Impact of Immunopathy and Coagulopathy on Multi-Organ Failure and Mortality in a Lethal Porcine Model of Controlled and Uncontrolled Hemorrhage

Abstract

Uncontrolled hemorrhage is a major preventable cause of death in patients with trauma. However, the majority of large animal models of hemorrhage have utilized controlled hemorrhage rather than uncontrolled hemorrhage to investigate the impact of immunopathy and coagulopathy on multi-organ failure (MOF) and mortality. This study evaluates these alterations in a severe porcine controlled and uncontrolled hemorrhagic shock (HS) model. Anesthetized female swine underwent controlled hemorrhage and uncontrolled hemorrhage by partial splenic resection followed with or without lactated Ringer solution (LR) or Voluven^® resuscitation. Swine were surveyed 6 h after completion of splenic hemorrhage or until death. Blood chemistry, physiologic variables, systemic and tissue levels of complement proteins and cytokines, coagulation parameters, organ function, and damage were recorded and assessed. HS resulted in systemic and local complement activation, cytokine release, hypocoagulopathy, metabolic acidosis, MOF, and no animal survival. Resuscitation with LR and Voluven^® after HS improved hemodynamic parameters (MAP and SI), metabolic acidosis, hyperkalemia, and survival but resulted in increased complement activation and worse coagulopathy. Compared with the LR group, the animals with hemorrhagic shock treated with Voluven^® had worse dilutional anemia, coagulopathy, renal and hepatic dysfunction, increased myocardial complement activation and renal damage, and decreased survival rate. Hemorrhagic shock triggers early immunopathy and coagulopathy and appears associated with MOF and death. This study indicates that immunopathy and coagulopathy are therapeutic targets that may be addressed with a high-impact adjunctive treatment to conventional resuscitation.

Keywords: MOF; coagulopathy; damage control resuscitation; immunopathy; mortality; swine; uncontrolled hemorrhagic shock.

Figure 1

Survival in a porcine model of hemorrhagic shock and fluid resuscitation. Percent survival (A) and survival time are shown. * p < 0.05 vs. sham, † p < 0.05 vs. H, and ‡ vs. H+LR, p < 0.05 using a log-rank test (A) and one-way ANOVA followed by Bonferroni posttests (B), respectively.

Figure 2

Coagulation disturbances after hemorrhagic and fluid resuscitation. Whole blood shear elastic modulus (G) parameter (A) and maximum amplitude (MA, (B)) were measured by thromboelastography. Fibrinogen concentrations (C), plasma prothrombin time (PT, (D)), activated partial thromboplastin time (aPTT, (E)), and were assessed using BCSTM XP system. * H+Voliuven/H+LR vs. H and † H+Voluven vs. H+LR, p < 0.05 using two-way ANOVA followed by Bonferroni posttests. # vs. BL, p < 0.05, using unpaired t-test (two-tailed). Data are presented as mean ± SEM. BL, baseline; PT, extrinsic pathway; aPTT, contact pathway.

Figure 3

Systemic inflammatory immune responses after hemorrhagic shock and fluid resuscitation. Serum hemolytic terminal complement activation was measured with CH50 assay (A), and blood levels of C3a (B), TNαF (C), IL-6 (D), and IL-8 (E) were assessed with ELISA. * vs. sham, † vs. H, and ‡ vs. H+LR, p < 0.05 using two-way ANOVA followed by Bonferroni posttests. Data are presented as mean ± SEM.

Figure 4

Multiple organ dysfunctions post-hemorrhagic shock and post-fluid resuscitation. Blood creatinine (A), aspartate aminotransferase (AST, (B)), muscle myocardium isoenzyme B (MMB, (C)), and creatine kinase (CK, (D)) were determined with Siemens Dimension Xpand Plus Chemistry 6 Analyzer. * vs. sham, † vs. H, ‡ vs. H+LR, p < 0.05 using two-way ANOVA followed by Bonferroni posttests. Data are presented as mean ± SEM.

Figure 5

Myocardial inflammatory responses after hemorrhagic shock and fluid resuscitation. Immunostaining and semiquantitative fluorescent intensity of C4d (A,B), C3 (C,D), C5 (E,F), C5b-9 (G,H), and IL-6 (I,J) in heart tissues were evaluated by immunohistochemistry. Scale bars = 50 μm. * vs. sham, † vs. H, and ‡ vs. H+LR, p < 0.05 using one-way ANOVA followed by Bonferroni posttests. Data are presented as mean ± SEM. Vol, Voluven. DAPI = 4′, 6′-diamidino-2-phenylindole.

Figure 6

Pulmonary/intestinal inflammatory responses after hemorrhagic shock and fluid resuscitation. Immunostaining and semiquantitative fluorescent intensities of C3 (A,B) and C5b-9 (C,D) in lungs and C3 (E,F) and IL-6 (G,H) in jejunum were evaluated by immunohistochemistry. Scale bars = 50 μm. * vs. sham, † vs. H, p < 0.05 using one-way ANOVA followed by Bonferroni posttests. Data are presented as mean ± SEM.

Figure 7

Histopathological changes in end organs after hemorrhagic shock and fluid resuscitation. Histopathological (H&E stain) photos and semiquantitative evaluations of the heart ((A,B), magnification = ×200, scale bars = 200 µm), lung ((C,D), magnification = ×200, scale bars = 200 µm), jejunum ((E,F), magnification = ×100, scale bars = 500 µm), kidney ((G,H), magnification = ×400, scale bars = 100 µm), and liver ((I,J), magnification = ×400, scale bars = 100 µm). Myocarditis is marked with white arrows (A), and the insert in panel (A) magnifies the region of the indicated box to show the mononuclear cells and polymorphic nuclear cells in the corresponding inflammatory infiltration foci. Data are presented as mean ± SEM. * vs. sham, † vs. H, and ‡ vs. H+LR, p < 0.05, using one-way ANOVA followed by Bonferroni posttests.

Figure 8

Correlation between circulating fibrinogen/CH50 and end organ injury after hemorrhage. Correlation analysis between fibrinogen/CH50 and liver i (A), kidney (B) and lung (C) organ injury was performed by using Spearman’s rank correlation. Data are shown as individual values with the correlation coefficient (rs).

Figure 9

Scheme of the experimental design.