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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 O 2 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., (© 2022. The Author(s).)

Background: Hemoglobin (Hb)-based oxygen (O 2 ) carriers (HBOCs) are being developed as alternatives to red blood cells and blood when these products are unavailable. Clinical trials of previous HBOC generations revealed side effects, including hypertension and vasoconstriction, that were not observed in preclinical studies. Large molecular weight (MW) polymerized bovine Hb (PolybHb) represents a new class of HBOC with promising results. We evaluated the safety profile of PolybHb after an exchange transfusion (ET) in guinea pigs (GPs). This study compares changes in indices of cardiac, inflammatory, and organ function after ET with high (R-state) and low (T-state) O 2 affinity PolybHb with high MW., Study Design and Methods: Guinea pigs underwent a 20% ET with PolybHb. To assess the implication of PolybHb ET on the microcirculation, hamsters instrumented with a dorsal window chamber were subjected to a similar volume ET., Results: T and R-state PolybHb did not induce significant alterations in cardiac function. T-state PolybHb induced mild vasoconstriction shortly after transfusion, while R-state did not have acute effects on microvascular tone., Conclusion: Large MW PolybHbs were found to be safe and efficacious in increasing O 2 carrying capacity and the O 2 affinity of the PolybHb did not affect O 2 delivery or extraction by tissues in relevant preclinical models. In conclusion, these results suggest that both T-state and R-state PolybHb are safe and do not impair O 2 delivery. The results are encouraging and support further evaluation of high MW PolybHbs and their future feasibility compared to allogenic blood in a trauma model., (© 2020 AABB.)

Alternatives to blood for use in transfusion medicine have been investigated for decades. An ideal alternative should improve oxygen (O 2 )-carrying capacity and O 2 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 O 2 -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 O 2 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 O 2 carriers by increasing blood viscosity in a concentration-dependent manner. Supplementation of O 2 -carrying capacity played a smaller role in maintaining cardiac function than increased blood and plasma viscosity.

There is a 0.138% incidence of adverse reactions related to blood transfusion. Transfusion-related acute lung injury, immunosuppression, fever, pathogen transmission, and hemolytic transfusion reactions are the most common ones. Synthetic oxygen carriers have been developed to deal with blood shortages and for use in the field where stored blood was not available. They were also designed to be pathogen free, including unknown viruses. In this study, we used Male Golden Syrian Hamsters implemented with a dorsal window chamber to determine how infusion of three different, genetically crosslinked recombinant acellular hemoglobin (rHb) solutions with different oxygen affinities and nitric oxide kinetics affect mean arterial pressure (MAP), heart rate (HR), kidney function, and kidney structure. We found that the administration of all three rHb solutions caused mild hypertension and bradycardia 30 minutes after infusion. However, acute changes in glomerular filtration rate (GFR) were not detected, even though histological analysis was performed 72 hours after treatment revealed some structural changes. All the rHb solutions resulted in hypertension 30 minutes after a 10% topload administration. Regardless of their properties, the presence of acellular Hb causes significant alterations to kidney tissue.

Malemide polyethylene glycol-conjugated Hb (MP4OX, Sangart Inc.), a high-affinity low-concentration acellular hemoglobin (P50 = 5 mmHg, 4.3 g/dl) solution, has been shown to optimize microvascular perfusion and target oxygen delivery to anoxic tissue. Microvascular perfusion during an acute hypoxic challenge in a transgenic anemic sickle cell disease mouse model was studied with MP4OX and saline. Arterioles were dilated in both groups. Functional capillary density (FCD) was maintained at a higher level with MP4OX. In conclusion, MP4OX treatment reduced the hypoxia-mediated decline in FCD, an effect in part due to higher arterial pressure resulting in increased microvascular perfusion pressures.

The giant extracellular hemoglobin (erythrocruorin) from the earth worm (Lumbricus terrestris) has shown promise as a potential hemoglobin-based oxygen carrier (HBOC) in in vivo animal studies. An important beneficial characteristic of this hemoglobin (LtHb) is the large number of heme-based oxygen transport sites that helps overcome issues of osmotic stress when attempting to provide enough material for efficient oxygen delivery. A potentially important additional property is the capacity of the HBOC either to generate nitric oxide (NO) or to preserve NO bioactivity to compensate for decreased levels of NO in the circulation. The present study compares the NO-generating and NO bioactivity-preserving capability of LtHb with that of human adult hemoglobin (HbA) through several reactions including the nitrite reductase, reductive nitrosylation, and still controversial nitrite anhydrase reactions. An assignment of a heme-bound dinitrogen trioxide as the stable intermediate associated with the nitrite anhydrase reaction in both LtHb and HbA is supported based on functional and EPR spectroscopic studies. The role of the redox potential as a factor contributing to the NO-generating activity of these two proteins is evaluated. The results show that LtHb undergoes the same reactions as HbA and that the reduced efficacy for these reactions for LtHb relative to HbA is consistent with the much higher redox potential of LtHb. Evidence of functional heterogeneity in LtHb is explained in terms of the large difference in the redox potential of the isolated subunits., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

The development of an alternative to blood transfusion to treat severe hemorrhage remains a challenge, especially in far forward scenarios when blood is not available. Hemoglobin level (Hb)-based oxygen (O2) carriers (HBOCs) were developed to address this need. Hemopure (HBOC-201, bovine Hb glutamer-250; OPK Biotech, Cambridge, MA), one such HBOC, has been approved for clinical use in South Africa and Russia. At the time of its approval, however, few studies aimed to understand Hemopure's function, administration, and adverse effects compared to blood. We used intravital microscopy to study the microcirculation hemodynamics (arteriolar and venular diameters and blood flow and functional capillary density [FCD]) and oxygenation implications of Hemopure administration at different Hb concentrations-4, 8, and 12 gHb/dL-compared to fresh blood transfusion during resuscitation from hemorrhagic shock. Experiments were performed in unanesthetized hamsters instrumented with a skinfold window chamber, subjected to hemorrhage (50% of the blood volume), followed by 1-hour hypovolemic shock and fluid resuscitation (50% of the shed volume). Our results show that fluid resuscitation with Hemopure or blood restored systemic and microvascular parameters. Microcirculation O2 delivery was directly correlated with Hemopure concentration, although increased vasoconstriction was as well. Functional capillary density reflected the balance between enhanced O2 transport and reduced blood flow: 12 gHb/dL of Hemopure and blood decreased FCD compared to the lower concentrations of Hemopure (P < .05). The balance between O2 transport and tissue perfusion can provide superior resuscitation from hemorrhagic shock compared to blood transfusion by using a low Hb concentration of HBOCs relative to blood., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The development of oxygen (O2)-carrying blood substitutes has evolved from the goal of replicating blood O2 transport properties to that of preserving microvascular and organ function, reducing the inherent or potential toxicity of the material used to carry O2, and treating pathologies initiated by anemia and hypoxia. Furthermore, the emphasis has shifted from blood replacement fluid to "O2 therapeutics" that restore tissue oxygenation to specific tissues regions. This review covers the different alternatives, potential and limitations of hemoglobin-based O2 carriers (HBOCs) and perfluorocarbon-based O2 carriers (PFCOCs), with emphasis on the physiologic conditions disturbed in the situation that they will be used. It describes how concepts learned from plasma expanders without O2-carrying capacity can be applied to maintain O2 delivery and summarizes the microvascular responses due to HBOCs and PFCOCs. This review also presents alternative applications of HBOCs and PFCOCs namely: 1) How HBOC O2 affinity can be engineered to target O2 delivery to hypoxic tissues; and 2) How the high gas solubility of PFCOCs provides new opportunities for carrying, dissolving, and delivering gases with biological activity. It is concluded that the development of current blood substitutes has amplified their applications horizon by devising therapeutic functions for O2 carriers requiring limited O2 delivery capacity restoration. Conversely, full, blood-like O2-carrying capacity reestablishment awaits the control of O2 carrier toxicity.

Significance: There has been a striking advancement in our understanding of red cell substitutes over the past decade. Although regulatory oversight has influenced many aspects of product development in this period, those who have approached the demonstration of efficacy of red cell substitutes have failed to understand their implication at the level of the microcirculation, where blood interacts closely with tissue., Recent Advances: The understanding of the adverse effects of acellular hemoglobin (Hb)-based oxygen carriers (HBOCs) has fortunately expanded from Hb-induced renal toxicity to a more complete list of biochemical mechanism. In addition, various unexpected adverse reactions were seen in early clinical studies. The effects of the presence of acellular Hb in plasma are relatively unique because of the convergence of mechanical and biochemical natures., Critical Issues: Controlling the variables using genetic engineering and chemical modification to change specific characteristics of the Hb molecule may allow for solving the complex multivariate problems of acellular Hb vasoactivity. HBOCs may never be rendered free of negative effects; however, quantifying the nature and extent of microvascular complications establishes a platform for designing new ameliorative therapies., Future Directions: It is time to leave behind the study of vasoactivity and toxicity based on bench-top measurements of biochemical changes and those based solely on systemic parameters in vivo, and move to a more holistic analysis of the mechanisms creating the problems, complemented with meaningful studies of efficacy.

Aim: Lumbricus terrestris (earthworm) erythrocruorin (LtEc) is a naturally occurring extracellular hemoglobin (Hb) with high molecular weight (3.6MDa), low autoxidation rate, and limited nitric oxide (NO) dioxygenation activity. These properties make LtEc a potential candidate for use as red blood cell (RBC) substitute, i.e. Hb-based oxygen carrier (HBOC). Previous studies have shown that small amounts of LtEc can be safely transfused into mice, rats, and hamsters without eliciting major side effects. Therefore, this study was designed to understand oxygen (O(2)) transport to tissues and systemic/microvascular hemodynamics induced by LtEc during anemic conditions., Main Methods: Hamsters fitted with dorsal window chambers were hemodiluted to 18% hematocrit (Hct) using 6g/dL dextran 70kDa (Dex70). Hemodilution was then continued to 11% Hct using 10g/dL LtEc, 6g/dL Dex70 or 10g/dL human serum albumin (HSA). Blood pressure, heart rate, blood gas parameters, microvascular hemodynamics, microvascular blood flow, functional capillary density (FCD), intravascular pO(2) and perivascular pO(2) were studied., Key Findings: LtEc maintained blood pressure without inducing vasoconstriction while increasing microvascular perfusion and FCD relative to Dex70 and HSA. LtEc increased blood O(2) carrying capacity and maintained systemic and microvascular parameters without decreasing arteriolar diameter or increasing vascular resistance with during extreme anemia. LtEc increased O(2) delivery compared to conventional plasma expanders., Significance: LtEc or synthetic molecules that replicate the characteristics of LtEc could be effective O(2) carriers with potential to be used in transfusion medicine to prevent tissue anoxia resulting from severe anemia., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Unlabelled: Widespread clinical use of acellular hemoglobin (Hb)-based O2 carriers (HBOCs) has been hampered by their ability to elicit both vasoconstriction and systemic hypertension. This is primarily due to the ability of acellular Hb to extravasate through the blood vessel wall and scavenge endothelial-derived nitric oxide (NO). Encapsulation of Hb inside the aqueous core of liposomes retards the rates of NO dioxygenation and O2 release, which should reduce or eliminate the vasoactivity of Hb. Our aim is to determine the extent of systemic and microvascular vasoactive responses (hypertension, vasoconstriction and hypoperfusion) after infusion of vesicle encapsulated Hbs, in which the encapsulated Hb is in either the deoxygenated or carbon monoxide (CO) state (HbV and COHbV, respectively). To investigate this hypothesis, we used the hamster window chamber model subjected to two successive hypervolemic infusions of HbV and COHbV solutions (each infusion represents 10% of the animal's calculated blood volume) at Hb concentrations of either 7 or 10 g/dL. The hypervolemic infusion model used in this study has all the regulatory mechanisms responsible for predicting the vasoconstrictive responses of HBOCs. The results of this study demonstrate the absence of vasoconstrictive and hypertensive responses upon single and multiple infusions of HbV and COHbV solutions. The HbV and COHbV solutions increased the plasma O2 carrying capacity. However, COHbV delivered low therapeutic levels of CO without inducing any microcirculatory disturbances., Significance: Vesicles containing Hb can be used as a new therapeutic agent in transfusion medicine to treat anemia and revert hypoperfusion.

Background: The hemoglobin of the earthworm Lumbricus terrestris (also known as erythrocruorin, or LtEc) is a naturally occurring high-molecular-weight protein assembly (3.6 MDa) that is extremely stable, resistant to oxidation, and transports oxygen similarly to human whole blood. Therefore, LtEc may serve as an alternative to donated human red blood cells. However, a suitable purification process must be developed to produce highly pure LtEc on a large scale that can be evaluated in an animal model to determine the safety and efficacy of LtEc., Study Design and Methods: We used tangential-flow filtration (TFF), an easily scalable and affordable technique, to produce highly pure LtEc from earthworms. The purity, yield, methemoglobin level, viscosity, colloid osmotic pressure, O(2) binding equilibria, and ligand-binding kinetics of the purified LtEc were measured in vitro. The purified LtEc product was then evaluated in hamsters using a hypervolemic infusion model to establish its basic biocompatibility and detect any changes in microcirculatory and systemic variables., Results: TFF was able to produce LtEc with high purity and yield (5-10 g/1000 worms). The purified LtEc product did not elicit hypertension or vasoconstriction when infused into hamsters., Conclusion: LtEc may be easily purified and safely transfused into hamsters in small amounts (0.5-1.5 g/dL final concentration in blood) without any noticeable side effects. Therefore, LtEc may serve as a very promising oxygen carrier for use in transfusion medicine., (© 2012 American Association of Blood Banks.)

A mathematical model was developed to study nitric oxide (NO) and oxygen (O(2)) transport in an arteriole and surrounding tissues exposed to a mixture of red blood cells (RBCs) and hemoglobin (Hb)-based O(2) carriers (HBOCs). A unique feature of this model is the inclusion of blood vessel wall shear stress-induced production of endothelial-derived NO, which is very sensitive to the viscosity of the RBC and HBOC mixture traversing the blood vessel lumen. Therefore in this study, a series of polymerized bovine Hb (PolyHb) solutions with high viscosity, varying O(2) affinities, NO dioxygenation rate constants and O(2) dissociation rate constants that were previously synthesized and characterized by our group was evaluated via mathematical modeling, in order to investigate the effect of these biophysical properties on the transport of NO and O(2) in an arteriole and its surrounding tissues subjected to anemia with the commercial HBOC Oxyglobin® and cell-free bovine Hb (bHb) serving as appropriate controls. The computer simulation results indicated that transfusion of high viscosity PolyHb solutions promoted blood vessel wall shear stress dependent generation of the vasodilator NO, especially in the blood vessel wall and should transport enough NO inside the smooth muscle layer to activate vasodilation compared to the commercial HBOC Oxyglobin® and cell-free bHb. However, NO scavenging in the arteriole lumen was unavoidable due to the intrinsic high NO dioxygenation rate constant of the HBOCs being studied. This study also observed that all PolyHbs could potentially improve tissue oxygenation under hypoxic conditions, while low O(2) affinity PolyHbs were more effective in oxygenating tissues under normoxic conditions compared with high O(2) affinity PolyHbs. In addition, all ultrahigh molecular weight PolyHbs displayed higher O(2) transfer rates than the commercial HBOC Oxyglobin® and cell-free bHb. Therefore, these results suggest that ultrahigh molecular weight PolyHb solutions could be used as safe and efficacious O(2) carriers for use in transfusion medicine. It also suggests that future generations of PolyHb solutions should possess lower NO dioxygenation reaction rate constants in order to reduce NO scavenging, while maintaining high solution viscosity to take advantage of wall shear stress-induced NO production. Taken together, we suggest that this mathematical model can be used to predict the vasoactivity of HBOCs and help guide the design and optimization of the next generation of HBOCs for use in transfusion medicine., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background: The objective of this study was to determine the role of plasma oxygen carrying capacity during resuscitation from hemorrhagic shock (HS)., Methods: Hemodynamic responses to small-volume resuscitation from HS with hypertonic saline followed by infusion of ultrahigh-molecular-weight tense-state polymerized hemoglobins (PolyHbs) were studied in the hamster window chamber model. HS was induced by withdrawing 50% of the blood volume (BV), and hypovolemic state was maintained for 1 hour. Resuscitation was implemented by infusion of hypertonic saline (3.5% of BV) followed by 10% of BV infusion of polymerized human Hb (PolyHbhum, P50=49 mm Hg), polymerized bovine Hb (PolyHbbov, P50=40 mm Hg), or human serum albumin (HSA), all at 10 g/dL. Resuscitation was monitored over 90 minutes., Results: PolyHbhum elicited higher arterial pressure, produced vasoconstriction, and decreased perfusion. In contrast, PolyHbbov and HSA exhibited lower blood pressure and partially restored perfusion and functional capillary density compared with PolyHbhum. Blood gas parameters showed a pronounced recovery after resuscitation with PolyHbbov compared with both PolyHbhum and HSA. Tissue PO2 was significantly improved in the PolyHbbov group, showing that the moderate increase in P50 of PolyHbbov compared with hamster blood (P50=32 mm Hg) was beneficial during resuscitation. However, an excessive increase in oxygen release between the central and peripheral circulation, as induced by PolyHbhum produced vasoconstriction and hypoperfusion, limiting the benefits of additional oxygen carrying capacity., Conclusions: Appropriately engineered PolyHb will enhance/reinstate oxygenation, without hypertension or vasoconstriction, to be used in situations where blood transfusion is not logistically feasible.

Background: To avoid unnecessary blood transfusions, physiologic transfusion triggers, rather than exclusively hemoglobin-based transfusion triggers, have been suggested. The objective of this study was to determine systemic and microvascular effects of using a perfluorocarbon-based oxygen carrier (PFCOC) to maintain perfusion and oxygenation during extreme anemia., Methods: The hamster (weight, 55-65 g) window chamber model was used. Two isovolemic hemodilution steps were performed using hydroxyethyl starch, 10%, at normoxic conditions to a hematocrit of 19% (hemoglobin, 5.5 g/dl), the point at which the transfusion trigger was reached. Two additional hemodilution exchanges using the PFCOC (Oxycyte) and increasing the fraction of inspired oxygen to 1.0 were performed to reduce the hematocrit to 11% (hemoglobin, 3.8 g/dl) and 6% (hemoglobin, 2.0 g/dl), respectively. No control group was used in the study because this concentration of hemodilution is lethal with conventional plasma expanders. Systemic parameters, microvascular perfusion, functional capillary density, and oxygen tensions across the microvascular network were measured., Results: At 6% hematocrit, the PFCOC maintained mean arterial pressure, cardiac output, systemic oxygen delivery, and oxygen consumption. As hematocrit was decreased from 11% to 6%, functional capillary density, calculated microvascular oxygen delivery, and oxygen consumption decreased; and the oxygen extraction ratio was close to 100%. Peripheral tissue oxygenation was not predicted by systemic oxygenation., Conclusions: The PFCOC, in conjunction with hyperoxia, was able to sustain organ function and partially provide systemic oxygenation during extreme anemia during the observation period. The PFCOC can work as a bridge until erythrocytes are available for transfusion or when additional oxygen is required, despite the possible limitations in peripheral tissue oxygenation.

NADH-localized fluorometry was used as a noninvasive technique to monitor changes in the energy state of intact tissue (muscle and connective tissue), without anesthesia, as a function of blood plasma O(2)-carrying capacity in the hamster window chamber model. Acute moderate isovolemic hemodilution was induced by two isovolemic hemodilution steps: in the first step, 6% 70-kDa dextran (Dex70) was used to induce an acute anemic state (18% Hct); in the second step, exchange transfusion of polyethylene glycol (PEG) maleimide-conjugated Hb (4 g/dl, PEG-Hb) or Dex70 (6 g/dl) was used to reduce erythrocytes to 75% of baseline (11% Hct). PEG-Hb had six copies of PEG (5 kDa) conjugated to each human Hb (0.48 g PEG/g Hb) through extension arm-facilitated chemistry. Systemic parameters, microvascular perfusion, functional capillary density, intravascular and interstitial Po(2), and intracellular NADH fluorescence were monitored. Mean arterial blood pressure after extreme hemodilution was statistically significantly reduced for Dex70 compared with PEG-Hb. The presence of PEG-Hb in the circulation maintained positive acid-base balance. While microvascular blood flows were not different, functional capillary density was significantly higher for PEG-Hb than Dex70. Arteriolar Po(2) was higher in the presence of PEG-Hb than Dex70, but tissue and venular Po(2) were not different. Cellular energy metabolism (intracellular O(2)) in the tissues was improved with PEG-Hb. Moderate hemodilution to 18% Hct (6.4 g Hb/dl) brings tissue O(2) delivery to the verge of inadequacy. Extreme hemodilution to 11% Hct (3.7 g Hb/dl) produces tissue anoxia, and high-O(2)-affinity PEG-Hb (Po(2) at which blood is 50% saturated with O(2) = 4 Torr, 1.1 g Hb/dl) only partially decreases anaerobic metabolism without increasing tissue Po(2).

Background: Maintenance of postsurgical tissue oxygenation depends on the ability of the specific tissue to recruit perfusion and oxygen (O(2)) supply. When native O(2)-carrying capacity is lacking, fluids to improve O(2)-carrying capacity based in hemoglobin (Hb) could prevent partially ischemic tissue hypoxia by increasing O(2) release from the remaining red blood cells., Study Design: Responses to facilitated O(2) transport after exchange transfusion with polymerized bovine Hb (PBH) were studied in a chronic partially ischemic tissue model, induced by large feeding arteriole ligation during hamster window chamber model implantation. PBH effects in microvascular perfusion and tissue oxygenation were studied after exchange transfusion of 40% of animal's blood volume. Experimental groups were defined by the concentration of PBH used, ie, PBH at 13 g/dL (PBH13); PBH at 4 g/dL in albumin solution to matching colloidal osmotic pressure (COP) (PBH4); and no PBH, only albumin solution at matching COP (PBH0)., Results: Restitution of O(2)-carrying capacity with PBH13 increased blood pressure and produced vasoconstriction compared with PBH4 and PBH0. On the other hand, PBH4 maintained blood pressure without substantial vasoconstriction, increased tissue partial pressure of O(2), arteriolar O(2) supply, and extraction to the partially ischemic tissue compared with PBH0 and PBH13., Conclusions: Results suggest the existence of an optimal concentration of low O(2)-affinity acellular Hb to increase oxygenation of partially ischemic tissue during anemic conditions., (Copyright 2010 American College of Surgeons. Published by Elsevier Inc. All rights reserved.)

Despite recent advances in the design of hemoglobin (Hb)-based oxygen carriers (HBOCs), vasoconstriction, presumably caused by nitric oxide (NO) scavenging, vessel wall hyperoxygenation, and/or extravasation, has been identified as the principal road block hampering commercial development of HBOCs. This study was designed to analyze systemic and microvascular responses to the molecular mass and plasma concentration of tense (T)-state polymerized bovine Hb (PolybHb) solutions. Experiments were performed using the hamster window chamber model subjected to successive hypervolemic infusions of T-state PolybHb solutions. PolybHb plasma concentrations were evaluated, namely, 0.5, 1.0 and 1.5 g/dl, respectively. Infusion of PolybHb solutions with molecular mass >500 kDa elicited hypertension and vasoconstriction proportional to the plasma concentration and inversely proportional to the PolybHb cross-link density. However, two high-molecular mass PolybHb solutions, PolybHb(40:1)(high) PolybHb(50:1)(high), did not elicit vasoconstriction at all concentrations studied, whereas PolybHb(50:1)(high) only elicited moderate hypertension at the highest concentration studied. In contrast, infusion of PolybHb solutions with molecular mass <500 kDa elicited significant hypertension and vasoconstriction compared with PolybHb solutions with molecular mass >500 kDa that was proportional to the plasma concentration and inversely proportional to the PolybHb cross-link density. We present promising results for highly cross-linked T-state PolybHb solutions with molecular mass >500 kDa [PolybHb(40:1)(high) PolybHb(50:1)(high)], which supports the concept that HBOC size/molecular mass influences its proximity to the vascular endothelium and molecular diffusivity. The hemodynamics of HBOC within the plasma layer surrounding the abluminal side endothelium regulates NO production and consumption, vessel oxygen flux, and extravasation. Although mechanistically attractive, neither of these hypotheses can be directly tested in vivo and will require further investigation.

Systemic and microvascular hemodynamic responses to hemorrhagic shock volume resuscitation with hypertonic saline followed by infusion of polymerized bovine hemoglobin (PBH) at different concentrations were studied in the hamster window chamber model to determine the role of plasma oxygen-carrying capacity and vasoactivity during resuscitation. Moderate hemorrhagic shock was induced by arterial controlled bleeding of 50% of blood volume (BV), and a hypovolemic state was maintained for 1 h. Volume was restituted by infusion of hypertonic saline (7.5% NaCl), 3.5% of BV, followed by 10% of BV of PBH at 2 different concentrations. Resuscitation was followed for 90 min and was carried out using 13 gPBH/dL (PBH13), PBH diluted to 4 gPBH/dL in albumin solution at matching colloidal osmotic pressure (PBH4), and an albumin-only solution at matching colloidal osmotic pressure (PBH0). Systemic parameters, microvascular hemodynamics, and functional capillary density were determined during hemorrhage, hypovolemic shock, and resuscitation. The PBH13 caused higher arterial pressure without reverting vasoconstriction and hypoperfusion. The PBH4 and PBH0 had lower MAP and partially reverted vasoconstriction. Only treatment with PBH4 restored perfusion and functional capillary density when compared with PBH13 and PBH0. Blood gas parameters and acid-base balance recovered proportionally to microvascular perfusion. Tissue PO2 was significantly improved in the PBH4 group, showing that limited restoration of oxygen-carrying capacity is beneficial and compensates for the effects of vasoactivity, a characteristic of molecular hemoglobin solutions proposed as blood substitutes.

O2-carrying fluids based on hemoglobin (Hb) are in various stages of clinical trials to determine their suitability as O2-carrying plasma expanders. Polymerized Hb solutions are characterized by their vasoactivity, low O2 affinity, oncotic effect, prolonged shelf life, and stability. Physiological responses to facilitated O2 transport after exchange transfusion with polymerized bovine Hb (PBH) were studied in the hamster window chamber model during acute moderate anemia to determine how PBH affects microvascular perfusion and tissue oxygenation. The anemic state [29% hematocrit (Hct)] was induced by hemodilution with a plasma expander (70 kDa dextran). After hemodilution, animals were randomly assigned to different exchange transfusion groups. Study groups were based on the concentration of PBH used, namely: PBH at 13 g Hb/dl [PBH13], PBH diluted to 8 (PBH8) or 4 (PBH4) g Hb/dl in albumin solution at matching colloidal osmotic pressure (COP), and no PBH (only albumin solution) at matching COP (PBH0). Measurement of systemic parameters, microvascular hemodynamics, capillary perfusion, and intravascular and tissue O2 levels was performed at 18% Hct. Restitution of O2-carrying capacity with PBH13 increased arterial pressure and triggered vasoconstriction, low perfusion, and high peripheral resistance. PBH4 and PBH0 exhibited lower arterial pressures compared with PBH13. Exchange transfused animals with PBH8 and PBH4 better maintained perfusion and functional capillary density than PBH13. Blood gas parameters and acid-base balance were recovered proportional to microvascular perfusion. Arterial O2 tensions were improved with PBH4 and PBH8 by preventing O2 precapillary release and increasing O2 reserve. Further studies to establish PBH optimal dosage, efficacy, safety, and its effect on outcome are indicated before Hb-based O2-carrying blood substitutes are implemented in routine practice.

Oxygen delivery and consumption after hemodilution with a perfluorocarbon-based oxygen carrier (PFCOC) was evaluated at sea level and at 2,600 m above sea level. Fifteen anesthetized rats were subjected to a two-exchange normovolemic hemodilution of 40% of the circulating blood volume each. First exchange was performed with a colloid solution. Second exchange was with 80% PFCOC and 20% colloid. Animals were then ventilated with 100% oxygen. Experiments were performed at barometric pressure of 1.0 atm (sea-level group, n=9) or 0.74 atm (2,600-m group, n=6). Blood gases, hematocrit, fluorocrit, and hemoglobin content were measured at baseline and 15 min after each exchange. After hemodilution, total arterial content was not modified by the PFCOC in either group. In contrast, arteriovenous oxygen difference increased significantly in both groups, as did the oxygen extraction ratio. In the second exchange, although total arterial content was similar between the two groups, the perfluorocarbon and plasma phases contributed significantly more at sea level. Arteriovenous oxygen difference was significantly less at sea level with a higher contribution from the perfluorocarbon and plasma phases. In conclusion, hemodilution with a PFCOC induced changes in oxygen delivery and consumption that differ with altitude. The 2,600-m group exhibited a higher oxygen extraction ratio and arteriovenous oxygen difference, with reduced oxygen delivery and unloading from both the fluorocarbon and plasma phase. Therefore, the efficacy of PFCOCs at 2,600 m above sea level is reduced, and altitude must be taken into account when PFCOCs are used.

Hemoglobin (Hb) solutions have been proposed as potential substitutes for erythrocytes to maintain oxygen-carrying capacity in situations in which blood is not available. This study investigated systemic and microvascular hemodynamics as well as tissue oxygenation and viability after an 80% exchange transfusion with an oxygen-carrying blood substitute based on polymerized bovine hemoglobin (PBH). Studies were carried in unanesthetized hamsters prepared with a window-chamber model for microcirculation evaluation. Heme iron-mediated injury to the tissue was analyzed by using deferoxamine (an iron chelator), which reduces free iron toxicity. Exchange transfusion led to a significant decrease in hematocrit (Hct) and an increase in plasma Hb, in addition to a significant decrease of arteriolar and venular diameters, flow velocity, and, therefore, microvascular blood flow. Capillary perfusion was severely compromised after exchange, but tissue pO2 increased above baseline, and oxygen extraction was reduced. Apoptotic and necrotic cells increased significantly after the exchange; however, this effect was only partially due to the toxicity of free iron. Iron therapy decreased the microvascular and oxygenation changes but did not fully reverse the adverse effects. Assessment of tissue viability after exchange suggests that chelation treatment in cases of large exchange transfusions with acellular Hb could be potentially beneficial.

Background: The effects of perfluorocarbon (PFC) emulsions administered at a nonhemodiluting dose were studied in the hamster window chamber model to determine the difference in ischemia-reperfusion injury associated with PFC delivery before and after an ischemic episode., Study Design: Ischemia was induced by compressing the periphery of the window chamber for 1 hour. Vessel diameter, red blood cell velocity, rolling and adherent leukocytes, and functional capillary density (FCD) were assessed by intravital microscopy. The animals received an infusion (10% blood volume) of PFC emulsion or equivalent volumes of saline, before or after ischemia. Two groups were studied in each experimental protocol: A, infusion after ischemia; and B, infusion before ischemia, where a fraction of the infused material stagnated in the ischemic zone during the occlusion time. Measurements were made before induced ischemia and at 0.5, 2, and 24 hours of reperfusion., Results: Animals treated with PFC after ischemia had substantially decreased leukocytes rolling and sticking in postcapillary venules and recovered functional capillary density and blood flow when compared with saline-treated controls. Conversely, administration of PFC before ischemia considerably reduced functional capillary density and increased leukocyte activation after reperfusion., Conclusions: Results indicate that PFC without stagnation within an ischemic zone attenuates postischemic reperfusion injury of striated skin muscle, presumably through the reduction of leukocyte-endothelial cell interactions. Accordingly, PFC effects on ischemia-reperfusion injury are determined mainly by the time of administration relative to the ischemic episodes.

Cell-free hemoglobin's (CFH) high affinity for nitric oxide (NO) could limit CFH's use as an oxygen-carrying blood replacement fluid because it scavenges NO, causing vasoconstriction and hypertension. However, the extent to which perivascular NO levels change following intravascular administration of hemoglobin (Hb) with different molecular dimensions correlates with vasoconstrictive responses in the microcirculation is unknown. The study objective was to determine vasoconstrictive effects following bolus infusions of (1) alphaalpha cross-linked Hb; (2) polymerized bovine Hb; or (3) polyethylene glycol-decorated Hb (PEG-Hb), by measurements of in vivo microvessel diameter, blood flow, perivascular NO concentration, and systemic hemodynamic parameters. All CFHs caused reductions in perivascular NO levels, not correlated to microvascular responses. PEG-Hb (largest molecular volume) maintained blood flow, while the others caused vasoconstriction and reduced perfusion. All solutions increased mean arterial pressure due to vasoconstriction and blood volume expansion, except for PEG-Hb, which increased blood pressure due to blood volume expansion and maintenance of cardiac output. In conclusion, perivascular NO reduction is similar for all Hb solutions because NO binding affinities are similar; however, effects on vascular resistance are related to the type of molecular modification, molecular volume, and oxygen affinity.

The development of an alternative to natural blood has evolved from the initial goal of replicating blood properties to the current objective of formulating a fluid that can be used to replace blood while preserving microvascular function and delivering oxygen. The properties of this fluid are counterintuitive and different from blood because it has high viscosity, oxygen affinity, and a low oxygen carrier concentration when compared with blood. The optimal oxygen carrier devised presently is poly-ethylene-conjugated human hemoglobin, a material demonstrated to be vasoinactive and void of the toxicities present in previous hemoglobin formulations. A feature of this material is that it is effective in small quantities, and therefore amplifies the equivalent supply of blood derived from blood donations.

A surface-modified polyethylene glycol-conjugated human hemoglobin (MP4) and alpha alpha-cross-linked human hemoglobin (alpha alpha Hb) were used to restore oxygen carrying capacity in conditions of extreme hemodilution (hematocrit 11%) in the hamster window model preparation. Changes in microvascular function were analyzed in terms of effects on capillary pressure and functional capillary density (FCD). MP4, at 1.0 +/- 0.2 g/dl blood concentration, significantly lowered mean arterial pressure (MAP) below baseline (99.6 +/- 7.6 mmHg) to 82.4 +/- 6.9 mmHg (P < 0.05) and decreased of FCD to 70 +/- 9%. alpha alpha Hb caused a greater recovery in MAP to 94.4 +/- 6.2 mmHg and lowered FCD to 62 +/- 8%. However, differences between alpha alpha Hb and MP4 in FCD were not statistically significant. Capillary pressures were in the ranges of 17-21 mmHg for MP4 and 15-19 mmHg for alpha alpha Hb, with both significantly lower than baseline (P < 0.05). Pressure in 80-microm-diameter arterioles was significantly increased with alpha alpha Hb relative to MP4 (P < 0.05). These results were compared with previous findings on the relation between capillary pressure and FCD; they supported the concept of a relationship between FCD and capillary pressure. Measurement of changes in arteriolar diameter, microvascular blood flow, and FCD show that there was no statistical difference between using alpha alpha Hb and MP4 in extreme hemodilution. Microvascular resistance in arterioles with a diameter range of 70-80 microm showed an increase relative to control with alpha alpha Hb, whereas MP4 caused a decrease.

The oxygen transport capacity of phospholipid vesicles encapsulating purified Hb (HbV) produced with a Po(2) at which Hb is 50% saturated (P 50 ) of 8 (HbV(8)) and 29 mmHg (HbV(29)) was investigated in the hamster chamber window model by using microvascular measurements to determine oxygen delivery during extreme hemodilution. Two isovolemic hemodilution steps were performed with 5% recombinant albumin (rHSA) until Hct was 35% of baseline. Isovolemic exchange was continued using HbV suspended in rHSA solution to a total [Hb] of 5.7 g/dl in blood. P(50) was modified by coencapsulating pyridoxal 5'-phosphate. Final Hct was 11% for the HbV groups, with a plasma [Hb] of 2.1 +/- 0.1 g/dl after exchange with HbV(8) or HbV(29). A reference group was hemodiluted to Hct 11% with only rHSA. All groups showed stable blood pressure and heart rate. Arterial oxygen tensions were significantly higher than baseline for the HbV groups and the rHSA group and significantly lower for the HbV groups compared with the rHSA group. Blood pressure was significantly higher for the HbV(8) group compared with the HbV(29) group. Arteriolar and venular blood flows were significantly higher than baseline for the HbV groups. Microvascular oxygen delivery and extraction were similar for the HbV groups but lower for the rHSA group (P < 0.05). Venular and tissue Po(2) were statistically higher for the HbV(8) vs. the HbV(29) and rHSA groups (P < 0.05). Improved tissue Po(2) is obtained when red blood cells deliver oxygen in combination with a high- rather than low-affinity oxygen carrier.

The O2-carrying blood substitute based on polymerized bovine hemoglobin (PBH) was used to determine efficacy in maintaining tissue Po2 after an 80% isovolemic blood exchange leading to a hematocrit of 19% [5.4 g Hb/dl from red blood cells (RBCs) and 6.3 g Hb/dl from PBH]. Effects were studied in terms of O2 delivery, O2 extraction, and tissue Po2 at the microcirculatory level at 1, 12, and 24 h after exchange transfusion in awake hamsters prepared with a window chamber model. At 1 h after exchange, arteriolar and venular diameters were decreased compared with baseline. Arteriolar diameter did not fully recover at 12 h after exchange, but venular diameter returned to normal. At 24 h after exchange, arteriolar and venular diameters were not different from baseline. Combining diameter and flow velocity data allowed us to calculate arteriolar and venular flows. At 1 h after exchange, arteriolar and venular flow was reduced compared with baseline. Arteriolar flow was lower at 12 h after exchange and recovered after 24 h. The number of capillaries with RBC passage [functional capillary density (FCD)] at 1 h after exchange with PBH was significantly lower than baseline. FCD remained decreased at 12 h; at 24 h after exchange transfusion, FCD was fully recovered. Tissue Po2 was maximal at 1 h after exchange and decreased progressively at 12 and 24 h after exchange. O2 release to the tissue was minimal at 1 h and increased at 12 and 24 h after exchange. These results suggest the impairment of tissue O2 metabolism after introduction of PBH into the circulation, which is mitigated as PBH concentration declines.

Development of a viable blood substitute began by focusing on recreating the oxygen-carrying capacity of blood, leading to the recognition that haemoglobin (Hb) is presently unequalled for this function. However, as human Hb is the only realistic source of this protein, the production of a blood substitute that solves transfusional blood availability problems and shortages must introduce a multiplying factor between supply of natural blood and blood substitute, while maintaining equivalency of function/efficacy. In other words, a unit of blood should produce several units of equivalent blood substitute. This expansion is now possible because of new understanding of how blood delivers oxygen in the microcirculation and the consequences of reducing oxygen-carrying capacity in haemorrhage. This information is used to provide improved resuscitation capacity and maintenance of tissue metabolism by tailoring the properties of a blood substitute to the task of maintaining microvascular function, rather than oxygen delivery capacity. Resuscitation in an organism that is haemorrhaging requires maintenance perfusion, a process directly linked to the maintenance of adequate levels of shear stress on the endothelium, induced by either increased blood/plasma viscosity or increased blood flow velocity in the microcirculation. This process must also be intimately coupled with the requirement that no portion of the tissue is anoxic. This disparate set of requirements can be satisfied with high viscosity Hb solutions that have high affinity for oxygen, a combination of properties that causes the microcirculation to remain functional, and a requirement that supersedes restoration of oxygen-carrying capacity in the treatment of haemorrhage.

Microcirculatory preservation is essential for patient recovery from hemorrhagic shock. In hemorrhagic shock, microcirculatory flow and pressure are greatly reduced, creating an oxygen debt that may eventually become irreversible. During shock, tissues become hypoxic, cellular respiration turns to anaerobic metabolism, and the microcirculation rapidly begins to fail. This condition requires immediate fluid resuscitation to promote tissue reperfusion. The choice of fluid for resuscitation is whole blood; however, this may not be readily available and, on a larger scale, may be globally insufficient. Thus, extensive research on viable alternatives to blood has been undertaken in an effort to develop a clinically deployable blood substitute. This has not, as of yet, achieved fruition, in part due to an incomplete understanding of the complexities of the function of blood in the microcirculation. Hemodynamic resuscitation is acknowledged to be contingent on a number of factors other than volume expansion. The circulation of whole blood is carefully regulated to optimize oxygen delivery to the tissues via shear stress modulation through blood viscosity, inherent oxygen-carrying capacity, cell-free layer variation, and myogenic response, among other variables. Although plasma expanders can address a number of these issues, hemoglobin-based oxygen carriers (HBOCs) introduce a method of replenishing the intrinsic oxygen-carrying capacity of blood. There continue to be a number of issues related to HBOCs, but recent advances in the next-generation HBOCs show promise in the preservation of microcirculatory function and limiting toxicities. The development of HBOCs is now focused on viscosity and the degree of microvascular shear stress achieved in order to optimize vasoactive and oxygen delivery responses by leveraging the restoration and maintenance of physiological responses to blood flow in the microcirculation. Blood substitutes with higher viscous properties tend to improve oxygen delivery compared to those with lower viscosities. This review details current concepts in blood substitutes, particularly as they relate to trauma/hemorrhagic shock, with a specific focus on their complex interactions in the microcirculation.

Nearly 21 million components of blood and whole blood and transfused annually in the United States, while on average only 13.6 million units of blood are donated. As the demand for Red Blood Cells (RBCs) continues to increase due to the aging population, this deficit will be more significant. Despite decades of research to develop hemoglobin (Hb) based oxygen (O2) carriers (HBOCs) as RBC substitutes, there are no products approved for clinical use. Lumbricus terrestris erythrocruorin (LtEc) is the large acellular O2 carrying protein complex found in the earthworm Lumbricus terrestris. LtEc is an extremely stable protein complex, resistant to autoxidation, and capable of transporting O2 to tissue when transfused into mammals. These characteristics render LtEc a promising candidate for the development of the next generation HBOCs. LtEc has a short half-life in circulation, limiting its application as a bridge over days, until blood became available. Conjugation with polyethylene glycol (PEG-LtEc) can extend LtEc circulation time. This study explores PEG-LtEc pharmacokinetics and pharmacodynamics. To study PEG-LtEc pharmacokinetics, hamsters instrumented with the dorsal window chamber were subjected to a 40% exchange transfusion with 10 g/dL PEG-LtEc or LtEc and followed for 48 hours. To study the vascular response of PEG-LtEc, hamsters instrumented with the dorsal window chamber received multiple infusions of 10 g/dL PEG-LtEc or LtEc solution to increase plasma LtEc concentration to 0.5, then 1.0, and 1.5 g/dL, while monitoring the animals' systemic and microcirculatory parameters. Results confirm that PEGylation of LtEc increases its circulation time, extending the half-life to 70 hours, 4 times longer than that of unPEGylated LtEc. However, PEGylation increased the rate of LtEc oxidation in vivo. Vascular analysis verified that PEG-LtEc showed the absence of microvascular vasoconstriction or systemic hypertension. The molecular size of PEG-LtEc did not change the colloid osmotic pressure or blood volume expansion capacity compared to LtEc, due to LtEc's already large molecular size. Taken together, these results further encourage the development of PEG-LtEc as an O2 carrying therapeutic.

At least a third of the blood supply in the world is used to transfuse 1-2 units of packed red blood cells for each intervention and most clinical trials of blood substitutes have been carried out at this level of oxygen carrying capacity (OCC) restoration. However, the increase of oxygenation achieved is marginal or none at all for molecular hemoglobin (Hb) products, due to their lingering vasoactivity. This has provided the impetus for the development of "oxygen therapeutics" using Hb-based molecules that have high oxygen affinity and target delivery of oxygen to anoxic areas. However it is still unclear how these oxygen carriers counteract or mitigate the functional effects of anemia due to obstruction, vasoconstriction and under-perfusion. Indeed, they are administered as a low dosage/low volume therapeutic Hb (subsequently further diluted in the circulatory pool) and hence induce extremely small OCC changes. Hyperviscous plasma expanders provide an alternative to oxygen therapeutics by increasing the oxygen delivery capacity (ODC); in anemia they induce supra-perfusion and increase tissue perfusion (flow) by as much as 50%. Polyethylene glycol conjugate albumin (PEG-Alb) accomplishes this by enhancing the shear thinning behavior of diluted blood, which increases microvascular endothelial shear stress, causes vasodilation and lowering peripheral vascular resistance thus facilitating cardiac function. Induction of supra-perfusion takes advantage of the fact that ODC is the product of OCC and blood flow and hence can be maintained by increasing either or both. Animal studies suggest that this approach may save a considerable fraction of the blood supply. It has an additional benefit of enhancing tissue clearance of toxic metabolites.

Perfluorocarbon (PFC) emulsions used as artificial oxygen carriers lack colloid osmotic pressure (COP) and must be administered with colloid-based plasma expanders (PEs). Although PFC emulsions have been widely studied, there is limited information about PFC emulsion interaction with PEs and blood. Their interaction forms aggregates due to electrostatic and rheological phenomena, and change blood rheology and blood flow. This study analyzes the effects of the interaction between PFC emulsions with blood in the presence of clinically-used PEs. The rheological behavior of the mixtures was analyzed in vitro in parallel with in vivo analysis of blood flow in the microcirculation using intravital microscopy, when PEs were administered in a clinically relevant scenario. The interaction between the PFC emulsion and PE with blood produced PFC droplets and red blood cell (RBCs) aggregation and increased blood viscosity in a shear dependent fashion. The PFC droplets formed aggregates when mixed with PEs containing electrolytes, and the aggregation increased with the electrolyte concentration. Mixtures of PFC with PEs that produced PFC aggregates also induced RCBs aggregation when mixed with blood, increasing blood viscosity at low shear rates. The more viscous suspension at low shear rates produced a blunted blood flow velocity profile in vivo compared to nonaggregating mixtures of PFC and PEs. For the PEs evaluated, human serum albumin produced minimal to undetectable aggregation. PFC and PEs interaction with blood can affect sections of the microcirculation with low shear rates (e.g., arterioles, venules, and pulmonary circulation) when used in a clinical setting, because persistent aggregates could cause capillary occlusion, decreased perfusion, pulmonary emboli or focal ischemia. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:796-807, 2013 [ABSTRACT FROM AUTHOR]

Erythrocyte free hemoglobin (Hb) induces vasoconstriction due to nitric oxide (NO) scavenging, limiting the NO available for vascular smooth muscle. The central objective of this study was to restore NO bioavailability using long-lived circulating NO-releasing nanoparticles (NO-np) to reverse the vasoconstriction and hypertension induced by polymerized bovine Hb (PBH) NO scavenging. PBH (13 g/dl) was infused in a volume equal to 10% of the animal blood volume. Intravascular NO supplementation was provided with an infusion of NO-np (10 and 20 mg/kg body wt). This study was performed using the hamster window chamber model to concurrently access systemic and microvascular hemodynamics. Infusion of PBH increased blood pressure and induced vasoconstriction. Treatment with 10 and 20 mg/kg NO-np reduced the blood pressure and vasoconstriction induced by PBH. Moreover, the higher dose of NO-np decreased blood pressure and induced vasodilation compared with baseline, respectively. Treatment with NO-np to decrease PBH-induced vasoconstriction increased methemoglobin levels and plasma nitrite and nitrate. In conclusion, NO-np counteracted both systemic hypertension and decreased the vasoconstrictor effects of PBH infusion, improving systemic and microvascular function. Based on the observed physiological properties, NO-np has clear potential as a therapeutic agent to replenish NO in situations where NO production is impaired, insufficient, or consumed, thereby preventing vascular complications. [ABSTRACT FROM AUTHOR]

Perfluorocarbons (PFCs) developed for the intravascular transport of oxygen have a higher solubility for gases compared to plasma and therefore can be used to transport and deliver a variety of bioactive gases for therapeutic purposes. Intravenous infusion of PFCs preequilibrated with carbon monoxide gas may provide a means to produce vasodilatation. A similar effect can be obtained by preloading PFC emulsions with nitric oxide (NO), as well as introducing unsaturated PFC emulsions in the circulation, since these can scavenge and redistribute NO. Volatile anesthetics delivered in conjunction with the intravenous presence of PFCs are more effective at lower dosages, and provide for a more uniform anesthetic effect in terms of duration of anesthesia. PFC emulsions are therefore proposed as vehicles for the transport and delivery of gases to the tissues, offering high accuracy of dosage, and avoiding uncertainties of gas delivery associated with gas inspiration via the lungs. [ABSTRACT FROM AUTHOR]

Potential shortages and perceived issues with the biosafety of blood have strengthened interest in the search for alternatives to blood. Currently available plasma expanders are effective in recovering pressure by volume expansion but are not safely deployed beyond the transfusion trigger. Conventional reasoning indicates that this limitation relates to oxygen carrying capacity reaching the threshold where it no longer can satisfy the metabolic demand of the organism; however, recent findings show that this limitation is also determined by other physical factors such as their viscosity and colloidal osmotic pressure. These fluid transport properties have a strong influence on the maintenance of microvascular perfusion and function in conditions of extreme hemodilution that obtain when resuscitation is continued beyond the transfusion trigger with plasma expanders. This realization leads to the possibility of developing new plasma expanders whose physical properties enhance microvascular function compensating for the decreased intrinsic oxygen carrying capacity in extreme hemodilution via enhanced microvascular perfusion. This review presents experimental, systemic and microvascular findings obtained using polyethylene glycol conjugated albumin solutions in extreme hemodilution and shock resuscitation in the hamster window chamber model, concluding that plasma expanders of this type can be effectively used beyond the transfusion trigger. [ABSTRACT FROM AUTHOR]

Isovolemic hemodilution to 11% systemic hematocrit was performed in the hamster window chamber model using 6% dextran 70 kDa (Dx 70) and 5% human serum albumin (HSA). Systemic and microvascular effects of these solutions were compared with polyethylene glycol (PEG)-conjugated 5% albumin (MPA) and PEG-conjugated 4.2% Hb (MP4). These studies were performed for the purpose of comparing systemic and microvascular responses of PEG vs. non-PEG plasma expanders and similar oxygen-carrying vs. noncarrying blood replacement fluids. Mean arterial blood pressure was statistically significantly reduced for all groups compared with baseline (P < 0.05), HSA, MPA, and MP4 higher than Dx 70 (P < 0.05). MP4 and MPA had a significantly higher cardiac index than HSA and Dx 70, in addition to a positive base excess. Microvascular blood flow and capillary perfusion were significantly higher for the PEG compounds compared with HSA and Dx 70. Intravascular Po2 for MP4 and MPA was higher in arterioles (P < 0.05) compared with HSA and Dx 70, but there was no difference in either tissue or venular Po2 between groups. Total Hb in the MP4 group was 4.8 ± 0.4 g/dl, whereas the remaining groups had a range of 3.6-3.8 g/dl. The hemodilution results showed that PEG compounds maintained microvascular conditions with lower concentrations than conventional plasma expanders. Furthermore, microvascular oxygen delivery and extraction in the window chamber tissue were significantly higher for the PEG compounds. MP4 was significantly higher than MPA (P < 0.05) and was not statistically different from baseline, an effect due to the additional oxygen release to the tissue by the Hb MP4. [ABSTRACT FROM AUTHOR]

A phospholipid vesicle encapsulating Hb [Hb vesicle (HbV)] has been developed as a transfusion alternative. One characteristic of HbV is that the O2 affinity [Po2 at which Hb is 50% saturated (P50)] of Hb can be easily regulated by the amount of the coencapsulated allosteric effector pyridoxal 5′-phosphate. In this study, we prepared two HbVs with different P50s (8 and 29 mmHg, termed HbV8 and HbV29, respectively) and observed their O2-releasing behavior from an occluded arteriole in a hamster skinfold window model. Conscious hamsters received HbV8 or HbV29 at a dose rate of 7 ml/kg. In the microscopic view, an arteriole (diameter: 53.0 ± 6.6 μm) was occluded transcutaneously by a glass pipette on a manipulator, and the reduction of the intra-arteriolar Po2 100 μm down from the occlusion was measured by the phosphorescence quenching of preinfused Pd-porphyrin. The baseline arteriolar Po2 (50–52 mmHg) decreased to about 5 mmHg for all the groups. Occlusion after HbV8 infusion showed a slightly slower rate of Po2 reduction compared with that after HbV29 infusion. The arteriolar O2 content was calculated at each reducing Po2 in combination with the O2 equilibrium curves of HbVs, and it was clarified that HbV8 showed a significantly slower rate of O2 release compared with HbV29 and was a primary source of O2 (maximum fraction, 0.55) overwhelming red blood cells when the Po2 was reduced (e.g., <10 mmHg) despite a small dosage of HbV. This result supports the possible utilization of Hb-based O2 carriers with lower P50 for oxygenation of ischemic tissues. [ABSTRACT FROM AUTHOR]

A surface-modified polyethylene glycol-conjugated human hemoglobin (MP4) and αα-cross-linked human hemoglobin (ααHb) were used to restore oxygen carrying capacity in conditions of extreme hemodilution (hematocrit 11%) in the hamster window model preparation. Changes in microvascular function were analyzed in terms of effects on capillary pressure and functional capillary density (FCD). MP4, at 1.0 ± 0.2 g/dl blood concentration, significantly lowered mean arterial pressure (MAP) below baseline (99.6 ± 7.6 mmHg) to 82.4 ± 6.9 mmHg (P <0.05) and decreased of FCD to 70 ± 9%. ααHb caused a greater recovery in MAP to 94.4 ± 6.2 mmHg and lowered FCD to 62 ± 8%. However, differences between ααHb and MP4 in FCD were not statistically significant. Capillary pressures were in the ranges of 17–21 mmHg for MP4 and 15–19 mmHg for ααHb, with both significantly lower than baseline (P < 0.05). Pressure in 80-μm- diameter arterioles was significantly increased with ααHb relative to MP4 (P < 0.05). These results were compared with previous findings on the relation between capillary pressure and FCD; they supported the concept of a relationship between FCD and capillary pressure. Measurement of changes in arteriolar diameter, microvascular blood flow, and FCD show that there was no statistical difference between using ααHb and MP4 in extreme hemodilution. Microvascular resistance in arterioles with a diameter range of 70–80 μm showed an increase relative to control with αα00Hb, whereas MP4 caused a decrease. [ABSTRACT FROM AUTHOR]

The O2-carrying blood substitute based on polymerized bovine hemoglobin (PBH) was used to determine efficacy in maintaining tissue PO2 after an 80% isovolemic blood exchange leading to a hematocrit of 19% [5.4 g Hb/dl from red blood cells (RBCs) and 6.3 g Hb/dl from PBH]. Effects were studied in terms of O2 delivery, O2 extraction, and tissue PO2 at the microcirculatory level at 1, 12, and 24 h after exchange transfusion in awake hamsters prepared with a window chamber model. At 1 h after exchange, arteriolar and venular diameters were decreased compared with baseline. Arteriolar diameter did not fully recover at 12 h after exchange, but venular diameter returned to normal. At 24 h after exchange, arteriolar and venular diameters were not different from baseline. Combining diameter and flow velocity data allowed us to calculate arteriolar and venular flows. At 1 h after exchange, arteriolar and venular flow was reduced compared with baseline. Arteriolar flow was lower at 12 h after exchange and recovered after 24 h. The number of capillaries with RBC passage [functional capillary density (FCD)] at 1 h after exchange with PBH was significantly lower than baseline. FCD remained decreased at 12 h; at 24 h after exchange transfusion, FCD was fully recovered. Tissue PO2 was maximal at I h after exchange and decreased progressively at 12 and 24 h after exchange. O2 release to the tissue was minimal at 1 h and increased at 12 and 24 h after exchange. These results suggest the impairment of tissue O2 metabolism after introduction of PBH into the circulation, which is mitigated as PBH concentration declines. [ABSTRACT FROM AUTHOR]

The oxygen transport capacity of nonhypertensive polyethylene glycol (PEG)conjugated hemoglobin solutions were investigated in the hamster chamber window model. Microvascular measurements were made to determine oxygen delivery in conditions of extreme hemodilution [hematocrit (Hct) 11%]. Two isovolemic hemodilution steps were performed with a 6% Dextran 70 (70-kDa molecular mass) plasma expander until Hct was 35% of control. Isovolemic blood volume exchange was continued using two surface-modified PEGylated hemoglobins (P5K2, P50 = 8.6, and P10K2, P50 = 8.3; P50 is the hemoglobin PO2 corresponding to its 50% oxygen saturation) until Hct was 11%. P5K2 and P10K2 are PEG-conjugated hemoglobins that maintain most of the hemoglobin allosteric properties and have a cooperativity index of n = 2.2. The effects of these molecular solutions were compared with those obtained in a previous study using MP4, a PEG-modified hemoglobin whose P50 was 5.4 and cooperativity was 1.2 (Tsai et al., Am J Physiol Heart Circ Physiol 285:H1411-H1419, 2003). Tissue oxygen levels were higher after P5K2 (7.0 ± 2.5 mmHg) and P10K2 (6.3 ± 2.3 mmHg) versus MP4 (1.7 ± 0.5 mmHg) or the nonoxygen carrier Dextran 70 (1.3 ± 1.2 mmHg). Microvascular oxygen delivery was higher after P5K2 and P10K2 (2.22 and 2.34 ml O2/dl blood) compared with MP4 (1.41 ml O2/dl blood) or Dextran 70 (0.90 ml O2/dl blood); however, all these values were lower than control (7.42 ml O2/dl blood). The total hemoglobin in blood was similar in all cases; therefore, the improvement in tissue PO2 and oxygen delivery appears to be due to the increased cooperativity of the new molecules. [ABSTRACT FROM AUTHOR]

The oxygen transport capacity of fluorocarbons was investigated in the hamster chamber window model microcirculation to determine the rate at which oxygen is delivered to the tissue in conditions of extreme hemodilution [hematocrit (Hct) 11%]. Hydroxyethlyl starch (HES 200; 200 kDa molecular mass) was used as a plasma expander for two isovolemic hemodilutions performed with 10% HES 200 until a Hct of 65%. A third step reduced the Hct to 75% of baseline and was performed with either HES 200 or a 60% perfluorocarbon (PFC) emulsion. Comparisons of HES 200 only-hemodiluted animals versus 4.2 g/kg PFC emulsion-hemodiluted animals were made at 21% and 100% normobaric oxygen ventilation. It was found that systemic and microvascular oxygen delivery was 25% and 400% higher in the PFC animals compared with HES 200 animals, respectively, showing that PFCs deliver oxygen to the tissue when combined with hyperoxic ventilation in the present experiments, with no evidence of vasoconstriction or impaired microvascular function. Oxygen ventilation (100%) led to a positive base excess for the PFC group (5.5 ± 2.5 mmol/1) versus a negative balance (-0.8 ± 1.4 mmol/l) for the HES 200 group, suggesting that microvascular findings corresponded to systemic events. [ABSTRACT FROM AUTHOR]