Effect of hypothermia on coagulatory function and survival in Sprague–Dawley rats exposed to uncontrolled haemorrhagic shock

doi:10.1016/j.injury.2011.11.016

Injury

Volume 44, Issue 1, January 2013, Pages 91-96

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Abstract

Background

Acute coagulopathy, hypothermia, and acidosis are the lethal triad of conditions manifested by major trauma patients. Recent animal studies have reported that hypothermia improves survival in animals subjected to controlled haemorrhagic shock. The objective of this study was to investigate the effect of hypothermia on coagulation in rats subjected to uncontrolled haemorrhagic shock.

Methods

Thirty-two male Sprague–Dawley rats were randomly divided into four groups: normothermia (control, group N), hypothermia (group H), hypothermic haemorrhagic shock (group HS), and normothermic haemorrhagic shock (group NS). Haemorrhagic shock was induced by splenic laceration. Capacity for coagulation was measured by rotation thromboelastometry (ROTEM^®), and was measured at baseline as well as the end of the shock and resuscitation periods. Survival was observed for 48 h post-trauma.

Results

Baseline parameters were not different amongst the groups. Rats exposed to hypothermia alone did not differ in coagulation capacity compared to the control group. Clot formation time (CFT) and maximal clot firmness (MCF) in group HS decreased as the experiment progressed. Maximal clot firmness time (MCFt) in groups H and HS was significantly prolonged during shock and resuscitation compared with that in group NS. In group NS, MCF did not change significantly, but MCFt was reduced compared with baseline. Group HS had poor survival when compared with normovolaemic groups.

Conclusion

Blood clotted less firmly in traumatic haemorrhagic shock, and hypothermia prolonged clotting. However, clot firmness maximised rapidly under normothermic haemorrhagic shock. Haemorrhage would continue for a longer time in hypothermic haemorrhagic shock. Survival of hypothermic shock was not significantly different compared to that of normothermic haemorrhagic shock.

Introduction

Trauma is the third greatest cause of mortality in Korea following neoplasm and cardiovascular disease, and the most common cause in individuals under 40 years of age.¹ Despite advances in trauma care, the leading cause of trauma death is exsanguination in up to 30% of cases.² In particular, coagulopathy, hypothermia, and acidosis are well known as the lethal triad contributing to trauma mortality.³

Traumatic haemorrhagic shock increases the rate of coagulation, which leads to the use of fibrinogen and reduced clot strength.⁴ Intravascular consumption of clotting components during continuous bleeding as well as blood dilution during massive transfusion and fluid resuscitation leads to post-traumatic coagulopathy.⁵ These haemostatic functional changes continue, causing alterations in platelet function and, finally, serious coagulopathy such as disseminated intravascular coagulation.⁴

Hypothermia is another important factor determining the prognosis of trauma patients. Trauma patients lose heat by evaporation through wet clothing in the field or by radiation and convection following exposure at the trauma scene. Body temperature is lowered by massive fluid resuscitation, transfusion of cold blood products, and irrigation of open wounds. Hypothermia is a huge contributing factor to mortality in trauma patients, regardless of injury severity and age.⁶ Patients who undergo rapid rewarming require less resuscitation fluid and have significantly less early mortality compared to those experiencing slow rewarming.⁷ For that reason, the Advanced Trauma Life Support Guidelines of the American College of Surgeons recommend that hypothermia should be avoided in trauma patients. Furthermore, warming of fluids to 39 °C before infusion and the use of a blood warmer are strongly recommended.⁸

Hypothermia itself is not only a strong determinant of poor prognosis in trauma patients but also has direct effects on clotting. Hypothermia attenuates the initiation phase of thrombin generation and fibrinogen availability.⁹ Severely injured patients with hypothermia and acidosis develop clinically significant bleeding, even though blood products such as red blood cells, plasma, and platelets are replaced.¹⁰

Recent experimental studies have reported that hypothermia improves survival in haemorrhagic shock.11, 12 However, in this work controlled haemorrhagic shock was induced by blood withdrawal, which is different to the clinical reality of uncontrolled bleeding, and hypothermia was induced only by external cooling.11, 12 In addition, the mechanisms eliciting these results were unclear. Artificial hypothermia therapy is effective for cardiac-arrest victims who remain comatose after restoration of spontaneous circulation.¹³ As previously stated, hypothermia clinically aggravates traumatic haemorrhagic shock in patients.

The objective of this study was to investigate the effects of mild hypothermia on coagulation capacity using rotational thromboelastometry and survival in a rat model of haemorrhagic shock with spleen injury.

Section snippets

Materials and methods

This study was approved by the Animal Care and Use Committee in Jeju National University (No. 2010-0045).

Thirty-two male Sprague–Dawley rats (weight, 350 ± 50 g; age, 9–10 weeks old; Charles-River, Montreal, Quebec, Canada) were used. The rats had unlimited access to food and water before the experiment.

Laboratory assessments

The mean body temperatures of each group during experiment are shown in Fig. 2. No significant differences in laboratory values were found at baseline amongst the four groups (Table 1). Haemorrhagic shock reduced bicarbonate, base excess, lactate, and haemoglobin, all of which differed significantly from values in non-shock groups at the end of the treatment period (Table 1). Lactate recovered by the end of resuscitation in groups HS and NS, and no difference was observed amongst the groups, as

Discussion

We investigated the effect of mild hypothermia on coagulation and survival in rats subjected to uncontrolled haemorrhagic shock. Rat subjected to mild hypothermia alone had no alteration in coagulatory function compared to the control group. Coagulopathy was observed only in rats subjected to haemorrhagic shock, and mild hypothermia significantly extended the time to maximal clot firmness. Survival was no different between groups HS and NS, and only group HS demonstrated poor survival compared

Conclusions

When hypothermia combined with traumatic haemorrhagic shock, coagulatory function deteriorated; platelet function decreased and it took longer time to form maximal clot during the shock and resuscitation period. Although clot firmness is weak in haemorrhagic shock with normothermia, it needs a shorter time to reach MCF. Haemorrhage might continue for a longer time in hypothermic haemorrhagic shock than in normothermic haemorrrhagic shock. Mortality in the hypothermic haemorrhagic shock group

Conflict of interest statement

None declared.

Acknowledgement

The authors thank Hye Jin Hyun for her excellent technical assistance.

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Effects of Seawater Immersion on Lethal Triad and Organ Function in Healthy and Hemorrhagic Shock Rats
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Citation Excerpt :
While VECs allow multiple mechanisms, including procoagulant and anticoagulant. Acidosis could damage VECs, progressively impairing platelet aggregation ability and clot formation, eventually leading to coagulation dysfunction.19,20 Additionally, hypothermia together with acidosis has an additive effect on aggravating coagulopathy, with hypothermia mainly prolonging the initiation phase, whereas acidosis prolongs the propagation phase of thrombin formation.
Marine casualties are increasing, and mortality from trauma associated with immersion in seawater is high. However, the associated pathophysiological characteristics remain unclear, limiting research into the early emergency treatment strategy.
Healthy and 50% hemorrhagic shock rats were soaked in 15°C and 21°C seawater for 2 h, 4 h and 6 h, respectively, and the effects on vital signs, internal environment, tissue metabolism, lethal triad, vital organ functions and survival were observed.
Immersion in seawater can cause death in healthy rats. Rats with hemorrhagic shock in 15°C seawater showed a lower survival rate than the corresponding groups in 21°C seawater. Moreover, compared with 21°C seawater, 15°C seawater played a more remarkable role in decreasing mean arterial pressure, heart rate, and respiration rate, increasing water content and decreasing Na⁺/K⁺-ATPase activity in the brain and lung; increase in plasma osmolality, Na⁺, K⁺, Cl⁻, and the occurrence of the lethal triad manifested by a decrease in core body temperature, pH, lactate, and an increase in coagulation parameters, as well as damage to cardiac, intestinal, hepatic, and renal functions in rats with hemorrhagic shock.
Immersion in seawater at low temperatures could be lethal to healthy rats, causing the occurrence of a lethal triad and damage to vital organs. Furthermore, 15°C-seawater had a more significant effect than 21°C-seawater on aggravating the imbalance of internal environment and tissue metabolism, resulting in a higher incidence of the lethal triad and thus aggravating the dysfunctions of vital organs, which eventually resulted in higher mortality in rats with hemorrhagic shock.
Impact of general anesthesia on rotational thromboelastometry (ROTEM) parameters and standard plasmatic coagulation tests in healthy Beagle dogs
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Hypothermia of 32 °C decreased fibrinogen synthesis by about 50% with no changes in fibrinogen breakdown measured by stable isotope technique in an in vivo swine model (Martini, 2007). Rats subjected to 33 – 34 °Celsius had no alteration in coagulatory function compared to the control group, measured with ROTEM at the temperature of 37 °C or 33 °C respectively (Park, Lee & Kim, 2013). This is similar to our findings, where ROTEM analysis and standard plasmatic coagulation tests were carried out at 37 °C and not corrected for the dogs’ actual rectal temperatures.
To assess the influence of general anesthesia on rotational thromboelastometry (ROTEM) and standard coagulation testing in healthy dogs
Prospective experimental study
10 healthy Beagle dogs
Dogs were administered methadone (0.2 mg/kg) intramuscularly. Anesthesia was co-induced intravenously 30 min later with midazolam (0.1 mg/kg) and propofol to effect, and maintained with sevoflurane. Crystalloids were administered at 5 ml/kg/h. Blood was sampled by direct venipuncture before induction (T0) and 3.5 h later (T3.5) and ROTEM parameters (ExTEM, InTEM, FibTEM, ApTEM), standard plasmatic coagulation tests (prothrombin time [PT], activated partial thromboplastin time [aPTT], fibrinogen concentration), hematology, ionized calcium, triglycerides, pH, lactate and body temperature were compared over time with Students t - test or Wilcoxon matched pairs signed-rank tests.
The following variables dropped significantly between T0 and T3.5: body temperature (p < 0.0001), hematocrit (p < 0.0001), platelet count (p < 0.01), pH (p < 0.01), triglycerides (p < 0.01), fibrinogen concentration (p < 0.01), ExTEM, FibTEM (p < 0.01) and ApTEM (p < 0.05) clotting times. Lactate concentration (p < 0.01), aPTT (p < 0.05) and FibTEM maximum clot firmness increased (p < 0.05). No changes were noted in ionized calcium, PT and InTEM values.
General anesthesia with concurrent hemodilution and hypothermia induced significant but clinically irrelevant changes in coagulation variables measured at 37 °Celsius. Blood samples from anaesthetized animals can be used for determination of coagulation status in dogs.
Efficacy of Different Fluid Resuscitation Methods on Coagulation Function of Rats with Traumatic Hemorrhagic Shock
2021, Journal of Surgical Research
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The whole MAP was maintained at 40 ± 5 mmHg by bloodletting and blood volume adjustment, and no fluid resuscitation was performed; In HES (n = 20), the THS rats were reinfused with 6% HES (H811486-250g Macklin, CA) until the MAP reached 50, 60, and 70 mmHg, respectively; In AR (n = 20), the rats experienced 3 times of blood loss of AR until the MAP reached 50, 60, and 70 mmHg, respectively; In AR + HES (n = 20), 6% HES and AR (CZ0120-500 mL, Lea gene, CA) was a positive control at a ratio of 1:2 when the MAP reached 50, 60, and 70 mmHg, respectively. The target blood pressure was recovered within 30 min, and blood was taken 4 h later.13,14 Standard coagulation parameters such as activated partial thromboplastin time (APTT), prothrombin time (PT, s), thrombin time (TT, s), international normalized ratio (INR), platelet count (Plt, × 109/L), fibrinogen concentration (Fib, g/L), fibrin degradation products (FDP, g/L), and D-dimer (D-D, μg/L) were obtained from the ACL TOP Hemostasis Testing Systems (Instrumentation Laboratory Company).
Fluid resuscitation is widely used for treating traumatic hemorrhagic shock. We focused on the efficacies of different fluid resuscitation methods on improving coagulation function of traumatic hemorrhagic shock (THS) rats.
Sprague-Dawley rats (n = 100) were randomly divided into 5 groups, namely, Sham group, THS group, acetic acid Ringer's fluid (AR) group, hydroxyethyl starch solution (HES) group, and AR + HES group. A THS rat model was established by left femoral bleeding. The effects of different fluid resuscitation methods on conventional coagulation function parameters, Rotational thromboelastometry parameters, platelet-derived microparticles and endothelial cell–derived microparticles content of the THS rats were detected by ACL TOP system, rotation thromboelastometry, and flow cytometry, respectively.
Using AR and HES alone had no significant effect on the coagulation function of THS rats, but the two in combination reduced the increases of thrombin time, prothrombin time, activated part thrombin time, international normalized ratio, fibrin degradation products, D-dimer and the decreases of platelet count and fibrinogen concentration induced by THS. The CT and CFT were significantly reduced, whereas α and MCF were increased in the THS rats in AR + HES group. The combination of AR and HES reversed the effect of THS on elevating platelet-derived microparticles and endothelial cell–derived microparticle levels. In addition, the coagulation was relatively the optimal in the AR, HES, and AR + HES groups when the mice were resuscitated to a mean arterial pressure of 60 mmHg.
AR combined with HES has a significant protective effect on coagulation function of THS rats when the mean arterial pressure reaches 60 mmHg.
Slow as Compared to Rapid Rewarming After Mild Hypothermia Improves Survival in Experimental Shock
2019, Journal of Surgical Research
Citation Excerpt :
These studies mostly showed a better outcome for the hypothermic animals.16,18-27 To our knowledge, only three studies demonstrated a less desirable outcome after experimental hypothermia.28-30 Using an established rat model of severe hemorrhagic shock,31 the aim of this study was to further investigate if slow rewarming is beneficial over rapid rewarming following mild hypothermia in terms of animal survival, physiological parameters, and organ injury.
Accidental hypothermia following trauma is an independent risk factor for mortality. However, in most experimental studies, hypothermia clearly improves outcome. We hypothesized that slow rewarming is beneficial over rapid rewarming following mild hypothermia in a rodent model of hemorrhagic shock.
We subjected 32 male Wistar rats to severe hemorrhagic shock (25–30 mmHg for 30 min). Rats were assigned to four experimental groups (normothermia, hypothermia, rapid rewarming [RW], and slow RW). During induction of severe shock, all but the normothermia group were cooled to 34°C. After 60 min of shock, rats were resuscitated with Ringer's solution. The two RW groups were rewarmed at differing rates (6°C/h versus 2°C/h).
Slow RW animals exhibit a significantly prolonged survival compared with the rapid RW animals (P < 0.05). Nevertheless, hypothermic animals show a significant survival benefit as compared to all other experimental groups. Whereas seven animals of the hypothermia group survived to the end of the experiment, none of the other animals did (P < 0.001). No significant differences were found regarding acid base status, metabolism, parameters of organ injury, and coagulation.
The results indicate that even slow RW with 2°C/h may be still too fast in the setting of experimental hemorrhage. Too rapid rewarming may result in a loss of the protective effects of hypothermia. As rewarming is ultimately inevitable in patients with trauma, potential effects of rewarming on patient outcome should be further investigated in clinical studies.
Treatment with a neutralising anti-rat interleukin-17 antibody after multiple-trauma reduces lung inflammation
2015, Injury
Citation Excerpt :
The recovered BALF was centrifuged at 3000 rpm for 10 min, and the supernatant was frozen at −80 °C until further analysis. Tissue samples from the middle lobe of the right lung was quickly removed and flushed with 0.01 mol/L cold phosphate buffer solution (PBS, pH 7.4) and immersed for 24 h in 10% buffered formalin for assessment of lung histological change [9–11]. Total mRNA was extracted from homogenised heart tissues by using of the TRIZOL Reagent® (Invitrogen, USA), and then used to synthesise cDNA with an RT Kit (Ferma, USA).
It has been well recognised that a deficit of numbers and function of CD4⁺CD25⁺Foxp3⁺ cells (Treg) is attributed to the development of autoimmune diseases and inflammatory diseases; additionally, IL-17-producing cells (Th17) have a pro-inflammatory role. The balance between Th17 and Treg may be essential for maintaining immune homeostasis and has long been thought as one of the important factors in the development/prevention of autoimmune diseases and inflammatory diseases. In our previous research, we explored that cytokines (IL-17) and the balance of Treg/Th17 had a significant relevance with tissue (lung) inflammation and injury in acute-phase after multiple-trauma.
To more verify whether an imbalance of Treg/Th17 is characteristic of rats suffering from multiple trauma.
Using IL-17 monoclonal antibody (IL-17mAb)-treated multiple-trauma rat, we tested the pathogenic role of IL-17 in the development of multiple-trauma. Rat models were treated respectively with IL-17mAb or rat IgG 2A isotype control or phosphate-buffered solution after model was established. Normal rats only received anaesthesia and cannulation were taken as sham. Rats in each group were killed respectively at the end of 1 h, 4 h, 8 h after injection. Collected serum and lung samples for assessment dynamically of MPO, IL-17, IL-6, and TGF-β-mRNA, and cytokine (IL-17, IL-6, TGF-β) and lung tissue for pulmonary histological analysis.
Neutralisation of IL-17 with anti-IL-17 can decrease serum IL-17 level and the IL-17-mRNA transcript level in lung, and ameliorate tissue inflammatory, defer disease course.
Our data suggest that IL-17 is crucially involved in the pathogenesis of multiple-trauma in rat, IL-17 inhibition might ameliorate the lung inflammation in acute-phase after multiple-trauma.
The imbalance between regulatory and IL-17-secreting CD4<sup>+</sup>T cells in multiple-trauma rat
2013, Injury
Citation Excerpt :
Plasma was obtained after centrifugation and stored at −80 °C for the measurement of cytokines (IL-17, IL-6, IL-2 and TGF-β). The proximal portion of the small intestine was quickly removed and flushed with 0.01 mol l–1 cold phosphate buffer solution (PBS, pH 7.4) and immersed for 24 h in 10% buffered formalin for assessment of intestinal histological change.23–25 Peripheral blood was prepared for flow cytometric analysis (FACScan), as described previously.26
It has been well recognised that a deficit of numbers and function of CD4⁺CD25⁺Foxp3⁺cells (Treg) is attributed to the development of auto-immune diseases, inflammatory diseases, tumour and rejection of transplanted tissue; however, there are controversial data regarding the suppressive effect of Treg cells on the T-cell response in auto-immune diseases. Additionally, interleukin-17 (IL-17)-producing cells (Th17) have a pro-inflammatory role. The balance between Th17 and Treg may be essential for maintaining immune homeostasis and has long been thought as one of the important factors in the development/prevention of auto-immune diseases, inflammatory diseases, tumour and rejection of transplanted tissue, but their role in multiple trauma remains unclear.
This study aims to investigate whether an imbalance of Treg and Th17 effector cells is characteristic of rats suffering from multiple trauma.
Sixty Sprague-Dawley (SD) rats were randomly divided into three groups. The control group (n = 20, group I) no received procedures (normal). The sham group (n = 20, group II) only received anaesthesia, cannulation and observation. The bilateral femoral shaft fractures with haemorrhagic shock groups (n = 20, group III). Rats in groups II and III were killed at the end of 4 h after models were established. Peripheral blood samples were collected for assessment of Treg cells, Th17 cells and cytokines (IL-17, IL-6, IL-2, transforming growth factor beta (TGF-β)) and intestine tissue was collected for intestine histological analysis.
We observed decreased Treg/Th17 ratios in CD4⁺T cells in rats with multiple trauma and a strong inverse correlation with disease activity (intestinal histological scores).
We suggest a role for immune imbalance in the pathogenesis and development of multiple trauma. The alteration of the index of Treg/Th17 cells likely indicates the therapeutic response and progress in the clinic.

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^☆: This study was presented at the 31st International Symposium on Intensive Care and Emergency Medicine in Brussels, Belgium, March 2011.

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Effect of hypothermia on coagulatory function and survival in Sprague–Dawley rats exposed to uncontrolled haemorrhagic shock☆

Abstract

Background

Methods

Results

Conclusion

Introduction

Section snippets

Materials and methods

Laboratory assessments

Discussion

Conclusions

Conflict of interest statement

Acknowledgement

Resuscitation

Transfus Med Rev

Am J Surg

J Thromb Haemost

Resuscitation

Am J Surg

Acta Anaesthesiol Taiwan

Metabolism

Volume replacement and microhemodynamic changes in polytrauma

Langenbecks Arch Surg

Major abdominal vascular trauma – a unified approach

J Trauma

Is hypothermia simply a marker of shock and injury severity or an independent risk factor for mortality in trauma patients? Analysis of a large national trauma registry

J Trauma

Is hypothermia in the victim of major trauma protective or harmful?

Ann Surg

Advanced Trauma Life Support® for Doctors

Advanced Trauma Life Support^® for Doctors