Acute lung injury can be a complication of hemorrhagic shock. Mechanisms of injury include neutrophil-derived inflammatory products that induce necrosis within the lung. Recent data has shown apoptosis, in addition to necrosis, as a pathway leading toward acute lung injury in shock models. This study quantitates apoptotic and necrotic cells in the lung after hemorrhagic shock. Mongrel pigs (20-30 kg) under general anesthesia (with pancuronium and pentobarbital) underwent instrumentation with placement of carotid and external jugular catheters. The animals were randomized to sham hemorrhage (n = 6) and to hemorrhagic shock (n = 7). The hemorrhagic shock group then underwent hemorrhage (40-45% blood volume) to a systolic blood pressure of 40-50 mm Hg for 1 hour. The animals were then resuscitated with shed blood plus crystalloid to normalization of heart rate and blood pressure. The animals were observed under general anesthesia for 6 hours after resuscitation, then sacrificed, and lungs were harvested. Lung injury parameters including histology (H&E stain), apoptosis [terminal deoxynucleotidy] transferase- mediated dUTP biotin nick end labeling (TUNEL)], and myeloperioxidase activity (spectrophotometric assay) were assessed. Hemorrhagic shock induced marked loss of lung architecture, neutrophil infiltration, alveolar septal thickening, hemorrhage, and edema in H&E staining. Furthermore, MPO activity, a marker for neutrophil infiltration and activation, was more than doubled as compared to controls (44.0 vs 20.0 Grisham units activity/g). Apoptosis (cell shrinkage, membrane blebbing, apoptotic bodies) and necrosis (cellular swelling, membrane lysis) in neutrophils, macrophages, as well as in alveolar cells was demonstrated and quantified by H&E staining use. Apoptosis was confirmed and further quantified by positive TUNEL signaling via digital semiquantitative analysis, which revealed a significant increase in apoptotic cells (16.0 vs 2.5 cells/hpf, shock vs control, respectively) and necrotic cells (16.0 vs 2.0 cells/hpf, shock vs control, respectively). Acute lung injury is a complex pathophysiologic process. Apoptosis in cells (neutrophils, macrophages, alveolar cells) is induced within the lung after hemorrhagic shock. The role of apoptosis in pulmonary dysfunction after hemorrhagic shock has yet to be determined.
PULMONARY DYSFUNCTION is a is common complication after hemorrhagic shock and causes significant morbidity and mortality in trauma patients. The etiology of acute lung injury is multifactorial and includes multiple organ ischemia and reperfusion injury after resuscitation. Central to this theme is the intestinal ischemia associated with hemorrhagic shock followed by reperfusion after resuscitation.1,2 This is associated with priming of the airway endothelial cells secondary to free oxygen radical release and the activation of neutrophils and subsequent migration into lung tissue.3-5 This event results in the pulmonary accumulation of neutrophils and secondary damage to the lung tissue due to the release of neutrophilassociated inflammatory products, namely reactive oxygen species, proteolytic enzymes, and multiple eicosanoids. This acute lung injury is characterized by alveolar capillary endothelial cell injury, increased capillary permeability, and hypoxia. In worst-case scenarios, these events lead to acute respiratory distress syndrome, or ARDS.
Numerous strategies have been developed to treat or prevent acute lung injury after hemorrhagic, as well as other forms of shock. These strategies include different resuscitation modalities, the use of anti-inflammatory agents,6-10 antioxidants,5,11 and antiadhesion molecule agents.12 However, with each of these different strategies, the lung injury in some cases is improved, but not to the point where pulmonary function is restored.
The process of programmed cell death, or apoptosis, in recent studies has been shown as a potential pathway leading toward acute lung injury in various shock models.13-15 Typical cellular death after shock or trauma is due to necrosis-a fundamental modality of cellular death, by which the cell membrane is damaged and the cellular content is released, therefore aggravating the inflammatory response after hemorrhagic shock. Apoptosis is different from necrosis. Apoptosis is characterized by cellular shrinkage, condensation of nuclear content and chromatin, apoptotic body formation, and noninflammatory phagocytosis by tissue macrophages.16- 18 Neutrophil apoptosis is believed to play a major role through which the duration of the inflammatory process is regulated. In addition, alveolar cell apoptosis is believed to play a role in the hypoxia associated with acute lung injury.
The purpose of this study is to demonstrate and quantitate the presence of apoptosis and necrosis in lung tissue in a clinically relevant model of hemorrhagic shock. In addition, the possible roles of apoptosis and the significance of apoptosis in alveolar cells and neutrophiis in lung tissue will be addressed.
Materials and Methods
Mongrel pigs weighing 20 to 40 kg were used and acclimated for 7 days in the Animal Care Facility at the University of Tennessee Health Science Center prior to surgical procedures. The experiment was approved by the Animal Care and Utilization Committee of the University of Tennessee Health Science Center. All care complied with the Principles of Laboratory Animal Care and the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, Bethesda, MD).
Animals underwent induction of anesthesia with intramuscular injections of ketamine and xylazine, and general anesthesia was maintained with continuous intravenous pentobarbital infusion. Right carotid artery and right external jugular vein catheters were inserted. The animals were monitored continuously during surgery with ECG, pulse oximetry, and continuous arterial blood pressure measurements.
The experiments were designed to mimic the clinical scenario of exsanguinating hemorrhagic shock followed by resuscitation. After instrumentation, the animals were randomized to sham hemorrhage (n = 6) and to hemorrhagic shock (n = 7). The hemorrhagic shock group underwent hemorrhage (44% to 55% blood volume) to a systolic blood pressure of 40 to 50 mm Hg and were maintained at that pressure with incremental blood loss for 1 hour. The animals were then resuscitated with shed blood plus crystalloid (normal saline) to normalization of heart rate and blood pressure. The animals were then observed under general anesthesia for 6 hours after resuscitation. After the 6-hour observation period, the animals then underwent bilateral thorocatomies, lungs were inspected grossly, and then harvested. The animals were then sacrificed with lethal injections of potassium chloride, pentobarbital, and pancuronium.
Tissue was then randomly biopsied from the harvested lungs and either immediately rinsed and placed in 10 per cent buffered formalin solution or frozen at -70C. The specimens were allowed to fix for 7 days. Sections were made and embedded in paraffin and cut into 6-μm sections for slides. All tissue slides underwent hematoxylin and eosin (H&E) staining. Apoptosis was determined by the terminal deoxynucleotidyl transferase-mediated dUTP biotin nick- end labeling (TUNEL) method (Roche Diagnostic TUNEL apoptosis kit). In addition, neutrophil presence and activation was detected by myeloperoxidase activity from frozen tissue samples by a spectrophotometer assay at 665-nm wavelength. H&E slides and TUNEL slides were then digitally analyzed. Cells undergoing apoptosis and necrosis were identified and quantitated per highpowered field.
Results
A total of 13 animals were randomized: 6 to the sham hemorrhage group, 7 to the hemorrhagic group. One animal died in the hemorrhagic group due to shock unresponsive to resuscitation.
In the hemorrhagic shock group, all animals had appropriate responses to hemorrhagic shock with profound hypotension and tachycardia. Resuscitation with shed blood plus crystalloid restored the animal’s vital signs to near baseline levels within a couple of hours after the hemorrhage. As expected, the sham hemorrhage group had no changes in blood pressure or heart rate during the 6-hour observation period (Fig. 1).
Myeloperoxidase Activity
Myeloperoxidase is an enzyme associated with the presence of activated neutrophils, The presence of elevated myeloperoxidase activity in tissue is therefore associated with tissue injury with subsequent migration of activated neutrophils into the tissue and release of myeloperoxidase and free oxygen radicals. In the sham hemorrhage group, myeloperoxidase activity was measured at 20 Grisham units activity per gram of lung tissue (Gu/g). This compares to the hemorrhagic shock group myeloperoxidase activity of 44 Gu/g (P
FIG. 1. Resuscitation endpoints. The control group maintained baseline heart rate and blood pressure during observation. After hemorrhage, the shock group was resuscitated to near baseline heart rate and blood pressure during the observation period.
FIG. 2. Hematoxylin an\d eosin staining of control group: normal- appearing lung parenchyma.
FIG. 3. Hematoxylin and eosin staining of hemorrhagic shock group: marked loss of lung architecture, neutrophil infiltration, alveolar septal thickening, and edema.
H&E Staining
Standard H&E staining of biopsied lung tissue was performed. Examination of slides of the sham hemorrhage group revealed normal- appearing lung parenchyma with open alveoli and thin-walled alveoli septi (Fig. 2). Examination of slides of the hemorrhagic shock group revealed marked loss of lung architecture, neutrophil infiltration, alveolar septal thickening, hemorrhage, and edema (Fig. 3).
Apoplosis and Necrosis Quantification
Apoptotic cells, described as those cells with cellular shrinkage, membrane blebbing, and the presence of apoptotic bodies, were identified on H&E staining. Necrotic cells, described as those cells with cellular swelling, nuclear and cellular membrane lysing, were also identified on H&E staining. Consecutive section slides were then stained for apoptosis presence by the TUNEL technique. The two consecutive slides (H&E and the corresponding TUNEL slide) were then digitalized for cellular identification and quantification. Those cells that were identified as undergoing apoptosis on H&E were confirmed by positive TUNEL staining in the digital analysis. Cells were then quantified per high-powered field (hpf) over five different sections of each of the four different animal lobes and then averaged. In the sham hemorrhage group, lung tissue averaged 2 necrotic cells per hpf and 2.2 apoptotic cells per hpf. This compares to 15 necrotic cells per hpf and 16 apoptotic cells per hpf in the hemorrhagic shock group. Comparing the sham hemorrhage group to the hemorrhagic shock group, there was a significant difference between the apoptotic (P
Discussion
In 1972, Kerr et al. first published an article describing the process of apoptosis.19 He described this novel physiological process of cells undergoing apoptotic cellular suicide, rapid energy- dependent cell shrinkage, and loss of their normal intercellular framework. Subsequently, those cells then exhibited chromatin condensation, nuclear fragmentation, cytoplasmic blebbing and fragmentation into small apoptotic bodies. Because no cytosolic contents are exposed during this process, noninflammatory phagocytosis of these apoptotic bodies occurs. Apoptosis maintains a homeostatic balance between cellular proliferation and death and varies from tissue to tissue. Apoptosis is important in developmental biology and in remodeling of tissues during repair. Dysregulation of apoptosis is associated with pathogenesis of numerous diseases including cancer, autoimmunity, heart and neurodegenerative diseases, and prolonged inflammatory processes. In some diseases, there is promotion of apoptosis leading to such diseases as organ dysplasia and neurodegeneration. In other diseases, there is inhibition of apoptosis leading to cellular proliferation dysfunction such as cancers and several inflammatory diseases.
Acute lung injury after hemorrhagic shock as well as other forms of shock was long attributed to the effects of neutrophils and neutrophil-derived inflammatory products. Initial strategies for alleviating this inflammatory injury, besides better resuscitation, were with anti-inflammatory agents such as steroids, different NSAIDS such as indocin, and the newer COX-2 inhibitors, and different immunomodulators such as adenosine. All of these agents have shown some improvement in diminishing the inflammatory process and somewhat improving pulmonary function.6-11 However, the body seemed to circumvent these inflammatory inhibitors and still cause significant pulmonary dysfunction.
The role of apoptosis in acute lung injury after shock is believed to bimodal.20,21 The “neutrophillic hypothesis” suggests that during acute lung injury, the release of various cytokines and hormones such as granulocyte colony stimulating factor and granulocyte/ macrophage colony stimulating factor prolong the survival of neutrophils by inhibiting apoptosis and thus prolonging the neutrophil life span. This continued presence of neutrophils and their inflammatory prodnets is further accentuated by the arrival of macrophages and their inflammatory by-products such as the cytokines TNF-alpha and IL-1. These products further enhance the inflammatory process and inhibit neutrophil apoptosis. Recent work has suggested the role of MAP kinases in neutrophil apoptosis. NF Kappa beta released from macrophages causes inhibition of neutrophil MAP kinases and thus inhibits apoptosis in neutrophils.22 The potential importance of apoptosis in the regulation of inflammation suggests therapeutic modalities may be used to alter the complex cascade of apoptosis to enhance neutrophil apoptosis in cases of acute lung injury after shock.22
The second hypothesis is the “epithelial hypothesis” of acute lung injury after shock. Epithelial cells such as alveolar cells type I and II are injured during shock and die by either necrosis due to irreversible damage or apoptosis. Alveolar cells that are not initially killed by the acute inflammatory process are stressed and either undergo repair and regain their function in oxygen absorption or undergo apoptosis. Stressed alveolar cells can sometimes undergo membrane changes causing Fas, which is a cell surface membrane protein normally located on the cytosolic side of the cellular membrane, to be transported to the extracellular side of the cellular membrane. Soluble Fas ligand is released from inflammatory cells, namely cytotoxic T cells, and binds to Fas, causing initiation of apoptosis. In addition, in a similar fashion, TNF released from macrophages during inflammation binds to a cell surface protein called TNF-receptor 1, causing apoptosis in alveolar cells. The end result of the initiation of apoptosis is the activation of the caspase pathway cascade and subsequent DNA fragmentation and cell death.3 The potential importance of apoptosis in the regulation of alveolar cell death is the theory that by inhibiting alveolar cell apoptosis, the cell will then repair itself and regain cellular function of oxygen absorption and carbon dioxide release. The use of surfactant protein A, nitrous oxide, and N- acetylcysteine in recent studies has shown promising results in increasing the survival of alveolar cells type I and II by inhibiting alveolar cell apoptosis.23-27
Acute lung injury is a typical response to a host of different pulmonary and systemic insults. The initial inflammatory response to the insult may well be adaptive and protective in nature with neutrophil recruitment to fight infection. At some point in time, the inflammatory process becomes excessive or dysregulated and becomes injurious to the tissues. The regulation of neutrophils is important in stopping the inflammatory process. Through combination therapies used to inhibit the inflammatory process as well as promote neutrophil apoptosis, the extent of neutrophil-induced damage might be controlled. In addition, to regain pulmonary function, alveolar cells must be protected by regulating the bombardment of inflammatory products, as well as inhibiting alveolar cell apoptosis. By doing so, alveolar cells might repair themselves and regain their function in oxygen transport.
The results of this study indicate that apoptosis does have a role in acute lung injury after hemorrhagic shock. Further studies will be needed to determine how modulating apoptosis of different cell lines, namely neutrophils and alveolar cells, will affect pulmonary function and recovery from acute lung injury.
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T. WRIGHT JERNIGAN, M.D., MARTIN A. CROCE, M.D., TIMOTHY C. FABIAN, M.D.
From the Department of Surgery, University of Tennessee Health Science Center, Memphis, Tennessee
Presented at the Annual Scientific Meeting and Postgraduate Course Program, Southeastern Surgical Congress, Atlanta, GA January 31-February 3, 2004.
Address correspondence and reprint requests to Timothy C. Fabian, M.D., Harwell Wilson Alumni Professor & Chairman, Department of Surgery, University of Tennessee Health Science Center, 956 Court Avenue, Suite G228, Memphis, TN 38163.
Copyright The Southeastern Surgical Congress Dec 2004
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