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This study aims to extend earlier Krogh Cylinder Models of an oxygen profile by considering axial diffusion and analytically solving Fick's Law Partial Differential Equation with novel boundary conditions via the separation of variables. We next prospectively collected a total of 20 animals, which were randomly assigned to receive either fresh or two-week-old stored red blood cell (RBC) transfusions and PQM oxygen data were measured acutely (90 min) or chronically (24 h). Transfusion effects were evaluated in vivo using intravital microscopy of the dorsal skinfold window chamber in Golden Syrian Hamsters. Hamsters were initially hemorrhaged by 50% of total blood volume and resuscitated 1-h post hemorrhage. PQM data were subsequently collected and fit the derived 2D Krogh cylinder model. Systemic hemodynamics (mean arterial pressure, heart rate) were similar in both pre and post-transfusion with either stored or fresh cells. Transfusion with stored cells was found to impair axial and radial oxygen gradients as quantified by our model and consistent with previous studies. Specifically, we observed a statistically significant decrease in the arteriolar tissue radial oxygen gradient after transfusion with stored RBCs at 24 h compared with fresh RBCs (0.33 ± 0.17 mmHg μ m-1 vs, 0.14 ± 0.12 mmHg μ m-1; p = 0.0280). We also observed a deficit in the arteriolar tissue oxygen gradient (0.03 ± 0.01 mmHg μ m-1 fresh vs. 0.018 ± 0.007 mmHg μ m-1 stored; p = 0.0185). We successfully derived and validated an analytical 2D Krogh cylinder model in an animal model of microhemodynamic oxygen diffusion aberration secondary to storage lesions., Competing Interests: Declaration of competing interest None Declared., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Hyperspectral imaging (HSI) provides a fast, reliable, and noninvasive way for the study of vascular microcirculation in animal models. Rapid hyperspectral imaging of large portions of the microcirculatory preparation is critical for understanding the function and regulation of vascular microcirculatory networks. This report presents the application of an off-the-shelf benchtop HSI linear scanning system to acquire larger field-of-view images of microcirculatory preparations. The HSI line detector was displaced perpendicular to the scanning direction to map larger areas, with a rate of displacement determined by the scanning rate and the exposure time. Analysis of the collected image was used to assess dynamic changes in the microcirculation. The system records dynamic changes in microvascular hemoglobin oxygen (HbO 2 ) saturation and vascular morphology during hypoxia and reoxygenation and has similar acquisition speeds to commonly referenced spectral-scanning HSI systems. In addition, the HbO 2 saturations collected via HSI closely correlate with those collected by phosphorescence quenching microscopy. The reported system enables dynamic functional imaging of the microcirculation for broad experimental and clinical applications. NEW & NOTEWORTHY This study presents a novel bench setup and algorithm to measure intravascular hemoglobin oxygen saturation in microcirculation. Wide-field hyperspectral imaging allows for rapid quantification of intravascular changes in hemoglobin saturation. The method described in this manuscript can expand the understanding of oxygen delivery to tissues in vivo.

Therapeutic agents that increase the Hb affinity for oxygen (O 2 ) could, in theory, lead to decreased O 2 release from Hb and impose a hypoxic risk to tissues. In this study, GBT1118, an allosteric modifier of Hb affinity for O 2 , was used to assess the impact of increasing Hb affinity for O 2 on brain tissue oxygenation, blood pressure, heart rate, O 2 delivery, and tolerance to hypoxia in Townes transgenic sickle cell disease (SCD) mice. Brain oxygenation and O 2 delivery were studied during normoxia and severe hypoxic challenges. Chronic treatment with GBT1118 increased Hb affinity for O 2 , reducing the Po 2 for 50% HbO 2 saturation (P50) in SCD mice from 31 mmHg to 18 mmHg. This treatment significantly reduced anemia, increasing hematocrit by 33%, improved cardiac output (CO), and O 2 delivery and extraction. Chronically increasing Hb affinity for O 2 with GBT1118 preserved cortical O 2 tension during normoxia, improved cortical O 2 tension during hypoxia, and increased tolerance to severe hypoxia in SCD mice. Independent of hematological changes induced by chronic treatment, a single dose of GBT1118 significantly improved tolerance to hypoxia, highlighting the benefits of increasing Hb affinity for O 2 and consequently reducing sickling of RBCs in blood during hypoxia in SCD. NEW & NOTEWORTHY Chronic pharmacologically increased hemoglobin affinity for oxygen in sickle cell disease mice alleviated hematological consequences of sickle cell disease, increasing RBC half-life, hematocrit, and hemoglobin concentration, while also decreasing reticulocyte count. Additionally, chronically increased hemoglobin affinity for oxygen significantly improved survival as well as cortical tissue oxygenation in sickle cell disease mice during hypoxia, suggesting that oxygen delivery and utilization is improved by increased hemoglobin affinity for oxygen.

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.)

Oxygen (O2) delivery facilitated by hemoglobin (Hb)-based O2 carriers (HBOCs) is a promising strategy to increase the effectiveness of chemotherapeutics for treatment of solid tumors. However, the heterogeneous vascular structures present within tumors complicates evaluating the oxygenation potential of HBOCs within the tumor microenvironment. To account for spatial variations in the vasculature and tumor tissue that occur during tumor growth, we used a computational model to develop artificial tumor constructs. With these simulated tumors, we performed a polymerized human hemoglobin (hHb) (PolyhHb) enhanced oxygenation simulation accounting for differences in the physiologic characteristics of human and mouse blood. The results from this model were used to determine the potential effectiveness of different treatment options including a top load (low volume) and exchange (large volume) infusion of a tense quaternary state (T-State) PolyhHb, relaxed quaternary state (R-State) PolyhHb, and a non O2 carrying control. Principal component analysis (PCA) revealed correlations between the different regimes of effectiveness within the different simulated dosage options. In general, we found that infusion of T-State PolyhHb is more likely to decrease tissue hypoxia and modulate the metabolic rate of O2 consumption. Though the developed models are not a definitive descriptor of O2 carrier interaction in tumor capillary networks, we accounted for factors such as non-uniform vascular density and permeability that limit the applicability of O2 carriers during infusion. Finally, we have used these validated computational models to establish potential benchmarks to guide tumor treatment during translation of PolyhHb mediated therapies into clinical applications., Competing Interests: The authors have declared that no competing interests exist.

Administration of hemoglobin-based oxygen carriers (HBOCs) into the systemic circulation is a potential strategy to relieve solid tumor hypoxia in order to increase the effectiveness of chemotherapeutics. Previous computational analysis indicated that the oxygen (O 2 ) status of the tumor and HBOC O 2 affinity may play a role in increased O 2 delivery to the tumor. However, no study has experimentally investigated how low- and high-affinity HBOCs would perform in normoxic and hypoxic tumors. In this study, we examined how the HBOC, polymerized human hemoglobin (PolyhHb), in the relaxed (R) or tense (T) quaternary state modulates O 2 delivery to hypoxic (FME) and normoxic (LOX) human melanoma xenografts in a murine window chamber model. We examined microcirculatory fluid flow via video shearing optical microscopy, and O 2 distributions via phosphorescence quenching microscopy. Additionally, we examined how weekly infusion of a 20% top-load dose of PolyhHb influences growth rate, vascularization, and regional blood flow in the FME and LOX tumor xenografts. Infusion of low-affinity T-state PolyhHb led to increased tissue oxygenation, decreased blood flow, decreased tumor growth, and decreased vascularization in hypoxic tumors. However, infusion of both T-state and R-state PolyhHbs led to worse outcomes in normoxic tumors. Of particular concern was the high-affinity R-state PolyhHb, which led to no improvement in hypoxic tumors and significantly worsened outcomes in normoxic tumors. Taken together, the results of this study indicate that the tumor O 2 status is a primary determinant of the potency and outcomes of infused PolyhHb.

The microcirculation is a complex network of vessels ranging from as large as 100 μm to as small as 5 μm. This complex network is responsible for the regulation of oxygen to the surrounding tissues and ensures metabolite washout. With a more complete understanding of the microcirculation's physiologic and pathologic tendencies, engineers can create new solutions to combat blood pathologies and shock-related diseases. Over the last number of decades a grown interest in the microcirculation has resulted in the development of fundamental techniques to quantify the microvasculature flow and the release of oxygen to tissues., Competing Interests: Disclosure The authors have nothing to disclose., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Resuscitation from hemorrhagic shock (HS) by blood transfusion restores oxygen (O2) delivery and provides hemodynamic stability. Current regulations allow red blood cells (RBCs) to be stored and used for up to 42 days. During storage, RBCs undergo many structural and functional changes. These storage lesions have been associated with adverse events and increased mortality after transfusion, increasing the need for improved RBC storage protocols. This study evaluates the efficacy of anaerobically stored RBCs to resuscitate rats from severe HS compared with conventionally stored RBCs., Methods and Results: Rat RBCs were stored under anaerobic, anaerobic/hypercapnic, or conventional conditions for a period of 3 weeks. Hemorrhage was induced by controlled bleeding, shock was maintained for 30 min, and RBCs were transfused to restore and maintain blood pressure near the prhemorrhage level. All storage conditions met current regulatory 24-h posttransfusion recovery requirements. Transfusion of anaerobically stored RBCs required significantly less RBC volume to restore and maintain hemodynamics. Anaerobic or anaerobic/hypercapnic RBCs restored hemodynamics better than conventionally stored RBCs. Resuscitation with conventionally stored RBCs impaired indices of left ventricular cardiac function, increased hypoxic tissue staining and inflammatory markers, and affected organ function compared with anaerobically stored RBCs., Conclusions: Resuscitation from HS via transfusion of anaerobically stored RBCs recovered cardiac function, restored hemodynamic stability, and improved outcomes.

Pure tense (T) and relaxed (R) quaternary state polymerized human hemoglobins (PolyhHbs) were synthesized and their biophysical properties characterized, along with mixtures of T- and R-state PolyhHbs. It was observed that the oxygen affinity of PolyhHb mixtures varied linearly with T-state mole fraction. Computational analysis of PolyhHb facilitated oxygenation of a single fiber in a hepatic hollow fiber (HF) bioreactor was performed to evaluate the oxygenation potential of T- and R-state PolyhHb mixtures. PolyhHb mixtures with T-state mole fractions greater than 50% resulted in hypoxic and hyperoxic zones occupying less than 5% of the total extra capillary space (ECS). Under these conditions, the ratio of the pericentral volume to the perivenous volume in the ECS doubled as the T-state mole fraction increased from 50 to 100%. These results show the effect of varying the T/R-state PolyhHb mole fraction on oxygenation of tissue-engineered constructs and their potential to oxygenate tissues.

Adaptation to hypoxia requires compensatory mechanisms that affect O 2 transport and utilization. Decreased hemoglobin (Hb) O 2 affinity is considered part of the physiological adaptive process to chronic hypoxia. However, this study explores the hypothesis that increased Hb O 2 affinity can complement acute physiological responses to hypoxia by increasing O 2 uptake and delivery compared with normal Hb O 2 affinity during acute severe hypoxia. To test this hypothesis, Hb O 2 affinity in mice was increased by oral administration of 2-hydroxy-6-{[(2 S )-1-(pyridine-3-carbonyl)piperidin-2yl] methoxy}benzaldehyde (GBT1118; 70 or 140 mg/kg). Systemic and microcirculatory hemodynamics and oxygenation parameters were studied during hypoxia in awake-instrumented mice. GBT1118 increased Hb O 2 affinity and decreased the Po 2 at which 50% of Hb is saturated with O 2 (P 50 ) from 43 ± 1.1 to 18.3 ± 0.9 mmHg (70 mg/kg) and 7.7 ± 0.2 mmHg (140 mg/kg). In a dose-dependent fashion, GBT1118 increased arterial O 2 saturation by 16% (70 mg/kg) and 40% (140 mg/kg) relative to the control group during 5% O 2 hypoxia. In addition, a GBT1118-induced increase in Hb O 2 affinity reduced hypoxia-induced hypotension compared with the control group. Moreover, microvascular blood flow was higher during hypoxia in GBT1118-treated groups than the control group. The increased O 2 saturation and improved blood flow in GBT1118-treated groups preserved higher interstitial tissue Po 2 than in the control group during 5% O 2 hypoxia. In conclusion, increased Hb O 2 affinity enhanced physiological tolerance to hypoxia, as evidenced by improved hemodynamics and tissue oxygenation. Therefore, pharmacologically induced increases in Hb O 2 affinity become a potential therapeutic approach to improve tissue oxygenation in pulmonary diseases characterized by severe hypoxemia. NEW & NOTEWORTHY This study establishes that pharmacological modification of hemoglobin O 2 affinity can be a promising and novel therapeutic strategy for the treatment of hypoxic hypoxia and paves the way for the clinical development of molecules that prevent hypoxemia., (Copyright © 2017 the American Physiological Society.)

Hemorrhage impairs myocardial contractile function and decreases oxygen delivery. This study investigates how polymerized bovine hemoglobin (PolyHb) solutions affect cardiac function after resuscitation from hemorrhagic shock (HS). Hamsters were hemorrhaged and resuscitated with PolyHb at 8.5 g/dL and 11.5 g/dL. Left ventricle (LV) function was assessed during shock and resuscitation using a miniaturize conductance catheter. PolyHb resuscitation had no beneficial effects in cardiac function; it increased cardiac afterload and systemic vascular resistance (SVR) of 46 and 116% for 8.5 and 11.5 g/dL, respectively. Study findings indicate that preclinical evaluation of cardiac function is essential to develop safe and efficacious alternatives to blood transfusion.

Background: Blood transfusion is used to treat acute anemia with the goal of increasing blood oxygen-carrying capacity as determined by hematocrit (Hct) and oxygen delivery (DO2). However, increasing Hct also increases blood viscosity, which may thus lower DO2 if the arterial circulation is a rigid hydraulic system as the resistance to blood flow will increase. The net effect of transfusion on DO2 in this system can be analyzed by using the relationship between Hct and systemic blood viscosity of circulating blood at the posttransfusion Hct to calculate DO2 and comparing this value with pretransfusion DO2. We hypothesized that increasing Hct would increase DO2 and tested our hypothesis by mathematically modeling DO2 in the circulation., Methods: Calculations were made assuming a normal cardiac output (5 L/min) with degrees of anemia ranging from 5% to 80% Hct deficit. We analyzed the effects of transfusing 0.5 or more units of 300 cc of packed red blood cells (PRBCs) at an Hct of 65% and calculated microcirculatory DO2 after accounting for increased blood viscosity and assuming no change in blood pressure. Our model accounts for O2 diffusion out of the circulation before blood arriving to the nutritional circulation and for changes in blood flow velocity. The immediate posttransfusion DO2 was also compared with DO2 after the transient increase in volume due to transfusion has subsided., Results: Blood transfusion of up to 3 units of PRBCs increased DO2 when Hct (or hemoglobin) was 60% lower than normal, but did not increase DO2 when administered before this threshold., Conclusions: After accounting for the effect of increasing blood viscosity on blood flow owing to increasing Hct, we found in a mathematical simulation of DO2 that transfusion of up to 3 units of PRBCs does not increase DO2, unless anemia is the result of an Hct deficit greater than 60%. Observations that transfusions occasionally result in clinical improvement suggest that other mechanisms possibly related to increased blood viscosity may compensate for the absence of increase in DO2.

Hypoxia is the major hindrance to successful radiation therapy of tumors. Attempts to increase the oxygen (O2) tension (PO2) of tissue by delivering more O2 have been clinically disappointing, largely due to the way O2 is transported and released by the hemoglobin (Hb) within the red blood cells (RBCs). Systemic manipulation of O2 transport increases vascular resistance due to metabolic autoregulation of blood flow to prevent over oxygenation. This study investigates a new technology to increase O2 delivery to a target tissue by decreasing the Hb-O2 affinity of the blood circulating within the targeted tissue. As the Hb-O2 affinity decreases, the tissue PO2 to satisfy tissue O2 metabolic needs increases without increasing O2 delivery or extraction. Paramagnetic nanoparticles (PMNPs), synthetized using gadolinium oxide, were coated with the cell permeable Hb allosteric effector L35 (3,5-trichlorophenylureido-phenoxy-methylpropionic acid). L35 decreases Hb affinity for O2 and favors the release of O2. The L35-coated PMNPs (L35-PMNPs) were intravenously infused (10 mg kg(-1)) to hamsters instrumented with the dorsal window chamber model. A magnetic field of 3 mT was applied to localize the effects of the L35-PMNPs to the window chamber. Systemic O2 transport characteristics and microvascular tissue oxygenation were measured after administration of L35-PMNPs with and without magnetic field. The tissue PO2 in untreated control animals was 25.2 mmHg. L35-PMNPs without magnetic field decreased tissue PO2 to 23.4 mmHg, increased blood pressure, and reduced blood flow, largely due to systemic modification of Hb-O2 affinity. L35-PMNPs with magnetic field increased tissue PO2 to 27.9 mmHg, without systemic or microhemodynamic changes. These results indicate that localized modification of Hb-O2 affinity can increase PO2 of target tissue without affecting systemic O2 delivery or triggering O2 autoregulation mechanisms. This technology can be used to treat local hypoxia and to increase O2 in tumors, enhancing the efficacy of radiation therapies.

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: Despite many advances in blood substitute research, the development of materials that are effective in maintaining blood volume and oxygen delivery remains a priority for emergency care and trauma. Clinical trials on hemoglobin (Hb)-based oxygen carriers (HBOCs) have not provided information on the mechanism of toxicity, although all commercial formulations have safety concerns. Specifically, it is important to reconcile the different hypotheses of Hb toxicity, such as nitric oxide (NO) depletion and oxidative reactions, to provide a coherent molecular basis for designing a safe HBOC., Recent Advances: HBOCs with different sizes often exhibit differences in the degree of HBOC-induced vasoactivity. This has been attributed to differences in the degree of NO scavenging and in the extent of Hb extravasation. Additionally, it is appears that Hb can undergo reactions that compensate for NO scavenging by generating bioactive forms of NO., Critical Issues: Engineering modifications to enhance bioactive NO production can result in diminished oxygen delivery by virtue of increased oxygen affinity. This strategy can prevent the HBOC from fulfilling the intended goal on preserving oxygenation; however, the NO production effects will increase perfusion and oxygen transport., Future Directions: Hb modifications influence NO scavenging and the capacity of certain HBOCs to compensate for NO scavenging through nitrite-mediated reactions that generate bioactive NO. Based on the current understanding of these NO-related factors, possible synthetic strategies are presented that address how HBOC formulations can be prepared that: (i) effectively deliver oxygen, (ii) maintain tissue perfusion, and (iii) limit/reverse underlying inflammation within the vasculature.

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.

Acclimatization to hypoxia requires time to complete the adaptation mechanisms that influence oxygen (O(2)) transport and O(2) utilization. Although decreasing hemoglobin (Hb) O(2) affinity would favor the release of O(2) to the tissues, increasing Hb O(2) affinity would augment arterial O(2) saturation during hypoxia. This study was designed to test the hypothesis that pharmacologically increasing the Hb O(2) affinity will augment O(2) transport during severe hypoxia (10 and 5% inspired O(2)) compared with normal Hb O(2) affinity. RBC Hb O(2) affinity was increased by infusion of 20 mg/kg of 5-hydroxymethyl-2-furfural (5HMF). Control animals received only the vehicle. The effects of increasing Hb O(2) affinity were studied in the hamster window chamber model, in terms of systemic and microvascular hemodynamics and partial pressures of O(2) (Po(2)). Pimonidazole binding to hypoxic areas of mice heart and brain was also studied. 5HMF decreased the Po(2) at which the Hb is 50% saturated with O(2) by 12.6 mmHg. During 10 and 5% O(2) hypoxia, 5HMF increased arterial blood O(2) saturation by 35 and 48% from the vehicle group, respectively. During 5% O(2) hypoxia, blood pressure and heart rate were 58 and 30% higher for 5HMF compared with the vehicle. In addition, 5HMF preserved microvascular blood flow, whereas blood flow decreased to 40% of baseline in the vehicle group. Consequently, perivascular Po(2) was three times higher in the 5HMF group compared with the control group at 5% O(2) hypoxia. 5HMF also reduced heart and brain hypoxic areas in mice. Therefore, increased Hb O(2) affinity resulted in hemodynamics and oxygenation benefits during severe hypoxia. This acute acclimatization process may have implications in survival during severe environmental hypoxia when logistic constraints prevent chronic acclimatization.

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.)

In a recent study, ultrahigh molecular weight (Mw ) glutaraldehyde-polymerized bovine hemoglobins (PolybHbs) were synthesized with low O2 affinity and exhibited no vasoactivity and a slight degree of hypertension in a 10% top-load model.(1) In this work, we systematically investigated the effect of varying the glutaraldehyde to hemoglobin (G:Hb) molar ratio on the biophysical properties of PolybHb polymerized in either the low or high O2 affinity state. Our results showed that the Mw of the resulting PolybHbs increased with increasing G:Hb molar ratio. For low O2 affinity PolybHbs, increasing the G:Hb molar ratio reduced the O2 affinity and CO association rate constants in comparison to bovine hemoglobin (bHb). In contrast for high O2 affinity PolybHbs, increasing the G:Hb molar ratio led to increased O2 affinity and significantly increased the CO association rate constants compared to unmodified bHb and low O2 affinity PolybHbs. The methemoglobin level and NO dioxygenation rate constants were insensitive to the G:Hb molar ratio. However, all PolybHbs displayed higher viscosities compared to unmodified bHb and whole blood, which also increased with increasing G:Hb molar ratio. In contrast, the colloid osmotic pressure of PolybHbs decreased with increasing G:Hb molar ratio. To preliminarily evaluate the ability of low and high O2 affinity PolybHbs to potentially oxygenate tissues in vivo, an O2 transport model was used to simulate O2 transport in a hepatic hollow fiber (HF) bioreactor. It was observed that low O2 affinity PolybHbs oxygenated the bioreactor better than high O2 affinity PolybHbs. This result points to the suitability of low O2 affinity PolybHbs for use in tissue engineering and transfusion medicine. Taken together, our results show the quantitative effect of varying the oxygen saturation of bHb and G:Hb molar ratio on the biophysical properties of PolybHbs and their ability to oxygenate a hepatic HF bioreactor. We suggest that the information gained from this study can be used to guide the design of the next generation of hemoglobin-based oxygen carriers (HBOCs) for use in tissue engineering and transfusion medicine applications., (Copyright © 2011 American Institute of Chemical Engineers (AIChE).)

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.

Recent clinical evaluation of commercial glutaraldehyde-polymerized hemoglobins (PolyHbs) as transfusion solutions has demonstrated several adverse side effects. Chief among these is the hypertensive effect. Fortunately, previous studies have shown that the hypertensive effect can be attenuated by removing free hemoglobin (Hb) and low-molecular-weight (low-MW) PolyHbs from the PolyHb mixture. In this work, polymerized human Hb (PolyhHb) solutions were synthesized in two distinct quaternary states with high MW and subjected to extensive diafiltration to remove free Hb and low-MW PolyhHb components (<500 kDa). The resultant PolyhHb solutions possessed high MW, distinct quaternary state, distinct reactivities with O(2) and CO, similar NO deoxygenating rate constants, distinct autoxidation rate constants, high viscosity, and low colloid osmotic pressure. To preliminarily assess the ability of PolyhHb solutions to oxygenate surrounding tissues fed by a blood vessel, we evaluated the ability of PolyhHbs to transport O(2) to cultured hepatocytes in a mathematical model of a hollow fiber bioreactor. The structure of individual hollow fibers in the bioreactor is similar to that of a blood vessel and provides an easy way to assess the oxygenation potential of PolyhHbs without the need for expensive and time-consuming animal studies. It was observed that PolyhHbs with low O(2) affinities were more effective in oxygenating cultured hepatocytes inside the bioreactor than high O(2) affinity PolyhHbs. Taken together, our results show that it is possible to synthesize high-MW PolyhHbs with no free Hb and low-MW PolyhHb components that are capable of transporting O(2) to cultured cells/tissues.

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).

We review the experimental evidence showing systemic and microvascular effects of blood transfusions instituted to support the organism in extreme hemodilution and hemorrhagic shock, focusing on the use of fresh vs. stored blood as a variable. The question: "What does a blood transfusion remedy?" was analyzed in experimental models addressing systemic and microvascular effects showing that oxygen delivery is not the only function that must be addressed. In extreme hemodilution and hemorrhagic shock blood transfusions simultaneously restore blood viscosity and oxygen carrying capacity, the former being critically needed for re-establishing a functional mechanical environment of the microcirculation, necessary for obtaining adequate capillary blood perfusion. Increased oxygen affinity due to 2,3 DPG depletion is shown to have either no effect or a positive oxygenation effect, when the transfused red blood cells (RBCs) do not cause additional flow impairment due to structural malfunctions including increased rigidity and release of hemoglobin. It is concluded that fresh RBCs are shown to be superior to stored RBCs in transfusion, however increased oxygen affinity may be a positive factor in hemorrhagic shock resuscitation. Although experimental studies seldom reproduce emergency and clinical conditions, nonetheless they serve to explore fundamental physiological mechanisms in the microcirculation that cannot be directly studied in humans., ((c) 2010 Elsevier Ltd. All rights reserved.)

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.)

Hemoglobin (Hb)-based O(2) carriers (HBOCs) constitute a class of therapeutic agents designed to correct the O(2) deficit under conditions of anemia and traumatic blood loss. The O(2) transport capacity of ultrahigh-molecular-weight bovine Hb polymers (PolybHb), polymerized in the tense (T) state and relaxed (R) state, were investigated in the hamster chamber window model using microvascular measurements to determine O(2) delivery during extreme anemia. The anemic state was induced by hemodilution with a plasma expander (70-kDa dextran). After an initial moderate hemodilution to 18% hematocrit, animals were randomly assigned to exchange transfusion groups based on the type of PolybHb solution used (namely, T-state PolybHb and R-state PolybHb groups). Measurements of systemic parameters, microvascular hemodynamics, capillary perfusion, and intravascular and tissue O(2) levels were performed at 11% hematocrit. Both PolybHbs were infused at 10 g/dl, and their viscosities were higher than nondiluted blood. Restitution of the O(2) carrying capacity with T-state PolybHb exhibited lower arterial pressure and higher functional capillary density compared with R-state PolybHb. Central arterial O(2) tensions increased significantly for R-state PolybHb compared with T-state PolybHb; conversely, microvascular O(2) tensions were higher for T-state PolybHb compared with R-state PolybHb. The increased tissue Po(2) attained with T-state PolybHb results from the larger amount of O(2) released from the PolybHb and maintenance of macrovascular and microvascular hemodynamics compared with R-state PolybHb. These results suggest that the extreme high O(2) affinity of R-state PolybHb prevented O(2) bound to PolybHb from been used by the tissues. The results presented here show that T-state PolybHb, a high-viscosity O(2) carrier, is a quintessential example of an appropriately engineered O(2) carrying solution, which preserves vascular mechanical stimuli (shear stress) lost during anemic conditions and reinstates oxygenation, without the hypertensive or vasoconstriction responses observed in previous generations of HBOCs.

Microvascular responses to blood volume restitution using red blood cells (RBCs) with modified hemoglobin (Hb) oxygen affinity were studied in the hamster window chamber model during resuscitation from hemorrhagic shock. Allosteric effectors inositol hexaphosphate and 5-hydroxymethyl-2-furfural were introduced into the RBCs by electroporation to decrease and increase Hb-oxygen affinity. In vitro P50s (partial pressure of oxygen at 50% Hb saturation) were modified to 10 and 50 mmHg (normal P50, 32 mmHg). Awake hamsters were subjected to hemorrhage of 50% of blood volume, followed by a shock period of 1 h, and then resuscitated with 25% blood volume with high or low P50 RBCs (hematocrit, 50%). After resuscitation, base excess was significantly lower than baseline in the high-P50 RBC group (HP50; 0.3 +/- 2 vs. 5.0 +/- 1.7 mM) and MAP was lower than baseline in the low-P50 RBC group (LP50; 93 +/- 6 vs. 109 +/- 6 mM). Arteriolar diameter and flow were significantly lower in the HP50. Functional capillary density in the HP50 was significantly lower than LP50 at 60 and 90 min after resuscitation. There was no significantly difference in arteriolar PO2. Tissue PO2, venular PO2, and oxygen delivery were higher in LP50 than in HP50. There was no significant difference in oxygen extraction. Oxygen extraction ratio (oxygen extraction/oxygen delivery) x 100 was significantly higher in HP50 than in LP50. These results suggest that lowering blood P50 in resuscitation provides improved microvascular function in comparison with higher P50.

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.

The development of volume replacement fluids for resuscitation in hemorrhagic shock comprises oxygen carrying and non carrying fluids. Non oxygen carrying fluids or plasma expanders are used up to the transfusion trigger, and upon reaching this landmark either blood, and possibly in the near future oxygen carrying blood substitutes, are used. An experimental program in hemorrhagic shock using the hamster chamber window model allowed to compare the relative performance of most fluids proposed for shock resuscitation. This model allows investigating simultaneously the microcirculation and systemic reactions, in the awake condition, in a tissue isolated from the environment. Results from this program show that in general plasma expanders such as Ringer's lactate and dextran 70 kDa do not sufficiently restore blood viscosity upon reaching the transfusion trigger, causing microvascular collapse. This is in part restored by a blood transfusion, independently of the oxygen carrying capacity of red blood cells. These results lead to the proposal that effective blood substitutes must be designed to prevent microvascular collapse, manifested in the decrease of functional capillary density. Achievement of this goal, in combination with the increase of oxygen affinity, significantly postpones the need for a blood transfusion, and lowers the total requirement of restoration of intrinsic oxygen carrying capacity.

Background: Hemoglobin (Hb) solutions are potential alternatives to blood transfusion when native oxygen (O(2))-carrying capacity is lacking. Polymerized bovine Hb (PBH) solutions are characterized by its vasoactivity, low O(2) affinity, oncotic effect, prolonged shelf life, and stability. Responses to facilitated O(2) transport, after exchange transfusion with PBH, were studied in the hamster window chamber model during acute extreme anemia to determine how PBH affects microvascular perfusion and tissue oxygenation., Study Design and Methods: An anemic state was induced by hemodilution with a plasma expander (70-kDa dextran). After hemodilution, animals were randomly assigned to exchange transfusion groups based on the concentration of PBH used, namely, PBH at 13 g(Hb) per dL (PBH13), PBH diluted to 8 or 4 g(Hb) per dL in albumin solution at matching colloidal osmotic pressure (COP; PBH8 and PBH4), and no PBH only albumin solution at matching COP (PBH0). Measurement of systemic variables, microvascular hemodynamics, capillary perfusion, and intravascular and tissue O(2) levels was performed at 11 percent Hct., Results: Restitution of O(2)-carrying capacity with PBH13 restored higher arterial pressure and triggered vasoconstriction, low perfusion, and higher peripheral resistance. Groups PBH4 and PBH0 had lower arterial pressures than Group PBH13. Groups PBH4 and PBH8 maintained higher perfusion and functional capillary density than Groups PBH13 and PBH0. In all groups, blood gas variables and acid-base balance were recovered proportional to microvascular perfusion. Arterial O(2) tensions were improved for Groups PBH4 and PBH8 by preventing O(2) precapillary release and increasing O(2) reserve., Conclusion: Further studies to establish acellular Hb optimal dosage, efficacy, safety, and effects on outcome are indicated before these solutions 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.

Responses to exchange transfusion using red blood cells (RBCs) with modified hemoglobin (Hb) oxygen (O(2)) affinity were studied in the hamster window chamber model during acute anemia to determine its role on microvascular perfusion and tissue oxygenation. Allosteric effectors were introduced in the RBCs by electroporation. Inositol hexaphosphate (IHP) and 5-hydroxymethyl-2-furfural (5HMF) were used to decrease and increase Hb-O(2) affinity. In vitro P50s (partial pressure of O(2) at 50% Hb saturation) were modified to 10, 25, 45, and 50 mm Hg (normal P50 is 32 mm Hg). Allosteric effectors also decreased the Hill coefficient. Anemic condition was induced by isovolemic hemodilution exchanges using 6% dextran 70 kD to 18% hematocrit (Hct). Modified RBCs (at 18% Hct in 5% albumin solution) were infused by exchange transfusion of 35% of blood volume. Systemic parameters, microvascular perfusion, capillary perfusion (functional capillary density, FCD), and microvascular Po(2) levels were measured. RBcs with P50 of 45 mm Hg increased tissue Po(2) and decreased O(2) delivery (Do(2)) and extraction (Vo(2)) and RBCs with P50 of 60 mmHg reduced FCD, microvascular flow, tissue Po(2), Do(2) and Vo(2). Erythrocytes with increased Hb-O(2) affinity maintained hemodynamic conditions, Do(2) and decreased tissue Po(2). This study shows that in an anemic condition, maximal tissue Po(2) does not correspond to maximal Do(2) and Vo(2).

Systemic and microvascular hemodynamic responses to transfusion of oxygen using functional and non-functional packed fresh red blood cells (RBCs) from hemorrhagic shock were studied in the hamster window chamber model to determine the significance of RBCs on rheological and oxygen transport properties. Moderate hemorrhagic shock was induced by arterial controlled bleeding of 50% of the blood volume, and a hypovolemic state was maintained for 1h. Volume restitution was performed by infusion of the equivalent of 2.5 units of packed cells, and the animals were followed for 90 min. Resuscitation study groups were non-oxygen functional fresh RBCs where the hemoglobin (Hb) was converted to methemoglobin (MetHb) [MetRBC], fully oxygen functional fresh RBCs [OxyRBC] and 10% hydroxyethyl starch [HES] as a volume control solution. Measurement of systemic variables, microvascular hemodynamics and capillary perfusion were performed during the hemorrhage, hypovolemic shock and resuscitation. Final blood viscosities after the entire protocol were 3.8 cP for transfusion of RBCs and 2.9 cP for resuscitation with HES (baseline: 4.2 cP). Volume restitution with RBCs with or without oxygen carrying capacity recovered higher mean arterial pressure (MAP) than HES. Functional capillary density (FCD) was substantially higher for transfusion versus HES, and the presence of MetHb in the fresh RBC did not change FCD or microvascular hemodynamics. Oxygen delivery and extraction were significantly lower for resuscitation with HES and MetRBC compared to OxyRBC. Incomplete re-establishment of perfusion after resuscitation with HES could also be a consequence of the inappropriate restoration of blood rheological properties which unbalance compensatory mechanisms, and appear to be independent of the reduction in oxygen carrying capacity.

Responses to exchange transfusion using red blood cells (RBCs) with normal and reduced flexibility were studied in the hamster window chamber model during acute moderate isovolemic hemodilution to determine the role of RBC membrane stiffness in microvascular perfusion and tissue oxygenation. Erythrocyte stiffness was increased by 30-min incubation in 0.02% glutaraldehyde solution, and unreacted glutaraldehyde was completely removed. Filtration pressure through 5-microm pore size filters was used to quantify stiffness of the RBCs. Anemic conditions were induced by two isovolemic hemodilution steps using 6% 70-kDa dextran to a hematocrit (Hct) of 18% (moderate hemodilution). The protocol continued with an exchange transfusion to reduce native RBCs to 75% of baseline (11% Hct) with either fresh RBCs (RBC group) or reduced-flexibility RBCs (GRBC group) suspended in 5% albumin at 18% Hct; a plasma expander (6% 70-kDa dextran; Dex70 group) was used as control. Systemic parameters, microvascular perfusion, capillary perfusion [functional capillary density (FCD)], and oxygen levels across the microvascular network were measured by noninvasive methods. RBC deformability for GRBCs was significantly decreased compared with RBCs and moderate hemodilution conditions. The GRBC group had a greater mean arterial blood pressure (MAP) than the RBC and Dex70 groups. FCD was substantially higher for RBC (0.81 +/- 0.07 of baseline) vs. GRBC (0.32 +/- 0.10 of baseline) and Dex70 (0.38 +/- 0.10 of baseline) groups. Microvascular tissue Po(2) was significantly lower for Dex70 and GRBC vs. RBC groups and the moderate hemodilution condition. Results were attributed to decreased oxygen uploading in the lungs and obstruction of tissue capillaries by rigidified RBCs, indicating that the effects impairing RBC flexibility are magnified at the microvascular level, where perfusion and oxygenation may define transfusion outcome.

The possibility of a plasma oxygen diffusion barrier implies a significant resistance to oxygen diffusion and the existence of capillary erythrocyte-associated transients of oxygen. This effect was analyzed by measuring intracapillary blood and tissue pO(2) in the hamster chamber window model using a noninvasive intravital microscopy palladium porphyrin phosphorescence decay technique for two set light excitations (high and low). Using high light excitation, intracapillary blood pO(2) was 13.7 +/- 6.1 mm Hg, and increased to 18.0 +/- 4.5 mm Hg for low light excitation. For high light excitation, intracapillary blood pO(2) peaks were in the range of 25-30 mm Hg, and the lowest values were in the range of 5-10 mm Hg. Reducing the excitation provided a more uniform pO(2) ranging 15-25 mm Hg. With temporal reduction in blood capillary pO(2), levels were correlated to the increase in phosphorescent amplitude that corresponded to plasma gaps. Tissue pO(2) measured at low light excitation in the proximity of capillaries was 23.1 +/- 1.8 mm Hg. In conclusion, low intracapillary blood pO(2) measurements at full hematocrit are an artifact, only observed when oxygen consumption by the measurement technique was excessive and/or absorption of the excitation light was increased by the absence of RBCs. These findings suggest that resistance to oxygen diffusion in plasma is a minor factor in tissue oxygenation by capillaries in the hamster model.

Systemic and microvascular hemodynamic responses to volume restoration from hemorrhagic shock were studied in the hamster window chamber model to determine the significance of blood rheological and oxygen transport properties. Moderated hemorrhage was induced by means of arterial controlled bleeding of 50% of the blood volume. The hypovolemic shock state was maintained for 1 h before resuscitation. The animals were resuscitated by infusion of 25% of blood volume using either fresh plasma or blood and were studied for 90 min. Transfusion was performed with either oxygen-carrying fresh red blood cells (RBCs) or non-oxygen-carrying RBCs whose hemoglobin was converted to methemoglobin (MetHb). Systemic parameters, including cardiac output, vital organ blood flow distribution, microvascular hemodynamics, and capillary perfusion (functional capillary density [FCD]), were measured during the resuscitation period. Fluorescent-labeled microspheres were used to measure organ blood flow (brain, heart, kidney, liver, lung, spleen, and window chamber). The blood viscosities at the end of the 90-min period were 2.4 cP after resuscitation with plasma, and 2.9 to 3.0 cP after blood transfusion (baseline, 4.2 cP). Resuscitation with RBCs with or without oxygen-carrying capacity had greater mean arterial pressure than did the plasma resuscitation group. The FCD was substantially higher for RBC transfusions (0.56% +/- 7% of baseline) compared with plasma (46% +/- 7% of baseline), and the presence of MetHb in the fresh RBC did not change the FCD or the microvascular hemodynamics. Oxygen delivery and extraction levels were significantly lower for resuscitation with plasma and MetHb-loaded RBCs compared with oxygen-carrying RBCs. The curtailed recovery of systemic and microvascular conditions after volume restitution with plasma seems to be due to the decrease in blood viscosity. Conversely, the restoration of blood rheological properties improves resuscitation independently of the restitution of oxygen-carrying capacity.

Objective: Acclimatization to reduced environmental oxygen includes erythropoietin-regulated increase in erythrocytes enhancing the blood's oxygen content. However, increased hematocrit levels result in elevated blood viscosity that might impair microcirculation and tissue oxygenation. To assess this oxygen supply to the skin, the authors used erythropoietin overexpressing transgenic mice (tg6) that develop excessive erythrocytosis in an oxygen-independent manner. These animals have been previously reported to elevate their blood viscosity 4-fold., Methods: The partial oxygen pressure (pO2) distribution was evaluated in microvessels as well as in subcutaneous interstitial tissue within a dorsal skinfold chamber of resting conscious mice using automated phosphorescence quenching., Results: Compared to wildtype (wt) animals, transgenic blood viscosity increased 4-fold but microvessel diameter was not altered. Despite sharing similar blood pO2 as the wt siblings, tg6 animals nearly doubled their oxygen content. Moreover, tg6 erythrocytes reduced hemoglobin's oxygen affinity by decreased 2,3-DPG levels and an increased Hill number. Transgenic arterioles and venules showed increased pO2 compared to wt controls whereas capillary and tissue pO2 were not altered., Conclusions: Excessive erythrocytosis does not elevate capillary oxygen delivery.

Background: Terminal lymphatic fluid is the compartment furthest removed from the oxygen supply, and therefore should present the lowest pO(2) in the tissue due to oxygen consumption by the tissue and the lymphatic vessel wall., Methods and Results: The distribution of pO(2) was determined in the tissue, the lymphatic microvessels, and arterioles and venules of the hamster chamber window model, which is studied without anesthesia with the tissue isolated from the environment. Lymphatic fluid pO(2) was measured with the phosphorescence oxygen quenching method. Small terminal lymphatic fluid pO(2) was 18.4 +/- 2.6 mmHg, and 18.0 +/- 2.4 mmHg in collecting lymphatics. Tissue pO(2) averaged 24.6 +/- 2.7 mmHg. The significant difference between tissue and intralymphatic pO(2) was due in part to the presence of an oxygen gradient across the lymphatic wall, which ranged from 3.7 +/- 1.3 mmHg for terminal lymphatics, to 6.0 +/- 1.2 mmHg for collecting lymphatics. This gradient is assumed to be due to the oxygen consumption by the cellular component of the lymphatic wall., Conclusion: The increased vessels wall gradient found in collecting lymphatics was reconciled by the findings that these microlymphatic vessels tend to be contiguous to the arterioles, whereas the terminal lymphatics are dispersed in the tissue. These findings indicate that terminal lymphatic present the lowest oxygen tension in the tissue, and therefore are the locations at risk to develop anoxia when the microvascular oxygen supply becomes limited.

Responses to exchange transfusion with red blood cells (RBCs) containing methemoglobin (MetRBC) were studied in an acute isovolemic hemodiluted hamster window chamber model to determine whether oxygen content participates in the regulation of systemic and microvascular conditions during extreme hemodilution. Two isovolemic hemodilution steps were performed with 6% dextran 70 kDa (Dex70) until systemic hematocrit (Hct) was reduced to 18% (Level 2). A third-step hemodilution reduced the functional Hct to 75% of baseline by using either a plasma expander (Dex70) or blood adjusted to 18% Hct with all MetRBCs. In vivo functional capillary density (FCD), microvascular perfusion, and oxygen distribution in microvascular networks were measured by noninvasive methods. Methylene blue was administered intravenously to reduce methemoglobin (rRBC), which increased oxygen content with no change in Hct or viscosity from MetRBC. Final blood viscosities after the entire protocol were 2.1 cP for Dex70 and 2.8 cP for MetRBC (baseline, 4.2 cP). MetRBC had a greater mean arterial pressure (MAP) than did Dex70. FCD was substantially higher for MetRBC [82 (SD 6) of baseline] versus Dex70 [38 (SD 10) of baseline], and reduction of methemoglobin to oxyhemoglobin did not change FCD [84% (SD 5) of baseline]. P(O2) levels measured with palladium-meso-tetra(4-carboxyphenyl)porphyrin phosphorescence were significantly changed for Dex70 and MetRBC compared with Level 2 (Hct 18%). Reduction of methemoglobin to oxyhemoglobin partially restored P(O2) to Level 2. Wall shear rate and wall shear stress decreased in arterioles and venules for Dex70 and did not change for MetRBC or rRBC. Increased MAP and shear stress-mediated factors could be the possible mechanisms that improved perfusion flow and FCD after exchange for MetRBC. Thus the fall in systemic and microvascular conditions during extreme hemodilution with low-viscosity plasma expanders seems to be, in part, from the decrease in blood viscosity independent of the reduction in oxygen content.

The rate of oxygen release from arterioles ( approximately 55 microm diameter) was measured in the hamster window chamber model during flow and no-flow conditions. Flow was stopped by microvascular transcutaneous occlusion using a glass pipette held by a manipulator. The reduction of the intra-arteriolar oxygen tension (Po2) was measured by the phosphorescence quenching of preinfused Pd-porphyrin, 100 microm downstream from the occlusion. Oxygen release from arterioles was found to be 53% greater during flow than no-flow conditions (2.6 vs. 1.7 x 10(-5) ml O2.cm(-2).s(-1), P < 0.05). Acute hemodilution with dextran 70 was used to reduce vessel oxygen content, significantly increase wall shear stress (14%, P < 0.05), reduce Hct to 28.4% (SD 1.0) [vs. 48.8% (SD 1.8) at baseline], lower oxygen supply by the arterioles (10%, P < 0.05), and increase oxygen release from the arterioles (39%, P < 0.05). Hemodilution also increased microcirculation oxygen extraction (33% greater than nonhemodilution, P < 0.05) and oxygen consumption by the vessel wall, as shown by an increase in vessel wall oxygen gradient [difference in Po2 between the blood and the tissue side of the arteriolar wall, nonhemodiluted 16.2 Torr (SD 1.0) vs. hemodiluted 18.3 Torr (SD 1.4), P < 0.05]. Oxygen released by the arterioles during flow vs. nonflow was increased significantly after hemodilution (3.6 vs. 1.8 x 10(-5) ml O2.cm(-2).s(-1), P < 0.05). The oxygen cost induced by wall shear stress, suggested by our findings, may be >15% of the total oxygen delivery to tissues by arterioles during flow in this preparation.

The objective of this work was to test the hypothesis that the limitation of nitric oxide (NO) availability accentuates microvascular reactivity to oxygen. The awake hamster chamber window model was rendered hypoxic and hyperoxic by ventilation with 10 and 100% oxygen. Systemic and microvascular parameters were determined in the two conditions and compared with normoxia in a group receiving the NO scavenger nitronyl nitroxide and a control group receiving only the vehicle (saline). Mean arterial blood pressure did not change with different gas mixtures during infusion of the vehicle, but it increased significantly in the NO-depleted group. NO scavenging increased the reactivity of microvessels to the changed oxygen supply, causing the arteriolar wall to significantly increase oxygen consumption. Tissue Po2 was correspondingly significantly reduced during NO scavenger infusion. The present findings support the hypothesis that microvascular oxygen consumption is proportional to oxygen-induced vasoconstriction. The effect of oxygen on vascular tone is modulated by NO. As a consequence, NO acts as a regulator of the vessel wall oxygen consumption. The vessel wall consumes oxygen in proportion to the local Po2, and an impairment of NO availability renders the circulation more sensitive to changes in the oxygen supply.

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.

Purpose of Review: The development of the phosphorescence quenching oxygen measurement technique has allowed for a simultaneous measurement of intra and perivascular partial pressure oxygen along arteriolar vessels in vivo. Mapping the microvascular distribution and oxygen gradients across the vascular walls using this high-resolution technique reveals the existence of large radial gradients between the vasculature and the tissue, with concomitant longitudinal oxygen loss. Mass balance analysis along vessel segments indicates that the vascular wall has a high rate of oxygen consumption. This review presents the current status of in-vivo studies on the partitioning of oxygen between blood, the vascular wall and the surrounding tissue, thereby positioning an oxygen sink between blood and tissue regulating oxygen release., Recent Findings: Induced vasoactivity (vasoconstriction and vasodilation) has been shown to modulate oxygen consumption of the vascular wall and directly affect the portion of oxygen available to the tissue. Inhibition of the endothelial layer of the vessel wall resulted in a decrease in the oxygen gradient across the vessel., Summary: The vascular wall is a sink for oxygen. The modulation of vessel wall oxygen consumption can substantially impact the amount of oxygen released to the tissue.

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.

A phospholipid vesicle encapsulating Hb [Hb vesicle (HbV)] has been developed as a transfusion alternative. One characteristic of HbV is that the O(2) affinity [Po(2) at which Hb is 50% saturated (P(50))] 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 P(50)s (8 and 29 mmHg, termed HbV(8) and HbV(29), respectively) and observed their O(2)-releasing behavior from an occluded arteriole in a hamster skinfold window model. Conscious hamsters received HbV(8) or HbV(29) at a dose rate of 7 ml/kg. In the microscopic view, an arteriole (diameter: 53.0 +/- 6.6 mum) was occluded transcutaneously by a glass pipette on a manipulator, and the reduction of the intra-arteriolar Po(2) 100 mum down from the occlusion was measured by the phosphorescence quenching of preinfused Pd-porphyrin. The baseline arteriolar Po(2) (50-52 mmHg) decreased to about 5 mmHg for all the groups. Occlusion after HbV(8) infusion showed a slightly slower rate of Po(2) reduction compared with that after HbV(29) infusion. The arteriolar O(2) content was calculated at each reducing Po(2) in combination with the O(2) equilibrium curves of HbVs, and it was clarified that HbV(8) showed a significantly slower rate of O(2) release compared with HbV(29) and was a primary source of O(2) (maximum fraction, 0.55) overwhelming red blood cells when the Po(2) was reduced (e.g., <10 mmHg) despite a small dosage of HbV. This result supports the possible utilization of Hb-based O(2) carriers with lower P(50) for oxygenation of ischemic tissues.