Safety and efficacy of human polymerized hemoglobin on guinea pig resuscitation from hemorrhagic shock

Abstract

For the past thirty years, hemoglobin-based oxygen carriers (HBOCs) have been under development as a red blood cell substitute. Side-effects such as vasoconstriction, oxidative injury, and cardiac toxicity have prevented clinical approval of HBOCs. Recently, high molecular weight (MW) polymerized human hemoglobin (PolyhHb) has shown positive results in rats. Studies have demonstrated that high MW PolyhHb increased O₂ delivery, with minimal effects on blood pressure, without vasoconstriction, and devoid of toxicity. In this study, we used guinea pigs to evaluate the efficacy and safety of high MW PolyhHb, since like humans guinea pigs cannot produce endogenous ascorbic acid, which limits the capacity of both species to deal with oxidative stress. Hence, this study evaluated the efficacy and safety of resuscitation from severe hemorrhagic shock with high MW PolyhHb, fresh blood, and blood stored for 2 weeks. Animals were randomly assigned to each experimental group, and hemorrhage was induced by the withdrawal of 40% of the blood volume (BV, estimated as 7.5% of body weight) from the carotid artery catheter. Hypovolemic shock was maintained for 50 min. Resuscitation was implemented by infusing 25% of the animal's BV with the different treatments. Hemodynamics, blood gases, total hemoglobin, and lactate were not different before hemorrhage and during shock between groups. The hematocrit was lower for the PolyhHb group compared to the fresh and stored blood groups after resuscitation. Resuscitation with stored blood had lower blood pressure compared to fresh blood at 2 h. There was no difference in mean arterial pressure between groups at 24 h. Resuscitation with PolyhHb was not different from fresh blood for most parameters. Resuscitation with PolyhHb did not show any remarkable change in liver injury, inflammation, or cardiac damage. Resuscitation with stored blood showed changes in liver function and inflammation, but no kidney injury or systemic inflammation. Resuscitation with stored blood after 24 h displayed sympathetic hyper-activation and signs of cardiac injury. These results suggest that PolyhHb is an effective resuscitation alternative to blood. The decreased toxicities in terms of cardiac injury markers, vital organ function, and inflammation following PolyhHb resuscitation in guinea pigs indicate a favorable safety profile. These results are promising and support future studies with this new generation of PolyhHb as alternative to blood when blood is unavailable.

Figure 1

Hemodynamics and hematological parameters. PolyhHb restored hemodynamics after hemorrhagic shock (HS) similar to fresh and stored blood. However, stored blood took a longer period of time to achieve a similar blood pressure compared to fresh blood and PolyhHb. Although, PolyhHb half-life was approximately 13 h and total hemoglobin was lower 24 h after resuscitation, PolyhHb restored hemodynamics at 24 h post-resuscitation. (a) Mean arterial pressure (MAP); (b) Heart rate (HR); (c) Total hemoglobin (tHb); (d) Hematocrit (Hct). Measurements were taken at baseline (BL), 50 min into hemorrhagic shock (shock) and 15 min, 2 h, and 24 h after resuscitation. Animals were awake at the 2 h and 24 h timepoints. *, p < 0.05. Statistical comparisons were completed using a two way- ANOVA; Tukey’s multiple comparison test.

Figure 2

Markers of liver function after resuscitation. These results indicate that animals resuscitated with stored blood presented an increased risk for liver damage at 24 h after resuscitation, while resuscitation with PolyhHb do not seem to drastically impair liver enzymes. (A) Aspartate aminotransferase (AST); (B) Alanine aminotransferase (ALT); (C) Alkaline phosphatase (ALP); (D)  Malondialdehyde (MDA); (E) Asymmetric dimethylarginine (ADMA); (F) Superoxide dismutase (SOD); (G) Vitamin C (Vit C); (H) Glycogen; (I) Liver chemokine ligand 1 (CXCL-1). Measurements were taken 24 h after resuscitation. *, p < 0.05 versus Fresh Blood; †, p < 0.05 versus Stored Blood. Statistical comparisons were completed using a one way- ANOVA; Tukey’s multiple comparison test.

Figure 3

Markers of kidney function after resuscitation. Stored blood and PolyhHb groups indicate changes in kidney function compared to the fresh blood group, as observed by increased serum creatinine. However, the level of dysfunction could be transient since BUN and U Ngal, both kidney damage markers, did not change when compared to the fresh blood group. (A) Serum creatinine; (B) Blood urea nitrogen (BUN); (C) Urinary neutrophil gelatinase-associated lipocalin (U Ngal). Measurements were taken 24 h after resuscitation. *, p < 0.05 versus Fresh Blood. Statistical comparisons were completed using a one way—ANOVA; Tukey’s multiple comparison test.

Figure 4

Markers of cardiac injury. Stored blood caused a greater degree of cardiac injury compared to fresh blood, since cardiac troponin increased for stored blood. However, resuscitation with stored blood or PolyhHb did not cause remarkable changes in cardiac inflammation. (A) Interleukin-6 (IL-6); (B) Tumor necrosis factor alpha (TNFα); (C) Monocyte chemoattractant protein-1 (MCP-1); (D) Troponin; (E) C-reactive protein; (F) Atrial natriuretic peptide (ANP). Measurements were taken 24 h after resuscitation. *, p < 0.05 vs. Fresh Blood; †, p < 0.05 versus Stored Blood. Statistical comparisons were completed using a one way—ANOVA; Tukey’s multiple comparison test.

Figure 5

Systemic inflammation, splenic inflammation, and catecholamines. Neither PolyhHb or stored blood presented higher systemic inflammation compared to fresh blood (the gold standard for resuscitation). However, stored blood increased catecholamine levels compared to fresh blood and PolyhHb suggesting higher sympathetic activation after resuscitation. (A) Interleukin-6 (IL-6); (B) Chemokine ligand 1 (CXCL-1); (C) Interleukin-1(IL-10); (D) Splenic CXCL-1; (E) Norepinephrine; (F) Epinephrine. Measurements were taken 24 h after resuscitation. *, p < 0.05 versus Fresh Blood; †, p < 0.05 versus Stored Blood. Statistical comparisons were completed using a one way—ANOVA; Tukey’s multiple comparison test.