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Abstract: Over 30% of trauma-related deaths are from massive hemorrhage with 90% of potentially preventable battlefield deaths occurring pre-hospital. Immediate resuscitation with whole blood is ideal but often limited to hospital and medical treatment facilities. Shelf-stable hemoglobin-based oxygen carriers (HBOCs) are designed to relieve the hypoperfusion and hypoxia of shock during the critical pre-hospital period. A new PEGylated human HBOC product, VS-101, with high oxygen affinity and hyperoncotic pressure, has been designed for hypovolemic resuscitation protocols at the point of injury. Thirty-six Sprague-Dawley rats underwent a severe, pressure-guided 45% total blood volume (TBV) hemorrhage. Shocked animals were randomly assigned to receive 20% TBV Lactated Ringers' (LRS), Plasma, Blood, or VS-101. Cardiovascular parameters, arterial blood gases, 8-hr survival, arteriolar diameters, and oxygenation of the spinotrapezius microvasculature were measured. Even compared with whole blood, VS-101 was the only group with survivors (67%) at the end of the 8-hr observation period. Mean survival times were 49, 95, 197, and 426 min for LRS, Plasma, Blood, and VS-101 (p < 0.05 vs all), respectively. VS-101 produced the highest spinotrapezius interstitial oxygenation and recovery of MAP with no evidence of hypertension or arteriolar vasoconstriction. Hypovolemic resuscitation with VS-101 was effective in stabilizing hemorrhagic shock in a simulated pre-hospital setting, which was associated with its combination of high oncotic pressure and oxygen carrying constituent. The lack of arteriolar vasoconstriction and hypertension suggests VS-101 is poised to pass critical safety and efficacy checkpoints for treatment of severe hemorrhage., (Copyright © 2025 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the Shock Society.)

Abstract: Blood products are the current standard for resuscitation of hemorrhagic shock. However, logistical constraints of perishable blood limit availability and prehospital use, meaning alternatives that provide blood-like responses remain an area of active investigation and development. VS-101 is a new PEGylated human hemoglobin-based oxygen carrier that avoids the logistical hurdles of traditional blood transfusion. This study sought to determine the safety and ability of VS -101 to maintain circulatory function and capillary oxygen delivery in a severe (50%) exchange transfusion (ET) model. Anesthetized, male Sprague Dawley rats were prepared for cardiovascular monitoring and phosphorescence quenching microscopy of interstitial fluid oxygen tension (P ISFo2 ) in the spinotrapezius muscle. Fifty-percent isovolemic ET of estimated total blood volume with either lactated Ringer's solution (LRS, n = 8) or VS -101 (n = 8) at 1 mL/kg/min was performed, and animals were observed for 240 min. VS -101 maintained P ISFo2 at baseline with a transient 18 ± 4 mm Hg decrease ( P < 0.05) in mean arterial pressure (MAP). In contrast, ET with LRS decreased P ISFo2 by approximately 50% ( P < 0.05) and MAP by 74 ± 10 mm Hg ( P < 0.05). All VS -101 animals survived 240 min, the experimental endpoint, while 100% of LRS animals expired by 142 min. VS -101 animals maintained normal tissue oxygenation through 210 min, decreasing by 25% ( P < 0.05 vs. baseline) thereafter, likely from VS -101 vascular clearance. No arteriolar vasoconstriction was observed following VS -101 treatment. In this model of severe ET, VS -101 effectively maintained blood pressure, perfusion, and P ISFo2 with no vasoconstrictive effects. Further elucidation of these beneficial resuscitation effects of VS -101 is warranted to support future clinical trials., Competing Interests: The study was funded by Vivosang, Inc., manufacturer of VS-101 and patent holder of another PEG-Hb product, MP4. AM, KV, and WMS are employed by Vivosang, Inc. Manuscript preparation was funded and executed by Song Biotechnologies, LLC. WHN and BKS are employees of Song Biotechnologies. FRS reports no conflicts of interest., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the Shock Society.)

Background: Hemorrhage is the leading cause of preventable, traumatic death. Currently, prehospital resuscitation fluids provide preload but not oxygen-carrying capacity-a critical blood function that mitigates microvascular ischemia and tissue hypoxia during hemorrhagic shock. Solutions containing polymerized hemoglobin have been associated with vasoactive and hypertensive events. A novel hemoglobin-based oxygen carrier, modified with PEGylation and CO moieties (PEG-COHb), may overcome these limitations., Objectives: To evaluate the systemic and microcirculatory effects of PEG-COHb as compared with the 6% hetastarch in a rat model of hemorrhagic shock., Methods: Male Sprague Dawley rats (N = 20) were subjected to severe, controlled, hemorrhagic shock. Animals were randomized to 20% estimated blood-volume resuscitation with either 6% hetastarch or PEG-COHb. Continuous, invasive, cardiovascular measurements, and arterial blood gases were measured. Microcirculatory measurements of interstitial oxygenation (PISFO2) and vasoactivity helped model oxygen delivery in the spinotrapezius muscle using intravital and phosphorescence quenching microscopy., Results: Hemorrhage reduced mean arterial pressure (MAP), arteriolar diameter, and PISFO2, and increased lactate 10-fold in both groups. Resuscitation with both PEG-COHb and hetastarch improved cardiovascular parameters. However, PEG-COHb treatment resulted in higher MAP (P < 0.001), improved PISFO2 (14 [PEG-COHb] vs. 5 [hetastarch] mmHg; P < 0.0001), lower lactate post-resuscitation (P < 0.01), and extended survival from 90 to 142 min (P < 0.001) as compared with the hetastarch group., Conclusions: PEG-COHb improved MAP PISFO2, lactate, and survival time as compared with 6% hetastarch resuscitation. Importantly, hypertension and vasoactivity were not detected in response to PEG-COHb resuscitation supporting further investigation of this resuscitation strategy.

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) ± additional 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 ± SEM; 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% ± 2, 59% ± 3, 52% ± 3, 49% ± 2, and 54% ± 2 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.

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.

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 ± SEM; 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.

Hemorrhage is the leading cause of potentially survivable trauma mortality, necessitating the development of improved therapeutic interventions. The objective of this study was to develop and characterize a reproducible clinically translatable nonhuman primate model of uncontrolled severe hemorrhage. Such a model is required to facilitate the development and meaningful evaluation of human-derived therapeutics. In Rhesus macaques, a laparoscopic left-lobe hepatectomy of 25% (n = 2), 50% (n = 4), or 60% (n = 6) was performed at T = 0 min, with no attempt at hemorrhage control until T = 120 min. A constant-rate infusion of normal saline was administered between T = 15 and 120 min to a total volume of 20 mL/kg. At T = 120 min, a laparotomy was performed to gain surgical hemostasis and quantify blood loss. Physiological parameters were recorded, and blood samples were collected at defined intervals until termination of the study at T = 480 min. Statistical analyses used Student t tests, with P < 0.05 considered statistically significant. Results are reported as mean ± SEM. The calculated percent blood loss for the 25% hepatectomy group was negligible (2.3% ± 0.2%), whereas the 50% and 60% hepatectomy groups exhibited 26.6% ± 7.1% and 24.9% ± 3.8% blood loss, respectively. At T = 5 min, blood pressure for the 25%, 50%, and 60% hepatectomy groups was reduced by 13.8%, 60.8%, and 63.2% from the respective baseline values (P < 0.05). In the 60% hepatectomy group, alterations in thromboelastometry parameters and systemic inflammatory markers were observed. The development of a translatable nonhuman primate model of uncontrolled hemorrhage is an ongoing process. This study demonstrates that 60% hepatectomy offers a significant reproducible injury applicable for the evaluation of human-derived therapeutics.

In our recent clinical study of damage control laparotomy, the abdominal compartment syndrome (ACS) emerged as an independent risk factor for postinjury multiple organ failure (MOF). We and others have shown previously that the ACS promotes the systemic production of proinflammatory cytokines. Our study objective was to develop a clinically relevant two-event animal model of postinjury MOF using the ACS as a second insult during systemic neutrophil priming to provoke organ dysfunction. Male adult rats underwent hemorrhagic shock (30 mmHg x 45 min) and were resuscitated with crystalloids and shed blood. The timing of postshock systemic neutrophil (PMN) priming was determined by the surface expression of CD11b via flow cytometry. Finding maximal PMN priming at 8 h, but no priming at 2 h (early) and 18 h (late), the ACS (25 mmHg x 60 min) was introduced at these time points. At 24 h postshock, lung injury was assessed by lung elastase concentration and Evans blue dye extravasation in bronchoalveolar lavage. Liver and renal injuries were determined by serum alanine aminotransferase, serum creatinine, and blood urea nitrogen. The ACS during the time of maximal systemic PMN priming (8 h) provoked lung and liver injury, but did not if introduced at 2 or 18 h postshock when there was no evidence of systemic PMN priming. The 24-h mortality of this two-event model was 33%. These findings corroborate the potential for the ACS to promote multiple organ injury when occurring at the time of systemic PMN priming. This clinically relevant two-event animal model of PMN organ injury may be useful in elucidating therapy strategies to prevent postinjury MOF.