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Eur J Cardiothorac Surg 2006;29:294-298
© 2006 Elsevier Science NL
a Department of Thoracic Surgery, Afyon Kocatepe University, School of Medicine, 03200 Afyon, Turkey
b Department of Anaesthesia, Kocatepe University, School of Medicine, Afyon, Turkey
c Department of Biochemistry, Kocatepe University, School of Medicine, Afyon, Turkey
Received 24 May 2005; received in revised form 6 December 2005; accepted 6 December 2005.
* Corresponding author. Tel.: +90 533 6471729; fax: +90 272 2172029. (Email: hesme{at}aku.edu.tr).
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Abstract |
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 Top Abstract 1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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Key Words: Remote injury • Lung • Liver • Heart
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1. Introduction |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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Ischemia is mainly a local event but, after revascularization, the mediators from the ischemic tissue enter the systemic circulation and affect other organ systems. It has been concluded that the systemic effects of IR injury are caused by activated neutrophils, the complement system, and proinflammatory and vasoactive mediators such as eicosanoids, nitric oxide, cytokines, and oxygen-free radicals [3–6]. The remote effects of IR are most frequently observed in the pulmonary, renal, hepatic and cardiovascular systems, and can result in the development of the systemic inflammatory response and multiple organ dysfunction syndromes, both of which account for 30–40% of the mortality in tertiary referral intensive care units [7,8].
Although the pulmonary injury that is formed by the effects of IR injury on remote organs has been previously studied, no previous study that investigated the effects of IR injury of lung on remote organs has been considered. We suggested that lung IR injury may produce reactive oxygen species (ROS) and induce systemic inflammatory response which may cause remote organ inflammatory response and dysfunction. For this purpose, we constructed IR injury in vivo rabbit model and investigated its effect on remote organ (heart and liver) injury. Myeloperoxidase (MPO) activity as indices of neutrophil recruitment, protein sulfhydryl (SH) level as indices of antioxidant status, thiobarbituric acid-reactive substances (TBARS) level as indices of lipid peroxidation, and protein carbonyl level as indices of protein oxidation were evaluated in lung, heart, and liver tissue.
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2. Materials and methods |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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2.1 Surgical procedure
The animals were sedated with 50 mg/kg ketamine HCL (Ketalar) and 15 mg/kg Xylazine (Rompun) i.m. as premedication. The ear artery and vein were located and the arterial and peripheral venous lines were inserted. Systemic arterial pressure, SpO2 and heart rate were continuously monitored (Datex Ohmeda Type F-CU8, Helsinki, Finland). The animals were anesthetized with 1% sevoflorane. An endotracheal tube was inserted in the trachea through a tracheostomy. The endotracheal tube was then connected to a volume-controlled ventilator (CWe SAR-830/P ventilator Ardmore, USA) and the animals were ventilated at a rate of 30 breaths/min, a tidal volume of 10 ml/kg, an inspired oxygen fraction of 1.0, and a positive end-expiratory pressure of 2 cmH2O. Sodium heparin (500 IU/kg) was administered through the ear peripheral vein. A heating pad was applied during anaesthesia in order to keep the body temperature at 38 °C. Under aseptic conditions, a left thoracotomy was made. The left lung was then mobilized, the pulmonary hilum was dissected, and perivascular and peribronchial tissues were removed. Ischemia was induced by applying a nontraumatic vascular clamp to the left lung hilum for 60 and 120 min in groups 2 and 3, respectively. After 3 min, the lung was inspected for signs of ischemia; the wound was covered with cotton soaked in sterile phosphate-buffered saline. After removal of the clamp, the left lung was inspected for restoration of blood flow for 60 min and rabbits in all groups were sacrificed and left lung, heart, and liver were harvested and stored for further analysis.
2.2 Tissue homogenization
A portion of each lung, heart, and liver was homogenized for all assays except MPO assay. Homogenization was performed in 1:10 (w/v) 0.1 M potassium phosphate buffer (pH 7.4) with a Ultra Turrax homogenizer (IKA T18 basic, Wilmington, NC, USA). After centrifugating the homogenates at 10,000 rpm, +4 °C for 10 min, the supernatants were removed and subjected to protein SH, TBARS, and protein carbonyl. For MPO assay, lung, heart, and liver tissues were first homogenized in 1:10 (w/v) 50 mM potassium phosphate buffer (pH 7.4). After centrifugation at 15,000 x
g, +4 °C for 10 min, the pellet was rehomogenized in an equal volume of 50 mM potassium phosphate buffer (pH 6.0) containing 0.5% HETAB and 10 mM EDTA. This homogenate was subjected to MPO assay.
2.3 Biochemical assays
The supernatants of homogenized tissue samples were analyzed to determine tissue concentrations of myeloperoxidase, TBARS, protein carbonyl, and protein SH. Tissue MPO activity was determined by the method of Suzuki et al. [9] and the results were expressed as units per milligram protein (x10–3
U/mg protein). The end product of lipid peroxidation, TBARS, was measured using thiobarbituric acid method so as to assess the degree of tissue lipid oxidation, as described by Okhawa et al. [10]. To evaluate the potential for oxygen radical scavenger activity in tissue, protein SH groups were measured spectrophotometrically using Ellman's reagent. This spectrophotometric analysis is based on the interaction of 5,5'-dithiobis-2-nitrobenzoic acid with the thiol-disulfide [11]. Protein oxidation in the tissue was assessed with a colorimetric assay that measured protein carbonyl content, through the interaction of the supernatant with dinitrophenylhydrazine, as described by Levine et al. [12]; tissue protein SH and carbonyl contents were expressed in relation to the tissue protein concentration that was measured using the biuret method [13]. TBARS, protein SH, and protein carbonyl levels were expressed in terms of the protein concentration of tissue samples, micromoles per gram wet tissue (µmol/g protein).
2.4 Statistical analysis
All parametric results were expressed as mean ± standard deviation for each group. Statistical analysis was performed with SPSS 11.5 software. Shapiro–Wilks test was performed to check the normality of the data before running tests. Each biochemical parameter of rabbits was compared using the one-way ANOVA. Tamhane and Tukey's significant difference test were used for comparisons within the groups. A p-value less than 0.05 was considered to be statistically significant.
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3. Results |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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4. Discussion |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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The liver, with its portal circulation, very extensive sinusoidal network, and associated slow flow rates, supports adhesion in postsinusoidal venules as well as in sinusoids. In fact, the sinusoids appear to be the dominant site of leukocyte sequestration in certain inflammatory conditions [14]. Evidence suggests that injury to the liver following a remote inflammatory insult occurs in a biphasic manner, much like that of local liver injury [15]. More specifically, the early phase of injury may be neutrophil independent, whereas the latter was neutrophil dependent. Support for this comes from studies of both remote and local hepatic injury that suggest the initiation of hepatocyte damage was mediated primarily by Kupffer cells [15,16]. Like other phagocytic cells, many antigens and inflammatory signaling molecules stimulate Kupffer cells to a degree of stimulation that is characterized by amplified inflammatory, cytotoxic, and chemotactic states [17].
In our study, we have shown that 60 min ischemia–60 min reperfusion was enough to increase the hepatic MPO activity almost threefold compared to sham-operated animals. Since no statistical significance was observed between different ischemia durations, it could be postulated that 60 min of ischemia is enough to elicit hepatic MPO production and subsequent changes. Increase in MPO activity was detected in a several hepatic disorders including hepatic IR injury and confirmed the recruitment of neutrophils [18]. We found that lung IR resulted in increased levels of hepatic tissue TBARS and protein carbonyls, indicating increased hepatic lipid and protein oxidation, respectively. The more prominent increase in TBARS and protein carbonyl levels in animals exposed to 120 min ischemia–60 min reperfusion, as opposed to those exposed to 60 min ischemia–60 min reperfusion, confirms the aggravating effects of prolonged ischemia on oxidative stress. Also, 60 min ischemia–60 min reperfusion caused decreased hepatic protein SH content, implying decreased potential for oxygen-radical-scavenger activity.
Grunenfelder et al. [19] reported that acute myocardial injury has been demonstrated as a remote sequela of severe lower torso ischemia–reperfusion due to proinflammatory events and remote myocardial injury after lower torso IR is present in both ventricles; however, the left ventricle seems to be more susceptible as assessed by albumin permeability. Weinbroum et al. [20] documented severe reduction in myocardial velocity of contraction and relaxation when the heart was exposed to an effluent exiting a liver that had previously undergone ischemia. These phenomena have been largely attributed to reactive oxygen species (ROS), which are abundant in the effluent that exits organs or areas that had been earlier subjected to IR [20]. "Stunning" of the heart following IR is a known clinical event that can lead to protracted and sometimes irreversible mechanical collapse. ROS were shown to alter myofibril function by inactivating creatine kinase or by disrupting intracellular calcium metabolism, which is essential for adequate functional myofibril activity [21]. The ROS-rich postischemia remote organ effluent could thus induce the sequential negative chronotropic and inotropic phenomena, both of which are energy dependent.
We found that the markers of oxidative stress such as carbonyl content of proteins increased after 60 min ischemia–60 min reperfusion while anti-oxidative activity markers such as SH levels of heart tissue proteins gained importance when ischemia time reached to 120 min. Our study also detected a significant increase in TBARS, indicating increased myocardial lipid oxidation, after 60 min ischemia–60 min reperfusion. The sham-operated animals also had MPO activity, but after 60 min ischemia–60 min reperfusion, an abrupt increase in MPO activity was detected. This increase reached maximum levels after 120 min ischemia–60 min reperfusion.
We have shown that lung IR injury causes deleterious effects in remote organs such as heart and liver but remote organ dysfunction that may be the result of these deleterious effects, may be argued. We have not studied remote organ dysfunction either biochemically (aspartate amino transferase, amino alanine transferase levels for liver and troponin levels for heart) or pathologically in current study which may be decided as the limitation of our study. However, oxidative stress is considered to be associated with many diseases, such as inflammatory, cardiovascular and hepatic diseases [22,23]. Remote organ oxidative stress that is shown in our study may present itself with organ dysfunction.
In conclusion, the data of the present study suggest that pulmonary IR induces liver and heart injury characterized by activated neutrophil sequestration and release of significant amounts of ROS. It confirmed that IR injury is a systemic phenomenon. Also, 60 min ischemia–60 min reperfusion is enough to elicit remote effects of IR injury, but it should be kept in mind that as IR duration increases, ROS-mediated deleterious changes will be observed. Since pulmonary IR injury is encountered in many clinical situations, such as pulmonary transplantation, cardiopulmonary bypass, pulmonary thromboembolectomy, bronchovascular sleeve resection, or total pneumothorax and massive pleural effusion, the danger of remote organ injury remains a devastating problem. Additional studies are still needed to elucidate the mechanisms that are involved in pulmonary IR-induced remote organ dysfunction and means of protection. Clinical significance of pulmonary IR-induced remote organ dysfunction should be further studied to guide thoracic surgeons in the management of patients with previous remote organ dysfunctions.
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References |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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, and microvascular perfusion. Hepatology 1999;30:137-142.[CrossRef][Medline]This article has been cited by other articles:
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  N. W. Hasaniya, S. Premaratne, W. W. Zhang, A. M. Razzuk, A. A. Abdul-Ghani, M. Siera, R. H. Dashwood, B. Eklof, L. R. Tinsley, and J. J. McNamara Ischemia-reperfusion Injury in the Lung: Quantitation Using Electron Microscopy Vascular and Endovascular Surgery, April 1, 2009; 43(2): 170 - 177. [Abstract] [PDF]
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