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Hypothermic Machine Perfusion Ameliorates Ischemia-Reperfusion Injury in Rat Lungs From Non-Heart-Beating Donors

Nakajima, Daisuke1; Chen, Fengshi1; Yamada, Tetsu1; Sakamoto, Jin1; Osumi, Akihiro1; Fujinaga, Takuji1,2; Shoji, Tsuyoshi1; Sakai, Hiroaki1; Bando, Toru1,2; Date, Hiroshi1,3

doi: 10.1097/TP.0b013e31822d8778
Basic and Experimental Research
Free

Background. The use of non-heart-beating donors (NHBD) has come into practice to resolve the shortage of donor lungs. This study investigated whether hypothermic machine perfusion (HMP) can improve the quality of NHBD lungs.

Methods. An uncontrolled NHBD model was achieved in male Lewis rats. Ninety minutes after cardiac arrest, HMP was performed for 60 min at 6°C to 10°C. The first study investigated the physiological lung functions during HMP and the lung tissue energy levels before and after HMP. The second study divided the rats into three groups (n=6 each): no ischemia group; 90-min warm ischemia+60-min HMP+120-min static cold storage (SCS) (HMP group); and 90-min warm ischemia+180-min SCS group. All lungs were reperfused for 60 min at 37°C. Lung functions were evaluated at given timings throughout the experiments. Oxidative damage during reperfusion was evaluated immunohistochemically with a monoclonal antibody against 8-hydroxy-2′-deoxyguanosine.

Results. The first study revealed that lung functions were stable during HMP. Lung tissue energy levels decreased during warm ischemia but were significantly increased by HMP (P<0.05). The second study confirmed that HMP significantly decreased pulmonary vascular resistance, increased pulmonary compliance, and improved pulmonary oxygenation. The ratio of 8-hydroxy-2′-deoxyguanosine positive cells to total cells significantly increased in the SCS group (P<0.01).

Conclusions. Short-term HMP improved lung tissue energy levels that decreased during warm ischemia and ameliorated ischemia-reperfusion injury with decreased production of reactive oxygen species.

1 Department of Thoracic Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan.

2 Department of Organ Preservation Technology, Graduate School of Medicine, Kyoto University, Kyoto, Japan.

The authors declare no funding or conflicts of interest.

Presented, in part, at the XXIII International Congress of the Transplantation Society, August 15–19, 2010, Vancouver, Canada.

3 Address correspondence to: Hiroshi Date, M.D., Ph.D., 54 Shogoin-Kawahara-Cho, Sakyo-Ku, Kyoto, Japan.

E-mail: hdate@kuhp.kyoto-u.ac.jp

D.N., T.Y., J.S., and F.C. participated in the performance of the research. D.N., T.Y., F.C., J.S., A.O., T.F., T.S., H.S., T.B., and H.D. participated in research design. D.N., F.C., T.B., and H.D. participated in the writing of the manuscript.

Received 7 February 2011. Revision requested 11 March 2011.

Accepted 12 July 2011.

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 non-heart-beating donors (NHBD) has been used to resolve this problem. Steen et al. (1) successfully performed lung transplantation from an uncontrolled NHBD in 2000. Since then, there have been several reports of successful lung transplantation from NHBD (2–6). Warm ischemia inevitably occurs in NHBD after cardiac arrest and may cause ischemia-reperfusion injury after transplantation. The acceptable period of warm ischemia is short (7–10), which thus limits the frequency of lung transplantation from uncontrolled NHBD. The inhibition of warm ischemia-reperfusion injury is, therefore, crucial to facilitate lung transplantation from uncontrolled NHBD.

Several strategies of organ preservation are used to improve graft function in kidney and liver transplantation. Hypothermic machine perfusion (HMP) is used to preserve kidneys and livers for transplantation with better results than static cold storage (SCS). HMP is associated with a reduced risk of delayed graft function and improved graft survival, in comparison with SCS (11, 12). HMP is based on the concept that oxidative energy production by mitochondrial electron transport would sustain at hypothermia (13). HMP continuously provides metabolic substrates for generation of adenosine triphosphate (ATP), and thus enables the graft to restore tissue energy. Kidneys and livers from NHBD are successfully improved by short-term HMP in animal models (14–17). On the other hand, the potential risk of HMP is the exposure to continuous oxygenation at hypothermia. Oxidative damage by reactive oxygen species (ROS) under oxygenated hypothermic conditions is demonstrated in studies on isolated cell systems (18). ROS cause DNA damage and produce various oxidatively modified products (19). Of these products, 8-hydroxy-2′-deoxyguanosine (8-OHdG) is one of the most popular markers for the evaluation of oxidative damage to DNA (20).

There have been no reports of success in preserving NHBD lungs with HMP. We stimulated a clinically relevant uncontrolled NHBD model in this study. This study investigated whether short-term HMP would improve lung tissue energy levels that decreased during warm ischemia and would reduce ischemia-reperfusion injury. Oxidative damage during reperfusion was evaluated immunohistochemically with a monoclonal antibody against 8-OHdG.

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RESULTS

Study I

Physiological lung functions (pulmonary compliance, airway resistance, mean pulmonary arterial pressure, and lung weight) were stable during 60 min of HMP (Fig. 1). Adenosine diphosphate (ADP), ATP, and total adenosine nucleotides (TAN) levels decreased during warm ischemia (TAN 4.31±0.61, ATP 1.03±0.21, ADP 0.81±0.18 nmol/mg) but significantly increased with HMP (TAN 5.93±0.59, ATP 2.64±0.23, ADP 1.12±0.13 nmol/mg, P<0.05; Fig. 2). The calculated energy charge (ATP+ADP/2)/TAN after HMP was significantly higher than that before HMP (after HMP; 0.545±0.061, before HMP; 0.333±0.028, P<0.01).

FIGURE 1.

FIGURE 1.

FIGURE 2.

FIGURE 2.

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Study II

Physiological Lung Functions During Reperfusion

All of the lungs in the three groups were reperfused successfully during 60 min of reperfusion. Pulmonary compliance was significantly better in the HMP group than that in the SCS group throughout the entire reperfusion period (P<0.05) and was similar to that in the no ischemia (NI) group (Fig. 3A). Pulmonary oxygenation was significantly better in the HMP group than that in the SCS group throughout the entire reperfusion period (P<0.01) and was similar to that in the NI group (Fig. 3B). Pulmonary vascular resistance in the NI group was significantly lower than that in the SCS group at the base line and 10 min of reperfusion (P<0.01; Fig. 3C). Pulmonary vascular resistance in the HMP group was significantly lower than that in the SCS group at the base line and 10 min of reperfusion, and tended to be lower than that of the SCS group at 50 and 60 min of reperfusion (P<0.05, and P<0.08, respectively; Fig. 3C). There were no significant differences between the three groups for lung weight gain (Fig. 3D).

FIGURE 3.

FIGURE 3.

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Oxidative Damage During Reperfusion

Nuclear staining with monoclonal antibody for 8-OHdG was weak in bronchial and alveolar epithelial cells in the NI group (Fig. 4A). Nuclear staining was moderate in bronchial and alveolar epithelial cells in the HMP group (Fig. 4B). Nuclear staining was not detectable in pulmonary arterial endothelial cells in either group. There was intense nuclear staining in not only bronchial and alveolar epithelial cells but also in pulmonary arterial endothelial cells in the SCS group (Fig. 4C). The ratio of 8-OHdG positive cells to total cells per high-power field significantly increased in the SCS group (NI group: 22.4%±11.5%, HMP group: 28.0%±7.0%, SCS group: 52.5%±9.4%, P<0.01; Fig. 4D).

FIGURE 4.

FIGURE 4.

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DISCUSSION

This study is the first report of preserving NHBD lungs by HMP and demonstrated that HMP ameliorated ischemia-reperfusion injury in NHBD lungs. HMP was performed safely without any side effects, and physiological lung functions were stable during HMP. Prolonged constant flow at hypothermia potentially injures pulmonary arterial endothelial cells due to increased vascular resistance and shear stress (21). Cypel et al. (22) recommended that maintenance perfusate flow rate for normothermic ex vivo lung perfusion was 40% of the estimated cardiac output to perfuse lungs. HMP that provided 25% of normothermic conditions resulted in complete perfusion of the liver without induction of endothelial injury (23). The flow rate for a rat weighting 300 g under normothermic conditions is approximately 10 mL/min to perfuse lungs, and the constant flow rate at hypothermia is estimated 2 to 3 mL/min. Dutkowski et al. (15) demonstrated that 60 min of short-term HMP improved rat livers injured by a combination of warm and cold ischemia. The current study found that 60 min of short-term HMP could resuscitate NHBD lungs injured by 90 min of warm ischemia. Pulmonary arterial pressure was stable during HMP, and therefore short-term HMP at a low flow rate potentially reduce pulmonary vascular resistance and endothelial sheer stress. Hypothermia reduces the metabolic rate with reduction of carbon dioxide production, which results in decreasing requirement for minute ventilation. Reduction of respiratory rate, so-called, lung rest would decrease the total amount of elastic stress imposed cooled lungs (24). Respiratory rate was reduced in conjunction with hypothermia during HMP, and the current study found that airway resistance and pulmonary compliance were stable during short-term HMP.

Normothermic ex vivo lung perfusion has been already studied and proved to enable organ viability assessment before transplantation, prolonged preservation, and resuscitation from injuries (25–28). However, the organ is metabolically active under normothermic conditions, and thus ex vivo lung perfusion requires that the physiological environment is 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 (24). The SCS is successful in maintaining organ viability of good donor lungs during more than 10 hr of cold ischemia but might eliminate the possibility of resuscitation from injuries in marginal donors. Although cold storage reduces the metabolic rate of lung tissues, the metabolism is not fully stopped in SCS (29). Dutkowski et al. (30) demonstrated that short-term HMP improved ATP levels in livers, which decreased during warm ischemia and cold ischemia. HMP could continue to provide the essential substrates for cell metabolism and restore lung tissue energy charge. Energy charge keeps the homeostasis and structural competence of the vasculature parenchymal cell interaction, and then marginal donor lungs can maintain the integrity of the structural elements necessary to recover from various injuries.

HMP can provide access for administration of cytoprotective and immunomodulatory drugs, and can perform pretreatment and reconditioning of marginal donor lungs (31, 32). Antioxidants and iron chelators, including glutathione, deferoxamine, or superoxide dismutase, are provided in the perfusate of HMP to reduce the risk of potential release of dangerous cytokine or ROS (16, 33, 34). The current study provided dibutyryl cyclic adenosine monophosphate (db-cAMP) and nitroglycerin (NTG) in the perfusate. The protective effects of db-cAMP and NTG for ischemia-reperfusion injury were described previously (35–37). A previous study demonstrated that intracellular cAMP levels decreased in NHBD lungs (38). db-cAMP elevates the intracellular levels of cAMP, which acts as a second messenger in suppressing vascular permeability, relaxing vascular smooth muscle, and suppressing neutrophil adhesion to the vascular wall. NTG works in vivo as an NO donor. NO stimulates production of cGMP, which acts as an intracellular second messenger in relaxing vascular smooth muscle, inhibiting platelet adhesion and aggregation, and adjusting vascular permeability.

Rauen et al. (18) conducted studies on isolated cell systems that suggested that production of ROS was increased by classical Fenton chemistry with increasing intracellular amounts of chelatable-free iron. On the other hand, several studies in animal models demonstrated that HMP resulted in minor oxidative damage (15, 39, 40). The present study evaluated oxidative damage during reperfusion immunohistochemically with a monoclonal antibody against 8-OHdG, which is one of the most common markers for the evaluation of oxidative damage to DNA. Oxidative damage in the HMP group was moderate in bronchial and alveolar epithelial cells, whereas oxidative damage in the SCS group was severe in bronchial, alveolar epithelial cells and pulmonary arterial endothelial cells. Mitochondrial respiration remained activated during HMP, so HMP possibly prevented the overload of oxygen on reperfusion for mitochondrial electron transport chain, and decreased production of ROS.

This study had several limitations. First, ischemia- reperfusion injury was evaluated not in vivo (e.g., lung transplantation model) but ex vivo using an isolated rat lung perfusion model. Ischemia-reperfusion injury is a multi- factorial inflammatory condition, and the acute phase of ischemia-reperfusion injury has been increasingly viewed as part of the innate immune response to the lack of vascular perfusion and oxygen (41). Therefore, future studies require to evaluate in vivo ischemia-reperfusion injury. Second, HMP was performed immediately after organ harvest, before SCS. Clinical practice may require some additional time for donor lungs to be transported from a donor hospital to a transplant center. Thus, short-term HMP may be effective after SCS, just before lung transplantation in a transplant center. However, this study used the same isolated rat lung perfusion model in HMP and reperfusion, which required time to prepare for reperfusion after HMP. Future studies will, therefore, investigate the protective effects of HMP for ischemia-reperfusion injury in large animal transplantation models mirroring clinical situations. Finally, the addition of db-cAMP and NTG in the perfusate results in an inability to conclusively prove that HMP alone was responsible for all of the benefits observed in this study. How much these additional additives contributed to the benefits of HMP would require further study.

In conclusion, short-term HMP with db-cAMP and NTG enabled the NHBD lungs to restore tissue energy, and ameliorated ischemia-reperfusion injury with decreased production of ROS. Short-term HMP can therefore be performed successfully at a low flow rate and a low respiratory rate and may be an ideal method of preservation that improves the quality of marginal donor lungs.

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MATERIALS AND METHODS

Animals

Male Lewis rats weighing 290 to 330 g (Japan SLC, Hamamatsu, Japan) were used in this study. All animals received human care in compliance with the Principals of Laboratory Animal Care, formulated by the United States National Society for Medical Research, and the Guide for the Care and Use of Laboratory Animals, prepared by the US 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 Ethical Committee of the Faculty of Medicine at Kyoto University, Japan.

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Study Design

Study I

HMP

Rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (50 mg/kg), intubated after tracheotomy, and ventilated with room air under the following conditions: respiratory rate of 60 cycles/min, inspiratory and expiratory positive-controlled pressure of +8 and +4 cm · H2O, and an inspiratory ratio of 50%. After a median abdominal incision, the diaphragm was incised, and then median sternotomy was performed. Cardiac arrest was induced by ventricular fibrillation to achieve a clinically relevant uncontrolled NHBD model. The lungs were kept deflated during warm ischemia. Ninety minutes after cardiac arrest, the ventilation was resumed with room air, and then HMP was performed for 60 min using an isolated rat lung perfusion model (Model 829; Hugo-Sachs Elektronik Harvard Apparatus). This model was described in previous studies (38, 42–45). The pulmonary artery and vein were cannulated directly and connected to the perfusion circuit. The HMP perfusate consisted of low potassium dextran (LPD) solution 50 mL with 8% albumin, NTG 2.5 mg, and db-cAMP 50 mg. The perfusate was driven by a roller pump at a constant flow of 2 to 3 mL/min, which was 10% of the estimated cardiac output (0.1 mL/min/g). The heart and lung block was harvested, and then placed in the artificial thorax. The lungs were ventilated with humidified air with negative pressure under the following conditions: respiratory rate of 40 cycles/min, peak inspiratory and expiratory chamber pressure of −8 and −4 cm · H2O, respectively, and an inspiratory ratio of 50%. The artificial thorax, the perfusion circuit and the airway were water jacketed to be controlled the temperature. The lungs were perfused at 37°C for 5 min to be stabilized, and then the temperature of the perfusate was decreased to 6°C over 10 min. The temperature of the perfusate during HMP was maintained between 6°C and 10°C. The perfusate drained from the pulmonary vein was deoxygenated in a glass deoxygenator with a gas mixture of nitrogen (92%) and carbon dioxide (8%). Blood gas analysis of the perfusate was performed at the baseline of HMP, and PO2 and PCO2 of the perfusate to the lungs were adjusted to 60±5 mm Hg and 40±15 mm Hg, respectively, to mimic the mixed venous blood in vivo. The PH, PO2, and PCO2 of the perfusate from the lungs at the baseline were 7.44±0.13 mm Hg, 208.8±5.5 mm Hg, and 23.2±6.0 mm Hg, respectively. The first study evaluated the physiological lung functions during HMP and the lung tissue energy levels before and after HMP. The rats were allocated into two groups: the left lung was collected 90 min after cardiac arrest in one group (before HMP, n=5) and after HMP in the other group (after HMP, n=5), immediately after flushing the pulmonary vascular bed with saline.

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Lung Tissue Energy Levels

The levels of ATP, ADP, and AMP were measured by high-performance liquid chromatography using a Shim-pack CLC-ODS column (15 cm×6.0 mm; Shimadzu, Japan) and 100 mM sodium phosphate buffer (PH 6.0) at a wavelength of 260 nm, as previously described in elsewhere (38).

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Study II

The rats were allocated into three groups (n=6 each): NI group, 90 min warm ischemia+60-min HMP+120-min SCS (HMP group), 90-min warm ischemia+180-min SCS group. In the NI group, the lungs were retrieved and immediately reperfused. Ninety minutes after cardiac arrest, the lungs in the SCS group were flushed with 20 mL of cold LPD solution, and then the lungs inflated with air were stored at 4°C for 180 min. HMP was conducted for 60 min in the HMP group 90 min after cardiac arrest, as described earlier, and then the lungs inflated with air were stored in LPD solution at 4°C for 120 min. All lungs were reperfused for 60 min at 37°C using an isolated rat lung perfusion model, in which physiological lung functions were evaluated on time during reperfusion (Fig. 5). The reperfusion perfusate contained heparinized rat blood obtained from two donor rats and saline with 4% albumin. The hematocrit was adjusted to the levels of approximately 15%, ranging 13% to 18%, and the PH was adjusted to between 7.25 and 7.35 with sodium bicarbonate. The lungs were reperfused at a flow rate of 10 mL/min and ventilated at a respiratory rate of 60 cycles/min, peak inspiratory and expiratory chamber pressure of −8 and −4 cm · H2O, respectively, and an inspiratory ratio of 50%. After reperfusion, the right upper lobe was retrieved to evaluate the oxidative damage during reperfusion immunohistochemically with a monoclonal antibody against 8-OHdG.

FIGURE 5.

FIGURE 5.

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Physiological Lung Function

The pulmonary arterial pressure, pulmonary airflow, and lung weight were all continuously recorded with a pressure transducer, a pneumotachometer, and a weight measurement system, respectively. Pulmonary vascular resistance (cm · H2O/mL · min) was defined as (pulmonary arterial pressure−pulmonary venous pressure)/perfusion flow. Dynamic airway resistance (cm · H2O/mL · sec) and dynamic pulmonary compliance (mL/cm · H2O) were calculated on an AT-compatible computer with the Pulmodyne software (Hugo Sachs Elektronik Harvard Apparatus, March-Hugstetten, Germany). Blood gas analysis of the perfusate to and from the lungs was performed at given time points.

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Immunohistochemistry for 8-OHdG

The Avidin-Biotin complex method was used for immunohistochemical staining. After deparaffinization of lung tissue sections, normal rabbit serum (diluted to 1:100; Dako, Kyoto, Japan) was used to block nonspecific binding. The sections were then incubated with primary anti-8-OHdG antibody 0.5 μg/mL (JaICA, Shizuoka, Japan) overnight at 4°C, biotin-labeled rabbit anti-mouse IgG serum (diluted to 1:300; Dako) for 40 min, and avidin-biotin-alkaline phosphatase complex (diluted to 1:100; Dako) for 50 min (20, 35, 46). Quantification of immunohistological data was performed blindly by two independent investigators (D.N. and J.S.) was shown as a mean of the ratio of 8-OHdG positive cells to total cells per high-power field within five representative fields per lung.

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Statistical Analysis

All data are presented as the mean±SD. The statistical analysis was performed by a one-way analysis of variance, Scheffe's post hoc multiple comparison test, and Student's t test. A P value less than 0.05 was considered to be statistically significant.

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ACKNOWLEDGMENT

The authors thank Yoshinobu Toda for his expert technical advice.

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Keywords:

Hypothermic machine perfusion; Non-heart-beating donor; Ischemia-reperfusion injury; Lung transplantation; 8-OHdG

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