Sepsis, the syndrome of microbial infection complicated by systemic inflammation, is a major public health problem associated with significant morbidity and mortality (1). Septic shock is frequently complicated by the syndrome of disseminated intravascular coagulation (DIC) because of a massive activation of the coagulation system. The coagulation and fibrinolytic systems appear to be influenced by the septic process largely independently, leading to a procoagulant imbalance between these systems. The resulting effects predispose to a procoagulant state with widespread fibrin deposition and increased levels of plasminogen activator inhibitor-1 (PAI-1), leading to inadequate fibrinolysis (2). Many studies imply that DIC is an important mediator in the multiple organ failure syndrome development (3). Moreover, an increasing DIC score is associated with worse outcomes and mortality in patients with serious infections (4).
It has become apparent that the prevalence of obesity has been steadily increasing worldwide (5). Approximately 30% of patients admitted to the intensive care unit are obese (body mass index, ≥30 kg/m2) and 7% are morbidly obese (body mass index, ≥40 kg/m2) (6). Obesity compromises the body's ability to adapt to stress or critical illness and is independently associated with increased morbidity and all-cause mortality (7). Abnormalities in coagulation and hemostasis represent a well-known link between obesity and thrombosis. Adipose tissue may release mediators that induce a chronic inflammatory state and alterations in coagulation, which contribute to a prothrombotic state (8). Several studies have shown that obese patients have higher plasma concentrations of all prothrombotic factors (fibrinogen, von Willebrand factor [vWF], and factor VII) compared with nonobese controls, with a positive association with central fat (9). Moreover, data indicate that pediatric obesity is associated with a hypofibrinolytic state, which might contribute to the increased thrombotic risk associated with this condition (10). In fact, plasma concentrations of PAI-1 have been shown to be higher in obese patients compared with nonobese controls and to be directly correlated with visceral fat (9).
Although obesity has been demonstrated to be a hypercoagulable and hypofibrinolytic state, its impact on endotoxin-induced DIC has never been studied. In this study, we aimed to determine if obesity impairs DIC in an acute endotoxic shock using minipigs.
MATERIALS AND METHODS
This is an ancillary work of a first study on the impact of obesity on macrocirculation, microcirculation, oxygenation, and metabolic status (11).
The Institutional Review Board for Animal Research approved the study (protocol CEEA Nord Pas-de-Calais 132012); care and handling of the animals were in accordance with National Institutes of Health guidelines.
Twenty adult male Yucatan miniature pigs (6 months old) were studied. Animals were submitted to a nutritional protocol according to Val-Laillet et al. (12). Animals were fed for 10 weeks. Two regimens were used: a standard diet (SD) designed to maintain a lean phenotype and a Western diet to obtain an obese phenotype. The SD (2,463 kcal/kg) had the formula and ration (41.5 g/kg 0.75 of live weight once a day at 9:00 AM). The Western diet (3,473 kcal/kg) was enriched with carbohydrates and lipids and offered ad libitum (one ration offered at 9:00 AM and calculated to exceed the daily consumption of the animals). For the experiment, animals were premedicated with intramuscular injection of ketamine (Ketamine 1000, 10 mg/kg of body weight; Virbac, Carros, France) and xylazine (Sedaxylan, 2.5 mg/kg of body weight; CEVA Santé Animale, Libourne, France). Then we used a 4% concentration of isoflurane (Aerrane; Baxter, Maurepas, France) for the intubation process once intubation realized the maintenance of anesthesia was performed with a continuous infusion of midazolam (Hypnovel, 1 – 2 mg/kg body weight per h; Roche, Meylan, France) for the whole experiment. Animals were connected to a constant-volume respirator for mechanical ventilation (Osiris 2; Taema, Antony, France). We chose to maintain ventilation similar in all animals without changing FiO2 or tidal volume during the experiment. Muscle relaxation was obtained by a continuous infusion of cisatracurium besylate (Nimbex, 2 mg/kg body weight per h; Hospira, Meudon la Forêt, France). Analgesia was achieved by a subcutaneous injection of buprenorphine (Vetergesic, 0.1 mg/kg body weight; Sogeval, Laval, France). Subcutaneous administration of buprenorphine in our animals is known to permit analgesia for 6 to 8 h and avoid intravenous bolus administration. After dissection of neck vessels, catheters were inserted in the right carotid artery for continuous blood pressure monitoring and blood sampling. An esophageal temperature probe measured the core temperature.
The study was carried out as described in Figure 1. During preparation period, animals received 500 mL 0.9% NaCl to prevent hypovolemia. When all preparations were completed, a 30-min period was allowed to stabilize the measured variables. Biologic measurements were taken during a 5-h period: at baseline after a stabilization period (T0) and at 30 (T30), 60 (T60), 90 (T90), 150 (T150), and 300 (T300) min. Four groups of five animals were studied: obese control, lean control, obese lipopolysaccharide (LPS), and lean LPS. After anesthesia, catheterization, and baseline collection (T0), control groups received an intravenous infusion of 50 mL of 0.9% NaCl for a 30-min period. Lipopolysaccharide groups were administered Escherichia coli LPS (serotype 055:B5; Sigma Chemical Co, St. Louis, Mo). The endotoxin was diluted in 50 mL of sterile 0.9% NaCl and infused for a 30-min period intravenously. All the animals received the same dose of endotoxin. This dose was calculated at a rate of 5 μg/kg per min according to the mean weight of lean pigs. Our model was a hypodynamic shock with a low level of resuscitation. The only resuscitation end point was mean arterial pressure (MAP). In the two groups submitted to endotoxin challenge, animals received 100 mL/h (from T30 to T300) of 0.9% NaCl. If MAP felt below 50 mmHg, a 50-mL infusion of 0.9% NaCl was given as rescue therapy every 15 min. Bolus infusions were performed to maintain MAP greater than 50 mmHg. We chose this model with a low level of resuscitation to avoid hemodilution. At the end of the study period, all animals were sacrificed with T61 administration (T61, 0.3 mL/kg of body weight; Intervet International GmbH, Köln, Germany).
Estimation of body fat percentage
Three body circumferences and leptin levels were measured at baseline according to Witczak et al. (13). Circumference measurements were obtained at the following three positions: ventral neck, midabdomen, and widest abdominal girth. Leptin levels were measured by radioimmunoassay (Multi-Species Leptin RIA Kit; Merck Millipore, Darmstadt, Germany).
Coagulation and fibrinolytic parameters: blood samples were collected from the arterial catheter in sterile tubes. No anticoagulant treatment was administered to the animals during the experiment. Leukocyte, hemoglobin, and platelet counts were obtained on EDTA anticoagulated blood. For coagulation assays, blood (four parts) was collected in tubes containing 3.8% sodium citrate (one part). Prothrombin time (PT) and fibrinogen levels were rapidly measured by standard procedures. Immunoassay methods were used to determine quantitative thrombin-antithrombin (TAT) complexes (Enzygnost TAT micro; Siemens, Munich, Germany), tissue-type plasminogen activator (t-PA) (Active porcine tPA functional assay ELISA kit; Molecular Innovations, Novi, USA), and PAI-1 (Active porcine PAI-1 functional assay ELISA kit; Molecular Innovations). Fibrin monomer (Liatest FM; Stago, Asnières, France) and D-dimer (Innovance DDI; Siemens, Marburg, Germany) were performed by immunoturbidimetric assay. Quantitative antithrombin (AT) activity was determined by chromogenic assay (Berichrom ATIII; Siemens, Marburg, Germany). Endothelial (vWF) and inflammation (tumor necrosis factor-α [TNF-α], interleukin-6 [IL-6], and IL-10) parameters were measured in serum as described in a previous study (11).
At the end of the experiment and immediately after euthanasia, kidneys were withdrawn for histologic study. Histology was performed in a blinded fashion in three animals of each group. After macroscopic examination, samples from each kidney were fixed (4% paraformaldehyde < 18 h). The samples were embedded in paraffin and sectioned (7 μm width). After deparaffinization and rehydration, sections were stained with Masson trichrome and evaluated in light microscopy.
We established a semiquantitative score for histologic abnormalities defined as the ratio: Score (%) = (number of glomerules damaged)/(number of glomerules examined) and (number of capillary sections thrombosed)/(number of capillary sections examined per damaged glomerules). At least 50 glomeruli were observed in each sample. The pathologist was blinded to the groups examined.
Statistical analysis was performed with GraphPad Prism 6 software. As the distribution was not normal (Shapiro-Wilk test), quantitative data were expressed using median and interquartile range. Considering the differences between groups for some parameters at baseline, values are expressed as a percentage of the first value. For multiple intergroup testing, we used the Kruskal-Wallis test with the Dunn multiple comparison test and the Mann-Whitney U test. Intragroup comparisons were performed using the Friedman test with the Dunn multiple comparison test. A P ≤ 0.05 was considered significant.
Comparison between obese and lean pigs at baseline
The estimation of body fat percentage of the experimental groups has already been reported (11). Coagulation and fibrinolytic data at baseline are illustrated in Table 1. Platelet count, fibrinogen, and vWF were significantly higher, whereas PT was significantly lower in obese pigs compared with lean pigs. Tumor necrosis factor-α, IL-6, and IL-10 levels (data not shown) were similar between obese and lean pigs at baseline.
Comparison between obese and lean control groups
All biological parameters remained stable throughout the study. Because there were no significant differences observed between the control groups, only the lean control group is illustrated in the figures.
Comparison between obese and lean LPS groups
We observed hypodynamic shock with a significant decrease of arterial pressure in all animals submitted to the endotoxin challenge. As already published, obese pigs developed a more severe hemodynamic failure with a more pronounced microcirculatory dysfunction (11).
Coagulation and fibrinolytic parameters
Variations in leukocyte, platelet count, PT, and fibrinogen are illustrated in Figure 2. In both groups receiving endotoxin, because of the endotoxin-induced capillary leak, hemoglobin count increased by 25% at 30 min and then remained stable until 300 min (data not shown). White blood cell counts rapidly decreased (−80% at 30 min) and remained very low throughout the entire study. A dramatic procoagulant response was demonstrated (Fig. 2). We observed a significant decrease of PT in both LPS groups. In the same way, circulating platelets promptly declined in obese LPS groups (443 [420 – 484] G/L at 0 min vs. 141 [65 – 161] G/L at 300 min; P < 0.001) and in the lean LPS group (434 [329 – 481] at 0 min vs. 161 [127 – 177] at 300 min; P < 0.001). The pronounced activation of the coagulation system was demonstrated by a decline in circulating fibrinogen in both LPS groups. A more severe decrease (−82%) in fibrinogen levels (5 [3.9 – 5.9] g/L at 0 min vs. 0.9 [0.7 – 2.7] g/L at 300 min; P < 0.05) was observed in the obese LPS group compared with the lean LPS group (−38%). Alterations in PT, platelet count, and fibrinogen were earlier and more pronounced in the obese LPS group.
Variations in TAT complexes, AT activity, fibrin monomer, D-dimer, t-PA, and PAI-1 are illustrated in Figure 3. Thrombin-antithrombin complex concentrations started to increase at 60 min to achieve a maximum level of 32 times the baseline value at 150 min in the obese LPS group and to achieve a maximum level of 18 times the baseline value at 300 min in the lean LPS group (Fig. 3). The increase of TAT complexes was earlier and significantly higher in the obese LPS group. These high concentrations of TAT complexes were associated with a significant decrease in AT activity in the obese LPS group. In the lean LPS group, we observed a nonsignificant decrease in AT activity. At 300 min, AT levels were significantly lower in the obese LPS group (70% [68% – 80%]) compared with the lean LPS group (85% [80% – 87%]; P = 0.03) and the lean control group (101% [93% – 114%]; P = 0.01). Next, we observed an increase in fibrin-related markers (fibrin monomer and D-dimer). The increase in fibrin monomer started earlier and was significantly higher at 90 min in the obese LPS group (124 [100 – 178] μg/mL) compared with the lean LPS group (46 [27 – 86] μg/mL; P = 0.02). Unfortunately, we could not interpret the results at 150 and 300 min in both LPS groups because some values were more than 200 μg/mL, the upper limit of measurement, despite dilutions. Nevertheless, the increase in fibrin turnover was highlighted by a significant increase in D-dimer in both LPS groups (P < 0.01 at 150 min compared with baseline). Activation of the fibrinolytic system, as indicated by increasing levels of t-PA, started within 30 min after the endotoxin challenge. The early increase in t-PA concentrations peaked at 90 min, whereafter t-PA decreased at 150 min until the end of the experiment. The peak concentration was significantly lower in the obese LPS group (5 [2 – 9] ng/mL) than in the lean LPS group (10 [8 – 17] ng/mL; P = 0.04). The release of PAI-1 into the circulation started at 150 min and peaked at the end of the study. The increase in PAI-1 concentrations was slightly higher at 300 min in the obese LPS group (481 [365 – 617] ng/mL) compared with the lean LPS group (355 [209 – 660] ng/mL; P = 0.66). All these changes were consistent with the classical endotoxin-induced sequence of coagulation and fibrinolysis activation.
The kidneys appeared macroscopically normal in the three studied piglets in the control group. In the two other groups, the organs appeared macroscopically enlarged and swollen.
In kidney sections from the lean control pigs, no lesion was observed. Nevertheless, fibrin thrombi were observed in glomerular capillaries of two obese control pigs (11.42% and 3.8% of glomeruli damaged, respectively). In the LPS groups, important lesions were observed in all kidney sections. Glomeruli showed signs of edema uniformly, and fibrin thrombi were mainly observed in glomerular capillaries. Results of the semiquantitative score are presented in Table 2. This semiquantitative score was higher in the obese LPS group and consistent with an increasing amount of microthrombosis (Fig. 4).
This was the first time that the impact of obesity was investigated in a large animal model of endotoxin-induced DIC. The main goal of this study was to determine whether obesity could impair DIC in an acute endotoxic shock using minipigs. The main result of our study was consistent with this hypothesis. We observed a prothrombotic state in obese pigs at baseline. Moreover, obese pigs developed a more severe DIC with a more pronounced procoagulant response. This finding might explain the more severe hemodynamic failure (11) and the increase in organ microthrombosis and tissue damage, contributing to the development of multiple organ failure.
Pigs were chosen as a clinically relevant species, resembling humans in coagulation reactions (14). Our model is a hypodynamic shock with a low resuscitation level to avoid hemodilution (hemoglobin count remained stable during the experiment). To have comparable groups, all animals were infused with the same endotoxin dose. So we determined a fixed dose by using the usual formula (LPS infusion of 5 μg/kg per min for a 30-min period) with the mean weight of lean pigs.
Activation of the coagulant system plays a pivotal role in the pathogenesis of sepsis and in the development of multiple organ failure. In our model, administration of endotoxin resulted in the same sequence with activation of coagulation and fibrinolysis and subsequent increase in PAI-1 concentrations. Experimental and human studies have documented that the activation of the coagulation cascade is triggered by tissue factor activation. Tissue factor complexed with factor VII induces prothrombin and thrombin generation. The coagulation activation was reflected by the increase in TAT complexes and the decrease in platelets, PT, and fibrinogen concentrations. The early increase in t-PA was consistent with an activation of fibrinolysis. This early increase preceded the detection of coagulation markers, reflected by increased TAT complex concentrations and decreased AT activity. It was followed and counteracted by a late increase in PAI-1 concentrations. These data are consistent with animal and human studies (15–17), suggesting that the increase in plasma PAI-1 concentrations was responsible for the attenuation of fibrinolysis. In our model, we observed a more important decrease in platelets, PT, and fibrinogen concentrations in the obese LPS group. The coagulation activation in obese pigs was reflected by a more pronounced decrease in AT levels. As a result, significantly higher plasma levels of TAT and fibrin monomer were found. This procoagulant response occurred earlier in the obese LPS group and was responsible for a marked increase in D-dimer. Concerning fibrinolysis, the t-PA increase was significantly less important and the increase in PAI-1 was slightly higher in obese pigs. The more pronounced imbalance between coagulation and fibrinolysis in obese pigs was associated with tissue damage prevailing in the kidney. Two explanations could be advanced in our model to explain this more important imbalance in obese pigs.
First, obese pigs presented a prothrombotic phenotype that could promote the endotoxin-induced DIC. In fact, at baseline, platelet count, fibrinogen, and vWF were significantly higher, whereas PT was significantly lower compared with lean pigs. These data are consistent with animal (18) and human studies (19, 20). Obese patients have higher plasma concentrations of all prothrombotic factors (9), and obesity is associated with a procoagulant state and endothelial dysfunction (21).
Second, several studies on the effects of endotoxin or TNF infusion in human volunteers (17, 22) and in animal models of septic shock (15, 16) indicated that these agents induce an imbalance between coagulation and fibrinolysis, resulting in a procoagulant state. Therefore, the more important proinflammatory response (TNF-α and IL-6) observed in obese pigs could play a key role in the more severe endotoxin-induced DIC. In fact, it has been proposed that the secretion of IL-6 by adipose tissue, combined with the actions of adipose tissue–expressed TNF-α in obesity, could underlie the association of insulin resistance with endothelial dysfunction, coagulopathy, and coronary heart disease (9). Tumor necrosis factor-α plays an important part in the early activation of the hemostatic mechanism and in the pathogenesis of DIC (23). Moreover, a TNF-α inhibitor can act as a protective drug in LPS-induced DIC in a dose-dependent manner (24). Plasma IL-6 is higher in patients with DIC than in those without DIC. Some data suggest that increases in IL-6 might give rise to hypercoagulable and hypofibrinolytic states, be a cause of DIC, and be related to prognosis and organ failure (25). Finally, immunoglobulins in the LPS-induced DIC model could significantly decrease plasma levels of TNF-α and IL-6 and improve hemostatic abnormality (26).
Our model presented some limits. We used the intravenous route for sepsis induction, whereas patients are still infected by the natural route. The temporal evolution of the aggression was imposed, whereas the pathological process of patients follows an individual natural progression. Finally, the endotoxin challenge was responsible for a more explosive proinflammatory response than in a septic shock. These limits, without questioning the validity of our pathophysiological model, could impair the comparability with DIC observed in human septic shock. Finally, the nutritional protocol represents an acute weight gain rather than chronic obesity. The question arises whether the model simulates true obesity-induced prothrombotic and inflammatory states or simply acute hypermetabolic response caused by a short-term hypercaloric boost. Moreover, there is increasing evidence that low-grade obesity may in fact be protective against sepsis-induced mortality (27). Based on the design of this study, the relationship between different obesity grading and hemostasis cannot be established.
In our porcine model, obese animals developed a more severe endotoxin-induced DIC that could be in relation to a prothrombotic state and an exaggerated proinflammatory response.
We thank M.H. Gevaert and R.M. Siminski (Laboratory of Histology, Universite de Lille, Lille, France) for technical support.
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Keywords:© 2015 by the Shock Society
Obesity; endotoxic shock; disseminated intravascular coagulation; fibrinolysis; inflammation; DIC; disseminated intravascular coagulation; IL; interleukin; TNF; tumor necrosis factor; vWF; von Willebrand factor; PT; prothrombin time; TAT; thrombin-antithrombin; PAI; plasminogen activator inhibitor; t-PA; tissue-type plasminogen activator; AT; antithrombin; MAP; mean arterial pressure; LPS; lipopolysaccharide; G/L; Giga per liter