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Initial topical cooling followed by backtable Celsior flush perfusion provides excellent early graft function in porcine single lung transplantation after 24 hours of cold ischemia
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Initial topical cooling followed by backtable Celsior flush perfusion provides excellent early graft function in porcine single lung transplantation after 24 hours of cold ischemia Bernhard Gohrbandt, MD,a,d Murat Avsar, MD,a Gregor Warnecke, MD,a Sebastian-Patrick Sommer, MD,b Axel Haverich, MD,a and Martin Strueber, MDc From the aHannover Thoracic Transplant Program, Department of Cardiac, Thoracic, Transplantation and Vascular Surgery, Hannover Medical School, Hannover; bDepartment of Thoracic and Cardiovascular Surgery, University Hospital Wuerzburg, Wuerzburg; c Department of Cardiac Surgery, University of Leipzig Heart Centre, Leipzig; and the dDepartment of Cardiovascular and Thoracic Surgery, Medical Centre of the Johannes Gutenberg University Mainz, Mainz, Germany.
KEYWORDS: preclinical lung transplantation; lung preservation; low potassium dextran (LPD); Celsior; graft function
BACKGROUND: Topical in situ cooling of the donor lungs is a prerequisite for procurement of non– heart-beating donor lungs and may be of interest for living related lung donation. METHODS: Twenty-four single lung transplants were performed in 4 groups of Landrace pigs (6 per group). Control LPD, control Celsior and topical cooling in situ, followed by LPD (exLPD) or Celsior (exCel) ex situ flush, were employed. All lungs were perfused antegrade with 1 liter of solution at 41C. Lungs were stored immersed in preservation solution for 24 hours at 41C. After transplantation of the left lung, the right recipient bronchus and pulmonary artery were clamped. RESULTS: Four of 6 animals each in the LPD and Celsior groups and all 6 animals in both the exLPD and the exCel groups survived the 7-hour reperfusion. The mean oxygenation index was favorably preserved in the exCel group at 7 hours after reperfusion (417 ⫾ 81) over all other groups (LPD 341 ⫾ 133, Celsior 387 ⫾ 86, exLPD 327 ⫾ 76; p o 0.0001). Pulmonary vascular resistance showed significantly lower values in the Celsior and exCel groups (LPD 1,310 ⫾ 620, Celsior 584 ⫾ 194, exLPD 1,035 ⫾ 361, exCel 650 ⫾ 116 dyn/s/cm5 at 7 hours after reperfusion; p o 0.0001). Consistently, the wet-to-dry lung weight ratio also indicated beneficial graft protection in the exCel group (LPD 8.1 ⫾ 0.8, Celsior 8.4 ⫾ 0.8, exLPD 7.5 ⫾ 1.0, exCel 3.1 ⫾ 0.9; p o 0.0001). CONCLUSION: Initial topical cooling followed by backtable perfusion is a sufficient technique for pulmonary graft preservation providing excellent post-transplant function. Celsior subsequent to in-situ topical cooling revealed the most beneficial results in this setting. This combined technique could advance non–heart-beating, living related lung lobe donation and, potentially, regular heart-beating lung donation. J Heart Lung Transplant 2013;32:832–838 r 2013 International Society for Heart and Lung Transplantation. All rights reserved.
More than 40,000 lung transplants have been reported to the registry of the International Society for Heart and Lung Transplantation (ISHLT) since 1989.1 The demand Reprint requests: Gregor Warnecke, MD, Hannover Thoracic Transplant Program, Department of Cardiac, Thoracic, Transplantation and Vascular Surgery, Hannover Medical School, Carl-Neuberg-Strasse 1, 30623 Hannover, Germany. Tel.: þ49 511 532 3373; fax: þ49 511 532 5625. E-mail address: [email protected]
for lung transplantation continues to increase and a considerable donor organ shortage remains. Lung donor criteria for acceptance have already been extended. Finding alternative sources of organs continues to be a major priority.1–3 Use of non–heart-beating organ donors (NHBDs) has been widely investigated, especially with regard to acceptable warm ischemia times, increased periods of in situ topical cooling, and issues of additional flush preservation.4–9
1053-2498/$ - see front matter r 2013 International Society for Heart and Lung Transplantation. All rights reserved. http://dx.doi.org/10.1016/j.healun.2013.06.005
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The concept of living related lung lobe donation and transplantation has been described. During graft recovery, the lobar graft may be exposed to warm ischemia in situ. Antegrade, retrograde or no-flush preservation has been employed backtable thereafter.3,10–14 Either topical cooling or flush perfusion of the lungs is accepted as safe and sufficient in NHB, living related lobar lung or deceased donor organ donation. Experimental data have revealed beneficial effects combining these approaches.8,9 The purpose of this study was to evaluate early graft function after 24 hours of cold ischemic time after topical cooling and subsequent ex situ antegrade flush preservation in comparison to conventional antegrade flush preservation only, aiming toward an effective strategy for living related donation. Because our own pre-clinical data reflect safe and sufficient graft preservation with Celsior (Cel),15–18 we evaluated two clinically employed preservation solutions: low-potassium dextran (LPD) solution and Celsior. We hypothesized that initial topical cooling followed by backtable Celsior flush perfusion is a preservation technique that offers good graft function.
Methods Animal care All animals received humane care in compliance with the “Principles of Laboratory Animal Care” and the Guide for the Care of Laboratory Animals, prepared by the Institute of Laboratory Animal Resources, National Research Council (National Academy Press, revised 1996), as well as ibeing n compliance with the European Convention on Animal Care.
Donor procedure As preservation solutions, either LPD (Perfadex; Vitrolife, Gothenburg, Sweden) or Celsior (Genzyme Germany, Neu-Isenburg, Germany) were used. LPD was supplemented by 0.3 ml tris hydroxymethyl aminomethane (THAM) buffer/liter. The components of the preservation solutions used are listed in Table 1. Animals were assigned to 4 groups of 6 animals each. Induction and maintenance of anesthesia was performed as described elsewhere.15,16,18 The donor animals (weight 27 to 32 kg) were ventilated in a pressure-controlled mode (inspiratory peak pressure 30 cm H2O, positive end-expiratory pressure [PEEP] 5 cm H2O, fraction of inspired oxygen [FIO2] 1.0, inspiration/expiration [I/E] ratio 1:1, ventilation rate 10 breaths/min). After median sternotomy, the pericardium and both pleural cavities were opened. The heart and lungs were isolated. Then 250 mg of methylprednisolone and 7,500 IU of heparin were administered systemically.
Control groups For LPD and Celsior controls, a cannula was inserted into the main trunk of the pulmonary artery and secured with a ligature. Simultaneously to cardiac inflow occlusion and drainage of the left heart, the pulmonary artery was clamped centrally of the
833 Table 1
Composition of the Preservation Solutions Used
Component þ
Na (mmol/liter) Kþ (mmol/liter) Cl− (mmol/liter) Ca2þ (mmol/liter) Mg2þ (mmol/liter) PO3− 4 (mmol/liter) SO2− 4 (mmol/liter) Histidine (mmol/liter) Mannitol (mmol/liter) Dextran-40 (g/liter) Glucose (g/liter) Glutamic acid (mmol/liter) Lactobionic acid (mmol/liter) Glutathione (mmol/liter) Osmolality (mOsm/liter) pH
LPD solution
Celsior
138 6 142 0 0 0.8 2 0 0 50 0.91 0 0 0 292 7.4
100 15 100 0.25 13 0 0 30 60 0 0 20 80 3 320 7.3
cannula. Subsequently, in-situ antegrade flush preservation via this cannula was initiated with 1 liter of 41C cold preservation solution. The perfusion pressure was controlled by a separate catheter and mean pressure was retained at 10 mm Hg by adjusting the static heights in which the containment was hung. Ventilation of the lungs was maintained throughout the entire preservation period. Finally, the trachea was clamped and the heart–lung block was excised.
Study groups Fibrillation of the heart was electrically induced, which led to circulatory arrest. Ventilation was stopped simultaneously. The atelectatic lungs were immersed in 3 liters of saline at 41C within the pleural cavities for 30 minutes. The trachea was clamped and the heart and lungs were then excised en bloc. After separating the heart and lungs the pulmonary artery was clamped. The lungs were antegradely flush preserved with 1 liter of either buffered LPD (exLPD) or Celsior (exCel) ex situ, approximately 3 to 5 minutes after stopping topical cooling. Although the lungs of the in situ groups were ventilated during the entire flush preservation, ventilation in the ex situ study groups was stopped prior to topical cooling by instillation of cold saline into the pleura. In all groups, the left lung was separated and the left main bronchus was clamped. Lung grafts of both in situ control groups were stored semi-inflated, in contrast to the lungs of both ex situ study groups, which were stored in an atelectatic state. All lungs were stored for 24 hours at 41C in the corresponding preservation solution and were then transplanted.
Porcine left-sided lung transplantation Recipient female pigs (weight 24 to 30 kg) were anesthetized and prepared as described elsewhere.15,16,18 The intubated animals were ventilated in a pressure-controlled mode (airway peak pressure 30 cm H2O, PEEP 5 cm H2O, I/E ratio 1:1, ventilation rate 10 breaths/ min) with FIO2 0.5 throughout the entire observation period. Recipient systemic arterial and central venous pressures were monitored by catheters inserted into the right carotid artery and jugular vein. Pulmonary artery hemodynamic was assessed via a
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The Journal of Heart and Lung Transplantation, Vol 32, No 8, August 2013 Assessment of parameters in 30-minutes intervals Contralateral clamping
24 hours ischemia Reperfusion
Stop of ventilation Topical cooling insitu 30 min
Bronchoscopy I Baseline parameters
Study groups
Graft recovery
Preparing of the recipient animal Transplantation
Antegrade flush perfusion
Bronchoscopy II
Termination
Stop of ventilation 0
Graft recovery Antegrade flush perfusion in-situ
Day 1
1
2
3
4
5
6
Control groups
7
8
9
10 [hours]
Day 2
Figure 1 Depiction of the experimental timeline. Pulmonary graft recovery of both the control and study groups was performed at day 1 followed by a 24-hour period of cold static ischemia and subsequent pulmonary transplantation at day 2. Swan–Ganz catheter. Cardiac output, extravascular lung water and intrathoracic total blood volume were recorded by a transpulmonary thermodilution catheter placed into the right femoral artery and connected to the recording device (PICCO; Pulsion Medical Systems AG, Munich, Germany). Access to the left pleura and the transplant procedure have been described previously.15,16,18 Fifteen minutes after reperfusion, the right pulmonary artery and right bronchus were clamped to warrant dependency on the grafted lung. The observation period was 7 hours after initiation of reperfusion. After the 7-hour observation period, or when systolic systemic blood pressure fell below 40 mm Hg, the observation period was terminated.
Statistical analysis Data analysis was executed with PASW for Windows, version 17.0.2 (SPSS, Inc., Chicago, IL). All data are expressed as mean ⫾ standard deviation (SD). Repeated measures analysis of variance (ANOVA) followed by Bonferroni’s post hoc test was applied to analyze continuous data. Non-continuous data were analyzed by 1way ANOVA. p o 0.05 was considered statistically significant. The findings from the 4 animals that died before the end of the 7hour reperfusion were included in the data analyses up to the point at which death occurred.
Results Assessment of hemodynamic parameters and lung function All hemodynamic parameters were recorded online. Dynamic ventilation parameters were measured continuously with a modified ventilator. Left atrial and central venous blood gas analyses were carried out after positioning of the catheters and in 30-minute intervals during the entire observation period. At the same intervals, the PICCO recording device was calibrated by 3 repeated bolus injections of 10 ml of 41C cold saline (0.9%) into the central venous catheter. At this point, the device calculated cardiac output and systemic vascular resistance online by pressure curve analysis and computed intrathoracic total blood volume and extravascular lung water (EVLW).19,20 Pulmonary vascular resistance (PVR) was calculated from cardiac output and pulmonary artery pressures. The experimental timeline of this model is depicted in Figure 1.
Animal survival Mean hemodynamic and respiratory parameters in donor animals before graft recovery were not different among groups. Four of 6 animals each from each of the in situ control groups survived the complete 7-hour observation period. The cause of death in all 4 animals that died before the end of 7 hours of reperfusion was right heart failure after profound pulmonary edema. The wet-to-dry ratio of the 4 dead control animals was higher (LPD, n ¼ 2, 8.93 ⫾ 0.58; Celsior, n ¼ 2, 8.92 ⫾ 0.89) compared with control animals that survived the entire observation period (LPD 7.62 ⫾ 0.43; Celsior 7.89 ⫾ 0.41). All animals of both ex situ study groups survived the entire observation period.
Cardiac output Wet-to-dry lung weight ratio Immediately after termination of the experiment the lingual segments of the transplanted lung were resected. This protocol was applied in all 4 groups to establish comparable conditions. All specimens were freshly weighed by a precision scale (increment 1 10−4 g) and then stored in an oven at 651C for 72 hours for complete drying. The dehydrated lung portion was again weighted using the same scale. The ratio of the weight before and after the drying procedure was calculated.
Mean cardiac output (Celsior 2.9 ⫾ 0.8 liters/min and exCel 2.6 ⫾ 0.3 liters/min at 7 hours after reperfusion) showed a trend toward higher values in both Celsior groups (p 4 0.05) compared with both LPD cohorts. The cardiac output in both Celsior groups declined somewhat throughout the observation period. Cardiac output in the LPD group (2.3 ⫾ 0.7 liters/min at 7 hours after reperfusion) remained significantly lower over time compared with all other groups (p o 0.001). Cardiac output in the exLPD
Gohrbandt et al.
Topical Cooling in Single Lung Tx
group (2.3 ⫾ 0.6 liters/min at 7 hours after reperfusion) revealed significantly lower levels compared with Celsior (p o 0.001) or exCel (p o 0.01). Mean cardiac output of both Celsior groups reflected equivalent results. Systemic vascular resistance and systemic arterial and central venous pressures were not statistically different among the groups.
significantly higher results compared with all other groups (p o 0.001).oFIG ID ¼ 34 Results of both control groups and exLPD were comparable over time. In the second half of the observation period the mean oxygenation ratios of all groups converged.
Wet-to-dry lung weight ratio
Pulmonary artery pressure Mean pulmonary artery pressures (LPD 41 ⫾ 9, Celsior 28 ⫾ 2, exLPD 34 ⫾ 5 and exCel 26 ⫾ 2 mm Hg at 7 hours after reperfusion) indicated significantly higher levels in both LPD groups in contrast to both Celsior groups over time (LPD vs Celsior and LPD vs exCel, p o 0.001; exLPD vs excel, p o 0.01; exLPD vs Celsior, p o 0.05).
Pulmonary vascular resistance Mean PVR (Figure 2) (LPD 1,310 ⫾ 620, Celsior 584 ⫾ 194, exLPD 1,035 ⫾ 361 and exCel 650 ⫾ 116 dyn/s/cm5 at 7 hours after reperfusion) was significantly elevated in the LPD group compared to the other groups (p o 0.001). oFIG ID ¼ 24 exLPD showed reduced results compared with LPD (p o 0.001), but elevated PVR levels in contrast to both Celsior groups (p o 0.001). Both Celsior groups showed comparable results at a lower level.
Lung compliance Dynamic lung compliance (LPD 16 ⫾ 4, Celsior 17 ⫾ 6, exLPD 21 ⫾ 4 and exCel 21 ⫾ 4 ml/mbar at 7 hours after reperfusion) reflected significantly improved results in the exCel group compared with both control groups (p o 0.001) and was equivalent to the exLPD group. Results in both control groups were inferior when compared with combined preservation.
Oxygenation index Mean oxygenation indices (Figure 3) (LPD 341 ⫾ 133, Celsior 387 ⫾ 86, exLPD 327 ⫾ 76 and exCel 417 ⫾ 81 at 7 hours after reperfusion) in the exCel group indicated 2500
Wet-to-dry lung weight ratio (Figure 4) was significantly reduced in the exCel group (3.1 ⫾ 0.9) compared with all other groups (LPD 8.1 ⫾ 0.8, Celsior 8.4 ⫾ 0.8 and exLPD 7.5 ⫾ 1.0; p o 0.001), although the latter 3 groups did not differ significantly from one another.
Extravascular lung water Extravascular lung water (EVLW; LPD 238 ⫾ 21, Celsior 256 ⫾ 36, exLPD 304 ⫾ 61 and exCel 193 ⫾ 42 at 7 hours after reperfusion) corroborated the results seen for wet-todry weight ratios (Figure 5). Except for LPD vs Celsior, all other differences were significant (p o 0.001 for LPD vs exCel, Celsior vs exCel and exLPD vs exCel; p o 0.05 for LPD vs exLPD and Celsior vs exLPD).
Bronachoalveolar lavage fluid Neutrophil cell count from bronchoalveolar lavage fluid (BALF) (Figure 6) obtained 2 hours after reperfusion, reflecting neutrophil infiltration of the graft, revealed a low baseline for native lungs. Neutrophil cell count was elevated in all groups with the lowest result in the exCel group. The results were not statistically significant.
Discussion We developed our pre-clinical porcine left lung transplantation model to assess pulmonary preservation strategies. The 24-hour period prior to implantation of the graft ensures a sufficiently injured graft, and thereby allows evaluation of graft preservation in small groups of animals.5,16,18,21
2000
ex-LPD ex-Cel
1500 1000 500
LPD
550
LPD Cel
pO2/FiO2
PVR [dyn*s*cm-5]
835
Cel
450
ex-LPD ex-Cel
350 250 150
0
Clamping of the right lung
Clamping of the right lung 0
1
2 3 4 5 6 Reperfusion time [h]
7
Figure 2 The LPD control group reveals a significantly increased pulmonary vascular resistance (PVR; p ¼ 0.001). Both Celsior groups show lower PVR (p ¼ 0.001) compared with the study group exLPD.
0
1
2 3 4 5 6 Reperfusion time [h]
7
Figure 3 Oxygenation index (PO2/FIO2) indicates enhanced gas exchange for the exCel study group (p ¼ 0.001) over the observation period. Oxygenation index of all other groups are comparable.
The Journal of Heart and Lung Transplantation, Vol 32, No 8, August 2013 80
Extravasal lung water
Since experimental results indicated improved and safe lung graft preservation by low-potassium dextran (LPD) solution 20 years ago, the majority of transplant programs have altered their preservation strategy to LPD solution. Besides the extracellular potassium concentration, dextran is considered to be the major active component in prevention of cell swelling.22,23 More recently, Celsior solution has revealed protective effects in pulmonary preservation, although it was originally considered a cardiac preservation solution. Because damage to the alveolocapillary membrane after lung transplantation is believed to be largely attributed to oxygen-derived free radicals, the glutathione contained in Celsior solution could act as a radical scavenger, providing a potential mechanism of improved preservation. It combines the general principles of hypothermic organ preservation with prevention of cell swelling, oxygen-derived free radical injury and depletion of high-energy triphosphates. Celsior had been used clinically as graft preservation in smaller clinical cohorts for pulmonary, hepatic, renal and pancreatic organ protection. It has been shown to be sufficient and safe as a standard protocol for each organ.22,24 In the current clinical stages of using NHBD lungs for human transplantation, there is no accepted standard procedure in the donation and recovery process. Heparinization has not been performed consistently. Mean warm
LPD Cel ex-LPD ex-Cel
400 300 200 100 Clamping of the right lung 0 0
1
2 3 4 5 Reperfusion time [h]
6
7
Figure 5 Extravascular lung water (EVLW) supported the observation seen in wet-to-dry weight ratio indicating a beneficial trend in the exCel study group (p o 0.001). EVLW was comparable between LPD and Celsior control groups (p o 0.05).
el in e
Figure 4 Seven hours after reperfusion, the wet-to-dry lung weight ratio from specimens represent a significantly decreased fluid accumulation over the post-transplant reperfusion period for the exCel study group (p ¼ 0.001), whereas the weight ratios between all other groups are comparable.
0
as
ex-Cel
el
ex-LPD
B
Cel
-C
LPD
20
ex
3.1±0.9
D
3
40
-L P
5
ex
7.5± 7.5±1.0
n.s. 60
el
8.1± 8.1±0.8
7
8.4± 8.4±0.8
LP D
9
BALF neutrophils [%]
Wet-to-dry lung weight ratio
p<0.001
C
836
Figure 6 Neutrophil cell counts from bronchoalveolar lavage fluid (BALF) obtained 2 hours after reperfusion reveal elevated values in all 4 groups, whereas presence of neutrophils was the lowest in the ex-Cel group. Baseline reflects neutrophil cell count in BALF obtained from native right lungs. There were no statistically significant differences between groups (n.s., not significant).
ischemic times have been ≤29 minutes. Regular antegrade and, occasionally, retrograde flush preservation were used. Early and mid-term survival and outcome up to 2 years are not inferior compared with cadaveric heart-beating lung donation.25,26 In the persent study we evaluated lung graft function following a consistent protocol in the setting similarly used as topical cooling in NHBD. Our study reflected beneficial results for the combined topical cooling and backtable flush preservation strategy. Similarly, approaches for graft preservation in living related lobar lung transplantation are widely divergent. Initiation of retro- or antegrade preservation or both follows a period of warm ischemia and mechanical manipulation attributable to dissecting the lobar structures. In addition, ischemic times of living related lobar lung grafts are very short compared with grafts recovered from deceased donors. The decreased tissue temperature may only be achieved by backtable flush preservation and very limited cold storage prior to placing the graft in the recipient’s chest for implantation, which later reveals a second warm ischemic period.10,12–14 Sufficient cooling of the entire graft may not be realized in this procedure. Pre-clinical data in several settings have indicated controversial effects of preservation by elevated temperatures of 401C or very low temperatures of o41C for flush solution and storage. Consecutively, aiming toward a rapid decrease in the graft temperature of between 41 and 81C may be recommended.27 This study was aimed at finding a feasible and capable cooling strategy within the pleural cavity and discovering the consequences for pulmonary protection, primarily in living related donation. Tissue temperature in atelectatic lungs decreases faster than in ventilated lungs if cooled externally. In addition, in heart-beating deceased donors, this strategy may offer beneficial effects in multi-organ donation. It allows for interrupting mechanical ventilation and immersing the atelectatic lungs in cold saline within the pleura until termination of organ flush perfusion and cardiac
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Topical Cooling in Single Lung Tx
recovery, rather than continuing ventilation of the lung with a room temperature gas/air mixture and thereby effectively warming it again after termination of cold flush during surgical recovery of the organ block. Topical cooling as separate preservation or in combination with flush techniques revealed sufficient pulmonary preservation in various settings pre-clinically. The vast majority of experiments mimic non–heart-beating donor (NHBD) settings.4,7–9,21,28,29 van de Wauwer and colleagues8 reported beneficial effects after retrograde compared with antegrade perfusion after a prolonged warm ischemia time of 1 hour and topical cooling for an additional 2.5 hours in an ex vivo model of 1-hour reperfusion. In this study, flush perfusion was administered antegradely, whereas retrograde perfusion was not assessed. The graft was not exposed to warm ischemia and the duration of topical cooling was limited to 30 minutes. However, this nearly reflects the clinical situation described by van de Wauwer et al, who reported short warm ischemic times of 29 minutes26 or DeVleeschauwer, who had a warm ischemic time of 15 minutes25 in contrast to extended warm ischemia for 41 hour as described in pre-clinical settings.8,9,28 Preclinical data also suggest not extending the warm ischemia time of atelectatic lungs to 41 hour, so as to ensure sufficient graft function.5 This suggests that lung graft function would not be adversely affected by warm ischemia times of ≤1 hour. Baretti and colleagues showed that atelectases of the lungs led to a significant maldistribution of the preservation solution, regardless of the route of delivery.30 In contrast, in our experimental setting, combined preservation by topical cooling of the atelectatic lung followed by ex situ antegrade flush preservation demonstrated better potential for early post-transplant graft function. The combined preservation improved functional graft parameters compared with the appropriate control group in our experiments. Compared with a previous study evaluating topical cooling in contrast to regular antegrade flush preservation,21 post-transplant graft function here was also improved by the combined strategy reflected by lower levels of pulmonary vascular resistance (PVR), elevated dynamic lung compliance and improved oxygenation index. Mechanisms for improved graft function achieved by the combination of topical cooling and backtable Celsior flush in comparison to all other experimental groups remain elusive. Locke et al suggested that topical cooling alone does not offer optimal lung preservation compared with antegrade flush perfusion with Euro-Collins.31 In their study, both strategies were combined to take advantage of the beneficial effects of each strategy employing extracellular preservation solutions. The combination of both preservation strategies investigated by Locke may reflect an additional effect contributing to an enhancement of graft preservation mirrored by functional parameters. Moreover, the combined preservation technique by means of in-situ topical cooling and ex-situ antegrade flush itself may result in a favorable lung preservation compared to the conventional antegrade flush only. This benefit may potentially be attributable to prevent the rewarming by continued ventilation with room temperature gas/air mixture. This effect
837 may also reveal differences between the two preservation solutions with regard to the components and diverse attributable effects. The significantly reduced wet-to-dry weight ratio and decreased extravascular lung water representing less fluid accumulation over the post-transplant reperfusion period and beneficially altered PVR, the significantly enhanced oxygenation index, and lung compliance were indicators of excellent graft preservation in the exCel group. Experimentally, Celsior had a superior effect on decreasing microvascular permeability as expressed by the reduced capillary filtration coefficient.32 The compounds of Celsior addressing radical scavenging, nutritional demands and pH leveling may support improved preservation of epithelial cell integrity and cellular protection,33 in addition to topical cooling in this setting. Although 2 animals in each control group died of right ventricular failure caused by the ischemia–reperfusion (IR)injury–associated increase in PVR, all animals in both study groups survived the entire observation period, indicating sufficient and improved preservation of the lung grafts after a prolonged ischemic time. This indicates safe and sufficient preservation by both combined strategies of topical cooling and ex situ antegrade flush preservation. We conclude that a combined preservation strategy may advance post-transplant lung graft function in living related lobar lung donation, but possibly also from standard cadaveric heart-beating donors or NHBDs. The application of this simple modification of lung preservation is feasible and could advance to the clinical setting.
Disclosure statement Perfadex solution was granted by Vitrolife, Sweden, and Celsior solution was donated by Genzyme Germany for this study performed by BG, MA, GW and SPS. BG was sponsored by Genzyme Europe for an invited lecture at the 5th ETCOSymposium in Riga, Letvia, in October 2008. SPS has received LPD solution from Vitrolife, Sweden, as a research support. There are no further financial or other relationships to disclose in the subject of this manuscript.
The authors thank Ms. Ilona Maeding for assisting the statistical analysis as a biostatistician. This work was supported by the German Research Foundation.
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32.
33.
compartments, extravascular lung water and liver function. Chest 1998;113:1070-7. Kutschka I, Sommer SP, Hohlfeld JM, et al. In-situ topical cooling of lung grafts: early graft function and surfactant analysis in a porcine single lung transplant model. Eur J Cardiothorac Surg 2003;24: 411-9. Thabut G, Vinatier I, Brugiere O, et al. Influence of preservation solution on early graft failure in clinical lung transplantation. Am J Respir Crit Care Med 2001;164:1204-8. Fischer S, Matte-Martyn A, de Perrot M, et al. Low-potassium dextran preservation solution improves lung function after human lung transplantation. J Thorac Cardiovasc Surg 2001;121:594-6. Karam G, Compagnon P, Hourmant M, et al. A single solution for multiple organ procurement and preservation. Transpl Int 2005;18: 657-63. DeVleeschauwer S, Wauters S, Dupont LJ, et al. Medium-term outcome after lung transplantation is comparable between brain-dead and cardiacdead donors. J Heart Lung Transplant 2011;30:975-81. van de Wauwer C, Verschuuren EAM, van der Bij W, et al. The use of non-heart beating lung donors category III can increase the donor pool. Eur J Cardiothorac Surg 2011;39:e175-80. de Perrot M, Keshavjee S. Lung preservation. Semin Thorac Cardiovasc Surg 2004;16:300-8. Steen S, Sjöberg T, Pierre L, et al. Transplantation of lungs from a nonheart beating donor. Lancet 2001;357:825-9. Steen S, Sjoberg T, Ingemannson R, et al. Efficacy of topical cooling in lung preservation: is a reappraisal due? Ann Thorac Surg 1994;58: 1657-63. Baretti R, Bitu Moreno J, Beyersdorf F, et al. Distribution of lung preservation solutions in parenchyma and airways: influence of atelectasis and route of delivery. J Heart Lung Transplant 1995;14: 80-91. Locke TJ, Hooper TL, Flecknell PA, et al. Preservation of the lung. Comparison of topical cooling and crystalloid pulmonary perfusion. J Thorac Cardiovasc Surg 1988;96:789-95. Roberts RF, Nishanian GP, Carey JN, et al. A comparison of the new preservation solution Celsior to Euro-Collins and University of Wisconsin solution in lung reperfusion injury. Transplantation 1999;67: 152-5. Xiong, Legagneux J, Wassef M, et al. Protective effects of Celsior in lung transplantation. J Heart Lung Transplant 1999;18:320-7.
Initial topical cooling followed by backtable Celsior flush perfusion provides excellent early graft function in porcine single lung transplantation after 24 hours of cold ischemia Bernhard Gohrbandt, MD,a,d Murat Avsar, MD,a Gregor Warnecke, MD,a Sebastian-Patrick Sommer, MD,b Axel Haverich, MD,a and Martin Strueber, MDc From the aHannover Thoracic Transplant Program, Department of Cardiac, Thoracic, Transplantation and Vascular Surgery, Hannover Medical School, Hannover; bDepartment of Thoracic and Cardiovascular Surgery, University Hospital Wuerzburg, Wuerzburg; c Department of Cardiac Surgery, University of Leipzig Heart Centre, Leipzig; and the dDepartment of Cardiovascular and Thoracic Surgery, Medical Centre of the Johannes Gutenberg University Mainz, Mainz, Germany.
KEYWORDS: preclinical lung transplantation; lung preservation; low potassium dextran (LPD); Celsior; graft function
BACKGROUND: Topical in situ cooling of the donor lungs is a prerequisite for procurement of non– heart-beating donor lungs and may be of interest for living related lung donation. METHODS: Twenty-four single lung transplants were performed in 4 groups of Landrace pigs (6 per group). Control LPD, control Celsior and topical cooling in situ, followed by LPD (exLPD) or Celsior (exCel) ex situ flush, were employed. All lungs were perfused antegrade with 1 liter of solution at 41C. Lungs were stored immersed in preservation solution for 24 hours at 41C. After transplantation of the left lung, the right recipient bronchus and pulmonary artery were clamped. RESULTS: Four of 6 animals each in the LPD and Celsior groups and all 6 animals in both the exLPD and the exCel groups survived the 7-hour reperfusion. The mean oxygenation index was favorably preserved in the exCel group at 7 hours after reperfusion (417 ⫾ 81) over all other groups (LPD 341 ⫾ 133, Celsior 387 ⫾ 86, exLPD 327 ⫾ 76; p o 0.0001). Pulmonary vascular resistance showed significantly lower values in the Celsior and exCel groups (LPD 1,310 ⫾ 620, Celsior 584 ⫾ 194, exLPD 1,035 ⫾ 361, exCel 650 ⫾ 116 dyn/s/cm5 at 7 hours after reperfusion; p o 0.0001). Consistently, the wet-to-dry lung weight ratio also indicated beneficial graft protection in the exCel group (LPD 8.1 ⫾ 0.8, Celsior 8.4 ⫾ 0.8, exLPD 7.5 ⫾ 1.0, exCel 3.1 ⫾ 0.9; p o 0.0001). CONCLUSION: Initial topical cooling followed by backtable perfusion is a sufficient technique for pulmonary graft preservation providing excellent post-transplant function. Celsior subsequent to in-situ topical cooling revealed the most beneficial results in this setting. This combined technique could advance non–heart-beating, living related lung lobe donation and, potentially, regular heart-beating lung donation. J Heart Lung Transplant 2013;32:832–838 r 2013 International Society for Heart and Lung Transplantation. All rights reserved.
More than 40,000 lung transplants have been reported to the registry of the International Society for Heart and Lung Transplantation (ISHLT) since 1989.1 The demand Reprint requests: Gregor Warnecke, MD, Hannover Thoracic Transplant Program, Department of Cardiac, Thoracic, Transplantation and Vascular Surgery, Hannover Medical School, Carl-Neuberg-Strasse 1, 30623 Hannover, Germany. Tel.: þ49 511 532 3373; fax: þ49 511 532 5625. E-mail address: [email protected]
for lung transplantation continues to increase and a considerable donor organ shortage remains. Lung donor criteria for acceptance have already been extended. Finding alternative sources of organs continues to be a major priority.1–3 Use of non–heart-beating organ donors (NHBDs) has been widely investigated, especially with regard to acceptable warm ischemia times, increased periods of in situ topical cooling, and issues of additional flush preservation.4–9
1053-2498/$ - see front matter r 2013 International Society for Heart and Lung Transplantation. All rights reserved. http://dx.doi.org/10.1016/j.healun.2013.06.005
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The concept of living related lung lobe donation and transplantation has been described. During graft recovery, the lobar graft may be exposed to warm ischemia in situ. Antegrade, retrograde or no-flush preservation has been employed backtable thereafter.3,10–14 Either topical cooling or flush perfusion of the lungs is accepted as safe and sufficient in NHB, living related lobar lung or deceased donor organ donation. Experimental data have revealed beneficial effects combining these approaches.8,9 The purpose of this study was to evaluate early graft function after 24 hours of cold ischemic time after topical cooling and subsequent ex situ antegrade flush preservation in comparison to conventional antegrade flush preservation only, aiming toward an effective strategy for living related donation. Because our own pre-clinical data reflect safe and sufficient graft preservation with Celsior (Cel),15–18 we evaluated two clinically employed preservation solutions: low-potassium dextran (LPD) solution and Celsior. We hypothesized that initial topical cooling followed by backtable Celsior flush perfusion is a preservation technique that offers good graft function.
Methods Animal care All animals received humane care in compliance with the “Principles of Laboratory Animal Care” and the Guide for the Care of Laboratory Animals, prepared by the Institute of Laboratory Animal Resources, National Research Council (National Academy Press, revised 1996), as well as ibeing n compliance with the European Convention on Animal Care.
Donor procedure As preservation solutions, either LPD (Perfadex; Vitrolife, Gothenburg, Sweden) or Celsior (Genzyme Germany, Neu-Isenburg, Germany) were used. LPD was supplemented by 0.3 ml tris hydroxymethyl aminomethane (THAM) buffer/liter. The components of the preservation solutions used are listed in Table 1. Animals were assigned to 4 groups of 6 animals each. Induction and maintenance of anesthesia was performed as described elsewhere.15,16,18 The donor animals (weight 27 to 32 kg) were ventilated in a pressure-controlled mode (inspiratory peak pressure 30 cm H2O, positive end-expiratory pressure [PEEP] 5 cm H2O, fraction of inspired oxygen [FIO2] 1.0, inspiration/expiration [I/E] ratio 1:1, ventilation rate 10 breaths/min). After median sternotomy, the pericardium and both pleural cavities were opened. The heart and lungs were isolated. Then 250 mg of methylprednisolone and 7,500 IU of heparin were administered systemically.
Control groups For LPD and Celsior controls, a cannula was inserted into the main trunk of the pulmonary artery and secured with a ligature. Simultaneously to cardiac inflow occlusion and drainage of the left heart, the pulmonary artery was clamped centrally of the
833 Table 1
Composition of the Preservation Solutions Used
Component þ
Na (mmol/liter) Kþ (mmol/liter) Cl− (mmol/liter) Ca2þ (mmol/liter) Mg2þ (mmol/liter) PO3− 4 (mmol/liter) SO2− 4 (mmol/liter) Histidine (mmol/liter) Mannitol (mmol/liter) Dextran-40 (g/liter) Glucose (g/liter) Glutamic acid (mmol/liter) Lactobionic acid (mmol/liter) Glutathione (mmol/liter) Osmolality (mOsm/liter) pH
LPD solution
Celsior
138 6 142 0 0 0.8 2 0 0 50 0.91 0 0 0 292 7.4
100 15 100 0.25 13 0 0 30 60 0 0 20 80 3 320 7.3
cannula. Subsequently, in-situ antegrade flush preservation via this cannula was initiated with 1 liter of 41C cold preservation solution. The perfusion pressure was controlled by a separate catheter and mean pressure was retained at 10 mm Hg by adjusting the static heights in which the containment was hung. Ventilation of the lungs was maintained throughout the entire preservation period. Finally, the trachea was clamped and the heart–lung block was excised.
Study groups Fibrillation of the heart was electrically induced, which led to circulatory arrest. Ventilation was stopped simultaneously. The atelectatic lungs were immersed in 3 liters of saline at 41C within the pleural cavities for 30 minutes. The trachea was clamped and the heart and lungs were then excised en bloc. After separating the heart and lungs the pulmonary artery was clamped. The lungs were antegradely flush preserved with 1 liter of either buffered LPD (exLPD) or Celsior (exCel) ex situ, approximately 3 to 5 minutes after stopping topical cooling. Although the lungs of the in situ groups were ventilated during the entire flush preservation, ventilation in the ex situ study groups was stopped prior to topical cooling by instillation of cold saline into the pleura. In all groups, the left lung was separated and the left main bronchus was clamped. Lung grafts of both in situ control groups were stored semi-inflated, in contrast to the lungs of both ex situ study groups, which were stored in an atelectatic state. All lungs were stored for 24 hours at 41C in the corresponding preservation solution and were then transplanted.
Porcine left-sided lung transplantation Recipient female pigs (weight 24 to 30 kg) were anesthetized and prepared as described elsewhere.15,16,18 The intubated animals were ventilated in a pressure-controlled mode (airway peak pressure 30 cm H2O, PEEP 5 cm H2O, I/E ratio 1:1, ventilation rate 10 breaths/ min) with FIO2 0.5 throughout the entire observation period. Recipient systemic arterial and central venous pressures were monitored by catheters inserted into the right carotid artery and jugular vein. Pulmonary artery hemodynamic was assessed via a
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The Journal of Heart and Lung Transplantation, Vol 32, No 8, August 2013 Assessment of parameters in 30-minutes intervals Contralateral clamping
24 hours ischemia Reperfusion
Stop of ventilation Topical cooling insitu 30 min
Bronchoscopy I Baseline parameters
Study groups
Graft recovery
Preparing of the recipient animal Transplantation
Antegrade flush perfusion
Bronchoscopy II
Termination
Stop of ventilation 0
Graft recovery Antegrade flush perfusion in-situ
Day 1
1
2
3
4
5
6
Control groups
7
8
9
10 [hours]
Day 2
Figure 1 Depiction of the experimental timeline. Pulmonary graft recovery of both the control and study groups was performed at day 1 followed by a 24-hour period of cold static ischemia and subsequent pulmonary transplantation at day 2. Swan–Ganz catheter. Cardiac output, extravascular lung water and intrathoracic total blood volume were recorded by a transpulmonary thermodilution catheter placed into the right femoral artery and connected to the recording device (PICCO; Pulsion Medical Systems AG, Munich, Germany). Access to the left pleura and the transplant procedure have been described previously.15,16,18 Fifteen minutes after reperfusion, the right pulmonary artery and right bronchus were clamped to warrant dependency on the grafted lung. The observation period was 7 hours after initiation of reperfusion. After the 7-hour observation period, or when systolic systemic blood pressure fell below 40 mm Hg, the observation period was terminated.
Statistical analysis Data analysis was executed with PASW for Windows, version 17.0.2 (SPSS, Inc., Chicago, IL). All data are expressed as mean ⫾ standard deviation (SD). Repeated measures analysis of variance (ANOVA) followed by Bonferroni’s post hoc test was applied to analyze continuous data. Non-continuous data were analyzed by 1way ANOVA. p o 0.05 was considered statistically significant. The findings from the 4 animals that died before the end of the 7hour reperfusion were included in the data analyses up to the point at which death occurred.
Results Assessment of hemodynamic parameters and lung function All hemodynamic parameters were recorded online. Dynamic ventilation parameters were measured continuously with a modified ventilator. Left atrial and central venous blood gas analyses were carried out after positioning of the catheters and in 30-minute intervals during the entire observation period. At the same intervals, the PICCO recording device was calibrated by 3 repeated bolus injections of 10 ml of 41C cold saline (0.9%) into the central venous catheter. At this point, the device calculated cardiac output and systemic vascular resistance online by pressure curve analysis and computed intrathoracic total blood volume and extravascular lung water (EVLW).19,20 Pulmonary vascular resistance (PVR) was calculated from cardiac output and pulmonary artery pressures. The experimental timeline of this model is depicted in Figure 1.
Animal survival Mean hemodynamic and respiratory parameters in donor animals before graft recovery were not different among groups. Four of 6 animals each from each of the in situ control groups survived the complete 7-hour observation period. The cause of death in all 4 animals that died before the end of 7 hours of reperfusion was right heart failure after profound pulmonary edema. The wet-to-dry ratio of the 4 dead control animals was higher (LPD, n ¼ 2, 8.93 ⫾ 0.58; Celsior, n ¼ 2, 8.92 ⫾ 0.89) compared with control animals that survived the entire observation period (LPD 7.62 ⫾ 0.43; Celsior 7.89 ⫾ 0.41). All animals of both ex situ study groups survived the entire observation period.
Cardiac output Wet-to-dry lung weight ratio Immediately after termination of the experiment the lingual segments of the transplanted lung were resected. This protocol was applied in all 4 groups to establish comparable conditions. All specimens were freshly weighed by a precision scale (increment 1 10−4 g) and then stored in an oven at 651C for 72 hours for complete drying. The dehydrated lung portion was again weighted using the same scale. The ratio of the weight before and after the drying procedure was calculated.
Mean cardiac output (Celsior 2.9 ⫾ 0.8 liters/min and exCel 2.6 ⫾ 0.3 liters/min at 7 hours after reperfusion) showed a trend toward higher values in both Celsior groups (p 4 0.05) compared with both LPD cohorts. The cardiac output in both Celsior groups declined somewhat throughout the observation period. Cardiac output in the LPD group (2.3 ⫾ 0.7 liters/min at 7 hours after reperfusion) remained significantly lower over time compared with all other groups (p o 0.001). Cardiac output in the exLPD
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Topical Cooling in Single Lung Tx
group (2.3 ⫾ 0.6 liters/min at 7 hours after reperfusion) revealed significantly lower levels compared with Celsior (p o 0.001) or exCel (p o 0.01). Mean cardiac output of both Celsior groups reflected equivalent results. Systemic vascular resistance and systemic arterial and central venous pressures were not statistically different among the groups.
significantly higher results compared with all other groups (p o 0.001).oFIG ID ¼ 34 Results of both control groups and exLPD were comparable over time. In the second half of the observation period the mean oxygenation ratios of all groups converged.
Wet-to-dry lung weight ratio
Pulmonary artery pressure Mean pulmonary artery pressures (LPD 41 ⫾ 9, Celsior 28 ⫾ 2, exLPD 34 ⫾ 5 and exCel 26 ⫾ 2 mm Hg at 7 hours after reperfusion) indicated significantly higher levels in both LPD groups in contrast to both Celsior groups over time (LPD vs Celsior and LPD vs exCel, p o 0.001; exLPD vs excel, p o 0.01; exLPD vs Celsior, p o 0.05).
Pulmonary vascular resistance Mean PVR (Figure 2) (LPD 1,310 ⫾ 620, Celsior 584 ⫾ 194, exLPD 1,035 ⫾ 361 and exCel 650 ⫾ 116 dyn/s/cm5 at 7 hours after reperfusion) was significantly elevated in the LPD group compared to the other groups (p o 0.001). oFIG ID ¼ 24 exLPD showed reduced results compared with LPD (p o 0.001), but elevated PVR levels in contrast to both Celsior groups (p o 0.001). Both Celsior groups showed comparable results at a lower level.
Lung compliance Dynamic lung compliance (LPD 16 ⫾ 4, Celsior 17 ⫾ 6, exLPD 21 ⫾ 4 and exCel 21 ⫾ 4 ml/mbar at 7 hours after reperfusion) reflected significantly improved results in the exCel group compared with both control groups (p o 0.001) and was equivalent to the exLPD group. Results in both control groups were inferior when compared with combined preservation.
Oxygenation index Mean oxygenation indices (Figure 3) (LPD 341 ⫾ 133, Celsior 387 ⫾ 86, exLPD 327 ⫾ 76 and exCel 417 ⫾ 81 at 7 hours after reperfusion) in the exCel group indicated 2500
Wet-to-dry lung weight ratio (Figure 4) was significantly reduced in the exCel group (3.1 ⫾ 0.9) compared with all other groups (LPD 8.1 ⫾ 0.8, Celsior 8.4 ⫾ 0.8 and exLPD 7.5 ⫾ 1.0; p o 0.001), although the latter 3 groups did not differ significantly from one another.
Extravascular lung water Extravascular lung water (EVLW; LPD 238 ⫾ 21, Celsior 256 ⫾ 36, exLPD 304 ⫾ 61 and exCel 193 ⫾ 42 at 7 hours after reperfusion) corroborated the results seen for wet-todry weight ratios (Figure 5). Except for LPD vs Celsior, all other differences were significant (p o 0.001 for LPD vs exCel, Celsior vs exCel and exLPD vs exCel; p o 0.05 for LPD vs exLPD and Celsior vs exLPD).
Bronachoalveolar lavage fluid Neutrophil cell count from bronchoalveolar lavage fluid (BALF) (Figure 6) obtained 2 hours after reperfusion, reflecting neutrophil infiltration of the graft, revealed a low baseline for native lungs. Neutrophil cell count was elevated in all groups with the lowest result in the exCel group. The results were not statistically significant.
Discussion We developed our pre-clinical porcine left lung transplantation model to assess pulmonary preservation strategies. The 24-hour period prior to implantation of the graft ensures a sufficiently injured graft, and thereby allows evaluation of graft preservation in small groups of animals.5,16,18,21
2000
ex-LPD ex-Cel
1500 1000 500
LPD
550
LPD Cel
pO2/FiO2
PVR [dyn*s*cm-5]
835
Cel
450
ex-LPD ex-Cel
350 250 150
0
Clamping of the right lung
Clamping of the right lung 0
1
2 3 4 5 6 Reperfusion time [h]
7
Figure 2 The LPD control group reveals a significantly increased pulmonary vascular resistance (PVR; p ¼ 0.001). Both Celsior groups show lower PVR (p ¼ 0.001) compared with the study group exLPD.
0
1
2 3 4 5 6 Reperfusion time [h]
7
Figure 3 Oxygenation index (PO2/FIO2) indicates enhanced gas exchange for the exCel study group (p ¼ 0.001) over the observation period. Oxygenation index of all other groups are comparable.
The Journal of Heart and Lung Transplantation, Vol 32, No 8, August 2013 80
Extravasal lung water
Since experimental results indicated improved and safe lung graft preservation by low-potassium dextran (LPD) solution 20 years ago, the majority of transplant programs have altered their preservation strategy to LPD solution. Besides the extracellular potassium concentration, dextran is considered to be the major active component in prevention of cell swelling.22,23 More recently, Celsior solution has revealed protective effects in pulmonary preservation, although it was originally considered a cardiac preservation solution. Because damage to the alveolocapillary membrane after lung transplantation is believed to be largely attributed to oxygen-derived free radicals, the glutathione contained in Celsior solution could act as a radical scavenger, providing a potential mechanism of improved preservation. It combines the general principles of hypothermic organ preservation with prevention of cell swelling, oxygen-derived free radical injury and depletion of high-energy triphosphates. Celsior had been used clinically as graft preservation in smaller clinical cohorts for pulmonary, hepatic, renal and pancreatic organ protection. It has been shown to be sufficient and safe as a standard protocol for each organ.22,24 In the current clinical stages of using NHBD lungs for human transplantation, there is no accepted standard procedure in the donation and recovery process. Heparinization has not been performed consistently. Mean warm
LPD Cel ex-LPD ex-Cel
400 300 200 100 Clamping of the right lung 0 0
1
2 3 4 5 Reperfusion time [h]
6
7
Figure 5 Extravascular lung water (EVLW) supported the observation seen in wet-to-dry weight ratio indicating a beneficial trend in the exCel study group (p o 0.001). EVLW was comparable between LPD and Celsior control groups (p o 0.05).
el in e
Figure 4 Seven hours after reperfusion, the wet-to-dry lung weight ratio from specimens represent a significantly decreased fluid accumulation over the post-transplant reperfusion period for the exCel study group (p ¼ 0.001), whereas the weight ratios between all other groups are comparable.
0
as
ex-Cel
el
ex-LPD
B
Cel
-C
LPD
20
ex
3.1±0.9
D
3
40
-L P
5
ex
7.5± 7.5±1.0
n.s. 60
el
8.1± 8.1±0.8
7
8.4± 8.4±0.8
LP D
9
BALF neutrophils [%]
Wet-to-dry lung weight ratio
p<0.001
C
836
Figure 6 Neutrophil cell counts from bronchoalveolar lavage fluid (BALF) obtained 2 hours after reperfusion reveal elevated values in all 4 groups, whereas presence of neutrophils was the lowest in the ex-Cel group. Baseline reflects neutrophil cell count in BALF obtained from native right lungs. There were no statistically significant differences between groups (n.s., not significant).
ischemic times have been ≤29 minutes. Regular antegrade and, occasionally, retrograde flush preservation were used. Early and mid-term survival and outcome up to 2 years are not inferior compared with cadaveric heart-beating lung donation.25,26 In the persent study we evaluated lung graft function following a consistent protocol in the setting similarly used as topical cooling in NHBD. Our study reflected beneficial results for the combined topical cooling and backtable flush preservation strategy. Similarly, approaches for graft preservation in living related lobar lung transplantation are widely divergent. Initiation of retro- or antegrade preservation or both follows a period of warm ischemia and mechanical manipulation attributable to dissecting the lobar structures. In addition, ischemic times of living related lobar lung grafts are very short compared with grafts recovered from deceased donors. The decreased tissue temperature may only be achieved by backtable flush preservation and very limited cold storage prior to placing the graft in the recipient’s chest for implantation, which later reveals a second warm ischemic period.10,12–14 Sufficient cooling of the entire graft may not be realized in this procedure. Pre-clinical data in several settings have indicated controversial effects of preservation by elevated temperatures of 401C or very low temperatures of o41C for flush solution and storage. Consecutively, aiming toward a rapid decrease in the graft temperature of between 41 and 81C may be recommended.27 This study was aimed at finding a feasible and capable cooling strategy within the pleural cavity and discovering the consequences for pulmonary protection, primarily in living related donation. Tissue temperature in atelectatic lungs decreases faster than in ventilated lungs if cooled externally. In addition, in heart-beating deceased donors, this strategy may offer beneficial effects in multi-organ donation. It allows for interrupting mechanical ventilation and immersing the atelectatic lungs in cold saline within the pleura until termination of organ flush perfusion and cardiac
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Topical Cooling in Single Lung Tx
recovery, rather than continuing ventilation of the lung with a room temperature gas/air mixture and thereby effectively warming it again after termination of cold flush during surgical recovery of the organ block. Topical cooling as separate preservation or in combination with flush techniques revealed sufficient pulmonary preservation in various settings pre-clinically. The vast majority of experiments mimic non–heart-beating donor (NHBD) settings.4,7–9,21,28,29 van de Wauwer and colleagues8 reported beneficial effects after retrograde compared with antegrade perfusion after a prolonged warm ischemia time of 1 hour and topical cooling for an additional 2.5 hours in an ex vivo model of 1-hour reperfusion. In this study, flush perfusion was administered antegradely, whereas retrograde perfusion was not assessed. The graft was not exposed to warm ischemia and the duration of topical cooling was limited to 30 minutes. However, this nearly reflects the clinical situation described by van de Wauwer et al, who reported short warm ischemic times of 29 minutes26 or DeVleeschauwer, who had a warm ischemic time of 15 minutes25 in contrast to extended warm ischemia for 41 hour as described in pre-clinical settings.8,9,28 Preclinical data also suggest not extending the warm ischemia time of atelectatic lungs to 41 hour, so as to ensure sufficient graft function.5 This suggests that lung graft function would not be adversely affected by warm ischemia times of ≤1 hour. Baretti and colleagues showed that atelectases of the lungs led to a significant maldistribution of the preservation solution, regardless of the route of delivery.30 In contrast, in our experimental setting, combined preservation by topical cooling of the atelectatic lung followed by ex situ antegrade flush preservation demonstrated better potential for early post-transplant graft function. The combined preservation improved functional graft parameters compared with the appropriate control group in our experiments. Compared with a previous study evaluating topical cooling in contrast to regular antegrade flush preservation,21 post-transplant graft function here was also improved by the combined strategy reflected by lower levels of pulmonary vascular resistance (PVR), elevated dynamic lung compliance and improved oxygenation index. Mechanisms for improved graft function achieved by the combination of topical cooling and backtable Celsior flush in comparison to all other experimental groups remain elusive. Locke et al suggested that topical cooling alone does not offer optimal lung preservation compared with antegrade flush perfusion with Euro-Collins.31 In their study, both strategies were combined to take advantage of the beneficial effects of each strategy employing extracellular preservation solutions. The combination of both preservation strategies investigated by Locke may reflect an additional effect contributing to an enhancement of graft preservation mirrored by functional parameters. Moreover, the combined preservation technique by means of in-situ topical cooling and ex-situ antegrade flush itself may result in a favorable lung preservation compared to the conventional antegrade flush only. This benefit may potentially be attributable to prevent the rewarming by continued ventilation with room temperature gas/air mixture. This effect
837 may also reveal differences between the two preservation solutions with regard to the components and diverse attributable effects. The significantly reduced wet-to-dry weight ratio and decreased extravascular lung water representing less fluid accumulation over the post-transplant reperfusion period and beneficially altered PVR, the significantly enhanced oxygenation index, and lung compliance were indicators of excellent graft preservation in the exCel group. Experimentally, Celsior had a superior effect on decreasing microvascular permeability as expressed by the reduced capillary filtration coefficient.32 The compounds of Celsior addressing radical scavenging, nutritional demands and pH leveling may support improved preservation of epithelial cell integrity and cellular protection,33 in addition to topical cooling in this setting. Although 2 animals in each control group died of right ventricular failure caused by the ischemia–reperfusion (IR)injury–associated increase in PVR, all animals in both study groups survived the entire observation period, indicating sufficient and improved preservation of the lung grafts after a prolonged ischemic time. This indicates safe and sufficient preservation by both combined strategies of topical cooling and ex situ antegrade flush preservation. We conclude that a combined preservation strategy may advance post-transplant lung graft function in living related lobar lung donation, but possibly also from standard cadaveric heart-beating donors or NHBDs. The application of this simple modification of lung preservation is feasible and could advance to the clinical setting.
Disclosure statement Perfadex solution was granted by Vitrolife, Sweden, and Celsior solution was donated by Genzyme Germany for this study performed by BG, MA, GW and SPS. BG was sponsored by Genzyme Europe for an invited lecture at the 5th ETCOSymposium in Riga, Letvia, in October 2008. SPS has received LPD solution from Vitrolife, Sweden, as a research support. There are no further financial or other relationships to disclose in the subject of this manuscript.
The authors thank Ms. Ilona Maeding for assisting the statistical analysis as a biostatistician. This work was supported by the German Research Foundation.
References 1. Christie JD, Edwards LB, Kucheryavaya AY, et al. The registry of the International Society for Heart and Lung Transplantation: 29th adult lung and heart lung transplantation report—2012. J Heart Lung Transplant 2012;31:1073-86. 2. Wilkes DS, Egan TM, Reynolds HY. Lung transplantation. Opportunity for research and clinical advancement. Am J Respir Crit Care Med 2005;172:944-55. 3. Maathuis MHJ, Leuvenink HGD, Ploeg RJ. Perspectives in organ transplantation. Transplantation 2007;83:1289-98. 4. Steen S, Ingemansson R, Budrikis A, et al. Succesful transplantation of lungs topically cooled in the non-heart beating donor for 6 hours. Ann Thorac Surg 1997;63:345-51. 5. van Raemdonck DEM, Jannis NCP, DeLyn PRJ, et al. Warm ischemic tolerance in collapsed pulmonary grafts is limited to 1 hour. Ann Surg 1998;228:788-96.
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