6 108 2,395 6 63 ... ... ...
2,598 6 140 2,558 6 56 2,770 6 95 2,820 6 103 2,699 6 109 2,586 6 11
2,560 6 89 2,650 6 78 2,779 6 122 ... ... ...
... 2,728 6 148 2,865 6 67 2,846 6 98c 2,661 6 133 2,712 6 71
5.0 6 0.3 4.0 6 0.4 4.1 6 0.3 ... ... ...
3.6 6 0.2 3.9 6 1.1 3.0 6 0.3 ... ... ...
4.2 6 0.4 4.4 6 0.1 3.6 6 0.3 ... ... ...
3.5 6 0.2 3.6 6 0.2 2.2 6 0.4c 3.6 6 0.2 3.8 6 0.3 4.6 6 1.3
3.5 6 0.2 2.7 6 0.1e 3.8 6 0.1 ... ... ...
... 3.7 6 0.3 4.2 6 0.4 3.7 6 0.1 3.6 6 0.4 4.2 6 1.1
b
Weight (g) 1: Deflated 2: Inflated room air 3: Ventilated room air 4: Inflated 100% O2 5: Ventilated 100% N2 6: Ventilated 100% O2 Temperature (°C)d 1: Deflated 2: Inflated room air 3: Ventilated room air 4: Inflated 100% O2 5: Ventilated 100% N2 6: Ventilated 100% O2
b Data are shown as the mean 6 the standard error of the mean from six experiments. The control (preischemic) value is 2,491 6 c d Significance: p , 0.05 versus control value (analysis of variance with repeated measurements). The control (preischemic) value is 4.0° 6 85 g. e Significance: p , 0.05 versus other groups (analysis of variance with factorial analysis). 0.4°C. a
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zoate; Orion Corporation, Farmos, Espoo, Finland). The animals were intubated using a cannula with a 3.5-mm inner diameter (Mallinckrodt Medical, Athlone, Ireland) through a cervical tracheostomy, and the lungs were ventilated using a Harvard rodent ventilator (model 683; Harvard Apparatus, Inc, South Natick, MA) with room air (respiratory rate, 30 breaths/min; tidal volume, 10 mL/kg of body weight; positive end-expiratory pressure, 2 cm H2O). The chest was opened through a median sternotomy. Thymic tissue was excised. Pleural cavities were opened. Both superior caval veins, the inferior caval vein, the ascending aorta, and the main pulmonary artery were encircled by individual ligatures. Heparin Novo, 700 IU/kg (sodium heparin, 5,000 IU/mL; Novo Nordisk, Bagsvaerd, Denmark), was administered through a marginal ear vein. The main pulmonary artery was cannulated through the right ventricular outflow tract using a 10-gauge catheter (Angiocath; Becton Dickinson Vascular Access, Sandy, UT). The pulmonary artery was isolated from the right ventricle by a ligature around the tip of the catheter just distal to the pulmonary valve, creating pulmonary ischemia. The animal was then sacrificed by ligating the ascending aorta and caval veins, which resulted in cardiac arrest. Both the endotracheal cannula and the pulmonary artery catheter remained in place until pulmonary flush. The cadaver was left at room temperature with sternal edges reapproximated using towel clips. The whole procedure was carried out under clean but not sterile conditions. In preliminary experiments, rabbits were sacrificed by intravenous injection through a marginal ear vein with 100 mg/kg of Nembutal (60 mg/mL of sodium pentobarbital; Abbott Laboratories, North Chicago, IL) inducing immediate cardiac arrest. This resulted in massive edema formation during pulmonary flush, even in control animals without ischemia (unpublished results). The edema was attributed to a direct pulmonotoxic effect of pentobarbital on the pulmonary vasculature. This method to sacrifice the animal was therefore abandoned.
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Bonaduz, Switzerland) connected to a pH meter (WTW pH-91, Weilheim, Germany), respectively. At the end of the flush, the heart-lung block was excised, and the pulmonary artery catheter and endotracheal cannula were removed, collecting the endotracheal edema fluid.
Assessment of Graft Function Immediately before the flush, the endotracheal cannula and the pulmonary artery catheter were connected to pressure transducers (Uniflow type 43-600F; Baxter, Uden, the Netherlands) and zeroed at this level. During the flush, peak airway pressure (AwP) and mean pulmonary artery pressure (mPAP) were continuously measured and recorded with an amplifier (Carrier amplifier AP-601G; Nihon Kohden, Tokyo, Japan) on a fourchannel recorder (heat writing recorder model WT-645G; Nihon Kohden). Flushing time (306 6 7 seconds) was recorded using a stopwatch (Hanhart Profile 1; Germany). Pulmonary vascular resistance (PVR) was calculated using the following formula: PVR 5 mean pulmonary artery flush pressure/(flush volume/flush time). At the end of the flush, the heart-lung block was weighed and dried in an oven (model HT 600; Heraeus, Hanau, Germany) at 150°C overnight to constant weight. Wet-to-dry weight ratio (W/D) was calculated as an estimate of the extent of lung edema.
Statistical Analysis Data are presented as the mean 6 the standard error of the mean. Differences within a group between control and ischemic values at successive intervals after death were calculated using one-way analysis of variance with repeated measurements followed by Scheffe´’s multiple comparison test [9]. Differences between groups at the same postmortem interval were compared using analysis of variance with factorial analysis (StatView SE1 Graphics [Abacus Concepts Inc, Berkeley, CA] on a Macintosh Performa 630 computer). Values of p less than 0.05 were accepted as significant.
Pulmonary Flush
Results
Both lungs were flushed through the pulmonary artery catheter by gravitational pressure at 30 cm H2O with 200 mL (680 mL/kg) of cold (3.7° 6 0.1°C) modified Krebs-Henseleit bicarbonate buffer (KHBB) solution (composition in millimoles per liter: NaCl, 118; NaHCO3, 25; KCl, 5.6; CaCl2, 2.9; MgCl2, 0.6; NaH2PO4, 1.2; and d-glucose, 11; pH, 7.4; osmolarity, 321 mOsm/L). The tip of the left atrial appendage was transected to allow free drainage of the flush solution prior to the start of the flush. During the flush, the lungs were ventilated with room air (respiratory rate, 30 breaths/min; tidal volume, 10 mL/kg of body weight; positive end-expiratory pressure, 2 cm H2O). The temperature and the pH of the KHBB solution were noted at the start of the flush using a rectal probe (type AR 1; Ellab Rødovre, Denmark) connected to a five-channel digital thermometer (Ellab, Copenhagen, Denmark) and a pH electrode (Hamilton Liq-Glass,
Values for mPAP, PVR, AwP, and W/D in all study groups are presented in Table 3. There was a gradual increase in both mPAP and PVR with longer ischemic intervals in all groups (mPAP, from 9.5 6 0.6 mm Hg to 15.2 6 1.7 mm Hg, and PVR, from 16.7 6 1.2 mm Hg z mL21 z s21 to 25.2 6 5.4 mm Hg z mL21 z s21, in group 1 [Defl] at 0 and 6 hours postmortem, respectively; p , 0.05). No significant differences were observed at any interval between groups 1 (Defl), 2 (Infl-RA), and 3 (Vent-RA), between groups 2 and 4 (Infl-O2), or between groups 3, 5 (Vent-N2), and 6 (Vent-O2) (see Table 3). Figure 1 shows AwP in groups 1 (Defl), 2 (Infl-RA), and 3 (Vent-RA). There was a sharp and constant increase in AwP in group 1 starting 2 hours after circulatory arrest (from 14.5 6 1.0 cm H2O [control value] to 53.7 6 12.2 cm H2O at 6 hours postmortem; p , 0.05). In contrast, the increase in AwP in groups 2 and 3 was much slower (20.9 6 0.5 cm H2O and 18.8 6 1.2 cm H2O at 6 hours after
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Table 3. Graft Variables at End of Cold Flush in All Study Groupsa Postmortem Interval (min) Group
30
60
120
240
360
480
10.8 6 0.3 12.0 6 0.5 10.8 6 0.3 ... ... ...
11.5 6 0.7 11.0 6 0.4 11.5 6 0.3 ... ... ...
10.5 6 0.6 12.0 6 1.1 11.8 6 0.3 ... ... ...
12.3 6 1.2 13.7 6 0.2c 13.0 6 0.6 12.7 6 0.5c,d 13.5 6 0.7c,d 12.7 6 1.1c,d
15.2 6 1.7c 14.2 6 0.7c 14.8 6 0.8c ... ... ...
... 15.3 6 0.5c 16.5 6 1.4c 14.3 6 0.3c,d 16.5 6 1.0c,d 16.5 6 0.8c,d
14.0 6 0.6 14.7 6 0.9 13.2 6 0.8 ... ... ...
17.3 6 1.1 13.5 6 0.8 14.3 6 0.6 ... ... ...
15.4 6 1.2 17.8 6 3.3 14.5 6 0.4 ... ... ...
17.6 6 1.6 19.8 6 0.6 17.6 6 1.3 17.4 6 1.2d 19.4 6 2.1d 20.4 6 3.8d
25.2 6 5.4c 23.5 6 1.9 24.6 6 2.8 ... ... ...
... 26.9 6 2.3c 36.5 6 7.2c 24.0 6 0.8c,d 34.7 6 4.1c,d 50.7 6 8.7c,d
15.0 6 0.8 15.2 6 0.6 16.1 6 0.4 ... ... ...
18.6 6 1.4 17.0 6 0.6 14.5 6 0.6g ... ... ...
21.8 6 2.5 15.0 6 0.6g 14.3 6 0.8g ... ... ...
36.0 6 8.3 18.8 6 0.8g 18.8 6 0.6g 16.3 6 0.5g 17.7 6 0.9d,g 18.6 6 0.9d,g
53.7 6 12.2c 20.9 6 0.5c,g 18.8 6 1.2g ... ... ...
... 21.1 6 1.6c 32.9 6 12.0c 21.5 6 1.1c,d 38.5 6 11.1c,d 26.3 6 5.7c,d
4.2 6 0.1 4.2 6 0.1 4.5 6 0.1 ... ... ...
3.7 6 0.1 4.3 6 0.1 4.7 6 0.1 ... ... ...
5.4 6 0.8 3.9 6 0.2 4.2 6 0.2 ... ... ...
6.9 6 0.7 4.1 6 0.1g 4.5 6 0.2g 4.4 6 0.4d,g 4.1 6 0.1d,g 4.8 6 0.4d,g
11.5 6 1.2c 5.2 6 0.3c,g 4.6 6 0.4g ... ... ...
... 5.6 6 0.4c 6.2 6 1.1c 5.2 6 0.5c,d 7.7 6 1.5c,d 7.2 6 1.5c,d
b
mPAP (mm Hg) 1: Deflated 2: Inflated room air 3: Ventilated room air 4: Inflated 100% O2 5: Ventilated 100% N2 6: Ventilated 100% O2 PVR (mm Hg z mL21 z s21)e 1: Deflated 2: Inflated room air 3: Ventilated room air 4: Inflated 100% O2 5: Ventilated 100% N2 6: Ventilated 100% O2 AwP (cm H2O)f 1: Deflated 2: Inflated room air 3: Ventilated room air 4: Inflated 100% O2 5: Ventilated 100% N2 6: Ventilated 100% O2 W/Dh 1: Deflated 2: Inflated room air 3: Ventilated room air 4: Inflated 100% O2 5: Ventilated 100% N2 6: Ventilated 100% O2
b Data are shown as the mean 6 the standard error of the mean from six experiments. The control (preischemic) value is 9.5 6 c d Significance: p , 0.05 versus control value (analysis of variance with repeated measurements). Significance: p 5 not significant, 0.6 mm Hg. e The control (preischemic) value is 16.7 6 group 2 versus group 4 and group 3 versus groups 5 and 6 (analysis of variance with factorial analysis). 21 21 f g h z s . The control (preischemic) value is 14.5 6 1.0 cm H2O. Significance: p , 0.05 versus group 1. The control 1.2 mm Hg z mL (preischemic) value is 3.6 6 0.1. a
AwP 5 peak airway pressure; ratio.
mPAP 5 mean pulmonary artery pressure;
death, respectively; p , 0.01 for group 2 and p 5 not significant for group 3 versus control value). The difference between groups 1 and 3 was significant starting at 1 hour and between groups 1 and 2, at 2 hours (see Fig 1). No significant differences were observed between groups 2 and 3 at any interval. In group 4 (Infl-O2), AwP was lower at 4 hours compared with group 2 (p , 0.05), but no significant difference was seen between groups 2 and 4 at 8 hours or between groups 3, 5 (Vent-N2), and 6 (VentO2) at 4 and 8 hours (Fig 2). Figure 3 shows the W/D in groups 1 (Defl), 2 (Infl-RA), and 3 (Vent-RA). There was a sharp and constant increase in W/D in group 1 starting 2 hours after cessation of circulation (from 3.6 6 0.1 [control value] to 11.5 6 1.2 at 6 hours postmortem; p , 0.001). In contrast, the increase in W/D in groups 2 and 3 was much slower (5.2 6 0.3 and 4.6 6 0.4 at 6 hours postmortem, respectively; p , 0.01 for group 2 and p 5 not significant for group 3 versus control value). The differences between group 1 and groups 2 and 3 were highly significant (p ,
PVR 5 pulmonary vascular resistance;
W/D 5 wet-to-dry weight
0.01 at 4 hours and p , 0.001 at 6 hours) (see Fig 3). No significant differences were observed between groups 2 and 3 at any interval, between groups 2 and 4 (Infl-O2), or between groups 3, 5 (Vent-N2), and 6 (Vent-O2) at 4 and 8 hours (Fig 4).
Comment Rapid cooling of perfused organs by in situ flush with a cold crystalloid solution forms the basis of any solidorgan preservation prior to transplantation. Lungs preserved in this way can be transplanted safely after up to 6 to 8 hours of cold ischemia. In the NHBD, however, there will always be a certain delay between (unexpected) circulatory arrest and the start of cold in situ flush of the organs. This critical interval, so-called warm ischemia, will rapidly lead to tissue and cellular damage [10, 11]. This might result in organ dysfunction jeopardizing the transplant recipient and remains the most difficult factor to overcome, especially after lung transplantation.
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Fig 1. Peak airway pressure (mean 6 standard error of the mean) in rabbit lungs (n 5 6) at end of cold (64°C) crystalloid flush at intervals after death comparing lungs left deflated (Defl) versus lungs inflated with room air (Infl-RA), versus lungs ventilated with room air (Vent-RA). (* 5 p , 0.05, Infl-RA [except 60 minutes] and Vent-RA versus Defl by analysis of variance; ∧ 5 p , 0.05 versus control value; ∧∧ 5 p , 0.01 versus control value.)
Indeed, besides extracorporeal membrane oxygenation, no valid alternative organ replacement therapy is available today in case of primary nonfunction of the pulmonary allograft. The period of inevitable warm ischemia in the NHBD, therefore, should be kept as short as possible. However, organizing organ retrieval and obtaining family consent for organ donation consume precious time. During this interval, organs have to be protected against cellular autolysis by preservation inside the dead body. The clinical use of lungs from NHBDs is still anecdotal [12, 13]. Nevertheless, transplantation of lungs retrieved from cadavers after cardiac arrest has been investigated in an
Fig 2. Peak airway pressure (mean 6 standard error of the mean) in rabbit lungs (n 5 6) at end of cold (64°C) crystalloid flush 4 and 8 hours after death comparing lungs inflated with room air (Infl-RA) versus lungs inflated with 100% oxygen (Infl-O2) and lungs ventilated with room air (Vent-RA), versus lungs ventilated with 100% nitrogen (Vent-N2), versus lungs ventilated with 100% oxygen (Vent-O2). (N.S. 5 not significant by analysis of variance; p 5 p , 0.05.)
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Fig 3. Wet-to-dry weight ratio (mean 6 standard error of the mean) in rabbit lungs (n 5 6) after cold (64°C) crystalloid flush at intervals after death comparing lungs left deflated (Defl) versus lungs inflated with room air (Infl-RA) versus lungs ventilated with room air (Vent-RA). (pp 5 p , 0.01, Infl-RA and Vent-RA versus Defl by analysis of variance; ppp 5 p , 0.001, Infl-RA and Vent-RA versus Defl by analysis of variance; ∧ 5 p , 0.05 versus control value; ∧∧ 5 p , 0.01 versus control value; ∧∧∧ 5 p , 0.001 versus control value.)
increasing number of animal transplant experiments during recent years [14 –19]. In the present study, we wanted to investigate graft function during delayed hypothermic crystalloid pulmonary flush at intervals after death. A gradual increase in mPAP and PVR over time was seen in all study groups. No intergroup differences were observed. It is well known that PVR is increased on reperfusion of the
Fig 4. Wet-to-dry weight ratio (mean 6 standard error of the mean) in rabbit lungs (n 5 6) after cold (64°C) crystalloid flush 4 and 8 hours after death comparing lungs inflated with room air (Infl-RA) versus lungs inflated with 100% oxygen (Infl-O2) and lungs ventilated with room air (Vent-RA) versus lungs ventilated with 100% nitrogen (Vent-N2) versus lungs ventilated with 100% oxygen (Vent-O2). (N.S. 5 not significant by analysis of variance.)
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allograft after a period of pulmonary ischemia [20]. The exact mechanism remains unclear. Hypoxic vasoconstriction, mediator-induced vasospasm, and microvascular plugging of blood elements have all been recognized as possible causes of increased resistance. Increased microvascular hydrostatic pressure and postischemic endothelial damage may then lead to permeability pulmonary edema and clinically apparent acute graft dysfunction after reperfusion [21]. In fact, these delayed flush experiments after a period of warm ischemia could be seen as blood-free reperfusion experiments, and therefore the findings could be interpreted in the same way. We observed a significant difference in AwP and W/D after hypothermic flush of lungs already subjected to a period of warm ischemia when comparing postmortem expanded versus nonexpanded lungs. The increase in group 1 (Defl) became evident as early as 2 hours after circulatory arrest. This suggests that warm ischemic tolerance in the atelectatic lung is limited to 690 minutes. Tolerance to warm ischemia could be extended to 4 hours if the lungs remained inflated or ventilated. No differences in W/D were found at 4 and 8 hours after death between postmortem inflation with room air (group 2 [Infl-RA]) versus oxygen (group 4 [Infl-O2]) and ventilation with room air (group 3 [Vent-RA]) versus nitrogen (group 5 [Vent-N2]) and oxygen (group 6 [VentO2]). In a study conducted by Ulicny and co-workers [15], no difference in early gas exchange was seen after canine single-lung transplantation and hilar occlusion of the native lung after a 4-hour period of postmortem donor ventilation with either 100% oxygen or 100% nitrogen. The authors therefore concluded, as we did in the present study, that the mechanics of ventilation after cessation of circulation appear to confer a functional advantage independent of a continued supply of oxygen. However, in a subsequent identical transplant study [19], postmortem ventilation with alveolar gas was inferior to ventilation with 100% oxygen. Further rat experiments by the same group showed that adenine nucleotides were well preserved after postmortem oxygen but not nitrogen ventilation [22], and this correlated well with light microscopy pulmonary cell viability quantified by pulmonary artery infusion with trypan blue vital dye [10]. This latter study suggested that a continued oxygen supply, not ventilation itself, is important to maintain aerobic metabolism and prevent cellular damage. We [6] also looked at rabbit pulmonary cell viability using trypan blue dye exclusion and came to the same conclusions. On the other hand, Koyama and colleagues [23] compared the effect of postmortem ventilation with room air versus oxygen versus nitrogen in a canine isolated reperfusion model and reported superior function in lungs ventilated with nitrogen. The authors concluded that reperfusion injury seen in oxygenventilated animals was mediated by oxygen free radicals. Briefly, conflicting data have been reported from metabolic, morphologic, and functional studies. The ideal gas mixture for delivery to ischemic pulmonary grafts during cadaveric storage remains an open question. We prefer to use KHBB as flush solution, a crystalloid
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with electrolytes of extracellular composition, without the addition of a prostanoid as vasodilator to examine solely the effect of increasing periods of warm ischemia on vascular resistance during pulmonary flush. It is well known that the high potassium concentration of an intracellular type of flush solution such as Euro-Collins [24] or University of Wisconsin [25] induces pulmonary vasoconstriction. In preliminary experiments, we also observed higher PVR in control animals (n 5 8) during flush with Euro-Collins solution compared with KHBB solution (52.2 6 7.6 mm Hg z mL21 z s21 versus 13.2 6 1.0 mm Hg z mL21 z s21, respectively; p , 0.0001). The absence of a high-molecular-weight osmotic-impermeant component in KHBB solution might have aggravated pulmonary edema in ischemic lungs. However, no significant difference in W/D was observed in an additional group of cadavers (n 5 7) with deflated lungs flushed 4 hours postmortem with low-potassium– dextran solution, a crystalloid with similar electrolyte composition as KHBB but with dextran 40 as an impermeant, compared with KHBB (6.0 6 0.6 versus 6.9 6 0.6, respectively; p 5 not significant). D’Armini and co-workers [26] investigated W/D in rat lungs ventilated postmortem with 100% oxygen for 4 hours followed by flush with modified Euro-Collins, University of Wisconsin, or Carolina rinse solution. The lungs were then fully expanded and stored in the same cold solution for another 4 hours. The W/D in lungs flushed with and stored in University of Wisconsin solution was significantly lower, with a value similar to that of fresh tissue. Another potential criticism of this NHBD model is the use of heparin in the donors before sacrifice [17]. We elected to heparinize the animals to exclude intravascular thrombosis as a cause of poor lung function during delayed hypothermic flush. In a supplemental group of nonheparinized animals (n 5 6), deflated lungs were flushed in an identical manner 4 hours after death. The PVR at the end of flush was significantly higher compared with that in heparinized animals (23.4 6 0.7 mm Hg z mL21 z s21 versus 17.6 6 1.6 mm Hg z mL21 z s21; p , 0.05). No significant differences, however, were observed in mPAP, AwP, and W/D between both groups. Finally, we also compared the effect of cold (3.5° 6 0.2°C) versus warm (22.5° 6 0.1°C) flush with KHBB solution in deflated lungs (n 5 6) 4 hours postmortem. No significant differences in mPAP, AwP, and W/D were found between groups. However, PVR was significantly higher after hypothermic flush (17.6 6 1.6 mm Hg z mL21 z s21 versus 12.1 6 1.2 mm Hg z mL21 z s21; p , 0.05). In this study, we measured relatively gross estimates of lung function during a very short period (65 minutes). Assessment of gas exchange at reperfusion with deoxygenated blood is probably the most reliable variable to evaluate the quality of lung preservation and permits clear differentiation between well-preserved and poorly preserved lungs. We have used these flush experiments as a rapid screening method to define the length of tolerable warm ischemia in all study groups. In later experiments (unpublished results) comparing the same study groups, we have investigated the effect of 4 hours’
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in situ warm ischemia in a rabbit isolated, pressurelimited, homologous blood reperfusion and room air– ventilated model (n 5 4 in each group). Arteriovenous oxygen pressure gradient after 1 hour of reperfusion was only 9 6 5 mm Hg in cadaver lungs that were left deflated during the ischemic interval versus 95 6 13 mm Hg in lungs that were inflated with room air, 96 6 7 mm Hg in lungs that were ventilated with room air, and 96 6 4 mm Hg in lungs ventilated with 100% nitrogen (p , 0.05, Defl versus all other groups; p 5 not significant, Infl-RA versus Vent-RA versus Vent-N2). This study therefore validates the conclusion of the present study that the prevention of postmortem alveolar collapse itself will confer a functional advantage independent of continued supply of oxygen. The exact mechanism of extended warm ischemic tolerance of the lung by preventing the alveolar space to collapse remains unclear. Release of surfactant from type II pneumocytes is known to be stimulated by inflation [27] and ventilation of lungs [28] and by mechanical stretch of isolated pneumocytes in culture [29]. Alveolar surfactant activity is reduced in the atelectatic lung [30]. Although no measurements of surfactant or surfactant activity were made in the present study, it is reasonable to speculate that repetitive or continuous alveolar expansion during warm ischemia in our study groups may have stimulated the release of pulmonary surfactant, thereby decreasing the alveolar surface tension, preventing damage to the alveolar-capillary membrane, and protecting against permeability pulmonary edema during flush [21]. From this study, we can conclude that in the NHBD (1) pulmonary edema will develop in atelectatic lungs if hypothermic flush is delayed for 2 hours after death, (2) postmortem inflation is as good as ventilation in protecting the lung against edema formation, thereby prolonging warm ischemic tolerance up to 4 hours, (3) postmortem inflation with oxygen or ventilation with nitrogen or oxygen is no different from that with room air, and (4) therefore, prevention of alveolar collapse appears to be the critical factor in protecting the lung from warm ischemic damage independent of continued oxygen delivery. Further studies are necessary to investigate whether lungs from human NHBDs will become a realistic alternative to expand the pulmonary donor pool. This work is supported by grant 7.0036.94 from the Nationaal Fonds voor Wetenschappelijk onderzoek—Levenslijn 1994. We thank Peter Lemmens, Magda Mathys, Kanigula Mubagwa, MD, and Anne Vancauwenbergh for expert technical and secretarial assistance.
References 1. Van Raemdonck D, Roels L, Verleden G, et al. Whence the lungs? Assessment of the use of lungs for transplantation from 156 consecutive donors. In: Cooper JD, Weder W, eds. Proceedings of the third international lung transplant symposium (Zurich, Switzerland, June 24 –25, 1993). 1993:40. 2. Starnes VA, Barr ML, Cohen RG. Lobar transplantation. Indications, technique, and outcome. J Thorac Cardiovasc Surg 1994;108:403–11.
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3. Couetil JPA, Tolan MJ, Loulmet DF, et al. Pulmonary bipartitioning and lobar transplantation. A new approach to donor organ shortage. J Thorac Cardiovasc Surg 1997;113: 529–37. 4. Starnes VA, Barr ML, Cohen RG, et al. Living-donor lobar lung transplantation experience. Intermediate results. J Thorac Cardiovasc Surg 1996;112:1284–91. 5. Van Raemdonck DEM, Jannis NCP, Rega FRL, De Leyn PRJ, Flameng WJ, Lerut TE. Delay of adenosine triphosphate depletion and hypoxanthine formation in rabbit lung after death. Ann Thorac Surg 1996;62:233– 41. 6. Kuang JQ, Van Raemdonck DEM, Jannis NCP, et al. Pulmonary cell death in warm ischemic rabbit lung is related to the postmortem alveolar oxygen reserve [Abstract]. J Heart Lung Transplant 1997;16:99. 7. Kuang JQ, Van Raemdonck DEM, Jannis NCP, et al. Pulmonary grafts should be inflated with 100% oxygen prior to hypothermic storage [Abstract]. J Heart Lung Transplant 1997;16:99. 8. Van Raemdonck DEM, Jannis NCP, Rega FRL, De Leyn PRJ, Flameng WJ, Lerut TE. External cooling of warm ischemic rabbit lungs after death. Ann Thorac Surg 1996;62:331–7. 9. Scheffe´ HA. A method for judging all contrast in the analysis of variance. Biometrika 1953;40:87–104. 10. D’Armini AM, Roberts CS, Griffith PK, Lemasters JJ, Egan TM. When does the lung die? I. Histochemical evidence of pulmonary viability after “death.” J Heart Lung Transplant 1994;13:741–7. 11. Alessandrini F, D’Armini AM, Roberts CS, Reddick RL, Egan TM. When does the lung die? II. Ultrastructural evidence of pulmonary viability after “death.” J Heart Lung Transplant 1994;13:748–57. 12. D’Alessandro AM, Hoffmann RM, Knechtle SJ, et al. Successful extrarenal transplantation from non– heart-beating donors. Transplantation 1995;59:977– 82. 13. Love RB, Stringham JC, Chomiak PN, Warner T, Pellett JR, Mentzer RM. Successful lung transplantation using a non– heart-beating donor [Abstract]. J Heart Lung Transplant 1995;14:S88. 14. Egan TM, Lambert CJ Jr, Reddick R, Ulicny KS Jr, Keagy BA, Wilcox BR. A strategy to increase the donor pool: use of cadaver lungs for transplantation. Ann Thorac Surg 1991;52: 1113–21. 15. Ulicny KS Jr, Egan TM, Lambert CJ Jr, Reddick RL, Wilcox BR. Cadaver lung donors: effect of preharvest ventilation on graft function. Ann Thorac Surg 1993;55:1185–91. 16. Buchanan SA, DeLima NF, Binns OAR, et al. Pulmonary function after non– heart-beating lung donation in a survival model. Ann Thorac Surg 1995;60:38– 46. 17. Umemori Y, Date H, Uno K, Aoe M, Ando A, Shimizu N. Improved lung function by urokinase infusion in canine lung transplantation using non– heart-beating donors. Ann Thorac Surg 1995;59:1513– 8. 18. Roberts CS, D’Armini AM, Egan TM. Canine double-lung transplantation with cadaveric donors. J Thorac Cardiovasc Surg 1996;112:577– 83. 19. Hennington MH, D’Armini AM, Lemasters JJ, Egan TM. Cadaver lungs for transplantation. Effect of ventilation with alveolar gas. Transplantation 1996;61:1009–14. 20. Allison RC, Kyle J, Adkins WK, Prasad VR, McCord JM, Taylor AE. Effect of ischemia reperfusion or hypoxia reoxygenation on lung vascular permeability and resistance. J Appl Physiol 1990;69:597– 603. 21. Novick RJ, Gehman KE, Ali IS, Lee J. Lung preservation: the importance of endothelial and alveolar type II cell integrity. Ann Thorac Surg 1996;62:302–14. 22. D’Armini AM, Tom EJ, Roberts CS, Henke DC, Lemasters JJ, Egan TM. When does the lung die? Time course of high energy phosphate depletion and relationship to lung viability after death. J Surg Res 1995;59:468–74. 23. Koyama I, Toung TJK, Rogers MC, Gurtner GH, Traystman RJ. O2 radicals mediate reperfusion lung injury in ischemic O2ventilated canine pulmonary lobe. J Appl Physiol 1987;63:111–5.
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24. Yamazaki F, Yokomise H, Keshavjee SH, et al. The superiority of an extracellular fluid solution over Euro-Collins’ solution for pulmonary preservation. Transplantation 1990; 49:690– 4. 25. 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. 26. D’Armini AM, Lemasters JJ, Egan TM. Studies of rat lung viability and adenine nucleotide metabolism after death. Ann Thorac Surg 1996;62:1448–53.
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27. Faridy EE. Effect of distension on release of surfactant in excised dogs’ lungs. Respir Physiol 1976;27:99 –114. 28. Oyarzun MJ, Clements JA. Control of lung surfactant by ventilation, adrenergic mediators, and prostaglandins in the rabbit. Am Rev Respir Dis 1978;117:879–91. 29. Wirtz HRW, Dobbs LG. Calcium mobilization and exocytosis after one mechanical stretch of lung epithelial cells. Science 1990;250:1266–9. 30. Oyarzun MJ, Stevens P, Clements JA. Effect of lung collapse on alveolar surfactant in rabbits subjected to unilateral pneumothorax. Exp Lung Res 1989;15:909–24.
DISCUSSION DR THOMAS M. EGAN (Chapel Hill, NC): That was a very nice study, and it adds to the body of literature supporting the hypothesis that lungs may be useful for transplantation even at intervals after death. I am a little curious about the group of lungs that were fully deflated. I gather from your manuscript that these lungs were deflated because, as part of the preparation, you opened both pleural spaces. I wonder if another control group might have been useful as well, that is, lungs maintained at functional residual capacity, because a fully deflated lung does not really mimic the clinical scenario of a cadaver unless the body dies of bilateral stab wounds to the chest. Do you think there would have been any difference had you studied lungs in a noninflated state, but lungs that were allowed to maintain volume at functional residual capacity? As I have said, this was an interesting study. It does suffer from one serious limitation, and that is that the duration of your perfusion was relatively short, and no gas exchange data could be accrued. DR VAN RAEMDONCK: Thank you, Dr Egan, for your kind remarks. It is well appreciated that your group has revived interest in the use of lungs from non– heart-beating donors. In regard to your first question about the group with deflated lungs, it is true that the lungs were deflated because we opened both pleural spaces after sternotomy. We started doing these experiments by killing the animals with an overdose of pentobarbital, and we observed that even in the control animals without any ischemia, we had a massive amount of lung edema. That is why we started to look for another method to kill the rabbits. Indeed, these lungs were not at functional residual capacity but below functional residual capacity. Before sacrifice, the lungs were ventilated with room air; as you know, room air is a gas mixture of 21% oxygen and 79% nitrogen. These lungs will not become fully deflated to the complete zero level as nitrogen
will not be absorbed. But indeed, as you suggested, we should study another group of animals with lungs at functional residual capacity. In regard to your second comment, we studied a relatively gross estimate of lung function during a very short period; the Flush was only 5 minutes. Gas exchange after reperfusion with deoxygenated blood is probably the most reliable variable to study the quality of lung preservation and can distinguish between well-preserved and poorly preserved lungs. We first used these flush experiments as a screening model to rapidly define the length of tolerable warm ischemia in all study groups. We have now extended these rabbit experiments in an isolated, pressure-limited, and homologous deoxygenated blood reperfusion and room air–ventilated model using the same study groups. After an ischemic period of 4 hours, we compared lungs that were deflated versus lungs that were inflated with room air and lungs ventilated with room air, and we looked at oxygenation capacity, defined as the arteriovenous oxygen pressure gradient. During a reperfusion period of 4 hours, there was a highly significant difference in oxygenation capacity between the lungs that were inflated with room air and ventilated with room air versus the deflated lungs. Also, the wet to dry weight ratio in the deflated lungs after 4 hours of reperfusion with homologous blood was significantly higher than in the lungs inflated with room air and those ventilated with room air. However, there was no significant difference in oxygenation capacity between the group ventilated with room air and the group ventilated with nitrogen, nor was there a difference in wet to dry weight ratio between these same two groups. We think this study confirms the conclusion of our present study that alveolar expansion itself, not continued oxygen supply, represents a functional advantage in postmortem lung preservation.