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Ann Thorac Surg 2004;77:1048-1055
© 2004 The Society of Thoracic Surgeons
a University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA
b San Raffaele Hospital, University of Milan, Milan, Italy
Accepted for publication August 28, 2003.
* Address reprint requests to Dr Egan, University of North Carolina at Chapel Hill, Medical School Wing C-Room 354, CB 7065, Chapel Hill, NC 27599-7065, USA.
e-mail: ltxtme{at}med.unc.edu
Presented at the Thirty-ninth Annual Meeting of The Society of Thoracic Surgeons, San Diego, CA, Jan 31–Feb 2, 2003.
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Abstract |
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METHODS: Thirty-six Sprague-Dawley rats underwent left lung transplant (six groups of 6). Lungs were transplanted immediately after arrest, or from non–heart-beating donors after 2 hours of oxygen-ventilation or no ventilation. Recipients were reperfused for 4 or 6 hours, then lungs were stained with a mouse anti-rat ICAM-1 monoclonal antibody, developed with avidin-biotin peroxidase to a biotinylated anti-mouse immunoglobin G antibody. Intercellular adhesion molecule-1 expression was graded by two masked observers as 0 = absent, 1 = weak, or 2 = strong in alveoli, arterioles, and venules. Explanted recipient left lungs served as negative controls, and positive controls were generated 6 hours after intraperitoneal injection of endotoxin. Intercellular adhesion molecule-1 expression above baseline among groups was compared by Fisher's exact test.
RESULTS: Constitutive expression of ICAM-1 was present in rat lung alveoli, with 24 of 35 controls staining weakly and 4 of 35 strongly positive in alveolar areas. Intercellular adhesion molecule-1 expression was not increased in transplanted lungs evaluated after 4 hours of reperfusion, even lungs retrieved from non–heart-beating donors. But when non–heart-beating donor lungs were assessed 6 hours after onset of reperfusion, ICAM-1 expression was significantly more apparent in alveolar and arteriolar areas, compared with controls and lungs transplanted immediately after arrest.
CONCLUSIONS: Lungs transplanted immediately after circulatory arrest do not sustain sufficient ischemia-reperfusion injury to upregulate ICAM-1. Onset of reperfusion is the signal for ICAM-1 expression, not the onset of ischemia or the total duration of ischemic and reperfusion time together. Strategies at reperfusion may minimize ICAM-1 expression.
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Introduction |
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We have also demonstrated that lung cells remain viable and retain ultrastructural integrity for hours after death and circulatory arrest [6, 7]. However, retrieval of lungs from circulation-arrested donors is associated with substantially increased capillary permeability within 2 hours of retrieval [8], and lung transplantation from circulation-arrested donors in animals is associated with accumulation of extravascular lung water [4], presumably related to ischemia-reperfusion injury (IRI).
Ischemia-reperfusion injury also occurs in the setting of lung transplantation from conventional organ donors, and is a recognized cause of morbidity and mortality following clinical lung transplantation [9]. Ischemia-reperfusion injury in the lung is a cascade of events that manifests in two phases: an immediate phase, which becomes apparent early after reperfusion, associated with increased endothelial permeability; and a later phase, which is associated with polymorphonuclear (PMN) leukocyte infiltration as a result of expression on endothelial cells of adhesion molecules (CAMs).
In this study, we focused on the expression of intercellular adhesion molecule-1 (ICAM-1), a potent PMN attractant that directs the migration of circulating PMNs from blood into the surrounding tissues [10]. We were particularly interested in when ICAM-1 upregulation became apparent with respect to the onset of warm ischemia in the non–heart-beating donor (NHBD) and the onset of reperfusion in the recipient. Immunohistochemical techniques were used to demonstrate ICAM-1 expression in lung tissue. To develop a scoring system to quantify ICAM-1 expression in transplanted lungs, experiments using intraperitoneal (IP) injection of endotoxin (lipopolysaccharide; LPS) were performed to develop ICAM-1–positive lung specimens.
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Material and methods |
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Left lung transplantation
Donor preparation
We used a cuff technique for rat left lung transplant as previously described [5]. Briefly, donor rats were anesthetized with IP Nembutal, 3.5 mg/100 g. A small laparotomy incision was made, and 600 U of heparin (Elkins-Sinn, Cherry Hill, NJ) was injected intrahepatically. After intubation through a tracheotomy with a 14-gauge catheter, the rat was sacrificed with an intrahepatic injection of Nembutal (30 mg/kg). Lungs were retrieved immediately after death (immed [n = 12]) or 2 hours after death from nonventilated or oxygen-ventilated NHBD cadavers (n = 12 each). Mechanical ventilation was established in ventilated donor rats using a Harvard rodent ventilator (model 683; Harvard Apparatus, Holliston, MA) that delivered 100% oxygen with a tidal volume of 1 mL/100 g at a rate of 25 breaths/min and a positive end-expiratory pressure of 5 cm H2O. The cuffs were constructed with 14-gauge and 16-gauge intravenous Teflon catheters (Becton Dickinson Vascular Access, Sandy, UT) for the pulmonary artery and the pulmonary vein, respectively. They consisted of a cylindrical body with a 1-mm extension as a "handle." Each vessel (artery and vein) was passed through its respective cuff, and the proximal end was everted and fixed with a circumferential ligature of 8-0 Prolene (Ethicon, Somerville, NJ). The lung was then stored immersed in cold saline solution (4°C) on ice before transplantation.
Recipient preparation
Recipient rats were anesthetized, intubated, and ventilated as described above. Anesthesia was maintained with halothane or Fluothane (0.2% to 0.4%), adjusted on the basis of heart rate and blood pressure. Surgery was facilitated by the use of an operating microscope (Zeiss, Dyonics, Woburn, MA). The left jugular vein was cannulated with a 16-gauge intravenous cannula, and Ringer's lactate solution (Travenol Laboratories Inc, Deerfield, IL) was infused with a Medfusion syringe infusion pump (Medex, Inc, Duluth, GA) at a rate of 3 to 4 mL/h. A left thoracotomy was performed in the fifth intercostal space. The inferior pulmonary ligament was divided, and the lung was wrapped in cotton gauze and gently retracted to expose the hilum. The hilar structures were dissected. The left bronchus was ligated at the pulmonary reflection with a 2-0 silk ligature and transected distally, resulting in collapse of the recipient left lung. The left pulmonary artery and pulmonary vein were clamped with microsurgical vascular clamps and encircled with 6-0 silk suture. A 2-mm incision was made proximal to the lung on the anterior wall of the pulmonary artery and pulmonary vein. The deflated donor lung was placed in the chest. The donor pulmonary artery cuff was inserted into the recipient and secured with the 6-0 silk ligature, and the recipient pulmonary artery was transected. The pulmonary vein anastomosis was performed in a similar fashion. The recipient left lung was then removed. The bronchial anastomosis was performed with 8-0 Prolene (Ethicon) running suture, and the left lung was inflated. The pulmonary vessels were unclamped, and reperfusion was restored. Recipient rats were anesthetized with halothane or Fluothane until sacrificed 4 or 6 hours after onset of reperfusion.
Immunohistochemistry
Tissue preparation
Frozen sections of the lower half of the transplanted left lung or the lower half of the explanted recipient left lung (control) were prepared by inflation through the bronchus with OCT freezing media (VWR, Torrance, CA) and normal saline solution (1:1). Inflated samples were then sliced, and the cut face was placed in plastic containers with OCT and frozen over dry ice. Blocks were then stored at -70°C until sectioned. Six-micrometer frozen sections of lung were cut and placed on Fisher Probe-on Plus slides (Fisher Scientific Inc, Pittsburgh, PA). Staining was accomplished using a Microprobe staining station (Fisher Scientific Inc) and commercially available ABC reagent kits (Vectostaining Elite Kit, ABC, Vector Labs, Burlingame, CA). Slides were thawed, and incubated with 0.3% H2O2 in methanol for 30 minutes to block endogenous peroxidase activity, then blocked in 10% horse serum, and incubated for 60 minutes at room temperature with a murine anti-rat ICAM-1 (1A29; Pharmingen, San Diego, CA) at a dilution of 1:40,000. As a negative control, the primary antibody was replaced with an irrelevant monoclonal antibody (mouse IgG1
, MOPC-21, Pharmingen) at the same dilution.
After incubation, slides were washed in buffer and incubated for 30 minutes with a secondary biotinylated antibody (horse anti-mouse IgG, H&L chain, 1:2, Vector Labs). The secondary antibody was rat absorbed to reduce nonspecific staining and cross-reactivity. Slides were washed and incubated with avidin-biotin peroxidase (ABC, Vector Labs) at room temperature for 7 minutes. Diaminobenzidine tetrahydrochloride (0.5% DAB) in H2O2 was added to visualize the antigen-antibody complex. Slides were counterstained with hematoxylin, hydrated, and coverslipped with Permount.
Intercellular adhesion molecule-1 expression quantification
Intercellular adhesion molecule-1 expression was quantified (2+ strong and uniform, 1+ baseline or endogenous, 0 negative) in two areas of lung tissue: alveolar tissue, reflecting ICAM-1 expression in alveolar endothelial capillaries, and larger vessels. Two individuals analyzed and scored all slides in a masked fashion to eliminate bias and lessen interobserver error. When there was interobserver disagreement, the particular specimen was re-reviewed by both observers and the disagreement resolved.
When larger numbers of transplanted and control lungs were evaluated from the transplant experiments, it became apparent that there were different degrees of ICAM-1 expression in arterioles and venules, differentiated by the presence of smooth muscle encircling endothelial-lined vessels. Accordingly these lung specimens were scored in three geographic areas: alveoli, arterioles, and venules. Sections represented the lower half of the transplanted left lungs, compared with the lower half of the explanted recipient left lung.
These experiments were approved by the University of North Carolina at Chapel Hill Animal Care and Use Committee. All animals received humane care in accordance with the "Guide for the Care and Use of Laboratory Animals" (National Institutes of Health publication 85-23, revised 1985).
Statistics
There is constitutive expression of ICAM-1 in different areas of rat lung, so we chose to express the number of samples above baseline expression as percentage above baseline in the geographic areas of lung evaluated. Because there was no difference in gas exchange characteristics among recipients of NHBD lungs on the basis of whether the lung was ventilated for 2 hours in the donor or not [5], recipients of NHBD lungs were grouped together and compared with recipients of lungs transplanted immediately. This was done to increase the power of the observation. Fisher's exact test (two-tailed) was used to compare ICAM-1 expression among five groups of lungs: control, explanted recipient left lungs (n = 35); immed (n = 6), evaluated 4 hours after reperfusion; immed (n = 6), evaluated 6 hours after reperfusion; NHBD lungs evaluated 4 hours after reperfusion (n = 12), and NHBD lungs evaluated 6 hours after reperfusion (n = 12). One control specimen was unavailable owing to technical misadventure.
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Results |
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removed 6 hours after IP injection of LPS uniformly stained negatively (negative controls; Fig 1C). The expression of ICAM-1 remained elevated in both the alveolar region and vasculature at 12 hours after LPS injection. However, by 24 hours, pulmonary arteriole and venule ICAM-1 expression was again similar to controls. The time course of ICAM-1 expression after IP injection of LPS and in sham-injected animals is depicted in Figure 2. Lungs from sham-injected rats were no different from controls at all times.
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instead of anti-ICAM-1 and uniformly stained negatively (negative controls). To increase power for nonparametric statistical analysis, the NHBD lungs were grouped according to reperfusion time, whether ventilated or not during the ischemic period. Intercellular adhesion molecule-1 expression is depicted in Figure 4 for these groups. Expression was significantly increased for NHBD lungs evaluated 6 hours after reperfusion compared with 4 hours after reperfusion in alveoli and arterioles. Lungs transplanted immediately after retrieval with only 1 hour of ischemic time had similar expression of ICAM-1 as controls, whether evaluated 4 or 6 hours after the onset of reperfusion. Expression of ICAM-1 in NHBD lungs evaluated after 4 hours of reperfusion was no different than controls.
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Comment |
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-granules of platelets, which translocates to the endothelial cell surface within minutes of an insult, and ICAM-1, which requires gene activation and mRNA translation for expression on the cell surface [10].
In the normal rat lung, low levels of ICAM-1 are constitutively expressed on the surfaces of type I pneumocytes and on capillary endothelial cells. Fingar and colleagues [15] demonstrated that the level of ICAM-1 expression in the lung microvasculature increased after stimulation with the cytokine tumor necrosis factor- in a time-dependent manner. Others have shown that intratracheal endotoxin administration increases ICAM-1 expression and that the level of expression was substantially decreased in the presence of antibodies to tumor necrosis factor- [16]. After intratracheal administration of LPS in rats, the level of mRNA for ICAM-1 peaked at 4 hours after injection and the level of protein detected peaked 6 hours after injection [17]. This is consistent with our observation that upregulation of ICAM-1 in alveolar regions and pulmonary vessels secondary to IP injection of LPS is apparent 6 hours after injection. The delayed increase in expression is because of the time required for mRNA transcription, translation into protein, and transport to the cell membrane for expression.
The current study sheds light on both the threshold and the trigger for increased expression (upregulation) of ICAM-1 in lung IRI. A timeline of our experiment is depicted in Figure 5. We established that transplantation of lungs retrieved from NHBDs 2 hours after circulatory arrest resulted in increased ICAM-1 expression 6 hours after the onset of reperfusion. Transplantation of lungs retrieved immediately after circulatory arrest was presumably an insufficient injury to cause significant upregulation of ICAM-1. Thus, there appears to be a threshold amount of ischemia for IRI to be sufficiently disruptive to endothelial homeostasis to trigger ICAM-1 upregulation.
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The striking degree of increased ICAM-1 expression in alveolar areas and arterioles led us to combine both NHBD groups for statistical analysis. It is conceivable that nonventilated NHBD lungs sustain a more severe injury on reperfusion than NHBD lungs ventilated with oxygen, although this was not apparent in the current study. We have previously demonstrated that canine lungs retrieved 4 hours after death from oxygen-ventilated cadavers function relatively well when transplanted compared with nonventilated lungs retrieved 4 hours after death [2–4]. However, it is clear that 2 hours of normothermic ischemia, whether ventilated or not, is sufficient to cause increased ICAM-1 expression on reperfusion in the rat.
Preventing CAM upregulation or its impact on lung function after IRI may be more practical and better tolerated than PMN depletion in the recipient. Establishing the threshold and trigger for CAM upregulation is important for evaluating interventions to inhibit or interfere with expression of CAMs in this setting. Both anti–P-selectin and anti–ICAM-1 antibodies reduced lung injury in mice given cobra venom factor to activate complement [18]. Polymorphonuclear leukocyte–depleted rats or rats administered a blocking anti–P-selectin antibody 10 minutes before reperfusion of a left lung transplant preserved for 6 hours had reduced graft PMN deposition and improved survival (to 30 minutes) compared with control animals [19]. DeMeester and coworkers [20] demonstrated attenuated IRI in a rat lung transplant model when lungs were flushed with anti–ICAM-1 antibody and recipients treated with anti–ICAM-1, anti-CD11a, and anti-CD18a. Toda and associates [21] used an antisense ICAM-1 oligodeoxyribonucleotide during lung preservation to inhibit ICAM-1 expression, which improved gas exchange and survival after rat lung transplant, confirming the important role of this CAM in conventional lung IRI. However, none of these studies addressed the issue of the nature of the stimulus for increased ICAM-1 expression.
Intercellular adhesion molecule-1 is one of several inflammatory proteins that are under control of the gene promoter Nf-B[22]. Nf-B–IB dissociation leads to transcription of a variety of genes in the inflammatory cascade. Although we did not measure any indices of Nf-B–IB dissociation in this study, it is tempting to speculate that Nf-B–IB dissociation occurs on reperfusion of ischemic lung tissue.
For NHBDs to be a practical solution to the lung donor shortage in clinical practice, there is a need to develop strategies to retrieve lungs from individuals who arrive at the hospital already deceased. Thus, there will be an obligatory interval of normothermic ischemia in the donor after circulatory arrest, until consent for retrieval is obtained and trained personnel are available to retrieve the organs. If genes are upregulated during this interval, then strategies to minimize IRI must be directed at the consequences of increased transcription of a variety of proteins involved in the inflammatory cascade of IRI. On the other hand, if reperfusion is the trigger for gene upregulation, as our study implies, then intervention at the time of organ retrieval or reperfusate modification offers substantial promise to mitigate reperfusion injury caused by gene upregulation.
In summary, we have shown that ICAM-1 is substantially upregulated in an NHBD lung transplant model after 6 hours of reperfusion, but not after 4 hours of reperfusion. Lungs transplanted with a very short ischemic time did not have sufficient IRI to result in increased ICAM-1 expression. Our findings imply that the trigger for increased ICAM-1expression caused by upregulation of ICAM-1 gene transcription is reperfusion and not the period of ischemia preceding reperfusion at the time of transplantation. Additional studies using molecular biologic techniques to more accurately quantify the degree of gene upregulation in lung IRI may help unravel more accurately the sequence of molecular events in IRI and provide opportunities for intervention to mitigate organ injury after transplantation.
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Appendix |
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I have a few questions for you. In your intracellular adhesion molecule (ICAM) upregulation that occurred 6 hours after reperfusion, do you have any correlative physiologic changes that you noticed, such as a change in the alveolar to arterial gradient or the pulmonary arterial pressure, or perhaps a wet-to-dry ratio, or even the gross histology? Was there any more diffuse alveolar damage seen in those animals compared with controls? Also, any theories as to why there is less ICAM upregulation in the venules than in the arterioles and in the alveoli? And then finally, we all know that hypoxia and ischemia upregulate nuclear factor-kappaB–dependent transcription, and do you think that if you had allowed the ischemic time interval to be greater than 6 or 8 hours, you may see the same ICAM upregulation irrespective of reperfusion?
Thank you. I enjoyed your paper very much.
DR DAVID SCHRUMP (Bethesda, MD): Nitric oxide as well as proteasome inhibitors I believe have been demonstrated to ameliorate ischemia-reperfusion injury. Do you have any information regarding regulation of ICAM-1 in this setting of either of those agents?
DR EGAN: Thank you for your questions.
Doctor Jones, we do not have physiologic data that differentiate lungs assessed at 4 hours after reperfusion or 6 hours after reperfusion, with this caveat, and that is we have myeloperoxi-dase data on some of these groups of lungs, and it appears that lungs retreived 6 hours after the onset of reperfusion did have elevated levels of myeloperoxidase activity, which would be consistent with increasing amounts of polymorphonuclear leukocytes. Unfortunately we do not have those data on all of these groups of animals. From a standpoint of the gross histologic assessment of the tissues, there was not a huge amount of polymorphonuclear leukocyte infiltration that was observed, and so that was clearly difficult to quantify. We have some data in human lungs that mRNA for ICAM-1 is in fact noted to be increased after the onset of reperfusion and is seen as early as 30 minutes after the onset of reperfusion using light cycler polymerase chain reaction.
I do not have a good explanation for the difference in ICAM-1 expression in venules and arterioles. We did not notice it in the endotoxin-injected rats. I will say that the histologic assessment of those lungs implies that the degree of injury of endotoxin is much more severe than the degree of injury we saw with transplantation of lungs even from non–heart-beating donors, and there may be a question of the degree of upregulation as well. We had to come up with a scoring mechanism, and it was clear when we started looking at the transplanted lungs that there seemed to be a difference between venules and arterioles, and, as a result, we elected to report it. I cannot explain it.
You asked a very important question I think about longer ischemic intervals and whether we have looked at that. We have not looked at that in this model. We have looked at the impact of longer ischemic intervals on cell death, and we know that oncerat lungs are ischemic for 4 hours and not ventilated that approximately 50% of the cells are dead by trypan blue exclusion, and so one might assume that something might happen with a longer time interval.
The question about nitric oxide is a very interesting one because clearly there are potentially mechanisms to downregulate this whole process of the inflammatory cascade in ischemia-reperfusion injury, and while we do not have any data on nitric oxide or nitroglycerin and ICAM-1 expression, there are some data that we allude to in the manuscript that references other work looking at the ability of nitric oxide to diminish ICAM-1 expression, and there are several instances in the literature of nitric oxide or nitric oxide donors that have subsequently resulted in reduced leukocyte infiltration after lung ischemia-reperfusion injury. I think that is a very fertile area for further investigation.
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References |
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B is activated during acute inflammation in vivo in association with elevated endothelial cell adhesion molecule gene expression, and leukocyte recruitment. J Inflamm 1995;45:283-296.[Medline]
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  H. Inokawa, M. Sevala, W. K. Funkhouser, and T. M. Egan Ex-vivo perfusion and ventilation of rat lungs from non-heart-beating donors before transplant. Ann. Thorac. Surg., October 1, 2006; 82(4): 1219 - 1225. [Abstract] [Full Text] [PDF]
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 A. Basit, J. Reutershan, M. A. Morris, M. Solga, C. E. Rose Jr., and K. Ley ICAM-1 and LFA-1 play critical roles in LPS-induced neutrophil recruitment into the alveolar space Am J Physiol Lung Cell Mol Physiol, August 1, 2006; 291(2): L200 - L207. [Abstract] [Full Text] [PDF]
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