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Corresponding author. Division of Environmental Medicine, National Defense Medical College Research Institute, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359-8513, Japan. Tel.: +81 4 2995 1626; fax: +81 4 2991 1612.
Affiliations
Division of Environmental Medicine, National Defense Medical College Research Institute, National Defense Medical College, Tokorozawa, Saitama, Japan
Department of Medical Engineering, National Defense Medical College, Tokorozawa, Saitama, JapanDivision of Biomedical Engineering, National Defense Medical College Research Institute, National Defense Medical College, Tokorozawa, Saitama, Japan
Department of Medical Engineering, National Defense Medical College, Tokorozawa, Saitama, JapanDivision of Biomedical Engineering, National Defense Medical College Research Institute, National Defense Medical College, Tokorozawa, Saitama, Japan
Crush syndrome (CS) has been reported in disasters, terrorist incidents, and accidents, and the clinical and pathologic picture has gradually been clarified. Few lethal and reproducible animal models of CS with use of a quantitative load are available. A new model is needed to investigate pathologic and therapeutic aspects of this injury.
Materials and methods
Using a device built from commercially available components, both hindlimbs of anesthetized rats were respectively compressed for 6 h using 3.6-kg blocks. The effects of trunk warming alone without compressed hindlimbs (Group A), non-warming at room temperature (Group B), whole-body warming including compressed hindlimbs (Group C), or warming of compressed hindlimbs alone (Group D) during compression were examined. Survival rates were compared and hematological and histologic analyses were performed at specific time points after compression release.
Results
Limb or whole-body warming significantly worsened the survival of rats. We found a much lower survival rate of 0%–10% in animals, in which the hindlimbs were warmed during compression (Groups C and D) at 12 h after compression release, compared with 90%–100% in animals without warming of the hindlimbs (Groups A and B). Groups C and D showed significantly enhanced hyperkalemia at ≥4 h after compression release and all blood samples from dead cases showed hyperkalemia (>10 mEq/L).
Conclusions
We developed a new lethal and reproducible rat CS model with a quantitative load. This study found that warming of compressed limbs worsened the survival rate and significantly enhanced hyperkalemia, apparently leading to cardiac arrest.
] in 1941 during World War II, based on the observation that victims pinned down among rubble in aerial bombing showed crush wounds and developed severe acute renal failure within a week. CS has subsequently been reported in disasters [
Turkish Study Group of Disaster An overview of morbidity and mortality in patients with acute renal failure due to crush syndrome: the Marmara earthquake experience.
Turkish Study Group of Disaster An overview of morbidity and mortality in patients with acute renal failure due to crush syndrome: the Marmara earthquake experience.
]. The high concentration of myoglobin released from necrotic muscle cells causes hyperglobulinemia, which progresses to acute renal failure with renal tubular necrosis [
Turkish Study Group of Disaster An overview of morbidity and mortality in patients with acute renal failure due to crush syndrome: the Marmara earthquake experience.
] used similar tourniquets in a rat model (survival rate, 25%). All of these studies used a special apparatus to create the animal CS model, and thus limited the reproducibility of conditions and findings from the model.
In surgery, systemic warming is recommended to prevent hypothermia and surgical site infection [
]. Furthermore, icing the compressed sites after compression release have retarded proliferation and differentiation of satellite cells at the early stage that might result in alleviation of ischemic–perfusion injury [
]. The ischemic tolerance time depends on ambient temperature. In this study, we happened to find that the warming of compressed hindlimbs substantially turned down the survival rates and increased blood potassium concentrations in the rat CS injury model. Therefore, the aim of this study was to establish lethal and non-lethal rat models of CS with controlling temperature of compressed hindlimbs.
2. Materials and methods
2.1 Experimental system components
We developed a new device (Fig. 1) to create a crush injury model. A pole 20 mm in diameter and 300 mm in height (PO-20-300; Sigma Koki, Tokyo, Japan) was vertically fixed to a magnetic base (MB-CB-PB; Sigma Koki) with a rod stand (RS-20-60; Sigma Koki). The magnetic base is firmly attached to a flat board (FB-545-50; Sigma Koki). A rectangular wooden board (Agathis, 250 × 80 × 10 mm) was fastened by three-pronged clamps (small double-leaf clamp, No. 91-2447-4; Sansyo, Tokyo, Japan). The clamp was fixed to the pole with a cross clamp connector (CCHN-20-12; Sigma Koki) at a right angle. The board and clamp move smoothly up and down the pole. The total weight of the loading part (including the board and clamp) is 340 g. Three interlocking blocks (Earthen Bricks, 198 × 98 × 30 mm and 1.2 kg each; Tokyo Electric Power Environmental Engineering, Tokyo, Japan) were loaded on the board and placed on the each hindlimb with the edge of the board on the thigh just caudal to the inguinal ligaments. Loads were placed flat using a level gauge (G-Director Level, ED-20GDLMR; Ebisu, Niigata, Japan). Using this experimental setup, a quantitative load could be used for limb compression.
All animal experiments were carried out according to the protocol approved by the Animal Experimentation Committee at the National Defense Medical College (Tokorozawa, Saitama, Japan). Ambient temperature was kept at 25 ± 2°C during the experiments. Male Sprague–Dawley rats weighing 290–320 g (9 wk old) were purchased from Japan SLC (Hamamatsu, Shizuoka, Japan) and given ad libitum access to food and water. Animals were lightly sedated in an ether-filled anesthesia box. After sedation, intraperitoneal anesthesia was performed using a 27-gauge injection needle. The anesthetic agent was a 5 mg/mL solution of pentobarbital sodium (Somnopentyl, 64.8 mg/mL; Kyoritsu Seiyaku, Tokyo, Japan) in physiological saline, injected at an initial dose of 50 mg/kg body weight. For blood sampling, a catheter (Atom Indwelling Feeding Tube for Infant 3 Fr; Atom Medical, Tokyo, Japan) was indwelled and fixed via the left intracarotid artery through a midline neck incision before the experiments were conducted. The catheter was filled with heparinized saline. The heparinized saline solution (10 U/mL) was made from 1 mL of heparin sodium solution (Novo-Heparin 1000 U/mL for injection; Mochida Pharmaceutical, Tokyo, Japan) and 99 mL of saline. After preparation, rats were placed on the experimental table in the supine position with hindlimbs open outward at 120°C. Wooden boards were placed along the inguinal ligament.
Saline (1 mL/kg body weight) was infused through the carotid artery and 10 mg/kg pentobarbital sodium was infused into the peritoneum cavity every hour. Eyelash reflex [
] were checked every 5 min, and additional pentobarbital sodium (10 mg/kg) was injected intraperitoneally as necessary. After 6 h of hindlimbs compression, the load was removed, and the compression was released.
To study the effects of controlling temperature of compressed hindlimbs during compression, animals were divided into four groups. To evaluate the effect of warming for trunk, compressed hindlimbs and the both, we planned four groups to be examined. In Group A (trunk warming alone without compressed hindlimbs), the head, neck, forearms, and body trunk were warmed using a heating mattress set at 40°C (KN-475-III-40; Natsume Seisakusho, Tokyo, Japan) during hindlimbs compression. In Group B (non-warming at room temperature), no heating mattress was used and the experiment was performed at ambient temperature. In Group C (whole-body warming including compressed hindlimbs), the head and neck, forearms, body trunk, and compressed hindlimbs were warmed using a heating mattress during compression. In Group D (warming of compressed hindlimbs alone), only the compressed hindlimbs were warmed during compression.
2.3 Survival analyses
To investigate the survival rate, a total of 40 rats were used (n = 10 in each of the four groups). During hindlimbs compression, temperature sensors were placed at the pharynx (sensor KN-91-E-1; Natsume Seisakusho), rectum (KN-91-E-2; Natsume Seisakusho), and ventral thigh (KN-91-E-2; Natsume Seisakusho). After hindlimbs compression for 6 h, rats were released to a cage to recover from anesthesia. Water and food were given ad libitum. Continuous observation was performed using a digital video camera (Handycam HDR-XR550; Sony, Tokyo, Japan). Based on the instructions given by the Institutional Animal Care and Use Committee of the National Defense Medical College, we sacrificed all animals after observation for 14 d.
2.4 Blood and histologic analyses
For blood and histologic analyses, a total of 63 rats were used (14 rats in each of the four groups and other seven rats for normal value measurement). After compression for 6 h, compression was released at an ambient temperature from the rats under continued anesthesia. Blood was drawn through the left carotid artery at the following time points: just before starting the 6-h compression and at 3 and 4 h after compression release (n = 7 in each of the four groups), and 6, 9, and 12 h after compression release or at the time of death (n = 7 in each of the four groups) to measure the concentration of blood potassium. Blood samples (0.3 mL each) were drawn using a heparin-coated syringe and analyzed immediately. In a separate experiment using total 28 rats (n = 7 in each of the four groups), a 4-mL sample was drawn at 4 h after hindlimbs compression in each group (n = 6) and centrifuged for serum separation (3000 rpm or 800 g, 15 min, centrifuge 5415 R; Eppendorf Japan, Tokyo, Japan). Creatine phosphokinase (CK) in this serum was measured immediately. The remaining serum sample was frozen and stored at −80°C. The myoglobin level was analyzed after unfreezing the serum.
Blood gas, electrolyte, and biochemical analyses were performed using a handheld blood analyzer (i-STAT 1 analyzer; Fuso Pharmaceutical Industries, Osaka, Japan). Glucose, blood urea nitrogen (BUN), creatinine (Cr), sodium+, potassium+, chloride−, ionized calcium, and hematocrit (Ht) were analyzed using an i-STAT CHEM8+ cartridge. Lactate and pH were analyzed with an i-STAT CG4+ cartridge.
CK was analyzed using an automatic biochemical analyzer (FUJI DRI-CHEM 7000V and FUJI DRI-CHEM slide CPK-PIII; Fujifilm Medical, Tokyo, Japan). Myoglobin was analyzed using a solid-phase enzyme-linked immunosorbent assay (Rat Myoglobin ELISA 2110-2-N; Life Diagnostics, West Chester, PA). After drawing a blood sample, 1000 U of heparin was injected through the carotid artery, and the animal was sacrificed under an overdose of anesthesia. The left jugular vein was cut down for depressurization. Perfusion fixation was performed at 160.3 cmH2O (120 mm Hg) with 10% neutralized formalin (060-01,667; Wako Pure Chemical Industries, Osaka, Japan) via the carotid artery. After rapid dissection, the sample was immersed in 10% neutralized formalin. Brain, lungs, heart, kidneys, and hindlimb muscles (adductor muscles) were fixed and sliced in the normal manner.
Samples (each n = 7) in all groups at 4 h after 6-h compression in four groups and samples at 24 h after 6-h compression in Groups A and B were embedded in paraffin and sliced at 4 μm thickness. Hematoxylin and eosin (HE) staining, anti-myoglobin staining (purified myoglobin polyclonal antibody, SIG-3120; Covance, Princeton, NJ) and anti-nitrotyrosine staining (anti-nitrotyrosine, SMC-154 C; StressMarq Biosciences, Victoria, Canada) were performed. Sample preparation and staining were performed by Mitsubishi Chemical Medience Corporation (Tokyo, Japan). The anti-myoglobin staining was performed because acute renal failure associated with myoglobin has been shown in many studies of CS [
To obtain normal value, seven rats were anesthetized. For blood sampling, a catheter (Atom Indwelling Feeding Tube for Infant 3 Fr) was indwelled and fixed via the left intracarotid artery through a midline neck incision. An amount of 4 mL of blood sample was obtained and measured for blood gas, electrolyte, and biochemical analyses as described previously for experimental groups. The normal values of CK and myoglobin were also analyzed as described previously. After drawing a blood sample, 1000 U of heparin was injected through the carotid artery, and the animal was sacrificed under an overdose of anesthesia. The left jugular vein was cut down for depressurization. Perfusion fixation was performed with 10% neutralized formalin via the carotid artery as described previously.
To measure each hindlimb-to-body weight ratio, hindlimb was disarticulated by Boyd's procedure [
Values are shown as the mean ± standard deviation. Multigroup analysis was performed using one-way analysis of variance (ANOVA) and one-way repeated measures ANOVA. Survival rate was analyzed by the Kaplan-Meier and log-rank methods with Bonferroni correction using StatMate IV software (ATMS, Tokyo, Japan). Values of P < 0.05 were considered statistically significant.
3. Results
3.1 Temperature analyses
No death occurred during anesthesia or compression of hindlimbs in the 40 rats. Temperatures at the monitored sites during 6-h compression and up to 3 h after release are shown in Figure 2. The value of temperature in four groups at each time points are calculated using one way ANOVA and the values of temperature in each group are calculated using one way repeated measures ANOVA and confirmed the statistical significance of variances. Pharyngeal temperature was initially 37°C and then elevated to 38°C in Groups A (trunk warming) and C (whole-body warming) during compression, gradually declining to 36°C after release. In Group D (hindlimbs warming), pharyngeal temperature remained at 34°C–35°C during compression and declined to 33°C after release. In Group B (ambient temperature), pharyngeal temperature remained at 32°C–33°C. At ≥1 h of compression, no significant difference in pharyngeal temperature was seen between Groups A and C, but the temperature was significantly lower in the other groups (P < 0.01; Fig. 2I).
Fig. 2Temperatures monitored at the (I) pharynx, (II) rectum, and (III) crushed hindlimb. The value of temperature in four groups at each time points are calculated using one-way ANOVA, and the values of temperature in each group are calculated using one-way repeated measures ANOVA and confirmed the statistical significance of variances.
Rectal temperature in Group C peaked at 38°C. In Groups D, A, and B, this temperature stayed at around 35°C, 34°C, and 32°C, respectively, and declined by 2°C–4°C after release. Most pairwise group comparisons using one-way ANOVA showed a significant difference from 1-h compression to 3 h after release (P < 0.05) except the difference between Groups A and D 3 h after release (Fig. 2II).
Hindlimb temperature remained at 36°C–38°C in Groups C and D, but declined to 32°C and 30°C in Groups A and B, respectively. All pairwise group comparisons using one-way ANOVA showed a significant difference from 1-h compression to 3 h after release (P < 0.05) except the difference between Groups A and D at 3 h after release (Fig. 2 III).
3.2 Survival analyses
Survival curves for each warming method are shown in Figure 3. No death occurred in Group A (trunk warming) during the 14 d of observation. One of 10 animals died in Group B (room temperature) at about 24 h after compression release. In Group C (whole-body warming), one animal died at 3 h after compression release, three at 4 h, one at 5 h, one at 10 h, one at 11 h, one at 21 h, and one at 23 h. Only one animal survived for 14 d. In Group D (hindlimb warming), two animals died at 3 h, two at 4 h, two at 7 h, one at 11 h, one at 13 h, one at 15 h, and one at 19 h; all rats in Group D died within 19 h. These results show that the warm compression of the hindlimbs in our model had a significant adverse effect on survival (10% and 0% versus 90% and 100%, P < 0.001). Paralysis of the compressed hindlimbs was observed just after release. The paralysis in Groups A and B animals that survived gradually improved but remained in the ankles and toes at 2 wk after the compression release.
Fig. 3Survival in the four thermal management groups (n = 10 for each group).
Sixty-three rats used in the hematological and histologic analyses. Blood (0.3 mL each) was drawn through the left carotid artery after 6-h compression and at 3, 4, 6, 9, and 12 h after compression release or at time of death to measure the blood concentration of potassium. No significant differences after 6 h of compression (mean, 3.5–4.4 mEq/L) and at 3 h after compression release (mean, 4.5–6.2 mEq/L) were seen among the four groups (Fig. 4). Although blood potassium levels were significantly increased in all groups at ≥4 h after compression release, levels were significantly higher in Groups C and D than in Groups A and B. Levels in Groups A and B never exceeded 7.0 mEq/L, and all rats used to evaluate their hyperkalemia survived. All blood samples from dead rats in Groups C and D showed hyperkalemia (>10 mEq/L), which seems to have caused cardiac arrest.
Fig. 4Changes in blood potassium concentrations in the four thermal management groups. Potassium in blood was increased in all groups at ≥4 h after compression release and was significantly higher in Groups C and D. Levels in Groups A and B never exceeded 7.0 mEq/L, and all rats survived. All blood samples from dead cases (■) in Groups C and D showed hyperkalemia (>10 mEq/L), which seemed to have caused cardiac arrest. Normal potassium level in blood was 2.8–3.9 mEq/L, which indicated red lines. (Color version of figure is available online.)
Normal hematological values of normal rat (n = 7) are also shown in Table 1. Blood analyses at 6 h of compression showed some variation, but no significant differences among the four groups except the values of BUN. At 4 h after compression release, BUN and Ht were significantly lower in Groups A and B than in Groups C and D; base excess (BE) was significantly higher in Groups A and B than in Groups C and D; pH was significantly decreased in all groups compared with the value after 6 h of compression; and Cr and lactate were significantly increased in all groups at 4 h after compression release, compared with the values after 6 h of compression (Table 1). Furthermore, blood CK and myoglobin levels were significantly increased from 100 ± 50 U/L (normal CK) and 45 ± 10 ng/mL (normal myoglobin) to >12,500 U/L and >950 ng/mL at 4 h after compression release in all four groups, respectively. CK values in Group B at 4 h after compression release were significantly lower than in Groups A, C, and D, and also myoglobin levels in Group B were significantly lower than those in Groups C and D (Fig. 5).
Table 1Hematological data for pH, BE, Ht, lactate, BUN, and Cr concentrations in the four study groups.
Fig. 5Hematological data for serum Cr phosphokinase and myoglobin concentrations in the four thermal management groups at 4 h after compression release. Values are shown as mean ± standard deviation (n = 7 for each group). (Color version of figure is available online.)
Histologic results from muscle samples (n = 7) at 4 h and 24 h after compression release in Group B and normal rats are shown as representatives of the four Groups in Figure 6. In muscle samples (n = 7) of all four Groups, muscle cell expansion and rupture of muscle fibers were recognized, and nitrotyrosine and myoglobin staining showed weakly positive results in the interstitial space of muscle tissue at 4 h after compression release. There were no substantial differences in each group and among the four groups. Expanded collapse, edema, and infiltration of neutrophils were observed in the interstitial space of muscles at 24 h after compression release, with congestion and infiltration of neutrophil invasion apparent in the walls of alveoli. Furthermore, nitrotyrosine staining was strongly positive in the interstitial space of muscle tissue (Fig. 6). There were no significant differences in the histologic observations of muscle samples at 24 h after compression release in Groups A and B, and all rats in Groups C and D died within 12 h after compression release by enhanced hyperkalemia.
Fig. 6Histologic findings in compressed muscle tissues in the rat CS model. Yellow arrows show collapsed muscle fibers. Black arrows show infiltration of neutrophils (HE staining), myoglobin (anti-myoglobin staining) and nitrotyrosine (anti-nitrotyrosine staining). Magnification ×400.
The aim of this study was to report on preparation of lethal and nonlethal animal model of CS and the effects of controlling temperature of compressed hindlimbs on the survival rate together with hematological and histologic examinations. Only minimum fluids were therefore administered to each group to minimize the influence of body temperature control. In many clinical reports about severe cases of CS, renal failure is said to be one of the main lethal factors [
Turkish Study Group of Disaster An overview of morbidity and mortality in patients with acute renal failure due to crush syndrome: the Marmara earthquake experience.
] Any renal failure with serum Cr >2.0 mg/dL was not observed (data not shown). One lower extremity-to-body weight ratio of human is reported to be 17.6%–20.4% [
]. We measured one hindlimb-to-body weight of rat as 7.70 ± 0.21% (n = 7). The anatomical difference may affect and make limitation for the animal model of CS. We successfully established lethal CS model because of hyperkalemia during acute phase. We found a much lower survival rate of 0%–10% in animals that received warming of the hindlimbs to 40°C during compression (Groups C and D) compared with 90%–100% in animals without warming of the hindlimbs (Groups A and B). There were no significant differences in the survival rate and the hematological data between Groups A and B at 4 h after compression release (Table 1 and Fig. 3). Hypothermia has been found to alleviate ischemia–reperfusion injury. In a canine gracilis muscle model of ischemia–reperfusion, Wright et al. [
] found that a continuous low temperature before ischemia and after reperfusion diminished muscle damage (infarct size) and vascular permeability and also showed that cooling to 21°C during reperfusion diminished muscle edema and provided an effective alternative treatment for reperfusion injury or compartment syndrome [
]. An icing the compressed sites after compression release have retarded proliferation and differentiation of satellite cells at the early stage that might result in alleviation of acute ischemic–reperfusion injury. However, the icing might be related to a delay in muscle regeneration [
This study suggested that warming of compressed limbs substantially worsened survival and increased blood potassium concentrations. No significant differences were seen among the four groups both after 6-h compression and at 3 h after compression release (Fig. 4). Blood potassium levels increased in all groups at ≥4 h after compression release and were significantly higher in Groups C and D. In rat electrocardiogram,P-wave disappeared when serum potassium concentration of ≥8.0 mEq/L [
]. Levels in Groups A and B never exceeded 7.0 mEq/L, and all rats survived. Furthermore, all blood samples from dead rats in Groups C and D showed hyperkalemia (>10 mEq/L), which seemed to cause cardiac arrest. Thus, the death in Groups C and D might cause because of cardiac failure by hyperkalemia >10 mEq/L.
Groups A and B showed improved survival and hematological results in BE, Ht, and BUN at 4 h after compression release compared with Groups C and D. Group A did not show improved hematological results at 4 h after compression release compared with Group B. No significant differences in pH, and lactate were seen among the four groups at 4 h after compression release.
We observed relatively minor rupture of muscle fiber, myoglobin, and nitrotyrosine staining in all four groups at 4 h after compression release and substantial rapture, myoglobin, and nitrotyrosine staining in both Groups A and B at 24 h after compression release. There were no significant differences in the rapture, myoglobin and nitrotyrosine staining between Groups A and B at 24 h after compression release. Furthermore, all rats in Groups C and D died within 12 h after compression release. Therefore, we present the histology in Group B as a representative to show crush injury at 4 and 24 h after compression release, although we did not perform grading the severity of injury or quantitative analyses for the histologic changes. Currently, we adopted Group B as a non-lethal and reproducible crush injury model, and Group D as a lethal and reproducible CS model for further study.
5. Conclusions
In summary, new lethal and reproducible rat CS model with a quantitative load and with controlled temperature of compressed hindlimbs was developed in this study. Rats that received warming of the hindlimbs to 40°C during compression exhibited a much lower survival rate and significantly enhanced hyperkalemia, apparently leading to cardiac arrest. In addition, this study may provide a non-lethal and reproducible CS injury model without warming of compressed hindlimbs.
Acknowledgment
We acknowledge the expertise and advice of Associate Prof Koichi Fukuda and we thank the personnel of the Institute of Laboratory Animals, Graduate School of Medicine, National Defense Medical College for expert care of animals.
None of the authors have any conflicts of interest to declare.
Author contributions: Conception and design: TN, MF, MaI, and MiI.
Analysis and interpretation: TN, MF, YY, MS, TM, and YK.
Data collection: TH, MF, MiI, YY, SO, YY, and YK.
Writing the article: TN, MF, and MaI.
Critical revision of the article: TN, MF, MaI, and SO.
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