Balance between oxygen transport and blood rheology during resuscitation from hemorrhagic shock with polymerized bovine hemoglobin

Abstract

Alternatives to blood for use in transfusion medicine have been investigated for decades. An ideal alternative should improve oxygen (O₂)-carrying capacity and O₂ delivery and support microvascular blood flow. Previous studies have shown that large-molecular diameter hemoglobin (Hb)-based oxygen carriers (HBOCs) based on polymerized bovine Hb (PolybHb) reduce the toxicity and vasoconstriction of first-generation HBOCs by increasing blood and plasma viscosity and preserving microvascular perfusion. The objective of this study was to examine the impact of PolybHb concentration and therefore O₂-carrying capacity and solution viscosity on resuscitation from hemorrhagic shock in rats. PolybHb was diafiltered on a 500-kDa tangential flow filtration (TFF) module to remove low-molecular weight (MW) PolybHb molecules from the final product. Rats were hemorrhaged and maintained in hypovolemic shock for 30 min before transfusion of PolybHb at 10 g/dL (PHB10), 5 g/dL (PHB5), or 2.5 g/dL (PHB2.5) concentration, to restore blood pressure to 90% of the animal's baseline blood pressure. Resuscitation restored blood pressure and cardiac function in a PolybHb concentration-dependent manner. Parameters indicative of the heart's metabolic activity indicated that the two higher PolybHb concentrations better restored coronary O₂ delivery compared with the low concentration evaluated. Markers of organ damage and inflammation were highest for PHB10, whereas PHB5 and PHB2.5 showed similar expression of these markers. These studies indicate that a concentration of ~5 g/dL of PolybHb may be near the optimal concentration to restore cardiac function, preserve organ function, and mitigate the toxicity of PolybHb during resuscitation from hemorrhagic shock.NEW & NOTEWORTHY Large-molecular diameter polymerized bovine hemoglobin avoided vasoconstriction and impairment of cardiac function during resuscitation from hemorrhagic shock that was seen with previous hemoglobin-based O₂ carriers by increasing blood viscosity in a concentration-dependent manner. Supplementation of O₂-carrying capacity played a smaller role in maintaining cardiac function than increased blood and plasma viscosity.

Keywords: blood substitutes; blood viscosity; cardiac function; hemorrhagic shock; rheology.

Fig. 1.

High concentration of polymerized bovine hemoglobin (PolybHb) sustains central hemodynamics compared with low concentration. A: mean arterial pressure (MAP) decreases during shock and is partially recovered during resuscitation. B: heart rate (HR) decreases during shock and does not fully recover in the 1-h resuscitation. Here, n = 7 animals per group. BL, Baseline; bpm, beats/min; PHB2.5, PHB5, and PHB10, 2.5, 5, and 10 g/dL PolybHb, respectively; R10 and R60, 10 and 60 min after beginning of resuscitation, respectively; SH, Shock. *P < 0.05 vs. Baseline.

Fig. 2.

Central hemodynamics following transfusion of high-concentration polymerized bovine hemoglobin (PolybHb). A: cardiac output (CO) recovers close to baseline values following 10 g/dL PolybHb (PHB10) transfusion. Average baseline CO was 69.5 mL/min. B: systemic vascular resistance (SVR) increases following resuscitation. Average baseline SVR was 11,000 dyn·s·cm⁻⁵. C: the high viscosity of PHB10 decreases systemic vascular hinderance (SVH) compared with 2.5 g/dL PolybHb (PHB2.5). Here, n = 7 animals per group. BL, Baseline; PHB5, 5 g/dL PolybHb; R10 and R60, 10 and 60 min after beginning of resuscitation, respectively; SH, Shock.

Fig. 3.

Cardiac mechanoenergetics following polymerized bovine hemoglobin (PolybHb) transfusion. A: stroke work (SW) recovers to baseline levels. Average baseline SW was 17,637 mmHg·µL. B: stroke work per volume ejected (SW/SV) is elevated with 10 and 5 g/dL PolybHb (PHB10 and PHB5, respectively), indicating better recovery of cardiac function and more pressure generation. Average baseline SW/SV was 101 mmHg. C: internal energy utilization (IEU) of the heart, an indicator of the heart’s potential energy, is increased after transfusion of PHB10 relative to 2.5 g/dL PolybHb (PHB2.5). Average baseline IEU was 8,619 mmHg·µL. Here, n = 7 animals per group. BL, Baseline; R10 and R60, 10 and 60 min after beginning of resuscitation, respectively; SH, Shock. *P < 0.05 vs. Baseline, †P < 0.05 vs. PHB2.5.

Fig. 4.

Volume requirements to resuscitate rats from hemorrhagic shock, as percentage of total blood volume (BV). Significantly higher volume of polymerized bovine hemoglobin (PolybHb) at 2.5 g/dL concentration (PHB2.5) is required to reach the mean arterial pressure target. Total blood volume is estimated as 7% of the animal’s body weight. Here, n = 7 animals per group. PHB5 and PHB10, 5 and 10 g/dL PolybHb, respectively; R10, R20, R30, R45, and R60, 10, 20, 30, 45, and 60 min after beginning of resuscitation, respectively. †P < 0.05 vs. PHB2.5.

Fig. 5.

Parameters of O₂ transport. Rows represent the O₂ transported by different elements of the blood, and each column represents a different parameter of oxygen transport. A–C: O₂ transported by the whole blood. D–F: O₂ transported by the red blood cells (RBCs). G–I: O₂ transported by the polymerized bovine hemoglobin (PolybHb). Here, n = 7 animals per group. BL, Baseline; PHB2.5, PHB5, and PHB10, 2.5, 5, and 10 g/dL PolybHb, respectively; PolyHb, polymerized hemoglobin; R10 and R60, 10 and 60 min after beginning of resuscitation, respectively; SH, Shock. *P < 0.05 vs. Baseline, †P < 0.05 vs. PHB2.5, ‡P < 0.05 vs. PHB5.

Fig. 6.

Markers of organ damage and function. A–C: kidney markers. D and G: lung markers. E and F: systemic markers. H and I: liver markers. Here, n = 7 animals per group. The average ± SE of these markers in unbled sham animals (n = 4) is indicated by the gray box surrounded with black dashed lines. ALT, alanine transaminase; AST, aspartate transaminase; BUN, blood urea nitrogen; Neut +, positive CD45 neutrophils; PHB2.5, PHB5, and PHB10, 2.5, 5, and 10 g/dL polymerized bovine hemoglobin, respectively; u-NGAL, urinary neutrophil gelatinase-associated lipocalin. *P < 0.05 vs. Sham, †P < 0.05 vs. PHB2.5, ‡P < 0.05 vs. PHB5.

Fig. 7.

Markers of organ neutrophil recruitment and inflammation. A: neutrophil recruitment in lungs is dependent on the polymerized bovine hemoglobin (PolybHb) concentration transfused. B–D: neutrophil recruitment in the spleen, in the liver, and systemically appears lowest for 5 g/dL PolybHb (PHB5). E and F: the inflammatory and anti-inflammatory responses appear to be equal and PolybHb concentration dependent. Here, n = 7 animals per group. The average ± SE of these markers in unbled sham animals (n = 4) is indicated by the gray box surrounded with black dashed lines. PHB2.5 and PHB10, 2.5 and 10 g/dL PolybHb, respectively. *P < 0.05 vs. Sham, †P < 0.05 vs. PHB2.5, ‡P < 0.05 vs. PHB5.

Fig. 8.

Markers of iron transport. A: plasma iron transport appears to be independent of the polymerized bovine hemoglobin (PolybHb) concentration transfused. B–E: markers of iron transport in the liver and spleen are dependent on the concentration of PolybHb transfused. F: heme metabolism is dependent on the concentration of PolybHb transfused. Here, n = 7 animals per group. The average ± SE of these markers in unbled sham animals (n = 4) is indicated by the gray box surrounded with black dashed lines. PHB2.5, PHB5, and PHB10, 2.5, 5, and 10 g/dL PolybHb, respectively. *P < 0.05 vs. Sham, †P < 0.05 vs. PHB2.5, ‡P < 0.05 vs. PHB5.