Chest ACCP Member Benefits
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Guest Access | Sign In via User Name/Password
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Article Archive
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (32)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Soncul, H.
Right arrow Articles by Kalaycioglu, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Soncul, H.
Right arrow Articles by Kalaycioglu, S.
(Chest. 1999;115:1672-1677.)
© 1999 American College of Chest Physicians

Role of Ischemic Preconditioning on Ischemia-Reperfusion Injury of the Lung*

Halim Soncul , MD; Eser Öz , MD and Sedat Kalaycioglu , MD

* From the Department of Thoracic and Cardiovascular Surgery (Drs. Soncul and Kalaycioglu) and the Department of Physiology (Dr. Öz), Gazi University Medical Faculty, Ankara, Turkey.


   Abstract
TOP
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Study objectives: Ischemia-reperfusion injury of the lung frequently occurs after cardiopulmonary bypass, after pulmonary thromboembolectomy, and especially during lung transplantation. The protective effects of preconditioning on the heart, liver, bones, and various other organs have been previously evaluated. In this comparative study, we used isolated guinea pig lungs to show the effects of preconditioning on lung ischemia.

Methods: The lungs (n = 10 in each group) were mounted on a modified Langendorff perfusion apparatus and perfused by Krebs-Henseleit solution for 30 min. We applied an ischemic preconditioning (5 min ischemia + 5 min perfusion, two times) in the experimental group. After 3 h of normothermic ischemia, the lungs were reperfused for 30 min. Pulmonary artery pressures and malondialdehyde (MDA) and glutathione (GSH) levels of the tissue and the perfusate were measured before and after the ischemic period and also at the end of reperfusion. Electron microscopic evaluation was done on randomly selected lungs of three animals in each group at the end of the experiment.

Results: Both MDA and GSH levels of tissue and perfusate decreased in the experimental group after reperfusion, although the reduction in GSH levels did not reach statistical significance. The increase in pulmonary artery pressure was lower in the preconditioning group after reperfusion.

Conclusions: Our data showed that ischemic preconditioning of the lung may have a protective effect in ischemic-reperfusion injury.

Key Words: ischemic injury • lung ischemia • preconditioning • reperfusion injury


   Introduction
TOP
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Because the shortage of donor organs still remains a major limiting factor in the widespread application of organ transplantation, there is an increasing interest in preservation of organs from ischemic and reperfusion injury for longer periods of times. Much more difficulty has been associated with lung preservation than with preservation of solid organs in vitro because of the unique structure of the lung, especially the close apposition of blood and air compartments in the alveoli.

In 1979, Fridovich first suggested that tissue injury related to temporary ischemia actually occurred during the initiation of reperfusion by the generation of reactive oxygen species.1 Since that time, the role of reactive oxygen species in ischemia-reperfusion injury has been examined by detecting byproducts of target molecule oxidation (lipid peroxidation and protein oxidation) and by determining the consumption of tissue antioxidants such as glutathione (GSH).2 3

Ischemic preconditioning was first described by Murry and colleagues on dog myocardium in 1986.4 Since its original description, the protective effects of ischemic preconditioning have been demonstrated in various species, including dogs, rabbits, rats, and guinea pigs.5 6 Many studies suggest that ischemic preconditioning achieved by brief periods of ischemia and reperfusion before a prolonged period of ischemia dramatically reduces the ischemic-reperfusion injury in solid organs such as the heart, liver, kidney, and bones. However, there are very limited data about the similar effects of preconditioning on lungs.7


   Materials and Methods
TOP
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Lungs were obtained from male guinea pigs (n = 20) weighing 340 to 480 gm. All animals received humane care in compliance with the "Guide for the Care and Use of Laboratory Animals," prepared by the Institute of Laboratory Animal Resources.8

The animals were anesthetized with urethane and received 200 U of heparin through the femoral vein. Sternotomy was performed after insertion of a no. 14 cannula into the trachea by an open tracheostomy. After cannulation of the pulmonary artery via the right ventricle, the lungs and the heart were rapidly harvested.

Perfusion Techniques
The lungs were mounted on a modified Langendorff perfusion apparatus. We inflated the lungs with room air and then began perfusion with a gassed (oxygen 95%, carbon dioxide 5%) Krebs-Henseleit solution, which is a well-known buffer widely used for isolated organ perfusion studies. The composition of the solution was as follows: NaHCO3, 25 mmol/L; NaCl, 118 mmol/L; KH2PO4, 1.2 mmol/L; KCl, 4.8 mmol/L; MgSO4, 1.2 mmol/L; CaCl2, 1.2 mmol/L; and glucose, 11.1 mmol/L. The Krebs buffer was pumped by a microtubing pump (model MP-3; Rikakikai Co, Ltd; Tokyo, Japan) at a rate of 15 mL/min at 37°C.

In the 30th minute of perfusion, we collected perfusate samples from the left atrium and excised one of the lung segments. We determined the malondialdehyde (MDA) and GSH levels in tissue and perfusate. After the perfusion had been stopped, the lungs were kept at 37°C in an isotonic saline bath for 3 h in the control group. In the experimental group, we stopped the perfusion for 5 min, perfused for 5 min, stopped again for 5 min, and reperfused for another 5 min prior to a 3-h ischemic period).

After 3 h of ischemia, we began reperfusion with the same buffer at 37°C. Mean pulmonary artery pressures were recorded and tissue pieces were excised at the beginning of reperfusion. After 30 min of reperfusion, the perfusate and tissue samples were collected while the pulmonary artery pressure was recorded. Pulmonary artery pressures were measured with the same cannula that was inserted into the pulmonary artery for perfusion (Fig 1 ).



View larger version (31K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 1. Experiment design and protocol. TS = collection of tissue samples; PS = collection of perfusate samples.

 
Data Analysis and Statistics
Pulmonary artery pressures (mm Hg) were measured with a patient monitor (Datascope 2001A monitor; Datascope Corp; Montvale, NJ) and a pressure monitoring kit (Viggo-Spectramed Inc; Oxnard, CA). The levels of tissue MDA (nmol/g of tissue), perfusate MDA (nmol/mL), tissue GSH (µmol/g tissue), and perfusate GSH (nmol/mL) were measured.9 10 Ultrastructural analysis was performed on three randomly selected pieces of tissue in each group at the end of the experiment. For the ultrastructural study, we used the uranyl acetate–lead citrate staining method (x 12,000) at the Department of Morphology, Hacettepe University Ankara.

The results are presented as mean (± SEM). The overall significance of differences between the groups was determined by the t test (two samples assuming equal variances) using data analysis software (Microsoft Excel 7.0; Microsoft Corp; Redmond, WA).


   Results
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Because we excised tissues for MDA and GSH determination, the lung sizes changed throughout the experiment. Therefore, we calculated the alterations of pulmonary artery pressure as the percentage change from the preischemic value in each experiment.

The percentage change in the mean pulmonary artery pressures, both after the ischemic period and after reperfusion, was greater in the control group. The difference between the control and experimental groups was not statistically significant (Table 1 and Fig 2 ).


View this table:
[in this window]
[in a new window]

  		Table 1. The Effect of Preconditioning on Ischemia-Reperfusion Injury*

 


View larger version (27K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 2. Pulmonary artery pressures (percentage change). Values are mean (± SEM).

 
In the preconditioning group, the mean tissue MDA level decreased noticeably when compared with the preischemic value, especially at the end of the reperfusion period (p = 0.003). The difference between the two groups after the reperfusion period was also significant (p = 0.0002; Table 1 and Fig 3 ).



View larger version (52K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 3. MDA levels in tissue (nmol/g of tissue) and perfusate (nmol/mL). *Significant difference (p = 0.003) between the indicated groups. {dagger}Significant difference (p = 0.0002) between the indicated groups. #Significant difference (p = 0.035) between the indicated groups. {ddagger}Significant difference (p = 0.016) between the indicated groups. Values are mean (± SEM).

 
Compared with the preischemic values, the MDA level of the perfusate increased significantly after reperfusion in the control group (p = 0.016). After reperfusion, the difference between the preconditioning and control groups was again significant (p = 0.035; Table 1 and Fig 3 ).

In the preconditioning group, the mean tissue GSH level showed a remarkable decrease after reperfusion when compared with the preischemic levels (p = 0.01). The difference between the groups was statistically significant after the reperfusion period (p = 0.05; Table 1 and Fig 4 ).



View larger version (50K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 4. GSH levels in tissue (µmol/g of tissue) and perfusate (nmol/mL). *Significant difference (p = 0.011) between the indicated groups. {dagger}Significant difference (p = 0.007) between the indicated groups. #Significant difference (p = 0.035) between the indicated groups. {ddagger}Significant difference (p = 0.01) between the indicated groups. Values are mean ± SEM.

 
The mean GSH level of the perfusate decreased significantly in the preconditioning group after reperfusion (p = 0.007). The difference between the two groups was also statistically significant after reperfusion (p = 0.01; Table 1 and Fig 4 ).

The ultrastructural analysis of the lung tissues in the control group showed a marked separation between the capillary endothelium and the alveolar epithelium, as well as plenty of empty cytoplasmic vacuoles with different sizes of pneumocytes (Fig 5 ).



View larger version (173K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 5. In the control group, the interalveolar septum is edematous and extravasation of leukocytes is evident. A = alveolar lumen; L = lymphocyte; arrows indicate interalveolar septum (lead citrate, original x6,500).

 
In the preconditioning group, the alveolar capillary membrane and cellular structures were nearly normal (Fig 6 ).



View larger version (112K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 6. In the preconditioning group, the interalveolar septum and alveolar capillary wall are normal. C = capillary membrane; A = alveolar lumen; E = erythrocyte; arrow indicates alveolocapillary membrane (lead citrate, original x6,500).

 

   Discussion
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It was initially thought that the lung was resistant to ischemic injury because of its dual pulmonary and bronchial arterial blood supply and its independent source of oxygen, available from the alveolar space.11 However, in the last 10 years, multiple studies have described the delicate alveolar-capillary membrane network as an extremely sensitive structure that is subject to ischemia-reperfusion injury in many experimental studies and clinical conditions.12

It is widely accepted that effective organ preservation is one of the keys to successful lung and heart-lung transplantation, especially when the allograft must sometimes be transported long distances. Although modern preservation techniques such as plegic solutions, provision of external substances, and temperature manipulation have revolutionized transplantation surgery, many investigators are still working toward a more reliable preservation method. Providing a longer ischemic period would permit safer procurement of lungs from greater distances.13 14

Ischemic preconditioning achieved by brief periods of ischemia and reperfusion before a prolonged period of ischemia was first described in 1986.3 This phenomenon has been widely studied in such solid organs as the kidneys, liver, and especially the heart. Most of the studies have shown that preconditioning plays a significant role in decreasing the extent of ischemic-reperfusion injury.15 16 Although the mechanism of this highly interesting phenomenon is still not clear, some of the mechanisms that have been suggested are adenosine receptor stimulation, release of A1 adenosine agonists, release of some protective proteins, activation of sodium and proton transport, and depletion of free radicals.17 18

The isolated perfused lung model used for our study was previously developed and used by a number of authors as a screening technique for the many factors affecting lung preservation and reperfusion injury.19 Previous experiments have demonstrated that lung function deteriorates after very long periods of time under hypothermic conditions.20 Because of the difficulties of keeping the standardized experimental environment for such long periods in our laboratory conditions, in the current experiment we opted to use 3 h of normothermic (37°C) ischemia.

In our study, lipid peroxidation associated with free radical generation was assessed by measuring tissue and perfusate MDA, which is a three-carbon product of lipid peroxidation. Tissue and perfusate levels of GSH were measured to show the defense mechanisms of tissue against free radical injury. Tissue damage was assessed by measuring pulmonary artery pressure, which is a relative parameter for pulmonary vascular resistance.

In this study, the MDA levels of tissue and perfusate increased in the control group and decreased in the preconditioning group after reperfusion. This may represent a protective effect of preconditioning from lipid peroxidation. The decrease of tissue and perfusate levels of GSH in the preconditioning group after reperfusion may suggest that tissue GSH is a defense mechanism for reperfusion injury and that this injury occurred by lipid peroxidation. From this point of view, the small decrease of tissue and perfusate levels may be due to consumption of tissue GSH. Although this decrease was not quite statistically significant, it could be argued that preconditioning of the tissue may activate some protective mechanisms.

Based on these data, it can be said that lung preconditioning may have a noticeable effect on ischemia-reperfusion injury of the ischemic lungs under normothermic conditions, probably by stimulating tissue defense mechanisms. Similar effects of preconditioning on other organs, especially the heart, have also been previously reported. Jenkinson and coworkers21 found that exposure of isolated rat lungs to hypoxia reoxygenation increases the concentration of the oxidized form of GSH in the lung perfusate during reoxygenation. Jackson and Veal22 also demonstrated that hypoxia reoxygenation decreased lung GSH in rabbit lungs. Both of these observations serve as a marker of hydrogen peroxide formation and a marker of oxidant stress during tissue reperfusion. In contrast with the study of Omar et al,23 the decrease in lipid peroxidation may suggest a free radical mechanism for the effect of preconditioning.

In conclusion, our study suggests that ischemic preconditioning achieved by brief periods of ischemia and reperfusion can significantly reduce the extent of ischemia-reperfusion injury of the lungs under normothermic conditions.


   Footnotes
 
Correspondence to: Halim Soncul, MD, Gazi Hastanesi Kalp Damar Cerrahisi ABD, Besevler 06510 Ankara, Turkey; e-mail: hs04-k@tr-net.net.tr, soncul@tip.gazi.edu.tr

Abbreviations: GSH = gluthatione; MDA = malondialdehyde

Received for publication June 26, 1998. Accepted for publication January 4, 1999.


   References
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Fridovich, I (1979) Hypoxia and oxygen toxicity. Adv Neurol 26,255-267[Medline]
  2. Eckenhoff, R, Dodia, C, Tan, Z, et al (1992) Oxygen-dependent reperfusion injury in the isolated rat lung. J Appl Physiol 72,1454-1461[Abstract/Free Full Text]
  3. Ayene, I, Dodia, C, Fisher, A (1992) Role of oxygen in oxidation of lipid and protein during ischemia/reperfusion in isolated perfused rat lung. Arch Biochem Biophys 296,183-190[CrossRef][ISI][Medline]
  4. Murry, CE, Jennings, RB, Reimer, KA (1986) Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation 74,1124-1136[Abstract/Free Full Text]
  5. Jennings, RB, Murry, CE, Reimer, KA (1991) Energy metabolism in preconditioned and control myocardium: effect of total ischemia. J Mol Cell Cardiol 23,1449-1458[CrossRef][ISI][Medline]
  6. Schott, N, Rohman, S, Braun, ER, et al (1990) Ischemic preconditioning reduces infarct size in swine myocardium. Circ Res 66,1133-1142[Abstract/Free Full Text]
  7. Du, ZY, Hicks, M, Winlaw, D, et al (1996) Ischemic preconditioning enhances donor lung preservation in the rat. J Heart Lung Transplant 15,1258-1267[ISI][Medline]
  8. Guide for the care and use of laboratory animals. Bethesda, MD: National Institutes of Health, 1985; Publication No. 85–23
  9. Kurtel, H, Granger, DN, Tso, P, et al (1992) Vulnerability of intestinal interstitial fluid to oxidant stress. Am J Physiol 263,573-578
  10. Casini, A, Ferrali, M, Pompella, A, et al (1986) Lipid peroxidation and cellular damage in extra-hepatic tissues of bromobenzene intoxicated mice. Am J Pathol 123,520-531[Abstract]
  11. Deffebach, M, Charan, N, Lakshminarayan, S, et al (1987) The bronchial circulation: small but vital attribute of the lung. Am Rev Respir Dis 135,463-471[ISI][Medline]
  12. Adkins, W, Taylor, A (1990) Role of xantine oxidase and neutrophils in ischemia-reperfusion injury in rabbit lung. J Appl Physiol 69,2012-2019[Abstract/Free Full Text]
  13. Hooper, TL, Locke, TJ, Fetherston, G, et al (1990) Comparison of cold flush perfusion with modified blood versus modified Euro-Collins solution for lung preservation. J Heart Transplant 9,429-434[ISI][Medline]
  14. Soncul, H, Kaptanoglu, M, Öz, E, et al (1994) The role of selenium added to pulmonary preservation solutions in isolated guinea pig lungs. J Thorac Cardiovasc Surg 108,922-927[Abstract/Free Full Text]
  15. Karck, M, Rahmanian, P, Haverich, A (1996) Ischemic preconditioning enhances donor heart preservation. Transplantation 62,17-22[CrossRef][ISI][Medline]
  16. Ogino, H, Smolenski, RT, Zych, M, et al (1996) Influence of preconditioning on rat heart subjected to prolonged cardioplegic arrest. Ann Thorac Surg 62,469-474[Abstract/Free Full Text]
  17. Ramasamy, R, Liu, H, Anderson, S, et al (1995) Ischemic preconditioning stimulates sodium and proton transport in isolated rat hearts. J Clin Invest 3,1464-1472
  18. Cohen, MV, Walsh, RS, Goto, M, et al (1995) Hypoxia preconditions rabbit myocardium via adenosine and catecholamine release. J Mol Cell Cardiol 27,1527-1534[CrossRef][ISI][Medline]
  19. Wang, LS, Yoshikawa, K, Mitoshi, S (1989) The effect of ischemic time and temperature on lung preservation in a simple ex-vivo rabbit model used for functional assessment. J Thorac Cardiovasc Surg 98,333-342[Abstract]
  20. Miyoshi, S, Shimokawa, S, Schreinemakers, H, et al (1992) Comparison of the University of Wisconsin preservation solution and other crystalloid perfusates in a 30-hour rabbit lung preservation model. J Thorac Cardiovasc Surg 103,27-32[Abstract]
  21. Jenkinson, S, Marcum, R, Pickard, J, et al (1988) Glutathione disulfide formation occuring during hypoxia and reoxygenation of rat lung. J Lab Clin Med 112,471-478[ISI][Medline]
  22. Jackson, R, Veal, C (1990) Effects of hypoxia and reoxygenation on lung glutathione system. Am J Physiol 259,518-524
  23. Omar, BA, Hanson, AK, Bose, SK, et al (1991) Ischemic preconditioning is not mediated by free radicals in the isolated rabbit heart. Free Radic Biol Med 11,517-520[CrossRef][ISI][Medline]



This article has been cited by other articles:


Home page
 Arch SurgHome page
S. Pasupathy and S. Homer-Vanniasinkam
Surgical Implications of Ischemic Preconditioning
Arch Surg, April 1, 2005; 140(4): 405 - 409.
[Abstract] [Full Text] [PDF]

Home page
 Eur Respir JHome page
C. S. H. Ng, S. Wan, and A. P. C. Yim
Pulmonary ischaemia-reperfusion injury: role of apoptosis
Eur. Respir. J., February 1, 2005; 25(2): 356 - 363.
[Abstract] [Full Text] [PDF]

Home page
 Ann. Thorac. Surg.Home page
D. G. Tang, D. R. Pavot, M. M. Mouria, D. M. Holwitt, and N. M. Cohen
Warm ischemia lung protection with pinacidil: an ATP regulated potassium channel opener
Ann. Thorac. Surg., August 1, 2003; 76(2): 385 - 390.
[Abstract] [Full Text] [PDF]

Home page
 Eur. J. Cardiothorac. Surg.Home page
M. C. Sivrikoz, B. Tuncozgur, M. Cekmen, K. Bakir, I. Meram, E. Kocer, B. Cengiz, and L. Elbeyli
The role of tissue reperfusion in the reexpansion injury of the lungs
Eur. J. Cardiothorac. Surg., November 1, 2002; 22(5): 721 - 727.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
T. Fukuse, T. Hirata, M. Omasa, and H. Wada
Effect of Adenosine Triphosphate-Sensitive Potassium Channel Openers on Lung Preservation
Am. J. Respir. Crit. Care Med., June 1, 2002; 165(11): 1511 - 1515.
[Abstract] [Full Text] [PDF]

Home page
 ChestHome page
S. Hammerschmidt, N. Buchler, and H. Wahn
Tissue Lipid Peroxidation and Reduced Glutathione Depletion in Hypochlorite-Induced Lung Injury
Chest, February 1, 2002; 121(2): 573 - 581.
[Abstract] [Full Text] [PDF]

Home page
 Eur. J. Cardiothorac. Surg.Home page
R. I. Gasparri, N. C.P. Jannis, W. J. Flameng, T. E. Lerut, and D. E.M. Van Raemdonck
Ischemic preconditioning enhances donor lung preservation in the rabbit
Eur. J. Cardiothorac. Surg., December 1, 1999; 16(6): 639 - 646.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Article Archive
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (32)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Soncul, H.
Right arrow Articles by Kalaycioglu, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Soncul, H.
Right arrow Articles by Kalaycioglu, S.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS