Nonhuman Primate (Rhesus Macaque) Models of Severe Pressure-Targeted Hemorrhagic and Polytraumatic Hemorrhagic Shock.

BACKGROUND: We endeavored to develop clinically translatable nonhuman primate (NHP) models of severe polytraumatic hemorrhagic shock. METHODS: NHPs were randomized into five severe pressure-targeted hemorrhagic shock (PTHS) ±â€Šadditional injuries scenarios: 30-min PTHS (PTHS-30), 60-min PTHS (PTHS-60), PTHS-60 + soft tissue injury (PTHS-60+ST), PTHS-60+ST + femur fracture (PTHS-60+ST+FF), and decompensated PTHS+ST+FF (PTHS-D). Physiologic parameters were recorded and blood samples collected at five time points with animal observation through T = 24 h. Results presented as mean ±â€ŠSEM; statistics: log transformation followed by two-way ANOVA with Bonferroni multiple comparisons, Wilcoxon nonparametric test for comparisons, and the Friedmans' one-way ANOVA; significance: P < 0.05. RESULTS: Percent blood loss was 40% ±â€Š2, 59% ±â€Š3, 52% ±â€Š3, 49% ±â€Š2, and 54% ±â€Š2 for PTHS-30, PTHS-60, PTHS-60+ST, PTHS-60+ST+FF, and PTHS-D, respectively. All animals survived to T = 24 h except one in each of the PTHS-60 and PTHS-60+ST+FF groups and seven in the PTHS-D group. Physiologic, coagulation, and inflammatory parameters demonstrated increasing derangements with increasing model severity. CONCLUSION: NHPs exhibit a high degree of resilience to hemorrhagic shock and polytrauma as evidenced by moderate perturbations in metabolic, coagulation, and immunologic outcomes with up to 60 min of profound hypotension regardless of injury pattern. Extending the duration of PTHS to the point of decompensation in combination with polytraumatic injury, evoked derangements consistent with those observed in severely injured trauma patients which would require ICU care. Thus, we have successfully established a clinically translatable NHP trauma model for use in testing therapeutic interventions to trauma.

Inflammatory Profile in Response to Uncontrolled Hemorrhage in a Non-Human Primate (Rhesus Macaque) Model.

BACKGROUND: Uncontrolled hemorrhage (UH), the leading cause of potentially survivable combat-related death, elicits a deleterious inflammatory response. Our group previously reported an increased secretion of pro-inflammatory cytokines in a novel non-human primate model of UH; however, to better understand the molecular profile of the inflammatory response to UH, we performed a comprehensive evaluation of inflammation at the proteomic and transcriptomic level. METHODS: Anesthetized rhesus macaques (n = 8) underwent UH by 60% left lobe hepatectomy T = 0 min. At T = 5 min, animals received 11 mL of 5% albumin followed by normal saline infusion to a total resuscitation volume of 20 mL/kg by T = 120 min. Blood (T = 0, 5, 20, 120, 480 min) was collected for qPCR and multiplex cytokine quantification. Results from each non-human primate (NHP) per time-point are shown. Statistical analysis by one-way ANOVA with repeated measures, P <0.05 was considered significant. RESULTS: Luminex analysis in serum revealed significant up-regulation compared with baseline of 8 cytokines/chemokines starting T = 120 min postinjury and significant down-regulation of 4 cytokines/chemokines as early as T = 20 min postinjury. Gene expression analysis in white blood cells uncovered 10 genes that were up-regulated greater than 3-fold compared with baseline and 29 genes that were down-regulated greater than 3-fold. CONCLUSION: The present study confirms the presence of systemic inflammation after UH at the proteomic and transcriptomic level providing insight into the inflammatory mediators that are involved as well as their kinetics following UH. The data demonstrates that NHP hemorrhage models may be suitable for evaluating therapeutics to control inflammation following hemorrhage.

Severe Hemorrhagic Shock Induces Acute Activation and Expansion of IL-8+/IL-10+ Neutrophils with Enhanced Oxidative Reactivity in Non-Human Primates.

BACKGROUND: Neutrophilic inflammation is a mediator of morbidity and mortality in response to hemorrhagic shock. Although injury-induced neutrophil margination has long been observed, the nature of neutrophils' role in the "second hit" paradigm remains to be fully elucidated. We sought to extensively characterize neutrophil phenotype and functionality in response to severe hemorrhage in non-human primates (NHPs). METHODS: NHPs (n = 8) were subjected to severe hemorrhagic shock and resuscitation. Blood was obtained at baseline (T = 0 min), end of shock (T = 60 min), end of resuscitation (T = 180 min), T = 360 min, and 24 h (T = 1440 min). Neutrophils were quantified by complete blood count and flow cytometry. IL-8 and IL-10 production was determined by intracellular flow cytometry. Oxidation of dihydrorhodamine-123 (DHR-123) was used to determine neutrophil oxidative bursts (untreated), priming (+fMLP), and burst capacity (+PMA/ionomycin) via microplate reader ex vivo. Data are reported as mean ±â€ŠSEM; statistical significance was measured using repeated measures ANOVA with Bonferroni adjustment. P < 0.05 is considered significant. RESULTS: CD45CD11bCD16 neutrophils doubled postinjury (P < 0.0001); this was due to activated IL-8/IL-10 neutrophils that increased in frequency in relation to resting IL-8IL-10 cells. At 24 h, the proportions of activated to resting neutrophils returned to baseline levels. Resuscitative measures initially decreased neutrophil oxidative output; however, oxidative bursts, priming, and burst capacity were significantly increased at 24 h (P < 0.0025, 0.0124, and 0.0118, respectively). CONCLUSION: These results demonstrate an acute expansion and phenotypic activation of circulating neutrophils postinjury followed by a return to homeostatic proportions within 24 h; paradoxically, phenotypically "resting" neutrophils at 24 h have significantly higher oxidative potential, predisposing for exaggerated inflammatory responses. These data are consistent with clinical literature and provide important functional insight into neutrophil-mediated shock pathology.