Lung transplantation has become a mainstay of therapy for end-stage lung diseases. However, there has been a progressive increase in the number of patients on the waiting list, which continually exceeds the number of available organs. The use of uncontrolled donated after cardiac death (DCD) donors has been employed to resolve this problem (1–3). Warm ischemia inevitably occurs in uncontrolled DCD donors and may cause ischemia-reperfusion injury after transplantation. Severe ischemia-reperfusion injury leads to primary graft dysfunction and remains a significant cause of early morbidity and mortality after lung transplantation (4). The inhibition of ischemia-reperfusion injury is therefore crucial to facilitate lung transplantation from uncontrolled DCD donors.
Warm ischemia impairs the mitochondrial electron transport chain, resulting in decreased adenosine triphosphate (ATP) production, and also decreases the efficacy of the mitochondrial antioxidant system (5, 6). Depending on its severity, the reintroduction of oxygen at reperfusion can lead to a significant production of reactive oxygen species (ROS), which induces the up-regulation of molecules on the cell surface and the release of proinflammatory mediators (4, 7).
Hypothermic machine perfusion (HMP) has been used to preserve kidneys and livers for transplantation, with better results than static cold storage (SCS) (8, 9). HMP is associated with a reduced risk of delayed graft function and improved graft survival compared with SCS. HMP is based on the concept that the oxidative energy production by the mitochondrial electron transport would be sustained under hypothermia (10). We previously demonstrated that short-term HMP, which helped recover the ATP production by the mitochondrial electron transport chain, ameliorated ischemia-reperfusion injury with decreased oxidative damage during reperfusion in an isolated rat lung perfusion model (11).
In the present study, we used a canine transplantation model mirroring the clinical situation to investigate whether short-term HMP could improve the mitochondrial function damaged by warm ischemia and decrease the oxidative damage and production of proinflammatory cytokines during reperfusion, thereby reducing ischemia-reperfusion injury.
Physiologic Lung Functions During HMP
The influent variables (temperature, solutes, pO2, and pCO2 levels) were stable during 120 min HMP. The temperature was maintained at a mean of 9.26°C±0.88°C, ranging from 7.9°C to 10.5°C. There was little variation in any solute during the HMP time (Na+ 144.93±0.70 M, K+ 5.49±0.28 M, and Ca2+ 0.85±0.03 M). The pH, pO2, and pCO2 levels were also maintained at means of 7.20±0.04, 113.73±1.03 mm Hg, and 37.67±5.67 mm Hg, respectively.
The dynamic pulmonary compliance was stable during HMP. The dynamic pulmonary compliance at baseline and after 120 min HMP were 25.77±7.18 and 26.46±7.10 mL/cm H2O, respectively (P=0.76; Fig. 1A). The pulmonary vascular resistance gradually decreased during HMP. The pulmonary vascular resistance after 60, 90, and 120 min HMP significantly decreased in comparison with that at the baseline of HMP (P<0.05; Fig. 1B).
Microthrombi in the Donor Lungs Just Before Transplantation
The biopsy specimens were collected from five donor lungs in the HMP group and four donor lungs in the SCS group. Residual microthrombi in the donor lungs just before transplantation were microscopically assessed to prove the washout effects of HMP. Residual blood cells or blood clots in the capillaries were observed more often in the SCS group (4 of 4 specimens; Fig. 1C) compared with the HMP group (0 of 5 specimens; Fig. 1D).
Lung Tissue ATP Levels
The lung tissue ATP levels were measured before cardiac arrest, after warm ischemia, and 4 hr after reperfusion to evaluate the mitochondrial function. In the HMP group, the lung tissue ATP levels, which decreased during warm ischemia, were significantly improved 4 hr after reperfusion (P<0.05; Fig. 2). Moreover, the lung tissue ATP levels 4 hr after reperfusion were significantly higher in the HMP group than in the SCS group (P<0.05; Fig. 2). The ATP levels before cardiac arrest and after warm ischemia were 6.33±0.79 and 2.68±1.07 nmol/mg dry weight, respectively. The ATP levels 4 hr after reperfusion in the HMP and SCS groups were 4.53±0.38 and 3.07±0.94 nmol/mg dry weight, respectively.
Oxidative Damage During HMP and Reperfusion
Malondialdehyde (MDA) is one of the most commonly used markers for lipid peroxidation (12). The MDA levels in the perfusate were measured at baseline and after 120 min HMP to assess the oxidative damage that occurred during HMP. HMP did not increase the MDA levels in the perfusate; the MDA levels at baseline and after 120 min HMP were 2.23±0.49 and 2.06±0.45 nmol/mL, respectively (P=0.69; Fig. 3A). The serum MDA levels were measured 4 hr after reperfusion to evaluate the oxidative damage that occurred during reperfusion. The serum MDA levels were significantly lower in the HMP group compared with the SCS group (HMP group 1.55±0.74 nmol/mL and SCS group 3.63±1.15 nmol/mL, P<0.05; Fig. 3B).
Proinflammatory Cytokine Levels in Bronchoalveolar Lavage Fluid After Reperfusion
The tumor necrosis factor (TNF)-α and interleukin (IL)-6 levels in the bronchoalveolar lavage (BAL) fluid were measured 4 hr after reperfusion. The TNF-α levels were significantly lower in the HMP group than in the SCS group (HMP group 5.83±3.22 pg/mL and SCS group 54.15±29.36 pg/mL, P<0.01; Fig. 3C). The IL-6 levels were also significantly lower in the HMP group compared with the SCS group (HMP group 1.55±0.74 pg/mL and SCS group 3.63±1.15 pg/mL, P<0.05; Fig. 3D).
Physiologic Lung Functions During Reperfusion
The lung oxygenation and dynamic pulmonary compliance were significantly better in the HMP group than those in the SCS group (P<0.01; Figs. 4A and B). The wet-to-dry lung weight ratio, indicating the severity of pulmonary edema, 4 hr after reperfusion was significantly lower in the HMP group than that in the SCS group (HMP group 7.09±0.77 and SCS group 12.03±4.05, P<0.05; Fig. 4C).
Histologic Findings of Ischemia-Reperfusion Injury
Severe interstitial and intraalveolar edema, hemorrhage, infiltration of inflammatory cells in the air space or vessel wall, and hyaline formation were detected in the SCS group 4 hr after reperfusion. The acute lung injury score was significantly lower in the HMP group in comparison with the SCS group (HMP group 22.6±6.80 and SCS group 44.6±4.45, P<0.01; Fig. 5).
The current study used a clinically relevant uncontrolled DCD model. We chose 4 hr warm ischemia to possibly expand the donor pool for lung transplantation, although the Madrid groups reported a maximum warm ischemic time of 2 hr (3, 13). The retrieval of lungs after cardiac death requires an intermediate period to be transported to the transplant center, so we added 12 hr SCS just before HMP. Dutkowski et al. suggested that 1 to 2 hr HMP should be performed during the recipient preparation without delay of the transplant procedure (10). We previously reported that 1 hr HMP significantly improved the rat lung tissue ATP levels, which had decreased during warm ischemia (11). In the current study, DCD lungs, which were injured by 4 hr warm ischemia and additional 12 hr cold ischemia, could be resuscitated by 2 hr HMP.
This study found that short-term HMP could be performed safely for DCD lungs, not inducing any significant amount of oxidative damage. We recently developed a reliable and reproducible technique for lung HMP in a large animal model, which demonstrated stable machine perfusion characteristics and excellent lung performance during 8 hr HMP (data not shown). The current study revealed that this technique could be used for reconditioning of ischemically damaged DCD lungs. None of the influent valuables showed spikes, and the dynamic pulmonary compliance was also maintained during the entire period of HMP. Oxidative damage under the exposure to oxygen at hypothermia has been demonstrated in studies on isolated cell systems (14), whereas several studies in animal models demonstrated that liver HMP resulted in minor oxidative damage (15, 16). The present study found that short-term lung HMP did not cause oxidative stress during the perfusion, which was indicated by the fact that the MDA levels in the perfusate did not increase during HMP.
Intravascular microthrombus formation, which results in an increase of intrapulmonary shunting and pulmonary vascular resistance, is one of the major causes of reperfusion injury in lung transplantation from DCD donors. The benefits of additional retrograde flushing have been shown in experimental lung transplantation (17–19). In the current study, a histologic examination of the donor lungs just before transplantation revealed fewer microthrombi in the HMP group compared with the SCS group. This indicated that most of the residual microthrombi wedged in the capillaries after the flushes were eliminated by HMP (9, 20). Ventilation during perfusion results in better distribution of the preservation solution. A reduction of minute ventilation decreases the total amount of elastic stress imposed on cooled lungs (21). Therefore, the current study adopted the ventilation mode reduced respiratory rate and tidal volume during HMP, which resulted in stable dynamic pulmonary compliance and the elimination of residual microthrombi.
The current study demonstrated that short-term HMP could improve the mitochondrial function following injury due to warm ischemia and decrease the oxidative damage and production of proinflammatory cytokines during reperfusion. Unlike other tissues that are transplanted, lung cells are able to maintain aerobic metabolism using the oxygen present in the alveoli during SCS (22). In the SCS group, the lung ATP levels, which decreased during warm ischemia, were improved a little, but the improvement was significantly lower than that in the HMP group. HMP could continue to provide the essential substrates for cell metabolism and restore the lung tissue ATP levels. The reintroduction of oxygen to impaired mitochondria at reperfusion leads to a significant production of ROS, which damage proteins, lipids, and DNA (6). The serum MDA levels after reperfusion were significantly lower in the HMP group compared with the SCS group. HMP possibly prevented the overload of oxygen on reperfusion for the mitochondrial electron transport chain by recovering the mitochondrial function before reperfusion and thus decreased production of ROS. Physical alterations of the plasma membrane caused by ROS activate Toll-like receptors (TLRs), which are expressed in endothelial cells and respiratory epithelial cells (7). The signal transduction mediated by TLRs results in the activation of nuclear factor-κB, inducing the production of proinflammatory cytokines and chemokines (7). Therefore, the significantly increased levels of TNF-α and IL-6 in the SCS group might have resulted from TLRs signaling in the pulmonary parenchymal cells, activated by the significant increase in lipid peroxidation.
Normothermic perfusion has already been studied and proven to enable organ viability assessment before transplantation, prolonged preservation, and resuscitation from injuries (23–27). It has been unknown which is more suitable for organ preservation, hypothermic perfusion, or normothermic perfusion. The organ is metabolically active under normothermic conditions; thus, normothermic perfusion might allow better reconstitution of the lung tissue ATP stores. However, normothermic perfusion requires that the physiologic environment is completely recreated with full nutritional support. Hypothermia decreases the metabolic rate of the organ and could be used as a means for lung rest in the acutely injured lung (21). This study demonstrated that HMP could continue to provide the essential substrates for cell metabolism and restore the lung tissue ATP levels under the slow metabolic rate conditions.
This study had several limitations. First, although we simulated a clinically relevant uncontrolled DCD model, cardiac arrest was induced by intravenous injection of potassium chloride. Such an abrupt cardiac arrest may have been removed from clinical reality, in that there was not an agonal phase, which is an important variable component of DCD (28). Second, the lung tissue ATP levels were measured after warm ischemia and reperfusion. It might be easier to prove the metabolic benefits of HMP if the ATP levels were measured just before and after HMP.
In conclusion, short-term HMP could resuscitate DCD lungs injured by prolonged ischemia and ameliorate ischemia-reperfusion injury. First, short-term HMP washed-out residual microthrombi in the donor lungs. Second, short-term HMP improved the ATP production by the mitochondrial electron transport chain, which led to the significant decrease in oxidative damage and production of proinflammatory cytokines after reperfusion compared with SCS.
MATERIALS AND METHODS
Beagles weighing from 9 to 13 kg (Kitayama Labes Co. Ltd., Hongo Farm, Yamaguchi, Japan) were used in this study. There was no significant difference in the beagles’ body weights between the two groups. All animals received humane care in compliance with the Principles of Laboratory Animal Care, formulated by the U.S. National Society for Medical Research, and the Guide for the Care and Use of Laboratory Animals, prepared by the U.S. Institute of Laboratory Animal Resources, and published by the National Institutes of Health (NIH Publication 85-23, revised 1996). The study was approved by the Ethics Committee of the Faculty of Medicine at Kyoto University (Kyoto, Japan).
The donor procedures, including anesthesia, induction of cardiac arrest, and antegrade and retrograde flushes of the lungs, were described in detail in a separate publication (29). Cardiac arrest was induced by the intravenous injection of potassium chloride (0.5 mEq/kg) without heparinization. Four hours after cardiac arrest, the donor lungs were retrieved and then were divided into two groups (n=5 each). The lungs in the SCS group were stored in an inflated state with oxygen fraction of 0.5 at 4°C for 14 hr using ET-Kyoto solution (Otsuka Pharmaceutical Factory, Inc., Tokushima, Japan) (30). The lungs in the HMP group were stored in an inflated state with oxygen fraction of 0.5 at 4°C for 12 hr using ET-Kyoto solution and then reconditioned by 2 hr HMP. In both groups, the left lung was then transplanted to a recipient as described previously (29). The transplanted lung was reinflated and mechanically ventilated with FiO2 of 1.0 and then reperfused for 4 hr to evaluate the posttransplantation lung functions. The right pulmonary artery was occluded with a tourniquet 45 min after reperfusion to specifically evaluate the functions of the transplanted lung. The pulmonary arterial pressure and peak airway pressure (PawP) were continuously monitored throughout the experiments. Dynamic pulmonary compliance was defined as tidal volume/(PawP-[positive end-expiratory pressure]) (mL/cm H2O). A blood gas analysis was performed using blood collected from the femoral artery at selected time points. Lung tissue biopsy samples collected from the left middle lobe 4 hr after reperfusion were weighed to obtain the wet lung weight, placed in an oven at 180°C for 24 hr, and then reweighed to obtain the dry lung weight. The wet-to-dry lung weight ratio was calculated to evaluate the presence of pulmonary edema.
Hypothermic Machine Perfusion
The lungs were placed in an XVIVO chamber (Vitrolife, Denver, CO). The pulmonary artery was cannulated directly and then connected to the perfusion circuit. The left atrium was left open, so that the left atrial pressure was always 0 mm Hg. The trachea was intubated and connected to the ventilator. Mechanical ventilation was started with FiO2 of 0.25, tidal volume of 10 mL/kg, frequency of 10 breaths/min, and positive end-expiratory pressure of 5 cm H2O. The perfusate, which contained STEEN solution (1500 mL) with methylprednisolone (500 mg) and heparin (10,000 IU), was driven by a centrifugal pump at a constant flow rate of 10% of the estimated cardiac output (CO=100 mL/kg). Deoxygenation of the perfusate was started with a gas mixture of nitrogen (86%), CO2 (8%), and oxygen (6%) to maintain the influent pCO2 of approximately 40 mm Hg. The temperature of influent was continuously monitored and maintained at approximately 10°C (31). The influent solute concentrations and pO2 and pCO2 levels were recorded every hour. The pulmonary arterial pressure and PawP were continuously monitored, and the physiologic lung functions (dynamic pulmonary compliance and pulmonary vascular resistance) during HMP were evaluated every 30 min. Recruitments were performed to ensure a PawP of 25 cm H2O every 30 min before each evaluation. Dynamic pulmonary compliance was defined as described above. Pulmonary vascular resistance was defined as ([pulmonary arterial pressure]-[left atrial pressure])/pulmonary arterial flow (mm Hg/L).
Lung Tissue ATP Levels
Lung tissue biopsy specimens were collected from the right lung before cardiac arrest and after warm ischemia and then were collected from the left upper lobe 4 hr after reperfusion. ATP levels were measured by high-performance liquid chromatography using a Shim-pack CLC-ODS column (15 cm×6.0 mm; Shimadzu, Kyoto, Japan) and 100 mM sodium phosphate buffer (pH 6.0) at a wavelength of 260 nm as described previously (32).
MDA levels were measured with the NWLSS Malondialdehyde Assay kit (Northwest Life Sciences Specialties, Vancouver, British Columbia, Canada) following the manufacturer’s protocol. MDA reacted with thiobarbituric acid, forming an MDA-TBA2 adduct that was measured at a wavelength of 532 nm.
Cytokine Levels in BAL Fluid
BAL was performed with 20 mL saline using a flexible bronchoscope wedged into the left lower bronchus. Collected samples were centrifuged at 1500g for 10 min at 4°C, and then the supernatant was stored at −80°C to evaluate the cytokine levels. TNF-α and IL-6 levels were measured with a Quantikine ELISA kit (R&D Systems, Inc., Minneapolis, MN) following the protocol developed by the manufacturer.
Histologic Evaluation of Microthrombi and Ischemia-Reperfusion Injury
Lung tissue biopsies were collected from the right lower lobe just before transplantation and the left lower lobe 4 hr after reperfusion. They were fixed in 10% buffered formalin, embedded in paraffin, and stained with hematoxylin and eosin. Five sections including capillaries were examined by blinded investigators (A.O. and J.S.) to evaluate the residual microthrombi in the donor lungs. The extent of ischemia-reperfusion injury was scored blindly by two investigators (A.O. and J.S.) using a four-point scale according to the combined assessment of edema (interstitial and intraalveolar congestion), hemorrhage, inflammatory cell infiltration, and hyaline membrane formation: 0=absent, 1=mild, 2=moderate, and 3=severe damage (33, 34).
All data are presented as means±standard deviation. The statistical analysis was performed using Student’s t test and a repeated-measures analysis of variance. P<0.05 was considered to be statistically significant.
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