Role of Hemorrhage in the Induction of Systemic Inflammation and Remote Organ Damage: Analysis of Combined Pseudo-Fracture and Hemorrhagic Shock Roman Pfeifer,1 Philipp Kobbe,2 Sophie S. Darwiche,3 Timothy R. Billiar,3 Hans-Christoph Pape2 1 Department
of Orthopaedic Surgery, University of Pittsburgh Medical Center, 3471 Fifth Avenue, Pittsburgh, Pennsylvania 15213, 2 Department of Orthopedic Surgery, University of Aachen Medical Center, Pauwelsstr.30, 52074 Aachen, Germany, 3 Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania
Received 20 April 2010; accepted 18 June 2010 Published online 5 August 2010 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jor.21214
ABSTRACT: This study was performed to analyze the role of hemorrhage-induced hypotension in the induction of systemic inflammation and remote organ dysfunction. Male C57/BL6 mice (6- to 10-week old and 20–30 g) were used. Animals were either subjected to pseudofracture [PF; standardized soft-tissue injury and injection of crushed bone, PF group: n = 9], or PF combined with hemorrhagic shock (HS + PF group: n = 6). Endpoint was 6 h. Systemic inflammation was assessed by IL-6 and IL-10 levels. Myeloperoxidase (MPO) and NFB activity in the lung and liver tissue were obtained to assess remote organ damage. The increases of systemic cytokines are similar for animals subjected to PF and PF + HS (IL-6: 189 pg/ml ± 32.5 vs. 160 pg/ml ± 5.3; IL-10: 60.3 pg/ml ± 15.8 vs. 88 pg/ml ± 32.4). Furthermore, the features (ALT; NF-B) of liver injury are equally elevated in mice subjected to PF (76.9 U/L ± 4.5) and HS + PF (80 U/L ± 5.5). Lung injury, addressed by MPO activity was more severe in group HS + PF (2.95 ng/ml ± 0.32) than in group PF (1.21 ng/ml ± 0.2). Both PF and additional HS cause a systemic inflammatory response. In addition, hemorrhage seems to be associated with remote affects on the lung. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J. Orthop. Res. 29: 270–274, 2011 Keywords: hemorrhagic shock; inflammation; lung injury; liver injury; pseudo-fracture
Patients with bilateral femur shaft fractures are known to have an increased rate of systemic complications such as SIRS and ARDS.1–5 Post-traumatic systemic inflammation leads to the activation of lung endothelium. It results in an increase of pulmonary capillary permeability and expression of adhesion molecules thus mediating the accumulation of PMH leucocytes in lung tissue.6 Similar mechanisms have also been confirmed in rodent models.1–35,7–9 An increase in systemic proinflammatory cytokines associated with remote organ dysfunction of the lung and liver has been described following bilateral femur fracture (BFF).7–10 To date, it is unclear whether this inflammatory response is caused by the fracture or the soft tissue injury components. Moreover the role of associated blood loss is unknown. We therefore analyzed the alterations in the systemic cytokine patterns of IL-6 and IL-10 and associated remote organ dysfunction in an established rodent pseudo-fracture (PF) model.
MATERIALS AND METHODS Animals This research protocol was approved by the local Institutional Animal Use and Care Committee (Protocol Number 0801799). Mice used in the experimental protocols were housed in accordance with National Institutes of Health (NIH) animal care guidelines. Male C57/BL6 mice (Charles Rivers Laboratories Inc, Walmington, MA, USA) 6- to 10-week old and weighing 20–30 g were maintained in the animal research center. A 12-h:12-h light:dark cycle and free access to laboratory food Correspondence to: Roman Pfeifer (T: 0049-241-8037041; F: 0049-241-8082415.; E-mail:
[email protected]) © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.
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and water were provided. Animals were anesthetized with pentobarbital (70 mg/kg IP) and isoflurane (Abbott Laboratories, Chicago, IL) was used to provide additional anesthesia in combination with pentobarbital as needed. Buprenex (Bedford Laboratories, Bedford, OH; 0.5 mg/kg) was injected subcutaneously prior to the experiment. Group Distribution Animals were randomly assigned to one of three experimental groups: Control animals (Control group: n = 6) were sacrificed directly after anesthesia to obtain physiological baseline levels. All other animals were either subjected to PF group (n = 9), or PF combined with hemorrhagic shock (HS + PF group: n = 6). All mice were anesthetized throughout the 6-h period until the end of the experiment. Pseudo-Fracture Model Donor mice were euthanized via overdose of pentobarbital and the long bones of the lower extremities were dissected and disarticulated at the ankle and the hip using aseptic technique. Femurs and tibias were removed from both hind limbs and crushed using a sterile mortar and pestle in the biohazard hood. The crushed bone fragments were solved with 2 ml phosphate-buffer solution (PBS), crushed again, and kept in a sterile tube. Recipient animals were anesthetized and both thighs were squeezed with a hemostat for 30 s to induce a soft tissue lesion. Then, 0.15 ml of bone mixture suspended in PBS was injected using a 1 cc syringe and an 18 G needle in the area of the soft tissue injury of each thigh. At the end of 6 h, mice were sacrificed and serum samples were obtained for cytokine and blood chemistry analysis. Organs were snap frozen in liquid nitrogen for molecular analysis. Induction of Pressure Controlled Hemorrhagic Shock Anesthesia was induced using pentobarbital (70 mg/kg) and inhaled isoflurane and maintained until the end of experiment.
ROLE OF HEMORRHAGE IN THE INDUCTION OF SYSTEMIC INFLAMMATION
Systemic Inflammation and Remote Organ Damage After 6 h, thoracotomy was performed and blood samples were obtained via cardiac puncture. Blood samples were centrifuged at 5,000 rpm for 10 min to separate the serum from cellular blood components and stored at −20◦ C until thawed for cytokine profile measurements. Livers and lungs were removed and snap frozen in liquid nitrogen for molecular assays. Serum IL-6 and IL-10 levels were quantified with ELISA kits (R&D System Inc., Minneapolis, MN). Hepatocellular damage following trauma was assessed by measuring serum alanine aminotransferase (ALT) using the veterinary chemistry analyser (Dri-Chem® , Fugifilm Corporation® , Asaka-shi saitama, Japan). Lung Myeloperoxidase Activity Lung tissue was snap frozen, thawed and homogenized in a lysis buffer. Myeloperoxidase (MPO)–enzymelinked immunosorbent assay kits (Cell Sciences, Canton, MA) were used to quantify MPO activity in the lung.
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Figure 1. Circulating serum IL-6 levels. Data are expressed as mean ± SE. PF and HS + PF showed increased (p < 0.05) levels at 6 h after induction of trauma. There was no statistical difference observed between these two study groups.
of anti-inflammatory cytokine IL-10 (PF: p = 0.011; HS + PF: p = 0.004; PF vs. HS+PF: p = n.s.), whereas, serum levels of IL-10 were more pronounced in animals subjected to HS + PF.
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Electrophoretic Mobility Shift Assay (EMSA) The liver was harvested, thawed and its NF-B activity measured by electrophoretic mobility shift assays using nuclear extracts prepared from liver tissue. Electrophoretic mobility shift assays were performed as described previously.8–10
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Statistical Analysis Group comparisons were assessed using the Kruskal–Wallace test followed by Mann–Whitney rank sum test. The null hypothesis was rejected for p < 0.05 (␣ = 0.05). Data were analyzed using SigmaStat® Version 3.1 (SPSS, Chicago, IL). All results are depicted as the mean ± standard error of the mean (SE).
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This surgical procedure was conducted per established sterile protocol. Unilateral groin incision (1 cm) was performed, and femoral artery was dissected and cannulated with a sterile PE-10 catheter, and flushed with small amount of heparin sulphate (Baxter Healthcare Corporation, Deerfield, IL). The arterial catheter was connected to a blood pressure monitor (Digi-Med® , Louisville, KY) and the mean arterial pressure (MAP) was recorded. Over a 12-min interval, blood was withdrawn via the arterial catheter until MAP reached 25 mmHg. Total withdrawn blood was recorded and mice were maintained at a 25 mmHg for 120 min. After 2 h of shock, the animals were resuscitated over 10 min with two times the maximal shed blood amount of Lactated Ringer’s solution. After resuscitation and recovery phase of 4 h, the animals were sacrificed. Serum samples were obtained for cytokine and blood chemistry analysis and organs were snap frozen in liquid nitrogen for molecular analysis.
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RESULTS Serum Cytokine Response Circulating IL-6 and IL-10 levels were measured to assess the magnitude of systemic inflammatory response to injury (Figs. 1 and 2). PF (189 pg/ml ± 32.5) and HS + PF (160.2 pg/ml ± 5.3) induce an inflammatory response characterized by significantly increased expression of IL-6 in both groups (PF: p = 0.001; HS + PF: p = 0.004; PF vs. HS + PF: p = n.s.). Similar pattern (PF: 60.3 pg/ml ± 15.8; HS + PF: 88 pg/ml ± 32.4) was observed analysing the levels
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Figure 2. Circulating serum IL-10 levels. Data are expressed as mean ± SE. PF and HS + PF showed increased (p < 0.05) levels at 6 h after induction of trauma. There was no statistical difference observed between these two study groups. However, levels following HS + PF were more pronounced. JOURNAL OF ORTHOPAEDIC RESEARCH FEBRUARY 2011
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Figure 3. Circulating serum ALT levels. Data are expressed as mean ± SE. PF and HS + PF showed increased (p < 0.05) levels at 6 h after induction of trauma. There was no statistical difference observed between these two study groups.
Liver Injury Liver cell injury was assessed by measuring circulatory ALT levels and NF-B (Figs. 3 and 4). ALT levels and NF-B activity showed significantly remote liver injury in both the PF (76.9 U/L ± 4.5) and HS + PF (80 U/L ± 5.5) groups (PF: p = 0.001; HS + PF: p = 0.002). There was no statistical difference between PF and HS + PF groups. (p = n.s.). Lung Injury Significant elevation of MPO activity was observed in animals with PF (1.21 ng/ml ± 0.2) and HS combined with PF (HS + PF; 2.95 ng/ml ± 0.32) groups (Fig. 5) (PF:
Figure 4. Hepatic nuclear extracts show that PF and HS + PF mice demonstrate comparable increased hepatic NF-B activation when compared with control animals. Also, no difference was observed between these study groups. JOURNAL OF ORTHOPAEDIC RESEARCH FEBRUARY 2011
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Figure 5. Lung tissue MPO activity. Data are expressed as mean ± SE. PF and HS + PF showed increased (p < 0.05) levels at 6 h after induction of trauma. Moreover, animals subjected to hemorrhagic shock demonstrated higher (p < 0.05) MPO activity when compared to PF and control mice.
p = 0.004; HS + PF: p = 0.002). However, mice sustained a PF with HS expressed significantly higher MPO activity when compared with PF group (p = 0.002).
DISCUSSION Trauma and HS initiate a complex systemic inflammatory response.11–14 Both pro- and anti-inflammatory cytokines correlate with injury severity.15–20 It appears that IL-6 is more closely associated with soft tissue injury than other cytokines.21–23 Hemorrhage or other tissue factors have been frequently used to induce a standardized systemic inflammatory response.10,24 A septic insult, often initiated by cell wall components or infection, is known to cause a reliable response.24 Others have used standardized fracture models and examined the systemic response.7,8 Our study group has previously demonstrated that local exposure of bone components (suspended bone fragments) to injured soft tissue induced a systemic inflammation and acute lung injury.25 These data suggest that cellular and soluble bone components stimulate a systemic inflammatory response. This model is different in that it uses a local stimulus that does not imply a local blood loss and therefore focuses on the inflammatory response induced by local factors. Our further investigation revealed that PF mimics the inflammatory response (serum IL-6
ROLE OF HEMORRHAGE IN THE INDUCTION OF SYSTEMIC INFLAMMATION
and IL-10 levels) and remote organ damage (liver and lung dysfunction) seen after BFF within 6-h post-injury (unpublished data). The present study has several limitations: (1) The true concentration of bone fragments and bone marrow injected in to the injured thigh musculature is unknown. We standardized the amount of long bones (femur and tibia) and PBS in order to minimize this effect. (2) Our study focuses on one single time point only; however, we performed pilot studies that clearly document a peak in the inflammatory response at 6 h after induction of PF. Moreover, our current results confirm an increase of IL-6 and IL-10 at this particular time point. A PF model was used in this experiment, because this model uses a standardized soft tissue injury and trauma severity, which is often not feasible when utilizing fracture models. Also, the hemorrhage can be controlled by induced blood loss through the femoral artery and no interference with fracture-induced hemorrhage occurs. Additionally, this model includes the ability to recover animals for longer observation, should this be required. Our main findings are as follows: (1) PF alone induces a similar inflammatory response (IL-6 and IL-10) as compared with PF and HS. (2) The features of liver injury are equally elevated in mice subjected to PF and HS + PF, whereas lung injury is more pronounced in animals sustained additional HS. Our group7 previously investigated the time course of cytokine release in BFFs and could demonstrate the role of fracture associated local soft tissue trauma.26 However, the role of fracture-associated hemorrhage in the induction of systemic inflammation and remote organ dysfunction is poorly investigated. Systemic hypotension (hemorrhage) leads to organ hypoperfusion, decreased oxygen delivery, and systemic ischemia.27,28 These are important pathogenic mechanisms of HS.27,28 Isolated HS is a well-known mediator of a systemic inflammation.10,28 Prince et al.34 reported increased IL-6 and IL-10 levels analysing the systemic inflammation in murine HS. Previous experimental results demonstrated that both hemorrhage and BFF are associated with adult respiratory distress syndrome (ARDS) related changes and marked liver injury.7,9,29 Our results do not confirm the expected cumulative increase in cytokine levels (IL-6 and IL-10). It may be surprising that an additional stimulus caused by HS does not lead to an increased cytokine release or additional damage of the liver. One possible explanation might be the previously reported depression of immune function that has been observed following combined trauma (soft tissue injury or bone fracture) and HS.30–32 In contrast to our results, the immunosuppression and a decreased cytokine release were shown at 24–72 h after injury.30–32 In the other hand, twohit models, especially those with combined HS, were also associated with attenuation of cytokine response and decreased immune function.33–35 According to these findings, we hypothesize that diminished cytokine production observed following combined PF and HS might
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be the consequence of priming of the immunologic cells with PF followed by the secondary hemorrhage. Alternatively, the magnitude of the second insult (HS) may not be large enough to induce an additional rise in IL-6 and IL-10 levels. If this was the case, however, the change in remote organ function (lung injury) in view of a lack of additional elevation of IL-6 and IL-10 levels is difficult to explain. In our study, the lung MPO activity was significantly elevated in animals with combined trauma (HS + PF) when compared with the PF group. This might be explained as the first manifestation of pulmonary damage after trauma.36–38 Depressed systemical oxygenation and systemic release of inflammatory mediators from injured lung tissue can promote additional liver and kidney dysfunction.36,37 Our results showed a significant increase of lung neutrophil migration without alterations of cytokine pattern. According to our previous investigations, we conclude that the observation period in the current study covers the peak time point of inflammation.7 The higher degree of lung damage in animals subjected to HS may be explained by local aggregation of inflammatory mediators within the lung tissue. This phenomenon might be associated with higher blood circulation through the lung. Thus, systemically released cytokines and activated immune cells during the shock period have to pass the lung tissue and interact with capillaries and endothelial cells.20 This results in migration of neutrophil granulocytes, permeability changes, and local inflammation leading to organ damage.6 An analysis of the dynamics of cytokine release and organ injury would provide more information about this complex mechanism. Moreover, the reduction of peripheral circulation during HS may affect the systemic spread and associated inflammation of locally administered cellular and soluble components of bone homogenate. In summary, this study demonstrates that additional severe bleeding in combination with a PF does not significantly alter the systemic cytokine pattern of IL-6 and IL-10. These results may originate from reported depressed immune function following combined trauma. Nevertheless, additional lung injury was observed after HS. Whether this additional affect is the result of more sustained general inflammation, or whether lungspecific pathways have been activated is subject to future studies. Moreover, long-term studies using the established PF model may provide more information about the dynamics of this inflammatory process.
ACKNOWLEDGMENTS Authors declare that they do not have financial and nonfinancial competing interests.
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