Anti-high mobility group box-1 (HMGB1) antibody attenuates kidney damage following experimental crush injury and the possible role of the tumor necrosis factor-α and c-Jun N-terminal kinase pathway.

BACKGROUND: Inflammation plays a crucial role in kidney damage after crush syndrome (CS). Several researchers report that high mobility group box-1 protein (HMGB1) may be the vital trigger in kidney damage, and tumor necrosis factor-α (TNF-α) and c-Jun N-terminal kinase (JNK) are involve in this pathophysiological process, but their biological roles are unclear. This study aimed to explore the relationship between HMGB1, JNK, and TNF-α in kidney damage. METHODS: The crush injury model was established using weight compression. The reliability of the crush injury model was determined by hematoxylin-eosin (HE) staining. Western blot was used to detect the expression of HMGB1, JNK, and TNF-α, and TUNEL was used to mark apoptotic cells in the renal cortex. RESULTS: The results showed that the highest expression of HMGB1 in muscle was 12 h after CS. JNK and TNF-α increased and peaked at 1 day after CS in kidneys. Western blot analysis revealed that anti-HMGB1 antibody could downregulate the expression of JNK and TNF-α. Anti-TNF-α could downregulate activation of JNK, and SP600125 could downregulate expression of TNF-α in the kidneys. In addition, anti-HMGB1 antibody, anti-TNF-α antibody, and SP600125 could reduce cellular apoptosis in the renal cortex. CONCLUSIONS: It is possible that JNK and TNF-α commonly contribute to kidney damage by assembling a positive feedback cycle after CS, leading to increased apoptosis in the renal cortex. HMGB1 from the muscle may be the trigger.

Low-Dose Sodium Nitrite Fluid Resuscitation Prevents Lethality From Crush Syndrome by Improving Nitric Oxide Consumption and Preventing Myoglobin Cytotoxicity in Kidney in A Rat Model.

OBJECTIVE: Crush syndrome (CS) is a serious medical condition characterized by muscle cell damage resulting from pressure. CS has a high mortality, even when patients receive fluid therapy. We examined whether administration of NaNO2-containing fluid can improve survival in a rat model of CS. DESIGN: The CS model was generated by subjecting anesthetized rats to bilateral hind limb compression with a rubber tourniquet for 5 h. Rats were then randomly divided into six groups: sham; CS with no treatment; CS with normal saline treatment; CS with normal saline + 25 mEq/L bicarbonate treatment; and CS with normal saline + 200 or 500 µmol/kg NaNO2. MEASUREMENTS AND MAIN RESULTS: Blood and tissue samples were collected for histological and biochemical analyses at predetermined time points before and after reperfusion. Ischemic compression of rat hind limbs reduced nitrite content in the crushed muscle, and subsequent reperfusion resulted in reactive oxygen species-induced circulatory dysfunction and systemic inflammation. Rats treated with 200 µmol/kg NaNO2 showed increased nitric oxide (NO) levels, blood circulation, and neoangiogenesis, decreased generation of reactive oxygen species, and suppression of the inflammatory response, leading to complete recovery. CONCLUSIONS: Treatment with 200 µmol/kg NaNO2 prevents muscle damage induced by ischemia reperfusion via the protective effects of NO and suppression of systemic inflammation, thereby increasing survival rates in CS.

Early Therapeutic Intervention for Crush Syndrome: Characterization of Intramuscular Administration of Dexamethasone by Pharmacokinetic and Biochemical Parameters in Rats.

Crush syndrome (CS) is the systemic manifestation of muscle cell damage resulting from pressure and crushing. It is associated with a high mortality rate, even when patients are treated with conventional therapy. We demonstrated the utility of intramuscular administration of dexamethasone (DEX) in disaster medical care by using a model of CS to characterize the pharmacokinetics and biochemical parameters. We compared intravenous (IV) and intramuscular (IM) injection. The IM sites were the right anterior limb (AL), bilateral hind limbs (bHL), and unilateral hind limb (uHL). DEX (5.0 mg/kg) was administered in sham-operated (sham, S-IV, S-AL, S-bHL, S-uHL groups) and CS rats (control, C-IV, C-AL, C-bHL, C-uHL groups). The survival rate in the IM groups was lower than that in the C-IV group. Survival was highest in the C-AL group, followed by the C-uHL and C-bHL groups. The blood DEX concentration of the C-AL group was similar to that in the C-IV group. The C-bHL and C-uHL groups had decreased blood DEX concentrations. Moreover, inhibition of inflammation was related to these changes. Administration of DEX to non-injured muscle, as well as IV administration, increased the survival rate by modulating shock and inflammatory mediators, consequently suppressing myeloperoxidase activity and subsequent systemic inflammation, resulting in a complete recovery of rats from lethal CS. These results demonstrate that injection DEX into the non-injured muscle is a potentially effective early therapeutic intervention for CS that could easily be used in transport to the hospital.

CHANGES OF LIPOPEROXIDATION AND ANTIOXIDATIVE ENZYMES DURING CRUSH-SYNDROME MODELLING.

Crush-syndrome (CS) is characterized by numerous pathological deviations due to the soft tissue damage and their further reperfusion. The aim of the study was to investigate pro- and antioxidative processes during different regimens of crush syndrome. The experiment was carried out on randomly selected 200-250gr. mass 50 laboratory rats using crush syndrome modeling classical method. Investigations were conducted at various stages of compression and decompression period. Activity of antioxidant enzymes - total ceruloplasmin, oxidized ceruloplasmin was determined in blood serum. LOO. and free oxygen species were as well determined with the use of relevant methods. According to our findings we can conclude that: - Lipoperoxidation intensity increases in compliance with crush syndrome duration; - Short-term (3-hour) compression causes enhancement of lipoperoxidation however, in further 1 hour decompression there is revealed a trend toward normalization of processes. Lipoperoxides and free oxygen species content decreases and the antioxidant enzymes activity is almost restored; - Long lasting compression (6 hours) leads to severe disorders in the body (total ceruloplasmin impaired production and after 6 hours from decompression antioxidant enzymes inactivation).

Oxidative stress may be involved in distant organ failure in tourniquet shock model mice.

Crush syndrome is characterized by prolonged shock resulting from extensive muscle damage and multiple organ failure. However, the pathogenesis of multiple organ failure has not yet been completely elucidated. Therefore, we investigated the molecular biological and histopathological aspects of distant organ injury in crush syndrome by using tourniquet shock model mice. DNA microarray analysis of the soleus muscle showed an increase in the mRNA levels of Cox-2, Hsp70, c-fos, and IL-6, at 3h after ischemia/reperfusion injury at the lower extremity. In vivo staining with hematoxylin and eosin (HE) showed edema and degeneration in the soleus muscle, but no change in the distant organs. Immunohistological staining of the HSP70 protein revealed nuclear translocation in the soleus muscle, kidney, liver, and lung. The c-fos mRNA levels were elevated in the soleus muscle, kidney, and liver, displaying nuclear translocation of c-FOS protein. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) analysis suggested the involvement of apoptosis in ischemia/reperfusion injury in the soleus muscle. Apoptotic cells were not found in greater quantities in the kidney. Oxidative stress, as determined using a free radical elective evaluator (d-ROM test), markedly increased after ischemia/reperfusion injury. Therefore, examination of immunohistological changes and determination of oxidative stress are proposed to be useful in evaluating the extent of tourniquet shock, even before changes are observed by HE staining.

Restraint stress aggravates rat kidney injury caused by a crush injury through endoplasmic reticulum stress.

BACKGROUND: The present study aimed to determine whether restraint stress aggravates kidney injury caused by a crush injury through endoplasmic reticulum stress (ERS). METHODS: In this study, Sprague-Dawley rat restraint stress, crush injury, and stressful injury models consisting of restraint stress and crush injury were established. An ERS inhibitor, Salubrinal (Sal), was administered intraperitoneally 30 minutes before induction of daily injury in the stressful injury group. At the end of the experimental procedures, plasma levels of noradrenaline and adrenaline, creatine phosphokinase, creatinine, and blood urea nitrogen were measured. Kidneys were harvested, and paraffin-embedded sections of kidney tissues were processed for hematoxylin-eosin staining and TUNEL assay to verify pathologic changes. Western blot was used to determine the protein levels of glucose-regulated protein 78, CCAAT/enhancer-binding protein-homologous protein, caspase 12, caspase 3, and MCP-1 in kidney specimens. RESULTS: Compared with crush injury, the most significant changes in kidney injury occurred in the stressful injury group, which was inhibited by Sal. The results suggested that restraint stress aggravates kidney injury caused by a crush injury, and the mechanism might involve ERS. Further study showed that double attacks induced a significant increase in the levels of glucose-regulated protein 78, CCAAT/enhancer-binding protein-homologous protein, caspase 12, and caspase 3, which was inhibited by Sal. The same changes were observed using the TUNEL assay. Double attacks also induced an increased expression of the proinflammatory cytokine, MCP-1, which was inhibited by Sal. CONCLUSION: Apoptosis and inflammation induced by ERS are important mechanisms by which restraint stress aggravates kidney injury caused by a crush injury.

[Coagulation and fibrinolytic activity of lymph in various pathological conditions (review of own and literature data)].

The review presents information by the author and his collaborators, as well as literature data on the coagulation of lymph in blood loss, convulsive syndrome, trauma, crush syndrome, bums, injections of thrombin and heparin, an experimental thrombosis and other pathological conditions. It is shown that the coagulation of lymph in the development of pathological conditions in tissues may outpace changes observed in the blood. We present evidence that dissolution of fibrin clots in the lymph tissue if damaged, is many times faster than in the blood.

Effect of free radical scavenger on c-jun activation in rats with crush syndrome.

OBJECTIVES: To evaluate the relationship between reactive oxygen species (ROS)-mediated kidney injuries and Jun N-terminal kinase (JNK) activity and the therapeutic effects of tempol in crush syndrome (CS) model rats. METHODS: Male Wister rats were randomly divided into sham operation group (SOG), CS groups (CS6G, CS12G and CS24G) and tempol treatment group (TG, a ROS scavenger). CS model rats were established by crushing the hind limbs of rats with 15 kg pressure for 6 hours, and inferior caval vein blood and kidney samples were harvested at 6, 12, 24 hours after removing crush pressure. In TG, 100 mg/kg tempol was intraperitoneally injected into CS model rats after withdraw of crush pressure. In SOG, rats were fixed on the board without any crush pressure. The activation of c-jun was determined by western blotting. Serological parameters and the content of malondialdehyde (MDA) in kidney tissues were determined by standard methods. RESULTS: Acute kidney injury reached the peak at 12 hours after the crush pressure. Compared with SOG, the content of phosphorylated c-jun was significantly higher in CSG and TG (p < 0.05), and the content of phosphorylated c-jun in the CSG was significantly higher than that in TG (p < 0.05). Interestingly, the changes of the MDA content in the kidney tissues of the 3 groups were similar to the changes of phosphorylated c-jun content. CONCLUSION: ROS-mediated phosphorylation of c-jun may play important roles in the acute kidney injury of CS rats. Tempol can inhibit the phosphorylation of c-jun and alleviate the acute kidney injury.

Sivelestat improves outcome of crush injury by inhibiting high-mobility group box 1 in rats.

Severe crush injury is associated with high mortality because of resulting hyperkalemia in early phase and multiorgan dysfunction in later phase. In this study, we investigated the effects of sivelestat administration 1 h before reperfusion on the outcome of crush injury. Crush injury was induced by 6 h of direct compression to both hindlimbs of anesthetized rats with blocks weighing 3.5 kg each side, followed by 3 h of reperfusion. Rats were randomly assigned to three groups. In the control group, rats were infused with normal saline at 1 mL/kg per hour throughout the experiment without compression. Rats in the positive control group were compressed for 6 h, followed by fluid resuscitation initiated 1 h before release with normal saline. The infusion rate was increased from 1 to 10 mL/kg per hour and continued for 4 h. Rats in the treated group underwent the same procedures as in the positive control group, but sivelestat was added to normal saline (concentration was adjusted to infuse 10 mg/kg per hour) during fluid resuscitation (for 4 h). Treatment with sivelestat significantly improved survival rate with P = 0.032. This was accompanied by lower serum high-mobility group box 1 (HMGB1) levels after 3-h reperfusion, attenuated lung injury (assessed using hematoxylin-eosin stain), and suppression of HMGB1 expression in the lung and the liver. These results suggest that treatment with sivelestat improves the outcome of crush injury, likely by inhibiting HMGB1 in rats.

The effect of hyperbaric oxygen in crush injuries and skeletal muscle-compartment syndromes.

Crush injuries represent a spectrum of injury to body parts as result of trauma. Presentations vary from minor contusions to limb-threatening damage. Typically, the injury involves multiple tissues, from skin and subcutaneous, to muscle and tendons, to bone and joints. In their most severe presentations, predictable complications--including osteomyelitis, non-union of fractures, amputations and failed flaps--occur in approximately 50 percent of the cases with standard of practice surgical and medical interventions. Skeletal muscle-compartment syndrome (SMCS) is another consequence of trauma, but in this situation the target tissues are muscles and nerve. Edema and/or bleeding within the confines of the fascial envelope can increase the pressure within the skeletal muscle-compartment. When the tissue fluid pressure within the compartment exceeds the capillary perfusion pressure to the muscles and nerves in the compartment, these tissues are rendered ischemic and manifest the signs and symptoms of SMCS. The SMCS, especially in its incipient stages before a fasciotomy is required, is a therapeutic challenge since no means to arrest its progression exist other than hyperbaric oxygen (HBO2). Unfortunately, HBO2 is woefully neglected as an adjunct for managing crush injury and SMCS. Strong arguments exist for its use based on evidenced-based information and how HBO2 mitigates the pathology of these conditions.

Systemic involvement of high-mobility group box 1 protein and therapeutic effect of anti-high-mobility group box 1 protein antibody in a rat model of crush injury.

Patients with crush injury often present systemic inflammatory response syndrome and fall into multiple organ failure. The mechanism by which the local tissue damage induces distant organ failure is still unclear. We focused on high-mobility group box 1 protein (HMGB1) as one of the damage-associated molecular pattern molecules that cause systemic inflammation in crush injury. We investigated involvement of HMGB1 and the effects of treatment with anti-HMGB1 antibody in a rat model of crush injury. Both hindlimbs of rats were compressed for 6 h and then released. In the crush injury group, the level of serum HMGB1 peaked at 3 h after releasing compression, followed by the increasing in the serum levels of interleukin 6 and tumor necrosis factor α. Hematoxylin-eosin staining showed substantial damage in the lung 24 h after the crush injury, with upregulation of the expression of receptor for advanced glycation end products, as revealed by immunohistochemical analysis. Intravenous administration of anti-HMGB1 antibody improved survival (n = 20 each group) and significantly suppressed serum levels of HMGB1, interleukin 6, and tumor necrosis factor α compared with the untreated crush injury group (n = 6-9 each group). Histological findings of lung damage were ameliorated, and the expression of receptor for advanced glycation end products was hampered by the treatment. These results indicate that HMGB1 is released in response to damage immediately after crush injury and acts as a proinflammatory mediator. Administration of anti-HMGB1 antibody reduced inflammatory reactions and improved survival by blocking extracellular HMGB1. Thus, HMGB1 appears to be a therapeutic target, and anti-HMGB1 antibody may become a promising novel therapy against crush injury to prevent the progression to multiple organ failure.

[Hepatoprotective properties of cytochrome C in traumatic toxicosis].

Experiments on rats showed that traumatic toxicosis (crush syndrome) was accompanied by disorders of both excretion and detoxication functions of the liver and a decrease in the energy potential of the liver. Systemic administration of cytochrome C (10 mg/kg) immediately after trauma and decompression increased the level of endogenous cytochrome C, recovered the pool of adenine nucleotides, normalized bromsulfaleine excretion from the blood, and decreased the content of toxic metabolites in the blood. The obtained experimental data show that cytochrome C possesses high hepatoprotective properties with respect to the development of traumatic toxicosis.

The influence of hypothalamic cytokine PRP on protein synthesis in brain subcellular compartments in crush syndrome.

Crush-syndrom (CS) was characterized by Bywaters E.G.L. in 1941 after London blitz. The soft tissues is followed by acute hemodynamic shock, myoglobinuria, acute renal insufficiency, and lethal endotoxicity. Data of CS pathogenesis study has shown that the largest changes in Crush occur during decompression and are accompanied by acute alteration of brain protein synthesis and strong morphological changes of brain structures. The period of decompression might be characterized by the proteolytic breakdown of the myoglobine and formation of toxic peptides. In our current work we have identified four newly formed peptides in the brain of the animals subjected to the experimental muscle tissue injury. Our investigations related with the CS experimental model have demonstrated that during the 2-hours compression protein synthesis was decreased in cytosol (32,7%) and mitochondria (49%), after 5-h compression there were registered non-significant changes in the level of protein synthesis. Intraperitoneal administration of Proline-rich peptide, ((PRP), 1 mcg/100g weight of rats), originating from proteolysis of C-terminal glycoprotein a neurophysin II along with vasopressin and oxytocin and transferring from the hypothalamus to the neurohypophysis by axonal transport, initiates activation of the protein synthesis in all studied cellular subcomponents of brain cells. The positive effect of the peptide is conditioned, most probably, by activation of the immune system and adaptation mechanisms, including mobilization of endogen-protective mechanisms of the organism.

Traumatic rhabdomyolysis (crush syndrome) in the rural setting.

BACKGROUND: Patients with traumatic rhabdomyolysis (crush syndrome)(CS) secondary to community beatings commonly present to a rural emergency department that has limited access to dialysis services. We describe a retrospective study of patients admitted with a diagnosis of CS to the emergency department of a government hospital in rural KwaZulu-Natal, between November 2008 and June 2009. OBJECTIVES: We assessed identification and management of these patients, considering: (i) early adverse parameters used to identify poor prognosis, (ii) the importance of early recognition, and (iii) appropriate management with aggressive fluid therapy and alkaline diuresis to prevent progression to renal failure. METHODS: Diagnosis was based on clinical suspicion and haematuria. Exclusion criteria included a blood creatine kinase level <1 000 U/l on admission. Data captured included demographics, the offending weapon, time of injury and presentation to hospital, and admission laboratory results. Outcome measures included length of time in the resuscitation unit, and subsequent movement to the main ward or dialysis unit, discharge from hospital, or death. RESULTS: Forty-four patients were included in the study (41 male, 3 female), all presenting within 24 hours of injury: 27 were assaulted with sjamboks or sticks, 43 were discharged to the ward with normal or improving renal function, and 1 patient died. CONCLUSIONS: Serum potassium, creatinine, and creatine kinase levels were important early parameters for assessing CS severity; 43 patients (98%) had a favourable outcome, owing to early recognition and institution of appropriate therapy - vital in the absence of dialysis services.

[Efficacy of cytoflavine in treating traumatic toxicosis].

Experiments on rats showed that traumatic toxicosis (crush syndrome) was accompanied with hypotension, bradycardia, reduction in temperature, breath frequency, and oxygen intake, and violation of acid-base buffer state with elevation of lactate acidosis in the blood serum that is typical of shock performance. Substrate combined antihypoxant cytoflavine (1.5 ml/kg) administered systemically to rats immediately after decompression, increased percentage of survived rats and recovered the main indexes of functional systems and acid-base buffer state 12 h after trauma, which provided general protection of rats against traumatic toxicosis.

The effect of antithrombin on pulmonary endothelial damage induced by crush injury.

After crush injury, patients often experience multiple organ dysfunction syndrome. In this study, we focused on vascular endothelial damage, which is believed to be a possible cause of multiple organ dysfunction syndrome, and revealed a pathological condition of distant organ failure. In particular, the lung is an especially prone target organ at the time of systemic inflammatory invasion after crush injury. We ascertained the effect of antithrombin (AT), which has recently attracted attention for its endothelial protective effects. Using a rat model of crush syndrome, we assessed severity of systemic inflammation and vascular endothelial damage through a blood test and degree of lung injury and centrally focused on morphological analysis of endothelium over time. Crush injury significantly elevated the blood concentration of tissue plasminogen activator-plasminogen activator inhibitor 1 complex, monocyte chemoattractant protein 1, and IL-6. Accumulation of active inflammatory cells (OX-42-positive cells) and expression of von Willebrand factor and vascular cell adhesion molecule 1 significantly increased in the lung 24 h after releasing crush. After 48 h, disarray of alveolar structure and alveolar hemorrhage appeared. Antithrombin administration significantly suppressed accumulation of inflammatory cells, expression of von Willebrand factor and vascular cell adhesion molecule 1, and mortality rate. Our research demonstrates that crush injury induces acute lung injury as distant organ failure, and it would seem that AT administration diminishes vascular endothelial damage and is effective against crush injury.

Screening for the formation of reactive oxygen species and of NO in muscle tissue and remote organs upon mechanical trauma to the mouse hind limb.

BACKGROUND: Until now, no systematic surveys exist in the literature on the early local and systemic generation of reactive oxygen species and of nitric oxide in response to muscle crush injury. Therefore, this study aims to evaluate the formation of reactive oxygen species and nitric oxide in different tissues (injured and contralateral muscle, liver, kidney, spleen and blood) that is induced by closed muscle trauma. METHODS: 5, 45 and 180 min after induction of blunt trauma to the mouse gastrocnemius muscle, animals were sacrificed, tissues harvested and homogenized, and analyzed for their content of glutathione, nitrate and thiobarbituric acid-reactive substances. RESULTS: The local formation of reactive oxygen species in the injured muscle started immediately upon induction of the mechanical trauma as indicated by changes in the glutathione redox balance. Liver and kidney did not show any response to trauma; however, a marked and immediate increase in the splenic nitrate content was detected, thus suggesting a specific nitric oxide-dependent response of splenic cells to injury. CONCLUSION: We conclude that immediately after the induction of trauma a formation of reactive oxygen species takes place at the site of crush injury. This might constitute the basis of further damage to the injured tissue by free radical-dependent mechanisms. The immediate formation of nitric oxide within the spleen upon muscle crush appears to represent a specific signalling mechanism of the body in response to distant organ injury.

[Leukocyte-modulating and antioxidant activity of blood serum and bronchoalveolar lavage fluid in the course of the decompression period of long crush syndrome].

We studied leukocyte-modulating activity (LMA) and antioxidant activity (AOA) of blood serum and bronchoalveolar lavage fluid (BAL) in the course of a postcompression period of crush syndrome (CS) in rats. CS was modelled by compression of the left back leg of the animal with metal vice (compression area 5 cm2, duration 4 hours). In CS decompression, LMA of both the serum and BAL was high. On decompression day 1 there was a compensatory growth of serum and BAL AOA, its fall on day 3 and 7 and recovery by day 21. Calculation of the coefficient of LMA to AOA shows imbalance between pro- and antioxidant systems with development of oxidant stress. Thus, prevalence of blood and lung LMA over AOA on intermediate stages of CS may cause stimulation of destruction in hepatic and pulmonary tissues with development of inflammatory reaction.

Production of consistent crush lesions in murine quadriceps muscle--a biomechanical, histomorphological and immunohistochemical study.

Poor healing of high-energy fractures is often associated with severe muscle damage. This may be partly due to the production, by the injured muscle, of inflammatory cytokines that somehow misdirect bone healing. In order to investigate this question, an animal model was established which embodies a controlled degree of muscle injury with a dose response to the energy absorbed, that can be characterised histologically. Using a custom crush jig, 60 CFLP mice had either 100 or 200 g masses dropped from a fixed height onto the quadriceps muscle, with mechanical measurement of the impact. Energy of impact was reliably and significantly different between the small and large impact conditions, though there was more variability when the large mass was used. Animals were sacrificed at day 2, 4, 8, 16, and 24 post-injury. Muscle histomorphometry at all time points and immunohistochemistry for IL-1beta, IL-6, and TNF-alpha up to day 8 were used as measures of muscle damage, inflammation and repair. Histological sections were analysed into areas of normal muscle fibres, damaged/regenerating muscle fibres and fibrous/inflammatory infiltrate. Early histological response was similar between the two groups; the large crush group displayed significantly greater areas of inflammatory infiltrate and damaged muscle at the later time points after day 8. In the large crush group, IL-1beta and IL-6 expression were significantly higher at day 2 and TNF-alpha was higher at day 8 when compared to the small crush group. The experiment demonstrated that more severe injury to muscle was reliably followed by increased inflammatory cytokine production and a greater degree of inflammation and fibrosis. Increased production of inflammatory cytokines such as TNF-alpha and IL-1beta in the damaged muscles may activate macrophages and recruit fibroblasts, promote scar formation and lead to delayed union or non-union of the adjacent fracture(s).

[Morphological changes of tissues after extensive soft tissue injury].

OBJECTIVE: To study the pathological morphological changes for diagnosing the cause of death of extensive soft tissue injury or crush syndrome. METHODS: The tissues were stained by HE and IHC. RESULTS: (1) The Mb positive rate was 60%, 75%, 95% respectively. (2) Both the HSP70 positive rate of hearts and brains were 90%. CONCLUSION: (1) The animal model of broad soft tissue injury was established. (2) Accumulated the pathological morphological data for diagnosing the cause of death of extensive soft tissue injury or crush syndrome.