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Compromised microcirculation and endothelial dysfunction are hallmarks of sickle cell disease (SCD). EAF PEG Haemoglobin (Hb) and EAF PEG Albumin (Alb) represent a novel class of semisynthetic colloidal supra plasma expanders that improve microcirculation. The therapeutic activity of supra plasma expanders to attenuate vaso-occlusion and restore the haemodynamic functions in patients with SCD has been investigated using NY1DD, a transgenic mouse model of mild SCD without anaemia. Vaso-occlusion and perturbation of haemodynamics are amplified in NY1DD by hypoxia-reoxygenation protocol. EAF P5K6 Alb and Alb T12 (Alb conjugated with 12 copies of antioxidant tempo) attenuate vaso-occlusion when infused at the start of reoxygenation. However, only EAF PEG Alb restores haemodynamics close to levels in control C57BL. EAF P5K6 Alb-T12, active plasma expander conjugated with antioxidant, completely clears vaso-occlusion and restores normal haemodynamics. EAF PEG Hb also completely clears vaso-occlusion and restores normal haemodynamics. Pretreating NY1DD with EAF PEG Hb protects it from hypoxia reoxygenation-induced damages. EAF P5K6 Alb T12 attenuates the endothelial dysfunction in S + S Antilles mice as reflected by the vasodilatory response of its arteries and arterioles to vaso-dilators. Active plasma expanders are novel therapeutics to restore normal haemodynamics in SCD patients to improve tissue oxygenation during episodes of painful vaso-occlusive crisis. Abbreviations: 2-IT: 2-immothiolane; Mal-T: 4-Maleimido tempo; Alb: human serum albumin (HSA); Alb-T12: human albumin conjugated with 12 copies of tempo; EAF: extension arm facilitated; EAF PEG Hb: extension arm facilitated PEGylated haemoglobin; EAF PEG Alb: extension arm facilitated PEGylated albumin; EAF P3K6 Hb: extension arm facilitated PEGylated haemoglobin conjugated with 6 copies of PEG3K; EAF P5K6 Alb T12: extension arm facilitated PEGylated albumin conjugated with 6 copies of PEG5K and 12 copies of tempo; Hb: haemoglobin; HAS: human serum albumin (Alb); PEG: polyethylene glycol; MP4: MalPEG Hb, is formulated at 4.2 g/dL in lactated Ringer's solution, a product of Sangart; SCD: sickle cell disease; NO: nitric oxide; SEC: size exclusive chromatography; Vrbc: red cell velocity; Q: volumetric flow rates, Q; SNP: sodium nitroprusside.

© 2016 Informa UK Limited, trading as Taylor & Francis Group. 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.

Background: Free tissue transplantations are lengthy procedures that result in prolong tissue ischemia. Restoral of blood flow is essential for free flap recovery; however, upon reperfusion tissue that is viable may continue to be nonperfused. To further elucidate this pathophysiology skeletal muscle microcirculation was investigated during reperfusion following 4-hour single arteriole occlusion. Materials and methods: A blunt micropipette probe was use to compress a single arteriole in the unanesthetized hamster (N = 20) dorsal skinfold chamber. Arteriole (n = 20), capillary (n = 97), and postcapillary venule (n = 16) diameters and blood flow were analyzed at 0, 30, 60, 120, 240 min and 24 hours of reperfusion after 4 hour occlusion. Results: Feeding arcade arterioles exhibited a brief (

We studied the extreme hemodilution to a hematocrit of 11% induced by three plasma expanders: polyethylene glycol (PEG)-conjugated albumin (PEG-Alb), 6% 70-kDa dextran, and 6% 500-kDa dextran. The experimental component of our study relied on microelectrodes and cardiac output to measure both the rheological properties of plasma-expander blood mixtures and nitric oxide (NO) bioavailability in vessel walls. The modeling component consisted of an analysis of the distribution of wall shear stress (WSS) in the microvessels. Our experiments demonstrated that plasma expansion with PEG-Alb caused a state of supraperfusion with cardiac output 40% above baseline, significantly increased NO vessel wall bioavailability, and lowered peripheral vascular resistance. We attributed this behavior to the shear thinning nature of blood and PEG-Alb mixtures. To substantiate this hypothesis, we developed a mathematical model of non-Newtonian blood flow in a vessel. Our model used the Quemada rheological constitutive relationship to express blood viscosity in terms of both hematocrit and shear rate. The model revealed that the net effect of the hemodilution induced by relatively low-viscosity shear thinning PEG-Alb plasma expanders is to reduce overall blood viscosity and to increase the WSS, thus intensifying endothelial NO production. These changes act synergistically, significantly increasing cardiac output and perfusion due to lowered overall peripheral vascular resistance.

Background: Infusion of large volume of fluid is practiced in the treatment of hemorrhagic shock although resuscitation with small fluid volumes reduces the risks associated with fluid overload. We explored the hypothesis that reduced Ringer's lactate (RL) volume restoration in hemorrhage is significantly improved by increasing its viscosity, leading to improved microvascular conditions. Methods: Awake hamsters were subjected to a hemorrhage of 50% of blood volume followed by a shock period of 1 hour. They were resuscitated with conventional RL (n = 6) or with RL whose viscosity was increased by the addition of 0.3% alginate (RL-HV) (n = 6). In both cases, the volume infused was 200% of shed blood. Results: After resuscitation, blood and plasma viscosities were 1.9 cp ± 0.18 cp and 1.0 cp ± 0.03 cp in RL and 2.5 cp ± 0.34 cp and 1.6 cp ± 0.05 cp in RL-HV. Mean arterial pressure was lower than baseline in RL. Arteriolar diameter and arteriolar and venular flow were significantly higher in RL-HV. Functional capillary density was significantly higher in RL-HV than RL. After 90 minutes of resuscitation, functional capillary density was lower than baseline in RL, whereas it was maintained in RL-HV. Arteriolar Po2 was higher in RL-HV than RL. Microcirculation O2 delivery and tissue Po2 were significantly higher in RL-HV. Conclusions: Increasing blood and plasma viscosities in resuscitation from hemorrhagic shock with increased viscosity RL improves microvascular hemodynamics and oxygenation parameters.

The effect of low and high viscosity hemodilution with plasma expanders on the extent of the cell free layer (CFL) width was analyzed in the microcirculation of the exteriorized cremaster muscle preparation of Sprague-Dawley male rats. Anesthetized animals were subjected to 40% hemodilution by blood volume, using 5% human serum albumin (HSA) or 6% Hetastarch (hydroxyethyl starch 670 kDa). Arterioles (n=5 for each treatment) were investigated. Mean arterial pressure, heart rate, vessel flow velocity and CFL width were measured at baseline and 5, 20 and 40 min post-exchange transfusion. Blood and plasma viscosity was determined from terminal blood collections. CFL width and pseudoshear rate, diameter and flow, normalized to baseline, were significantly elevated at all post-exchange assessments. Peripheral vascular resistance decreased. The increase of the CFL width was greater with HSA by comparison with Hetastarch hemodilution (p

Introduction This study analyzed the systemic and microvascular hemodynamic changes related to increased nitric oxide (NO) availability during the early phase of hemorrhagic shock. Hemodynamic responses to hemorrhagic shock were studied in the hamster window chamber. Materials and Methods Exogenous NO was administered in the form of nitrosothiols (nitrosylated glutathione, GSNO) and was given prior the onset of hemorrhage. Moderate hemorrhage was induced by arterial controlled bleeding of 50% of the blood volume, and the hypovolemic shock was followed over 90 min. Results Animals pre-treated with GSNO maintained systemic and microvascular conditions during hypovolemic hemorrhagic shock, when compared to animal treated with glutathione (GSH) or the Sham group. Low concentrations of NO released during the early phase of hypovolemic shock from GSNO mitigated arteriolar vasoconstriction, increased capillary perfusion and venous return, and improved cardiac function (recovered of blood pressure and stabilized heart rate). GSNO's effect on resistance vessels influenced intravascular pressure redistribution and blood flow, preventing tissue ischemia. Discussion Increases in NO availability during the early phase of hypovolemic shock could preserve cardiac function and microvascular perfusion, sustaining organ function. Direct translation into a clinical scenario may be limited, although the pathophysiological importance of NO in the early phase of hypovolemia is clearly highlighted here.

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.

Ischemia reperfusion (IR) injury, occurring during heart attacks, hemorrhagic shock, and bypass and transplant surgeries, impairs microcirculatory function and nitric oxide (NO) synthesis. We report the regulation of endothelial and inducible NO synthase (eNOS and iNOS) proteins as a consequence of the application of continuous mode diagnostic frequency ultrasound application following IR injury.Animals were assigned to one of five groups for microcirculatory assessment or Western blot analysis (WB) as follows: (1) IR+iNOS inhibition (1400W); and (2) IR+1400W+ultrasound for microcirculatory assessment, (3) Control; (4) IR; and (5) IR+ultrasound for WB. Functional capillary density and microvascular diameter, flow velocity, and flow were monitored for microcirculatory assessment. Skin tissue samples were harvested for WB. 2.49MHz continuous ultrasound was used for application.Both the inhibition of iNOS alone and iNOS inhibition with ultrasound irradiation positively influenced the microcirculation of observed animals relative to baseline values. Ultrasound exposure resulted in a significant production of eNOS protein in skin tissue harvested 24h into reperfusion (p0.01). iNOS levels from the same tissue of ultrasound exposed animals were found to be significantly decreased 0.5h into reperfusion (p0.05).Protection from lasting IR injury effects in the microcirculation, with continuous mode diagnostic frequency ultrasound, results from augmented eNOS protein levels during late reperfusion. Ultrasound inhibited iNOS protein production during early reperfusion may also confer protection from IR injury.

The circulation is adapted to specific levels of blood viscosity resulting in a balance that simultaneously sets peripheral vascular resistance, blood pressure and cardiac output, factors in part mediated by the production of nitric oxide by the endothelium. Although it is generally perceived that decreasing blood viscosity is beneficial for cardiovascular function, small increases of blood viscosity in normal healthy experimental subjects significantly improve cardiovascular function. These changes are within the normal variations of viscosity due to the variations of hematocrit in the healthy population. Hemodilution reduces blood viscosity, which is proposed to be physiologically beneficial. However, in extreme hemodilution, increased plasma viscosity via the use of viscogenic plasma expanders sustains microvascular and tissue function at significantly reduced levels of oxygen delivery. Studies in hemorrhagic shock resuscitation using oxygen carrying and non-carrying red blood cells show that restoration of blood viscosity is as important as restoration of oxygen carrying capacity. It is concluded that although hemodilution is indicated for reducing abnormally high blood viscosities, it is beneficial to increase plasma viscosity when hematocrit is reduced. Furthermore small increases in hematocrit may be beneficial due to the related increase in blood viscosity, independently of the increase of oxygen delivery capacity.

Summary Systemic and microvascular hemodynamic responses to hemorrhagic shock resuscitation with hypertonic saline (HTS, 7.5% NaCl) followed with a small volume of plasma expander were studied in the hamster window chamber model to determine the role of plasma expander viscosity in the acute resuscitation outcome. Moderate hemorrhagic shock was induced by arterial controlled bleeding of 50% of blood volume (BV) and the hypovolemic state was maintained for 1 h. Volume restitution was performed by infusion of HTS, 3.5% of BV followed by 10% of BV plasma expanders. Resuscitation was followed for 90 min. The experimental groups were named based on the plasma expanders infused after the HTS, namely: [ Hextend ], Hextend ® (6% Hetastarch 670 kDa in lactated electrolyte solution, 4 cp), [ Hextend+V ], Hextend ® with viscosity enhanced by the addition of 0.4% alginate, 8 cp, and [ NVR ] no volume resuscitation as control group. Measurement of systemic parameters, microvascular hemodynamics and capillary perfusion were performed during hemorrhage, shock and resuscitation. Restitution with Hextend yielded the higher mean arterial pressure (MAP), followed by Hextend+V and NVR . Increasing plasma viscosity did not increase peripheral vascular resistance. Functional capillary density (FCD) was higher for Hextend+V than Hextend and NVR . The level of restoration of acid–base balance correlated with microvascular perfusion and was significantly improved with Hextend+V when compared to Hextend and NVR . These results suggest the importance of restoration of blood rheological properties through enhancing plasma viscosity, influencing the re-establishment of microvascular perfusion during small volume resuscitation from hemorrhagic shock.

Our objective was to determine the effects of ischemia reperfusion (IR) treatment with continuous-mode diagnostic frequency ultrasonography exposure by assessing leukocyte-endothelial cell interactions, in terms of frequency and relative proportions of rolling and firmly attached leukocytes.Studies were carried out in the awake hamster chamber window preparation. Tourniquet ischemia was implemented by compressing a circular ring on the chamber window tissue for 4 hours, followed by reperfusion. Animals were randomly assigned into one of four groups as follows: IR; IR + ultrasonography; IR + N(omega)-nitro-L-arginine methyl ester (L-NAME); and IR + L-NAME + ultrasonography. Venules were exposed to epi-illumination, videorecorded, and leukocytes were categorized as "rolling"; flowing with endothelial contact; or "immobilized" cells, and counted during digital video-playback in 100-microm length segments.Leukocyte interaction with venular endothelium substantially decreased, during longterm reperfusion (p0.05) with ultrasonography exposure. Nitric oxide production inhibition, after L-NAME treatment, and ultrasonography exposure resulted in additional earlier substantially decreased leukocyte-endothelial cell interactions (p0.05).Venular function improvement, after IR damage, is a primary benefit derived from continuous-mode diagnostic frequency ultrasonography exposure. Although decreased interaction of adherent leukocytes can also be attributed to enhanced arteriolar flow, reduced interaction of rolling leukocytes is an immediate consequence of ultrasonography exposure.

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.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.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.

Administration of fluids to maintain or restore intravascular volume is a common intervention after hemorrhagic shock, but there is uncertainty whether the choice of fluid significantly influences outcome. Systemic parameters, microvascular perfusion, and functional capillary density were used to characterize resuscitation from hemorrhagic shock with hydroxyethyl starch (HES) of different molecular weights. Studies were made in the hamster window chamber model to determine their effects on blood rheological properties, restoration of perfusion and coagulation changes. Moderate hemorrhagic shock was induced by controlled arterial bleeding of 50% of blood volume, and hypovolemia was maintained for 1 h before resuscitation. Twenty-five percent of blood volume was restituted, and recovery was followed over 60 min. Low-molecular weight (MW) HES (L-HES) 130 kd, degree of substitution (DS) 0.40, and high-MW HES (H-HES) 670 kd, DS 0.75, were used as resuscitation fluids. Microthrombi formation was induced by endothelial laser irradiation. H-HES improved systemic conditions, microcirculatory flow, and metabolic recovery after resuscitation when compared with L-HES. Mean arterial pressure was significantly improved after resuscitation with H-HES compared with L-HES, but lower than baseline and the sham group. Thrombus formation was impaired in both groups after resuscitation compared with sham. There was no difference in microthrombi formation between low- and H-HES for medium and large laser endothelial injuries. Our results indicate that fluid resuscitation with HES may increase the risk of bleeding, but not necessarily caused by the properties (MW and DS) of the colloid. Impairment of thrombus formation seems to be in part related to altered hemodynamics and transport inherent to hemodilution, leading to lowered platelet availability due to hemodilution and increased shear stress at the vessel wall when plasma viscosity is increased. The HES MW does not seem to be a factor in compromising platelet adherence on stimulated endothelium. The longer initial intravascular persistence of H-HES might result in longer-lasting volume effects.

TetraPEGylated canine Hb, [SP (succinimidophenyl)-PEG5K]4-canine-Hb, with PEGylation at its four reactive cysteine residues (α111 and β93) has been prepared and characterized. The hydrodynamic volume and the molecular radius of (SP-PEG5K)4-canine-Hb are intermediate to those of di- and hexaPEGylated human Hb as expected. However, the COP (colloidal osmotic pressure) of tetraPEGylated canine Hb is closer to that of hexaPEGylated human Hb than to that of diPEGylated human Hb. The O2 affinity of tetraPEGylated canine Hb is higher than that of canine Hb and comparable with that of hexaPEGylated Hb. The O2 affinity of tetraPEGylated canine Hb is not responsive to the presence of DPG (diphosphoglycerate) or chloride, but it retains almost full response to L-35, an allosteric effector that interacts at the αα-end of the central cavity. The tetraPEGylated canine Hb is vasoinactive in hamster in 10% top load infusion studies. It is also essentially non-hypertensive in an extreme exchange haemodilution protocol in hamster just as di- and hexaPEGylated human Hb. The O2 delivery by tetraPEGylated canine Hb is comparable with that of hexaPEGylated Hb but not as efficient as diPEGylated Hb. These results demonstrate that PEGylation-induced solution properties of PEG [poly(ethylene glycol)]–Hb conjugates are dictated by the level and chemistry of PEGylation and the interplay of these plays a critical role in tissue oxygenation. The studies imply the need to establish the right level (and/or pattern) of PEGylation and O2 affinity of Hb–PEG adducts in designing O2-carrying plasma volume expanders, and this remains the primary challenge in the design of PEGylated Hb as blood substitutes.

Oxygen transport from blood to the mitochondria is dependent on oxygen gradients. The interstitial or extracellular pO(2), measured by the phosphorescence-decay method, is indicative of these driving forces and the amount of oxygen available to the mitochondria. Diverse protocols for sampling tissue pO(2) show that measurements sampling only interstitial pO(2) levels provide a reliable measurement of the tissue pO(2) level. Present findings lead to the hypothesis that tissue has a fairly uniform interstitial fluid pO(2) level and that local inhomogeneity due to the presence arteriolar and venular vessels is smoothed out by the steep gradients at the microvascular walls.

Systemic parameters and microvascular and capillary hemodynamics were studied in the hamster window chamber model before and after hyaluronan degradation by intravenous injection of Streptomyces hyaluronidase (100 units, 40–50 U/ml plasma). Glycocalyx permeation was estimated using fluorescent markers of different molecular size (40, 70, and 2,000 kDa), and electrical charge. Systemic parameters (blood pressure, heart rate, blood gases) and microhemodynamics (vascular tone, velocity, and blood flow) remained statistically unchanged after injection of hyaluronidase, compared with inactivated hyaluronidase. Conversely, capillary hemodynamics were drastically affected. Functional capillary density, the capillaries perfused with red blood cells (RBCs), decreased by 35%, capillary Hct of the remaining functional capillaries increased from 16 to 27%, and penetration of 70-kDa fluorescent marker increased. Furthermore, plasma-only perfused capillaries statistically increased 30 min after hyaluronidase. The decrease in functional capillary density accounted for an increased RBC flux in the remainder of the capillaries, since the same number of RBCs had to traverse a reduced number of capillaries. Flux balances showed a reduction from baseline of 11% for the RBC flux and 20% for the plasma flux after treatment. These discrepancies are within the margin of error of the techniques used and could be explained by accounting for RBC over-velocity compared with plasma. These findings suggest that the decrease in the glycocalyx leads to capillary perfusion impairments.

Objective: To determine how top loads with different doses of carbon monoxide (CO)-saturated saline solutions (CO-saline) affect microvascular and systemic hemodynamics and to delineate the corresponding biochemical mechanisms. Design: Prospective study. Setting: University research laboratory. Subjects: Male Golden Syrian hamsters. Interventions: Hamsters implemented with a dorsal window chamber were given different volumes (characterized as percent of blood volume, BV) by intravenous injection of CO-saturated saline. Measurements and Main Results: Hamsters were observed until 90 mins after infusion of CO-saline solution. In the 20% BV CO-saline infusion group, observation was extended until 180 mins. Systemic variables measured included mean arterial pressure, heart rate, systemic arterial blood gases, and cardiac output and index. Microvascular hemodynamic measurements included vessel diameter, red blood cell velocity, and functional capillary density. Cyclic guanosine monophosphate (cGMP) content in the chamber tissue was measured by enzyme immunoassay. 10% BV of CO-saline increased flow maximally in the microcirculation at 30 mins after infusion (207% in arterioles and 238% in venules, p

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.

Summary The response to exchange transfusion with red blood cells (RBCs) saturated with carbon monoxide (CO) in amelioration of microvascular function and providing tissue protection in hemorrhagic shock resuscitation was investigated in the hamster chamber window model. Shock was induced by the withdrawal of 50% of blood volume (BV). Blood volume was restored 1 h after hemorrhage with a single volume infusion (resuscitation) of 25% BV with fresh RBCs (saturated or unsaturated with CO) suspended in human serum albumin (HSA). Hemorrhage, shock and resuscitation were monitored continuously in terms of mean arterial pressure (MAP), microvascular blood flow, capillary perfusion and systemic gas parameters. Eight hours after resuscitation, Annexin V and propidium iodide (PI) were injected into the window chamber to study tissue viability, and labeled cells were observed by using intravital epifluorescence microscopy. TUNEL staining was performed on the tissue to confirm in vivo results. Systemic and microvascular restoration were not different with or without CO up to 90 min after resuscitation. CO concentration decreased over 90 min, increasing oxygen carrying capacity and gradually reoxygenating the tissue. CO saturated blood partially mitigated cell injury at 8 h after resuscitation. The precise cellular mechanisms involved require further elucidation. CO is a novel experimental strategy to improve tissue viability and requires the appropriated preclinical studies to confirm its efficacy.

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) αα 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.

Awake hamsters equipped with the dorsal window chamber preparation were subjected to hemorrhage of 50% of the estimated blood volume. Initial resuscitation (25% of estimated blood volume) with polymerized bovine hemoglobin (PBH) or 10% hydroxyethyl starch (HES) occurred in concert with an equivolumetric bleeding to simulate the early, prehospital setting (exchange transfusion). Resuscitation (25% of estimated blood volume) without bleeding was performed with PBH, HES, or autologous red blood cells (HES-RBCs). Peripheral microcirculation, tissue oxygenation, and systemic hemodynamic and blood gas parameters were assessed. After exchange transfusion, base deficit was -8.6 +/- 3.7 mmol/L (PBH) and -5.1 +/- 5.3 mmol/L (HES) (not significant). Functional capillary density was 17% +/- 6% of baseline (PBH) and 31% +/- 11% (HES) (P < 0.05) and arteriolar diameter 73% +/- 3% of baseline (PBH) and 90% + 5% (HES) (P < 0.01). At the end, hemoglobin levels were 3.7 +/- 0.3 g/dL with HES, 8.2 +/- 0.6 g/dL with PBH, and 10.4 +/- 0.8 g/dL with HES-RBCs (P < 0.01 HES vs. PBH and HES-RBCs, P < 0.05 PBH vs. HES-RBCs). Base excess was restored to baseline with PBH and HES-RBCs, but not with HES (P < 0.05). Functional capillary density was 46% +/- 5% of baseline (PBH), 62% + 20% (HES-RBCs), and 36% +/- 19% (HES) (P < 0.01 HES-RBCs vs. HES). Peripheral oxygen delivery and consumption was highest with HES-RBCs, followed by PBH (P < 0.05 HES-RBCs vs. PBH, P < 0.01 HES-RBCs and PBH vs. HES). In conclusion, the PBH led to a correction of base deficit comparable to blood transfusion. However, oxygenation of the peripheral tissue was inferior with PBH. This was attributed to its negative impact on the peripheral microcirculation caused by arteriolar vasoconstriction.

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.

The effect of small hematocrit (Hct) increases on cardiac index (cardiac output/body wt) and oxygen release to the microcirculation was investigated in the awake hamster window chamber model by means of exchange transfusions of homologous packed red blood cells. Increasing Hct between 8 and 13% from baseline increased cardiac index by 5–31% from baseline ( P < 0.05) and significantly lowered systemic blood pressure ( P < 0.05). The relationship between Hct and cardiac index is described by a second-order polynomial ( R2 = 0.84; P < 0.05) showing that Hct increases up to 20% from baseline increase cardiac index, whereas increases over 20% from baseline decrease cardiac index. Combining this data with measurements of blood pressure allowed to determine total peripheral vascular resistance, which was a minimum at 8–13% Hct increase and was described by a second-order polynomial ( R2 = 0.83; P < 0.05). Oxygen measurements in arterioles, venules, and the tissue at 8–13% Hct increase were identical to control; thus, as a consequence of increased flow and oxygen-carrying capacity, oxygen delivery and extraction increased, but the change was not statistically significant. Previous results with the same model showed that the observed effects are related to shear stress-mediated release of nitric oxide, an effect that should be also present in the heart microcirculation, leading to increased blood flow, myocardial oxygen consumption, and contractility. We conclude that a minimum viscosity level is necessary for generating the shear stress required for maintaining normal cardiovascular function.

The increase of plasma and blood viscosity is usually associated with pathological conditions; however, elevation of both parameters often results in increased perfusion and the lowering of peripheral vascular resistance. In extreme haemodilution, blood viscosity is too low and insufficient to maintain functional capillary density, a problem that in experimental studies is shown to be corrected by increasing plasma viscosity up to 2.2 cP. This effect is mediated by mechanotransduction-induced nitric oxide (NO) production via shear stress in the endothelium as shown by microelectrode perivascular measurements of NO concentration. Moderate elevations of blood viscosity by increasing haematocrit ( approximately 10%) result in comparable reductions of blood pressure and peripheral vascular resistance, an effect also NO-mediated as it is absent after Nomega-nitro-L-arginine methyl ester treatment and in endothelial nitric oxide synthase-deficient mice. These findings show that the rheological properties of plasma affect vessel diameter in the microcirculation leading to counterintuitive responses to the changes in blood and plasma viscosity. Application of these findings to haemorrhagic shock resuscitation leads to the concept of hyperosmotic-hyperviscous resuscitation as a modality for maintaining the recovery of microvascular function.

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 cell-free layer between the erythrocyte column and the vessel wall is an important determinant of hydrodynamic resistance in microcirculatory vessels. The authors report a method for continuous measurement of the width of this layer.The light intensity of a linear array of pixels perpendicular to the vessel axis is continuously determined from a video image of a microcirculatory vessel. A threshold level based on Otsu's method is used to establish the interface between the cell-free layer and the erythrocyte column. To test the method, video images at 750-4500 frames/s were obtained from venules and arterioles in rat spinotrapezius muscle at normal and reduced arterial pressures before and after induction of erythrocyte aggregation with Dextran 500. The current measurements were compared to manual measurements of the same images.Values obtained by the manual and the new methods were in agreement within the 95% confidence limit by the Bland-Altman analysis and within 90-95% range by the correlation coefficient (R2). The more frequent measurements reveal substantial, rapid variations in cell-free layer width and changes in mean values with alteration of arterial pressure and red cell aggregability.A new, computer-based technique has been developed that provides measurements of rapid, time-dependent variations in the width of the cell-free layer in the microcirculation.

Objective: To determine whether resuscitation with polyethylene glycol conjugated bovine serum albumin (2.5% weight/volume) infused at 16 mL/kg/hr (PEG-BSA-16) or at 24 mL/kg/hr (PEG-BSA-24) for 1 hr improves microcirculatory conditions in endotoxemia compared with dextran 70 (6% weight/volume) infused at 24 mL/kg/hr (Dex). Design: Prospective study. Setting: University research laboratory. Subjects: Male Golden Syrian hamsters. Interventions: Hamsters implemented with a skinfold window chamber were given an intravenous injection of lipopolysaccharide and resuscitated within 10 mins with Dex, PEG-BSA-16, or PEG-BSA-24. Measurements and Main Results: Hamsters were observed during 24 hrs after lipopolysaccharide injection. Systemic variables measured included mean arterial pressure, heart rate, and systemic arterial blood gas. Microvascular function was characterized by measuring vessel diameter; red blood cell velocity; functional capillary density (FCD); Po2 in arterioles, venules, and tissue; and perivascular nitric oxide concentration 6 hrs after lipopolysaccharide injection. At 6 hrs, animals with no treatment had the lowest FCD (6.7 ± 5.7% of baseline). PEG-BSA provided significantly improved microvascular conditions as shown by restoration of FCD. Recovery of FCD was related to improved microvascular flow and perivascular and tissue Po2, normalization of shear rate, and decreased perivascular nitric oxide concentration. These effects were related to improved fluid retention using PEG-BSA-24 as evidenced by the significantly lower hematocrit at 24 hrs after resuscitation. Nitric oxide at 6 hrs after induction of sepsis achieved perivascular millimolar concentrations, which were reduced to normal values by PEG-BSA-24 treatment. At 6 hrs there were significant differences in FCD, tissue Po2, and perivascular nitric oxide concentration following PEG-BSA treatment by comparison with Dex treatment, although there were no differences in systemic variables between Dex and PEG-BSA groups. Conclusions: PEG-BSA produces improved microcirculatory conditions in the treatment of endotoxemia when compared with dextran 70.

Nitric oxide (NO) is an important signaling molecule modulating diverse processes such as vasodilation, neurotransmission, long-term potentiation, and immune responses. The endothelium contributes a significant fraction of NO from endothelial NO synthase (eNOS). The objective of this work was to analyze the role of eNOS in the modulation of oxygen supply to the tissues and in adaptation to maintain oxygenation uncompromised. Oxygen delivery and consumption were measured in the microcirculation of homozygous mutant endothelial nitric oxide synthase-deficient (eNOS(-/-)) and wild-type mice. Animals were implanted with a dorsal window chamber, allowing us to assess the intact microvascular system. Hemodynamics and oxygen tension were assessed in the microcirculation of conscious animals. The eNOS(-/-) mice had significantly higher blood pressure and lower heart rate (146 +/- 8 mm Hg, 401 +/- 17 bpm) than wild type (127 +/- 6 mm Hg, 428 +/- 20 bpm). Microvascular hemodynamic parameters were not significantly different between groups. The eNOS(-/-) animals delivered less oxygen to the microcirculation and released more oxygen to the tissue; both differences were statistically significant compared to wild type. The arteriolar vessel wall oxygen gradient, a measure of vascular smooth muscle cells and endothelial cell wall oxygen consumption, was significantly lower for eNOS(-/-) than for wild type, suggesting that the inhibition of eNOS is an antianoxia (oxygen sparing) mechanism. Finally, the findings of the study support the argument that NO availability limits oxygen consumption by the tissue.

Hematocrit (Hct) of awake hamsters and CD-1 mice was acutely increased by isovolemic exchange transfusion of packed red blood cells (RBCs) to assess the relation between Hct and blood pressure. Increasing Hct 7–13% of baseline decreased mean arterial blood pressure (MAP) by 13 mmHg. Increasing Hct above 19% reversed this trend and caused MAP to rise above baseline. This relationship is described by a parabolic function ( R2 = 0.57 and P < 0.05). Hamsters pretreated with the nitric oxide (NO) synthase (NOS) inhibitor Nω-nitro-l-arginine methyl ester (l-NAME) and endothelial NOS-deficient mice showed no change in MAP when Hct was increased by 19% of baseline) led blood viscosity to increase >50%, overwhelming the NO effect through a significant viscosity-dependent increase in vascular resistance, causing MAP to rise above baseline values.

Rational development of adjunct or anti-disease therapy for severe Plasmodium falciparum malaria requires cellular and molecular definition of malarial pathogenesis. Nitric oxide (NO) is a potential target for such therapy but its role during malaria is controversial. It has been proposed that NO is produced at high levels to kill Plasmodium parasites, although the unfortunate consequence of elevated NO levels might be impaired neuronal signaling, oxidant damage and red blood cell damage that leads to anemia. In this case, inhibitors of NO production or NO scavengers might be an effective adjunct therapy. However, increasing amounts of evidence support the alternate hypothesis that NO production is limited during malaria. Furthermore, the well-documented NO scavenging by cell-free plasma hemoglobin and superoxide, the levels of which are elevated during malaria, has not been considered. Low NO bioavailability in the vasculature during malaria might contribute to pathologic activation of the immune system, the endothelium and the coagulation system: factors required for malarial pathogenesis. Therefore, restoring NO bioavailability might represent an effective anti-disease therapy.

A phospholipid vesicle encapsulating Hb [Hb vesicle (HbV)] has been developed as a transfusion alternative. One characteristic of HbV is that the O2affinity [Po2at 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 HbV8and HbV29, respectively) and observed their O2-releasing behavior from an occluded arteriole in a hamster skinfold window model. Conscious hamsters received HbV8or HbV29at 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 Po2100 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 HbV8infusion showed a slightly slower rate of Po2reduction compared with that after HbV29infusion. The arteriolar O2content was calculated at each reducing Po2in combination with the O2equilibrium curves of HbVs, and it was clarified that HbV8showed a significantly slower rate of O2release compared with HbV29and was a primary source of O2(maximum fraction, 0.55) overwhelming red blood cells when the Po2was reduced (e.g., 2carriers with lower P50for oxygenation of ischemic tissues.

The microvascular distribution of oxygen was studied in the arterioles and venules of the awake hamster window chamber preparation to determine the contribution of vascular smooth muscle relaxation to oxygen consumption of the microvascular wall during verapamil-induced vasodilatation. Verapamil HCl delivered in a 0.1 mg/kg bolus injection followed by a continuous infusion of 0.01 mg·kg−1·min−1caused significant arteriolar dilatation, increased microvascular flow and functional capillary density, and decreased arteriolar vessel wall transmural Po2difference. Verapamil caused tissue Po2to increase from 25.5 ± 4.1 mmHg under control condition to 32.0 ± 3.7 mmHg during verapamil treatment. Total oxygen released by the microcirculation to the tissue remained the same as at baseline. Maintenance of the same level of oxygen release to the tissue, increased tissue Po2, and decreased wall oxygen concentration gradient are compatible if vasodilatation significantly lowers vessel wall oxygen consumption, which in this model appears to constitute an important oxygen-consuming compartment. These findings show that treatment with verapamil, which increases oxygen supply through vasodilatation, may further improve tissue oxygenation by lowering oxygen consumption of the microcirculation.

We tested the hypothesis that high-viscosity (HV) plasma in extreme hemodilution causes wall shear stress to be greater than low-viscosity (LV) plasma, leading to enhanced production of nitric oxide (NO). The perivascular concentration of NO was measured in arterioles and venules and the tissue of the hamster chamber window model, subjected to acute extreme hemodilution, with a hematocrit (Hct) of 11% using Dextran 500 ( n = 6) or Dextran 70 ( n = 5) with final plasma viscosities of 1.99 ± 0.11 and 1.33 ± 0.04 cp, respectively. HV plasma significantly increased the periarteriolar, perivenular, and tissue NO concentration by 2.0, 1.9, and 1.4 times the control ( n = 7). The NO concentration with LV plasma was not statistically different from control. Arteriolar shear stress was significantly increased in HV plasma relative to LV plasma in arterioles but not in venules. Aortic endothelial NO synthase (eNOS) protein expression was increased with HV plasma but not with LV plasma. There was a weak correlation between perivascular NO concentration and the locally calculated shear stress induced by the procedures, when blood viscosity was corrected according to Hct values previously determined in studies of microvascular Hct distribution. The finding that the periarteriolar and venular NO concentration in HV plasma was the same although arteriolar shear stress was significantly greater than venular shear stress maybe be due to differences in vessel wall metabolism between arterioles and venules and the presence of NO transport through the blood stream in the microcirculation. Results support the concept that in extreme hemodilution HV plasma maintains functional capillary density through a NO-mediated vasodilatation.

The O2-carrying blood substitute based on polymerized bovine hemoglobin (PBH) was used to determine efficacy in maintaining tissue Po2after 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 O2delivery, O2extraction, and tissue Po2at 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 Po2was maximal at 1 h after exchange and decreased progressively at 12 and 24 h after exchange. O2release to the tissue was minimal at 1 h and increased at 12 and 24 h after exchange. These results suggest the impairment of tissue O2metabolism after introduction of PBH into the circulation, which is mitigated as PBH concentration declines.

The aim of this study was to test the effects of using a high-viscosity fluid after small-volume hyperosmotic resuscitation from hemorrhagic shock and to compare this to hyperosmotic followed by hyperoncotic resuscitation. Studies were made in the awake hamster window chamber preparation with the animals subjected to hemorrhage of 50% of blood volume and resuscitated with a small volume of a 7.5% NaCl solution, which was followed within minutes by infusion of 25% of withdrawn volume of either 0.7% or 0.8% alginate solutions (A0.7%, 7.6 cp; and A0.8%, 10.2 cp) or 5% hydroxyethyl starch (HES 5%, 2.1 cp). All modalities of resuscitation returned blood pressure to near baseline values in 5 min, which remained elevated after 90 min with A0.7% and A0.8% but returned to near shock values in 15 min with HES 5%. Microvascular flow and functional capillary density (FCD) followed the same pattern, being significantly higher for the alginate solutions than HES 5% after 90 min. Plasma viscosity 90 min after resuscitation was 2.1 and 2.6 cp for A0.7% and A0.8%, respectively, and 1.1 cP for HES 5%. There was an apparent directly proportional relationship between the concentration of alginate and blood pressure recovery, with blood pressure near normal with A0.8%, and approximately 20 mmHg lower with A0.7%. The recovery of microvascular flow and FCD, although showing a trend toward being more effective with A0.8%, was not significantly different from A0.7% but statistically different and improved relative to HES 5%. The high-viscosity fluids provide a novel small-volume method of resuscitation that maximizes microvascular perfusion for extended periods until surgical control of bleeding is possible. Results show that high-plasma-viscosity resuscitation provides a more consistent and prolonged resuscitation than hyperoncotic treatment. The increase in viscosity presents a gradual recovery in blood pressure and may be used as an alternative for small-volume hypotensive resuscitation, increasing tissue perfusion while potentially limiting hemorrhage in vascular injuries of the major blood vessels.

Our aim was to determine the efficacy of polyethylene glycol-conjugated human albumin (MalPEG-Alb) in restoring circulatory volume after 1 h of hemorrhagic shock. Experiments were performed in the awake condition in the hamster skin fold preparation. Microhemodynamic parameters and tissue Po2 were assessed with intravital microscopy and the use of the phosphorescence quenching technique. One hour after shock induction by withdrawal of 50% of the blood volume, animals were resuscitated with MalPEG-Alb (n = 6). Systemic and microhemodynamic parameters following resuscitation were identical to those obtained with the same protocol using MalPEG-Hb (1). However, parameters related to microvascular oxygen distribution were significantly lower in the MalPEG-Alb group compared with the previous data from the MalPEG-Hb group in that tissue oxygen partial pressure was 5 +/- 2 mmHg (vs. 8 +/- 3 mmHg, P < 0.05), oxygen delivery was reduced to 60 +/- 27% (P < 0.05), and oxygen consumption was reduced to 69 +/- 28% (P < 0.05). Both molecules were matched in composition (4.2 g/dL) and surface chemistry. MalPEG-Alb colloid osmotic pressure was 37 mmHg (vs. 49 mmHg for MalPEG-Hb), and viscosity was 2.7 cP (vs. 2.5 cP for MalPEG-Hb). The present results show that both solutions are efficacious plasma expanders and that the hemoglobin-based solution provides improved oxygen distribution and tissue Po2 in the hamster chamber model.

The microvascular distribution of oxygen was studied in the arterioles and venules of the awake hamster window chamber preparation to determine the contribution of vascular smooth muscle contraction to oxygen consumption of the microvascular wall during arginine vasopressin (AVP)-induced vasoconstriction. AVP was infused intravenously at the clinical dosage (0.0001 IU·kg−1·min−1) and caused a significant arteriolar constriction, decreased microvascular flow and functional capillary density, and a substantial rise in arteriolar vessel wall transmural Po2 difference. AVP caused tissue Po2 to be significantly lowered from 25.4 ± 7.4 to 7.2 ± 5.8 mmHg; however, total oxygen extraction by the microcirculation increased by 25%. The increased extraction, lowered tissue Po2, and increased wall oxygen concentration gradient are compatible with the hypothesis that vasoconstriction significantly increases vessel wall oxygen consumption, which in this model appears to constitute an important oxygen-consuming compartment. This conclusion was supported by the finding that the small percentage of the vessels that dilated in these experiments had a vessel wall oxygen gradient that was smaller than control and which was not determined by changes in tissue Po2. These findings show that AVP administration, which reduces oxygen supply by vasoconstriction, may further impair tissue oxygenation by the additional oxygen consumption of the microcirculation.

Blood losses are usually corrected initially by the restitution of volume with plasma expanders and subsequently by the restoration of oxygen-carrying capacity using either a blood transfusion or possibly, in the near future, oxygen-carrying plasma expanders. The present study was carried out to test the hypothesis that high-plasma viscosity hemodilution maintains perfused functional capillary density (FCD) by preserving capillary pressure. Microvascular pressure responses to extreme hemodilution with low- (LV) and high-viscosity (HV) plasma expanders and an exchange transfusion with a polymerized bovine cell-free Hb (PBH) solution were analyzed in the awake hamster window chamber model ( n = 26). Systemic hematocrit was reduced from 50% to 11%. PBH produced a greater mean arterial blood pressure than the nonoxygen carriers. FCD was higher after a HV plasma expander (70 ± 15%) vs. PBH (47 ± 12%). Microvascular pressure spanning the capillary network was higher after a HV plasma expander (16–19 mmHg) compared with PBH (12–16 mmHg) and a LV plasma expander (11–14 mmHg) but lower than control (22–26 mmHg). FCD was found to be directly proportional to capillary pressure. The use of a HV plasma expander in extreme hemodilution maintained the number of perfused capillaries and tissue perfusion by comparison with a LV plasma expander due to increased mean arterial blood pressure and capillary pressure. The use of PBH increased mean arterial pressure but reduced capillary pressure due to vasoconstriction and did not maintain FCD.

In this study, we assessed the magnitude of the erratic deviations in the radial position of red blood cells (RBCs) in the laminar flow regime of arterioles in a hamster window preparation and the intraluminal Po2 profile to determine whether this variability affects the intraluminal distribution of oxygen in conditions of normal hematocrit and hemodilution. A gated image intensifier was used to visualize fluorescently labeled RBCs in tracer quantities and obtain multiple measurements of RBC radial and longitudinal positions at time intervals on the order of 5 ms within single arterioles (diameter range 40–95 μm). RBCs in the velocity range of 0.3–14 mm/s exhibit a mean coefficient of variation of velocity of 16.9 ± 10.5% and a SD of the radial position of 1.98 ± 0.98 μm. Both quantities were inversely related to hematocrit, and the former was significantly lowered by hemodilution. Our experimental results presented very similar values and shape compared with the intraluminal oxygen profile derived theoretically for normal hematocrit, suggesting that shear-augmented diffusion due to the measured radial displacement of RBCs did not significantly affect oxygen diffusion from blood into the arteriolar vessel wall. Po2 profiles in the arterioles assumed an increasingly parabolic configuration with increasing levels of hemodilution.

Microcirculatory changes in the window chamber preparation in Syrian golden hamsters, secondary to chronic hypoxia adaptation, are presented herein. Adaptation was attained by keeping animals in a 10% oxygen environment for 1 wk and 5% the following week. The following groups were studied: group 1, adapted to chronic hypoxia and kept in a 5% oxygen environment throughout the experiment; group 2, adapted to chronic hypoxia and kept in a 21% oxygen environment 24 h before and during the experiment; and group 3, control. Adaptation caused venule enlargement and hematocrit increase (68.6 ± 2.44 in group 1, 70 ± 2.66 in group 2, and 43.27 ± 2.30 in group 3; P < 0.05). Whereas heart rate decreased in adapted animals, blood pressure remained constant. Group 1 presented alkalosis, hypocapnia, and hypoxemia. The adapted groups had decreased blood flow velocity in arterioles and veins. We found no difference in microvasculature oxygen tension between groups 2 and 3; however, the number of capillaries with flow was markedly reduced in group 1 but significantly increased in group 2. Our findings suggest that, as an adaptation to hypoxia, erythropoiesis may prove beneficial by increasing blood viscosity and shear stress, leading to vasodilatation, in addition to the increase in oxygen-carrying capacity. Calculations show that oxygen extraction in the tissue of the window chamber model was significantly lowered in adapted animals breathing 5% oxygen, but was unchanged from the control when breathing 21% oxygen, even though blood hemoglobin content was increased from 14.5 ± 0.07 g/dl at control to 21.04 ± 1.24 g/dl in the adapted animals ( P < 0.05).

The microvascular effects and hemodynamic events following exposure to normobaric hyperoxia (because of inspiration of 100% O2) were studied in the awake hamster window chamber model and compared with normoxia. Hyperoxia increased arterial blood Po2to 477.9 ± 19.9 from 60.0 ± 1.2 mmHg ( P < 0.05). Heart rate and blood pressure were unaltered, whereas cardiac index was reduced from 196 ± 13 to 144 ± 31 ml · min–1· kg–1( P < 0.05) in hyperoxia. Direct measurements in the microcirculation showed there was arteriolar vasoconstriction, reduction of microvascular flow (83% of control, P < 0.05), and functional capillary density (FCD, 74 ± 16% of control), the latter change being significant ( P < 0.05). Calculations of oxygen delivery and oxygen consumption based on the measured changes in microvascular blood flow velocity and diameter and estimates of oxygen saturation corrected for the Bohr effect due to the lowered pH and increased Pco2showed that oxygen transport in the microvascular network did not change between normal and hyperoxic condition. The congruence of systemic and microvascular hemodynamics events found with hyperoxia suggests that the microvascular findings are common to most tissues in the organism, and that hyperoxia, due to vasoconstriction and the decrease of FCD, causes a maldistribution of perfusion in the microcirculation.

Cardiac output (CO) measurements based on indicator dilution, microspheres, thermodilution and ultrasonic sensors are not suitable for small animals, because of limited blood volume, high heart rates and small caliber vessels that do not allow probe placement within the heart. We developed a modified thermodilution method to measure CO in awake animals weighing less than 100 g. Under anesthesia, the animal is instrumented with a jugular vein catheter placed proximal to the subclavian vein and a temperature probe in the carotid artery with the thermocouple positioned at the aortic arch. Two days after implantation, room temperature saline is injected (150 microl) into the jugular catheter and the temperature change recorded. This system uses the temperature probe as a digital feedback control: (1) to minimize recirculation error; (2) to adjust baseline temperature, thereby increasing sensitivity to small changes in temperature; and (3) to stabilize animal core temperature. The system was calibrated using a laboratory bench model with anatomically scaled components. CO was measured (n=29) in 16 hamsters (65-115 g), and was linearly related to body weight. Cardiac index (CI=CO/weight) was 197.0 +/- 18.8 (ml/min)/kg. Repeated measurements were made. This technique allows correlating systemic flow changes to be correlated to those measured in the microcirculation of window chamber preparations.

An automated system for p O 2 analysis based upon phosphorescence quenching was tested. The system was calibrated in vitro with capillary samples of saline and blood. Results were compared to a conventional measuring procedure wherein p O 2 was calculated off-line by computer fitting of phosphorescence decay signals. P O 2 measurements obtained by the automated system were correlated ( r 2 = 0.98) with readings simultaneously generated by a blood gas analyzer, accuracy being highest in the low (0–20 mm Hg) and medium p O 2 ranges (21–70 mm Hg). Measurements in in vivo studies in the hamster skin-fold preparation were similar to previously reported results. The automated system fits the phosphorescence decay data to a single exponential and allows repeated p O 2 measurements in rapid sequence.

A phospholipid vesicle encapsulating hemoglobin (Hb vesicle, HbV) has been developed to provide O2-carrying capacity to plasma expanders. Its ability to restore systemic and microcirculatory conditions after hemorrhagic shock was evaluated in the dorsal skinfold window preparation of conscious hamsters. The HbV was suspended in 8% human serum albumin (HSA) at Hb concentrations of 3.8 g/dl [HbV(3.8)/HSA] and 7.6 g/dl [HbV(7.6)/HSA]. Shock was induced by 50% blood withdrawal, and mean arterial pressure (MAP) at 40 mmHg was maintained for 1 h by the additional blood withdrawal. The hamsters receiving either HbV(3.8)/HSA or HbV(7.6)/HSA suspensions restored MAP to 93 ± 14 and 93 ± 10 mmHg, respectively, similar with those receiving the shed blood (98 ± 13 mmHg), which were significantly higher by comparison with resuscitation with HSA alone (62 ± 12 mmHg). Only the HSA group tended to maintain hyperventilation and negative base excess after the resuscitation. Subcutaneous microvascular blood flow reduced to ∼10–20% of baseline during shock, and reinfusion of shed blood restored blood flow to ∼60–80% of baseline, an effect primarily due to the sustained constriction of small arteries A0 (diameter 143 ± 29 μm). The HbV(3.8)/HSA group had significantly better microvascular blood flow recovery and nonsignificantly better tissue oxygenation than of the HSA group. The recovery of base excess and improved tissue oxygenation appears to be primarily due to the increased oxygen-carrying capacity of HbV fluid resuscitation.

Background Heparanase is the major enzyme involved in degradation of endothelial heparan sulfates, which is associated with impaired endothelial nitric oxide synthesis. However, the effect of heparan sulfate chain length in relation to endothelial function and nitric oxide availability has never been investigated. We studied the effect of heterozygous mutations in heparan sulfate elongation genes EXT 1 and EXT 2 on endothelial function in vitro as well as in vivo. Methods and Result Flow‐mediated dilation, a marker of nitric oxide bioavailability, was studied in Ext1 +/− and Ext2 +/− mice versus controls (n=7 per group), as well as in human subjects with heterozygous loss of function mutations in EXT 1 and EXT 2 (n=13 hereditary multiple exostoses and n=13 controls). Endothelial function was measured in microvascular endothelial cells under laminar flow with or without si RNA targeting EXT 1 or EXT 2 . Endothelial glycocalyx and maximal arteriolar dilatation were significantly altered in Ext1 +/− and Ext2 +/− mice compared to wild‐type littermates (glycocalyx: wild‐type 0.67±0.1 μm, Ext1 +/− 0.28±0.1 μm and Ext2 +/− 0.25±0.1 μm, P Ext1 +/− 15.2±1.4% and Ext2 +/− 13.8±1.6% P P EXT 1 and EXT 2 under flow led to significant upregulation of endothelial nitric oxide synthesis and phospho‐endothelial nitric oxide synthesis protein expression. Conclusions Our data implicate that heparan sulfate elongation genes EXT 1 and EXT 2 are involved in maintaining endothelial homeostasis, presumably via increased nitric oxide bioavailability.

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 toxic side effects of early generations of red blood cell substitutes have stimulated development of more safe and efficacious high-molecular-weight polymerized hemoglobins, poly(ethylene glycol)-conjugated hemoglobins, and vesicle-encapsulated hemoglobins. Unfortunately, the high colloid osmotic pressure and blood plasma viscosity of these new-generation materials limit their application to blood concentrations that, in general, are not sufficient for full restoration of oxygen-carrying and -delivery capacity. However, these materials may serve as oxygen therapeutics for treating tissues affected by ischemia and trauma, particularly when the therapeutics are coformulated with antioxidants. These new oxygen therapeutics also possess additional beneficial effects owing to their optimal plasma expansion properties, which induce systemic supraperfusion that increases endothelial nitric oxide production and improves tissue washout of metabolic wastes, further contributing to their therapeutic role.

Phospholipid vesicles encapsulating purified hemoglobin (HbV) were developed to provide O2-carrying capacity to plasma expanders. Microvascular perfusion was determined for HbV with different O2affinity (P50= 9, 16, and 30 mmHg) prepared by coencapsulating pyridoxal 5′-phosphate (PLP) at the molar ratios of [PLP]/[Hb] = 0, 0.5, and 3, respectively (cf. hamster blood, P50: 28 mmHg), and suspended in 8 g/dl human serum albumin (HSA). Eighty percent of the red blood cell (RBC) mass of conscious Syrian golden hamsters fitted with dorsal skinfold windows was substituted with either of the HbV-HSA suspensions, washed hamster RBC suspended in HSA (RBC-HSA), and HSA alone. All three HbV-HSA groups and RBC-HSA groups showed stable blood pressure and heart rate, which could not be sustained with HSA alone. Only the HbV (P50= 9)-HSA group showed an increase in arterial O2tension (89.8 ± 14.7 mmHg, baseline 58.4 ± 4.0 mmHg) because of hyperventilation, and microvascular perfusion was decreased, indicating that facilitated O2unloading of HbV by decreasing the O2affinity (increasing P50) with PLP as an allosteric effector is important. Microvascular perfusion and microvascular and interstitial O2tensions in the HbV (P50= 16 and 30)-HSA groups were significantly higher than those in the HSA group. The O2release rate from the HbV was 18–32 s−1vs. 4.4 s−1for RBC. Functional capillary density was improved from 17 to 41% on average by decreasing P50from 30 to 16 mmHg, which appears to be an optimal value for the P50in this system.

The unanesthetized hamster dorsal skinfold preparation was used to monitor diameters and blood flow rates in resistance arteries (small arteries, A0: diameter, 156 ± 23 μm) and capacitance vessels (small veins, V0: 365 ± 64 μm), during 45 min of hemorrhagic shock at 40 mmHg mean arterial pressure (MAP) and resuscitation. A0 and V0 vessels constricted significantly to 52 and 70% of the basal values, respectively, whereas precapillary arterioles (A1–A4, 8–60 μm) and collecting venules (VC–VL, 26–80 μm) did not change or tended to dilate. Blood flow rates in the microvessels declined to 0 and V0 diameters even 2 h after resuscitation (71 ± 14% and 81 ± 18%, respectively, of basal value), whereas other vessels did not change significantly. The behavior of A0 diameter coincided with the incomplete recovery of blood flow rates in all the vessels (ca. 50%) according to Poiseuille’s law, and the incomplete recovery of functional capillary density (ca. 75%). Resuscitation with 8% human serum albumin in saline (HSA) tended to show higher levels of A0 constriction and A4 dilation and lowered blood flow rates. Resuscitation with SAB restored tissue[Formula: see text] 27 ± 10 mmHg after 2 h, which was near control levels (28 ± 5 mmHg), whereas resuscitation with HSA caused tissue [Formula: see text] to remain significantly depressed (6 ± 2 mmHg), and flow rates were significantly lower than resuscitation with SAB. These results indicate that response of the A0 vessels is the crucial determinant of blood flow in the observed area. The constriction of A0 may help sustain MAP, and constriction of V0 may enhance blood redistribution from the skin to the vital organs under the hypotensive condition.