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Ann Thorac Surg 2004;77:438-444
© 2004 The Society of Thoracic Surgeons
a Center for Experimental Surgery and Anesthesiology, Catholic University of Leuven, Leuven, Belgium
b Laboratory for Pneumology, Catholic University of Leuven, Leuven, Belgium
c Department of Thoracic Surgery, University Hospital Gasthuisberg, Leuven, Belgium
* Address reprint requests to Dr Van Raemdonck, Department of Thoracic Surgery, University Hospital Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium.
e-mail: dirk.vanraemdonck{at}uzleuven.be
Presented at the Thirty-ninth Annual Meeting of The Society of Thoracic Surgeons, San Diego, CA, Jan 31–Feb 2, 2003.
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Abstract |
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METHODS: Domestic pigs were anesthetized and divided into three groups (n = 6 in each group). In the control group (HBD), lungs were flushed, explanted, and further stored in low potassium dextran solution (4°C) for 4 hours. In the two study groups pigs were sacrificed by myocardial fibrillation and left untouched for 1 hour. Chest drains were then inserted for topical lung cooling (6°C) for 3 hours (NHBD-TC3) or 6 hours (NHBD-TC6). The left lung in all groups was then prepared for evaluation. In an isolated circuit lungs were ventilated and reperfused through the pulmonary artery. Hemodynamic, aerodynamic, and oxygenation variables were measured 35 minutes after onset of controlled reperfusion. Wet-to-dry weight ratio was calculated.
RESULTS: No significant differences were observed among the three groups in pulmonary vascular resistance (p = 0.38), mean airway pressure (p = 0.39), oxygenation index (p = 0.62), and wet-to-dry weight ratio (p = 0.09).
CONCLUSIONS: These data confirm that 1 hour of warm ischemia does not affect the pulmonary graft from NHBD compared with HBD. The preharvest interval can be safely extended up to 7 hours postmortem by additional topical cooling of the graft inside the cadaver. This technique may facilitate distant organ retrieval in NHBD.
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Introduction |
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After the initial publication by Egan in 1991 many groups [6–12] have now reported data sustaining the hypothesis that transplantation of lungs from a NHBD might be one strategy to resolve the organ shortage. For many years we have been exploring in our laboratory the possibility of using lungs from these donors. In previous rabbit studies we have investigated the effect of postmortem cadaver lung inflation, ventilation, and cooling [13–16] on the catabolism of adenine nucleotides [17, 18], on pulmonary cell viability [15], and on graft function [19].
Warm ischemic tolerance in the lung after cardiac arrest appears to be limited to 1 hour [5, 8, 9, 19]. After this period lungs should be protected against (further) tissue degradation in order not to compromise the viability of the donor organ.
In a recent study we have shown that 3 hours of topical cooling starting 1 hour after death was more effective to protect the pulmonary graft inside the NHBD compared with postmortem ventilation and was not different from cold flush in the heart-beating donor [20]. A sufficient length of in situ preservation is necessary however to organize distant organ retrieval.
In the present study we hypothesized that prolonging the interval of intrapleural cooling from 3 to 6 hours would not affect the viability of the pulmonary graft.
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Material and methods |
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Animals were premedicated with an intramuscular injection of 2.5 mL zolazepam/tiletamine (Zoletil 100; Virbac s.a., Carros, France) and 2.5 mL xylazine (Xyl-M 2%; V.M.D. nv/sa, Arendonk, Belgium). The animals were intubated with an endotracheal tube (nr 7.5 [SIMS Portex, Hythe, Kent, UK]) and ventilated (Titus, Dräger, Germany) with an inspired oxygen fraction (FiO2) of 0.5, tidal volume of 10 mL/kg, and a frequency of 20 breaths per minute. Anesthesia was maintained with isoflurane (0.6% to 1% [Forene; Abbott Laboratories, Queensborough, Kent, UK]). Muscle relaxation was controlled with an intermittent bolus of pancuronium bromide (2 mg/mL [Pavulon; Organon Teknika, Boxtel, Netherlands]).
Preservation of the heart-beating donor lungs
After median sternotomy thymic tissue was excised. The pleural cavities and the pericardium were opened. The superior and inferior caval veins, the ascending aorta, and the pulmonary artery trunk were encircled. Sodium heparin 10,000 IU (Heparin Rohrer 5,000 IU/mL; Rhône-Poulenc Rohrer, Brussels, Belgium) was administered. The main pulmonary artery was cannulated with a 24F catheter (DLP, Grand Rapids, MI) and secured by a purse string suture in the right ventricular outflow tract. The ascending aorta was ligated and the pulmonary artery was isolated from the right ventricle by ligature around the tip of the catheter just distal to the pulmonary valve, creating pulmonary ischemia. All remaining ligatures were tightened, the left atrial appendage was incised for venting, and the lungs were flushed with 50 mL/kg cold (4°C) Perfadex with addition of 0.6 mL/L (2 g/5 mL) of the buffer solution Trometamol (Addex-THAM, Kabi, Sweden) and CaCl2 (1.2 mL/L, 11 mEq/g). After this flush ventilation was stopped, catheters were removed, and the heart-lung block was excised and immersed in Perfadex (4°C) for 4 hours, adjusted in the same manner as described above.
Preservation of the nonheart-beating donor lungs
The pigs in the nonheart-beating donor groups were sacrificed by myocardial fibrillation induced with a subxyphoidal needle puncture using a square pulse generator (amplitude ranged between +15 V to -15 V, a currency of not more than 300 mAmp and a frequency of 50 Hz). Blood pressure dropped immediately below 20 mm Hg and the animals were declared dead after 5 minutes. The endotracheal tube was disconnected from the ventilator and left open to the air. The animals were left untouched for 60 minutes at room temperature (21°C). After this 1 hour interval of in situ warm ischemia, chest drains (16F Trocar catheter; Argyle, Sherwood Medical, Tullamore, Ireland) were inserted in the thoracic cavity [21], two in each pleural space (one deep drain for inflow and one superficial drain for outflow; Fig 2, A).
Through these chest drains cold (6°C) saline solution was infused and continuously recirculated with a roller-pump from a reservoir placed in an ice basket. The whole system (pleural cavities, tubing, and reservoir) was filled with approximately 6 L of saline. To ensure that the lungs were well immersed in cold saline, a 5 cm H2O pressure overflow system was connected to the outflow drains (Fig 2, B).
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Preparation of the heart-lung block
At the end of the ischemic interval the lungs in all three experimental groups were prepared in the same manner for evaluation in an isolated reperfusion circuit. The right lung was extracted and discarded. The pulmonary artery trunk was cannulated through the right ventricular outflow tract using a 36F catheter. Special attention was given to remove all clots in the NHBD groups. Major clots were removed under direct vision. Thereafter a suction tip catheter was introduced deeply into the branches of the pulmonary artery to remove smaller clots.
The ascending aorta was ligated and the pulmonary artery was isolated from the right ventricle by ligature around the tip of the catheter just distal to the pulmonary valve. The left atrium was cannulated with a 36F catheter through the apex of the left ventricle and secured by a purse-string around the apex. Finally a tube (nr 6.5) was placed in the trachea and tightened.
Preparation of the perfusate
Autologous blood (± 450 mL) was withdrawn from each animal at the moment of sacrifice through a catheter in the left, internal jugular vein and collected in an empty sterile bag (1,000 mL NaCl 0.9%; Baxter, Lessines, Belgium) containing 2 mL of heparin (2 mL/L, 5,000 IU/mL). This whole blood was centrifuged for at least 10 minutes at 5,600 rpm using a cell-saving device (Sequestra 1000; Medtronic, Parker, CO). Thereafter white blood cells were removed using a leukocyte filter (Imugard III-RC; Terumo Europe N.V., Haasrode, Belgium). The red blood cell concentrate was then diluted to a hematocrit of approximately 15% with Perfadex and albumin. Thirty minutes before reperfusion the perfusate was finalized by adding CaCl2 (1.2 mL/L, 11 mEq/g), Trometamol buffer solution (0.3 mL/L, 2 g/5 mL), heparin (2 mL/L, 5,000 IU/mL), nitroglycerin (2 mL/L, 50 mg/50 mL [Nitro-Pohl]), and sodium bicarbonate (4.5 mL/L, 0.8 mol/L [Baxter]).
Isolated reperfusion circuit
The perfusate (± 1,400 mL) was placed in a hardshell reservoir (Vision; GISH Biomedical, Irvine, CA) and circulated using a centrigal pump (Bio-Medicus; Medtronic, Minneapolis, MN). It then passed an adult gas exchanger (Vision; GISH Biomedical), operating as a deoxygenator with a gas mixture of CO2 (8%), O2 (6%), and N2 (86%). The heating element of the gas exchanger was connected to a heater/cooler system (Bio-Cal, Medtronic). On the inflow cannula, pulmonary artery flow (PAF [FF 100T 10-mm probe connected to the MFV-3200 electromagnetic flow meter; Nihon Kohden, Tokyo, Japan]), pulmonary artery pressure (PAP [Uniflow type 43-600F; Baxter, Uden, Netherlands, connected to a monitor type 78534A, Hewlett Packard]), and temperature of the perfusate were recorded online.
Pulmonary effluent was drained from the left atrium. The left atrial pressure (LAP) was kept at 0 mm Hg by regulating the height of the outflow cannula. At this point temperature of the outflowing perfusate was recorded, reflecting the temperature of the lung parenchyma.
During reperfusion the lung was ventilated by gradually increasing the tidal volume up to 0.140 L with a frequency of 14 breaths per minute (FiO2 of 0.5 and 5 cm H2O PEEP). Mean airway pressure was continuously monitored.
Pulmonary artery pressure and temperature of the inflowing perfusate were gradually increased to a maximum of 15 mm Hg and 37.5°C respectively. Thirty-five minutes after onset of reperfusion the function of the left lung was assessed during 20 minutes.
Graft variables
During this 20 minutes interval five consecutive measurements of PAF, PAP, LAP, and mean airway pressure (mAwP) were recorded and five blood samples were taken from the outflowing blood to analyze pO2 (ABL 4 Radiometer A/S, Copenhagen, Denmark). Mean values of the five measurements were calculated for all variables.
Pulmonary vascular resistance (PVR) was calculated using the formula PVR = [PAP (mm Hg) - LAP (mm Hg)] x 80/PAF (L/min) and was expressed in Dynes x sec x cm-5. Oxygenation index (OI) was calculated using the formula OI = [mAwP (mm Hg) x FiO2 (%)]/pO2 (mm Hg).
At the end of the isolated perfusion the left lung was separated from the heart, weighed, and dried overnight in an oven (model HT 600; Heraeus, Hanau, Germany) at 150°C to constant weight. Wet-to-dry weight ratio was calculated as an estimate of the amount of lung edema.
Statistics
Data analysis was performed using the software package Statistica 6.0 (Statsoft, Tulsa, OK). All data are expressed as mean ± SEM. As it was not possible to check for the normality of the variables with only six observations per group, we used nonparametric tests. We performed a Kruskal-Wallis test to look for differences among the three groups. Whenever a significant difference was observed the Mann-Whitney test was used for comparison between two groups.
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Results |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsDiscussionReferences |
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Comment |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsDiscussionReferences |
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Protection of the pulmonary graft inside the NHBD cadaver is nevertheless important to minimize tissue degradation by anaerobic catabolic processes that will affect early and late graft function after transplantation. An efficient technique for organ preservation inside the cadaver will also help to prolong the preexplantation interval thereby giving the transplant team the opportunity to ask for family consent and to organize organ retrieval. In the literature we have found different opinions regarding the preferred NHBD protocol. Some authors have suggested that ventilation [9, 12, 22–24] is the preferred technique to preserve the graft inside the NHBD donor, others are more in favor of topical cooling [7, 11, 21]. In a recent study performed in our laboratory comparing both techniques we have demonstrated that topical cooling was more efficient in protecting the pulmonary graft from the NHBD [20]. In that study an interval of 3 hours was used for topical cooling identical to the NHBD-TC3 group in this study.
In experimental settings lungs from heart-beating donors may function very well after long-term cold preservation, even up to 48 hours [25]. The question that rose after our previous study was how long topical cooling in the NHBD could be safely continued. The length of this interval will determine if it will even be possible to retrieve organs from donors at a distance without compromising the viability of the graft. In 1997 Steen and associates [26] already reported that topical cooling for 6 hours was realistic. In this study, however, no warm ischemic interval preceded the start of topical cooling. Also, in these experiments the lungs were cooled with the chest open and the elegant technique of topical cooling through chest drains was not yet implemented [21]. This simple technique can be performed by any trained physician working in an emergency room or intensive care unit.
In our study, we looked at graft viability during short-term (± 55 minutes) reperfusion of the lung measuring pulmonary vascular resistance, mean airway pressure, oxygenation ratio, and wet-to-dry weight ratio. No differences were observed between both study groups indicating that topical cooling can be safely extended from 3 to 6 hours. We also included a control group of heart-beating donors and did not find any difference in graft function compared with the study groups. This confirms the findings from our previous study [19] that a 1 hour warm ischemic interval before cooling is well tolerated. In this experimental setup therefore we did not include a positive control group with a 1 hour warm ischemic interval only.
In our NHBD study groups we did not administer heparin before death. According to the Maastricht NHBD classification [27] it is not possible to give heparin in category I (death on arrival) and category II (failed resuscitation) donors. In our opinion these two NHBD categories have the largest potential to increase the number of grafts with good quality.
From experience we know that there is nearly no clotting in the more peripheral microvasculature of the lung after death. Apparently the endothelial cells of the pulmonary vasculature are able to produce anticoagulant factors that prevent clotting in the microvasculature of the lung. We could not find any data in the literature to sustain this hypothesis. In preliminary experiments we did not extract clots in the main pulmonary artery and left atrium. This resulted in a high pulmonary vascular resistance at onset of reperfusion. Once we began to rigorously extract these clots with suction, pulmonary vascular resistance at onset of reperfusion dropped. With longer intervals of topical cooling exceeding 6 hours clots became very organized and were difficult to remove resulting in poor outcome in the isolated reperfusion model. When one would like to prolong the topical cooling interval beyond 6 hours it might be useful to add a trombolytic agent during reperfusion [28].
In our experimental protocol we performed an assessment during 20 minutes starting 35 minutes after onset of reperfusion. This 35-minute time interval is necessary to allow the lung parenchyma to gradually reach 37.5°C because only at this point the lung can be safely ventilated (PEEP 5 cm H2O and maximum tidal volume). Ventilation of a cold lung may damage the lipid bilayers of the cellular membranes thereby destructing the pulmonary parenchyma [29]. A weakness of this study however is the short evaluation period, giving us only an idea of early graft function. In further experiments we cooled the lungs again after assessment as described by Steen [21] in his first successful clinical case report of lung transplantation from NHBD. We then reevaluated these lungs in our isolated reperfusion model 20 hours later. Graft function was comparable to the initial function 4 hours after death [30]. From that study it appears that this short evaluation on itself does not damage the lung. To examine the effects of reperfusion in an ex vivo setting we are now testing these lungs in a porcine transplantation model.
So far only a few centers succeeded in performing transplantation of lungs from NHBD [21, 31, 32]. We are convinced that many others will follow. The biggest concern will remain the ethical issues on the use of organs from NHBD. In Belgium we have the advantage of an opting out law regarding organ donation, meaning that everyone is considered to be a donor unless registered otherwise in the National Database that can be accessed by every transplant coordinator. Careful and respectful handling is very important in considering the use of lungs from NHBD. Insertion of chest drains can be performed after informed consent of the relatives. Providing further information to the public to accept these postmortem handlings will be necessary. Studies like these may be helpful to convince other colleagues working in the emergency department or intensive care unit that the use of lungs from NHBD is a possible answer to alleviate the organ shortage.
In summary in this isolated pig lung reperfusion model we have found that 1 hour of warm ischemia does not affect the pulmonary graft from NHBD, that postmortem topical cooling inside the cadaver is as effective as immediate cold flush, and that the preharvest interval can be safely extended up to 7 hours. This technique may facilitate distant organ retrieval in NHBD.
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Acknowledgments |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsDiscussionReferences |
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Discussion |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsDiscussionReferences |
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DR REGA: Thank you, Dr Egan, for your comment and questions. Regarding your question about the flow rate, we have to stress that we only evaluated the left lung in our setup. The flow over the left lung was about 1.2, 1.3 L, which we consider to be physiologic because in a pig of that weight an average flow of 3, 3.5 to 4 L is noted. There is of course a difference between a pulsatile flow and a continuous flow. We used in our evaluation model a centrifugal pump that sends a continuous flow through the pulmonary vasculature.
Regarding your second question on low tidal volumes, we considered that the left lung in our pig model is about one third in height and length compared with the right lung, so we gave it about 33% of the total tidal volume we used in vivo and then we come to 0.14 L.
DR EGAN: Your pulmonary vascular resistance was a little bit high compared with what you would expect in a conventional animal. Do you have any explanation for that and have you tried agents to reduce that?
DR REGA: We didn't try agents to reduce the pulmonary vascular resistance. One explanation might be that we run on continuous flow and not on pulsatile flow.
DR W. ROY SMYTHE (Houston, TX): I have a quick question. It's such a short time interval of controlled reperfusion and with leukocyte-free blood, as I understand, in the reperfusate.
DR REGA: Indeed.
DR SMYTHE: Does your experimental wet-to-dry lung ratio really mean anything compared with what you would see in an orthotopic clinical posttransplant reperfusion situation, as in that situation blood is not leukocyte depleted and the reperfusion duration is indefinite?
DR REGA: This is a very good remark. In other types of experiments we extended the reperfusion interval to 4 hours and we could not find any difference in pulmonary vascular resistance. It remained constant. In some experiments we cooled the lungs down after assessment and we reevaluated the same lungs some hours later. We did not notice any difference in the parameters we measured. To really follow a receptor-induced response we need to perform transplantation experiments. And that's what we are doing right now. We are setting up an experimental transplantation protocol to evaluate this.
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This article has been cited by other articles:
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  C. Van De Wauwer, A. P. Neyrinck, N. Geudens, F. R. Rega, G. M. Verleden, E. Verbeken, T. E. Lerut, and D. E.M. Van Raemdonck Retrograde flush following topical cooling is superior to preserve the non-heart-beating donor lung Eur. J. Cardiothorac. Surg., June 1, 2007; 31(6): 1125 - 1133. [Abstract] [Full Text] [PDF]
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A. P. Neyrinck, C. Van De Wauwer, N. Geudens, F. R. Rega, G. M. Verleden, P. Wouters, T. E. Lerut, and D. E.M. Van Raemdonck Comparative study of donor lung injury in heart-beating versus non-heart-beating donors. Eur. J. Cardiothorac. Surg., October 1, 2006; 30(4): 628 - 636. [Abstract] [Full Text] [PDF]
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