Crush syndrome, also known as traumatic rhabdomyolysis, is the systemic manifestation of muscle crush injury. It is often seen in victims of natural disasters such as earthquakes and in other situations including war and vehicular accidents and is associated with high morbidity and mortality (1–3). The main causes of death from crush syndrome result from its systemic effects such as hypovolemic shock and hyperkalemia in the early phase and multiorgan dysfunction and sepsis occurring in the later phase (1, 3). Current treatment of crush syndrome generally focuses on avoiding life-threatening conditions in the early phase of the syndrome such as fluid resuscitation, forced diuresis, and renal replacement therapy for counteracting hemodynamic collapse, electrolyte disturbances, and renal failure (3–5). However, treatments for the later phase are currently limited.
High-mobility group box 1 (HMGB1) is a nuclear protein involved in gene transcription and stabilizes nucleosome formation. It is released passively from necrotic and injured cells, but actively from immune cells such as macrophages and mononuclear cells (6, 7). High-mobility group box 1 binds to receptors, including the receptor for advanced glycation end products and Toll-like receptors (TLR-2, TLR-4, and TLR-9), resulting in translocation of nuclear factor kappa κB, which subsequently leads to release of cytokines and activation of neutrophils (6). Previous studies have demonstrated that HMGB1 plays a crucial role in the pathogenesis of systemic acute inflammation such as sepsis (6, 8, 9) and nonsepsis (10–14) and has become a key therapeutic target (7, 10, 12, 13, 15, 16).
The neutrophil elastase (NE) inhibitor, sivelestat, was reported to attenuate acute lung injury (ALI) induced by endotoxins (15, 17) and mesenteric ischemia-reperfusion injury (18). Sivelestat was also reported to inhibit nuclear factor kappa κB, thereby decreasing the release of cytokines including HMGB1 (19). Accordingly, we hypothesized that sivelestat may potentially improve the outcome of crush injury by inhibiting HMGB1. To examine this hypothesis, we investigated the effects of sivelestat on the outcome of crush injury and HMGB1 expression in the lung, liver, and in the systemic circulation.
MATERIALS AND METHODS
Animals used in the present study were maintained in accordance with the Guiding Principles for Care and Use of Animals in the Field of Physiological Science set by the Physiological Society of Japan. The experiments were approved by the Animal Research Committee of Gifu University. Male Sprague-Dawley rats weighing 280 to 350 g were used for the experiments. Anesthesia was initiated with an intraperitoneal injection of sodium pentobarbital (50 mg/kg body weight; Abbott Laboratories, Abbott Park, Ill) and was maintained by continuous intravenous infusion of sodium pentobarbital at 10 mg/kg per hour. Animals were fixed in a supine position; catheters were inserted into both external jugular veins for fluid and drug infusion and the right carotid artery for arterial blood pressure monitoring and blood sampling.
Induction of crush injury
Crush injury was induced according to previous reports with some modifications (13, 20–22). Briefly, both hindlimbs of rats were compressed with a specific apparatus designed for inducing crush injury. It includes two plastic plates with a 3.5-kg weight hung on each plate. Crush injury was induced by direct compression of both hindlimbs, which were placed under the plastic plates, for 6 h (6-h compression), followed by 3-h reperfusion. Normal saline was continuously infused via the jugular venous catheter at 1 mL/kg per hour for the first 5 h of compression and was then increased to 10 mL/kg per hour for the subsequent 4 h.
Rats were randomly assigned to three groups as shown in Figure 1. In the positive control group (n = 30), rats underwent hindlimb compression for 6 h followed by 3-h reperfusion, with fluid infusion as described above. The treated group (n = 30) underwent the same procedures as applied in the positive control group except for an addition of sivelestat to normal saline, where the concentration was adjusted to infuse sivelestat at 10 mg/kg per hour. The control group (n = 10) had no crush injury induction, and normal saline was infused at 1 mL/kg per hour throughout the experiment. For survival rate measurement, all catheters were removed after 3-h reperfusion. Rats were then returned to individual cages, allowed access to food and water ad libitum, and were monitored for 7 days.
Serum biochemistry and cytokine level
Blood samples were collected using the arterial catheter at different time points: before compression (0 h, control group n = 6, positive control group n = 15, treated group n = 12) and 3, 24, and 48 h after reperfusion. Ten rats in the positive control group and six rats in the treated group died before 48 h after reperfusion; thus, the numbers of rats at 48 h were six in the control group, five in the positive control group, and six in the treated group. Arterial blood gas samples were immediately analyzed (VetStat Electrolyte and Blood Gas Analyzer, IDEXX Laboratories, Inc., Westbrook, Me). Blood was centrifuged at 10,000 revolutions/min for 10 min, and supernatants were collected and stored at −40°C for further biochemical (VetTest Chemistry Analyzer; IDEXX Laboratories, Inc) and cytokine assays. Serum concentration of cytokines was measured using available commercial kits for HMGB1 (Shino-Test, Kanagawa, Japan), interleukin 6 (IL-6), and tumor necrosis factor α (TNF-α) (R&D Systems, Minneapolis, Minn). All assay procedures were carried out in accordance with the manufacturer’s instructions.
Neutrophil elastase activity was measured using the method described by Yoshimura et al. (23). N-methosuccinyl-Ala-Ala-Pro-Val p-nitroaniline (Sigma-Aldrich, St Louis, Mo) was used for the substrate of NE because this compound is not hydrolyzed by cathepsin G. The hydrolysis of the substrate was measured by spectrophotometric method. Serum samples were incubated with 1 mM substrate in 0.1 M Tris-HCl buffer (pH 8.0) containing 0.5 M NaCl at 37°C for 24 h. After incubation, the amount of released p-nitroaniline measured by the absorbance at 405 nm reflected the NE activity.
Hematoxylin-eosin staining and immunohistochemistry
Rats were transcardially perfused with normal saline and fixed using 4% paraformaldehyde solution at 3, 24, and 48 h after reperfusion with three rats in each group. The lung and the liver were harvested for histological study. Tissues that had been fixed in 4% paraformaldehyde were subsequently embedded in paraffin and then cut into 3-μm-thick sections for hematoxylin-eosin or immunohistochemistry staining. For immunohistochemistry staining, the sections were deparaffinized and rehydrated. Heat-induced epitope retrieval was performed by 1-min autoclaving at sub-boiling temperature. The sections were then incubated in 3% H2O2, washed, and blocked with 2% bovine serum albumin for 60 min. The slides were then incubated overnight with the primary antibody (rabbit anti-HMGB1 polyclonal antibody ab18256; ABCAM, Cambridge, Mass). The primary antibody was visualized using the biotinylated polyclonal goat anti–rabbit immunoglobulin (Dako, Ltd, Carpinteria, Calif) and the Vectastain Elite ABC Kit (Vector Laboratories Inc, Burlingame, Calif) according to the manufacturer’s instructions. The slides were then counterstained with hematoxylin.
A pathologist blinded to group assignment performed the morphological analysis. Five random fields on each slide were digitized with a microscope under ×400 magnification (Olympus Digital Camera DP70; Olympus Optical Co, Ltd, Tokyo, Japan). Levels of lung injury were determined by the technique of Murakami et al. (24). Briefly, 24 areas in the lung parenchyma were graded on a scale of 0 to 4 (0, absent and appears normal; 1, light; 2, moderate; 3, strong; 4, intense) for congestion, edema, infiltration of inflammatory cells, and hemorrhage. The mean score for each of the parameters was then used for analysis purposes.
Results are expressed as mean ± SEM. Two-way analysis of variance was used for analysis of data, with time course and groups as the two factors. If the F ratio was significant, the Tukey-Kramer post hoc test was used to compare differences among groups. Kaplan-Meier survival curves were calculated and compared using the log-rank test. Statistical significance was accepted at P < 0.05.
Survival curves are shown in Figure 2. All rats in the control group survived. However, the numbers of surviving animals in the positive control group at 24 h were 36.7% (11/30) and 66.7% (20/30) in the treated group. At 7 days, the survival rates of the positive control and treated groups were 26.7% and 53.3%, respectively. The survival curve of the treated group was significantly different compared with the positive control group, P = 0.032.
Figure 3 shows changes in mean arterial pressure after induction of crush injury. Although there was no difference in baseline mean arterial pressure between the groups (control group 120.0 ± 2.6 mmHg, positive control group 119.9 ± 2.1 mmHg, treated group 115.7 ± 3.0 mmHg), reperfusion significantly decreased mean arterial pressure, and this decrease in the positive control group (–31.8 ± 2.2 mmHg) was significantly larger compared with the treated group (–22.6 ± 2.9 mmHg), P < 0.05.
Serum biochemistry, blood gas, and cytokine level
Table 1 shows serum biochemistry data before and after crush injury. Crush injury–induced muscle injury was demonstrated by a significantly increased serum creatine kinase concentration compared with baseline level, which increased at 3 and 24 h after reperfusion and then returned to baseline levels at 48 h. Serum potassium also significantly increased and peaked at 3 h after reperfusion. Aspartate aminotransferase and alanine aminotransferase also increased after reperfusion, reached peak values at 24 h, and then decreased, but were still significantly higher at 48 h. Blood urea nitrogen (BUN) and creatinine also increased after reperfusion; BUN peaked at 24 h, and creatinine peak at 3 h, and then returned to baseline levels at 48 h. No significant difference was seen in serum biochemistry between the positive control and treated groups.
Crush injury caused metabolic acidosis as shown by decreases in bicarbonate and base excess. These changes were compensated for by hyperventilation as shown by a decrease in PCO2 and an increase in PO2, and then pH was relatively maintained (Table 2). No difference was seen in the pH and arterial blood oxygenation between the positive control and treated groups. To clarify the effects of sivelestat itself, three anesthetized rats without compression were enrolled with sivelestat administration. Results showed that sivelestat did not have any effects in blood pressure, renal function, enzyme levels, and blood gas exchange.
After reperfusion, NE activity and HMGB1 level immediately increased and peaked at 3 h and then decreased to baseline levels at 24 h (Fig. 4, A and B). The peak NE activity and HMGB1 concentration of the treated group were significantly lower compared with the positive control group, P < 0.05. Conversely, the time course of changes in serum IL-6 and TNF-α levels was later compared with HMGB1 level, where significant increases were found at 48 h (Fig. 4, C and D). No difference was seen in IL-6 and TNF-α levels between the positive control and treated groups.
Lung morphology and immunohistochemistry
Hematoxylin-eosin staining of the lung revealed that crush injury induced lung edema, congestion, microhemorrhage, and infiltration of inflammatory cells (neutrophils) in the lung parenchyma (Fig. 5). Quantity analysis showed that these morphological changes of ALI were most severe in the first 24 h after crush injury, and treatment with sivelestat significantly ameliorated crush injury–induced ALI, especially improved the lung congestion, infiltration, and hemorrhage (Table 3).
Typical HMGB1 expression in the lung at three different time points is shown in Figure 6A. Averaged HMGB1 expression in the lung and liver is shown in Figure 6, B and C. High-mobility group box 1 expression in the lung and the liver was highest at 3 h and gradually decreased thereafter. High-mobility group box 1 expression was significantly lower in the treated group compared with the positive control group at 3, 24, and 48 h in the lung and at 3 h in the liver, P < 0.05.
The major findings of the present study are, first, that treatment with sivelestat improved survival rate in crush-injured rats. Second, treatment with sivelestat suppressed HMGB1 expression in the lung and the liver, decreased serum HMGB1 concentration, and suppressed the NE activity after crush injury.
It is well known that crush injury results in systemic insults, such as hypotension, acute renal failure, and ALI (4, 13, 21, 25). In the present study, hypotension, renal and liver dysfunction, and hyperkalemia developed after reperfusion. In addition, metabolic acidosis and its compensation by hyperventilation were observed. These results are consistent with previous reports (2, 4, 20, 22, 25). Although no significant difference was seen in hemoglobin concentration between the groups, it tended to increase at 3 h in the positive control group compared with the control group, suggesting the possibility that loss of serum in the positive control group was more severe than in the treated group, resulting in a greater decrease in arterial pressure in the positive control group. Histological data in lung edema might support these data.
It has been reported that neutrophil activation plays an important role in the pathogenesis of ischemic-reperfusion injury (1, 14). Neutrophil-derived toxic products, especially NE, are considered important in the pathogenesis of ALI and acute respiratory distress syndrome, by degrading microvascular and lung tissue components and neutrophil transvascular migration (16, 18, 25). The NE inhibitor, sivelestat, has been reported to improve outcome of ALI in endotoxin (15, 17, 26) and ischemia-reperfusion injury models (16, 18). Sivelestat was reported to improve lung function in patients with acute respiratory distress syndrome and with systemic inflammatory response syndrome (SIRS), although its efficacy was controversial (27, 28). Our data show that NE activity significantly increased at 3 h after crush injury and was completely inhibited with sivelestat treatment.
In the present study, a significant decrease in PCO2 was accompanied by a significant increase in PO2 at 3 h, suggesting that a compensatory hyperventilation occurred. The decrease in PCO2 was still observed at 24 h in the positive control group, but this was not accompanied by an increase in PO2. This suggests a deteriorated gas exchange through the pulmonary capillary-alveolar membrane in the positive control group. This conclusion is supported by lung histology, showing that lung injury became more prominent at 24 h after crush injury; demonstrated by interstitial edema, congestion, diffuse parenchyma microhemorrhage, and infiltration of inflammatory cells. Acute lung injury score was highest in the first day after crush injury, and treatment with sivelestat improved ALI score that resulted in improvement of blood gas exchange, thereby improved survival rate.
High-mobility group box 1 plays a key role in the pathogenesis of a variety of diseases and conditions in which SIRS exists (6, 8, 9, 14, 29). Systemic inflammatory response syndrome is closely associated with the activation of leukocytes, especially neutrophils, and HMGB1 is considered to be a chemoattractant. This indicates that suppression of HMGB1 can improve SIRS, and therefore, HMGB1 has recently become a therapeutic target (10, 12, 13, 29). Accordingly, we examined HMGB1 expression using immunohistochemistry.
Treatment with sivelestat reduced the peak serum HMGB1 level and expression of HMGB1 in the lung and liver, suggesting that sivelestat suppressed HMGB1 systematically. Notably, tissue HMGB1 expression peaked at 3 h, and this expression paralleled the serum HMGB1 level. This finding indicates that HMGB1 released into the circulation originated not only from compressed muscle tissue, but also from other sources such as the lung and liver. However, the peak time point of IL-6 and TNF-α expression was different compared with HMGB1. This difference may have resulted from the fact that the inflammatory response became prominent in crushed muscle tissue, which resulted in a significant sequestration of inflammatory cells and a massive release of cytokines after 1 day of crush injury. Further investigation of the late immune response may be required in this model.
In conclusion, sivelestat-suppressed HMGB1 and neutrophil activation improved outcomes of crush injury.
Although administration of HMGB1 itself was lethal (8), and anti-HMGB1 treatment improved outcome of crush injury (13), there are no available data that relate HMGB1 to inflammation cascade in crush injury. To clarify this, further study is required in the future.
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rhabdomyolysis; neutrophil elastase inhibitor; acute lung injury; systemic inflammatory response syndrome; ischemia-reperfusion injury; multiorgan dysfunction syndrome