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Ann Thorac Surg 2000;69:1532-1536
© 2000 The Society of Thoracic Surgeons

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Original articles: General thoracic

Apoptosis induced by ischemia and reperfusion in experimental lung transplantation

^a Division of Thoracic Surgery, University Hospital Zurich, Zurich, Switzerland
^b Department of Pathology, University Hospital Zurich, Zurich, Switzerland

Address reprint requests to Dr Schmid, Division of Thoracic Surgery, University Hospital, CH-3010 Berne, Switzerland

	Abstract

Top Abstract Introduction Material and methods Results Time course of apoptosis... Comment References

 
Background. Apoptosis is a distinct form of single-cell death in response to injury. Time course of apoptosis in lung parenchymal cells during posttransplant reperfusion and the influence of oxygen content during preservation on apoptosis of parenchymal cells are studied.

Methods. Orthotopic syngenic single left lung transplantation was performed in male Fischer (F344) rats after 18 hours of cold ischemia (n = 5 in all groups). Apoptotic cells were stained by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) technique. Strictly TUNEL-positive pneumocytes were counted on anonymized slides by a pathologist on 100 fields (x400) per specimen (mean ± SEM).

Results. The peak of apoptotic pneumocytes occurred 2 hours after reperfusion (16.8 ± 2.2 pneumocytes/100 fields [p/100f]; p = 0.000012 vs controls, lungs fixed after 18 hours of ischemia), whereas the lowest level of apoptotic pneumocytes was seen in lungs fixed after harvest (1.4 ± 0.51 p/100f) and lungs not undergoing reperfusion (2.8 ± 0.49 p/100f). Four hours after reperfusion, the number of apoptotic pneumocytes was lower than 2 hours after reperfusion (13.6 ± 3.1 p/100f; p = 0.00032 vs controls), with a further decline at 8 hours (6.4 ± 1.5 p/100f) and 12 hours after reperfusion (4.0 ± 1.2 p/100f). Interestingly, lungs inflated with N₂ before storage revealed a significantly lower level of TUNEL-positive pneumocytes 2 hours after reperfusion (8.8 ± 2.0 p/100f) compared with lungs inflated with 100% O₂ (p = 0.0052).

Conclusions. Apoptosis of pneumocytes after posttransplant lung reperfusion is a very early event. Prolonged hypothermic preservation without reperfusion, however, does not lead to an elevated rate of apoptotic pneumocytes in lung grafts.

	Introduction

Top Abstract Introduction Material and methods Results Time course of apoptosis... Comment References

 
Ischemia/reperfusion injury has been studied extensively as one of the major problems in the early phase after lung transplantation. Prolonged ischemia results in poor organ function after reperfusion, and cell death may occur due to preservation injury. The most severely damaged cells eventually become necrotic. However, cells injured by ischemia and reperfusion may undergo a specific form of cell death termed apoptosis [1]. This energy-dependent process may typically occur when injury to the cell is beyond repair but not as severe to induce necrosis, which is defined as damage to the cell membrane leading to complete loss of the cell’s functions.

A number of studies demonstrate that reperfusion after ischemia induces apoptosis in solid organs. Bardales and colleagues [2] stated that extensive apoptosis of type II pneumocytes occurs in the resolution phase of acute lung injury. However, little is known on the frequency and the time course of apoptotic pneumocytes in the lung after transplantation.

In this study, we investigated the time course of apoptosis after prolonged preservation in a model of syngenic left lung transplantation in rats.

	Material and methods

Top Abstract Introduction Material and methods Results Time course of apoptosis... Comment References

 
Animals and surgical procedure
Weight-matched male Fischer (F344) rats (200 to 250 g) underwent syngenic orthotopic single left lung transplantation using a cuff technique for the vessel anastomoses [3] and a conventional running suture for the bronchial anastomosis. All animals received humane care in compliance with the European Convention of Animal Care. The protocol was approved by the local animals study committee.

Donor procedure
Animals were anesthetized by intraperitoneal administration of pentobarbital (50 mg/kg) and heparinized (500 IU/kg). A tracheotomy was carried out and the animals were ventilated through a cannula with 100% O₂ by a Harvard rodent ventilator (Harvard Apparatus, South Natick, MA) at a tidal volume of 10 mL/kg. After cutting the inferior vena cava and left appendix of the heart, a small silicon tube was inserted into the main pulmonary artery. Both lungs were flushed with 20 cc of low-potassium dextrane (LPD) solution [4] (Perfadex; XVIVO AB, Uppsala, Sweden) at a pressure of 20 cm H₂O. The trachea was tied in end-inspiration. After removal of the heart-lung block, 14-gauge cuffs were placed around the pulmonary artery and vein, and the vessels were inverted and tied onto the cuff. The lung was stored in LPD solution at 1°C until implantation.

Recipient procedure
The recipient was anesthetized by breathing Halothane in a glass chamber followed by intubation. Anesthesia was maintained with Halothane 2%. A left lateral thoracotomy was performed in the fourth intercostal space. The left hilum was dissected. After clamping the pulmonary artery and vein with removable microclips, the pulmonary vein was opened, flushed with heparinized saline solution, and the cuff was inserted and fixed with 6-0 silk. In the same technique, the pulmonary artery was anastomosed. The native left lung was removed and the bronchial anastomosis performed with a running over-and-over suture with 9-0 Monosof (Tyco Healthcare, Wollerau, Switzerland). The lung was first inflated and then reperfused. A chest tube was inserted and the thoracotomy closed. The chest tube was removed after restoration of spontaneous breathing.

Operation time for implantation was 50 ± 4 minutes and did not differ between groups. Warm ischemic time in all animals was 21 to 24 minutes, with no statistical differences between groups.

Sacrifice
The animals were anesthetized by intraperitoneal administration of pentobarbital (50 mg/kg) and heparinized (500 IU/kg). Animals were ventilated with 100% O₂ by a tracheotomy, and the lung was flushed through the pulmonary artery with 20 cc of saline solution. The heart-lung block was excised and the lungs were fixed overnight at room temperature with 10% buffered formalin. Formalin was instilled through a tube inserted in the trachea to ensure that all lungs were expanded equally with a defined pressure of 20 cm H₂O.

Study groups
Each group consisted of five cases. For evaluation of the time course of apoptosis, six groups were studied. In groups I to IV, transplantation was carried out after 18 hours of cold (1°C) ischemia, and animals were sacrificed 2 hours (I), 4 hours (II), 8 hours (III), and 12 hours (IV) after reperfusion. For evaluation of the effect of ischemia alone, lungs in group V were fixed after 18 hours of cold ischemia. Lungs in group VI were flushed at harvest and instantly fixed with formalin as described above.

One additional group (VII) with sacrifice 2 hours after reperfusion was studied for evaluation of the influence of oxygen inflation during storage on posttransplant apoptosis and compared with group I. To wash out O₂, donor lungs were ventilated with 100% N₂ for 5 minutes during flush and were stored inflated with N₂ under the same condition as group I.

Histological assessment
Sections (4 µm) of formalin-fixed, paraffin-embedded lung tissue were stained after a modified method of Gavrieli and associates [5], based on the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick-end labeling (TUNEL) at sites of DNA breaks. The tissue sections were routinely deparaffinized and rehydrated, shortly rinsed in Tris-buffered saline (TBS; 0.15 mol/L NaCl, 50 mmol/L Tris-HCI, pH 7.4), and digested with proteinase K (Boehringer, Mannheim, Germany) at a concentration of 30 µg/mL TBS for 20 minutes at room temperature (RT). After being washed in distilled water for 10 minutes, sections were equilibrated for 15 minutes with TdT reaction buffer (200 mmol/L potassium cacodylate, 2.5 mmol/L CoCl₂, 0.25 mg/mL BSA, 25 mmol/L Tris-HCI, pH 6.6). The sections were then covered with the TUNEL mix (62.5 U/mL calf thymus TdT (Boehringer), 3.75 mmol/mL digoxigenin-11-dUTP in TdT reaction buffer) and incubated for 1 hour at 37°C in a water-saturated atmosphere. The sections were then washed for 10 minutes with 2 x SSC at 40°C, rinsed with TBS, and preincubated with blocking buffer (2% BSA, 0.3% Triton X-100 in TBS) for 30 minutes (RT). For the detection of digoxigenin, sections were treated for 1 hour with alkaline phosphatase-labeled Fab fragments of affinity-purified sheep anti-digoxigenin antibodies (1 U/mL blocking buffer, RT). After a further washing step with TBS, the red-colored reaction product was developed under eye control using naphthol AS-BI phosphate and New Fuchsin as substrate. Endogenous alkaline phosphatase was blocked by levamisole. After counterstaining with hemalum, coverslips were mounted with glycerol/gelatin. In addition, slides of 4-µm thickness were stained with hematoxylin-eosin.

The slides were analyzed by a blinded pulmonary pathologist. TUNEL-positive pneumocytes were counted at 400x in 100 microscopic fields per lung. Only cells lining the alveolar wall with positive nuclear and no cytoplasmatic staining were regarded as apoptotic pneumocytes (Fig 1). No apoptotic cells found within the interstitium or in the alveoli were counted. Cells containing cytoplasmatic staining indicative of phagocytosis of apoptotic nuclei by macrophages were not considered. Internal lymphoid tissue and separate tonsil tissue served as positive controls. Slides treated with buffer instead of TdT were included as negative controls.

View larger version (146K): [in this window] [in a new window]   Fig 1. Transplanted rat lung (x400) with a TUNEL-labeled pneumocyte (arrow). Inset reveals a positively stained pneumocyte (arrow) and an apoptotic endothelial cell (arrowhead). Only apoptotic pneumocytes were used for analysis.

	Results

Top Abstract Introduction Material and methods Results Time course of apoptosis... Comment References

 
Negative and positive controls
No apoptotic cells were detected on slides treated with buffer instead of TdT, which were used as negative controls. As positive controls, slides with internal lymphoid tissue and tonsil tissue were stained, demonstrating a high number of TUNEL-positive cells.

	Time course of apoptosis after lung transplantation

Top Abstract Introduction Material and methods Results Time course of apoptosis... Comment References

 
The slides of group V lung grafts, which served as controls, revealed only few apoptotic pneumocytes (2.8 ± 0.49 pneumocytes/100 fields [p/100f]) after 18 hours of cold ischemia without reperfusion. The lowest level of apoptotic pneumocytes was seen in group VI lung grafts, which were analyzed directly after harvest (1.4 ± 0.51 p/100f; p = 0.58 vs group V).

The peak of apoptotic pneumocytes (Fig 2) was observed in lungs studied 2 hours after reperfusion (group I: 16.8 ± 2.2 p/100f; p = 0.000012 vs group V). Four hours after reperfusion, the number of apoptotic pneumocytes was slightly less than 2 hours after reperfusion (group II: 13.6 ± 3.1 p/100f; p = 0.00032 vs group V). This decline was more pronounced 8 hours (group III: 6.4 ± 1.5 p/100f; p = 0.18 vs group V) after reperfusion, and 12 hours after reperfusion; no statistically significant difference between these lungs and lungs fixed without reperfusion was observed (group IV: 4.0 ± 1.2 p/100f; p = 0.65 vs group V). The decline after 8 and 12 hours was statistically significant as compared with either group I (2 hours; p 0.01) and group II (4 hours; p 0.05).

View larger version (17K): [in this window] [in a new window]   Fig 2. Time course of apoptosis after reperfusion. The peak of apoptotic pneumocytes occurred 2 hours after reperfusion, with a rapid decline during the first 12 hours after reperfusion. (Control = lungs fixed after 18 hours of storage without reperfusion.) Data are mean ± SEM.

	Comment

Top Abstract Introduction Material and methods Results Time course of apoptosis... Comment References

 
Our results indicate that apoptosis in lung grafts with severe reperfusion injury is an early event. The peak in the number of apoptotic pneumocytes is seen 2 hours after reperfusion. Ischemia alone results in a slight but statistically insignificant elevation in the number of apoptotic pneumocytes as compared with lungs fixed directly after harvest. Whereas the influence of nitrogen inflation before storage is deleterious to organ function [6], it does not elevate the number of apoptotic pneumocytes 2 hours after reperfusion in this model.

Since the 1950s, embryologists know the importance of single-cell death in vertebrate ontogeny [7]. Focal apoptosis is necessary for the development of lumina in tubular structures and interdigital clefts, and for the involution of phylogenetic vestiges. Cell death in mature individuals was regarded only as a nonphysiological noxious event. In 1972, Kerr and associates [1] published a pioneering paper elucidating the importance of physiologic cell deletion as the counterpart to mitosis in the regulation of tissue kinetics. Apoptosis, an energy-dependent, gene-directed, distinct form of multifocal single-cell death, plays a major role in embryogenesis, tissue kinetics, neoplasia [8, 9], and maturation of the immune system. In contrast to necrosis, where a cluster of cells is damaged by an external injury, individual cells ‘fall-off’ (apoptosis) due to physiological or nonphysiological stimuli. Apoptosis is characterized morphologically by nuclear condensation and shrinkage of the cytoplasm followed by fragmentation of nuclear chromatin. Multiple fragments of cytoplasm including condensed nuclear chromatin are then engulfed rapidly by adjacent cells. Typically, inflammation is sparse or absent. In necrosis, general cell swelling and swelling of organelles precedes membrane disruption, which leads to inflammation in the surrounding tissue and infiltration of leukocytes.

In tissues bruised by chemical substances or radiation, an elevated amount of apoptosis is seen, which was first observed by Kerr in 1971. Polunovsky and coworkers [10] showed that apoptosis occurs during repair after acute lung injury, and Bardales and associates [2] demonstrated that in the resolution phase of acute lung injury, an extensive number of type II pneumocytes undergo apoptosis. Kerr speculated that ischemia may induce dense clusters of apoptotic cells adjacent to necrosis and described apoptosis of hepatocytes in response to ischemic organ injury [11]. Subsequently, it has been shown that apoptosis is seen in all organs undergoing ischemia and reperfusion [12–18].

We found a relatively small number of apoptotic pneumocytes at any given time point of examination in transplanted lungs after severe ischemia/reperfusion injury. However, already in 1972, Kerr stated that the process of apoptosis is completed rapidly [1]. He recognized that the vast majority of apoptotic bodies (AB) are in the cytoplasm of intact cells, suggesting that an apoptotic cell is rapidly phagocytosed and that AB may form and disappear within hours. In this context, it is important to note that, similar to the significance of a small number of mitotic figures as sign for cell proliferation, the finding of relatively few apoptotic cells in histological tissue sections suggests a quite extensive cell "drop-out." Whereas these kinetics have not been proved for lung tissue, it is likely that this process is equal in lungs because apoptosis seems to be regulated in all tissues by the same genes.

To our knowledge, the only calculation of the time course of apoptosis for a single cell was done by Bursch and associates [19]. This group determined the half-life time of AB in rat hepatocytes by logarithmic regression to be less than 120 minutes and of the visible stages of apoptosis to be less than 3 hours. This supports the concept that even a low number of apoptotic cells at a given time point are indicating a substantial rate of cell loss.

Recently, Noda and colleagues [15] published the results of a rat model of small intestine ischemia-reperfusion injury. The peak in DNA fragmentation and stained cells by TUNEL technique was seen 1 hour after reperfusion with a slight drop 3 hours after reperfusion. Six hours after reperfusion, only few apoptotic cells were seen.

Sasaki and coworkers [13, 20] used a model of portal vein and hepatic artery clamping for 30 or 60 minutes in rats. The peak of apoptotic cells was seen 3 hours after reperfusion, and the number of cells stained by TUNEL correlated well with the number of AB, which almost completely disappeared within 24 hours after reperfusion.

In the present study, we found a slight and statistically not significant elevation of the number of apoptotic pneumocytes in lungs stored for 18 hours and fixed without reperfusion, as compared with lungs fixed immediately after harvest. In a rabbit model of cardiac ischemia, Gottlieb and associates [12] demonstrated that 4.5 hours of continuous ischemia does not result in nucleosomal cleavage, whereas after 4 hours of ischemia and 30 minutes of reperfusion, all animals showed a typical nucleosome ladder in DNA electrophoresis, indicating apoptotic cell death. Shah and associates [16] demonstrated that in small intestinal grafts, storage for 24 hours alone did not elevate the number of apoptotic cells; however, 1 hour after reperfusion, crypt apoptosis was moderate to severe.

Surprisingly, lungs inflated with nitrogen and stored anoxic did not reveal an elevated number of apoptotic pneumocytes 2 hours after reperfusion. In an ex vivo model of ischemia and reperfusion in rabbit lung, it has been shown that nitrogen inflation is deleterious to the graft, resulting in rapid formation of edema [6]. Therefore, we expected more apoptotic cells after anoxic storage with nitrogen inflation. On the other hand, it could be speculated, because apoptosis is an energy-dependent process, that the onset of apoptosis is delayed under anoxic storage conditions. The peak of apoptotic cell death may occur later.

We conclude that apoptosis in the transplanted rat lung is an early event and that a significant number of pneumocytes undergo apoptosis after reperfusion, that the peak in the number of apoptotic pneumocytes occurs as early as 2 hours after reperfusion, and that ischemia alone does not lead to an increase in apoptotic pneumocytes as compared with normal lungs.

	Acknowledgments

 
This work was supported by grants from Olga Mayenfisch-Stiftung and Hartmann-Müller-Stiftung.

	References

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1. Kerr J.F.R., Wyllie A.H., Currie A.R. Apoptosis. Br J Cancer 1972;26:239-257.[Medline]
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6. Weder W., Harper B., Shimokawa S., et al. Influence of intraalveolar oxygen concentration on lung preservation in a rabbit model. J Thorac Cardiovasc Surg 1991;101:1037-1043.[Abstract]
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8. Schulte-Hermann R, Bursch W, Kraupp-Grasl B, Oberhammer F, Wagner A. Programmed cell death and its protective role with particular reference to apoptosis. Toxicol Lett 1992;64–65:569–74.
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14. Schumer M., Colombel M.C., Sawczuk I.S., et al. Morphologic, biochemical, and molecular evidence of apoptosis during the reperfusion phase after brief periods of renal ischemia. Am J Pathol 1992;140:831-838.[Abstract]
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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Author home page(s): Walter Weder Ralph A. Schmid Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Stammberger, U. Articles by Schmid, R. A. Search for Related Content PubMed PubMed Citation Articles by Stammberger, U. Articles by Schmid, R. A.